Light as an Energy Source and Information Carrier in Plant Physiology

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Light as an Energy Source and Information Carrier in Plant Physiology Edited by

Robert C. Jennings and

Giuseppe Zucchelli University of Milan Milan, Italy

and

Francesco Ghetti and

Giuliano Colombetti CNR Institute of Biophysics Pisa, Italy

Plenum Press New York and London Published in cooperation with NATO Scientific Affairs Division

Proceedings of a NATO Advanced Study Institute on Light as Energy Source and Information Carrier in Plant Photophysiology, held September 26 - October 6, 1994, in Volterra, Italy

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ISBN-13: 978-1-4613-8039-9

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© 1996 Plenum Press, New York

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PREFACE

A NATO Advanced Study Institute on "Light as Energy Source and Information Carrier in Plant Photo physiology" was held at Volterra, Italy, from September 26 to October 6, 1994, in order to consider the fundamental role that light plays in plant growth and development. This book summarises the main lectures given at this meeting which concentrated on both photochemical energy conversion and signalling (photosensing) aspects.

Light harvesting and conversion into chemical energy in photosynthesis occurs at the level of chlorophyll/carotenoid containing photosystems in plants. Pigments are non­covalently bound to a variety of polypeptides which serve as a specific scaffolding, necessary to determine the energy coupling between pigments and thus allowing rapid excitation energy trasfer from the antenna to the special reaction centre chlorophylls. Data from transient, time resolved spectroscopies, in the femtosecond and picosecond domain, together with model calculations, suggest that this process occurs in the 20-100 picosecond time span. The special ~ll u~ture of reaction centre complexes, ensures rapid primary charge separation, probably in the order of 1-3 picoseconds, with subsequent charge stabilisation reactions proceeding in the hundreds of picoseconds range. The recently resolved crystallographic structure of LHCII, the principal antenna complex of plants, allows precise determination of pigment-pigment distances and thus permits calculation of approximate chlorophyll-chlorophyll Forster hopping rates, which are in good agreement with time resolved measurements.

A number of lectures dealt with the complex phenomenon of light induced inhibition of photosynthetic activity (photoinhibition) and the protective mechanisms plants have evolved to counteract this. These processes occur mainly at the level of photo system II with reaction centres of this photo system being particularly sensitive to high iight fluxes. Quenching mechanisms, which thermally degrade excited states and which appear to be mainly localised in the external antenna complexes of photo system II, are effective in reducing excited state levels many-fold, in a fashion which is modulated by the light flux itself. The fact that the chlorophyll spectral forms are not organised as an energy "funnel" with respect to reaction centres in photosystem II increases the effectiveness of such control mechanisms.

The effect of ultraviolet radiation on both terrestrial and aquatic ecosystems was also dealt with. Increases in ultraviolet fluxes, particularly interacting with other environmental stress factors, can induce significant damage in plants, with photosystem II being particularly sensitive. The effect of ultraviolet radiation on the vertical distribution phytoplankton in the water column may be significant.

Photosensing processes are based on a variety of strategies which detect either time­integrated light quantity, spectral quality, light direction via intracellular light gradients in which such optical phenomena as absorption, refraction, intereference and dichroism are involved. Action spectroscopy is the principal technique which permits identification of a photoreceptor though problems associated with host-induced absorption shifts, absorption

v

screening and scattering can be considerable. Genetic and molecular biological manipulations can also be an important tool in identifYing photoreceptors as demonstrated by recent studies on the different morphogenetic roles of the family of phytochrome types via the use of specific phytochrome mutants. Phytochrome, by far the best characterised photomorphogenic photoreceptor, has ideal absorption characteristics to sense the dramatic changes in the terrestrial light environment within and underneath plant canopies. Progress has been made in recent years in understanding phytochrome phytochemistry. Phototransformation involves Z{l5)--E{l5) isomerisation which occurs within a few picoseconds. Subsequent changes in the a-helical folding near the N-terminus may be important in determing the biochemical and physiological activity of the phototransformed molecule.

vi

R.C. Jennings G. Zuc~helli F. Ghetti G. Colombetti

Milano, Italy Pisa, Italy

CONTENTS

Photosynthesis: An Overview ............................................. . G. Forti

Photosynthetic Electron Transfer and Energy Transduction in Plants . . . . . . . . . . . . . .. 17 D. R. Ort and J. Whitmarsh

Specific Features of Excitation Migration in Photosynthesis ..................... 31 A. Yu. Borisov

Biochemistry and Molecular Biology of Pigment Binding Proteins . . . . . . . . . . . . . . .. 41 R. Bassi, E. Giuffra, R. Croce, P. Dainese, and E. Bergantino

Spectral Heterogeneity and Energy Equilibration in Higher Plant Photosystems ..... 65 R. C. Jennings, G. Zucchelli, L. Finzi, and F. M. Garlaschi

Photosynthetic Reaction Centers ........................................... 75 P. Mathis

Photoinhibition of Photosynthesis .......................................... 89 N. R. Baker

Nonphotochemical Quenching of Chlorophyll Fluorescence ..................... 99 P. Horton

Regulation of Excited States in Photosynthesis of Higher Plants .................. 113 J.-M. Briantais

Chirally Organized Macrodomains in Thylakoid Membranes. Possible Structural and Regulatory Roles .................. : ................................ 125

G. Garab

Interaction ofUV Radiation with the Photosynthetic Systems .................... 137 J. F. Bornman

Molecular Basis of Photoreception .......................................... 147 F. Lenci, N. Angelini, and A. Sgarbossa

Photomorphogenic Systems ............................................... 159 W. R. Briggs, E. Liscum, P. W. Oeller, and J. M. Palmer

vii

Overview of Photos en sing in Plant Physiology ................................ 169 W. Haupt

Mechanisms of Photoreception: Energy and Signal Transducers .................. 185 D.-P. Hader

Light Signal Transduction Mediated by Phytochromes .......................... 197 D. Sommer and P.-S. Song

Light Penetration into the Canopy of Terrestrial Ecosystems ..................... 219 M. G. Holmes

Light Penetration and Effects on Aquatic Ecosystems ........................... 231 D.-P. Hader

Interception of Light and Light Penetration in Plant Tissues ..................... 243 M. G. Holmes

Photosensory Transduction in Flagellated Algae ............................... 263 R. Marangoni, E. Lorenzini, and G. Colombetti

Action Spectroscopy ..................................................... 275 F. Ghetti and G. Checcucci

Photoregulation of Fungal Gene Expression .................................. 285 E. Cerda-Olmedo and L. M. Corrochano

Phototropism in Phycomyces .............................................. 293 E. Cerda-Olmedo and V. Martin-Rojas

What Can Errors Contribute to Scientific Progress? ............................ 301 W. Haupt

Index ................................................................. 311

viii

Light as an Energy Source and Information Carrier in Plant Physiology

PHOTOSYNTHESIS: AN OVERVIEW.

Giorgio Forti

Centro di Studio CNR sulla Biologia Cellulare e Molecolare delle Piante. Dipartimento di Biologia dell'Universita di Milano, Via Celoria 26, Milano, Italy.

INTRODUCTION

Oxygenic photosynthesis of green plants and cyanobacteria utilizes water as an electron donor, light energy and CO2 to generate carbohydrates and other organic substances, according to the overall equation :

(1)

The photochemical system involved, which is bound to the photosynthetic membranes (the thylakoids) utilizes two photochemical reactions in series to transfer electrons against the electrochemical gradient from H20 (Em= 810m Y at pH 7) to the iron-sulphur protein ferredoxin (Fd, Em= -420 mY at pH 7). Reduced ferredoxin is then utilized by a membrane bound flavoprotein to reduce NADP (Em= -320 mY at pH 7). The electrochemical work of 1.23 ev is accomplished through the cooperation of two photochemical reactions. Electron transport is coupled to the synthesis of ATP from ADP+Pi (inorganic OIthophosphate).

The stable products of photosynthetic electron transport ATP and NADPH are then utilized to activate and reduce CO2 to the level of carbohydrates by a muItienzyme system present as a dense protein solution in the stroma of the chloroplasts (or in the cytoplasm of cyanobacteria) where the thylakoids are embedded. This latter process will not be discussed in this paper, which is limited to the photochemical events and electron transport producing NADPH and ATP.

In green plants, including the unicellular green eukariotes, the overall process of photosynthesis is therefore accomplished within the chloroplast (see fig. 1), through the cooperation of the events occurring within and on the surface of the thylakoids (light absorption and excitation energy migration to the reaction centres, primary photochemical reactions, electron transport and ATP synthesis) and those occurring in the stroma (C02

assimilation).

Light as an Energy Source and Information Carrier in Plant Physiology Edited by Jennings et al.. PleI11Ull Press, New York, 1996

LIGHT ABSORPTION AND UTILIZATION: THE "PHOTOSYNTHETIC UNIT".

The photosynthetic pigments involved in oxygenic photosynthesis are the chiorophylls a and b (chla and chlb) in green plants, and chi a and the phycobilins (phycocyanin and phycoerithrin, respectively, in Cyanobacteria and the red algae). The structures of chla, chlb and the chromophore of phycocyanin are shown in fig.2, and the absorption of the photosynthetic pigments (dissolved in organic solvents) are compared with the solar spectrum on the Earth surface in fig.3. It can be seen that the photosynthetic pigments absorb energy through most of the visible part of the solar spectrum, suggesting the evolutionary adaptation of the photosynthetic apparatus to the ambient conditions. It must be considered also that the pigments in the photosynthetic apparatus of plants are bound to different specific proteins in macromolecular complexes, and these are organized in an ordered topology in the photosynthetic membranes (the thylakoids). As a consequence of this, the absorption spectra of the pigments "in vivo" are narrowed and slightly red-shifted as compared to their spectra in organic solvents, and a spectral heterogeneity of the light absorbing chlorophylls is observed (see Jennings, this volume). In the case of higher plants, thanks to the peculiar anatomy of the leaves the high light scattering of the tissues increases the light path through the pigment containing cells, and even radiation of wavelengths between 500 and 600 nm, poorly absorbed by the chlorophylls, is efficiently absorbed and utilized (Garlaschi et aI. , 1989) (see fig. 4).

10) Granum

SIroma IO_IlOe

Grana lamellae

Figure 1. (a) Cut-away representation ofa chloroplast showing three-dimensional structure. (b) Section ofa chloroplast in the cytoplasm ofa spinach leaf:(c) a single granum within a chloroplast showing the stacks of thylakoids and the interconnecting stromal thylakoids. Cw, cell wall; cm, cytoplasmic membrane; m, mitochondrion; ce, chloroplast envelope; g, granum, consisting of stacks of thylakoids; s, stroma: st, starch granule.

2

Structure of bacteriochlorophyll

Chromopnore of phycocyanin Struc:ure oi chlorophyll a

COOHCOOH I I

CH, CH, CH, CH, I I I I

CH M CII, CH, M CH,

M. I I - M_I -!.-.!IHO...L-J O~~M!'iJl.7' N~l ·~r W'~O

c o °a " If) D ~

: I ; I ,...Y .... I Figure 2. The stlUctures of bacteriochlorophyll, the chlorophylls a and b and the chromophore of phycocyanin.

_ Chloroph lib Solar spectrum

400 500 600 700 Wavelength (nm)

Figure 3. The spectlUm of solar radiation reaching the Earth surface and the absorption spectlUm of photosynthetic pigments.

3

1.0

t-

I 0.5

450 550 650 Wavelength ( nm)

\ • I I I \ \ , I \ \ \ • I \ \ \ • I

I I

• \

750

Figure 4. The absorplance (incident minus trasmitlcd radiation, I-T) spectra of a spinach leaf and of an equivalent chloroplast suspension (on a chlorophyll per unit area basis). Full dots: leaf containing 29.5 Ilg Chi cm·2; empty squares: chloroplast. The spectra were corrected for light scattering and reflectien. See Garlaschi et aI., 1989, from which this figure is taken).

The photochemical utilization of the light absorbed involves two steps: the migration of the excitation energy (in the form of the excited singlet state of the chi molecule) within a large array of properly packed pigment molecules (the light absorbing "antenna") which eventually brings the excited state to a special chla pair (the "reaction centre", RC) where the singlet is oxidized by an electron acceptor in the primary photochemical reaction. The oxidized RC chla is then reduced by a primary electron donor, so closing the turnover of the RC, and the light dependent charge separation is performed. The primary acceptor is then reoxidized by a chain of electron carriers of progressively higher midpoint potential (Em), while the primary donor is reduced by a chain of electron donors. The RC centre requires that the primary acceptor and the primary donor are regenerated in the proper redox state (respectively oxidized and reduced) to perfonn the next photochemical charge separation. A scheme of the functioning of a RC is shown in fig.S; more details are found in the contributions by Ort and by Mathis (this volume).

Figure 5. Scheme of primary photochemical reaction at a RC (see text). AI,DI: primary electron acceptor and donor, respectively; A2, D2; secondary acceptor and donor, respectively.

The concept of the differentiated role of chi as light harvesting antenna and RC performing photochemistry was established by the classical experiments of Emerson and Arnold (Emerson & Arnold, 1932), who demonstrated that upon illumination of Chlorella cells with light flashes of a few us duration, the maximal O2 yieldlflash (in the steady-state) was obtained only if an adequate dark period (of ca. 20 ms) was allowed between flashes.

4

They interpreted correctly their observations as indicating that the products of the very fast photochemical reactions must be utilized by kinetically much slower chemical reactions ("dark reactions") to perform the overall photosynthetic process. They thus defined the "photosynthetic unit" (PU) as the complex structure including antenna pigments, reaction centres, electron carriers and the enzyme systems required to perform O2 evolution (and CO2 assimilation). In Emerson and Arnold's experiments, the size of the PU was of ca. 2400 chi molecules/ O2 evolved x flash (under conditions of maximum oxygen yieldlflash). While the concept of PU is a basic one to understand photosynthesis, the size of PU has been found to vary in different plants and different growth conditions: in general, growth under low light intensity induces a larger antenna for each RC, and vice-versa.

THE EMERSON EFFECT AND THE HILL SCHEME OF PHOTOSYNTHETIC ELECTRON TRANSPORT.

The quantum yield of photosynthesis (defined as the ratio electron transportedlhv absorbed) or its reciprocal quantum requirement, is not constant as a function of the absorption spectrum of the photosynthetic pigments: the classical observations of Emerson and collaborators (Emerson, 1957) established that at the far-red end of the chi absorption spectrum the quantum yield drops rather abruptly (above 685 nm; the so-called red drop), and that the superimposition of a beam oflower lambda produces a superadditive effect (the "Emerson enhancement"). Such enhancement was also observed when the long and shorter lambda lights were separated by a dark interval of a few seconds (Myers & French, 1960), an observation which rules out the possibility of a cooperation between excited states produced by the absorption of different oscillators, and indicate that the enhancement effect must involve much more stable chemical intermediates.

Emerson's observations are easily interpreted in the frame of the "Z scheme" of Hill (Hill & Bendall, 1960) (see an up-to-date version of it in fig.6): two photochemical reactions are operating in series, and the electron acceptor of one of them (reaction 2) is reoxidized by the other reaction (reaction 1), through a chain of electron carriers, in sequence according to the electrochemical gradient. The two photochemical reactions are catalyzed by two different RCs, each one collecting excitation energy on its antenna consisting of ca. 300 chi molecules, organized in several chi-protein complexes (see Bassi, this volume). The functional unit consisting of a RC and its antenna is defined a photosystem (PS); the Emerson photosynthetic unit, defined by experiments in vivo where photosynthesis was measured as O2 evolution, consists of PS2, PS I, the electron transport chain connecting them and the enzyme system performing CO2 assimilation. The absorption spectrum of PS2, which oxidizes water, is largely overlapping but not identical with that of PS 1, which reduces ferredoxin then NADP and finally CO2. PSI absorption extends into the far-red up to 720-730 nm, where PS2 has negligible absorption. These spectral differences account for the Emerson enhancement effect. While light of A>ca. 690 nm is very poorly absorbed by PS2 and therefore has very little efficiency for O2 evolution, it activates PS I thereby reducing ferredoxin and reoxidizing all the electron carriers of the intersystem chain. As a consequence, the addition of shorter wavelength radiation activating PS2 will produce a cooperative effect (superadditive electron transport from H20 to ferredoxin). Such effect has a spectrum with peaks at the wavelengths where PS2 absorption exceeds PS 1 absorption. This can be observed in vivo (Emerson, I 957;Canaani & Malkin, 1984) as well as with isolated thylakoids (Govindjee et aI., 1964).

The basic criterium for establishing the Z scheme of electron transport (the Hill scheme) has been the observation that certain electron carriers, such as cytochrome f (Duysens et aI., 1961) and plastoquinone (see review by Debus, 1992) are oxidized by PSI activation and reduced by PS2 activation. This property has been used to establish whether a newly discovered electron carrier belongs to the intersystem electron transport chain.

5

Ul ..... o >

-1.0

-0.5

0.0

+0.5

+1.0

* Chla I

:\ * P680 , Chla

t \ ~~, I Pheo £ Fe·S_o_o : 1 + H+ lc~t:so_o~ -

- H out • out ,- L'FNR o~ Ie. I Vc?)" I C

I 1 i Cyclic (?,) NADPo2 o. i pathway I g , A.. ,/ I o~

I .... 0 _ PO ( POOl), I Q; B 02 0 :c I I) ~

I I 0;:"2

~ ~2-°2

e

Figure 6. Scheme of the electron transport pathway in o~"ygenic photosynthesis. For explanations see text. On the left, the Em potentials of the electron carriers are represented. ASC, ascorbate; MDA, the radical monodehydroascorbate produced in the l-electron oxidation of ascorbate.

At the reducing side of PS2, a molecule of pheophytin (pheo) is reversibly reduced by the RC chla, named P680 after the peak wavelength of its bleaching upon oxidation (Debus, 1992;Witt, 1990; see also the contributions by art and by Mathis, this volume). This reaction is performed in few ps; ca. 400-600 ps are then needed to transfer the electron from pheo- to the D2 protein bound plastoquinone molecule, Qa (Schatz et a!., 1987). Qa is a one electron acceptor, so its reduction is a one quantum-one electron process (see Mathis, this volume; also the reviews Debus, 1992; and Renger, 1993). The next carrier is a plastoquinone (Qb) molecule reversibly bound to the D1 protein of the RC (the same polypeptide which binds P680). Qb accepts two electrons produced by two successive photochemical events, and when fully reduced it is protonated and PQH2 dissociates from the RC, because the binding constant to D1 of the protonated species PQH2is much smaller than that of PQ. The Qa-Qb electron transfer is the step where the transition from a one electron ~o two electron transfer occurs. PQH2 rapidly diffuses into the lipid phase of the membrane, and constitutes a pool common to many PS2-PS 1 units (Cramer & Knaff, 1989). It is reoxidized by the complex cytochrome f-cytochrome b6-Rieske iron-sulfur centre. The oxidation of plastoquinol is the rate limiting step of photosynthetic electron transport (it has an half-time· of ca. 10 ms), releases protons into the lumen and is controlled by the difference ofH+ electrochemical gradient between the internal (lumenal) and the external (stromal) side of the thylakoids. The electrons can be recycled across the membrane by the two haem groups of cyt.b563 (so named after the peak of its a band), a process which re-reduces PQ

6

and as a result transfers more protons from the stromal to the lumenal side of the membrane (Cramer & Knaff, 1989; see also Ort, this volume), contributing to the creation of the proton electrochemical potential which is the source of energy used for the synthesis of ATP coupled to electron transport (see below). Cyt.f(Em==365 mY, see fig.6) is reoxidized by the copper protein plastocyanin (PC, Em=380 mY), which is present in solution in the thylakoid lumen and can bind to PSI, where it is reoxidized by the photoxidized RC in about 15-20 ~lS. The RC chI a of PSI, (P700, after the absorption peak of its bleaching upon oxidation) is photooxidized in the ps time scale (Shuvalov et aI., 1986; see also Mathis, this volume). The primary acceptor is a chla molecule, which becomes reduced to the anion radical; this is reoxidized by a molecule of phylloquinone. Three iron-sulfur centres are membrane bound intermediates before the reduction of the iron-sulfur protein ferredoxin (Fd), which is in solution in the chloroplast stroma (Forti & Grubas, 1985). Fd forms a one/one complex with the thylakoid bound flavoprotein ferredoxin-NADP reductase (FNR) (Foust et al., 1969), which simultaneously binds NADP. The electron transfer to bound NADP occurs probably in two steps through the intermediate anionic radical of FAD, the prosthetic group ofFNR.

On the oxidizing side ofPS2, the primary donor to PS2 is the tyr residue 161 of the Dl protein (see reviews Debus, 1992; Witt, 1990; Renger, 1993). Its one electron oxidation occurs in the ns time scale; tyr 161 is then reduced by the Mn containing water oxidation complex. It has been shown that when single turnover, saturating flashes are fired on dark adapted green cells or isolated thylakoids, O2 evolution has a periodicity of 4, starting however at the third flash (Kok et aI., 1970). These observations were interpreted to indicate that each turnover of PS2 is required to advance from a state, So, to So+': S4 oxidizes 2 molecules of H20, thus returning to So. In the dark, an equilibrium must exist between S, and So, with a ratio of ca. 4 S,/So, to account for the observation that ca. 4 times more O2 is produced by the third flash than by the 4th. The periodicity of O2 production is damped with increasing number of flashes, and is completely lost usually after 20-25 flashes. This is due to the failure to utilize the flash (misses) and double hits (advances from So to So+2 during the flash) statistically distributed among the large number of PS2 units. The "S state" indicates the number of positive charges accumulated in the Mn enzyme: when 4 positive charges are accumulated (state S4, requiring 4 turnovers of PS2 starting from So), two H20 molecules are oxidized and one O2 molecule is produced. These established facts indicate that dioxygen and not atomic 0 is the product of water oxidation in photosynthesis, and that 4 quanta are required by PS2 to perform the reaction; this means that not less than 8 quanta are required for the overall process which requires an equal number of photochemical events in PS I.

It has been found that other electron acceptors, alternative to Fd, can be reduced at the reducing side of PS 1: one of them is O2, in the so called Mehler reaction, after the name of its discoverer. Univalent reduction of O2 generates the anion radical O2-. The enzyme superoxide dismutase (SOD), present both in thylakoid bound form and in solution in the chloroplast stroma, disproportionates O2- to yield 0 2- and hydrogen peroxide. The overall stoichiometry of such electron transport system is the uptake of one O2/4 electrons transported across the chain. This electron transport pathway is the same as that reducing NADP up to the Fd step, and is coupled to ATP synthesis (Forti & Jagendorf, 1961). The univalent reduction of O2 at the reducing side of PS 1 is a slow reaction (ca. 15 to 20 times slower than NADP reduction). However, the H20 2 formed in the process involving SOD reacts rapidly with ascorbate (which is always present in rather high concentrations in the chloroplast stroma) through the catalysis of ascorbate peroxidase (Miyake & Asada, 1992), producing the radical monodehydroascorbate, MDA (Miyake & Asada, 1992). The latter is also produced by the direct reaction of ascorbate with O2-. MDA is an efficient electron acceptor from PS 1 (Forti & Ehrenheim, 1993): electron transport from H20 to MDA occurs at a rate of about 50% the rate of NADP reduction and competes with NADP for electrons at the reducing side of PS 1 (Forti & Ehrenheim, 1993). The coupling of this electron transport system to ATP formation occurs with the same stoichiometry as in the case of

7

NADP reduction (Forti & Elli, 1995), as would be expected because the same electron transport system and the same photochemical reactions are involved.

Ascorbate has a dual function in this system: (a) as a scavenger of the harmful oxygen species (02- is a very reactive substance, which inactivates many enzymes and structures of the photosynthetic apparatus ), and (b) as a catalyst of electron transport coupled to ATP formation.

Reduced ferredoxin (and possibly other reductants generated by PSI) may be reoxidized by the intersystem electron carriers; cyt.b6 and PQ are the most likely candidates for this function. In this way, a cyclic electron transport around PS 1 is set on, dependent only on PSI photochemical reaction and therefore activated also by light absorption in the far-red end of chI spectrum. This process is coupled to ATP formation in isolated thylakoids (Arnon, 1977) (it is called cyclic photophosphorylation) and was demonstrated to occur in vivo under conditions where the electron flow from the reducing side of PS2 is inhibited at the level of Qa- by a specific inhibitor (Forti & Parisi, 1963), but not under physiological conditions.

PHOTOPHOSPHORYLA TION.

The charge separation of the photochemical reactions of PS2 and PS 1 produce an electric potential difference across the thylakoids, negative on the outer surface (Witt, 1979) because the electron acceptors are located close to the stromal side, whilst the donors are close to the lumenal side of the membranes. Furthermore, the proton-producing reactions of electron transport release the H+ into the lumenal water space (which is a continuum within each chloroplast), whilst the proton binding reactions (the reduction of Qb) take up protons from the stromal side. This is due to the topology of the electron carriers as they are organized in the architecture of the thylakoids. The result of such sovramolecular organization is that the photochemical reactions and the following electron transport are coupled to the formation of an electrochemical potential of protons across the membranes, which are intrinsically very impermeable to H". Such potential is utilized by the membrane bound enzyme ATP synthase for the synthesis of ATP from ADP and inorganic phosphate (Pi), according to the chemiosmotic theory of Mitchell (Mitchell, 1977).

The electrochemical potential of protons can be described by the equation

(2)

(where \1' is the electric potential; the other symbols have their usual meaning) . .1.\11 is formed in the ps time scale both at PS2 and PS 1 RCs, and the two PSs

contribute to the same extent to it (Witt, 1979) . .1.\1' values above 200mV are observed a few ns after the beginning of illumination (Witt, 1979). A slower formation of membrane potential is due to the electron recycling by the cyt b6-cyt f complex (see Ort, this volume). However" the electric potential in the thylakoids decays rather rapidly due to the inward diffusion of anions (Witt, 1979), mainly cr which is transported through a specific channel (Schonknecht et aI., 1988). In the steady state, .1.\1' is usually in the range of 10-30 mY, while most of the .1.G H is accounted for by .1.PH .

The synthesis of ATP is defined by the reaction catalyzed by the ATP synthase:

ADP+Pi~ATP+H20+ H+. This reaction is endergonic, and the value of its .1.Go is =7.6 Kcal/mole, at pH 7.4. Such an ufavourable thermodynamic situation is overcome in the thylakoids (as well as in mitochondria) by the fact that ATP synthase is asymmetrically

8

located across the membrane, and couples the synthesis of ATP to the translocation of protons from the lumenal to the stromal side. The overall reaction is therefore:

(3)

The loss of the proton electrochemical potential is coupled in this reaction to the increase of the chemical potential of ATP synthesis (Mitchell, 1977), and ATP synthesis requires that ~GATP+~GH<O. The opposite reaction, i.e. ATP hydrolysis, occurs when ~GATP>~GH. In this case, protons are translocated into the lumen. Equilibrium is attained when the ATP/ADP+Pi ratio is such that the thermodynamic potential of this system is equal to the proton electrochemical potential. From what was just said and from the fact that proton uptake into the lumen is coupled to electron transport with a defined stoichiometry (at two levels: the oxidation of H20 and the reduction and reoxidation of PQ; see Witt, 1979;), it follows that the size of ~GH controls the rate of electron transport. Inhibition of the electron transport rate by ~GH ("photosynthetic control") can be explained in thermodynamic terms by the fact that PQH2 oxidation comes closer to equilibrium (though never close to it) as the ~GH increases. However, the hypothesis could also be made that lumenal proton concentration may regulate the catalytic activity of electron carriers, such as those of the cyt b6-f complex. The mechanism of "photosynthetic control" is therefore still uncertain.

The coupling of A TP synthesis to photosynthetic electron transport is linked to the impermeability of thylakoids to protons, which makes possible the formation and stability of ~GH , and to the peculiar properties of the ATP synthase of thylakoids. This enzyme is made of two components: one (CFa) is a 4-subunits (4 polypeptides) strongly lipophilic moiety crossing the membrane, the other (CF]) consists offive different polypeptides (a, ~, y, 8, c) and protrudes out of the stromal surface of the thylakoids (Boekema et aI., 1988). The active enzyme is made of3-(a), 3(B), 1 (y), 1(8), l(c) subunits (Boekema et aI., 1988). CFa has the function of proton transfer from the lumen, where it becomes protonated at the low pH value, to the surface of the membrane where it is linked to CF]. CF] binds ADP at the catalytic site only when a threshold value of ~GH is attained; in the presence of Pi, ATP is formed and released into the outer space (the stroma in intact chloroplasts) together with the protons transported across the membrane by CFa. ATP is thus made available for CO2

assimilation in the stroma. One important feature of the ATPase-A TPsynthase of thylakoids is that its activation

requires the conversion of an inactive form into the active one. This activation is endergonic and requires a ~GH value higher than that required for A TP synthesis. The activation is accompanied by the release from the enzyme of a ADP molecule tightly bound to an allosteric site (Junesch & Graber, 1985). The activation reaction is rapidly reversible (Junesch & Graber, 1985; Fromme & Graber, 1990) when ~GH decreases, so that the enzyme is inactive when ~GH is below the threshold required for ATP synthesis. This feature therefore prevents ATP hydrolysis which would otherwise occur. However, the enzyme can be stabilized in its active form upon reduction of an -S-S- bridge (by thiol compounds). Under these conditions, i.e. the enzyme stabilized in its active form, ATP is hydrolyzed at a high rate. The hydrolysis is the complete reversal of the ATP synthesis reaction, including the proton translocation across the membrane, in this case into the lumen. The hydrolysis of ATP generates therefore ~pH (Junesch & Graber, 1985;Fromme & Graber, 1990). ATP hydrolysis can occur in vivo by this mechanism, and the regulation of the redox state of the -S-S-/-SH of the enzyme is performed in the chloroplasts by the ferredoxin-thioredoxin reductase-thioredoxin system (see review by Ort & Oxborough, 1992).

The stoichiometry of protons translocated across the membrane/ ATP synthesised (see equation 3) is still under debate, though 4 seems to be likely. On the other hand, the ratio of H+ translocated inside the lumen/electron transported from H20 to NADP is also uncertain,

9

though most experimental results indicate that it should be close to 2 (Witt, 1979). If these ratios are correct, they would be in agreement with the stoichiometry of ATPINADPH =1 found in most experiments over the last 30 years. Only a few reports of values between 1 and 1.3 can be found in the literature, and they need to be corrected for low levels of the simultaneously occurring Mehler reaction and/or cyclic electron transport around PSI. Such corrections are rather uncertain, because of the experimental difficulty of estimating very low levels of the rates of the interfering reactions, with the accuracy necessary to correct the ratio ATPINADPH observed, not to mention the theoretical difficulty of attributing a stoichiometry to ATP formation in cyclic electron transport. The problem of the ATPINADPH ratio is obviously important to understand the quantum requirement of photosynthesis, as the requirement of 3 ATP/2 NADPH for the assimilation of one CO2 is well established. This implies that the extra ATP needed must be supplied by utilizing more photons. In fact, a large number of measurements of quantum requirement in vivo or in intact chloroplasts assimilating CO2 reported in the literature indicate that not less than 10 quanta/C02 are required. This would be compatible with the utilization of 8 quanta to perform the reduction of2 NADP and the coupled synthesis of2 ATP, and 2 more quanta to produce the extra ATP required.

Phosphorylation coupled to the electron transport triggered by the Mehler reaction seems to be the most likely mechanism to generate ATP in the chloroplasts stroma (where CO2 assimilation occurs) at the rate required for steady state photosynthesis to proceed. The Mehler reaction is known to activate ascorbate oxidation, the production of monodehydroascorbate and the fast electron transport with MDA as acceptor for PS 1 (Miyake & Asada, 1992; Forti & Ehrenheim, 1993) which is coupled to ATP synthesis (see above). Furthermore, it occurs at rates compatible with the observed rates of overall photosynthesis (Forti & Ehrenheim, 1993), whilst the rates of cyclic phosphorylation observed in isolated thylakoids are rather low (Arnon, 1977), and their measure in vivo under physiological conditions is not feasible. Moreover, the functioning of cyclic electron transport around PS I in cyanobacteria has been challenged (Myers, 1987). The real physiological relevance of photophosphorylation coupled to cyclic electron transport around PS I is therefore doubtful.

The switch at the reducing side of PS I from NADP reduction to O2 and MDA reduction is easily understood in terms of the depletion of NADP when NADPH cannot be reoxidized because of the lack of ATP which prevents the formation of 1,3 bis­phosphoglycerate (the electron acceptor for NADPH in the Calvin cycle). As soon as the Mehler reaction plus MDA reduction generate ATP, NADPH is reoxidized and electron transport is switched back to NADP.

"LOCALIZED" AND "DELOCALIZED" PROTON POOLS IN PHOTOPHOSPHORYLATION.

The chemiosmotic theory of Mitchell (Mitchell, 1977) holds that the energy for ATP synthesis in oxidative and in photosynthetic phosphorylation is provided by the proton electrochemical potential established across the energy transducing membranes. In its original and simplest formulation, L\GH is established (as a consequence of electron transport) 'between the internal and the external water phases at the two sides of the membranes. This theory has been supported by a large number of experiments (see Witt, 1979;Cramer & Knaff. 1989). Evidence was provided that several thousands PS I-PS2 units cooperate to replenish one proton pool available to ATP synthase (Hangarter & Ort, 1985) and that just one gramicidin ion channel can dissipate L\GH created by several hundred PS 1-PS2 units (Witt, 1979).

However, a number of observations have lent support to the idea that the proton electrochemical potential relevant to A TP synthesis may be established between the external

10

medium and proton "domains" within the thylakoids, rather than the internal (lumenal) water phase. The basic facts are the following. The presence of buffers permeating into the internal phase does not delay the onset of A TP synthesis under flash illumination of thylakoid suspensions (Hangarter & Ort, 1985; Dilley, 1991), if the suspensions are kept in low salt media in the presence of ca. 1 mM Ca2+ , whilst it does if the thylakoids are kept in high-salt media (200 mM NaCI) or depleted ofCa2+ (see review by Dilley, 1991). These observations have been interpreted to indicate that membrane-localized protons are used for ATP synthesis (see equation 3) before being equilibrated with the lumenal H+ pool. The role of Ca2+ seems to be a structural one, preventing the membrane localized H+ from diffusing into the lumen (Dilley, 1991). The transfer of protons from the sites of production in PS2 (which is localized mostly in the stacked regions of thylakoids) and at the level of the cyt b6-f complex (where PQH2 is oxidized) to the CFo-CF1 complex must occur through a If' conduction system, probably a series of reversibly protonated-deprotonated groups closely associated in the membranes. The reversible transition from the "localized" to the "delocalized" conditions is, according to this hypothesis, regulated by Ca2+ availability imd the ionic composition of the lumenal medium (Dilley, 1991).

LIGHT ABSORPTION, EXCITATION ENERGY TRANSFER TO THE REACTION CENTRES AND THE REGULATION OF ENERGY DISTRIBUTION AMONG THE PHOTO SYSTEMS.

Light absorption by the ChIs in the Soret region generates the second excited singlet, which decays very rapidly through thermal equilibration producing the first singlet, also attained upon light absorption in the red region of the spectrum. Because of the extremely rapid decay of the second singlet, it is the first singlet which is mostly utilized photochemically by PS 1 and PS2.

The energy absorbed by the antennae ChIs can "migrate" within the antennae due to the transfer of the singlet excited state from one molecule to the other and can reach the RCs where it can be utilized in the primary photochemical reaction. The mechanism of excitation energy transfer described by Forster (see Borisov, this volume) involves unidirectional transfer through induced resonance. Its velocity is proportional to the integral of the overlapping of the emission spectrum of the donor molecule with the absorption spectrum of the acceptor molecule. The transfer velocity is inversely proportional to the 6th power of the distance between donor and acceptor, measured by the distance between the two oscillating dipoles. It is also a function of the cosine of the angle between the two dipoles and the line connecting their centres. The Forster mechanism applies after thermal equilibration of the excited state. It still operates efficiently when the distance between donor and acceptor is in the range of 50 to 100 A

The probability for the exciton to be on a molecule having energy E rather than on one having energy Eo is determined by the Boltzmann' factor, exp«E-Eo)/kB T), where kB is Boltzmann's constant. This distribution of energy refers to equilibrium at constant temperature.

The probability of excitation energy utilization by anyone of the molecular processes which compete for the same pool of excited states can be defined in terms of the ratio of the kinetic constant of that process to the sum of the kinetic constants of all the processes (Butler & Kitajima, 1975). So, the probability of energy transfer is

(4)

where kT is the rate constant for energy transfer, kD for thermal decay, and kF for fluorescence emission. In the same way, the fluorescence yield is

11

(5)

where kp is the rate constant for the primary photochemical reaction (which includes in this case kT)' PS 1 is not fluorescent at physiological temperature; its fluorescence can be observed at low temperatures. The chi fluorescence measured at room temperature is therefore emitted mostly by PS2, and provides a very useful method to investigate the state and the kinetics of the PS2 reaction centre, the size and condition of its antenna and changes in the distribution of energy between the two photosystems. When all the primary acceptor Qa is oxidised, as is the case in dark adapted chloroplasts, the fluorescence is at its minimal level, Fa. Upon illumination, Qa is progressively reduced. In the presence of an inhibitor of its reoxidation by the intersystem chain (such as DCMU), Qa becomes totally reduced, and fluorescence reaches its maximum level, Fm. In the presence of very strong light Fm is approached even in the absence of the inhibitor, though not attained. The difference Fm-Fa= Fv is the "variable fluorescence". The increase of fluorescence upon illumination is a measure of the progress of Qa reduction, and is indicated as the fluorescence induction curve; the area above the fluorescence induction is a measure of Qa available as the electron acceptor (see Briantais, this volume). The ratio FvlFm is therefore a useful indicator of PS2 photochemical activity. Independently of the redox state of Qa, the fluorescence may decrease if a quencher is formed in the antenna or at the RC; the quencher will dissipate thermally the absorbed energy, a situation indicated by the increase of kD in equation 5. Both fluorescence and photochemistry ofPS2 will be decreased by the rise ofkD (see equation 5). In the case of excitation energy transfer from PS2 to PS 1 which is not fluorescent at room temperature, PS 1 itself will act as a quencher for PS2 fluorescence. This provides useful method for the investigation of the distribution of energy between the two photosystems and its regulation.

The very important observation by Joliot and Joliot (Joliot & Joliot, 1964) that the rise of fluorescence from Fa to Fm is sigmoidal, has been interpreted to indicate that cooperativity exists between PS2 units. The fluorescence rise curves could be represented by the equation

fv = (l-a)(l-[Qa])/(l-a(I-[Qa])) (6)

where a represents the probability for exciton migration from a PS2 unit having the RC "closed" (Qa fully reduced) to a neighbor unit with open RC. The value of alfa was estimated by fitting the fluorescence rise curves to the model, and was found to be in the range of 0.4 to 0.6. The velocity of PS2 turnover could accordingly be described by the equation

v = I J3[Qa]l(l-a(I-[QaJ)) (7)

where /3 is the optical cross section of PS2 and I is the photon flux absorbed. This equation was used to calculate the velocity ofNADP reduction by isolated thylakoids, and was found to describe accurately the measured velocity (Jennings & ZuccheJli, 1986). The cooperativity among several PS2 units is an obvious advantage in terms of the efficiency of PS2 photochemistry inasmuch as it allows the utilization of photons absorbed by PS2 units having closed centres.

Any departure from the equal distribution of photons to the photosystems decreases the quantum yield of photosynthesis, because the electron transport system requires the two photochemical reactions in series to transfer one electron from H20 to NADP. To achieve equal distribution of the absorbed energy to PS2 and PSI they should have equal absorption cross section throughout the spectrum, or their difference of absorption should be in some way compensated. It is known that this is not the case. The distribution of the pigment protein complexes is imbalanced in favor of PS2 antenna, which receives the excitation

12

energy from ca. 60% of the pigment molecules, versus 40% belonging to PS 1 antenna (Thornber et ai., f987).

A regulation of the size of the antennae has been reported by Bonaventura and Myers (Bonaventura & Myers, 1969). They observed that when Chlorella cells performing steady­state photosynthesis are suddenly transferred to an illumination regime imbalanced in favor of PS2 (;A..=650 nm), two types of fluorescence changes occur: a very rapid (in the ms time scale) increase due to the over-reduction of Qa, followed by a slow decrease requiring several minutes (5 to 10). They described this slow transition as the "state 1 (high fluorescence)-state 2 (low fluorescence)" transition. The state 1 was restored upon illumination with a 710 nm beam (absorbed mainly by PS 1) in several minutes. These reversible state I-state 2 transitions were seen as an adaptation of the relative size of the antennae of the two photosystems to the prevailing illumination conditions.

The biochemical mechanism of the state I-state 2 transition is understood as due to the activity of a thylakoid bound protein kinase which is activated when the PQ pool is over­reduced, and phosphorylates a threonine residue close to the N-terminal of LHC II (the major chi a-b protein complex which belongs to the antenna ofPS2), exposed on the stroma side of the membranes (see review by Allen, 1992). Upon phosphorylation of LHCII, a decrease (15-25%) of PS2 fluorescence and photochemistry is observed, and a corresponding increase of PS 1 photochemistry (Forti & Fusi, 1990). The decrease of fluorescence concerns Fo and Fm to the same extent (Allen, 1992), and their ratio does not change. A fraction of the phosphorylated LHC II has been shown to migrate from the grana partitions to the stroma-exposed membranes, where PSI is concentrated (see Allen, 1992; also Bassi, this volume). As a consequence of the increased ratio of PSI to PS2 photochemical activity, PQH2 is reoxidized and the kinase becomes inactive. A thylakoid bound phosphatase dephosphorylates LHC II, which migrates back to the grana partitions where it is reintegrated into the PS2 antenna. The time course of LHC II phosphorylation­dephosphorylation and the accompanied changes of PS2 fluorescence and photochemical activity are in fair agreement with the kinetics of state I-state 2 transitions in vivo.

REGULATORY PHENOMENA AT THE LEVEL OF PS2 ACTIVITY.

The turnover of the PS2 reaction centre can be regulated at two levels: (a) the photochemical reaction itself, or the reactions on its oxidizing and/or reducing side; (b) the transfer of excitation energy from the antenna.

It has been reported that PS2 photochemical activity is inhibited by protons produced during the activity itself, and that the inhibition is reversed by lipophilic uncouplers and by the presence of ADP+Pi (Finazzi et ai., 1992). These observations indicate that membrane localized protons produced during H20 oxidation inhibit PS2 activity and that such protons are available to the ATP synthase for ATP synthesis coupled to .1GH utilization. This autoregulation of PS2 through the protons produced by its activity seems to concern the reaction centre rather than the antenna, because it does not affect the level of Fo nor of Fm but only the velocity of fluorescence rise from Fo to Fm, i.e. the rate of PS2 primary photochemistry. The mechanism of inhibition ofPS2 by protons is not known.

It thus appears that both the proton-producing reactions of photosynthetic electron transport, H20 oxidation and PQH2 oxidation, are regulated by the protons that they produce, either localized within the membrane or released into the lumen.

In the case of PQH2 oxidation (the rate limiting reaction of photosynthetic electron transport) the regulatory mechanism has been tentatively explained in terms of the approach to the thermodynamic equilibrium when PQH2 oxidation and the coupled proton translocation into the lumen are working against high value of .1GH. However, this explanation could not apply to PS2. It is attractive to think (especially in the case of PS2) that the regulation of the electron transport rate by protons may operate through a

13

regulation of the turnover rate of the electron carriers, involving the conformational change of some protein component. The reaction rate and/ or pathway would in this way be effectively modified when the system is still very far from equilibrium.

MECHANISMS OF PROTECTION AGAINST EXCESSIVE EXCITATION.

The primary photochemical reactions of photosynthesis have rate constants exceeding by several orders of magnitude those of the electron transport reactions and of the enzymatic reactions of CO2 assimilation. As a consequence of this fact, high concentrations of chI excited states in the antennae give rise to the generation of chI triplet in relevant concentration. The latter, reacting with O2, leads to the formation of different chemical species harmful to the photosynthetic apparatus. Among these are the formation of O2

singlet, 0; and OH radical, which are species very reactive with proteins, lipids and the pigment molecules themselves, leading to inactivation of the system and eventually to bleaching of the pigments. The protective function of carotenoids has been clearly recognized (Demming-Adams, 1990), and the general pathway for protection is through the thermal dissipation of the energy absorbed in excess of the kinetic capacity of the electron transport system, as is discussed in other chapters in this volume.

Of course, thermal dissipation of the energy absorbed is useful when the excited states in the antennae are present in excess of the amount that can be utilized photochemically, while it would be deleterious to the efficiency of the system if energy were dissipated in competition with photochemical utilization. In fact, the onset of thermal dissipation as a function of incident light intensity has been the object of contrasting reports. In sunflower leaves it was observed only at high light intensity (Demming-Adams, 1990), whilst rather low intensity produced thermal dissipation in pea leaves (Genty et aI., 1990). A mechanism for turning on and off the thermal dissipation has been found to be dependent upon the value of .1GH across the thylakoid membrane (Wright & Crofts, 1970; Krause et aI., 1982). Such dissipation competes with photochemical utilization of energy as well as with fluorescence emission (see equation 5); for this reason, it has been defined "high energy quenching" of fluorescence, qE. The phenomenology and mechanisms of qE are discussed by Briantais and by Horton (this volume). It will only be mentioned here that qE can be easily distinguished experimentally from the photochemical quenching (qP) of fluorescence, which is due to the presence of the electron acceptor Qa. In its presence, photochemistry competes successfully with fluorescence, because kp»kr (see equation 5). When dark adapted leaves (or isolated chloroplasts) are exposed to a short light flash of high intensity (oversaturating with respect to electron transport rate), Qa becomes fully reduced and the Fm level of fluorescence is attained transiently. Continuous illumination at rather high intensity causes the decrease of fluorescence, both Fm and Fo. The recovery of the original Fm level occurs in ca. 1 min in the dark (the time required for dissipation of .1pH), or it can be observed in isolated chloroplasts in a few ms upon dissipation of .1GH by an uncoupler. The qE is due to the formation of quenchers in the antennae ofPS2 (see Briantais, this volume), which efficiently dissipate the energy absorbed in excess of the kinetic capacity of electron transport, so protecting the photosynthetic apparatus from inactivation. However, the fraction of excitation energy which is transferred to the reaction centres has been reported as being utilized with unimpaired efficiency for photochemistry (Genty et aI., 1989; also Briantais, this volume). So, the estimate of Fm,-FJFm, (where Fm, and Fs are, respectively, Fm in the quenched state and the steady-state fluorescence) has been claimed to be a measure of the quantum efficiency of the absorbed photons. The mechanism of qE is still unknown; however, in intact leaves it has been observed that fluorescence quenching is correlated with the de­epoxidation of the carotenoid violaxanthin to yield zeaxanthin (Demmig-Adams, 1990). This process, however, is not an absolute requirement for qE (Demmig-Adams, 1991).

14

PHOTO INHIBITION.

Exposure of the leaves to high light intensity, especially if the rate of electron transport and CO2 assimilation is limited by low temperature and other stress factors, causes inhibition of photosynthesis, a phenomenon defined photoinhibition (see Baker, this volume). This phenomenon is slowly reversible in vivo, and its reversal depends on de novo protein synthesis. In isolated thylakoids and in PS2 preparations photoinhibition has been shown to be followed by the cleavage of the reaction centre protein D 1, that which binds P680 and Qb.

REFERENCES.

Allen J.F. (1992) Biochim. Biophys. Acta 1098: 275-335. Arnon DJ. (1977) in "Encyclopedia of Plant PhysioL" New sec (Trebst A & Avron M. eds), voLS.pp 7-56.

Springer-Verlag, Berlin. Boekema E.1., Schmidt G. & Graber P. (1988) Z. Naturforsch.43 c:219-225. Bonaventura C. & Myers 1. (1969) Biochim. Biophys. Acta 189: 366-383. Butler W.L. & Kitajima M. (1975) Biochim. Biophys. Acta 399: 72-85. Canaani O. & Malkin S. (1984) Biochim.Biophys.Acta 766:513-524. Cramer WA & KnaffD.B. (1989) "Energy Transduction in Biological Membranes". Springer-Verlag. Debus RJ. (1992) Biochim.Biophys. Acta 1102: 269-352. Demmig-Adams B. (1990) Biochim. Biophys Acta 1020: 1-24. Demmig-Adams B. (1991) in "Current Res. Photosyn1." (Baltscheffsky M. ed), vol. II, pp 357-364. Kluwer

Acad.PubL The Netherlands. Dilley R (1991) in "Current Topics in Bioenergetics" VoLI6. (Lee c.P. ed.), pp 265-315. Academic Press,

San Diego. Duysens L.N.M.. Amesz 1. & Kamp B.M. (1961) Nature 190: 510-51 I. Emerson R & Arnold W. (1932) J.Gen.Physiol. 15: 391-420. Emerson R (1957) Science 125: 746. Finazzi G., Ehrenheim A.M. & Forti G. (1992) Biochim. Biophys. Acta 1142: 123-128. Forti G. & Jagendorf A.T. (1961) Biochim. Biophys. Acta 54: 322-330. Forti G. & Parisi B. (1963) Biochim. Biophys. Acta 71: 1-6. Forti G. & Grubas P.M.G. (1985) FEBS Letters 186: 149. Forti G. & Fusi P. (1990) Biochim.Biophys. Acta 1020: 247-252. Forti G. & Ehrenheim AM. (1993) 1183: 408-412. Forti G. & Elli. G. (1995) Plant Physiol. in press Foust G.P.,Mayhew S.G. & Massey V. (1969) 1.BioI.Chem. 244: 964-970. Fromme P. & Graber P. (1990) Biochim. Biophys. Acta 1016:29-42. Garlaschi F.M., Zucchelli G. & Jennings RC. (1989) Pho1. Res. 20:207-220. Genty B .. Briantais J.M. & Baker N.R (1989) Biochim. Biophys. Acta 990: 87-92. Genty B., Harbison 1., Briantais 1.M. & Baker N. (1990) Photosyn1. Res. 25: 249-257. Govindjee, Govindjee R & Hoch G. (1964) Plant Physiol. 39: 10-14. Hangarter R & Ort D,R (1985) Eur. 1. Biochem. 149: 503-510. Hill R & Bendall F. (1960) Nature 186: 136-137. Jennings RC. & Zucchelli G. (1986) Arch. Biochem. Biophys. 246: 108-113. Joliot P. & Joliot A (1964) C.R Acad. Sc. Paris. 1.258: 4622-4625. Junesch U. & Graber P. (1985) Biochim. Biophys. Acta 809:429-434. Kok B., Forbush B. & McGloin M (1970) Photochem. Photobiol.l1:457-475. Krause G.H .. Vernotte C. & Briantais J.M. (1982) Biochim. Biophys. Acta 679: 116-124. Mitchell P. (1977) Febs Letters 78:1-20. Miyake C. & Asada K. (1992) Plant Cell Physiol. 33: 541-553. Myers J & French C.S. (1960) 1.Gen.Physiol. 43: 723-736. Myers 1. (1987) Photosynth. Res. 14: 55-69. Ort D.R & Oxborough K. (1992) Ann.Rev.Plant Physioi. 43: 269-291. Renger G. (1993) PhotRes. 38: 229-247. Schatz G H. Brock H & Holzwarth AR (1987) PNAS. USA 8414-8418. Schonknecht G .. Hedrich R. Junge W. & Raschke K. (1988) Nature 336:589-592. Shuvalov VA Nuijs AM., van Gorkom H.J. Smit H.W.J. & Duysens L.N.M. (1986) Biochim. Biophys.

Acta 850:319-323. Thornber 1.P., Peter G.F. & Nechustai R (1987) Physiol. Plant. 71: 236-240.

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Witt H.T. (1979) Biochim.Biophys.Acta 505: 355-427. Witt H.T. (1990) in "Curr. Res. in Photosyn.". (M. Baltscheffsky ed.), vol.1, pp 837-840. Kluwer Acad.

Pub!.. The Netherlands. Wright C.A. & Crofts A.R. (1970) Eur. 1. Biochem. 17: 319-327.

16

PHOTOSYNTHETIC ELECTRON TRANSFER AND ENERGY TRANSDUCTION IN PLANTS

Donald R. Ort and John Whitmarsh

Photosynthesis Research Unit, USDAIARS & Department of Plant Biology, University of Illinois, Urbana, IL 61801 USA

INTRODUCTION

The photosynthetic membranes of plants perform a remarkable feat. They convert a portion of the energy available in light into the chemical energy of ATP and NADPH. In this way photosynthetic membranes provide a stable form of energy that can be used at later times for energy-requiring biochemical processes, such as the reduction of C02 to carbohydrate. The first step in photosynthetic energy transformation is the absorption of light by the antenna array, resulting in the conversion of the transient energy stored in electromagnetic radiation into the excited state of pigment molecules. The excited state energy residing in the antenna system is short lived and must migrate rapidly to reaction center complexes, where it drives primary charge separation. The energy stored in the reaction centers by charge separation drives a series of oxidation/reduction reactions within the thylakoid membrane that ultimately convert the energy into the chemical free energy of ATP and NADPH. These energy conversion reactions are achieved by the cooperative interaction of four major protein complexes located in the thylakoid membrane. Three of these complexes, photo systems I and II (PS I and PS II) and the cytochrome bf complex (Cyt bf) are involved in light-driven electron and proton transfer. The fourth protein complex (ATP synthase) produces ATP from ADP and phosphate. In this introductory overview we have two goals. First is to introduce the players, that is the components of the chloroplast thylakoid membrane that are responsible for the basic reactions of photosynthetic electron transfer and energy transduction. Second we will track the energy transformations that ultimately result in the conversion of light energy into stable chemical forms. For the sake of clarity, we focus on general concepts and reference mostly review articles from which the interested reader can launch into the primary literature. More detailed descriptions of photosynthesis are available in references (Cramer & Knaff ,1991; Ort,.1994; Ort & Yocum, 1995; Walker, 1992; Whitmarsh & Govindjee, 1995).

Discussions of the biochemical and physiological aspects of photosynthesis are given in references (Nobel, 1991; Taiz & Zeiger, 1991).

Light as an Energy Source and Information Carrier in Plant Physiology Edited by Jennings et aI., Plenum Press, New York, 1996 17

STRUCTURE AND MORPHOLOGY OF THE CHLOROPLAST

All of the fundamental energy conversion processes of photosynthesis in higher plants take place within a subcellular organelle known as the chloroplast (Staehelin, 1986). Chloroplasts occur most abundantly in leaf mesophyll cells where they typically number between 50 and 200 per cell, depending on the species, growth conditions and developmental stage of the leaf. Mature chloroplasts are generally lens shaped and range in size from about 1-3 ~m across by 5-7 ~m in their longest dimension. Chloroplasts are bounded by two distinct envelope membranes of which the inner envelope membrane contains specific transporters that mediate the flow of photosynthate from the chloroplast to the cytoplasm where sucrose synthesis takes place.

The photosynthetically-active, chlorophyll-containing membranes of higher plant chloroplasts are flat lamellar vesicles called thylakoids. The thylakoid membrane is vesicular, defining a closed space with an outer water space (stromal phase) and an inner water space (lumen). These lamellar vesicles are frequently tightly appressed to one another; forming structures known as grana stacks. Although the extent of this "stacking" is variable, thin­section electron micrographs show the high incidence of surface contacts in mesophyll cell chloroplasts of both C3 and C4 plants. Stacked membranes are altogether absent in the specialized bundle sheath cell chloroplasts of some types of C4 plants.

Figure 1 shows a cut-away view of a chloroplast showing the appearance of thylakoid membranes in both two and three dimensions. As revealed in the drawing, the simple and relatively familiar two-dimensional appearance of thylakoid organization belies the highly intricate three-dimensional structure of these membranes. The central feature of three­dimensional organization of the thylakoid membrane is that of multiple, tilted membrane planes arranged helically around the surface of a cylindrical core of stacked flat lamellar vesicles or discs (Fig. 1). These tilted membranes, the stroma lamellae, form attachments to the individual discs such that there is a continuous internal lumen between the two

Figure 1. <;hloroplast thylakoid membrane architecture. This drawing of a sectioned chloroplast shows the relationship ofthylakoid membranes viewed in two dimensions compared with the appearance viewed in three dimensions. The drawing shows that the appressed membranes, often called grana stacks, seen in two dimensions arisc from viewing in cross-section a cylindrical core of grana discs and that the helically arranged membrane sheets are the origin of the unappressed or stroma membranes visible in two dimensions. The thylakoid membranes are surrounded by the stromal phase where the reactions of CO2

reduction, starch synthesis, fatty acid synthesis, chlorophyll synthesis and numerous other biosynthetic pathways critical to plants occur. The chloroplast is bounded by two envelope membranes (Reprinted from Ort, 1986).

18

membrane types. Neighboring grana stacks are connected by narrow bridges between the tilted membranes thereby further extending the interconnection of lumen spaces (Mustardy & Janossy, 1979; Paolillo & Falk, 1966).

The glycerol lipid composition of the thylakoid membrane is markedly different from that of other membranes in plants (Murphy, 1986). About 70% of the lipid forming the thylakoid bilayer is contributed by two unusual and highly unsaturated glycolipids, monogalactosyldiacyl-glycerol and digalactosyldiacyl-glyceroL These glycolipids, which are also found in certain photosynthetic bacteria and cyanobacteria, are exceedingly rare in non­photosynthetic membranes. Nevertheless, by virtue of being the major glycerol lipids of thylakoid membranes, these two galactolipids are the most abundant bilayer lipids in the biota. Diacylsulfoquinovosylglycerol is another glycerol lipid apparently unique to photosynthetic membranes. Phosphatidylglycerol, while frequently encountered in a wide range of membrane types, is distinquished in thylakoid membranes by the C 16: 1 trans fatty acid tails. In fact, there is a question of whether phosphatidylcholine is a genuine thylakoid lipid or a contaminant from other plant cell members. In addition to the glycerol lipids of the bilayer, thylakoid membranes contain an abundance of the lipid chlorophyll in amounts equivalent to nearly O.IM (i.e. on the order of 109 chlorophyll molecules per chloroplast).

LIGHT ABSORPTION

It is axiomatic that only light that is absorbed can stimulate photochemical reactions. The action spectrum of higher plant photosynthesis includes light at wavelengths between about 350 and 700 nm. About 55 to 60% of the sunlight incident on the earth's surface falls within this photosynthetically active wavelength interval. All of the pigments that participate in the absorption of light for photosynthesis in higher plants are located within the thylakoid membrane and fall into two general classes of compounds, chlorophylls and carotenoids. Chlorophylls in plants have two chemically distinct forms designated a and b, which have complex ring structures that place them in a group of related compounds that include the pigments of hemoglobin and cytochromes. Plants appear green by virtue of their chlorophyll, which intensely absorbs red and blue light, scattering green light. Carotenoids, which occur at in much lower amounts in plants than chlorophyll, are linear polyenes of which f3-carotene is a common example.

The first step in photosynthesis is the absorption of light, an energy conversion reaction that transforms electromagnetic energy into excited state energy in an antenna molecule by rearrangement of electron orbitals. The formation of this excited state occurs on an exceptionally rapid time scale (~1O-15 s). A basic feature of the photosynthetic apparatus is that the vast majority of light-absorbing pigments within the thylakoid membrane act as a large antenna to intercept light (see Bassi et aI., and Jennings et al. this volume), rapidly transferring the energy to specialized reaction centers that initiate photochemistry. This array of antenna pigments serve to increase the absorption cross-section of a reaction center in two ways. First, the absorption cross section increases in proportion to the number of pigment molecules in the antenna associated with each reaction center. For example, in crop plant species, there are typically 250 antenna chlorophyll molecules associated with each reaction center. Second, the antenna contains a number of different spectral species of pigments which broaden the absorption spectrum of the antenna array and thereby increase the likelihood of efficiently capturing photons over a wider range of wavelengths (see Jennings et al. this volume).

An essential feature of the antenna system is that it must efficiently transfer energy to the photosynthetic reaction center with which it is associated. The pigment molecules that make up the antenna system are bound to membrane proteins. Because of the proximity of other antenna molecules within the protein/pigment array, the excited state energy has a high probability of being transferred to a near neighbor pigment. The transfer of excited state

19

energy between antenna molecules is due to the interaction of the transition dipole moment of the molecules (Knox, 1995). The rate of transfer is dependent on the distance between the transition dipoles of the donor and acceptor molecules, the relative orientation of the transition dipoles, and the overlap of the emission spectrum of the donor molecule with the absorption spectrum of the acceptor molecule (see Borisov, this volume). Photosynthetic antenna systems are very efficient at this process. Under optimum conditions, over 90% of the absorbed quanta are transferred from the antenna system to the reaction center within a few hundred picoseconds. The exceptionally high efficiency of energy transfer in photosynthetic antenna arrays arises from the precise orientation of the pigments within the antenna that is created by the organization of the pigment-binding proteins (Kiihlbrandt et ai., 1994).

ELECTRON AND PROTON TRANSPORT: STRUCTURE AND FUNCTION

The next step in energy transformation occurs within reaction centers, which trap the excited stated state energy of the antenna system (see Mathis, this volume). The excited state energy localized within the antenna system provides the energy for the primary photochemical reaction of photosynthesis - the transfer of an electron from a donor molecule to an acceptor molecule. Both the donor and acceptor molecules are attached to the reaction center protein complex. The energy stored in the primary charge separation reaction drives the subsequent oxidation/reduction reactions, which are energetically downhill. In oxygenic photosynthetic organisms, two different reaction centers work in series - photo system II and photosystem I. Electrons are transferred from photosystem II to the photosystem I reaction center by intermediate carriers. The net reaction is the transfer of electrons from a water molecule to NADP+, thereby producing NADPH. The three major protein complexes within the thylakoid membrane that are responsible for the conversion of energy stored as the excited state of a pigment molecule into a transmembrane electrochemical potential and NADPH are shown in Fig. 2. These three complexes, PS II, PS I, and Cyt bf, are the key players in the light-driven electron and proton transfer reactions of photosynthesis.

The PS II complex from a typical higher piant contains at least fifteen different polypeptides (currently of uncertain stoichiometry) which, as with all of the intra-thylakoid complexes, are a mixture of chloroplastic and nuclear gene products (see Bassi et ai., this volume). These polypeptides bind and properly orient on the order of 200 chlorophyll a molecules, 100 chlorophyll b molecules, 50 carotenoid molecules, 2 plastoquinones (QA, QB), 1 iron, 2 pheophytin a molecules, 1 or 2 cyt b559 molecules, 4 manganese ions, and an undetermined number of chloride and calcium ions. The redox components, tyrosine, P680, pheophytin, QA and QB are bound to two key polypeptides, D 1 and D2, that form the heterodimeric reaction center core of PS II (Hansson & Wydrzynski, 1990; Ghanotakis & Yocum, 1990; Debus, 1992).

The PS II reaction center drives two chemical reactions - the oxidation of water and the reduction of plastoquinone. To accomplish this, the PS II complex must link the one­electron charge transfer events of the reaction center with the four-electron oxidation of two water molecules and the two-electron reduction of plastoquinone. Both of these reactions are coupled to proton transfer. The pathways of electrons and protons is not completely characterized and there is much about these reactions that we do not understand. Photochemistry in PS II is initiated by charge separation between P680 and pheophytin, creating P680+lPheo-. Primary charge separation takes about 3 picoseconds (Fig. 3). Recombination of the primary charge separation is avoided by transferring the electron within 200 picoseconds from pheophytin to a plastoquinone molecule (QA) that is

20

Sue...... ....,...,--__ I"---;;~,___,.

$ynl,...l.

LUMEN

Figure 2. This stylized drawing of a chloroplast depicts the functions of the major protein complexes of the thylakoid membrane and the coupling of their activities to the photosynthetic carbon reduction cycle which takes place in the chloroplast stroma. Illustrated is the concept of light-driven linear electron flow coupled to the net accumulation of protons in the thylakoid lumen which is in turn used to drive the reversible ATP synthase in the direction of net ATP formation. In addition to the energy stored in ATP formation, energy derived from absorbed light is also stored by the reactions of the thylakoid membrane in the formation of NADPH. Photosynthetic carbon reduction is shown as a three stage cycle. Carbo:>.:ylation: a molecule of CO2 is covalently linked to a carbon skeleton. Reduction: energy in the form of ATP and NADPH is used to form simple carbohydrate. Regeneration: energy in the form of ATP is used to regenerate the carbon skeleton for carbo:>.:ylation. OEC, o:-,:ygen-evolving complex; P680, reaction center chlorophyll ofPS II; Ph, pheophytin acceptor of PS II; QA & QB, quinone acceptors of PS II; PQ & PQH2' plastoquinone and reduced plastoquinone; cyt, cytochrome; FeS, Rieske iron sulfur protein; PC, plastocyanin; P7CJO, reaction center chlorophyll ofPS I: Ao, primary acceptor ofPS I; FeS, bound iron sulfur acceptors ofPS I; Fd, soluble ferredoxin; FNR, ferredoxin-NADP reductase. (Reprinted from Ort, 1994).

permanently bound to PS II. Although plastoquinone normally acts as a two-electron acceptor, it works as a one-electron acceptor at the QA-site. The electron on QA- is then transferred to another plastoquinone molecule that is loosely bound at the QB-site. Plastoquinone at the QB-site differs from QA in that it works as a two-electron acceptor, becoming fully reduced and protonated (forming plastoquinol) after two photochemical turnovers of the reaction center. This secondary quinone acceptor acts to connect the single electron transfer events of the reaction center with the pool of free plastoquinone in the membrane by operating as a two electron gate, allowing the passage of electrons out of PS II only in pairs via plastoquinol. The proposed operation of the two-electron gate of photo system II i's depicted in Fig. 4. Plastoquinol at the QB-site is loosely associated with the quinone-binding site. Its dissociation vacates the quinone-binding site, thereby completing the cycle.

Photosystem II is the only known protein complex that can oxidize water, resulting in the release of O2 into the atmosphere. Energetically, water is a poor electron donor. The oxidation-reduction midpoint potential (Em,7) of water is +0.82 V (pH 7). In PSII this

21

-1.2

-0.8

-0.4

~ o 2:. 0.0

E W

0.4

0.8

1.2

<£~O~':PS Pheo

PSII

J 200 ps

°A ~O 1 ms 100-600JlS B ~ PO

H 01 ms hv 2_ I ) Mn_

o 100-800 JlS Tyr - fP68O\ 2 200ns~

Figure 3. Photosystem II electron transport pathways and rates. The vertical axis shows the midpoint potential of the electron carriers. The heavy vertical arrow show light absorption. P680+ is the electronically excited state ofP680. Abbreviations are as given in Figure 2. (Reprinted from Whitmarsh & Govindjee, 1995).

Figure 4. The two-electron gate of PS II. Photosystem II centers must reconcile the 2 electron reduction of plastoquinone with the 1 electron charge transfer events of the reaction center. This figure traces the sequence of events that occur during plastoquinone reduction. The operation of the two-electron gate is discussed in the text. (Reprinted from Ort. 1986).

reaction is driven by the oxidized reaction center, P680+ (the midpoint potential of P680/P680+ is estimated to be + 1.2 V at pH 7). How electrons are transferred from water to P680+ remains a mystery. It is known that P680+ oxidizes a tyrosine on the DI protein and that Mn plays a key role in water oxidation. Four Mn ions are present in the water oxidizing complex. X-ray absorption spectroscopy shows that Mn undergoes light-induced oxidation. Water oxidation requires two molecules of water and involves four sequential turnovers of the reaction center. Each photochemical reaction creates an oxidant that removes one electron. The net reaction results in the release of one O2 molecule, the deposition of four protons into the inner water phase, and the transfer of four electrons to the QB-site, producing two molecules of plastoquinol that are released into the hydrophobic core of the photosynthetic membrane.

22

Photosynthesis in plants requires the cooperative operation of two reaction centers -electrons extracted from water by PS II are transferred to PS I. The PS I reaction center catalyzes the oxidation of plastocyanin, a small soluble eu-protein found in the thylakoid lumen, and the reduction of ferredoxin, a small FeS protein located the stroma phase (Golbeck, 1992). Reduced ferredoxin is a potent reductant that provides the electrons for NADPH formation. The PS I reaction center is a heterodimer (Krauss et aI., 1993) that provides the ligands for the primary electron donor P700 (a chlorophyll dimer), the primary electron acceptor, Ao (a chlorophyll monomer), Al (phylloquinone) and Fx (a Fe4S4 center). The final electron acceptors of PS I are two Fe4S4 centers that are bound to a peripheral protein. The reaction center is served by an antenna system that consists of a couple of hundred chlorophyll molecules (mainly chlorophyll a). In contrast to photosystem II, many of the antenna chlorophyll molecules in PS I are bound to the reaction center proteins, and so far as is known, PS I electron transfer is not coupled to proton translocation. The electron transfer reactions and rates following primary charge separation in PS I are shown in Fig. 5.

-1.2

-O.S

2' -0.4

~ J 0.0

0.4

O.S

hv

200J.LS I PC",

P700

Figure 5. Photosystem I electron transport pathways and rates. The vertical axis shows the midpoint potential of the electron carriers. Abbreviations are given in the legend of Figure 2. (Reprinted from Whitmarsh & Govindjee. 1995).

In addition to converting excited state energy into redox free energy, the electron transfer reactions of PS II and PS I contribute to the electrochemical potential across the thylakoid membrane that drives phosphorylation. The energy is converted by two types of reaction: (I) The release of protons during the oxidation of water by photo system II and the translocation of protons from the outer aqueous phase to the inner aqueous phase by the coupled reactions of photosystem II and the cytochrome bf complex in reducing and oxidizing plastoquinone on opposite sides of the membrane. This creates a concentration difference of protons across the membranes (LipH = pHin - pHout). (2) Primary charge separation by PS II and PS I drives an electron across the photosynthetic membrane, creating an electric potential across the membrane (NP = \fin - \fout). Together, these two forms of energy make up the proton electrochemical potential across the photosynthetic membrane (Li~H+) which is related to the pH difference across the membrane and the

23

electrical pot~ntial difference across the membrane by the following equation:

J..lH+ = F ~qt - 2.3 RT ~pH

where F is the Faraday constant, R is the gas constant, and T the temperature in Kelvin. The light-driven oxidation/reduction reactions of the two photo systems are

interconnected through the catalytic activity of the Cyt bf complex (Cramer & Black, 1987; O'Keefe, 1988). Both photosynthetic and respiratory membranes of prokaryotes and eukaryotes contain closely related protein complexes that function to oxidize a low potential quinol and reduce a high potential metalloprotein (Cramer & Black, 1987). In the case of higher plant chloroplasts, the cytochrome bf complex catalyzes the energetically downhill reaction of oxidizing plastoquinol produced by PS II and reducing plastocyanin. In comparison to the photosystems, the cytochrome bf complex is structurally simpler, consisting of single copies offive polypeptides. The redox-active components consist of two b-type cytochrome hemes (cytochrome b6), a c-type cytochrome (cytochrome f), and a Fe2S2 center (Rieske iron-sulfur cluster). In addition to its role in connecting the electron transfer reactions of PS II with those of PS I, the Cyt bf complex plays a central role in storage of energy that is subsequently used to synthesize ATP. A portion of the redox free energy that is available from the energetically downhill reactions catalyzed by the Cyt bf complex is captured in transmembrane pH difference that is created because the reduction and protonation of plastoquinone occurs on the stromal side of the membrane whereas the oxidation and proton release occurs on the lumenal side of the thylakoid (see Fig. 2).

The proposed Q cycle mechanism for the oxidation of plastoquinol by the cytochrome bf complex is somewhat intricate. It must reverse what was achieved by the two electron gate of PS II that produced plastoquinol. The two electron oxidation of plastoquinol by the Cyt bf complex is coupled to the one electron reduction of plastocyanin. Operation of the Q­cycle relies on a binding site for plastoquinol near the inner surface of the thylakoid membrane as well as a binding site for plastoquinone near the outside surface (Fig. 6). Upon transfer of an oxidizing equivalent from PS I via plastocyanin, one electron is removed from the high potential carriers of the bf complex. Plastoquinol bound to the Cyt bf complex reduces the Rieske iron sulfur center and the more highly reducing semiquinone is then capable of transferring an electron to the nearest cytochrome b6 heme. The electron on the Rieske FeS center is transferred to cytochrome f, and then to plastocyanin. The reduced plastocyanin debinds from the Cyt bf complex, diffuses through the lumen and eventually donates an electron to PS I. The second electron is transferred from the inner b-heme to the cytochrome b6 heme located near the outer edge of the membrane. Since this electron is electrically uncompensated, it contributes to the membrane potential formed across the thylakoid membrane. The electron on cytochrome b6 reduces a plastoquinone bound near the stromal side of the membrane, forming plastosemiquinone. A second turnover of the Cyt bf complex results in an identical series of events, except in this case cytochrome b6 reduces the plastosemiquinone to plastoquinol. Thereafter, the plastoquinol dissociates from the site and is replaced by plastoquinone from the membrane pool. The reactions for two consecutive turnovers are shown in Fig. 6. The Q-cycle achieves the movement of 4Ir across the membrane for the net oxidation of one plastoquinol (i.e., two plastoquinols are oxidized and one plastoquinone is reduced).

PHOTOPHOSPHORYLATION AND COUPLING FACTOR

As depicted in Fig. 2, there are two redox couples within the photosynthetic electron transfer sequence that result in the accumulation of protons within the thylakoid lumen and thereby result in the formation of a pH difference across the membrane. Water and

24

1st turnover

2nd tu mover

Figure 6. The Q cycle model for the oxidation plastoquinol by the Cytochrome b6f complex. The details of the proposed mechanism are given in the text. Overall the Q cycle oxidizes two plastoquillOls, reduces one plastoquinone and thereby translocates 4W for every 2e- reaching P700 +. (Reprinted from Ort, 1986).

plastoquinol are alike in the respect that both are hydrogen donors. Therefore oxidation yields hydrogen ions. Since the oxidation reactions take place at the inner side of the thylakoid membrane, and the reduction of plastoquinol takes place near the outer side of the membrane where protons are taken up from the stroma phase, a transmembrane pH difference is generated. This proton chemical potential, together with the electric potential generated by uncompensated charge transfer across the membrane, provides the energy for ATP synthesis. Fig. 2 illustrates an important concept in photosynthetic energy coupling, that the transmembrane electrochemical potential difference is an intermediate form of energy storage between two otherwise independent processes, electron transfer and ATP formation.

The enzymatic coupling of the transmembrane electrochemical potential with the energy-requiring reaction of ADP phosphorylation is performed by a reversible ATP synthase or coupling factor which is located in the thylakoid membrane (Cramer & Black, 1987). This is Ii structurally complex multisubunit enzyme complex for which the catalytic mechanism is poorly understood despite more than two decades of active research attention. Structurally the enzyme complex has two distinct domains. Five subunits make up the hydrophilic domain that protrudes into the stroma and contains the catalytic sites which are involved in ADP binding and phosphorylation. The integral membrane portion of the chloroplast coupling factor complex contains four different polypeptides. While it is clear that this portion of the enzyme is involved in conducting protons across the thylakoid membrane to the active site located in the hydrophilic domain, it is undoubtedly an

25

oversimplification to consider the membrane-localized domain of the enzyme simply as a noncatalytic proton channel.

Current thinking about the catalytic mechanism of ATP synthesis has its origins in the "binding change" or "alternating site" model proposed by Paul Boyer (1979). The basic trim eric configuration of the a13 pairs of CF I depicted in Fig. 2 has helped promote the notion of three functionally identical catalytic sites. According to a three site binding change model (Boyer, 1979), three catalytic sites "rotate" sequentially through three distinct states, such that each site is in a different state from the other two at any given time. Briefly, the release of ATP from the first site (C I , Fig. 7) is coincident with the tight binding of ADP + Pi at the second site (C2). Simultaneously, the third site (C3) is converted from an open form, which has a very low affinity for binding nucleotides, to a form capable of loose nucleotide binding. A fundamentally important energetic concept introduced by the alternating site model is that the actual formation of A TP on the enzyme occurs with an equilibrium constant near unity and the major energy requirement for net ATP formation is involved with releasing the newly synthesized A TP from the enzyme.

Figure 7. The three-site alternating site model for the catalytic mechanism of the FI ATP synthase as proposed by Boyer. The details of the proposed mechanism are given in the text. (Reprinted from Ort & Oxborough, 1992).

The nonuniform and unpredictable light environment that plants encounter necessitate that the catalytic activity of the chloroplast ATP synthase be carefully regulated (Ort & Oxborough, 1992). Otherwise, the thermodynamically-favored hydrolysis of ATP could quickly drain chloroplast and possibly cellular energy pools when light is limiting or absent. On the other hand, these regulatory controls must respond if energy is to be stored efficiently as light levels increase. Current evidence suggests that there is a regulatory hierarchy consisting of three principal components: the electrochemical potential across the thylakoiO membrane, the oxidation state of a cysteine bridge within the hydrophilic domain of the ATP synthase, and the interactive binding of ATP, ADP and Pi to the enzyme complex. These controls act together to allow catalytic inactivation of the A TP synthase at night and dynamic modulation of activity as light levels change during the day.

26

SUMMARY OF PHOTOSYNTHETIC ENERGY TRANSFORMATIONS

The foregoing discussion described the structure of the photosynthetic membrane and the molecular events that convert light energy into chemical free energy. Camouflaged within these reactions are numerous steps that transform energy from one form to another. Since photosynthesis is fundamentally a series of energy transformation reactions, it is worthwhile to track the intermediate forms of energy and their locations within the thylakoid vesicle (Fig. 8). First is the conversion of a photon to an excited electronic state of an antenna pigment molecule located in the antenna system. The electronic excited state is transferred over the antenna molecules. Some of the excited state energy is converted back into photons and emitted as fluorescence, while some is converted into heat. However, much

ELECTRON CARRIERS

Light Absorption

t ANTENNA SYSTEM

Photochemistry

-t

Electron Ti'ansfer Proton and Electron Transfer

Redox Electrochemical

Energy

Electron Transfer

Chemical Bond (NAD?H) Energy (AT?)

"-,.. (CARBOHYDRATE)/

MEMBRANE VESICLE

AT? SYNTHASE

Figure 8. Energy Transformation in Photosynthesis. Photosynthesis is shown as a series of reactions that transform energy from one form to another. The different forms of energy are shown in boxes and the direction of energy transformation is shown by the arrows. The energy transforming reaction is shown by italics in the arrows. The site at which the energy is stored is shown in capital letters outside the boxes. The primary photochemical reaction, charge separation, is shown in the oval. Details of these reactions are given in the text. (Reprinted from Whitmarsh & Govindjee, 1995).

of the excited state energy is "trapped" by reaction centers and where it provides the energy for the primary photochemical reaction of photosynthesis - the transfer of an electron from a donor molecule to an acceptor molecule. The subsequent electron transfer reactions are energetically downhill, as electrons are transferred from photo system II to the PS I reaction center by intermediate carriers. The net reaction is the reduction of NADP+, produCing NADPH. In the photosynthetic process, much of the energy initially provided by light energy is stored as redox free energy in NADPH, to be used later in the reduction of carbon. In addition, the electron transfer reactions concentrate protons inside the membrane vesicle. In this process the electron transfer reactions convert redox free energy into an electrochemical

27

potential of protons. The energy stored in the proton electrochemical potential can in tum be used by the ATP synthase to covalently attach a phosphate group to ADP forming the terminal anhydride bond of ATP. Protons pass through the ATP-synthase protein complex that transforms electrochemical free energy into a type of chemical free energy known as phosphate group-transfer potential (or a high-energy phosphate bond) (Klotz, 1967). The net effect of the light reactions is to convert radiant energy into redox free energy in the form of NADPH and phosphate group-transfer energy in the form of ATP. Ultimately, the NADPH and ATP formed by the light reactions provides the energy for the photosynthetic carbon reduction cycle that occurs in chloroplast stroma (Fig. 2). The carbon reduction cycle' involves the transfer and rearrangement of chemical bond energy.

CONCLUDING REMARKS

Photosynthesis provides the energy for nearly all life on our planet. Due. to human activity the global atmosphere is currently undergoing a rapid and irreversible change that will have an increasingly strong influence on world climate. Increased levels of atmospheric carbon dioxide that may bring higher temperatures and redistribution of rainfall, as well as an increase in UV-B radiation incident on the earth, portend an environment that will have a profound, yet poorly understood impact on photosynthesis (Bowes, 1993). Over the past two decades our knowledge of photosynthesis has grown tremendously. We know most if not all of the players and the structures of many of the proteins have been resolved and progress on others is promising. The success of efficient transformation systems has provided a powerful tool to study protein function and structure. However, our understanding of photosynthesis is still limited, we are not able to alter the photosynthetic process in a manner that increases productivity or overcomes environmental adversity. Currently, parallel research efforts focus on revealing fundamental mechanisms of photosynthesis at the molecular level and on understanding the integration of the photosynthetic processes in the overall physiology. The goal is to blend the molecular and physiological knowledge to understand the role of photosynthesis in the growth and development of plants. On the horizon is the exciting possibility of using new found knowledge to design and create plants with improved photosynthetic performance.

REFERENCES

Bowes, G. (1993) Ann. Rev. Plant Physiol. and Plant Mol. BioI. 44; 309-332. Boyer, P.D. (1979) in "Membrane Bioenergetics", (C.P. Lee, G. Schatz, & L. Ernster, eds.), pp. 461-479.

Addison-Wesley, Reading, MA. Cramer, W.A. & Black, M.T. (1987) in "The Light Reactions". (1. Barber, ed.), pp. 447-493. Elsevier

Science Publishers. Cramer, W.A. & Knaff, D.B. (1991) in "Energy Transduction in Biological Membranes". Springer-Verlag,

New York. Debus, R.J. (1992) Biochim. Biophys. Acta 1102; 269-352. Ghanotakis, D.F. & Yocum, C.F. (1990) Annu. Rev. of Plant Physiol. Plant Mol. BioI. 41; 255-276. Golbeck, J.H. (1992) Annu. Rev. Plant Physiol. Plant Mol. BioI. 43; 293-324. Hansson, O. & Wydrzynski, T. (1990) Photosynth. Res. 23; 131-162. Klotz, I.M. (1967) "Energy Changes in Biochemical Reactions"'. Academic Press, New York, NY. Knox, R. (1995) "Excitons". VCH Publishers/American Institute of Physics, New York. Krauss, N., et al. (1993) Nature 361; 326-331. Kiihlbrandt, W., D.N. Wang, & Fujiyoshi. Y. (1994) Nature 367; 614-621. Murphy, D.1. (1986) Biochim. Biophys. Acta 864; 33-94. Mustardy. L.A. & A.G.S. JanosS)' (1979) Plant Science Letters 16; 281-284. Nobel, P.S. (1991) "Physicochen~ical and environmental plant physiology". Academic Press, San Diego,

California. O'Keefe, D.P. (1988) Photosynth. Res. 17; 189-216.

28

Ort, D.R (1986) in "Photosynthesis III - Photosynthetic Membranes and Light-Harvesting Systems", (L.A. Staehelin and C.J. Arntzen, eds.), pp. 143-196. Springer-Verlag.

Ort, D.R & Oxborough, K. (1992) Annu. Rev. Plant Physiol. Plant Mol. BioI. 43; 269-291. Ort, D .R. (1994) in "Encyclopedia of Agricultural Science" Academic Press, New York. Ort, D.R & C.F. Yocum (1995) "Ox-ygenic Photosynthesis: The Light Reactions". Kluwer Publisher,

Dordrecht, The Netherlands. Paolillo, D.l & Falk, RH. (1966) American Journal of Botany 53; 173-180. Staehelin, LA (1986) in "Photosynthesis III - Photosynthetic Membranes and Light Harvesting Systems",

(L.A. Staehelin and c.A. Arntzen, eds.), pp. 1-84. Springer-Verlag, Heidelberg, Germany. Taiz, L. & E. Zeiger (1991) "Plant Physiology". The Benjamin/Cummings Publishing Co., Inc., Redwood

City. California. Walker, D. (1992) in "Energy, Plants and Man". Packing Publishing Ltd., Sheffield, UK. Whitmarsh, l & Govindjee (1995) in "Encyclopedia of Applied Physics", G.L. Trigg, VCH

Publishers/American Institute of Physics, New York.

29

SPECIFIC FEATURES OF EXCITATION MIGRATION IN PHOTOSYNTHESIS.

AYu. Borisov

AN. Belozersky Institute ofPhys.-Chem. Biology Moscow State University 119899 Moscow, Russia.

INTRODUCTION.

Energy migration is a very general phenomenon. It proceeds between oscillating systems in mechanics, acoustics and electricity, it takes place in our world, in planet systems and between molecules in the microworld; this last one is the topic of this article.

Two similar pendulums hanging on the same horizontal cord make a good example of energy migration. If one pushes either of the pendulums, its amplitude decays while the second pendulum which was initially quiet becomes oscillating with increasing amplitude.

We have to make two important digressions before going into the realm of dye molecules.

a. As a first approach let us proceed to electric phenomena. A typical example is so called electromagnetic induction phenomenon, i.e. energy migration between radio coils which are present in every radio or TV. Michael Faraday was the first to observe such a phenomenon in simple experiments with big wire coils. From his experiments he concluded that the effect of induction of electric power from one coil to another depends on: a) the number of turns of wire in each of two coils, and we shall call it "the coil strength", b) the intercoil distance, c) coil mutual orientation.

Thus generalizing Faraday's findings to the molecular realm we may infer that the phenomenon of inter-molecular energy migration depends on the energy coupling between two systems:

1) the strength of the donor molecule - G1 (d), 2) the strength of the acceptor molecule, - G2 (a), 3) the intersystem distance - G3 (R),

4) mutual orientation of the systems - G 4 (j) , 5) dielectric properties of the media in the close vicinity especially between the

systems - G 5 (c) .

Light as an Energy Source alld h!fonnatioll Carrier in Plant Physiology Edited by Jennings et al., Plennm Press, New York, 1996 31

In addition mutual tuning (resonance degree) of interacting systems should be involved here as a m_ultiplier, but we shall do it in the following in a specific "Forster" way. Taking into account the advantageous fact that all the above mentioned parameters act independently, they may be involved in the theory as the product of independent functions:

(1)

One should not be surprised by the seemingly strange aspect of this formula. Although it differs much from the one we know from the Forster theory we shall see below that it is just the same.

b. Formula (1) may be regarded as the general case of those formulas comprising the Forster theory or the theory of inductive resonance (TIR) which are its approximations for some definite conditions which we will elucidate below.

The goal of this work is to understand explicitly the intrinsic features of the Forster theory, bearing in mind: a) the limits of its application, b) some peculiar features of biological objects, especially those from photosynthesis, as compared with "chaotic" physical or chemical systems.

We now proceed step by step to the essentials of the theory of induction resonance (TIR) which, as suggested above, is divided into a series of multipliers. They may be introduced in the following way. Let us consider the elementary act of an excitation transfer from donor "D" to acceptor "A" molecules:

In every energy transfer the rate of the process should apparently playa crucial role. It is proved in the theory of quantum state change, that in the presence of disturbed interaction the rate of quantum state transfer (in our case: from state D +A to state D+A*) is equal to:

k = 27r fdE w2

m h (2)

where W is the energy of a disturbed interaction, and dE is the differential energy

transition. Formula (2) gives another general description of the same phenomenon. The five Gi

functions offormula (1) are represented by W. We will not use formula (2). The only thing to be borrowed from it in reference to

formula (I) is that for a weak molecular interaction the interaction energy is squared!

TRANSITION DIPOLES OF DYES.

An important approximation of the Forster theory is that mutually coupled molecular transitions ( D ~ D* and A ~ A*) are considered as dipolar and consequently

W represents the energy of dipole-dipole interaction between donor ( D ~ D' ) and acceptor

(A~A') molecules. The strengths of molecular transitions So ~ S] are simple functions of their absorption

(emission) capacities in these optical bands. The transition dipole moment represents a vectorial alteration of the molecular electric

dipole, when a molecule undergoes a particular transition. If we consider absorption of a

32

photon (So~ Sl) this then represents the absorption transItIOn dipole moment. For fluorescence (SI ~ So) is the fluorescence transition dipole moment. An important point is that according to Einstein's law (Einstein, 1917), in the Si ~ Sj transitions the absorption and

emission abilities of a molecule are strictly proportional. In physics, energy is known to be equal to the product of interacting dipole moments. Therefore in formula (2) the square of the interaction energy W2 can be represented by the square of the product of transition dipoles of the donor and acceptor molecules (Forster, 1948; 1959; Agranowich & Galanin, 1982).

Gl(d)G2(a)G3(R)ocW2 oc[Pa Pdf OCR-3[fdv8a(V)V-lj.R-3[Jdv8d(V)V-lj (3)

where pa and Pd are the acceptor and donor transition dipoles, respectively; 8( v) are the molar extinctions of molecules; v is the frequency; R is the interchromophore distance.

In homogeneous atoms and small molecules, which have sharp absorption and emission lines, the So ~ SI and Sl ~ So transitions are equal in magnitude and oppositely directed. In more complex dye molecules, in particular in chlorophylls and other photosynthetic pigments, they are somewhat different.

Comments.

a) To use the dipole approximation of transition moments was a very fiuitful idea, but one should be entirely sure that it fits well in every particular case. This means that the lengths of both interacting dipoles must be considerably (at least 3 times) shorter than the interdipole distance. In classical physics the dipole length of a molecule is formally defined as the dipole moment divided by the electron charge. In the context of this definition the /B/Chl transition dipole (about 5 Debye) length is approximately one angstrom. However the quantum mechanical theory has proved that amplitudes of local charges often amount to a portion of the electron charge. Thus, if one takes quite a different approach to the problem, the dipole length in IB/Chls may be longer, at most, as the frame of their 1t-electron double bond system, i.e. about 6-7A . In this case it is imperative that the interchromophore distance exceed some 12 - 15A. However the question still requires clarification.

b) A red shifted position of the long wavelength absorption bands in IB/Chls favors a buildup of their net quantum absorption due to the "v-2" multiplier in formula (3) and even "v-4" in the final formulae (5,9). Correspondingly, B/Chl transition moments (",,5 Debye) are a bit greater than those for most of the "usual" dye molecules. Recently Pearlstein (Pearlstein, 1991) has obtained theoretical evidence that they may be about 8 Debye.

DIPOLE TUNING.

Forster elegantly avoided involvement of a special "tuning" function (Forster, 1948). In formula (3) he united both donor and acceptor transition dipoles under the same overlap integral! He replaced 8d( v) V-I by the "emission strength"of the same molecule according to the Einstein law (Einstein, 191 7):

(4)

Thus formula converts (3) into,

(5)

33

Now in formula (5) ca(v) V-I and ,;1 Fd(V) v- 3 terms represent the donor and acceptor dipole strengths, while the integral of their product is responsible for their mutual tuning. This factor apparently reaches a maximum, provided the peaks of these two bands coincide. In homogeneous dye molecules the Stokes shift between the absorption and fluorescence spectra reduces the tuning factor.

Comments.

As stated above, the Stokes shift slightly hampers energy migration between homogeneous molecules, especially in core complexes of photosynthetic RCs where excitations usually spend the greatest portion of their in vivo life. Thus, it apparently plays a negative role here. The Stokes shift is mostly determined by the following factors:

a) Molecules can absorb photons with a somewhat greater energy than the corresponding Sj state requires. Then excess energy is converted into a vibrational quanta (phonons), i.e. into thermal energy which delocalizes in surrounding molecules in less than a few picoseconds, and thus is lost for the subsequent electronic processes. This IQcal heating means inevitable loss of the corresponding portion of electronic energy. In dye molecules this factor can as a rule account for Stokes shifts up to and no more than 40-50 cm-1

("" 2 - 3 nm for /B/Chls see Connolly et al.; 1982; Seely et aI., 1973; Zucchelli et aI., 1992). b) Being excited, a molecule necessarily changes the distribution of its valence

1t-electrons. This may entail some conformational change in the chromophore structure if it possesses flexibility. Such a change also signals a loss of some electronic energy and thus contributes to the Stokes shift. Luckily, flat tetrapyrrol rings have conjugated bonds nearly throughout. Therefore their construction is very rigid and this type of energy loss is negligible.

c) The surrounding molecules (especially polar ones and those with polar groups) may respond to the above changes in the charge distribution in an excited molecule thus assimilating a portion of electronic energy. But Nature has taken special care of it: in photosynthetic membranes the nearest surroundings of /B/Chls are formed by very neutral, hydrophobic parts of lipids (tails) and membrane proteins (0.- and /3-helixes) (Zuber & Brunisholz, 1991). So this excitation energy loss is also small.

d) In /B/Chls the long wavelength absorption peak is readily shifted to longer wavelengths, even due to weak interactions with the closest polypeptides and mutual interactions. This enables /B/Chl molecules to form in vivo series of distinct spectral forms which widen the spectral range of efficient light absorption and greatly facilitate excitation delivery from vast antenna ensembles to their related RCs by optimizing the overlap integral in eq.(5). Even in "shallow" PS2 this gain reaches "" 1.5 (Jennings et aI., 1993). Such bathochromic shifts are known for many dyes, but hardly any of them can produce such great shifts as in Bchls In Bchls the spectral shifts are record-breaking: from 770 nm in neutral organic solutions to 880-890 nm in vivo. Due to this remarkable feature of Chis one RC can efficiently serve many hundreds of antenna pigments!

MUTUAL ORIENTATION OF MOLECULES G4G>.

We have already mentioned this multiplier by including in formula (1) an "angle" function which determines mutual orientation of two interacting dipoles. It helps if the space angles are known. For ideal dipoles the value ofG4 (j) could be calculated, using the well

known formula:

G4 (j) == G4 (i,y,k) = (cos (i) - 3cos (y)cos (k)r (6)

34

The angles i, y and k are independent in the three-dimensional space, but if two dipoles are positioned in a plane, the following equality is evident: i + Y + k = 180 0 . For randomly

oriented molecular ensembles Forster suggested using an "averaged" value of G 4 (j) which is equal to:

2/3 in three dimensional space (Forster, 1948; 1959)

in two dimensional space! (Campillo et aI., 1977)

The lower value is 1.75-fold greater than the upper one, though in both cases G 4 (j) varies from zero (orthogonal dipoles) to four (coaxial dipoles). This is because in two-dimensional structures the portion of more advantageous mutual orientations (with G 4 (j) l> 2.0) is noticeably greater than in three-dimensional structures.

Comments.

a) Although being an annoying obstacle to the theory, the fact that all dye molecules are apparently three-dimensional entities is a very advantageus circumstance!

In TIR, as has been indicated in many studies, the ratio between maximal and minimal (4.0/0.0) G4 (j) values is infinite and correspondingly, the error in its applications seems to be infinitely great. Fortunately, all dye molecules are three-dimensional bodies and therefore their "club" shaped 7t-electronic transition dipoles are also three-dimensional.

This means that in none of the mutual positions of the two dipoles their products may be equal to 4.0, neither can it reach Gmin=O.O even in their orthogonal position

The latter conclusion is more important, as now one can use TIR for measuring interchromophore distances or excitation jumptimes with reasonable precision.

We calculated the maximal and minimal values of G 4 (j) for porphyrins. Starting from the experimental fact that maximum polarization in this class of molecules is,

Pm .. , = 0.42 ± 0.02 (Clayton et al. 1968, Goedheer, 1973)

instead of the theoretical value of 0.50, we obtained the upper and lower limits for the G 4 (j) function respectively in coaxial and orthogonal mutual positions of interacting molecules (Borisov & Vinogradov, 1992):

theory for unidirectional dipoles: calculation for BChis and Chis:

4.00 l> G4 (j) l> 0.00 3.59 l> G 4 (j) l> 0.29

b) The extensive studies by many laboratories have proved that i) antenna !B/Chls lie in a very thin layer in the membrane (Zuber & Brunisholz, 1991); ii) their red transition dipoles lie in this plane within some 20-25° (Breton, 1974; Vermeglio & Clayton, 1976; Abdurakhmanov et aI., 1978). Thus these antenna ensembles are close to two-dimensional

systems and consequently their averaged G 4 (j) must be greater than 2/3 calculated by Forster for liquid and solid solvents. Taking this circumstance into account and bearing in mind the above calculated ratio of maximal to minimal G4 (j) values: 3.52 : 0.29 ~ 12,we suggest using in the systems with an unknown precise structure the mean averaged value:

1 "Two dimentional space" implies not only that all molecules are in the same plane, but also that their transition dipoles lie in that plane

35

G4 (j) = 1.0

In this case the maximal possible error in G4 (j) would be about 3.59, but the error in the calculation of the interchromophore distance will be much smaller (see below).

INTERACTION WITH THE NEAREST ENVIRONMENT Gs (m).

The best media for electromagnetic propagation or interaction is a total absence of any media, at least in the immediate vicinity of the interacting systems. In real media, electromagnetic forces and interactions decrease noticeably, up to about hundreds of times in constant fields or at low frequencies. In the optical region the corresponding factor is proportional to the squared refractive coefficient (n) but, taking into account the

W2 multiplier in formula (3) we can conclude that in TIR:

(7)

Therefore for energy migration to be efficient it should proceed in the media with the lowest possible "11 "value. In thousands of condensed media yet known 1.28 <l 11 <l 2.40

(Handbook 1981); that correspond to 2.68 <l n4 <l 33.2 .

Comments

The lowest values of" n" are peculiar to neutral molecules, with all their local charges compensated and immovable and external electrons fixed along ordinary (not double!) bonds, like saturated carbohydrates. In photosynthetic membranes the nearset surroundings of /B/Chl are formed by lipid tails and transmembrane parts of proteins. The lipid tails are simply saturated hydrocarbons and consequently they will have the lowest values of n .

Now, what about" 11" value in proteins? Few data available in the literature are as a rule about 1.50 - 1.65 (Armstrong et aI., 1947; Putzeys & Brostreaux, 1936). This is due to the presence of charged and aromatic amino acids in all proteins. But extensive studies in Zuber's laboratory have revealed (Zuber. 1987; Zuber & Brunisholz, 1992) that these types of amino acids are predominantly located in their outer C- and N-terminals while the transmembrane a- and l3-helixes are formed from nonpolar and alifatic amino acids. Thus all the micro environment around /B/Chls in vivo is formed from the matter which must have the lowest possible nvalue, i.e. about

in vivo n == 1.28 - 1.30; n4 == 2.7

THE CRITICAL DISTANCE OF EXCITATION MIGRATION

Now it is high time to combine all the multipliers discussed above into what we call the basic Forster formula:

(8)

here L1 r m is the mean excitation jumptime from donor to acceptor molecules; R is the mean distance between donor and acceptor chromophores; Fd (v) is the normalized fluorescence

36

spectrum of donor molecules; By definition, the critical distance for excitation migration (Rer) is that when rate

constant of excitation migration k nr' and of its trivial deactivation in donor molecules,

(r~) -I, are equal. Thus with k m = (r~) -I, from formula (8) we obtain:

r~ G 4 (j)' f dvFd(v) &.(v) v-4

r r n 4

(9)

Notice that this formula may be used both for concrete G 4 (j) values if known, and for

averaged values G 4 (j) in other cases. If one divides formula (9) in its general presentation and in its particular form with

R = ReT then most of their terms would be mutually cancelled and thus one obtain another formula in TIR:

(10)

This important formula enables one to calculate the mean duration of an excitation living jumptime I'u i from the donor molecule. For homogeneous excitation migration, once the

rate constants are equal for both directions this formula also gives the mean number of excitation jumps to proceed.

Comments

ReT is a constant, but unfortunately it is different in every particular donor/acceptor system, even in every solvent used which changes the r~ value. It is reasonable for

photosynthetic applications to introduce this constant in the manner recommended by R.S.Knox. This author associated ReT not with the real, but with the intrinsic radiative lifetime r r of donor molecules which is practically the same for /B/Chls in different media (Knox, 1968).

ReT was calculated for BChl in vivo to be equal to 80 A (Reed et a1.1968) for n = 1.5

and G 4 (j) = 2/3. For G 4 (j) = 5/4 (precise two- dimensional case) it was calculated as 89 A (Campillo et al 1977). For Chla in vivo Rer was calculated in a number of laboratories but mostly for different r~ therefore its values were widely scattered within 47-70 A (for review

see Knox, 1977). The problem of Rer was revised (Borisov & Zuber, 1992) bearing in mind

i) the increased G4 (j) value from 2/3 to 1.0; ii) decreased n from 1.5 to 1.3, i.e. n4 = 2.7 as it was deduced in the previous chapter. The revision yielded:

Bchla in vivo: ReT ~ 100 ± loA (Borisov & Zuber, 1992).

b) Now we can estimate maximal possible errors in the determination of interchromophore distances (R):

coaxial chromophores. This chromophore pair has maximal G; value (3.59). In the

formula (10) Rer ~ G~/6 = 3.591/6 = 1.24. Thus, by using G~!6 = lone makes underestimation ~ 24 %.

37

Orthogonal chromophores. These chromophores are characterized by a minimal G4

value (0.29). In the formula (10) Rer == Gl!6 = 0.291/6 == 0.81 == (1.24)-1. Thus, by using

Gt = lone makes overestimation == 1.24 = 24 %. Other mutual orientations apparently yield smaller errors. Notice that the corresponding error estimations for other (not porphyrin) dye molecules are close to the above calculated.

c) In biological applications one often deals not with pure molecular solutions, but with small sub cellular particles, membrane fragments, pigment-protein complexes and other homogeneous polymolecular associates, each having a limited number of chromophores. In such objects chromophore distribution can not be regarded as chaotic, although sometimes the difference is not very significant. If possible, it is better to know something about real molecular mutual positions in such objects. If this is not possible, it is desirable to check that the chromophores are not positioned at the extreme angles to each other. Even if this cannot be proved, the maximal possible error in the estimation must be within 0.23-0.24 as it demonstrated above.

GENERAL REMARKS.

TIR was first developed by T. Forster in 1947 and then generalized by Dexter (Dexter, 1952) on the basis of quantum mechanics. Despite some attacks and discussions, it has developed as a powerful, muIti- functional tool, especially in physics, chemistry, molecular biology and photosynthesis. On the other hand we should not overestimate and extend it to research areas greater than it really covers. There has been a tendency in photosynthesis to use TIR in all cases, particularly because the alternative exciton theory is still not developed to the extent that allows quantitative calculations. Three independent criteria (small interaction energy; jumptime » Frank-Condon rearrangement time; dipole length « R) all lead to rather similar estimations: homogeneous and slightly heterogeneous IS/Chls TIR usage appear to be reasonable for interchromophore distances R 2: lSA. In the R <lISA region one apparently gets into the exciton realm.

Does this produce real problems? This appears to be the case. It is possible that in excitonic states, when an excitation is delocalized over at least two molecules, these molecules may radically change their basic optical properties: absorption, emission, transition dipoles etc. This may produce very serious effects. At least one of them deserves serious study. If we assume that the interchromophore distance between two or several dye molecules corresponds only to the transition region between intermediate (excitonic) and weak (inductive resonance) interactions, then it seems plausible that they absorb photons as individual molecules, but after the absorption act, an excited electron becomes spread over a part or all of the whole ensemble. This means that the length of such an "antenna", i.e. its "dipole length" may be greater and correspondingly its critical distance Rer may become greater than for an individual molecule. Such a mechanism, if it exists, may produce important effects in energy migration between ensembles with different numbers of molecules - the smaller group (or a single molecule in the limit) would be preferentially populated by electronic excitations.

REFERENCES.

Abduraklunanov LA., Ganago A.O., Erokhin Y.E., Solov'ev A.A (1978) Biochim. Biophys. Acta, 546:183-186

Agranowich V.M., Galanin M.D. (1982) "Transfer of elecrtronic excitation in condensed media", North-Holland, Amsterdam

Armstrong S.H., Budka M.J., Morrison M. & Hasson, M.P. (1947) 1. Organic Chem., 69: 1747-1752 Borisov A., Zuber H. (1992) in "Research in Photosynthesis" (N.Murata ed.), yoU, pp 117-120,

38

Kluwer Academic Publishers. Borisov AY., Vinogradov AY. (1992) BioI. Membranes, 6(4), pp 569-586, Hartwood Acad. Publ.

GmbH Breton, 1. (1974) Biochim. Biophys. Res. Comm., 59:1011-1017. Campillo A.J., Hyer RC., Monger T.G., Parson W.W., Shapiro S.L. (1977) Proc. Natl. Acad. Sci.

USA, 74:1997-2001. Clayton RK., Reed D.W., Zankel K.L. (1968) Proc. Natl. Acad. Sci. USA, 61: 1243-124.9. Connolly J.S, Jansen AF., Samuel E.B. (1982) Photochem. Photobiol., 36:559-576. Dexter D.L. (1953) J. Chern. Phys., 21:836-850. Einstein A (1917) Phys. Z., 18:121-129. Forster T. (1948) Ann. Physik .. 2:55-67. Forster T. (1959) Disc. Faraday Soc., 27:7-12. Goedheer J. (1973) Biochim. Biophys. Acta, 292:665-676 Handbook of Chern. Phys. (1981) 61 edition, pp 341-390. CRC-Press, Robert Weast ed. Boca Raton,

Florida. Jennings R, Bassi R, Garlaschi F.M., Dainese P., Zucchelli G. (1993) Biochemistry, 32:3203-3210 Knox RS. (1968) J. Theoret. BioI., 21:244-259 Knox RS. (1977) in "Primary Processes of Photosynthesis", (Barber 1. ed.) pp. 55-97, ElsevierlNorth

Holland Biomedical Press, Amsterdam. Pearlstein RM. (1991) in "Chlorophylls", (Scheer H. ed.), pp. 1047-1078, CRC Press, Boca Raton Putzeys P. & Brostreaux 1. (1936) Bull. Soc. Chim. BioI., 18: 681-688. Seely G.R (1973) 1. Theor. BioI., 40:173-187 Vermeglio A, Clayton R (1976) Biochim. Biophys. Acta, 449:500-515. Zuber H. (1987) in "Light Reactions", (1. Barber cd.) pp 197-259, Elsevier Science Publ., Amsterdam Zuber H., Brunisholz R. (1991) in "Chlorophylls", pp 627-703, CRC Press, Boca Raton, Florida. Zucchelli G., Jennings R:C., Garlaschi F.M. (1992) Biochim. Biophys. Acta, 1099:163-169.

39

BIOCHEMISTRY AND MOLECULAR BIOLOGY OF PIGMENT BINDING PROTEINS.

Roberto Bassi, Elisabetta Giuffra, Roberta Croce, Paola Dainese l and Elisabetta Bergantino l

Universita di Verona, FacoIta di Scienze, Strada Le Grazie 37134 Verona, Italia. IUniversita di Padova, Dipartimento di Biologia via Trieste 75-35121 Padova, Italia.

INTRODUCTION

ChlorophylIs and carotenoids are components of every pigment-protein complex in the photosynthetic apparatus of higher plants. They function in light harvesting, photoprotection, and down-regulation of PSII photochemistry. In the folIowing we summarize the present knowledge on the localization and function of chlorophyll a, chlorophylI band carotenoids within the photosynthetic apparatus of higher plants. This subject was previously reviewed (Bassi et a1.l990, Jansson 1994, Jennings et ai. 1995)

Pigment-proteins of both PSI and PSII have a dual origin in that they are in part coded by the plastid genome and synthesized within the chloroplast. Alternatively they can be coded by the nuclear genome, synthesized in the cytoplasm as higher molecular mass precursors and then imported into the chloroplast where they are processed to the mature size and assembed into pigment proteins within the thylakoid membrane. ChlorophylI a­proteins of PSI and PSII core complexes belong to the first group while chlorophylI alb proteins of both PSI and PSII antenna systems belong to the second group. In the folIowing, after a short description of the supramolecular complexes, we will proceed to summarise the present knowledge of core complex proteins while antenna proteins, which are largely homologous to each other, will be discussed subsequently.

PHOTOSYSTEM I AND PSII SUPRAMOLECULAR COMPLEXES

PSI is a muItisubunit complex that is located in the unstacked, stroma-exposed membranes where it forms the large PFu particles (10.3 x 12.5 nm) visible by freeze fracture E.M. (Simpson, 1983). It is composed ofa core complex (PSI core) and a light harvesting component (LHCI). A particle containing both these components with a chl1P700 ratio of about 210 and a chI alb ratio of 6-6.5 can be obtained by solubilization with anionic detergents and sucrose gradient ultracentrifugation (MulIet et aI., 1980; Bassi & Simpson,

Light as an Energy Source and Infomlation Carrier in Plant Physiology Edited by Jennings et al., Plenum Press, New York, 1996 41

1987; Nechustai et aI., 1987). Such a preparation contains at least 15 polypeptides. Similar preparations in smaller yield can be obtained by SDS-PAGE or Deriphat-PAGE (Bassi et aI., 1985; Peter et aI., 1991). The pigment moiety of the PSI-LHCI complex from spinach chloroplasts (Anderson et al., 1983) includes chi a and b, 13 -carotene and the xanthophylls, lutein and violaxanthin; neoxanthin, a component of PSII chlorophyll-proteins is, however, absent (table 1). This is consistent with the analysis of isolated stroma lamellae obtained by mechanical fractionation (Henry et aI., 1983).

PSII is located in the stacked membranes of granal chloroplasts where it forms the large EFs (11.7 x 15.5 nm) freeze-fracture particles with the core complex. The outer LHCII is arranged in the complementary fracture face (PFs) to form 9.0 x 10.3 nm particles (Simpson 1978, Miller & Kushman, 1979). PSII is composed of a chi a binding core complex and several surrounding chi alb proteins which constitute the outer antenna. The whole PSII complex can be prepared as stacked membranes free of other thylakoid complexes (Berthold et aI., 1981). When solubilized, the complex splits into the core complex and the outer antenna components that can be separated by sucrose gradient ultracentrifugation or PAGE (Bassi et aI., 1987, Dainese & Bassi, 1991, Peter & Thornber, 1991). The PSII membranes and the isolated core complex can catalyze electron transport from water to quinone analogues or other electron acceptors (for a review see Satoh, 1985). Carotenoids are present in both the PSII core complex and the antenna moieties. 13-carotene is bound mainly to the PSII core complex whereas the xanthophylls are components of the antenna system.

Table 1. Pigment composition of PSI proteins.

ChI a ChI b J3-carotene Lutein Neoxanthin Violaxanthin Reference PSI-LHCI 186 24 27 12 0-2 9 a, b, c PSI-CORE 100 14 b, c LHCI* 86 24 13 24 01 9 d Lhcal ND ND ND + + e Lhca2 ND ND ND + ND e Lhca3 ND ND ND + ND e Lhca4 ND ND ND + ND e

* Calculated from the difference between PSI-LHCI complex and PSI core complex (Sieferman-Harms, 1985). I The neoxanthin value was set to 0 since stroma membranes were shown to lack this pigment (Henry et aI., 1982). a. Anderson et aI., 1983a; b. Begins & nelson, 1975; c. Setifet aI., 1980, d. Siefermann-Harms, 1985, e. Thornber et aI., 1993.

CHLOROPLAST ENCODED CHLOROPHYLL A 13-CAROTENE PROTEINS OF CORE COMPLEXES.

Photosystem I core complex

This is a chi a binding complex which has an apparent size of 250 kDa in non­denaturing PAGE (Bassi et aI., 1985; Bassi & Simpson, 1987; Peter & Thornber, 1991). This particle binds P-700 and can photoreduce NADP+ in the presence of ferredoxin and ferredoxin-NADP reductase (Bruce & Malkin, 1988). All pigments are bound to the two major protein subunits, the products of psaA and psaB chloroplast genes, with P700 being located at the interface between the two (Goldbeck, 1990). Together, the subunits bind about 90 chi a (Bassi & Simpson, 1987) and 14 13-carotene molecules (Bengis & Nelson 1975, Rawyler et aI., 1980, Anderson et aI., 1983, Haworth et aI., 1983, Siefermann-Harms, 1984). The organization of 13-carotene in PSI is unknown yet. However, it is expected to be elucidated soon since the structure of PSI from Synechococcus is resolved from 3.8 A diffraction crystals (Witt et aI., 1987; Krauss et aI., 1993).

42

Photosystem II Core Complex

The core complex binds the electron transport cofactors Mn2+, P680, phaeophytin, Qa, in addition to 50-55 antenna chI a molecules, and carotenoids (table 2). The two sets of cofactors are bound to distinct polypeptides, the electron transport components being bound to the Dl and D2 polypeptides while the antenna pigments are located on the two homologous CP43 and CP47 proteins. Additional subunits of the core complex include the two cytochrome b559 subunits and the three oxygen-evolving enhancers (O.E.E.).

Chlorophyll binding proteins in PSII include the Dl and D2 polypeptides encoded by psbA and psbD respectively. Both proteins are composed of 353 amino acid residues in most species, however their apparent molecular weight as determined by SDS-P AGE is 32 and 34 kDa respectively. The isolated complex, consisting ofDI and D2 polypeptides and cyt b559, is photochemically active (Namba & Satoh 1987). Bound pigments include 4 (Namba & Satoh, 1987) or 6 chi a molecules and two pheophytins (Gounaris et a!., 1990, Kobayashi et a!., 1990). The number of l3-carotene molecules is also under debate. One (Namba & Satoh, 1987) and two (Van Dorsen et a!., 1987, Gounaris et a!., 1990, Kabayashi et a!., 1990) molecules per RC have been reported. However, spectroscopically distinct l3-carotene forms can be induced by the interaction between carotenoid molecules in dimeric RC complexes, supporting the view of one l3-carotene per DI-D2-cytb 559 complex (Newell et al. 1992). The organization of l3-carotene in this complex can only be hypothesized on the basis of the homology with the situation of spheroidene in Rh. sphaeroides. If the homology holds, 13-carotene should be located in D2 near the accessory chI a which lies between the pheophytin andP680 (Yates et a!., 1988; Feher et aI., 1991).

Table 2. Pigment composition ofPSII proteins (molecules per polypeptide). The values refer to experimentally determined stoichiometry. Values within brakets refer to the figure obtained assuming that 12 chlorophyll molecules bind to each pigment-protein as suggested by sequence homology to LHCII complex resolved by electron crystallography (Kiihlbrandt et a!., 1994)

ChI a Chlb l3-carotene Lutein Neoxanthin Violaxanthin Reference DIID2 6+2 phaeo. 1 (2) c, d, f heterodimer CP47 25 2 0.4 a, c, d, f CP43 25 3 1 a, b, h PSII core -56 5 a, b,h CP29 6 (9) 2 (3) 0.1 (0.15) 1.3 (2) 0.5 (0.7) 1 (1.5) a,h CP26 6 (8) 3 (4) 0.1 (0.15) 1.3 (1.7) 0.4 (0.5) 0.5 (0.7) a,g CP24 3 (7) 2 (5) 0.2 (0.15) 1.2 (2.8) 0.5 (1.3) a,g LHCII 7 5 2 0.4-1 * 0.05-0.02 a, e Total psn 160 70 10 2.8 7 3.7 a membranes

* Calculated from Bassi et a!., 1993. a. Bassi et a!., 1992; b. Delepelaire & Chua, 1979; c. Gounaris et aI., 1990; e. Kiihlbrandt et a!., 1994; f. Namba & Satoh, 1987; G. Peter & Thornber, 1991; h. de Vitry et a!., 1984.

CP43 and CP47

Due to the unequivocal location of PSII reaction center in the DI-D2-Cyt b559 complex (Namba & Satoh, 1987) both CP43 and CP47 must be considered as a part of the light harvesting system. They bind both chI a and carotenoids (table 2), and are encoded by the psbB and psbC genes which are located in the chloroplast genome close to the psbA and psbD genes which encode the Dl and D2 proteins. The deduced protein sequences ofCP47

43

(508 residues) and CP43 (461 residues) are well conserved, with homology being 94 or 95% between higher plant proteins and 72 or 77% with the cyanobacteria proteins respectively (Vermaas et aI., 1987). In the light of the significant homology between the two proteins, a common structure with six transmembrane helices can be hypothesized (Holschul et aI., 1984; Morris & Hermann, 1984; Bricker, 1990). The two proteins are thought to bind 20-25 chi a molecules each (de Vitry et aI., 1984; Satoh, 1985), though lower values have been suggested (Glick & Melis, 1989) on the basis of functional measurements in developing plant material and mutants lacking chI b or by biochemical measurements (Barbato et aI., 1991). The lower values should be viewed with caution as a recent finding shows that intermittent light-grown plants change not only their subunit stoichiometry but also in the number of chI molecules per polypeptide (Marquardt & Bassi, 1993). A more complete study on pigment binding to PSII core subunits is certainly needed.

The major carotenoid of CP43 and CP47 is I)-carotene, but lutein may be present in low amounts (table 1) (Bassi et aI., 1993). Delepelaire and Chua (1979) reported there were more I)-carotene molecules in CP43 than in CP47 (probably 5 and 3 respectively). The organization of this pigment in the complex is unknown but the orientation of I)-carotene in CP43 and CP47 is certainly different. I)-carotenes in CP43 are largely parallel to the plane of the membrane and those in CP47 are perpendicular. The absorption maxima of I)-carotene in CP43 were found to be blue shifted by 10-15 nm as compared to those of CP47 (Breton & Satoh,1987).

NUCLEAR ENCODED CHLOROPHYLL AlB PROTEINS OF THE ANTENNA SYSTEM

Light harvesting complex I

In higher plants, LHCI splits into two moieties, LHCI-680 and LHCI-730 according to their fluorescence emission maxima at 77 K (Lam et aI., 1984). Most authors agree that LHCI polypeptides are the product of four lhca genes (Hoffman et a1.1987, Knoetzel et ai. 1992). The first report of an isolated LHCI was as a high molecular weight complex from Chlamydomonas reinhardtii (Wollman & Bennoun, 1982). In this organism the complex appears to be different from the higher plants in that the fluorescence emission of the whole complex is at 705 rather than 735 nm. Moreover, the number ofLHCI polypeptides is higher (7-10 vs 4-5 in higher plants) (Bassi et aI., 1992). There are no rigorous measurements available for the pigment-protein stoichiometry in either LHCI-730 and LHCI-680 due to pigment loss when dissociating individual proteins from the LHCI complex. Indirect evidence suggests that each LHCI polypeptide may bind 8-10 chi molecules (Bassi et aI., 1992). The composition of higher plants LHCI is summarized in table 1.

The reported chi alb ratios for LHCI have been ranged widely from 1.4 to 3.0 (Bassi & Simpson, 1987, Bassi et aI., 1985). The lower values are probably due to the fact that some pigment proteins like LHCI-680 and CP24 are prone to loss of chi a when subjected to PAGE. The number of chlorophyll molecules bound to LHCI-680 in a PSI unit is approximately 30-35 (Bassi & Simpson, 1987). Based on this value, the carotenoid composition ofLHCI can be calculated (table 1). Determinations on the individual pigment­proteins dissociated from the PSI-LHCI complex is qualitative (Thornber et aI., 1993), simply confirming that violaxanthin and lutein are present while neoxanthin is absent in LHCI. The presence of I)-carotene has not been reported from purified LHCI; however, since its concentration in PSI is decreased by half on removal of LHCI from a PSI-LHCI complex, I)-carotene may be a component of LHCI similar to the minor pigment-proteins CP29, CP26, and CP24 ofPSII (Bassi et aI., 1993).

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The major LHCII complex

The light-harvesting chlorophyll alb protein of psn of higher plants photosystem IT (LHCn) is the most studied pigment protein and therefore it will be discussed in some detail. In fact, its sequence homology to many other proteins in both photosystems of higher plants, as well as with algae having light harvesting and/or photoprotection functions, makes it the most useful model for the organization of pigments in antenna systems.

The major LHcn complex was the first chI-protein described. It constitutes about one third of the total thylakoid protein and binds half of the total chlorophyll. This protein is mostly, if not completely, present in a trimeric form in the membranes (Peter & Thornber, 1991). , HPLC analysis of purified LHCn shows that it contains, besides chI a and chI b, the xanthophylls lutein, neoxanthin, and violaxanthin. The presence of ~-carotene in LHCn has been suggested in earlier reports (Braumann et aI., 1982; Lichtenthaler et aI., 1982), but the analysis of a purified preparation (Bassi et aI., 1993 ) ruled this out.

While HPLC pigment analysis accurately determines the relative pigment amounts, the pigment-protein stoichiometry is the object of intense debate. Biochemical determinations yielded chlorophyll per LHCn monomer values of 7 (Ryrie et aI., 1980), 12 (Dainese & Bassi, 1991), or 15 (Butler & Kilhlbrandt, 1987). Electron crystallography at 6 A resolution (Kilhlbrandt & Wang, 1991) confirmed the higher value, but recent determination at 3.4 A supported the value of 12 or 13 (Kilhlbrandt et aI., 1994). On the basis of the 1. 4 molar ratio between chI a and b, determined for highly purified complex, 7 chI a, 5 chI b and two lutein molecules can be assigned to each LHen monomer (Bassi et aI., 1993, Juhler et aI., 1993). Two additional pigments have been found in LHen preparations: violaxanthin (V) and neoxanthin (N). V has always been found in variable sub stoichiometric amounts, i.e., ten times less with respect to lutein (Setif et aI., 1980; Siefermann-Harms 1984). The case of N is less clear, since it can be found in amounts of one molecule per LHen monomer; however, the amount is variable in different preparations (Peter & Thornber, 1991, Bassi et aI., 1993). This suggests that both V and N may be located more peripherally than lutein in the complex.

Lutein, (or the related xantophyll Loroxantin) seems to be essential for LHCn accumulation and stability as shown in lutein deficient mutants (Chunaev et aI. 1991). Neoxanthin and violaxanthin may not occupy specific positions in the complex, or are not present in each monomer. In fact, LHen is an heterogeneous protein: not only can several apoproteins be resolved from the LHen complex by denaturing electrophoresis (Spangfort & Andersson, 1989; Di Paolo et aI., 1990; Sigrist & Staehelin, 1992), but also a number of LHen subpopulations can be resolved by IEF (Larsson et aI., 1987, Bassi et aI., 1988, Bassi & Dainese., 1992). This is consistent with the large number of highly homologous genes which have been found in several species (Dunsmuir, 1985, McGrath et aI., 1991) and which fall into three types known as Lhcb 1, Lhcb2 and Lhcb3 (Jansson et aI., 1992).

Interesting results were obtained from reconstitution experiments of LHCn complexes. It was found that adding purified pigment to the denatured apoprotein, chI a and b were indispensable to reassembly, however, in the absence of xanthophylls the stability of the complex was significantly decreased (Plumley & Schmidt, 1987; Paulsen et aI., 1990). Since neoxanthin can substitute to some extent for lutein (Plumley & Schmidt, 1987), the possibility that neoxanthin (and perhaps also violaxanthin) actually occupy specific sites in some components of the heterogeneous LHCn complex cannot be excluded.

Ucb] and 2 but not Lhcb3 gene products can be phosphorylated in the stroma exposed N-terminal domain leading to a detachment of part of the LHCn antenna from psn and migration from grana to stroma exposed membranes where it can transfer energy to PSI. This process, called state] - state 2 transition, is beyond the scope of this review and has been exaustively described in recent articles (Allen 1991, Allen 1995).

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The minor pigment-proteins of PSII antenna

Beside LHCII, three additional chi alb-xanthophyll proteins are present in the PSII complex. They are CP29, CP26, and CP24, named from their apparent mass in non­denaturing (green) gels. They are encoded by the nuclear genes Lheb4, Lheb5 and Lheb6 respectively, and synthesized in the cytoplasm as higher molecular weight precursors and imported into the chloroplast. They are subsequently inserted into the thylakoids and form pigment-protein complexes which have a higher chi alb ratio than LHCII. They are present in PSII in equal amounts; however, together they bind only 15% of the total PSII chlorophyll (vs. 63% by LHCII).

Comparison of the primary sequences of CP29, CP26, and CP24 show that they are highly homologous to each other and to other the gene products. This suggests they have a molecular structure similar to that described for LHCII. Nevertheless, recent results showed that they are distinct in their carotenoid content, most noticeably binding more than 80% of the PSII violaxanthin, strongly suggesting they have an important role in the regulation of energy transfer to PSII core complex.

The pigment-protein complex CP29 was the first minor complex to be distinguished from LHCII (Machold & Meister, 1979). Its apparent mass is slightly higher (31 kDa) than that of the major LHCII polypeptides both in denaturing and green gels (Camm & Green, 1980, Bassi et ai., 1987). The protein is N-terminally blocked, but partial sequencing was obtained from spinach, maize, and tomato (Henrysson et ai., 1989) showing that it is coded by the Lheb4 gene which was recently isolated and sequenced (Morishige & Thornber, 1991). This is the largest the gene, coding for a mature protein of approximately 257 amino acids, and is extremely well conserved between maize and barley not only in the mature protein but also in the pre-sequence region (Morishige & Thornber, 1991). The larger size is due to a 42-residue long insertion, which is not included in other the genes; the insertion is located just before the first transmembrane helix. This complex has a chi alb ratio of 2.2-2.8 (Dainese et ai., 1990, Dainese & Bassi, 1991, Peter & Thornber, 1991), and contains lutein, violaxanthin, and neoxanthin as additional pigments (Peter & Thornber, 1991, Bassi et al 1993). Values offour (Barbato et al. 1989, Irrgang et ai., 1991), eight (Dainese & Bassi, 1991), and ten (Henrys son et aI., 1989) chi molecules per polypeptide have been reported. For a long time it was thought that CP29 was not phophorylated in higher plants. However, it is now clear that CP29 can be heavily phosphorylated (Bergantino et ai. 1995) in the N­terminal, stroma exposed domain by a kinase different from that of LHCII. The physiological conditions leading to CP29 phosphorylation are the excess of light leading to overreduction of plastoquinone and potential photoinhibition (Bergantino et al. 1995). The phosphorylation of CP29 leads to changes in the protein conformation and spectral properties (Croce, R. & Bassi,R., unpublished results).

The CP26 pigment-protein complex has been described in maize and spinach as having an intermediate chi b content between that of CP29 and that of LHCII (Bassi et aI., 1987, Dunahayet ai., 1987). Its pigment component includes violaxanthin, lutein, and neoxanthin, as well as chi a and chi b in a 2.2 ratio (Dainese & Bassi, 1991). Lower (l.8) and higher (2.7) chi alb ratio values have also been reported (Barbato et aI., 1989, Thornber et ai., 1993). The binding of between 9 and II chi molecules per polypeptide has been determined (Dainese & Bassi, 1991, Peter & Thorber, 1991). In urea gels, two closely migrating apoproteins are resolved, in similar amounts, with apparent masses of 28 and 29 kDa (Bassi et aI., 1987). Both polypeptides are N-terminally blocked and therefore the actual molecular weights and the maturation sites are not known. Antibodies against oligopeptides obtained from the Lheb5 gene recognize the CP26 apoprotein (Allen & Staehelin, 1992), implying that this is the coding gene. This has been confirmed by direct protein sequencing.

CP24 is the product of the Lheb6 gene as shown by N-terminal sequencing (Morishige et ai., 1990). Lheb6 genes have been sequenced from tomato and spinach, (Schwartz & Pichersky, 1990, Spangfort et aI., 1990) coding for a 210 amino acid protein. The

46

carotenoid content is qualitatively similar to that of CP29 and CP26 except for the absence ofneoxanthin, while very divergent values have been reported for the chI alb ratio (1.6-0.8) and the chI to polypeptide ratio (5-13) (Dainese & Bassi, 1991, Peter & Thornber, 1991). This latter difference is presumably due to the preferential loss of chI a during PAGE. Its red absorption peak is at 675.5 nm and the fluorescence emission at 681.5 nm (Jennings et aI., 1993a, b). The characteristics of the protein with regard to molecular mass, pigment composition, and immunological cross-reactions are similar to the LHCI-680 component Lhea2, thus leading to the suggestion, in earlier reports, that they were the same protein (Bassi et aI., 1987). This hypothesis was later disproved by using monoclonal antibodies (Di Paolo et aI., 1990) and N-terminal sequencing (Morishige & Thornber, 1991). However, a polypeptide very similar to CP24 is present in LHCI as recently found in Chlamydomonas reinhardtii (Bassi et aI., 1992) and the absence of neoxanthin is a characteristic that CP24 shares with LHCI polypeptides (Henry et aI., 1983, Nechustai et aI., 1987).

Table 1 shows the composition of the PSII pigment-proteins. The actual number of pigment molecules present in each pigment-protein complex is more difficult to determine. Loss of pigments during isolation and/or difficulties in accurately determining protein concentration in the small amount of highly purified pigment-protein available are the chief causes for differences reported in the pigment to protein ratios for each complex. Moreover, it has been consistently reported that CP26, CP24, and CP29 polypeptides bind a lower number of pigment molecules than LHCII (Henrysson et aI., 1989, Barbato et aI., 1991, Dainese & Bassi, 1991, Irrgang et aI., 1991), although the stoichiometry was different for each individual pigment. These results should be reconsidered in the light of the strong homology between all the the genes as well as in the light of results from electron crystallography. In fact, the amino acid residues coordinating eight out of 12-13 chlorophyll molecules that have been identified in LHCII (Kiihlbrandt et aI., 1994) are markedly conserved in the sequences of the Lhcb and Lhea gene products so far identified. The only exception being a histidine residue located close to the C-terminus of the the gene missing in CP24. Therefore, the number of chI molecules for each Lhc polypeptide can be estimated as being between 7 and 12. The difference between these two figures being due to the pigments whose coordinating aminoacid residue has not been identified yet (Kiihlbrandt et al 1994). The number of carotenoids bound to individual chlorophyll alb proteins assuming 12 chlorophylls per protein, as determined by biochemical measurements and E.M. cristallography of LHCII (Dainese & Bassi 1991, Kiihlbrandt et aI. 1994), is reported in brackets in Table 2.

In the three minor chI alb proteins, lutein is present in more than one (probably two) molecules per polypeptide, whereas neoxanthin is always present in sub stoichiometric amounts. The most noticeable feature in these proteins is the high content in violaxanthin (and/or its de-epoxidation products antheraxanthin and zeaxanthin obtained in high light conditions) (Peter & Thornber, 1991, Bassi et aI., 1993, Thayer & Bjorkman, 1993, Ruban et aI., 1994).

Chlorophyll-proteins and Non-Photochemical Quenching of Chlorophyll Fluorescence

The three pigments violaxanthin, antheraxanthin and zeaxanthin participate in the xanthophyll cycle which has been shown to protect reaction center II from photoinhibition by thermally dissipating excess excitation energy (Demmig-Adams, 1990). Different locations have been proposed for the NPQ process namely PSII RC and the antenna proteins.

i) PSII RC. It has been proposed that the quenching is due to either the inhibition of the donor side with the formation ofP680+ (Weiss & Berry, 1987) or to over-reduction ofQA (Vass et al 1992), both P680+ and triplets formed through QA overreduction being fluorescence quenchers. However, Yerkes and Crofts (1992) showed that an active PSII is not required for NPQ and QAis unlikely to reach the degree of reduction required for such

47

models (Meunier & Bendall, 1993). ii) antenna proteins. Location of NPQ within the outer antenna was supported by the

finding that the quenching operated at its normal rate and amplitude not only when PSII photochemistry was bloched on either the acceptor side by DCMU or on the donor side by NH20H (Yerkes & Crofts, 1992) but also when the proton gradient was mantained by ATP-hydrolysis (Gilmore & Yamamoto 1992). In order to protect Rcn by dumping excitation energy before it gets to the RC, the quencher must be in the antenna and act through a reaction in the subpicosecond range in order to compete successfully with primary photochemistry. ~ong the different proteins composing the antenna, LHCII is the most abundant and has been the main candidate for the location of the quencher. Horton and colleagues have been the major supporters of this hypothesis (Horton et al. 1991, Ruban and Horton, 1992, Mullineaux et al. 1993). These authors suggested that light scattering changes in chloroplasts concomitant to the formation of delta pH (Horton et al. 1991, Ruban and Horton 1992) reflect an aggregation of LHcn leading to the formation of new chlorophyll bands and fluorescence quenching similarly to the quenching observed in isolated tHcn at low detergent concentration {Ide et al 1987, Bassi et al. 1991, pH (Horton e~ al. 1991, Ruban & Horton 1992). However, the correlation between theMg++ induced aggregation phenomena in chloroplasts and isolated LHCII is uncertain since they have opposite effect in the fluorescence yield of the sample. Moreover, the suggestion that the quenching depends on formation of new aggregation induced-chlorophyll absorption forms is contraddicted by the finding that fluorescence yield of chlorophyll forms absorbing between 650 and 690 nm is constant (Jennings et al. 1994).

The location of xantophyll cycle pigments within the pigment-protein complexes is important for understanding the molecular mechanism of photoprotection. However, this is not an easy task since structural studies are not available yet, and therefore only tentative hypotheses can be proposed on the basis of the following data: a) the xanthophyll cycle pigments are present in purified pigment-proteins in variable substoichiometric amounts, depending on the isolation procedure (Peter & Thornber, 1991, Bassi et aI., 1993, Ruban et al., 1994).

b) the size ofV+A+Z pool is strongly increased in plants adapted to a condition of high light, while antenna size decreases in the same conditions (reviewed in Demmig-Adams, 1990).

c) violaxanthin is de-epoxidized, in light stress conditions, within 1-2 seconds (Bilger et al. 1989).

d) the xanthophyll cycle pigments are present and the cycle is operating in plants which have a reduced antenna system and even in the chlorina f2 mutant, which lacks all peripheral antenna proteins (Dainese et aI., 1992, Leverenz et aI., 1992, Marquardt & Bassi, 1993, Krol et al., 1994 ).

e) V to Z conversion in do f2 and in other mutants lacking violaxanthin binding proteins do not correspond to the operation of the photoprotection mechanism. (Leverenz et al., 1992, Krol et aI., 1994, Bassi et aI., unpublished results).

Since the establishment of photoprotection through NPQ is likely to require a close contact between zeaxanthin and chlorophyll in order for the. energy transfer to take place (Leverenz et aI., 1992), it seems unlikely that violaxanthin could be freely accessible to the de-epoxi<Jase enzyme if deeply buried within the hydrophobic core of the complex, in a site similar to that identified for lutein in LHCII (Kiihlbrandt et aI., 1994). The operation of the xanthophyll cycle in mutants lacking some or all of the chlorophyll alb proteins (Bassi et al., unpublished results) was observed to induce a rapid de-epoxidation to a higher extent than WT. This result can be interpreted as the result of a higher accessibility of the violaxanthin to the de-epoxidase. In these conditions, however, photoprotection cannot take place. The mechanism of xanthophyll dependent NPQ is therefore likely to operate into two sequential steps: the first being the formation of zeaxanthin induced by the trans-membrane pH gradient (Gilmore & Yamamoto, 1992). This is likely to operate within the thylakoid lipids

48

surrounding chlorophyll-proteins. The second step is the actual energy quenching, and is most likely operating within chi alb proteins, through the tight coupling between chI a and zeaxanthin. The mechanism of reversible xanthophyll entry into the pigment-proteins is not yet clear; however, low pH has been shown to increase accessibility of chI a to the solvent (Siefermann-Harms & Ninnemann, 1982) and a DCCD binding site has been identified on the lumenal side in LHCII and CP26 (Jahns & Junge, 1990, Ruban & Horton et aI., 1992, Walters et al. 1994) within a shared sequence homology with proton translocating proteins (Ruban & Horton, 1992). It is therefore possible that the trans pH gradient may act by inducing a conformational change of the polypeptide, which allows zeaxanthin to come into contact with chlorophylls. It still must be explained how violaxanthin (and possibly neoxanthin) bind to minor chi alb proteins. A tentative hypothesis is based on the observation (Tremoliere et aI., 1994) that CP29, CP26, and CP24 tightly bind between 4 (CP26) and 10 (CP24) times more lipids (mainly MGDG) than LHCII or the PSII core complex. When considering both the de-epoxidase's absolute requirement for MGDG in order to be activated (Yamamoto et aI., 1977), and the inverse relationship between the size of the xanthophyll pool (and operation of the cycle ) with the presence of chi alb proteins, it can be hypothesized that xanthophyll cycle pigments may be dissolved in the lipids tightly bound to the complexes rather than bound to specific sites within the polypeptide. Irrespective of the way xanthophyll cycle pigments bind to CP26, CP29, and CP24, it is interesting that it has consistently been proposed that these proteins have a peri central location in PSII units, thus acting as connecting antennas between the major LHCII complex and the PSII core complex (Harrison & Melis, 1991, Peter & Thornber, 1991, Bassi & Dainese, 1992, Jansson, 1994). The location of xanthophyll cycle pigments in these proteins is consistent with their action as switches for excitation energy to be either funnelled to RCII or diverted to heat dissipation.

A somehow different model has been recently proposed by Crofts and Yerkes (1994). these authors suggest that chlorophyll dimers, formed within CP26 and CP29, might be the Non Photochemical Quenchers. In this model, the Glu131 residue may act as a pH dependent ligand releasing, at low pH, a chi molecule in order it to form a dimer with quenching characteristics with a closely located chlorophyll partner.

STRUCTURE OF CHLOROPHYLL AlB PROTEINS.

LHCll.

Our knowledge of chlorophyll alb proteins is mainly based on the structure of the major antenna complex, LHCII, which has been determined at 3.4A resolution by electron crystallography of two dimensional crystals (Kiihlbrandt et al. 1994) Fig. 1. The structure of other members of the Lhc family can only be deduced from that of LHCII, on the basis of sequence homology and spectral properties (Fig 2). LHCII is an integral membrane protein with an helix-loop-helix organisation. The apoprotein is 232 aminoacids long and each polypeptide binds 12 chlorophyll molecules (7 chi a· and 5 chi b), 2 luteins and non stoichiometric amounts of violaxanthin and neoxanthin (Kiihlbrandt et al. 1994, Bassi et al. 1993). Three transmembrane domains with a-helix conformation are connected by two hydrophilic loops on either sides of the membrane, while N-terminal and C-terminal peptides are exposed, respectively, on the stromal and lumenal spaces, each bearing a small helix domain. The N-terminal one is fully hydrophilic, thus it protrudes into stroma space, while the lumen-exposed one is amphyphylic, thus it lies on the membrane surface. The B helix is the nearest to the N-terminal, it is 51A long, starts at Pr055 and stops at Gly 89 after 9.5 turns. The A helix, on the C-terminal side, is a 43 AA long, extending from Pro 170 to lie 199 in 8 turns. These two domains form an X shaped structure: they are tilted by 32° relative to the membrane normal and are held together by ion pairs formed by charged

49

Figure 1. Overall view ofLHCII monomer as resolved from electron crystallography (Kiihlbrandt et al. 1994). Carotenoids extend from the stromal to the lumenal surface of the molecule forming a cross brace near helices A and B. The two rings of each putative lutein molecule are indicated by arrows. Porphirin rings drawn with open lines refers to putative chI b molecules, with filled lines to putative chI a molecules.

residues Arg70 and Arg 185, Glu65 and Glu180. The two buried ion pairs probably provide a strong attractive force between the two helices, and are likely to play a major part in stabilising the protein in the membrane. The first 24 residues of helices A and B are related by an axis of local 2-fold symmetry running perpendicular to the membrane plane. The C helix, smaller than A and B, runs from Ser123 to Ile143 in 5.5 turns, over a length of 31A, with a tilt angle of 9° relative to the membrane normal. The D helix is amphypathic, it starts at Pro205 and stops at Ala214. The electron density ofN-terminal peptide is weak and it is impossible to actually resolve the structure, but the presence of a small helix in the N­terminal region can be hypothesised by using the secondary structure prediction algorithms. This helix starts at the Argl up to Pro9. The description of the loop regions between the helices is more difficult because of the low electron density obtained.

LHCII is a chlorophyll binding protein and the 3.4 A resolution allows us the understanding of the chromophores location. At this resolution, the very small structural differences between chI a and b as well as those between the three xanthophyll species present in the complex, cannot be distinguished. Two carotenoid molecules can be resolved at the centre of the complex on either side of helices A and B with an angle of 50° to the membrane normal. These can be only tentatively identified as luteins based on their 2: 1 stoichiometric amounts. Luteins form an internal cross branch in the centre of the complex, providing a direct, strong link between the peptide loops on both surfaces. The porphyirin rings can be tentatively attributed to chI a or b on the basis of their proximity to the carotenoids. This is because the energy transfer from chI b to chI a is much faster (ps) than the formation of chI triplets, which takes several ns. It follows that triplet quenching by carotenoids is only required for chI a (Kiihlbrandt et al. 1994).This makes it possible to assign the 7 chlorophyll molecules closest to the carotenoids to chi a. The identification of chi ligands is also very interesting. Two histidines (His68 and His212), three amides (Gln131, Gln197, AsnI83), three charge-compensated glutamates, forming ion pairs with arginines either in the same helix (Glu139-ArgI42) or in another helix (Glu65-ArgI85, GluI80-Arg70) can be identified. Remaining chi molecules are probably bound to the peptide carbonyls with or without involving a water molecule. On the basis of the above described LHCII structure, and of the primary sequence analysis structure, models can be inferred for the related chlorophyll proteins. In the following the structure of homologous chlorophyll-proteins will be described as for comparison with that ofLHCII. Substitutions of

50

pigment ligands will be described referring to the residue numbering according to Kiihlbrandt et al.(l994). Other structural features will be described on the basis of the residue position within each polypeptide in maize as deduced from cDNA sequencing (Dainese et aI. 1995, Bergantino et aI. 1995).

CP29

The polypeptide is 257 aminoacids long. It binds 6 chi a and 2 chI b, Its chlalb ratio is 2.8-3.0 (Fig.2b). CP29 is longer that LHCII and mainly differs in the N-terminal region, where it shows a 42 aminoacid insertion which has no counterpart in other Lhc proteins. In this portion of the molecule, two helices can be predicted on the basis of sequence analysis: the first, very close to the N-terminus, runs from Lysl0 to Va1I6, the second, longer, runs from Glu61 to Ala76. The sequence identity between CP29 and LHCII is 34.3%, but most substitutions are homologous especially in the helix regions. The residues which are of major importance in maintaining the structure such as chi-ligands and the buried ionic pairs, are conserved in CP29 sequence. Only the glutamine in the C helix, which binds chI b6, is changed into a Glutamic acid residue. Accordingly, CP29 has a lower chI b content, as from biochemical data (Herryson et aI. 1987, Dainese & Bassi, 1991). An additional substitution, Asnl83His, occurs in helix A . This residue was shown to be a chlorophyll a ligand (Kiihlbrandt et al 1994). This substitution should not prevent the pigment binding in this position since histidine is a well known ligand for porfirins.

CP26

CP26 is 247 aminoacids long. It binds 7chl a and 3 chI b (Fig.2c). and shows (48.7%) identity with respect to LHCII. Helix A and the C-terminal region are very similar, while major differences can be found in helix C: the identity in this region is very low, but the substitution is conservative. Like CP29, CP26 lacks the glutamine residue in helix C and therefore cannot coordinate a chlorophyll b molecule in this position.

A small helix can be predicted in the N-terminal part of the molecule by secondary structure analysis, starting at Ser23 untiIJ Ala27.

Among chlorophyll alb proteins, CP26 and CP29 have the characteristic of binding DCCD to a Glu residue in helix C, probably Glu 131, concomitant with inhibition of NPQ (Jahns & Junge 1990, Ruban et al. 1992, Walters et aI. 1994). It is tempting to suggest that changes in excitation energy dissipation are correlated to protonation of Glu 131 and reorganization of pigment binding in this lumenal portion of the molecule (Crofts & Yerkes, 1994).

CP24

The polypeptide of CP24 is 210 aminoacids long, and is therefore the smallest within the Lhcb group (Fig.2d). Its pigment complement has been determined to be the lowest (3 chI a and 2 chI b) with respect to the other Lhcb members, however, this could be due to the instability of CP24 structure, due to the lack of the C-terminus. Sequence homology and absorption spectra rather suggest 6 chI a and 4 chI b for polypeptides. The identity respect to LHCII sequence is 29.2%. CP24 loses the C-terminal and also the D helix. Within the N­terminal region an helix can be predicted between AlaI and Trp9. Two chlorophyll ligands are lost due to the C-terminal deletion including helix D and the Gln197Glu substitution in A helix. Conservative changes Asn 183His and Glu65Asp are observed for the chI a2 and chI a4 ligands.

51

B

52

A

pO V N N

NAWAYATNFVPGK 220 2:tO

pD l l S H '-------' T

TI FOTFGGSJ.

CP29

stroma

lumen

LHCIJ

Q

l' HA 5 ..., K PNGLYDLGG

F 110 E Gt: AVWfKAGSOIFS -

W LT

, S S

c

pD l F S G ---_--' N

NlLTVISGAAERTPSL

D

CP24

stroma CP26

lumen

Figure 2. A-D: Plan of the Lhcbl (LHCII); Lhcb4 (CP29); Lhcb5 (CP26) and Lhcb6 (CP24) from Zea mays polypeptides in the thylakoid membrane. Letters on black background indicate Chi side chain ligands. Glutamate side chains that are presumed to act as chi ligands are connected by a line to the chgarge compensating arginines close to them, marked by open circles. Open, capital, letters A, B, C, D indicate the membrane and surface bound helices. The plan was obtained on the basis of :(i)the structure of pea LHCII (Kiihlbrandt et aI., 1994), (ii) secondary structure analysis and (iii) biochemical data on pigment to protein ratio (Dainese and Bassi 1991). Maize sequences were deduced from cDNA: LHCII, Matsuoka et aJ. 1992; Lhcb4, Lhcb5 Lhcb6,( Dainese et al. 1995).

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EVOLUTION OF THE CHLOROPHYLL AlB ANTENNA PROTEINS

The antenna ChI alb-binding proteins of higher plants and green algae includes an extended family coded in the nuclear genome, extending to the fucoxantin-Chl alc antenna proteins (FCPs) of chromophyte algae and the Early Light-Induced Proteins (ELIPs) of higher plants. Following a recently proposed nomenclature (Jansson et aI., 1992), the chlorophyll alb binding proteins are represented by 10 gene types: Lhea 1 - 4 (LHCI) and Lheb 1 - 6 (LHCII). Most of the genes have been cloned and sequenced by using cDNA libraries, and related to the encoded polypeptide by tryptic peptide sequencing. To date, an almost complete set of Lhe DNA sequences is available in tomato, Lyeopersieon eseulentum, while only few sequences (mainly belonging to photo system II) have been reported for other species.

The protein sequences deduced from the aligned DNA sequences of the family reveal a modular pattern (Fig. 3), where some structure motifs are almost identical in all members and others appear to have evolved much more rapidly (Green et aI., 1991). Three hydrophobic regions, predicted to give rise to three transmembrane helices, can be recognized accordingly in the protein structure determinations (see above). All members share two highly conserved regions including the first and third transmembrane helix and the stroma exposed, beta tum regions preceding them. The two conserved regions share considerable sequence similarity, suggesting that they arose from gene duplication. The three dimensional structure has been reported by electron cristallography at 3.4 A of resolution for pea LHCII (Lhebl or Lheb2) (Kiihlbrandt et aI., 1994), confirming that the protein does have three transmembrane helixes with the aminoterminus exposed on the stromal side and the C-terminus extending in the thylakoid lumen. Because of the high degree of sequence conservation in the family, it is reasonable to conclude that all the family members will have the same overall fold (see above).

Lhe genes are substantially divergent (60-70%), indicating that the gene duplication that gave rise to them occurred very early in evolution. Most of the data available for LHCII (Lhebl and Lheb2), the most abundant protein of photo system II. These two genes display only 15% of divergence, mainly due to 14 distinctive N terminal aminoacid residues. This reveals a more recent duplication event, probably at'the time of divergence between fern and seed plants (Green et aI., 1991). Lhebl genes are present in multiple copies in the genome of the angiosperm and dicot species investigated and encode identical or almost identical products, as a probable result of an additional, very recent duplication process. A strong degree of conservation is observed for Lheb I and Lheb2 at the interspecific level (Identity> 90%) (Jansson, 1994). The other genes, on the basis of the available data, seem to be present as single or duplicated copies, quite highly conserved at the interspecific level. In the following we present the results obtained by comparing sequences obtained from maize: we have cloned the cDNA coding for the minor antenna apoproteins of photosystem II (CP29, CP26, CP24, or Lheb4, Lheb5, Lheb6) (Bergantino et aI., in prep.; Dainese et aI., in prep.). The deduced aminoacid sequences have been aligned with the corresponding genes already described (and analyzed for the protein structure: see above). CP29 differs from the other LHC proteins in having an insertion of 42 aminoacids close to the first predicted (l- helical region. CP29 was isolated from maize in single copy and is the longest the described (262 a.a. as mature protein). Maize CP29 is highly conserved at the interspeCific level. Identity values of the mature protein are 86.8% and 88.7% versus the Arabidopsis and barley corresponding sequences, respectively. It has been demonstrated that the maize CP29 can be reversibly phosphorylated in response to photoinhibitory conditions (cold light stress), giving rise to a different electrophoretic form (Bergantino et aI., 1995). Phosphorylation occurs at one or two sites within the N terminal, stromal exposed region. The Lheb5 gene, coding for the CP26 polypeptide, was isolated in only one copy from tomato, barley and Scots pine. In maize we isolated two different cDNA Lheb5 clones, which share high homology (97%) in the coding region. Regarding CP24 (Lheb6), the

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smallest of LHC proteins, only one cDNA clone was isolated from maize, while two genes were sequenced in tomato. Maize CP24 has a much shorter transit peptide when compared to other species; the protein sequence displays a considerable similarity with tomato (86%) and spinach (85%) counterparts.

The evolutionary origin of the Lhc polypeptides is not known, since there is no discernible similarity between the LHC and the pigment binding proteins oflower organisms. On the basis of the strong homology between the first and third transmembrane helices among all members, the ancestral gene could have originated from a duplication event followed by gene fusion. The recently reported sequence and structure prediction of PsbS. a gene encoding a 22 KDa intrinsic protein of PSII able to bind chlorophyll (Green & Pichersky, 1994), would suggest that the entire family of three-helix proteins may have arisen from a two helix protein that was duplicated to give a four helix protein (as PsbS), and subsequent loss of most of the fourth helix.

PHYLOGENETIC RELATIONSHIPS BETWEEN LHC PROTEINS

Multigene families are sets of genes that have descended by duplication and divergence from common ancestral sequences. A phylogeny of a family can be inferred by compari~g the protein or nucleotide sequences of the different members.

The main interest of the phylogenetic approach is the possibility of classifYing the objects of study not simply on the basis of their similarity, but of their parental relationships, which result from the evolutionary process. In the case of the Lhc family, an almost complete set of sequenced genes is available for tomato. The trees reported so far have been obtained either by similarity dendograms (Jansson, 1994) or by phylogenies whose underlying algorithms have not been specified (Green et ai, 1991). In another species, the soybean (Glycine max), Demmin et al. (1989) reported a phylogenetic maximum likelihood analysis carried out at the DNA level (107 bp). However, the analysis was done only on the Cab 1-5 genes (Lhcb 1.1-5). With a similar analysis at the interspecific level (10 plant species, 25 Cab genes of the Lhcb1 and Lhcb2 types) the current taxonomic classification of six mono cot and dicot taxa was verified.

We show here a first attempt to infer the phylogeny for the different members of the higher plants LHC family by a parsimony approach. All parsimony methods try to minimize the number of steps at each individual character required to explain a given tree topology (reviewed by Felsenstein, 1982). The main advantages of this class of methods may be summarized as follows:

1) no information is lost in the process of phylogeny reconstruction (i.e., data are not reduced to a distance matrix of distances);

2) there is no assumption of a molecular clock (this is of particular importance in the case of gene families, where different genes could evolve at different rates);

3) it is possible to obtain a "physical" estimate of the tree branch length (that is, the number of character state steps define the branch length);

4) there is no need to know the ancestor sequence, as the simple addition of an outgroup sequence to the data allows the obtaining of a polarized tree.

The tomato LHC protein sequences array (Fig. 3), aligned by the PILEUP program of the GCG package (Jansson, 1994) was used as data set. The protein sequences of one maize Lhcb1 gene (Viret et ai., 1990), and of the minor complexes Lhcb4, Lhcb5 (clone 2) and Lhcb6 (unpublished data) were subsequently added by aligning them to the tomato counterparts . The phylogenetic analysis was carried out at the aminoacid sequence level. Trees were generated according to a maximum parsimony criterion using the PROTP ARS program. The characteristics and assumption of the algorithm are described in the program documentation. Majority rule consensus trees were constructed using the CONSENSE program and confidence statements on branches were estimated by running PROTP ARS on

56

100 bootstrap replicates obtained by the SEQBOOT program. All these programmes belong to the PHYLIP 3.5e computer package ofFelsenstein (1993).

Trees were polarized using the barley ELIP HV58 cDNA deduced protein sequence (Grimm et al. 1989).

The network drawn in Fig. 4a was obtained by a partially reduced matrix of 245 aa, where the residues before the first conserved N-terminal hydrophilic domain (boxed in Fig. 3) and the 37 aa present only in the Lheb4 genes were removed. This shortening made it possible to at least partially reduce the number of computations for assigning character states. A previous analysis was done on the full matrix (333 characters) in order to confirm that the same topology of the tree was obtained and to assign the correct bootstrap (BP) values. The branch lengths are proportional to the number of state changes, and the BP values are reported at each node.

Overall, the network identified groupings well supported at the statistical level by high BP values. As expected, the maize sequences clustered in all cases (BP=IOO) with the tomato counterparts. The Lhebl-2-3-5 grouping was supported in all cases (BP=100), with Lheb5 being the most divergent. Lhea genes failed to cluster together; Lheal occupies an external position with respect to the other Lhea, which cluster with Lheb6 (BP =98), while Lheb4 is located at an intermediate position. Though we did not carry out an exhaustive analysis of the individual character changes, the main feature of the network is the absence of a clear divergence between the photosystem I and II antennae, with the exception of the well defined Lhebl-2-3-5 cluster.

When the ELIP outgroup sequence is added, the same network is converted to a polarized tree. Fig. 4b Lheb4, Lheb6 and the Lhca genes form now a cluster supported by a BP value= 60, while among the internal branching there is the pairing of Lhea2 and Lhea3 and the position of Lheb4 is the less supported. The two main clusters of the tree display approximately the same amount of divergence with respect to the root.

This analysis represents a first attempt to depict the evolutionary relationships of the different members of the the multi gene family on a basis of a defined parsimony optimality criterion for protein sequences (Felsenstein, 1993). To our knowledge, with the exception of a maximum likelihood analysis applied only to the Lhcb I and Lhcb2 genes of soybean (Demmin et al., 1989), no such examples are given in the literature.

The same tree topology was statistically well supported with both 333 and 245 characters in the input matrix, and the addition of the ELIP as outgroup sequence allowed a polarization of the tree without substantial changes of the member relationships. Indeed, though ELIPs are distant relatives of the the genes and their role in the photo system physiology is not clear, they are considered members of the same gene families (Green & Pichersky, 1994). An additional assay was done on a reduced matrix of 169 characters, which included only the sequence portions which are the most conserved among all the Lhe genes and which give rise to protein secondary structures (boxed in Fig. 3), thus removing the hydrophilic connectors. The tree obtained yielded the Lheb 1-2-3-5 cluster again, but was quite different in the clustering of the Lheb4-6 and Lhea genes (data not shown). An analysis limited to the most conserved sequence portions could be recommended in the case of very distant sequences, and could allow, in the future, the carrying out of extensive analysis, including the polypeptides from lower plants and algae. However, we consider the phylogeny based on the full and most informative matrix more correct because the hydrophilic comlector regions were also quite easy to align.

Indeed, many shared derived (synapomorphic) characters are found in the connector regions (fig.3). This last observation could partially account for the clustering of the Lheb4 and Lheb6 genes with the Lhea genes. A somewhat related result of protein sequence similarity was reported by Jansson (1994): considerable sequence similarity exists between Lheal and Lheb4 within a region of aproximately 50 a.a. which includes the greater part of the hydrophilic connector between helices 1 and 2. Moreover, at the N-terminus, Lheb4 contains a sequence motif (WFPG) proposed to be LHC I-specific. The calculations of

57

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overall sequence identity between the two polypeptides showed that Lheb4 is equally related to Lheb5 and Lheal, less related to the other Lhea and even less to the other Lheb genes. In addition, Lheal appeared to be more related to Lheb4 than to the other Lhea proteins. These findings fit very well with the position of Lheb4 and Lheal in our phylogeny. Regard.jng Lhcb6, considered the most divergent of the the family, its clustering to the Lhca group has already been depicted in an the tree by Green et al. (199 I ).

With the exception of the LhebI and Lheb2 genes, which are closely related (about 85% homology), are present in multiple copies in the genomes and probably arose from recent duplication events (Green et aI., 199 I), the deeper nodes of the tree are short with respect to the more recent nodes. Most of -the polypeptides of both photo system antennae diverged in earlier times, and this happened in a way that is probably more related to their physiological role than to their location in the respective photosystems. Thus, it is possible

58

to postulate that at least the minor complexes Lhcb4 and Lheb6 share some common functions with the-Lhca polypeptides. This can also be put in relation to the discovery that the red alga Porphyridium, which does not have chlorophyll b, does have several polypeptides that cross-react with both barley anti-LHC I (Lhea genes) and diatom anti-FCP (Fucoxantin-Chl alc proteins) antibodies (Wolfe et ai, 1994). This finding implies that the ancestor of the the proteins was present prior to the divergence of the eukaryotic algae into rhodo-, chromo- and chlorophyte divisions but could also support the apparent lack of early differentiation between photo system I and II antennae in higher plants, which would therefore be composed of "ancient" polypeptides which evolved, indipendently, physiological functions somewhat common to the two photosystems.

OTHER CHLOROPHYLL AND/OR CAROTENOID BINDING PROTEINS

Besides the relatively well characterized polypeptides described above, recent reports suggest that other carotenoid binding proteins may exist in the photosynthetic apparatus:

1. PsbS

The gene coding for a 22kDa protein, previously reported to be important in PSII assembly (Hundal et aI., 1990), has been sequenced (Wedel et aI., 1992, Kim et aI., 1992) and homology with Lheb genes was recognized. On the basis of hydrophobicity plots, psbS is hypothesized to have four rather than three transmembrane helices (as other the genes). Purified psbS.has been reported not have any chlorophyll molecule associated with it (Ljungberg et ai. 1986, Bowlby 1990). However, Funk et aI.(1994) reported chi a and b binding in a preparation enriched in PsbS . Still, it is not clear which is the role of this proteins in PSII an antenna function being rather unlikely due to its low abundance (less than 1% ofPSn chi).

2. LHClIe and ELiPs

Following several earlier reports (Krishnan & Gnanam, 1979, Irrgang et ai., 1990) the finding of a low molecular mass chi alb protein, LHCIIe, which is greatly enriched in xanthophylls, has been reported (Peter & Thornber, 1991). To date, other labs have not been able to confirm this interesting report. The possibility exists that the above result could be due to the presence of ELIPs, a newly discovered class of proteins transiently synthesized during light exposure of dark grown plants (Grimm et ai., 1989, Hundal et ai., 1990) or after photoinhibition (Adamska et ai., 1992) but also present at low levels in fully greened leaves (Droppa et ai., 1987, Marquardt & Bassi, 1993). Their deduced sequence shows a MW between 14 and 17 kDa (Meyer & Kloppstech, 1994). N-terminal sequencing of LHCIIe is needed in order to verify this hypothesis. ELIP's sequences show homology to the LHC polypeptides and to the carotenoid binding protein of green algae (Levy et ai., 1991). Recent results suggest that ELIPs are binding sites for photoconvertable xanthophylls and may replace or complement a deficient xanthophyll binding capacity under conditions of light stress (Krol et at, 1994). This view is supported by the cloning of an ELIPs like gene from Dunaliella called Cbr. The 17 kDa gene product is synthesized transiently during V to Z deepoxidation and becomes associated with a LHCII fraction which, thereafter is shown to contain zeaxanthin (Levy et ai., 1993, Lerst et ai. 1991). The isolation ofELIPs and Cbr in their putative pigment binding form is needed in order to clarify this point.

59

CONCLUSIONS

Chlorophylls and carotenoids are present in all of the photosystem I and II pigment­proteins. The chloroplast encoded subunits of the PSII core are chi aJ [3-carotene complexes while the nuclear encoded subunits of the antenna system are chi alb/xanthophyll complexes. Each individual pigment-protein of the antenna system has its own peculiar carotenoid composition in addition to the common lutein component. Lutein, in addition to its photoprotection function, appears to have an important structural role in the assembly of LHCII and probably also of other LHC gene products. The functional meaning of the complex topological distribution of the other xanthophylls is not clear; however, the location of xanthophyll cycle pigments (Y, A, Z) in the minor pigment-proteins CP24, CP26, and CP29, which connect the major antenna LHCII to the PSII core complex, strongly suggests that the photoprotection mechanism of zeaxanthin-dependent-non-photochemical-quenching is located therein. The carotenoid distribution in PSI is less clear but the data so far suggests that the situation is not too different from PSII.

Acknowledgments

We would like to thank the Italian Ministry of Agriculture and Forestry for financial support to R. B. (grant n0349-7240-9I) and Werner Kiihlbrandt (EMBL, Heidelberg) for providing the LHCII coordinates before the release to data bank.

Abbreviations

A, antheraxanthin; chi, chlorophyll; CP, chlorophyll-protein; DCCD, dicyclohexilcarbodiimmide; Deriphat, Lauryl -D imminopropionidate, Efs, endoplasmic face, stacked; ELIP, early light induced protein; E.M., electron microscopy, HPLC, High Performance liquid chromatography, LHCI, II, Light Harvesting Complex of PSI, II; PAGE, poliacrylamide gel electrophoresis, MGDG, monogactosyldiacylglycerol, NPQ, nonphotochemical quenching; O.E.E., oxygen evolving enhancee; PSI, II, Photosystem I, 11; PFu, periplasmic face, unstacked, SDS, Sodium duodecylsulphate, Y, violaxanthin; Z. zeaxanthin.

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63

SPECTRAL HETEROGENEITY AND ENERGY EQUILIBRATION IN HIGHER PLANT PHOTOSYSTEMS

Robert C. Jennings, Giuseppe Zucchelli, Laura Finzi and Flavio M. Garlaschi

Centro C.N.R. Biologia Cellulare e Molecolare delle Pi ante, Dipartimento di Biologia, Universita di Milano, Via G. Celoria 26, 20133 Milano, Italy.

INTRODUCTION

Higher plant thylakoids contain a number of pigment binding proteins which are organised into two photo systems known as photosystem I (PSI) and photo system II (PSII). The major light harvesting pigments are chlorophylls (chis) of which about 200-2S0 are associated with each photosystem, though this number is somewhat variable depending on the growth conditions. Light energy is absorbed by these antenna pigments and transferred rapidly to the reaction centre primary electron donor, a special chi dimer or monomer, where photochemistry occurs. Energy transfer times between neighbouring antenna chis occur in the range of 0.2-Sps (for review see Jennings et aI., 1995). The time between light absorption and primary photochemistry is in the range 100-S00ps with the time constant for reduction of the primary pheophytin electron acceptor probably around 3ps. The reader is referred to chapters by Bassi et al. for details of pigment-protein biochemistry, Borisov for descriptions of energy transfer mechanisms, and Mathis for details on reaction centres.

For a detailed understanding of energy transfer processes in antenna and between antenna and reaction centres precise knowledge is required of the following parameters (a) interchromophore distances (b) mutual dipole-dipole orientation and ( c) absorption and fluorescence energy levels and spreads of the pigment bands. The interchromophore distances are only accessible by high resolution crystallographic structures as recently reported for LHCII (Kiihlbrandt et aI., 1994). To some extent, dipole orientation may also be investigated by crystallographic techniques though polarisation spectroscopies are also needed. In the case of the energy levels and spreads of pigment bands, which for example are needed for calculation of the overlap integrals (see Borisov, this volume) only spectroscopic techniques may be employed. These latter parameters are also of particular interest in understanding the distribution of excitation energy in antenna chI-protein complexes at thermal equilibrium.

In the present chapter the absorption bands of chi molecules bound to chi-protein complexes will be examined. The physical principles which lead to broadening of absorption

Light as an Energy Source and Infonnation Carrier in Plant Physiology Edited by Jelmings et 01., Plenum Press, New York, 1996 65

bands at physiological temperatures wilI be introduced. Individual protein-bound pigment absorption -characteristics will be discussed with special reference to LHCII. FinaIly this information for the various pigment-protein complexes wiIl be used to determine the equilibrium excited state population in the antenna and reaction centre complexes of PSII and the conclusions discussed in terms of the physiological importance.

SPECTRAL BROADENING OF PIGMENT ABSORPTION BANDS: HOMOGENEOUS AND INHOMOGENEOUS BAND BROADENING.

Extensive studies both of an experimental and theoretical nature indicate that at very low temperatures, close to absolute zero, the absorption spectrum of a single pigment site is very different from that with which we are familiar at higher temperatures (Reddy et aI., 1992; Hayes et aI., 1988). Thus the absorption spectrum of a single pigment site is composed of a very narrow zero phonon line, associated with the purely· electronic transition, and a relatively broad phonon side band associated with interactions between the excited state electrons and low frequency vibrations (v = 10-100cm- l ) of the protein matrix to which the pigment is bound. The phonon side band is to the high energy side of the zero phonon line and is energetically separated from it by an amount which is approximately equal to the parameter known as the optical reorganisation energy (Sv) where S is the strength of the electron-phonon coupling (linear) and v is the average frequency of the protein matrix phonons. Studies in recent years using hole burning spectroscopies suggest that v for chis bound to chi-protein complexes to be 20-30cm-1 (Hayes et aI., 1988). The relative intensity of the of the zero phonon line with respect to the phonon side band yields a value for S. Once again from hole burning studies S values for antenna chis come out in the range 0.5-1 (Hayes et aI., 1988; Reddy et aI., 1994). The parameter Sv is therefore important in understanding the thermal broadening of absorption bands. In the following we discuss spectral broadening at temperatures far from absolute zero, with special reference to the room temperature (RT) situation. At these higher temperatures absorption spectra are dominated by the phonon side band with the zero phonon line structure being completely washed out. In the case of a single, average, low frequency protein phonon, which seems to be case for protein-bound chi, equation 1 is a very useful expression which helps in understanding thermal broadening:

FWHMfiorn = (8 In 2) S V2 coth(hCVrnJ 2kBT

(1)

where FWHMhom is the temperature-sensitive half band width of a gaussian shaped absorpion band and the subscript stands for homogeneous; h is the Planck constant; kB is the Boltzmann constant and T is the absolute temperature.

For v values of20-30cm-1 and T;:: 70K the expression simplifies to

2 kB FWHMhorn = (l6In2)-Svrn T hc

(2)

Thus with these expressions one can start to understand how the band width associated with a particular pigment site broadens as the temperature rises. It has however become apparent in recent years that spectral broadening of protein-bound pigments involves another factor. This is the so-called inhomogeneous broadening and is due to the fact that the protein matrix is not a rigid, static scaffolding. Fluctuations take place within the protein which have the effect of modifying the each pigment binding site in a statistical way. This in turn modifies the absorption energy levels, giving rise to a distribution of energy levels associated

66

with each pigment site. This site distribution function, being of a statistical nature, is gaussian in shape: It is thought to be largely independent of temperature (assuming that the protein structure does not change) and is "frozen" into samples at cryogenic temperatures.

If one assumes that the homogeneously broadened band is gaussian in shape, which is reasonable for phonon side bands at temperatures well above 0 Kelvin, and taking the inhomogeneous broadening to be gaussian, one can write

222 FWHM = FWHMhom + FWHMinh (3)

This situation is represented schematically for an hypothetical pigment absorption band in figure 1.

0.6 rI''it " D

" 0 0

0

0 u () n

O. 0

n

0 u

0

0.4

0.3

0.2

0.1

J 400 500 600 700

Wavelength (nm)

Figure 1. Schematic representation of the homogeneous (temperature dependent) and inhomogeneous (site fluctuations) broadening components giving rise to the overall width of a pigment absorption band. The continuous curves show a spread of homogeneously broadened bands while the discontinuous curve is the site distribution function which determines the probability density of each homogeneous band. The overall absorption band width (not shown) is a convolution of both these components and is clearly broader than either one taken separately.

67

_:: }.~- ".~"0~ 1 1 l A .95 ~

.83

c .~.59 a. 0.55 rn

.0 «.4

_:: } 4,-

1.1] .96

.83

.59

.55

.41

.28

.14

cO .. _ Q r "'JV B

o O~~~~~~~~~~~~~ 530 64 j 653 664 675 686 698 709 720 630 641 653 564 675 685 698 709 720

Wavelength (nm) Wavelength (nm)

Figure 2. Gaussian sub-band decomposition description of the Qy absorption band of spinach LHCII at 70K

(A) and room temperature (B). The plot of the residuals «A - B)/ In, where A is the measured absorption, B is the sum of the gaussian sub-bands. n is the number of data counts) is shown above. In this way six chla and 2 chlb absorption bands are identified and the Qy bands (around IOnm at room temperature) are much narrower than for solvent extracted chlorophyll

Very few techniques are available to examine protein-bound pigment absorption characteristics at R T. One approach is that of spectral decomposition in terms of gaussian bands. In principle this can give information on the wavelength position, band width and relative band intensity. The main disadvantage of the technique is that more than one sub­band description may be possible in spectra which have little structure. This may in part be overcome by comparing decomposition data at R T with spectra measured at cryogenic temperatures, where spectral structure is greater. Results should also be compared with other spectroscopies such as circular dichroism and linear dichroism which can yield information on the approximate wavelength position of transitions, preferably at RT. These aspects will be illustrated below for LHCII, the principal outer antenna chI-protein complex (see Bassi et aI., this volume).

In figure 2 the spectral decomposition for LHCII at 72K and R T are presented. These descriptions give the minimum sub-band number required to adequately describe the absorption spectrum at both temperatures. Major sub-bands with maxima at 643nm, 649nm, 657nm, 663nm, 670nm and 684nm are present at RT. At 72K the 684nm band is largely suppressed in a temperature sensitive mechanism which is presently under investigation. The two shorter wavelength bands are thought to represent chI b, while the other sub-bands are probably: all chI a spectral forms. With the exception of the 684nm band (temperature sensitive) rather similar conclusions concerning the wavelength positions have been found by Hemelrijk et al. (1992) from low temperature studies of linear dichroism and circular dichroism spectra.

An interesting feature of this decomposition description is that the FWHM values at RT are between 9.5-11nm (>:::;220 cm"), which are approximately half that of chI in organic solvents.

68

50000

45000

40000

'" 35000 ~ J:

~ 30000

25000

20000

50 1 00 150 200 250 300 Temperature (K)

Figure 3. Thermal broadening analysis ofthe 677nm band ofLHCII. From the straight line one can determine both the inhomogeneous band width and the optical reorganisation energy (Svm) according to equations 2 and 3.

In figure 3 a thermal broadening analysis of the 677nm band is presented. From equations 2 and 3 it is clear that in the case of homogeneous broadening assuming linear coupling to a single phonon mode, the FWHM2 is linear with temperature. This situation is confirmed in figure 3. The optical rearrangement energy (Sv), calculated from the slope, is near 11 cm-I and the FWHMinh approximately 140 em-I. These values are in excellent agreement with recent hole burning results for a very weak band absorbing near 680nm in LHCII at 4 K (Reddy et aI., 1994).

From equation 3 the FWHMhom (RT) can be calculated and has a value of approximately 7.5 nm (160cm-I). The FWHMinh is near 6.5nm (140cm-I). This shows that to understand the band width at RT both the homogeneous and inhomogeneous components must be considered. The situation seems to be rather similar for the other chI a spectral forms in LHCII (Zucchelli et al. in preparation).

It is worth noting that the Stokes shift, important in understanding energy transfer rates, is approximatly equal to 2Sv (Hayes et aI., 1988), for homogeneously broadened bands. Thus for pigments bound to LHCII we may conclude that the Stokes shift is approximately lnm (22cm-I). This is much less than for chi dissolved in organic solvents, where Stokes shift values are close to 6nm. This has important implications for the calculation of energy transfer dynamics, as exciton transfer in the energetically "downhill" direction will be slower than would be expected using the in vitro values, while it will be faster than expected for "uphill" energy transfer.

PSII ANTENNA: SHALLOW OR DEEP FUNNEL?

From the above considerations it is clear that chI-protein complexes contain a number of chi spectral forms which absorb at different wavelengths. It has often been suggested that these spectral forms may be spatially distributed with respect to P680 to form an "energy

69

o ... ..;

~I

~I !I ~I ~I ~I ~I ~I ~I ~I

:1 ~I

~J ~I

~I

~I

~I

690 680 670 660 650 640

WAVELENGTH (nm)

0102 3.9

CP43 12.3

CP47 12.4

CP24 3.0

CP26 6.3

CP29 6.9

LHC 55.3

Figure 4. Wavelength positions of the absorption sub-b~ds at room temperature for all the chlorophyll protein complexes comprising PSII. Nmnbers represent the absorption-weighted population of excited states calculated at T=300K for thermal equilibrium (equation 4). The right hand column of numbers is the excited state population in each type of chlorophyll-protein complex.

funnel", with the red-most absorbing forms located close to the RC and the shorter wavelength forms localised predominately in the outer antenna. The presence of chI b only in the outer antenna complexes (LHCII, CP29, CP26, CP24) and not in the core or RC complex seemed to support this idea. Calculations show that this kind of organisation may increase the rate of energy flow from antenna to RC many times (Seely, 1973a; Seely, 1973b; Fetisova et aI., 1985). Very rapid energy transfer would in turn increase the quantum efficiency of primary charge separation. For PSII antenna this concept has been directly investigated recently by an analysis of the absorption and fluorescence spectra of all the isolated chI-protein comprising the antenna system (Jennings et aI., 1993; 1994). In particular the absorption spectra at RT ofLHCII, CP29, {:P26, CP24, CP43, CP47 were described in terms of a minimum number of gaussian bands required to give a good fit. As discussed by Bassi et al. (this volume) LHCII, CP29, CP26,CP24 constitute the outer antenna while CP43 and CP47 are more directly in contact with the RC complex. The data from this analysis are summarised in the energy level diagram of figure 4, where it can be seen that all complexes have similar chla spectral forms at this level of spectral resolution. The presence of chlb (between 643nm and 649nm) is evident only in the four outer antenna complexes. By inspection of these data it is evident

70

outer antenna

.29 kt

core antenna

.07kt

Reaction coordinate

Figure 5. Standard free-energy differences for excited-state transfer in PSII between outer antenna, core antenna and the D I1D2/cytb559 complex. For calculation details see text. Energy differences are expressed in terms of the thermal energy (kB T) at room temperature. The figure shows clearly how PSII is a very shallow energy funnel.

that longer wavelength spectral forms are present in significant amounts in complexes both of the outer and core antenna.

In figure 4 data are also included for the D11D2/cytb559 complex which binds P680. Also in this case it is clear that the RC complex is not particularly enriched in long wavelength chi forms. Thus it is evident that a "deep antenna funnel" is not present in PSI!.

In order obtain more precise information on this, the population of excited states in all chi-protein complexes has been calculated. This was achieved using the Boltzmann distribution expression:

lffiij

Ni = gi e-kBT

Nj gj (4)

where NilNj are the excited state population values of spectral forms i and j; the gi, gj are weighting factors associated with the intensity of each absorption band with respect to the entire PSII pigment system. These were calculated from the sub-band areas after normalising each chi-protein complex to its overall stoichiometry in PSII (Jennings et a!., 1993). L\Eij is the energy gap between sub-bands i and j. These may be approximated by the energy levels of the absorption maxima or more correctly by the energy levels of the so-called mirror symmetry points for absorption and fluorescence spectra. kB is the Boltzmann constant and T the absolute temperature.

Data are presented in Table 1 for the calculated excited state population at equilibrium for the chi-protein complexes, together with the total absorption of each complex in PSII. The S * / A ratio is a good indicator of excited state density in the different complexes. It is therefore clear that energy is expected to be distributed rather evenly over the entire antenna system with slightly higher amounts in the core and RC complexes. The free energy differences (L\GO) for exciton transfer between outer antenna, core antenna and the RC complex can be calculated from these S*/A values using the expression L\Go=-RT lril<eq (fig.

71

5). These values indicate that the energy gradient (LlGo~-O.3kB T) is much less than the thermal energy (kBT at room temperature). Thus energy flow between these three compartments is readily reversible at RT.

Table 1. Absorption (A) and room temperature excited state population (S) in PSII in terms of the chl~protein complexes. The excited state population values have been determined on the basis of a Boltzmann excitation distribution amongst the spectral forms present in the chI-protein complexes (for details see Jennings et a1.1993; 1994)

Chi-protein complex A (% total) S (% total) S/A LHCII 55.3 48.8 0.88 CP29 6.9 7.4 1.07 CP26 6.3 5.9 0.94 CP24 3.0 2.6 0.87 CP43 12.3 14.2 1.15 CP47 12.4 15.6 1.26 DIID2/cytb559 3.9 5.0 l.28

It is interesting to compare these free energy differences with what would be expected for a "deep antenna funnel" in which all the short wavelength forms are concentrated in the outer antenna and all long wavelength forms in the core. In this situation the energy gap would be 2-3kBT i.e. an order of magnitude greater than is found. Thus the PSII antenna is a very shallow energy funnel.

SLOW TRAPPING IN PSII.

While psn has almost the same number of antenna chI molecules as PSI, the time between photon absorption and primary photochemistry (trapping time, 'tt,) for PSII is much longer ('ttr PSII ~ 300ps, 'tt, PSI ~ 80ps) (Roelofs et aI., 1991; Leibl et aI., 1989; Turconi et aI., 1994, Karukstis & Sauer, 1983; Schatz et aI., 1988; Holzwarth et aI., 1993). In fact PSII is the slowest trapping of all photo systems, including bacterial ones. The physical reasons for slow PSII trapping may be discussed under two general headings.

(a) Shallow antenna energy funnel.

As discussed above the antenna ofPSII is organised as a very shallow energy funnel. If the spectral forms were organised as a deep funnel the trapping rate would be much greater. Model simulations suggest that a deep funnel may accelerate the trapping rate by up to approximately five times (Seely, 1973a; 1973b).

(b) Shallow photochemical trap.

The trapping rate may be approximated by kt, = kRC Np· 1 where kRC is the primary charge separation rate (kRC ~0.3pS·l) and Np· 1 is the probability ofP680 being excited. In the case of230 chI antenna molecules which are isoenergetic with P680, Np•1 = 11230 = 0.0043. However as the antenna is energetically somewhat higher than P680 the energy distribution must be taken into account. This can be done using the Boltzmann expression as described above. In this way it comes out that Np· 1 = 0.013. Thus one calculates 'tt,= 430ps, which is in approximate agreement with experiment.

It is useful to compare this value with that for PSI, where P700 constitutes a deep trap, i.e. is energetically well below the antenna. Due to this factor the value for Np•1 is

72

considerably greater (Np-l = 0.04) for P700. Assuming a similar value for kRe as we have used for PSII (kRC ",0.3 pS-l), Ttr is calculated to be l40ps.

From this rough comparison ofPSII and PSI trapping it is evident that slow trapping in PSII is associated with the relatively low Np-1 value which is in tum due to the fact that P680 is a shallow trap.

DOES SLOW TRAPPING IN PSII HAVE A PHYSIOLOGICAL FUNCTION?

From the preceding section it is evident that slow exciton trapping in PSII is associated with (a) a very shallow energy funnel in the antenna and (b) P680 being a shallow trap. In the following it will be demonstrated that these two characteristics of PSII are important factors in minimising photoinhibition.

In this context the importance of the shallow antenna funnel can be understood in terms of the mechanism(s) of non-photochemical quenching, known to protect PSII from photoinbition (see Briantais, Horton, this volume). These quenching mechanisms seem to be mainly localised in the outer antenna complexes (Horton, this volume). From the equilibrium excited state population data of Table 1 it is clear that the probablity of an exciton residing on one of the four outer antenna complexes is near 0.65. In the case of a deep antenna funnel (~G = 2-3ksT) this probablity would be an order of magnitude lower. Thus it is clear that the shallow antenna funnel is an important factor in determining the high physiological efficiency of quenching processes in the outer antenna.

To illustrate the importance of the shallow trap properties we can write the following simple equilibrium equation to represent exciton equilibration between the primary rection centre donor (RC) and antenna (A).

* ~ * A +RC ~ A+RC

For P680 (~G = -0.3ks T at RT) Keq = 1.35. In the case of a deep trap (~G =- 2-3ks T) Keq comes out in the range 8-20. Thus in PSII energy is strongly repartitioned towards the antenna and away from P680. This feature is expected to minimise photoinhibition as the probability of an exciton residing on P680 (Np-1 = 0.013) will be low. This means that when traps are closed (QA reduced) photoinhibitory damage due to triplet formation on P680 (Telfer et aI., 1994) will be greatly reduced with respect to the deep trap situation. The Np-1

value for PSII is about 3-4 times smaller than for PSI. It is therefore clear that the shallow trap ofPSII significantly reduces photodamage.

Thus we may conclude that the thermodynamic properties of the antenna-trap system of PSII which lead to slow trapping are important in minimising photo inhibitory damage. Slow trapping however means an inherently lower photochemical efficiency of primary charge separation (<Pes). The approximate relation between trapping time and <Pes is given by

where T is the overall excited state lifetime in the absence of RC trapping. For PSII this is about 2.5 ns. Thus we calculate for PSII (Ttr '" 0.5 ns) that <Pes = 0.86. This is a high value and could only be marginally improved by increasing the trapping rate. Thus the price in terms of a reduced <Pes that psn pays to minimise photo inhibition is rather a low one.

73

REFERENCES

Fetisova, Z.G., Borisov, A.Y. & Fok, M.V. (1985) J. Theor. BioI. 112,41-75. Hayes, J.M., Gillie, J.K, Tang, D. & SmaIL G.J. (1988) Biochim. Biophys. Acta 932, 287-305. Hemelrijk, P.W., Kwa, S.L.S., Van Grondelle, R & Dekker, J.P. (1992) Biochim. Biophys. Acta 1098, 159-

166. Holzwarth, A.R, Schatz, G., Brock, H. & Bittersmann, E. (1993) Biophys. J. 64,1813-1826. Jennings, RC., Bassi, R & ZuccheIli, G. (1995) in Photoinduced electron transfer (Mattay, J., ed.),

Springer-Verlag, Berlin, Heidelberg, in press. Jennings, Re., Bassi, R, Garlaschi, F.M., Dainese, P. & Zucchelli, G. (1993) Biochem. 32,3203-3210. Jennings, Re., Garlaschi, F.M., Bassi, R, Zucchelli, G., Vianelli, A. & Dainese, P. (1993) Biochim.

Biophys. Acta 1183, 194-200. Jennings, RC., Garlaschi, F.M., Finzi, L. & Zucchelli, G. (1994) Lith. J. Phys. 34, 293-300. Karukstis, KK & Sauer, K (1983) Biochim. Biophys. Acta 722, 364-371. Kuhlbrandt, W., Wang, D.N. & Fujiyoshi, Y. (1994) Nature 367, 614-621. Leibl, W., Breton, J., Deprez, J. & Trissl, H.W. (1989) Photosynth. Res. 22, 257-275. Reddy, N.RS., Lyle, P.A. & Small, G.J. (1992) Photosynth. Res. 31, 167-194. Reddy, N.RS., Van Amerongen, H., Kwa, S.L.S., Van Grondelle, R & Small, G.J. (1994) J. Phys. Chem.

98,4729-4735. . Roelofs, T.A., Gilbert, M.,Shuvalov, V.A. & Holzwarth, A.R (1991) Biochim. Biophys. Acta 1060,237-

244. Schatz, G., Brock, H. & Holzwarth, A.R (1988) Biophys. J. 54, 397-405. Seely, G.R (1973) J. Theor. BioI. 40, 173-187. Seely, G.R (1973) J. Theor. BioI. 40, 189-199. Telfer, A., Bishop, S.M., Phillips, D. & Barber, J. (1994) J. BioI. Chem. 269, 13244-13253. Turconi, S., Weber, N., Schweitzer, G., Strotrnann, H. & Holzwarth, A.R (1994) Biochim. Biophys. Acta

1187, 324-334.

74

PHOTOSYNTHETIC REACTION CENTERS

P. Mathis,

DBCMfSBE, CEA Saclay, 91191 Gif-sur Yvette Cedex, France

INTRODUCTION

Photosynthesis is the process by which living organisms ensure the conversion of solar energy into chemical energy, essentially in the form of carbohydrates which are used for their growth and reproduction. It is a very complex process which involves the cooperative function of a large number of proteins. The initial reactions are realized by large proteins named "reaction centers". These proteins can be considered as microscopic solar cells which are located in a membrane and ensure photo-induced charge separation with a quantum yield close to unity.

Photosynthesis takes place in many classes of living organisms, both procaryotic and eucaryotic. In a first kind of classification, we have to distinguish organisms which can use water as a source of electrons and evolve oxygen in the process. They are named "oxygenic" organisms. Some of them are procaryots (cyanobacteria) and others are eucaryots (plants, algae). Those organisms which cannot oxidize water perform a so-called anoxygenic photosynthesis. They are procaryots and they include four categories: purple bacteria, green non-sulphur bacteria, green sulphur bacteria, heliobacteria. Considering their structure and function, reaction centers can be classified in three categories: purple bacteria (green non­sulphur bacteria have very similar reaction centers), photosystem-II (PS2) from oxygenic organisms, and type I reaction centers, including photosystem-I (PSI) from oxygenic organisms, green sulphur bacteria and heliobacteria.

These reaction centers are coupled with other components of photosynthesis as sketched in Fig. 1 (not shown here is the coupling of reaction centers with the so-called antenna, which absorbs light and conveys electronic excitation to the reaction centers). In purple bacteria, electron transfer is essentially cyclic and serves as a proton pump; this creates an electrochemical potential which is used by another membrane protein, the ATP­synthase, which' synthesizes ATP from ADP and inorganic phosphate. The electron cycle involves another large membrane protein, the b-c complex, which is also found in oxygenic photosynthesis and in respiratory electron transfer. It should be added that electron transfer in purple bacteria is not purely cyclic and that photosynthetic electron transfer includes some input from reduced electron donors (such as sulphur compounds) and some output (e.g. through the respiratory pathway), In contrast, linear electron transfer is dominant in oxygenic photosynthesis; the two classes of reaction centers (PS2 and PS 1) cooperate in transferring electrons from water to NADP+. Electron transfer is also coupled to trans-

Light as an Energy Source and Iriformation Carrier in Plant Physiology Edited by Jennings et aI., Plel1ll11l Press, New York, 1996 75

Pt -.. I --NADP' W

P* I· hv

hvj' -\.-\' w

Net e· transfer and proton pump

Figure 1. In purple bacteria, reaction centers serve mainly as a proton pump, coupled to light-induced electron transfer (left; P: primary electron donor). In o:\-ygenic photosynthesis, two reaction centers function in series for electron transfer from water to NADP+; energy is stored both as redox energy and as a proton gradient (right).

membrane proton transfer, so that energy IS stored In two forms: redox energy and membrane electrochemical potential.

REACTION CENTER OF PURPLE BACTERIA: A MODEL

Basic functional properties.

The reaction center of purple bacteria transfers electrons from cytochrome ~ (Em # + 340 mY) to ubiquinone (Em # 0 mY: complex and pH-dependent) (Deisenhofer & Norris, 1993). This reaction is endergonic and is made possible by the energy of a photon. Photon absorption induces electronic excitation of a bacteriochlorophyll dimer P to its lowest excited singlet state. Excited P (P*) is highly reactive: it transfers an electron to a primary electron acceptor (a monomeric bacteriochlorophyll) which transfers to another acceptor (bacteriopheophytin), then to a first quinone QA, and then to a second quinone QB. Since P has been oxidized, we are left with a cation radical P+, which then accepts an electron from a secondary electron donor (cytochrome ~). At the level of the reaction center, the ubiquinone QB is the terminal acceptor. Since reduction of a quinone requires two electrons, and two protons, the reaction center has to turn-over twice : Q + e- ~ Q- ; Q- + e- + 2 H+ ~ QH2. The reduced ubiquinone leaves the reaction center, diffuses in the membrane and transfers electrons to the cytochrome b-c complex.

The reaction kinetics are given approximately in Fig. 2 for forward, normal reactions, and for wasteful back-reactions (there are variations from species to species, and also with some environmental factors such as pH, temperature, ... ). The primary reactions have to be very fast, in order to compete with possible paths for energy "loss", such as fluorescence, internal conversion an so on. It is remarkable that, at the level of an isolated reaction center, the quantum yield is nearly 100 %, i.e. each photon induces a charge separation to the state (cyt C2+, quinone reduced). This is possible only if forward reactions are all much faster than potential wasteful back reactions.

Reaction center structure

It is remarkable that the structure of the reaction center is known at the atomic level from two species of purple bacteria: Rhodopseudomonas (Rps.) viridis and Rhodohacter (Rh.) sphaeroides (Deisenhofer and Michel, 1989; Feher et aI., 1989). Both structures are very similar and, for simplicity, they will not be differentiated in this presentation (one important difference between these reaction centers: in Rh. sphaeroides, a soluble

76

p

. l-l00ms

\~l) 10-100..as \ Q

~ 0 A Q . 0 ·

Figure 2. Schematic structure of the cofactors in reaction centers of purple bacteria, and half-times of a few electron transfer reactions. P: primary donor (dimer of bacteriochlorophyll); Behl: bacteriochlorophyll; BPheo: bacteriopheophytin; QA, QB: quinones.

cytochrome c2 is the direct electron donor to p+ instead of a bound tetraheme cytochrome in Rps. viridis). The reaction center of Rps. viridis has been purified and crystallized, permitting X-ray crystallography with a resolution of 2.3 A. The reaction center comprises four different polypeptides. Two of them, named Land M are homologous and hydrophobic. Each contains five transmembrane alpha-helices. They are assembled in the form of a heterodimeric structure which holds all pigments and redox centers involved in primary charge separation. These are organized in a pseudo-symmetrical manner, with two species located on the symmetry axis, at the interface between Land M polypeptides: the primary donor P (a dimer of bacteriochlorophylls), and an iron atom Fe2+ with unknown function. The other cofactors are organized into two "branches",. each comprising one monomeric bacteriochlorophyll, one pheophytin and one quinone. On the cytoplasmic side of the reaction center, a third sub-unit named H seems to cap the L, M pair, without carrying any cofactor. A fourth subunit is located on the peri plasmic side: it is a tetraheme cytochrome Q,

with the four hemes arranged in a nearly linear manner. This structure has been analyzed in great detail since it is the best basis available for

understanding the structure-function relationships in photosynthetic reaction centers (for reviews see Deisenhofer & Norris, 1993). It may be pointed out, however, that the present state of this 3-D structure is ambivalent: on one hand it provides an essential structural model and it is accurate enough to provide a good basis for interpreting most spectroscopic data and several aspects of electron transfer, and also for deciding which amino-acids should be modified for useful site-directed mutagenesis. On the other hand, however, the structure is largely insufficient. This insufficiency has two aspects (which are not unique to the 3-D structure of the reaction center):

• the structure is not accurate enough for predicting electron or proton transfer rates (this will be illustrated below in comparing electron transfer along the two branches of the reaction center);

the reaction center structure is well-defined and relatively rigid. The protein has indeed one essential function of scaffolding for pigments and redox centers. Structural changes, however, are essential for electron transfer. This can be realized from the equation which describes electron transfer rates (according to the Marcus theory, in cases where electron transfer is coupled to low frequency vibrations) (Marcus & Sutin, 1985):

kET = A exp(-~R) exp(-( L\Go + '/..)2/4 '/.. kT) (1)

77

(R is the edge to edge distance between electron donor and acceptor; T is temperature; and k is the Boltzmann contant; _~Go is the free energy change in going from the initial to the final state; A., the reorganisation energy, is related to the structural changes which are associated with electron transfer). The equation clearly shows the importance of distance, and hence of correct scaffolding, for electron transfer (in proteins the value of f3 is usually 1.4 A-l) (Moser et aI., 1992). The "driving force" _~GO is determined by the nature of the redox centers. When D and A are the electron donor and acceptor, with redox potentials ED and EA, one can write:

(2)

It should be clear that the redox potentials are not those which are measured in vitro for isolated species. What has to be considered are the in situ potentials, which are strongly affected by the protein, by means of specific interactions, dielectric properties, etc. These effects are very distance-dependent and influenced by local protein properties: their prediction thus requires a high accuracy for the 3-D structure.

Reorganization energy is also an essential parameter, as seen from equation 1. Its value can sometimes be determined experimentally, from a curve of kET versus _~Go, which should display a maximum when _~Go = A. (Fig. 3). This approach, however, can be used only in a few favorable cases. The reorganization energy can also be calculated, in cases when the structure of the donor-acceptor couple is known in detail both before and after electron transfer. This is another limitation of the present 3-D structure: not only is it not accurate enough, but it should ideally be obtained in various stages of the light-induced electron transfer process. Obviously this is out of reach, but several spectroscopic methods such as differential Fourier-transform infra-red spectroscopy and resonance Raman spectroscopy provide very important information (Breton et aI., 1994; Lutz & Mantele, 1991). NMR might also provide information on the differential dynamics of sites in the reaction center.

Structure-function relationships in reaction centers of purple bacteria

A large number of problems are raised at that level, and only a few of them will be briefly envisioned here.

1. What is P? Why a dimer? The dimeric nature of P was recognized very early, on the basis of the circular dichroism and absorption properties ofP, and ofEPR spectroscopy ofP+. The hypothesis has been nicely demontrated by X-ray crystallography. A few essential questions are still discussed with respect to P: what is the extent of electronic interaction within the dimer and between the dimer and the neighbouring bacteriochlorophyll molecules ? This question is connected with the precise nature of the lowest excited state ofP, which is of obvious importance for the primary electron transfer, a key step in the reaction center function. What is the charge distribution within the radical cation state p+ ? From ENDOR and EPR data, this state is known to be more or less symmetrical (Rautter et aI., 1994). The asymmetry of P may be related to the asymmetry of electron transfer among the active and inactive' branches.

The redox potential of the P/P+ couple is of obvious importance for electron transfer. It is somewhat higher than the corresponding bacteriochlorophyll in solution.

2. What are the primary steps of electron transfer? Picosecond absorption spectroscopy has shown, a few years ago, that the excited state of P decays in about 2 ps (Rh.sphaeroides, room temperature) and that a bacteriopheophytin is reduced with approximately the same kinetics (Martin et aI., 1986). The function of the monomeric bacteriochlorophyll located between P and the bacteriopheophytin has remained an object of

78

debate. However, there are now firm arguments for believing that electron transfer takes place relatively slowly from P* to the bacteriochlorophyll (2 ps) and much faster from reduced bacteriochlorophyll to bacteriopheophytin (300 fs), so that there is normally no significant amount of reduced bacteriochlorophyll after flash excitation (ArIt et aI., 1993). The factors which determine these reaction rates remain to be understood.

3. Properties of QA and QB' QA behaves like practically all redox cofactors in proteins: it is always bound to its site, essentially provided by the M polypeptide, and it works as a one-electron carrier, receiving one electron from the bacteriopheophytin (in about 200 ps) and transferring it to QB. The chemical nature of QA varies in bacteria: ubiquinone-l 0 in Rb. sphaeroides, menaquinone 10 in Rps. viridis. QB is always ubiquinone - 10, but its behaviour is rather different:

• it operates as a 2-electron carrier: QB + e- ---+ QB-; QB- + e- + 2 H+ ---+ QB H2 • the semi-quinone is firmly bound to the site (on polypeptide L), but the oxidized

form and the fully reduced form (hydroquinone) are weakly bound. QBH2 leaves its site on the reaction center and diffuses in the membrane to its oxidation site on the cytochrome b-c complex. The reaction center site thus becomes vacant for binding a neutral (oxidized) ubiquinone.

• there are well-known inhibitors which bind competitively with ubiquinone at the QB site. These inhibitors belong to the same family as herbicides which bind at the equivalent site in PS2 of plants.

The precise properties of the protein which permit these behaviours of QA and Os remain to be understood (Deisenhofer & Norris, 1993; Deisenhofer & Michel, 1989).

4. Electron transfer from QA to QB; proton uptake. This reaction displays properties very different from other electron transfer steps within the reaction center, in particular its strong dependence on temperature and pH (within limits). These properties are probably related to the large reorganisation energy due to QB reduction and to proton uptake. Considerable effort is being expended to try to understand how protons are involved, particularly for the full reduction of QB, and how they can reach the QB site: is there a specific hydrophilic channel, or do protons migrate in the protein from one amino­acid to another; in that case is there a well-defined path or multiple possibilities?

It seem reasonable that the Fe2+ atom, which is positioned mid-way between QA and QB, should be implicated in electron transfer. There is, however, no positive argument in favor of that role: it even seems that Fe2+ can be replaced by many metal ions without impairment of electron transfer.

5. What permits kforward » kbackward? In general terms it is clear that in order to have a high quantum yield for electron transport the overall rate of the forward reactions should be much greater than for the back reactions in reaction centers. In the bacterial reaction center this is achieved at the level of pheophytin oxidation when the forward reaction to QA has a time constant of around 200 ps while the back reaction to p+ is about IOns. Distance is a key factor for rapidity of electron transfer, but we may consider here that the distances from H- to or to p+ are about the same. We also consider that the back­reaction is to the ground electronic state of P. The difference in the rate can be understood on the basis ofthe Marcus theory (Marcus & Sutin, 1985) and of the respective values of-A GO and 'A for the two reactions (Gunner & Dutton, 1989; equation 1):

H- (Em = - 600 mY) to QA (Em = - 200 mY) : _AGO = 400 meV; 'A = 600 meV H- (Em = - 600 mY) to p+ (Em = + 500 mV): -AGO = 1100 meV; 'A = 200 meV

If we assume that both reactions have equal rates under optimum conditions (-AGo = 'A), it appears that the first reaction is not far from the maximum rate (_AGo is not very different

79

from A; the reaction nearly corresponds to the top of the curve, where kET changes very little with _~GO), but that the second reaction corresponds to the "inverted region", very far from the optimum, where kET decreases when _~Go increases (Fig. 3). It is thus understandable that the forward reaction is faster than charge recombination. In addition, it is quite clear that in all cases a forward reaction (with a positive value of _~GO) will be faster than the immediate reverse reaction (which is always endergonic).

o 10910 k/k""",

A=IOOOmeV

-I

As400meV

-2

-3

-4

1000 1200

Figure 3. Prediction, according to the Marcus theory, of the relative rate of electron transfer as a function of -AGo for the reaction (see equation I). The curves are drawn for T = 294 K and for three values of the reorganisation energy A.

6. Why does electron transfer take place on the L branch rather than on the M branch? This question is difficult to answer at present. The knowledge of the structure, the limited amount of thermodynamic data (redox potentials) and a few attempts of site-directed mutations do not lead to a simple answer. It is probable that the large difference in electron transfer efficiency (perhaps a factor of 50 between P* and either bacteriopheophytin) is the result of many factors which reinforce each other. This is a field where much more research needs to be done (Deisenhofer & Michel, 1989; Deisenhofer & Norris, 1993).

7. Electron transfer from cytochrome f to p+. As mentioned above, the reaction center of purple bacteria induces a nearly cyclic electron transfer in which p+ is re-reduced by cytochrome~. The structural and functional aspects of that reduction are very different in the two situations encounterd in nature:

• in Rps. viridis (and many other species), a tetraheme cytochrome is firmly found to the reaction center and directly reduces p+. Two hemes have a relatively high potential (+380 and +310 mY) and two others have a much lower redox potential (+10 and -60 mY). Under physiological conditions, the two low-potential hemes are permanently oxidized: they cannot contribute to electron transfer and their function is not known. p+ is reduced in

80

about 200 ns by the proximal (highest-potential) heme, which is itself reduced by the second high-potential heme in a time of around 2 J..ls. It is probable that the second high-potential heme is reduced by soluble cytochrome c2. This tetraheme cytochrome is an interesting example in which several electron transfer steps can be studied under several redox conditions and as a function of temperature, revealing unexpected properties (Ortega & Mathis, 1993).

• in Rh. sphaeroides (and a few other species), a soluble cytochrome c2 is in equilibrium between two states: free in solution or bound to the reaction center (Venturoli et aI., 1993). The kinetics of electron donation thus display a biphasic behaviour, with a fast first-order phase (half-time around 0.6 J..ls) for the pre-formed complex and a second-order reaction for reaction centers devoid of bound cytochrome at the time of P oxidation. We recently used this system to study the effect of _~Go on the reaction rate, as predicted by the Marcus theory. This was made possible by the availability of mutated reaction centers in which the Em of P/p+ was varied by changing the number of histidine residues which establish hydrogen bond with carbonyl groups of the special pair (Lin et aI., 1994). It should be pointed out that this is a good example of tuning by the protein to the redox potential of a redox cofactor.

8. The triplet state of P. The singlet excited state ofP reacts very quickly, by electron transfer, in about 2 ps, and there is thus no time for a formation of 3p by intersystem crossing. The triplet state can be populated, however, when electron transfer from bacteriopheophytin to QA is blocked (usually if QA is reduced prior to excitation). In that case the radical pair (p+ H-) lasts for about IOns before recombining, a time long enough for the pair to evolve into its triplet state. The triplet sub-levels (which can become energetically separated in a magnetic field) are very differently populated and the EPR spectrum of 3p, as recorded at low temperature, is highly characteristic (Hoff, 1979; Budil & Thumauer, 1991). At room temperature, the triplet 3p disappears very quickly (in less than 20 ns) by energy transfer to a carotenoid molecule in the reaction center. This transfer is blocked at low temperature and it never takes place in Rps. viridis, for energetic reasons, the energy of 3p being lower than that of the carotenoid triplet.

PS2 REACTION CENTER OF OXYGENIC PHOTOSYNTHESIS

The PS2 reaction center can be described as a light-activated water oxidase/plastoquinone reductase. It brings about the oxidation of water into molecular oxygen. The photoinduced reaction abstracts electrons and pulls to the right the redox couple:

Em, 7 = + 0.81 Volt

It is well known that the inverse reaction takes place in respiration. This basic function immediately imposes two important characteristics of PS2, if we consider it as a microphotocell:

• it needs to have a positive pole at a high oxidizing potential, much higher than +0.81V;

• it needs to include some storage device, in order to accumulate the succesive loss of four electrons, due to four successive light-induced reactions. It has indeed been found experimentally, in agreement with thermodynamic predictions, that water is not oxidized in four one-electron reactions but rather in one single four-electron reaction.

It was recognized in the 1980's that the PS2 reaction center (Fig. 4) has many aspects in common with its counterpart of purple bacteria (Mathis & Rutherford, 1987). It has an inner core made of two hydrophobic polypeptides named Dl and D2 (equivalent and

81

somewhat homologous to L and M). Its acceptor side includes, as in purple bacteria, a pheophytin, a quinone QA (one-electron carrier) and a quinone QB (two-electron carrier) displaceable by competitive inhibitors, and with a Fe2+ atom presumably located between QA and QB. These analogies lead to the conclusion that the PS2 structure, for which there are no crystallographic data available, is similar to that of purple bacteria as sketched in Fig. 4.

c_~ Figure 4. Schematic structure of the PS2 reaction center of o"1'genic photosynthesis. Pheo: pheophytin; Tyr: tyrosine residue. The figures are MW in kDa of a few polypeptides of the reaction center.

It should be realized, however, that PS2 displays very important differences from its bacterial counterparts, which can probably be associated with its ability to oxidize water, as mentioned above. The aspects similar to those found in purple bacteria (including rates of electron transfer on the acceptor side) need not be repeated and I shall insist on some specific characters (see for reviews Debus, 1992; Rutherford & Nitschke, 1994).

1. Polypeptides. PS2 has no subunit directly equivalent to the H subunit. It contains, however, a large number of polypeptides which are encoded in part by the nuclear and in part by the chloroplastic genomes (in eucaryots). In addition to Dl and D2, there are at least eleven polypeptides present in the membrane, which are, more or less obviously, part of the reaction center, (without considering the PS2 antenna, see Bassi et aI., this volume), and at least three polypeptides (of 16, 23 and 33 kDa) are peripheral and involved in maintaining the water oxidizing catalytic site. It is possible to isolate an inner core ofPS2, with a limited capacity in terms of electron transfer since QA is lost, which might be called the "reaction center". It includes five polypeptides: Dl and D2, the two subunits of cytochrome bSS9, and a very small hydrophobic polypeptide (4kDa) named subunit I.

2. Non-hemic iron, Fe2+. By contrast with what happens in purple bacteria, this atom can be oxidized to Fe3+. The couple has an Em around + 400 mV (pH-dependent). Fe3+ is a very good electron acceptor for QA-. There are also differences in the Iiganding, which may be of physiological relevance: instead of four histidines and one glutamate (bidentate), there are only four histidines available in PS2. The other ligands are perhaps provided by bicarbonate (HC03-). This bicarbonate can be displaced, resulting in a block of electron transfer from QA to QB. This could be a site of regulation of electron transfer, although there is no physiological evidence for it.

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3. Extra loop on Dl: site of cleavage. Although the precise organization of polypeptides in PS2 is not known, several attempts have been made to model the folding of D} and D2, hypothesizing that both contain five transmembrane alpha-helices. Such modelling shows that the D} polypeptide includes between helices labelled IV and V, on the stroma side, and close to the binding site for QB, an extra loop which has no equivalent in purple bacteria. It has been shown that the loop contains a site (between aminoacids 240 and 248) where proteolytic cleavage takes place during D} tum-over associated with photo damage (there is no equivalent tum-over in purple bacteria). It should also be added that a few other loops also exist on the lumenal side, where they could contribute to binding the water oxidizing catalytic machinery.

4. Properties of P-680. Dimer or monomer ? P680 is very difficult to study and it should be stressed that most spectroscopic data on that species are very ambiguous. The redox potential of the P-6801P-680+ couple has only been estimated: no redox titration can be made in reaction centers above + } V. The oxidized form can only be detected a.s a transient species, in flash experiments. In all attempts to oxidize P-680 in steady-state, under continuous light, it is not certain whether P-680+ accumulates or oxidizes neighbouring pigment molecules. P-680 can be oxidized in flash experiments, as detected by absorption changes around 680 nm (disappearance of chlorophyll !! absorption) or 820 nm (appearance of a chlorophyll radical cation).

Another way to study P-680 is to populate its triplet state by the same mechanism as presented above for P in purple bacteria: when electron transfer from pheophytin to QA is not possible, recombination within the radical pair populates a triplet state which can be studied by EPR at low temperature. Two aspects of this reaction are fairly surprising:

o the yield of 3p-680 is very low when QA is singly reduced to the radical-anion state and it rises greatly when QA is either fully reduced (two electrons) or displaced from its site.

o the orientation ofP-680 has been studied by measuring the EPR spectrum of 3p-680 in oriented membranes. It appears that the gz axis, which is perpendicular to the macrocycle plane, is oriented very close (at 30°) to the membrane normal, which means that the tetrapyrrolic plane is close (30°) to the membrane plane, instead of being perpendicular as in purple bacteria.

These properties are not yet understood. Neither is it understood why P-680 has such a high redox potential, and why its absorption maximum is so close to that of chlorophyll !! in the antenna (see Jennings et aI., this volume, for a possible biological significance). Several hypotheses can be envisioned, ranging from P-680 having a structure similar to that of P in purple bacteria, or being a dimer of chlorophyll !! with a weak interaction, or a monomeric chlorophyll !! with the same position as the "accessory" bacteriochlorophyll on the L branch in bacteria (Rutherford & Nitschke, 1994).

5. Electron donor to P-680+: tyrosine Z. The immediate donor to P-680+ has been identified as a tyrosine residue (named TyrZ) of the polypeptidic backbone of D}. It donates to P-680+ in about 50 ns. It is supposed that TyrZ undergoes a large decrease in pK upon oxidation and that a proton is transferred to a neighbouring amino-acid. The oxidized species, TyrZ·, is mainly known from its EPR properties, known as Signal II. TyrZ has a counterpart on D2, named TyrD, which is not an efficient electron donor to P-680+ in the main flow of electron. It is usually kept oxidized (a fairly unusual property for a high­potential species): it is supposed to be oxidized by P-680+ and to play a role in the biosynthetic pathway which builds the manganese cluster, presumably by oxidizing Mn2+.

6. Catalytic site for water oxidation. Water oxidation is the major functional property of the PS2 reaction center: it is a process of fundamental importance and also one which is very poorly understood. The subject can only be briefly introduced here (for reviews, see

83

Debus, 1992; Rutherford et aI., 1992), but it cannot be ignored in a description of photosynthetic reaction centers.

The water oxidizing machinery includes the following elements: four manganese atoms, which are the major site for the storage of holes (electron deficits) produced by successive electron transfers to TyrZ+; parts of the Dl and D2 subunits which serve three functions: scaffolding the cluster of four Mn, electron donation by means of at least one residue (presumably histidine), and proton movements which compensate the positive charges on redox active centers; Ca2+ and CI- ions, which are essential for the function of the catalytic site. All these elements are important and their structure/function properties are the field of very active research. Of special interest is the structure of the manganese cluster and the state of oxidation of the different atoms. These questions are mostly studied by EPR and by spectroscopy of X-ray absorption, together with the synthesis of artifical clusters which would permit us to understand the biologically active system. The mechanism of water oxidation itself is totally unknown.

7. Cytochrome b559' In all organisms, the PS2 reaction center includes a tightly bound cytochrome, named cytochrome b559. This cytochrome remains in the smallest PS2 reaction centers which have been obtained, so it is certainly a bona-fidae component, closely associated with the D1-D2 core. Flash experiments have not demonstrated any role of cytochrome b559 in the normal flow of electron transfer from water to plastoquinone. There is no counterpart to this species in other types of reaction centers.

What is the function of cytochrome b559? A function can be searched for along several lines:

• a purely structural function: it is required for the assembly of the reaction center; • a redox function associated with the process of buiding-up the manganese cluster

from Mn2+ ions present in the cellular fluid; • a protective role, associated with the reduction of an excess of positive charges,

specially when the water oxidizing machinery is not working perfectly.

8. Photodamage to PS2. It is well established that light has damaging effects on PS2, as described in several chapters of this volume (see Baker, Horton, this volume. See also Baker & Bowyer, 1994). These effects take place even under low light; they are increased under high light and UV has additional effects (see Bornman, this volume). These effects are certainly much less pronounced in purple bacteria. Polypeptide Dl is a major site of damage, resulting in its proteolytic cleavage either on the stromal or on the lumenal side of the membrane. The molecular mechanisms of damage are the subject of considerable controversy, as is also the biological significance of these processes. It would seem natural that the damage originates on the donor side ofPS2, because of the very oxidizing character ofP-680+ and Tyr Z+, of the production of oxygen, and of possible mistakes in the function of the water oxidation process which could induce the formation of peroxides or radicals. This view, however, is contradicted by many data showing that damage can also result from acceptor side effects.

PSI AND BACTERIAL ANALOGS

The PS 1 reaction center of plants follows the same principles of construction and operation as previously discussed: a couple of hydrophobic polypeptides (named PSI-A and PSI-B) carry the redox centers involved in primary electron transfer (Fig. 5); transmembrane electron transfer involving several steps, the first of which are very rapid, etc. (Golbeck & Bryant, 1987, Setif, 1992). The PS 1 reaction center complex of a thermophilic cyanobacterium, Synechococcus elongatlls, has been crystallized and its structure has been

84

P700 : cl\lorophyll a dlmer Ao chloroi>/'yU a A, : i>/'vtloqu none F. : F. and F. : 4Fe-4S elmors

Figure 5. Schematic structure of the PSI reaction center, a few major polypeptides of which are named PSI­A to F. Ao: chlorophyll; AI: phylloquinone; FX, FA, FB: 4Fe-4S centers.

elucidated at 6 A resolution, permitting location of a few redox centers in the protein (Krauss et aI., 1993).

The major characteristic of PS 1 is that it operates at low redox potentials, in order to transfer electrons from small proteins at an Em of about + 360 m V, to other small proteins, ferredoxin or flavodoxin, which are very reducing, with an Em around - 350 mY. Similar properties are found in several classes of bacteria, the green sulphur bacteria and the heliobacteria, which will not be described in detail (see Nitschke et aI., 1990; Nitschke & Rutherford, 1991; Buttner et aI., 1992; Nitschke et aI., 1994).

1. Sequence of electron carriers. The primary donor, named P-700, is a dimer of chlorophyll ~. The P-7001P-700+ redox couple has a relatively low Em, + 500 mY. The dimer is held at the interface between the PS I-A and B subunits. Close to P-700, there are four chlorophyll ~ molecules which are organized similarly to monomeric bacteriochlorophylls and bacteriopheophytins in purple bacteria. They probably playa similar role, two of them being the primary electron acceptors and two being on an inactive branch. These assignments are very tentative, however. The reaction center also contains two molecules of phylloquinone which have not been located in the X-ray electron density map. One of the phylloquinones is the next electron acceptor (the 2nd or the 3rd in the series), and the other one has no known function. It can be guessed that the redox potential of the active phylloquinone must be around - 800 m V, which is very low for this class of molecules. The low potential could be due to a destabilisation of the radical anion in a hydrophobic environment.

The successive electron acceptors are three 4Fe - 4S centers, named FX, FB, FA FX has a very low potential (about - 730 mY). It has the very unusual property that its cysteine ligands are on the two large subunits (PSI-A and B). FB and FA (Em about - 590 and - 540 m V, respectively) are held by another subunit, named PS I-C. The path and kinetics of electron transfer between the phylloquinone, FX, FB and FA have not been elucidated, in spite of much effort, probably because there is not a unique well-defined sequence. It is nevertheless clear that the terminal acceptors in the reaction center are the Fe-S centers FA and FB, where an electron presumably arrives in 1 J..t.s or less.

85

2. Large core antenna. The polypeptide pair PS I-A and B which make up the reaction center core have a large size (MW around 83 kDa). In addition to the redox centers, they include a large number of pigment molecules with an antenna function (about 100 chlorophyll ~ and 10 (3-carotene). This peculiarity is fairly disturbing for spectroscopic studies of electron transfer reactions. It has been suggested that the large size of PSI-A and B is due to their genes being the result, during evolution, of a fusion of genes of the core polypeptides and of the core antenna in a PS2-like precursor (Nitschke et aI., 1994).

3. Electron donor and electron acceptor to PSt. The PSI reaction center has the peculiarity of interacting with soluble proteins on both sides of the membrane (Fig. 5). The electron donor to P-700+ is usually a small copper protein named plastocyanin (it can be replaced in some cases by a cytochrome f). The docking of plastocyanin, which permits a fast electron transfer (tlh about 12 I1s), may be facilitated by one subunit of the reaction center, PS I-F, which extends into the lumenal volume.

On the stromal side, electrons are picked to the F A,FB couple by a small soluble ferredoxin (a protein with a 2Fe-2S center) (this protein also has a substitute, flavodoxin). After its reduction, ferredoxin contributes to several cellular functions, the most important of which is to reduce NADP+ with the help of an enzyme named NADP+-reductase. The docking of ferredoxin involves two subunits of the reaction center, PS 1-D and PS I-E (Fig. 5).

CONCLUSIONS

Methods of study

Progress in understanding the structure and the functional properties of reaction centers requires the concerted use of many methods:

• biochemical methods: to isolate reaction centers; to learn their composition in terms of proteins and cofactors; to modify them in a desired manner; to identify protein-protein interactions; etc. When possible, X-ray crystallography brings essential structural information.

• spectroscopic methods of all kinds. Of special interest are: flash absorption spectroscopy, vibrational spectroscopies, magnetic resonance (EPR, NMR and their cross­products), X-ray absorption spectroscopy, etc.

• molecular biology is of special interest in permitting the sequencing of polypeptides and to make selective modifications or deletions.

• chemists bring essential contributions in synthesizing models which permit the understanding of the properties of redox centers and of light-induced electron transfer. Theoretical aspects are also important for electron transfer, energy transfer, molecular dynamics, etc.

This list is far from being exhaustive: it merely attempts to illustrate that reaction centers are a field where multidisciplinary research operates in a very active manner.

General model for reaction centers

A survey of all reaction centers clearly shows that they can be classified into a few categories, which obey common principles for structure and function (a core of two hydrophobic polypeptides; trans-membrane positioning; successive electron transfer steps; etc.). They also make use ofa small variety of pigments and redox-active components. Each class of reaction centers displays little variation, an indication that the constraints are very strong at their level. This evolutionary stability is particularly striking when one compares

86

the PS 1 and PS2 reaction centers of plants and of cyanobacteria, which have a wide evolutionary difference but very similar reaction centers.

An important recent finding has been the observation that the PS 1 type reaction center of green sulphur bacteria and of heliobacteria has a homodimeric protein structure: it is made of two identical subunits, instead of two homologous ones. It will be exciting to learn whether this homodimeric structure goes with complete symmetric properties for the cofactors, or if there is an asymmetry as is demonstrated in purple bacteria.

Stability toward ligh-induced damages

Photosynthetic reaction centers are focal points where an enormous amount of excitation energy arrives (during day time). How can they resist such a high flow of energy ? Two reasons may be proposed for their stability:

1. In most cases where photodamage have been studied in vitro, the triplet state of a ~igment was found to be the source of damage, probably after reaction with 02 to form 02. The high yield of charge separation in reaction centers results in a very short life for

the singlet excited state of chlorophyll-like pigments and a low yield of triplet state formation. In the antenna, any triplet state formed is rapidly quenched by carotenoids for harmless internal conversion into heat.

2. Radicals are also a frequent source of degradation. They are necessarely formed during the normal function of reaction centers. They are however restricted to specific volumes which are screened from unwanted access by the protein thickness.

For both mechanisms, PS2 displays unique properties because the lifetime of the chlorophyll !! singlet excited state is relatively long, and because radical species at the water oxidizing site are not screened for the lumenal medium. These are probably the reasons why PS2 degradation is prevented by additional quenching of excited states (as discussed in other chapters of this volume) and is efficiently repaired in case of damage.

REFERENCES

ArltT., Schmidt, S., Kaiser, W., Lauterwasser, c., Meyer, M., Scheer, H. & Zinth,W. (1993) Proc. Natl. Acad. Sci. USA, 90:11757-11761

Baker, N.R & Bowyer, J.R (1994) Photoinhibition of photosynthesis. Bios Scientific Publishers Breton, J., Burie, J.R, Berthomieu, C., Berger, G. & Nabedryk, E. (1994) Biochemistry, 33:4953-4965 Budil, D.E. & Thurnauer, M.C. (1991) Biochim. Biophys. Acta, 1057:1-41 Biittner, M., Xie, D.L., Nelson, H., Pinther, W., Hauska. G. & Nelson, N. (1992) Proc. Natl. Acad. Sci.

USA, 89:8135-8139 Debus, R (1992) Biochim. Biophys. Acta ,1102: 269-352 Deisenhofer, J. & Norris, J.R (1993) The photosynthetic reaction center, Vol. 1 and 2, Academic Press Deisenhofer, J. & Michel, H. (1989) EMBO J., 8:2149-2169 Feher, G., Allen, J.P., Okamura, M.Y. & Rees, D.C. (1989) Nature, 339:111-116 Golbeck, J.H. & Bryant, D. (1987) Curro Top. Bioenerg., 16:13-57 Gnnner, M.R & Dutton,P.L. (1989) J. Am. Chern. Soc., 111:3400-3412 Hoff, AJ. (1979) Physics Reports, 54:75-200 Krauss, N., Hinrichs, W., Witt, I., Fromme, P., Pritzkow, W .. Dauter, Z., Betzel, C., Wilson, K.S.,

Witt,H.T. and Saenger,W. (1993) Nature, 361:326-331 Lin, X, Murchison, H.A, Nagarajan, V., Parson, W.W., Allen, J.P. & Williams, J.C. (1994)

Biochemistry, 91: i13517-13523 Marcus, RA & Sutin, N. (1985) Biochim. Biophys. Acta, 811:265-322 Martin, J.L., Breton, J., Hoff, AJ., Migus, A. & Antonetti, (1986) Proc. Natl. Acad. Sci. USA, 83:957-

961 Moser, C.C., Keske. M., Warncke, K.. Farid. RS. & Dutton, P.L. (1992) Nature, 335:796-802 Mathis, P. & Rutherford, A.W. (1987) in "Photosynthesis" (J. Amesz, ed.), pp 63-96, Elsevier Science

Publishers Lutz, M. & Miintele. W. (1991) in "Chlorophylls", (H. Scheer. ed.), CRC Press, Boca Raton, 855-902 Nitschke, W., Setif, P., Liebl, U., Feiler. U. & Rutherford, AW. (1990) Biochemistry, 29: 11079-11088

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Nitschke, W. & Rutherford, AW. (1991) Trends Biochem. Sci., 16:241-245 Nitschke, W., Mattioli, T. & Rutherford, A.W. (1994) in "Origin and Evolution of Biological Energy

Conservation" (H. Baltscheffsky, ed.) VCH New York, in press Ortega, 1.M. & Mathis, P. (1993) Biochemistry, 32: 1141-1151 Rautter, 1., Lendzian, F. & Lubitz, W. (1994) Biochemistry, 33:12077-12084 Rutherford, AW. & Nitschke, W. (1994) in "Origin and Evolution of Biological Energy Conservation"

(H. Baltscheffsky, ed.) VCH New York, in press Rutherford, A.W., Zimmermann, 1.L. & Boussac, A. (1992) in" The Photosystems: Structure,

Function and Molecular Biology", (Barber J., ed.), Elsevier. Amsterdam, 1992, chap. 5 Setif, P. (1992) in "The Photosystems: Structure, Function and Molecular Biology", (Barber 1., ed.),

Elsevier, Amsterdam, chap. 12 VenturoJi, G., Mallardi, A, & Mathis P. (1993) Biochemistry, 32: 13245-13253

PHOTO INHIBITION OF PHOTOSYNTHESIS

Neil R. Baker

Department of Biology, University of Essex, Colchester, C04 3 SQ u.K.

INTRODUCTION

Photoinhibition of photosynthesis is a widespread phenomenon in oxygenic photosynthetic organisms that can result in large decreases in photosynthetic productivity. In the widest context photoinhibition of photosynthesis can be defined as the light-induced decrease in CO2 assimilation, which would include the effects of photo-oxidative radical damage to many components of the photosynthetic apparatus that can occur in environmentally stressed organisms at high irradiances. However, quite frequently photoinhibition of photosynthesis is used to refer specifically to light-induced damage to the PSII reaction centre, which precedes the onset of the more severe radical-induced damage to other components of the photosynthetic apparatus. In the past decade it has become established that an essential, intrinsic feature of the photosynthetic apparatus is the light­induced decrease in the quantum efficiency of photosynthesis as irradiance increases; when leaves are exposed to increasing photon flux densities an increasing proportion of the absorbed energy is lost as heat, thereby reducing the quantum efficiency of the photosynthetic processes. Although this light-induced decrease of photosynthetic efficiency is not associated with damage to components of the photosynthetic apparatus and is, in "fact, a mechanism to protect from the damaging effects of high light, it does constitute a light­dependent depression of photosynthetic potential and warrants consideration in any treatise on photoinhibition of photosynthesis. In 1956 Kok defined photoinhibition as the light­dependent reduction in photosynthetic efficiency and this still perhaps provides the most useful working definition of photoinhibition of photosynthesis for leaves and whole organisms. In this chapter consideration will be given to the molecular basis of photoinhibition, . the physiological and environmental factors associated with the onset and recovery from photoinhibition and the consequences of photoinhibition to plant productivity.

LIGHT RESPONSE BEHAVIOUR OF PHOTOSYNTHESIS

The typical response of photosynthesis (C02 assimilation) to increasing irradiance is

Light as all Energy Source and Information Carrier in Plant Physiology Edited by Jennings et af.. Plenum Press. New York. 1996 89

shown in Figure 1 A. Initially photosynthesis increases linearly with irradiance, and it is in this region of the curve that the maximum quantum yield or efficiency of C02 assimilation (<pmax, given by the slope of the line) is expressed. At higher irradiances the relationship between absorbed light and photosynthesis becomes non-linear and consequently the quantum efficiency decreases. Eventually photosynthesis becomes light saturated and the rate of photosynthesis remains constant with increasing irradiance, although it should be

CI)

·iii

~ ! s::. a. '0 Q)

iii a:

I.

I I

I

I I

I I

I I

I

Absorbed Ilght not used lor phoIosynlheala

potential energy loss to leal

potenUal energy loss to leal

after photoinhibition

Absorbed PPFD

A

B

c

Figure 1. A: Response of the rate of photosynthesis (C02) assimilation to absorbed light (photosynthetically active photon flux density, PPFD). The maximum quantum efficiency of C02 assimilation (4)ma.,) is determined from the initial slope of the response curve. The shaded area above the light dosage response curve and the extrapolated linear portion of the curve (dashed line) indicates the amount of absorbed light that is not used for photosynthesis. B: The dashed line represents a theoretical (Blackman) response where photosynthesis operates at the maximum quantum efficiency until light saturation occurs and photosynthesis is limited by the dark reactions. The shaded area enclosed by the Blackman curve and the actual light dosage response curve of photosynthesis (solid line) represents the absorbed light energy that could potentially be used for photosynthesis, but is lost due to intrinsic characteristics and regulatory processes of the photosynthetic apparatus. C: The effect of severe photoinhibition on the light dosage response curve (lower solid line) is shown. The shaded area between the normal and photoinhibited curves represents the absorbed light energy lost to photosynthesis due to the photoinhibition of the system.

noted that the quantum efficiency of photosynthesis continues to decrease with increased photon absorption. In the region of the curve between <Pmax and the point oflight saturation, changes occur within the thylakoids that result in a smaller proportion of absorbed light being used for photochemistry due to an increase in the rate at which energy absorbed by the antennae of PSII is dissipated as heat. This light-induced quenching of excitation energy occurs in the light-harvesting antennae ofPSII and is associated with a decrease in the pH of the thylakbid lumen during formation of the thylakoid llpH and the consequent conversion of the carotenoid violaxanthin to zeaxanthin via the xanthophyll cycle (for a discussion of the mechanisms involved see articles by Horton and Briantais, this volume). This phenomenon has been demonstrated in a simple experiment where the <Pmax of a dark-adapted red

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campion It!af, determined at a PPFD of 34 !lmol m-2s- i , was found to be 0.05L However after the leaf was-exposed to a PPFD of 1500 !lmol m-2 S-i for 30 minutes, which was saturating for photosynthesis, and then transferred to back to 34 !lmol m-2 S-i, <Pmax had dropped to 0.044, a decrease of 14% (Genty et al., 1989). This light-induced decrease in <Pmax was fully reversible by 60 min dark treatment (Genty et af., 1989). Assuming that at light saturation photosynthesis is not limited by the rate of supply of ATP and reductants to the photosynthetic carbon reduction cycle, the potential loss of absorbed light energy from the leaf due to the intrinsic characteristics and regulatory processes of the photosynthetic apparatus, of which the light-induced increase in excitation energy dissipation as heat is a major component, is shown in Figure IB.

It is now widely accepted that this light-induced dissipation of excitation energy as heat is a protective mechanism by which excitation energy in excess of that required for photochemistry is prevented from reaching and damaging the reaction centres ofPSII. When P680, the reaction centre chlorophyll a ofPSII, exists in the oxidised state (P680+), primary charge separation is prevented and consequently a potentially damaging situation arises. Photodamage to PSI reaction centres is not considered to be major problem in this context since P700, the reaction centre chlorophyll a of PSI, will quench excitation energy efficiently in both the P700 and P700+ states (Nuijs et aI., 1986). If the light-induced quenching mechanism associated with the PSII antennae is unable to effectively dissipate excess excitation energy as heat, as might be the case if leaves are exposed to irradiances well above those normally experienced or if photosynthetic electron transport were inhibited by decreases in the rate at which ATP and reductants are consumed by metabolic processses, then overexcitation of the PSII reaction centres can occur and photodamage result. The consequences of severe photodamage to PSII for photosynthesis are shown in Figure 1 C; the maximum quantum efficiency will decline with prolonged exposure to stress conditions, as will the light-saturated rate of photosynthesis if the loss of active PSII complexes is sufficiently great to become limiting for C02 assimilation at the saturating light level. Consequently, photodamage to PSII can result in decreases in use of absorbed light energy for photosynthesis over the whole irradiance range. However, it should be emphasised that the leaves of many plants can experience severe environmental stress which markedly restrict CO2 assimilation, but yet do not exhibit photodamage to PSII reaction centres because they have the capacity to quench very large excesses of excitation energy via zeaxanthin in a process that is fully reversible when leaves are placed in the dark (Demmig-Adams & Adams, 1992). A particularly good example of this is seen during dehydration of French bean and sunflower leaves, where PSII antenna quenching increased dramatically with suppression of CO2 assimilation, but was independent of whether photorespiration, which acts as an additional sink for reductants, was operative (Comic, 1994). It should be noted that the onset of such stress-induced, light-dependent quenching of excitation energy can result in decreases in photosynthetic efficiency over a wide irradiance range which will be indistinguishable, in the context of photosynthesis, from any decreases in efficiency resulting from photodamage to PSII reaction centres.

PHOTODAMAGE TO PSII REACTION CENTRES

A schematic model for the PSII complex is shown in Figure 2 (see also Bassi, this volume). The multi subunit complex consists of at least twenty proteins, ranging in size from 4 to 50 kDa, that are encoded by genes located on both the chloroplast and nuclear genomes (for review see Vermaas, 1993). The largest PSII proteins are the chlorophyll-binding primary antenna proteins CP47 and CP43 (ca. 47 and 43 kDa), a 33 kDa manganese stabilising protein and another two chlorophyll-binding proteins of 32 and 34 kDa, known as D 1 and D2. Cytochrome b559 is also a component of PSII preparations. The D 1 and D2 proteins bind a chlorophyll a molecule which when excited will transfer an electron to the

91

stroma

Figure 2. Schematic model for photosystem II. D 1 and D2 are the core proteins of the reaction centre that bind P680, QA, QB and two pheophytin molecules (pheo). Tyrz is a tyrosine residue on the D I protein that transfers electrons from the manganese-containing complex (4 Mn) to P680. CP43 and CP47 are antennae pigment complexes binding chlorophyll a molecules. Cyt bS59 is cytochrome bSS9• Proteins labelled 17, 23 and 33 (designating molecular mass in kDa) are extrinsic membrane proteins and are involved· in water oxidation.

adjacent pheophytin (see Mathis, this volume). This special chlorophyll is called P680 after its absorption maximum at 680 nm. The DIID2 heterodimer also binds pheophytin, which is the immediate, metastable electron acceptor from P680, and the two primary, stable quinone electron acceptors, QA and QB. The immediate electron donor to P680 is a tyrosine residue, Tyrz, of the Dl protein.

On transfer of excitation energy from antennae chlorophylls to P680 (see Jennings et aI., this volume), primary charge separation occurs as the excited P680 transfers an electron to pheophytin. The electron is then transferred from pheophytin to the primary quinone acceptor, QA> and then to the secondary quinone acceptor, QB· The semiquinone, QB-' is then further reduced by a second turnover of the reaction centre, producing the doubly reduced QB2-, which becomes protonated to plastoquinol and dissociates from the reaction centre. Oxidised P680 (P680+) has an extremely high oxidising potential and will oxidise the neighbouring Tyr z residue on the D 1 protein, which will then be reduced by electron transfer from water via the Mn cluster.

Photoinactivation of the PSII reaction centre can occur by two independent mechanisms, associated with the acceptor and donor sides of PSII respectively, that both result in inhibition of electron transfer through PSII and subsequent degradation of the D 1 protein (Styring & Jegerschbld, 1994; Telfer & Barber, 1994). Acceptor side inhibition will occur under high light conditions when the plastoquinone pool is fully reduced, and consequently there is a lack of oxidised plastoquinone to bind to the QB site on the D 1 protein. In this state QA will become doubly reduced on a second turnover of the reaction centre to form QA2-, then becomes protonated to form QAH2 and is released from the QA­binding site on the D 1 protein. With the QA site vacated, excitation of P680 will result in the formation of the radical pair, P680+Pheophytin-. Recombination of these radicals will result in the formation of the triplet state of P680, which will react with oxygen to form singlet oxygen.' Singlet oxygen is potentially damaging to protein, and is thought to react with the Dl protein thus triggering the degradation of the Dl (Aro et ai. , 1993; Styring & Jegerschbld, 1994; Telfer & Barber, 1994). Acceptor side photoinactivation would be expected to occur in leaves when the rate of consumption of the products of electron transport is decreased, as would occur when C02 assimilation is restricted, and the processes which quench excitation within the PSII antennae do not have the capacity to dissipate the excess excitation.

Donor side photoinhibition ofPSII will occur when water oxidation is inhibited and the

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highly reactive P68o+ and Tyrz+ are formed. P680+ will oxidise neighbouring molecules. Oxidation of accessory chlorophylls and /3-carotene and degradation of D 1 have been found to occur under conditions favouring P68o+ formation (Telfer & Barber, 1994). Loss of donor side electron transport and consequent photodamage to the D 1 protein has been linked to the generation of a large H+ electrochemical potential gradient across the thylakoid and the possible associated release of Ca2+ from the water oxidising complex (Ohad et al., 1994). Although donor side photoinhibition has been frequently observed in in vitro experiments, it is has not yet been demonstrated to be a feature of photoinhibition in leaves operating under physiological conditions.

D1 DEGRADATION AND REPAIR

Degradation of the D 1 protein is generally a consequence of photoinactivation of PSIl. It has been suggested that the highly oxidising chemical species generated at the PSIl reaction centre under photoinhibitory conditions could be responsible for cleavage of peptide bonds in the Dl protein (Andersson et al., 1994). However, Dl degradation is now generally considered to result from protease activity primarily because (i) the inactivation of PSII electron transport occurs much more rapidly than Dl degradation (Ohad et aI., 1985; Virgin et al., 1988), (ii) D 1 degradation can be prevented in thylakoids in which photoinactivation of PSII has occurred at low temperature (Aro et aI., 1990), (iii) Dl degradation can occur in the dark at 20-25°C in thylakoids which have had PSII photoinactivated at low temperature (Andersson et al., 1994), and (iv) inhibitors of serine­type prot eases reduce the rate of D 1 degradation during photoinhibitory treatment of PSII preparations (Andersson et al., 1994). At present the protease(s) responsible for Dl degradation has not been identified, but it is thought to associated with the PSII core complex (Andersson el al., 1994). The mechanism by which photodamaged Dl is recognised from native Dl by the proteolytic enzyme is not yet known.

It has been suggested that photoinactivation and recovery of PSII can occur in the absence of Dl degradation and resynthesis since loss of PSII activity has been found to occur in a number of systems in the absence of D 1 degradation or when D 1 synthesis is inhibited (Long et al., 1994). However, it is not evident that the photoinactivation of PSII resulting from the generation of singlet O2 from triplet P680 or the accumulation ofP680+, as discussed above, has actually occurred in these cases, and the decreases in PSII photochemical activity may well be attributable to other phenomena, such as increased quenching of excitation energy by antenna pigments.

The D 1 protein is encoded in the chloroplast genome by a gene designated psbA, and its synthesis in the chloroplast is regulated by nuclear-encoded factors that are synthesised in the cytoplasm and imported into the chloroplast (Rochaix, 1992). Photodamage to Dl is thought to occur in the granal regions of the thylakoids, where the bulk of the PSII is located. However, synthesis of the D 1 precursor protein is primarily associated with polyribosomes associated with stromal (non-appressed) thylakoids (Ohad et al., 1994). It has been speculated that photoinactivated PSII complexes migrate from appressed to non­appressed membrane regions, where insertion of newly synthesised D 1 into partially assembled PSIt complexes will occur, resulting in the repair of the damaged PSII complex; repaired PSII complexes then migrate back to appressed membrane regions (Adir et al., 1990). To date little is known of the mechanisms associated with the repair of photodamaged PSII complexes, however phosphorylation (Aro et al., 1993), and possibly acylation (Mattoo & Edelman, 1987), of Dl may well be involved in the process of Dl turnover and repair of photo inactivated PSII complexes (Ohad et al., 1994).

An important, and often overlooked, fact when considering photoinhibition is that in non-stressed leaves the D 1 protein is the most abundantly synthesised thylakoid protein and is continually being turned over in the absence of any detectable inefficiency in PSI!

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photochemistry. This has been clearly shown for maize leaves grown in a controlled environment cabinet at 25°C under a moderate PPFD of 230 IlmOI m-2 S-I and in the field (Nie & Baker, 1991; Stirling et at., 1991). Consequently, it can be argued that the process of PSII-driven water photooxidation in leaves, operating under normal physiological conditions, must inevitably carry the possibility of generating highly oxidising radicals at the reaction centre which will be damaging for the 01 protein. Under ambient growth conditions the rate of photodamage to the D 1 population must be matched by the rate of repair of the photo damaged PSII complexes in order to maintain a constant, photochemically competent PSII population. However, if the rate of damage exceeds the capacity for repair, as might occur when severe environmental stresses are imposed under high light conditions, or the synthesis of Dl is inhibited by low temperature, then the number of photochemically competent PSII complexes will decrease with a consequent loss of photosynthetic competence. It would appear that such a situation may be found only rarely in leaves in the natural environment (see below), and is much more prevalent in contrived controlled laboratory experiments in which leaves are exposed to much more severe stress conditions than would normally be experienced during growth.

PHOTOINHIBITION IN THE NATURAL ENVIRONMENT

A considerable body of information demonstrates that light-induced inhibition of photosynthesis occurs regularly in the field (Long et aI., 1994). Perhaps the most convincing demonstrations of photoinhibition in the field have been made from comparisons of photosynthetic performance in exposed and shaded leaves. Leaves of maize (Farage & Long, 1987) and oil-seed rape (Farage & Long, 1991) canopies exhibited significant decreases in the maximum quantum efficiency of photosynthesis when experiencing low temperatures and high PPFDs in the mornings, which were not observed in leaves of the crop experiencing similar temperatures but that had been artificially shaded. Similarly, exposed leaves of holly (Groom et at., 1991) and snow gum (Ball et at., 1991) trees exhibited lower maximum photosynthetic quantum efficiencies than leaves that were shaded either by other leaves or artificially during the winter. Shaded leaves of a water stressed sorghum canopy were also found to exhibit significantly greater maximum photosynthetic quantum efficiencies than exposed leaves (Ludlow & Powles, 1988). Many examples can be found in the literature of light-induced decreases in the ratio of variable to maximal fluorescence (FvlFm) of leaves exposed to stress conditions that indicate the occurrence of a photoinhibition of the maximum quantum efficiency ofPSII photochemistry (Long et at., 1994). There is no doubt that photoinhibition of photosynthesis occurs regularly in many field situations. However it is not yet clear what type of photoinhibition is occurring. Is it photoinactivation and photodamage to PSII reaction centres or light-induced increases in antennae quenching?

This question has proved to be difficult to resolve, primarily due to the practical difficulties of studying the detailed characteristics of photoinhibition under field conditions. A number offield studies have reported decreases in Fv/Fm ofleaves for range of species in different habitats with increasing PPFD during the morning, followed by increases in FvlFm as PPFD decreased in the afternoon (Long et aI., 1994). In a study of leaves of a winter wheat crop in south-east England FvlFm was found to decrease with increasing PPFD during the morning on days when the temperature was low and PPFD was high, but not on days with higher temperatures and lower PPFDs (Groom & Baker, 1992). The FvlFm values were maximally depressed around midday and then increased throughout the afternoon as the PPFD decreased, despite the fact that temperature was often decreasing in the afternoons. The possibility that these light-dependent decreases in FvlFm that occur during the morning were the result of photodamage to PSII reaction centres was examined by isolating thylakoids from leaves and determining their ability to bind 3-(3,4-dichorophenyl)-1,I-dimethylurea (DCMU). DCMU binds specifically to the QB binding site on the Dl

94

protein, consequently decreases in the ability of DCMU to bind to the thylakoids indicate damage to the Dl protein. No significant changes in the capacity of the thylakoids to bind DCMU were found throughout the mornings when FvIFm was decreasing, indicating that the light-induced depressions in FvlFm at low temperatures experienced during the morning cannot be due to photodamage to PSII reaction centres (Groom & Baker, 1992). Other circumstantial evidence supports this conclusion. The rapid recovery from the morning depressions in FvlFm during the afternoon as PPFD decreases, but when temperatures are often falling, suggests that a relaxation of light-dependent quenching of excitation energy in the antennae is occurring rather than a repair of damaged PSII reaction centres. The degradation and removal of damaged D 1 protein and synthesis of new D 1 protein is severely impaired by temperature reduction (Aro et al., 1991; Bredenkamp & Baker, 1994). Although these data suggest that photo inhibition of photosynthesis induced in the winter wheat crop by low temperatures is not attributable to photodamage of D 1, it cannot yet be assumed that this is not the mechanistic basis of photoinhibition in the field for other species, particularly for those growing close to their geographical limits. Recent field studies of a maize crop during late spring in south-east England have shown that during periods :when FvlFm of the leaves was severely depressed (below 0.5), very rapid synthesis ofDl protein was observed (Fryer, Andrews & Baker, unpublished data), even at temperatures that have been shown in the laboratory to severely inhibit D 1 protein synthesis in maize chloroplasts (Bredenkamp & Baker, 1994). This may indicate that under field chilling conditions maize leaves may experience increased degradation, and possibly a net loss, of D 1 protein. This question warrants further study.

It is now widely accepted that photoinhibition of PSII is associated with decreases in C02 assimilation in the field. However, it is difficult to assess what the consequences of such photoinhibition are for the overall photosynthetic productivity of a plant or canopy in the natural environment. The predominant effect of photo inhibition is to reduce photosynthetic efficiency at limiting light levels, although reductions in the light saturated rate of photosynthesis are also prevalent when severe damage to the PSII population occurs. The linear relationship between the absorption of light and the production of dry matter that has been observed for a number of crops indicates that crop photosynthetic productivity is normally limited by light, and supports the argument that most of crop photosynthesis is performed at light levels below those required to saturate photosynthesis (Baker & Ort, 1992). Consequently even moderate levels of photoinhibition, when there are decreases in the maximum quantum efficiency of photosynthesis, but not in the light saturated rate of photosynthesis, would be expected to significantly influence canopy carbon gain. Studies on a willow canopy have shown that mild photoinhibition occurs regularly on clear days even when temperature and water status are optimal for photosynthesis and, using simulation modelling, it was estimated that approximately 10% of the potential carbon gain would be lost by this photoinhibition on clear days (Ogren & Sjostrom, 1990). Modelling of the effects of diurnal changes in photoinhibition in leaves of a crop canopy in southern England on the carbon gain have predicted a 9% loss in C02 uptake by the canopy (Long et aI., 1994). This model assumed a 50% decline in the maximum quantum efficiency of C02 assimilation (<I>max) ofleaves exposed to full sunlight by mid-afternoon with a recovery to approximately 10% of the control value by dusk, and predicted that the major loss in potential carbon gain would occur during the early afternoon.

The relationship between photoinhibition and dry matter production in the field is difficult to demonstrate empirically. However, studies of an oil':seed rape canopy in south­east England during the winter have implicated photoinhibition as a major factor in the depression of dry matter accumulation (Farage & Long, 1991). During the autumn period (from October 1 until December 1), dry matter production increased linearly with canopy light interception; the gradient of this relationship giving the efficiency of the crop canopy at converting incident light energy to dry matter (conversion efficiency), which was determined as 1.81 g MJ·\. This value for the conversion efficiency is relatively high with respect to the

95

potential maximum of2.51 g MJ-l for a C3 crop. In late December, the relationship between dry matter-production and intercepted radiation changed. Although a linear relationship was still maintained between these parameters, the conversion efficiency had decreased to 0.46 g MJ-I and did not begin to increase until late March. The light conversion efficiency of crop stands has generally been assumed to be constant (Monteith, 1981), although the conversion efficiency will decrease as plants enter into a reproductive state (Marshall & Wiley, 1983). However, the development of reproductive organs could not be responsible for the decline in the conversion efficiency observed for the oil-seed rape canopy in late December. The changes in conversion efficiency for the crop canopy observed throughout the period from October to April were closely correlated with changes in 4>max of individual leaves (Farage & Long, 1991). Since the conversion efficiency and 4>max are derived independently, it can be argued that 4>max of individual leaves within the canopy is a major determinant of the productivity of the canopy. Also, it is evident from shading experiments that depressions in 4>max of the leaves during the winter could in part be attributable to chill-induced photoinhibition (Farage & Long, 1991). Consequently, it can be concluded that photoinhibition was an important factor in determining the dry matter production of this rape crop. A similar analysis of a maize crop in south-east England during the early summer when chilling-dependent photoinhibition was prevalent also showed a strong linear dependence of the conversion efficiency on 4>max (Long et al. 1992).

REFERENCES

Adir. N., Schochat, S. & Ohad, I. (1990) J. BioI. Chern,. 265: 12563-12568. Anderrson, B., Ponticos, M., Barber, J., Koivuniemi, A, Aro, E-M, Hagman, A., Salter, AH., Dan­

Hui, Y. & Lindahl, M. (1994) in "Photoinhibition of Photosynthesis: from Molecular Mechanisms to the Field", (Baker, N.R & Bowyer, J.R eds), pp. 143-159, Bios Scientific Publishers, Oxford.

Aro, E-M., Hundal, T., Carlberg. I. & Andersson, B. (1990) Biochim. Biophys. Acta, 1019: 269-275. Aro, E-M., Virgin, I. & Andersson, B. (1993) Biochim. Biophys. Acta, 1143: 113-134. Baker, N.R & art, D.R (1992). in "Crop Photosynthesis: Spatial and Temporal Detenninants",

(Baker, N.R & Thomas, H. eds.), pp. 289-312, Elsevier Science Publishers B.V., Amsterdam. Ball, M.C., Hodges, V.S. & Laughlin, G.P. (1991) Funct. Ecol., 5: 663-668. Bredenkamp, GJ. & Baker, N.R (1994) Plant Cell Environ., 17: 205-210. Comic, G. (1994) in "Photoinhibition of Photosynthesis: from Molecular Mechanisms to the Field",

(Baker, N.R & Bowyer, J.R eds.), pp. 297-313, Bios Scientific Publishers, Oxford. Demmig-Adams, B. & Adams, W.W. (1992) Annu. Rev. Plant Physiol. Plant Mol. Bioi,. 43,599-626. Farage, P.K. & Long, S.P. (1987) in "Progress in Photosynthesis", Vol. IV, (Biggins, J. ed.), pp. 139-

142, Martinus NijhoffPublishers, Dordrecht. Farage, P.K. & Long, S.P. (1991) Planta, 185: 279-286. Genty, B., Briantais, J-M. & Baker, N.R (1989) Biochim. Biophys. Acta. 990, 87-92. Groom, Q.J. & Baker, N.R (1992) PlantPhysiol., 100: 1217-1223. Groom, Q.J., Baker, N.R & Long, S.P. (1991) Physiol. Plant.. 83: 585-590. Kok, B. (1956) Biochim. Biophys. Acta, 21, 234-244. Long, S.P., Farage, P.K.. Aguilera. C. & Macharia. I.M.N. (1992) in "Trends in Photosynthesis

Research", (Barber, J., Guerro, M.G. & Medrano, H. eds.) pp.345-356, Intercept, Andover. Long, S.P., Humphries, S. & Falkowski, P.G. (1994) Annu. Rev. Plant Physiol. Plant Mol. Bioi,. 45:

633-662. Ludlow, M.M. & Powles, S.B. (1988) Aust. 1. Plant Physiol. 15: 178-194. Marshall, B. & Wiley, RW. (1983) Field Crops Res. 7: 141-160. Mattoo, AK. & Edelman, M. (1987) Proc. Natl. Acad. Sci. USA 84: 1497-1501 Monteith, J.L. (1981) in "Physiological Processes Limiting Plant Productivity" (Johnson, C., ed.) pp.

23-38, Butterworths, London. Nie, G-y' & Baker, N.R (1991) Plant Physiol,. 95: 184-191. Nuijs, A.M .. Shuvalov, V.A., van Gorkom, H.1., Plijter, J.J. & Duysens, L.N.M. (1986) Biochim.

Biophys. Acta, 850: 310-318. Ogren, E. & Sjostrom, M. (1990) Planta, 181: 560-567. Ohad, I., Kyle, D.J. & Hirschberg, 1. (1985) EMBO 1.,4: 1655-1659. Ohad, 1., Keren, N., Zer, H., Gong, H., Mor, T.S., Gal. A, Tal, S. & Domovich, Y. (1994). in

"Photoinhibition of Photosynthesis: from Molecular Mechanisms to the Field" (Baker, N.R &

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Bowyer. I.R eds.), pp. 161-177, Bios Scientific Publishers, Oxford. Rochaix, 1-0. (1992). in "Plant Gene Research: Cell Organelles", (Hermlann, RG., ed.), pp. 249-274,

Springer-Verlag, Vienna. Stirling, C.M. , Nie, G-Y., Aguilera, C., Nugawela, A., Long, S.P. & Baker, N.R (1991) Plant Cell

Environ,. 14: 947-954. Styring, S. & legerscMld, C. (1994) in "Photoinhibition of Photosynthesis: from Molecular

Mechanisms to the Field", (Baker, N.R. & Bowyer. 1.R eds.), pp. 51-73, Bios Scientific Publishers, Oxford.

Telfer, A. & Barber, 1. (1994) in "Photoinhibition of Photosynthesis: from Molecular Mechanisms to the Field", (Baker, N.R & Bowyer, 1.R eds.), pp. 25-49, Bios Scientific Publishers, Oxford.

Vermaas, W. (1993) Annu. Rev. Plant Physiol. Plant Mol. BioI.. 44,457-481. Virgin, I., Styring, S. & Andersson, B. (1988) FEBS Lett, 233: 408-412.

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NONPHOTOCHEMICAL QUENCHING OF CHLOROPHYLL FLUORESCENCE

Peter Horton

Robert Hill Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, PO Box 594, Western Bank, Sheffield S 10 2UH, UK.

INTRODUCTION

Measurement of chlorophyll fluorescence is an invaluable method for examining the interaction of photosynthetic organisms with their environment and for the non-invasive probing of the metabolism of the chloroplast (Krause & Weis, 1991; Horton & Bowyer, 1990). Fluorescence, most of which is emitted from PSII chlorophylls at room temperature, can be quenched from its maximum value, F m, as found ih a healthy dark adapted sample, either photochemically or nonphotochemically. Whereas photochemical quenching occurs due to the process of energy consumption in photosynthetic electron transport, the quenching of fluorescence nonphotochemically in general terms reflects a change in "state" of the photosynthetic apparatus - thus, its measurement has provided evidence of the presence of, on the one hand, regulatory mechanisms, and on the other, inhibition and damage to the thylakoid membrane under stress conditions. Nonphotochemical quenching is measured most often as the decrease in the Fm value to some new value Fm', and this can be recorded in any given steady state using modulated fluorimetry, in the laboratory or in the field, by application of a brief saturating light pulse to close PSII centres (Quick & Horton, 1984; Schreiber et aI., 1986). If the change in Fm is normalised on Fm', the value of qN then approaches direct proportionality to the rate of energy dissipation (Demmig-Adams, 1990; Bilger & Bjorkman, 1990); however, this is only strictly correct if the quenching process is of the Stern-Volmer type. Alternatively, qN can be calculated by normalising the change in Fm on the Fv', and in this case has a range of 0 to 1 (Schreiber et al., 1986; van Kooten & Snel, 1990); the disadvantage of this calculation is that Fo' needs to be measured to determine Fv', something quite difficult in the field, and furthermore, the qN value becomes very insensitive to increases in energy dissipation rate at high values. For example, a difference between. qN of 0.90 and 0.94 calculated in this way may indicate a 30% increase in energy dissipation. In some sections of the literature the terms NPQ, SVN and KD have been used to describe nonphotochemical quenching calculated by dividing by Fm', reserving

Light as an Energy Source and Information Carrier in Plant Physiology Edited by J<!1mings et al., Plenum Press, New York, 1996 99

the term qN only for normalising on Fv'. Since in all cases it is the same biological processes that are being measured, this distinction is both arbitrary and unnecessary.

Nonphotochemical quenching arises due to three processes which have been called qE, qT and qI (table 1, see also Briantais, this volume). These can be distinguished by their kinetics of relaxation in a darkened sample and by their responses to various inhibitors (Demmig & Winter, 1988; Horton & Hague, 1988; Quick & Stitt, 1989; Walters & Horton, 1991). Quenching due to state transitions, qT was the first to be characterised, and although many details remain to be understood it is widely accepted that it arises due to phosphorylation of the peripheral antenna of PSII, the light harvesting complex II (LHCn) (Allen, 1992). It also is believed that this process is responsible for adjusting the relative absorption cross sections of PSII and PSI necessary to maximise quantum efficiency, although examination of whole leaf photosynthesis has failed to provide any evidence of such a role (Andrews et aI., 1993). Nevertheless, as expected, qT saturates at very low light intensities and is only a significant component of qN in strictly light-limiting conditions (Horton & Hague, 1988; Walters & Horton, 1991). It has been further suggested that the state transitions enable the rates of linear and PSI cyclic electron transport to be adjusted, since an increased cellular demand for ATP has been correlated with increased LHcn phosphorylation (Fernyhough et aI., 1983). Similarly, a more effective state transition has been observed at elevated temperature where other membrane reorganisations occur (Timmerhaus & Weis, 1990), and could also be explained by a higher ATP demand under stress conditions. However, in both cases a significant photosynthetic role of the redistribution of energy has not been directly demonstrated. Therefore, there remains an attractive alternative explanation that thylakoid protein phosphorylation and the state transition are only the first steps in the signalling of changes in composition of the thylakoid membrane, rather than being involved in the short-term regulation of photosynthesis (Allen, 1992).

The other forms of qN are induced whenever the intensity of absorbed light is in excess of that which can be used with maximum quantum yield by the photosynthetic electron transport chain. In particular, qE, the rapidly relaxing form, increases in a manner which closely matches the decline in the quantum yield as the light intensity increases, and it should be considered as a mechanism that gets rid of at least a part of the excess excitation energy absorbed by the PSII anterina. The effects of qE on PSII efficiency are clearly revealed by the maintenance of high qP, despite a reduced quantum yield of electron transport (Weis & Berry, 1987; Genty et aI., 1989). Equally, it is important to realise that qE does not result in the de-excitation of all the excess energy, since qP does eventually fall when the light intensity becomes increasingly saturating for photosynthesis. Protection from photoinhibition of PSII is provided by qE since an excessive rate of excitation of the reaction centre is the cause of its inactivation; it has been estimated that the onset of photoinhibition occurs when qP falls below 0.6 (0quist et aI., 1992), showing that the capacity of qE to provide photoprotection can be exceeded. The importance of qE should be viewed in two ways: firstly, at around the growth light intensity, almost complete protection from photoinhibition results, since, without qE, significant photoinhibition would arise when the inflexion point on the irradiance curve is exceeded; secondly, once qE is saturated, its protective role is one of reducing the quantum yield for photoinhibition (Horton et a1., 1989), and therefore, ·the saturating value of qE, which varies by a factor of 4 (expressed as a AFmlFm') between species lmd growth conditions (Johnson et aI., 1993, Demmig-Adams & Adams, 1992) governs the rate of photo inhibition during periods of light saturation of photosynthesis.

The third component of qN, qI, becomes significant only under conditions of light saturation; therefore it monitors photoinhibition. However, there is a great deal of uncertainty about the origin and physiology of qI (Krause, 1988). Quenching was formerly associated with a photo-damaged reaction centre; for example, it has been shown that treatment of thylakoid membrane in vitro results in quenching that correlates with the

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Table 1. Properties of different types of nonphotochemical quenching. General features of quenching were derived from numerous studies on isolated chloroplasts and leaves. Items in brackets 0 refer to indirect effects of ascorbate and DTT through there effects of the xanthophyll cycle, and fluoride through its inhibition of protein phosphatase. Interactions between quenching types have been ignored (e.g. inhibition of qE stimulates qI; DTT inhibits zeaxanthin-related qI but may stimulate qI arising from PSII damage).

qE qT qI

Cause ilpH LHCII phos numerous zeaxanthin

dark stable ilpH LHCII conformation

PSII inactivation

Relaxation 5-10 s 5-10 min 30 min-hours [usually] [days]

Inhibitors uncouplers none (DIT) antimycin A photosynthesis

DCCD uncouplers (DIT)

Stimulators Mg (Fluoride) photosynthesis inhibitors dibucaine stress

zeaxanthin (ascorbate)

Mechanism nonradiative LHCII nonradiative (proposed) decay (chi or zea) detachment decay

PSII charge transfer to PSI PSII charge recombination recombination

P680 or chi oxidation

irreversible inactivation of PSII that precedes its turnover (Cleland et aI., 1986). However, qI is found in vivo under high light conditions when there is no evidence for loss of PSII reaction centres (Baker et aI., 1994); this qI commonly reverses in less than 1-2 hours, and can overlap with relaxation of qT. It has been proposed therefore that the qI formed under most conditions (i.e. excluding severe stress) results from a kind of protective photoinhibition, or a sustained down-regulation of PSII (Oquist et aI., 1992). Because of its slow relaxation kinetics, it can have an important negative effect on photosynthesis during a diurnal cycle (Baker et aI., 1994; Ogren, 1994). As will be discussed further below, this kind of qI may merely be a more stable form of qE. These complexities mean that extreme care has to be taken when analysing and interpreting the kinetics of relaxation of qN in vivo.

PROPERTIES OF ENERGY DEPENDENT QUENCHING, qE

Relationship between qE and ApH

The formation of qE is obligatorily dependent on the presence of a proton gradient across the thylakoid membrane (Briantais et aI., 1979). Useful information about qE has

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been obtained by simultaneous measurement of ApH (using methods such as the quenching of the fluorescence probe 9-aminoacridine) and qE in isolated chloroplasts (Noctor et al., 1991). For example, it was shown that the rates of formation and relaxation ofqE are both slower than those for the ApH, suggesting that a "conformational" change in the thylakoid membrane is involved in quenching (figure 1). The kinetic relationship between qE and ApH is variable: in light activated chloroplasts (see below) qE relaxes with a half-time of 2 min compared to 15 sec for ApH; upon lowering the temperature, qE can persist for several minutes after relaxation of ApH. In fact, in leaves, it seems that either strong illumination or persistent illumination results in the formation of a sustained form of qE that is uncoupled from the bulk phase ApH and so resembles qI (Walters & Horton, 1991; Gilmore & Bjorkman, 1994). The notion of a "conformational" basis for qE is in agreement with the conclusion made by Krause (1973) following observation of the 535 nm light scattering change that also accompanies qE (figure 2). This absorbance change was thought to be a direct result of the effects of ApH formation on macroscopic aspects of thylakoid structure, such as membrane thickness, but it has been possible to inhibit AA535 (with antimycin A) without affecting ApH (Noctor et aI., 1993); in fact in both leaves and isolated chloroplasts, AA535 correlates with qE (figure 2). Therefore, AA535 seems to be an indicator of a change in conformation of a membrane component that is an essential part of qE (Ruban et aI., 1993a; Bilger & Bjorkman, 1994); at present its exact origin is unknown.

Secondly, it has been possible to record steady-state titrations of qE vs estimated lumen acidity, with an apparent pK of approx 4.5 being obtained. Further, it was shown that the pK value can be shifted by alteration in experimental conditions: mild heating and dibucaine cause qE to be formed at a much reduced pH gradient (Krause et aI., 1988); antimycin A greatly increases the pH requirement so that qE is almost completely inhibited because a saturated ApH can not be achieved (Noctor & Horton, 1990). These observations show that a component obligatorily involved in qE behaves in a manner comparable to an enzyme whose activity is regulated through change in "Km". Hence, the general characteristics of qE and its relationship to ApH allow a mechanistic model of the process to be formulated.

Other features of qE

The other important features of qE include the fact that it quenches both the Fo and Fm levels of fluorescence in a manner that quantitatively resembles that caused by addition of chemical quenching agents such as dinitrobenzene to thylakoids (Rees et aI., 1990); this behaviour shows qE to be caused by an almost perfect Stern-Volmer quencher, although a more detailed mathematical analysis shows this not to be a completely accurate description (Walters & Horton, 1993). Quenching giving rise to qE can still be observed in samples frozen to 77 K in the quenched state, a fact which of course complicates the use of this method to estimate state transitions. A detailed analysis shows that qE preferentially quenches the longer wavelength emitters of PSII, and a quenching spectrum has a peak near 700 nm (in contrast qP quenches shorter wavelength emitters of the PSII core) (Ruban & Horton, 1994). As referred to already, qE is associated with absorbance changes, principally near 535 nm; this change has been shown to be well correlated to qE both in leaves and in isolated chloroplast, but can be separated from ApH (antimycin blocks qE and AA535 but not ApR, whereas dibucaine reduces ApH but does not effect qE or AA535). Other absorbance changes in the Soret and Qy band of chlorophyll have also been reported to accompany qE formation (Ruban et aI., 1992a; Horton & Ruban, 1994).

Sites ofqE

Quenching could occur either in the PSII antenna, or in the reaction centre, or both (Horton & Ruban, 1992, Horton et al., 1994). The facts that PSII is a shallow trap and that

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chlorophyll fluorescence measuring beam

actinic light

DCMU

Figure 1. Comparison of the kinetics of the formation and relaxation of qE and the 6pH as measured by 9-aminoacridine fluorescence. Chloroplasts were isolated from dark-adapted (left) or light treated (right) spinach leaves and given actinic light to induce qE and DCMU to induce collapse of 6pH and relaxation of qE. Redrawn from Noctor et aI., 1991.

"r-----O o

.F • olP' o

tt# " r 0

Chloropidl!il

" 0 o o. 011 o.

S I •• <1. 0'

o "'~ oo~

.. •

o I Z l 4!t 0 I t 1

A~3~ • 1000 . .

Figure 2. Correlation between the 535 nm absorption change and the extent of qE. For chloroplasts, the level of qE was controlled by titration with antimycin A at constant 6pH for light treated (open circles) and dark-adapted (closed circles) chloroplasts. For leaves, data is taken at different time points during induction of quenching in high light grown plants (open circles) or shade grown (closed circles). Data taken from Noctor et al. (1993) and Ruban et aI., 1993a.

excitation energy equilibrates between the antenna and reaction centre (see Jennings et aI. this volume) makes it difficult to establish the site of quenching from steady-state fluorescence methods, since a quencher located anywhere in PSII would have the same effect (for example on Fo). However, for quenching in the reaction centre, models have been proposed that involve specific involvement of the components of charge separation itself which would confer a unique signature on qE. In these models it is proposed that the low pH inactivates the electron donor side ofPSII by causing release ofCa2+ (Kreiger & Weis, 1993), increasing the lifetime of P680+ which results either in a direct quenching or in a nonradiative charge recombination with QA- (Kreiger et ai., 1992. This process can be observed quite readily in isolated thylakoids and in PSII BBY particles (Crofts et aI., 1991; Rees et ai., 1992; Kreiger et ai., 1992), but the features of quenching seem to be different in

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important respects from qE: ascorbate is an inhibitor of donor side related quenching since it provides electron to reduce P680+, whereas ascorbate often stimulates qE; this quenching does not lower F 0 and does not quench fluorescence at 77 K, both of which arise with qE. Moreover, charge recombination as mechanism for qE has been excluded following direct measurement of the kinetics of heat emission (Mullineaux, 1994). Therefore, it has been concluded that this mechanism does not explain qE; however, it is possible that donor-side inhibition could account for quenching under some circumstances, when the antenna is modified or under more severe stress, and may even be involved in the photoinhibition­related qI.

The alternative site of qE is in the PSII antenna; some of the evidence in favour of this has already been outlined above. Of course, the PSII antenna consists of least eight different chlorophyll binding proteins (see Bassi et aI., this volume), and the question is whether qE occurs specifically to anyone of these. In part the answer to this question comes from the fact that qE is associated with the xanthophyll cycle carotenoids, pigments found only in the peripheral antenna complexes, LHCII and not in the core proteins CP47 and CP43 (Peter & Thornber, 1991; Bassi et aI., 1993; Ruban et aI., 1994a; Lee & Thornber, 1995). However, LHCII itself consists of six gene products and 4 different pigment protein' complexes (Jansson, 1994); the major complex LHCIIb binds 60% of the PSII chlorophyll where the minor complexes, LHCIIa, c and d together comprise about 20%

THE XANTHOPHYLL CYCLE

Distribution of xanthophyll cycle carotenoids in the thylakoid membrane

The xanthophyll cycle describes the reversible de-epoxidation of violaxanthin to zeaxanthin via the intermediate antheraxanthin. In dark-adapted leaves most of the xanthophyll cycle pool exists as violaxanthin, but upon exposure of leaves to light conditions that start to saturate photosynthesis, zeaxanthin accumulates; this conversion is considered to result from the activation of the violaxanthin epoxidase by the acidification of the thylakoid lumen associated with formation of the LlpH. Methods which allow separation of the pigment-protein complexes of the thylakoid membrane under non-denaturing conditions have shown that the xanthophyll cycle carotenoids are bound to the LHC both of PSII and PSI (Thayer & Bjorkman, 1992; Lee & Thornber, 1995). A number of studies have shown that the xanthophyll cycle carotenoids are enriched in the three minor LHCII complexes of PSII but there is some uncertainty over the amount of these carotenoids bound to the bulk LHCIIb, values ranging from near 50% to less than 5% (Bassi et aI., 1993; Ruban et aI., 1994a; Lee & Thornber, 1995). In contrast, the content of lutein is constant at 2 per LHCIIb monomer, strongly suggesting that these are the carotenoids seen within the complex in the structural model of LHCII derived from electron crystallography (Kiihlbrandt et aI., 1994); most likely the xanthophyll cycle carotenoids are bound to the periphery of this complex, explaining the variability in the data for different preparations. The high content of xanthophyll cycle carotenoids in the minor LHCII means that they could replace one of the two luteins assumed to be present in these complexes. The availability of the violaxanthin for de-epoxidation is variable across the LHCII components, with LHCIIa (CP29) showing the lowest de-epoxidation state of the 3 minor complexes. There is disagreement over the extent of de-epoxidation of the violaxanthin associated with LHCIIb; in one study it was concluded that it was totally unavailable for de-epoxidation (Bassi et aI., 1993), whereas in others the conversion to zeaxanthin was similar to that observed in the minor complexes (Ruban et aI., 1994b); it has been suggested that the enigma of "violaxanthin availability" (describing the fact that in many plants only about 60% of the violaxanthin can be epoxidised) can be explained by the amount of violaxanthin bound to LHCIIb and to LHCI (Pfundel & Bilger, 1994), but this notion is not supported by the available data.

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Role of the xanthophyll cycle in qE

Although the xanthophyll cycle had been characterised in some detail over 20 years ago, it was the work of Demmig-Adams that first established its role in photoprotection and more specifically in the qE and qI components of qN (Demmig-Adams, 1990; Demmig­Adams & Adams, 1992). Convincing correlations were found between the extent of qN in leaves and their content of zeaxanthin under a wide range of conditions and in a variety of plant species. Similar results were later obtained in isolated chloroplasts where it was shown that even better correlations result if antheraxanthin is included as having equivalent quenching strength to zeaxanthin (Gilmore & Yamamoto, 1992b). The most straight forward interpretation of this data was that zeaxanthin (and antheraxanthin) was the quencher responsible for qE. However, it has been shown that qE can be formed, both in isolated chloroplasts and in leaves, in the absence of zeaxanthin and with only trace amounts of antheraxanthin (Adams et a!., 1990; Noctor et a!., 1991). These observations have been explained by the proposition that "zeaxanthin-independent quenching" occurs by a different mechanism (Demmig-Adams, 1990), perhaps in the PSII reaction centre. Whilst this explanation can not be discounted, the characteristics of qE (e.g. sensitivity to antimycin A; relative quenching ofFo and Fm; correlation with ~A535) with and without the presence of zeaxanthin are identical (N octor et a!., 1991; 1993). The only significant difference was found to be the level of ~pH required (Noctor et a!., 1991); qE required a smaller pH in the presence of zeaxanthin, and direct assessment from the pH dependency for quenching showed a shift from 4.5 to 6.0 (Rees et a!., 1992). For this reason, it was suggested that zeaxanthin was an activator of quenching, and that low pH and zeaxanthin therefore act synergistically to cause qE (Horton et a!., 1991). A similar conclusion was reached by Gilmore & Yamamoto (1992b) who showed that qE was linearly related to the product of lumen acidification and (zeaxanthin + antheraxanthin).

It is important to consider the ways in which the properties of violaxanthin and zeaxanthin differ (table 2). The removal of the epoxide group has three effects: firstly, the polarity of the end-groups is decreased; secondly, whilst violaxanthin is twisted, the zeaxanthin molecular is planar; thirdly, the number of C dQuble bonds is increased from 9 to 11. The last has profound implications for the energetics of the carotenoid molecule, for example, as seen in the different absorption ~ectra of violaxanthin and zeaxanthin. All carotenoids have a first excited singlet state (2 Ag) which is "forbidden" i.e. it can not be populated by absorption from the ground state because of the low oscillator strength. It has been shown that the energy level of this state is dependant on the number of conjugated double bonds in the carotenoid molecule (DeCoster et al., 1992); for violaxanthin this was estimated to be 15200 cm-1 (equivalent to 660 nm), decreasing to 14200 cm-1 (705 nm) for zeaxanthin (Frank et aI., 1994). Thus it was suggested by Owens and co-workers that the energy level of zeaxanthin could allow energy transfer from singlet excited chlorophyll in LHCII to this carotenoid, but not to violaxanthin (Owens et a!., 1992; Owens, 1994). It has been shown that the probability for radiationless decay of the 21 Ag state is high, thereby explaining quenching of chlorophyll fluorescence. However, energy transfer from chlorophyll to carotenoid cannot occur by Forster transfer, but instead van der Waals contact is required to enable transfer by the Dexter exchange mechanism. Therefore, an attractive idea js that the ~pH is required to protonate residues on a chlorophyll protein complex, causing a structural change which brings the chlorophyll into contact with zeaxanthin. At present this proposal remains a hypothesis since there is proof neither that fluorescence can be quenched by this mechanism even in vitro nor that it accounts for qE. Obviously, it can not easily explain how strong quenching can occur in the absence of zeaxanthin; the energy level of antheraxanthin is approximately equal to chlorophyll a in LHCII (Frank et a!., 1994), but its low concentration makes it unlikely that it could account

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Table 2. Properties of violaxanthin and zeaxanthin that may explain the role of the xanthophyll cycle in qE. The difference in energy levels arise directly from the increase in number of double bonds in the conjugate chain. The differences in polarity and conformation contribute to the differences in aggregation, and may control LHCII structure.

Property viol zea qE mechanism

2 Ag energy level 15200 em' 14200 em' direct transfer from chI (660 nm) (705 nm)

Polarity high low indirect (LHCII conformation)

Conformation twisted planar indirect (LHCII conformation)

for the high level of zeaxanthin-independent quenching frequently observed. It should be borne in mind that the 2 I Ag energy level of lutein is also equal to chlorophyll sin~e it too has 10 conjugated double bonds; the high concentration of this carotenoid in LHCII could readily explain the quenching seen in the absence of qE. Activation of qE by zeaxanthin would then be the introduction of a more efficient quencher.

It has been suggested that rather than the energy level effects, the differences in polarity and conformation of zeaxanthin and violaxanthin provide the explanation for the role of the xanthophyll cycle in the control of qE (Ruban et al., 1993b). Thus, the activation of qE seen in the presence of zeaxanthin could result from effects of these pigments on membrane structure, or more specifically the structure of LHCII. In addition to the circumstantial evidence based on light activation, there is direct experimental support for this idea; thus the quenching induced upon protonation of isolated LHCII is controlled by the ratio of zeaxanthin to violaxanthin, and most significantly, the latter seemed to behave as a quenching inhibitor (Ruban et at, I 994b); it is therefore possible that de-epoxidation is necessary to remove violaxanthin rather than to provide zeaxanthin. In terms of the simple kinetic model for qE described above, violaxanthin would then be viewed as stabilising the unquenched, unprotonated state ofLHCII in a manner somewhat similar to antimycin A.

THE ROLE OF PROTEIN STRUCTURAL CHANGE IN qE

If the xanthophyll cycle has only an indirect role in qE, then an alternative quencher has to be found. It should be pointed out that quenching of chlorophyll fluorescence can be easily induced in simple in vitro system; almost complete quenching of chlorophyll fluorescence can be observed upon addition of water to a solution in organic solvent, and quenching at high concentration is a well-known phenomenon. It has been estimated that the chlorophyll concentration in LHCII is approx 6 M (Kiihlbrandt & Wang, 1991) providing many opportunities for quenching through chlorophyll-chlorophyll interactions. The environment provided by the polypeptide chain presumably specifically prevents high levels of non-radiative energy dissipation, and therefore it is easy to imagine how a change in protein structure resulting from protonation of key amino acid residues could lead to quenching. As mentioned above, quenching in isolated LHCII can be induced by acidification; generally, such quenching is associated with formation ofLHCII aggregates. It was shown by Arntzen and co-workers that aggregation of LHCII results in a massive quenching of chlorophyll fluorescence (Burke et al., 1978). This phenomenon has been characterised in more detail (Ruban & Horton, 1992; 1995a; Horton & Ruban, 1994; Ruban et al., I 994b), and is associated with the formation of a number oflong wavelength emitters; absorption change from a small fraction of pigments also occurs together with changes in pigment orientation. Changes in chlorophyll-protein H bonding and xanthophyll orientation

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have also been observed (Ruban et aI., 1995b). It is believed that aggregation confers upon LHCII a unique macrostructure that gives rise to an enhanced CD (Garab et aI., 1988). These properties ofLHCII have been linked to the observations of xanthophyll cycle control ofqE to formulate the LHCII aggregation model for qE (Horton et aI., 1991; 1994). In this model qE is induced because protonation induces a change in protein structure that resembles that found upon aggregation observed in vitro; the xanthophyll cycle carotenoids are modulators of this change in LHCII organisation.

Some clarification of the essential features of this model are required: firstly, there is quite good evidence that LHCII exists as a macroaggregate in vivo (e.g. Garab et aI., 1988); secondly, it is of course unlikely that the kind of aggregates formed in vitro could exist in vivo because of the limitation on protein-protein interactions given by the overall structure of the PSII unit; similarly, the model does not specifY the scale of the changes in LHCII structure involved, and therefore it includes alterations localised within a LHCII complex resulting from protonation as well as interactions between complexes. Thirdly, the model considers the whole of LHCII, both the bulk LHCIIb and the minor LHCII components, LHCIIa, LHCIIc and LHCIId. Fourthly, the model specifies the way in which the ·L\pH triggers qE, but not the identity of the quencher itself; either a chlorophyll-chlorophyll interaction or a chlorophyll-xanthophyll interaction (i.e. causing the direct quenching mechanism through the 21Ag state) could be involved (Horton & Ruban, 1994; Horton et al.,1994).

There is experimental evidence to support the general aspects of the LHCII model. Analysis of 77 K spectra show preferential quenching of excitation in LHCII (Ruban et aI., 1991; 1993b; Ruban & Horton, 1994), and the change in excitation spectra upon qE formation resembles an LHCII absorption spectrum. Changes in the absorption spectrum in the presence of qE occur in the chlorophyll and carotenoid bands (Ruban et aI., 1992a; Horton & Ruban, 1994), and strongly resemble those observed for quenching of LHCII fluorescence in vitro (figure 3). In fact, the qE difference absorption spectrum, including the 535 nm band mentioned above can be simulated by the sum of the spectra for chlorophyll and xanthophyll aggregation. These data clearly establish that the properties of chlorophyll in LHCII are changed when qE is present in a manner consistent with the quencher being chlorophyll itself. However, the fact that changes in xanthophyll spectrum are observed, suggests that chlorophyll/xanthophyll interactions are involved in qE, and these changes may be reflecting the increased proximity of these pigments required for chlorophyll to zeaxanthin energy transfer.

ROLE OF THE MINOR LHCII COMPONENT IN qE

There is evidence which points to a significant role for the minor LHCII components in the formation of qE. Firstly, because they bind only about 15 % of PSII chlorophyll their presence can not be explained in terms of light collection; therefore in all models of PSII it is assumed that they have a role linking LHCIIb to the PSII core. Secondly, as discussed above, these complexes are enriched in the xanthophyll cycle pigments. Thirdly, LHCIIa and LHCIIc have been shown to bind Ca (Irrgang et aI., 1991) and possibly Cu (Arvidsson et aI., 1993) respectively, suggesting some kind of 'enzymatic' function involved in the regulation of light harvesting. Fourthly, they contain hydrophobic carboxy amino acids which can react with DC CD leading to the inhibition of qE (Ruban et aI., 1992b; Walters et aI., 1994). Examination of the pattern of labelling of peptides in LHCIIc has lead to the conclusion that at least two residues react with DCCD, one on helix B (glu 110) and one on the D helix (glu 218) (Walters, unpublished data); both of these face the lumen side of the complex. Both residues are also present in LHCIIb although the specificity of DC CD labelling for LHCIIc and LHClIa is retained both in vivo and in the isolated complex. These proton binding sites

107

A B I,

0.01 " 0.00 I, , , I ,

-.; ... c 0.00 r .s -0.01 e-O

'" .0 -0.01 <C

-0.02

-0.02

400 440 480 520 560 400 440 480 520 560

Wavelength, run

Figure 3. Absorption difference spectra for qE in leaves (solid line) and chloroplasts (dashed line) from spinach (A) and aggregation of purified chlorophyll a and zeaxanthin (B). For S, pigments were dissolved in ethanol and water added to induce aggregation.

may be the primary sites by which the thylakoid proton gradient initiates qE. Protonation of carboxy-amino acids in the hydrophobic domain of the minor LHCII proteins could initiate quenching through several possible mechanisms: it may trigger a conformational change in the polypeptide that results in movement of chlorophylls towards eachother to create a quencher, possibly in different complexes, or to bring chlorophyll closer to zeaxanthin (Horton & Ruban, 1994); it may alter the local electric field around the zeaxanthin or chlorophyll molecules so as to promote energy transfer (Owens, 1994); if the carboxyamino acids are chlorophyll binding sites, it may directly cause relocation of chlorophyll to give rise to quenching as above (Crofts & Yerkes, 1994). At present it is not possible to distinguish between these alternatives, and it is even too soon to conclude that protonation of these residues is an important part of the mechanism of qE (as opposed to indicating only the existence of proton channelling around photosysh:m II). Further structural and functional studies on this intriguing group of proteins is required.

The location of the site of qE in LHCIIc and/or LHClIa is consistent with all of the evidence that has pointed to the involvement of LHCII. Acidification of LHCIIb in vitro induces quenching that has the same inhibitor sensitivity as qE in thylakoids (Ruban et al., 1994b), but the minor complexes also show these properties (Ruban, unpublished data); therefore, the behaviour of LHClIb in vitro should not be used as evidence of its involvement in qE in vivo. Equally, however, there is no evidence that excludes the possibility of qE occurring in LHCIlb. It has been argued that the weaker qE seen in the chlorophyll b-less mutant of barley, and in the LHCIIb-less intermittent light grown plants is evidence for involvement of the bulk LHCII in quenching (Lokstein et al., 1994). However, the fact that quenching is still observed has been used to argue the opposite viewpoint, and it has been suggested that qE is larger in the presence of LHCIIb only because of an increased connectivity within the PSII antenna that gives a greater possibility for energy transfer to the quencher (Briantais, 1994). A more systematic study of the relationship between antenna composition and qE capacity is needed before any firm conclusions can be reached. In fact, the variation in the capacity of qE between different plant species and between different growth conditions within a species is currently unexplained. The increased xanthophyll cycle carotenoid to chlorophyll ratio in high light grown plants is associated with increased quenching (Demmig-Adams & Adams., 1992), but the changes in pigment composition may only reflect the loss of the relatively xanthophyll cycle deficient LHCIlb; thus it could be change in LHCII composition not xanthophyll cycle content that maximises quenching. The loss of the peripheral LHClIb, enriched in the lhcb2 gene product (27 kDa polypeptide) has been reported to occur in high light (Maenpaa & Andersson B, 1989), and it is interesting

108

that this peripheral LHCII shows a decreased tendency for in vitro aggregation compared to the inner complexes (Spangfort & Andersson, 1989).

THE RELATIONSHIP BETWEEN qE AND MORE SUSTAINED FORMS OF qN

As already referred to, when the conditions for formation of qE are extreme, either in excess light and/or after extended periods of illumination, qN becomes increasingly irreversible. Whilst a portion of this quenching certainly results from damage to PSII, it is also clear that this form of qN resembles qE. A more stable form of qE could occur through a variety of mechanisms: a persistence of the pH gradient through continued maintenance of cell energy charge in darkness (Gilmore & Yamamoto, 1992a; Gilmore & Bjorkman, 1994); a more stable conformation of LHCII (Ruban et aI., 1993a); a stably protonated membrane domain (Horton & Ruban, 1994). This kind of sustained qE is associated with operatiolJ. of the xanthophyll cycle, and therefore with light activation as described above. Whether sustained qE can be observed without the accumulation of zeaxanthin has not been explored. In some circumstances this kind of qN can be the major one, although generally it only represents 10-30% quenching of Fm. It is therefore not possible to conclude that it has an important role in photoprotection since the amount of energy dissipated is rather small. However, it has been argued that sustained qE is merely indicating a state of the thylakoid (light-activated) that is adapted to induce energy dissipation both rapidly and fully in response to illumination (Ruban et al., 1993a); the decreased FvlFm associated with this adaptation is thus the price that has to be paid for enabling this response. Moreover, because the relaxation time of this state is so slow, losses in potential photosynthesis can occur during a diurnal cycle since in periods of low light PSII in "over-regulated"; recent field studies suggest that this is perhaps the most significant form of "photoinhibition" (Baker et al., 1994; see also Baker, this volume). Obviously, the photoprotection by even long-lived quenching states is advantageous compared either to damage to the photosynthetic reaction centre proteins (which requires disassembly, proteolysis, new synthesis, and re-assembly for repair), or to photooxidation of pigments and lipids, which, if unchecked, would lead to cell and leaf death.

The fact that the capacity for very high levels of rapidly inducible quenching is not found in all plant materials suggests that the switch from maximum quantum yield to high levels of energy dissipation requires a dynamic range greater that achievable within a fixed membrane composition; species-dependent adjustments would then be expected to sacrifice quantum yield to achieve high levels of photoprotection, but this appears to not be the case; instead, such species adapted to high light have the capacity to show strong light activation. Similarly, acclimation to high light environments gives rise to a thylakoid composition which can also optimally respond to light-dependent stress. However, it is not clear what features of the acclimation of shade prevent the attainment of high qE, and why the capacity to form high levels of quenching is not constitutive. As discussed above, the adaptation and acclimation to high light have been shown to be associated with the presence of elevated xanthophyll cycle pool sizes and/or altered LHCII composition. Understanding the relationship between the acclimation of the photosynthetic membrane and the regulation of energy dissipation has to be a major objective for future research.

CONCLUSIONS

The discovery of nonphotochemical quenching of chlorophyll fluorescence and the general understanding its origins, together with the development of new types of fluorimeters designed specifically for its detection has revolutionised the study of the physiological aspects of photosynthesis. It is now possible to evaluate the performance of

109

plants to a high level of sophistication and accuracy in any environment, and fluorescence analysis wil1 increasingly form an essential part of all ecological studies. In order to achieve the final aim of increasing the level of photoprotection in crop plants it is necessary to identifY the molecular basis of quenching, and to understand the factors which control its dynamics and capacity. To achieve this will require the same advanced biophysical methods needed to probe the basic processes of energy transfer in photosynthesis, coupled to an understanding not just of the structure of individual PSII proteins but also the dynamic aspects of protein-protein, pigment-protein and pigment-pigment interactions.

ACKNOWLEDGEMENTS

I wish to thank all my colleagues Kate Maxwell, Conrad Mullineaux, Andy Pascal, Sasha Ruban, Robin Walters and Andy Young for many stimulating discussions about nonphotochemical quenching. This work is supported by both the Biotechnology and Biological Sciences Research Council and the Natural Environmental Research Council of the UK.

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185 Maenpaa, & Andersson B. (1989) Z. Naturforsch. 44:403-406 Mullineaux C.W., Ruban AV., & Horton P. (1994) Biochim. Biophys. Acta, 1185:119-123 Noctor G., Horton P. (1990) Biochim. Biophys. Acta 10 16:228-234. Noctor G., Rees D., Young A. & Horton P. (1991) Biochim. Biophys Acta 1057:320-330 Noctor G., Ruban A.V., & Horton P. (1993) Biochim. Biophys. Acta. 1183:339-344 Ogren E. (1994) in "Photoinhibition of Photosynthesis", (Baker N.R,& Bowyer J.R, eds), Bios

Scientific Pubs, Oxford UK, pp 433-447 Oquist G., Chow W.S., & Anderson 1.M. (1992) Planta, 186:450-460 Owens T.G., Shreve AP., & Albrecht AC (1992) in "Research in Photosynthesis",( Murata N. cd),

Vol 4, Kluwer Academic Pubs, Dordrecht, pp179-186 Owens T.G. (1994) in "Photoinhibition of Photosynthesis", (Baker N.R, & Bowyer J.R cds), Bios

Scientific Pubs, Oxford UK, pp 95-109 Peter G.F., & Thornber J.P. (1991) J. BioI. Chern., 266: 16745-16754 Prondel E., & Bilger W. (1994) Photosynth. Res. 42:89-110 Quick W.P., & Horton P. (1984) Proc. Roy. Soc. B, 220:371-382 Quick W.P" & Stitt M. (1989) Biochim. Biophys. Acta ,977:287-296 Rees D., Noctor G., & Horton P. (1990) Photosynth. Res., 25: 199-211 Rees D., Noctor G, Ruban AV .• Crofts J., Young A, & Horton P. (1992) Photosynth. Res., 31:11-19. Ruban AV., & Horton P. (1992) Biochim. Biophys. Acta, 1102:30-38 Ruban A.V., & Horton P (1994) Photosynth. Res. 40:181-190 Ruban AV., Rees D., Noctor G., Young A, & Horton P. (1991) Biochim. Biophys. Acta, 1059:355-

360 Ruban AV., Rees D., Pascal AA. & Horton P. (I992a) Biochim. Biophys. Acta, 1102:39-44 Ruban AV., Waiters RG., & Horton P. (1992b) FEBS Lett. 309:175-179 Ruban A.V., Young AJ., & Horton P. (I993a) Plant. Physiol. 102:741-750 Ruban AV., Horton P., & Young AI. (l993b) I. Photobiol. Photobiochem. B BioI., 21:229-234. Ruban AV., Young AI., Pascal A.A.. & Horton P. (1994a) Plant Physiol. 104:227-234 Ruban AV., Horton P., & Young AI. (1994b) Biochim. Biophys. Acta ,1186:123-127 Ruban AV., Dekker I.P., Horton P., & van Grondelle R .(1995a) Photochem. Photobiol., in press Ruban A V., Horton P., & Robert B. (1995) Biochemistry, 34:2333-2337 Schreiber U., Schliwa U., & Bilger W. (1986) Photosynth. Res. 10:51-62 Spangfort M., & Andersson B. (1989) Biochim. Biophys. Acta, 977: 163-170 Thayer S.S., & Bjorkman O. (1992) Photosynth. Res. 33:213-225 Timmerhaus M., & Weis E. (1990) in "Current Research in Photosynthesis", (M BaItscheffsky, cd.), Kluwer Acad Pubs, Netherlands, vol II, pp771-774 van Kooten 0., & Snel I.F.H. (1990) Photosynth. Res., 25:147-150 Walters RG., & Horton P. (1991) Photosynth. Res., 27: 121-133 Walters RG., & Horton P. (1993) Photosynth. Res. 36: 119-139. Walters RG, Ruban A.V., & Horton P. (1994) EuT. I. Biochem., 226:1063-1069 Weis E., & Berry 1. (1987) Biochim. Biophys. Acta, 894:198-208

III

REGULATION OF EXCITED STATES IN PHOTOSYNTHESIS OF HIGHER PLANTS

Jean -Marie B riantais Laboratoire d'Ecologie Vegetale Bat. 362 Universite Paris-Sud 91405 Orsay-cedex France

BASIC STATEMENTS

Regulation of excited chlorophyll concentration in photosynthetic membranes occurs at the level of both light absorption and exciton-deactivation processes i.e.: photochemistry, fluorescence and internal conversion. In a dark adapted leaf, the yield of photochemistry is high; the quantum yield of linear electron flow through the two photosystems measured in dim light is close to 0.1 Ilmole O2 evolved per IlEinstein absorbed. Therefore the quantum yield of charge separation and its stabilization in each photosystem reaches 0.8: (0.lx4e­x2photosystems) indeed to evolve one O2 molecule, four electrons have to go through two photoreaction in series. When photochemistry is maximal, the PSII associated fluorescence yield is around 2.5%, reaching about 11% when the photochemistry is nil. The yield of PSI fluorescence is 0.5% and, in contrast to PSII fluorescence, it does not change with the extent of the photochemistry.

Photochemistry and internal conversion, which compete with fluorescence, are named quenchers of fluorescence; they determine respectively the so called photochemical (qP) and non-photochemical (qN) quenchings (see appendix 1 and 2)

The photosynthetic apparatus is equipped to allow plants to grow in dim light but in the natural environment, plants are subjected to large variations ofirradiance with a maximum of around 2000 ~lEinstein m-2 S-l which over-saturates photosynthesis. It is when irradiance is over-saturating for the photosynthetic processes that photo inhibition occurs (see Baker, this volume). Nevertheless we will see that the photosynthetic apparatus has developed a number of regulatory mechanisms, mainly at the level of PSII, which reduce considerably the occurence of photoinhibition.

It has been shown that when the irradiance increases, the quantum yield of linear electron flow decreases, being around 0.3 when photosynthesis is saturated. This decrease is not only due to a diminution in the number of open photochemical traps, but, to a large extent, to an enhancement of heat deactivation.

In the case of PSII, which exhibits an increase of fluorescence emission parallel to the decrease of photochemistry due to trap closure (Delosme et aI., 1959; Bennoun and Li, 1973), chlorophyll fluorescence has been extensively used to follow, in vivo, using the "light doubling" method introduced by Bradbury & Baker (1981), variations of quantum yield of

Light as an Energy Source and Information Carrier in Plant Physiology Edited by Jennings et al., Plenum Press, New York, 1996 113

PSII photochemistry and of internal conversion (Schreiber et al.1986). Figure 1 shows a basic experiment on a leaf which shows that PSII fluorescence is controlled by both photochemical and non-photochemical quenchings. (see appendix 2)

Some years ago it was shown (Weis et al. 1987; Genty et al. 1989) that PSII chlorophyll fluorescence yield can measure the quantum yield of linear photosynthetic electron flow. Indeed, as shown in figure 2, in many conditions (varying light intensity, CO2

concentration, during induction of photosynthesis) a linear relationships is obtained between the fluorescence parameter ~F/Fm and the quantum yield of O2 evolution or the quantum yield of CO2 assimilation in the absence of photo respiration.

Such a proportionality between the yield of PSII (q,psn) measured by fluorescence and the quantum yield of linear electron flow through the two photosystems has also been verified by the linear relationship observed between q,psn and <PPSI by Genty et at (I 990b) (see figure 3). Therefore, in the last few years, changes of PSII chlorophyll fluorescence have been extensively utilized to determine in vivo variations of the yields of the various PSII pathways of excitation utilization. Direct measurements of heat deactivation have also been performed, mainly using .the photoacoustic method (Malkin & Cahen, 1979) and more recently by thermal radiometry (Malkin et al.,1991). The principle of these methods is to follow changes in amplitude of the thermal signal due to illumination with modulated light. In the photo acoustic method, variations of modulated pressure are measured. These are due both to gas exchange (photobaric signal) and heat deactivation (photothermal) processes. These two components can be separated by a multifrequency analysis, the photobaric signal decreases as the frequency of the modulated light is enhanced. These methods have given data consistent with those which measure variations of photochemistry and fluorescence. One difficutly of thermal measurement is to be able to discriminate quantitively what is due to PSI and PSIl (for a review see Malkin 1994).

Fm

1 min. '------'

Fm

CI) u c: CI) u '" CIl '-0 :l ;;:

"0 CI) ... Fo ... :; "0 0 ~

L2 t t t I mol. t L 1 t L3 Ii L 1

Time

Figure 1. Experimental protocol for the determination of Fo and Fm during photosynthetic induction of a

dark adapted barley leaf. ml = modulated analytical light; Ll=actinic light (1000 J-tE.m-2.s-I); L2=1 s pulse

of saturating actinic light (7000 J-lEm-2s- I); L3=near infar-red light (20 J-lE m-2 s-I).Upward and downward arrows indicate light on and off respectively (from Genty et al. 1990, Photosynth Res, 26: 133-139.).

114

1.0 1.0

• • • 0.8 • 0.8

0

E "- 0.6 0.6 .e-

O ~ l7

.e- 0.4 0.4 <j

0.2 0.2

0.0 0.02 0.04 0.06

Quantum yield (mal CO2 .mol-1 photon)

Figure 2. The relationship between the quantum yield of CO2 assimilation and the photochemical yield of PSII, i.e. 6<j>F/tV Fm (0, 0 ,~) and the fluorescence photochemical quenching, qQ ,( e, II, ~), for a maize leaf at steady state as a function oflight intensity (e, 0), in a range of atmospheric CO2 concentration (II, D) or during photosynthetic induction in air (~, 8).

0.11

0.6 UI D-

B- 0.4

0.2

0.0 0.0 0.2 0.4 0.8 0.11 1.0

¢PSI

Figure 3. Relationship between the qua tum yields of PSI (tV PSI) and PSII( tV PSII) over a range of irradiances in a barley leaf exposed to an atmosphere containing 2% (0) or 20% (~) ox-ygen.tV PSII= ~FlFm', tV PSI= fraction of non-oxidized P700.

MODULATIONS OF EXCITED STATES IN VIVO

Variations of the input

Variations of antenna size may occur with environmental conditions of growth. a) Irradiance and quality of the light. Structural, organizational and functional

changes of the photosynthetic apparatus depending upon the light intensity in shade and sun plants have been studied extensively (Boardman, 1977). Generally speaking, with increasing growth irradiance, light harvesting complexes of PSI and PSII decline in parallel with PSI and with PSII reaction center concentration. In some cases, such as Dunaliella tertiolecta (Sukenick et aI., 1988), a differential accumulation of individual LHCI and LHCII

115

apoproteins occurs. In high-light grown cells of this algae, LHCII contains a lower amount of chlorophyllb than in cells grown in low light.

An alteration in the balance of red/far red light causes substantial structural, organizational and functional change in higher plant chloroplasts. An enrichment in far red light occurs for shade plants growing under a vegetation canopy. The essential effect of this is an increase of Photo system II over Photosystem I ratio, but according to Melis (1984) and Kim et al. (1993) in higher plants the size of the functional antenna of individual PSII and PSI complexes does not change substantially.

b) Temperature. It has been observed by Schreiber and Berry (1977) that temperatures higher than 40°C induce an irreversible increase ofFo. This phenomenon was interpreted as due to a disconnection of the LHCII from PSII centers. This conclusion is supported by the absence of this effect in the chlorina f2 mutant of barley which lacks LHCII. In natural conditions such high temperature can be reached in stress conditions such as strong light plus water-stress which closes stomata. Meanwhile Havaux (1993) clearly showed that higher plants which are adapted to high temperatures develop hardening leading to a shift to higher temperatures of the disconnection ofLHCII.

Another effect of increasing temperature on antenna size is on the expression of LHCn biosynthesis in certain mutants. For example, a mutant of barley, chlorina 104, is deficient in LHCII when grown at low temperatures (10° C), but it has normal content of this light harvesting complex when it is grown at 25 ° C (Knetzel & Simpson, 1992).

PSII connectivity. Changes in exciton input at constant incident irradiance occurs in most PSII units depending upon the degree of photochemical trap aperture. As the number of closed PSII units increases the rate of photochemical closure of the remaining open traps increases. This has been interpreted as being due to exciton transfer between neighbouring PSII units densely packed in certain zones ofthylakoid membranes named grana-stacks. This phenomenon has been directly observed measuring the rate of O2 evolution (Joliot & Joliot 1964). Consequently, as shown by Bennoun and Li (1973), there are anti parallel changes in PSII variable fluorescence and of PSII photochemistry with trap closure, which are equally influenced by the connectivity. This exciton transfer between PSII units can explain the sigmoidal shape of the PSII fluorescence induction in the presence of inhibitors ofQA to QB electron flow (as triazines or substitued ureas). Figure 4 shows the relationship between the increase of fluorescence yield and the amount of QA reduced (measured by the complementary area over the fluorescence induction) in two states: PSII packed in certain zones of the membrane (curves 2 and 3), PSII homogeneously distributed in all the membrane (curve 1). Recently Trissl et al. (1993) and Lavergne & Trissl (1995) have extensively discussed the meaning of the fluorescence induction sigmoidicity.

States transitions. (For a recent review of this topic see Allen, 1992). State 1- State 2 transitions account for 20-30% opposite changes in antenna sizes of the two photosystems. This phenomenon occurs in green plants as well as in cyanobacteria an red algae and was first described by Bonaventura and Meyer (1969). Light mainly absorbed by PSII (or dim white light) induces a decrease of PSII antenna cross section and a corresponding increase of PSI antenna size. Many experimental data have suggested that State 2 corresponds to the detachment from PSII of a fraction of peripheral LHCII or phycobilisome antenna after phosphorylation of these complexes with subsequent migration and association with PSI. Therefore State I-State2 equilibrium is determined by the relative activities of a kinase and a phosphatase.The kinase activity is redox-controlled and is activated by the imbalance of electron transport through the two photosystems when plastoquinones associated to the cytocrome b6f complex are reduced. However it should be noted that in Cyanobacteria, Vernotte et al. (1990) have suggested that the State2 adaptation is not associated with a decrease ofPSII antenna size but corresponds to an increase in spill-over from PSII to PSI,

116

w u z w U III W a:: o :::> ....J LL

W ....J m =:! a:: <{ >

COMPLEMENTARY AREA

Figure 4. Variable fluorescence versus complementary area over the fluorescence curve, during induction in

the presence of DCMU: 1O-5M, in thylakoids isolated from pea leaves and which have been preincubated various time in a medium containing MgCIz :5 mM . No MgCI2 :(1), plus MgCI2 , 5 min: (2) , plus MgCIz, 30 min: (3) (from Briantais et aI. 1984, Biochim Biophys Acta , 766 : 1-8)

i.e. a quenching of PSII excitons by PSI. Indeed in a mutant of Synechocystis lacking phycocyanin the state transition controlled by the redox state of plastoquinone pool still occurs and can be correlated, as in the wild type with structural changes of PSII associated EF particles which become randomly distributed in State2. For state-transitions in red algae and cyanobacteria various models have been proposed and reviewed by Biggins & Bruce (1989).Among evidence that State 2 transition (and according to Horton & Hague, 1988, the so called quenching qT) is associated to a decrease in PSII antenna size (and not a direct PSII quenching) in higher plants and green alga is the fact that State 2 decreases by the same percentage Fo and Fm and these decreases are not accompanied by a proportional change in the average lifetime of fluorescence. This is in contrast to changes observed when PSII and PSI distribution in the membrane is modified by cation concentration (see table 1). It should be pointed out that State 2 in vivo and phosphorylation ofthylakoid induce the same effect.

Table 1. The effect ofa Statel-State2 transition, protein phosphorylation, at 5 and 2 mM

Mg2+ and cation (Mg2+) depletion on the total fluorescence yield (F), average life time (t mean) and absorption (u) at Fm. (u is the fraction of incident light absorbed by PSII and it is proportional to the inverse of the slope of the 't mean- F relationship).

State Phosphorylation Cation Transition Depletion

5mMMi+ 2 mMMg2+

~F 18 27 47 57 ~'tmean 8 5 21 48 ~a 13 26 30 17

The ratio (Fm-Fo)lFm also called FvlFm = kp/(kp+kF+kD) is routinely used to determine the intrinsic quantum efficiency of PSII. Therefore State transitions which change by the same percentage Fo and Fm, does not affect the quantum yield ofPSII open traps.

117

Variations of the exciton output.

Exciton annihilation. At very high photon density (> I013photon cm-2 pS-I) the concentration of excited states becomes large enough to have a probability for two SI excited states to meet and to processes according to the following reactions:

S1 + SI ~ So + Sn; Sn ~ SI or

SI + 1; ~ So + Tn; Tn ~ Tl

where SI is the first excited singlet state, So the ground state, T is the excited triplet, Sn and Tn are higher excited states than SI and TI respectively. This phenomena induces a quenching of chlorophyll fluorescence as fluorescence comes from SI to So deactivation.

At physiological irradiances (maximum 2000 /-LE m-2 S-I which is equivalent to 1.2xI05 photon cm-2 pS-I) this phenomenon does not occur. Meanwhile in order to study fast primary events of photo conversion the use of intense brief pulses can be necessary and therefore it is safe to verify if annihilation is occuring or not. As an example, in initial determinations of chlorophyll fluorescence lifetimes with laser pulses, the lifetime values obtained where significatly affected by annihilation (Campillo et aI., 1976).

Meanwhile annihilation process is an interesting tool to investigate antenna size. Indeed . from the intensity of the ps pulse at which annihilation starts to occur the size of pigment domains where excitons can interact through a fast exciton transfer can be calculated (for a quantitative understanding see Paillotin et al.; 1979; van Grondelle & Amesz, 1986). Intuitively it is obvious that the closer two excited chromophores are, the higher is the probability of a collision (Forster law). In chloroplasts with closed PSII reaction centers the diffusion range of the singlet exciton appears to be limited more by its life time than by any topological barrier that might prevents its migration.

Deactivations at physiological irradiances. As previously described, the photochemical use of excitons by the photosynthetic apparatus is very high, but it is well established that the quantum yield of photosynthetic linear electron flow in vivo, measured at steady state, either by O2 evolution or CO2 assimilation, (in the absence of photorespiration) decreases as irradiance increases.

It has been experimentally shown (see figure 5c) that over a broad range ofirradiances (except in very dim light; see Hormann et al.; 1994) the quantum yield oflinear electron flow is proportional to the quantum yield ofPSII photochemistry measured by dFIFm where dF is the increase of fluorescence yield induced by a saturating pulse from the fluorescence yield before the pulse and Fm the maximum yield of fluorescence reached under this brief, saturating pulse (see figure 1). This linearity is not surprising according to the data of Bennoun & Li (1973), (see also the section on connectivity). Figures 5 a and b show that dFIFm (figure 5c) depends on both photochemical quenching (qp), which measures the number of open traps, and by non-photochemical quenching, which decreases FvIFm, a measure of the intrinsic quantum yield of an open trap; the value of FvIFm in higher plants of around 0.8 in dark adapted leaves decreases to 0.4 when photosynthesis is saturated. Therefore, a regulation of PSII photochemical deactivation, depending upon the utilization of the products of PSII photo conversion, occurs essentialy through an increase of heat deactivation which adjusts PSII yield to the rate limiting step of electron draining.

In vivo, the linearity between dFIFm and the quantum yield of linear electron flow is supported at various levels of qN. This suggests that qN affects all PSII centers in the same way.

118

1.0 fJJ D

0.8 a •• -roo • cJJ

0.6 e6 fo;,t. A ... 0- ..

0.'" a .. ~

0.2

0.0

0.8 b OJ D

E 0.6 .reo ° Lo.. :# ) 0.'" ..

~A eA

0.2

0.0

0.8 C

E 0.6 Lo..

~ OA <l

O •. tv-''-----.-~-----.....J .00 0.05 0.10

Quantum yield (mol C02·mol photon-1)

Figure 5. Relationships between (a) photochemical-quenching coefficient qP ; (b) intrinsic quantum efficiency of an open PSI! trap FvlFm and (c) ~FlFm ,quantum yield of PSI! photochemistry and the apparent quantum yield of CO2 uptake, measured in non-photorespiratory conditions in a bean leaf. (0):

different CO2 concentrations,l% O2, PFD 340 llE.m-2s- l ; (0) different COz concentrations, 1% O2 , PFD

720 !!E m-2 s-l; (fI., {I.}: different PFD ,1500 1l.l-1 CO2 , 21% O2 ; (D): different PFD, 370 mll-1 CO2 ,1% O2• (from Comic and Briantais 1991, Planta , 183: 178-184 ).

Non-photochemical quenching contains different phenomena. Horton & Hague (I 988) showed that the time course of the dark relaxation of qN can be decomposed into, at least, three exponential components that were denoted qT, qE and qI. These contributions have also been correlated with the irradiance range at which they saturate: qI, qT in low light, qE at an intensity which saturates photosynthesis and qI at higher intensities.

a) qT quenching. qT quenching occurs in dim light and its half-time of dark-relaxation is around five minutes. It has been interpreted as a State 2 transition, as qT dark relaxation is blocked by NaF, a phosphatase inhibitor (Horton.& Hague, 1988; Hodges et aI., 1989). This interpretation is also consistent with the non-linearity of the quantum yield of electron flow versus ~FIFm relationship, specifically observed under dim white light (Horman et aI., 1994). Indeed, a State' 2 transition will not affect FvIFm, then only qP will slightly decrease and ~FIFm, the apparent quantum yield of PSII, will decrease less than the apparent quantum yield of electron flow. Meanwhile this interpretation of qT as a State 2 transition is somewhat unclear as at low white light intensity the level of reduced plastoquinones may be very low. It should be noted that in higher plants the amplitude of this phenomenon is only 20% of the total qN.

Note that if qT is a State 2, i.e. a decrease of PSII antenna, it is improper to call it a quenching. It is a change of the exciton input and not exciton deactivation.

119

A possible discrepancy arises from reports showing that in barley mutant (Lokstein et aI., 1993) and in intermittent light grown pea leaves (Briantais, 1994), both lacking LHCII, a qN component has very similar dark relaxation kinetics to that observed for qT in plants possessing LHCII. However, as concluded by Walter & Horton (1991), the kinetic analysis of qN dark relaxation may not be sensitive enough for discriminating all the various components of qN.

b) qE quenching. This component of qN, called qE (E for energization), can be assayed in vitro by its cancellation upon uncoupler addition or, in vivo, by its rapid relaxation (half-time around 10 s). In contrast to unicellular green algae (Falkowsky et al., 1986) and cyanobacteria (Demmig-Adams & Adams,1992), where state transitions are well developed, qE is the major qN component in higher plants at physiological irradiances. As previously shown, (figure 5c) it decreases by two fold the optimun quantum yield of PSII when photosynthesis is saturated.

The molecular mechanism of qE is still controversial and is discussed in more detail by Horton (this volume). Here the discussion will be restricted to data which suggest that qE can have different origins (except the requirement of proton gradient) depending upon the presence or not of the light harvesting antenna LHCII. Indeed a qN component very similar to qE is built up in plants which lack this antenna complex. We will also discuss whether the same type of qE is obtained in vivo and in isolated thylakoids

Weis et aI. (1987) have proprosed that energization of the thylakoid membrane induces the formation of inactive PSII centers of the quenching sink type. The quenching could be

induced by accumulation of centers in the P+680 QA-state (Weis & Lechtenberg, 1989; Schreiber & Neubauer, 1990) or from a rapid cyclic electron flow around PS II (Horton & Lee, 1985; Schreiber & Neubauer, 1987; Ramm & Hansen, 1993). Thus, qE in vivo would be the same phenomenon as the qN induced by chemical acidification of isolated thylakoids and PSII enriched preparations (Krieger & Weis, 1990; Krieger et aI., 1992). According to recent results of Krieger & Weis (1993), acidification of the thylakoid lumen induces a

reversible disassociation of Ca++ from the water splitting enzyme and the subsequent inhibition of the electron donor side of PSII which is known to block PSII fluorescence at the Fo level (Butler et aI., 1973). This was observed by Krieger et al., (1992). This is in contrast to the marked quenching ofFo accompanying qE in vivo (see figure1 and Bilger & Schreiber, 1986; Rees et aI., 1990; Genty et al., 1990a; 1992; Demming-Adams, 1990). Genty et ai. (1992) reported also that in chloroplast which develop upon illumination a qE quenching ofFo and Fm similar to that observed in leaves, the kinetics of the dark relaxation of the quenching is not affected by addition of a strong reductant (dithionite). This suggests

that accumulation of P680+ is not involved in qE developed by these chloroplasts. Moreover, to develop this quenching, the chloroplasts needed to be supplemented with ascorbate, a reducing mediator, which reverses qN induced by chemical acidification (Crofts & Horton, 1991; Rees et aI., 1992). Direct evidence showing that qE does not involve a significant conversion ofPSII centers into inactive sinks has been recently obtained by Genty (personal communication) who found that in protoplasts isolated from barley leaves, the same oxygen yield per single tum over saturating flash and per open PSII trap was measured in the presence and absence of non-photochemical quenching.

The decrease of Fo induced by qE in LHCII containing leaves is accompanied by a decrease of the average fluorescence lifetime (Genty et aI., 1992) and as shown in figure 6 and table 2, it is consistent with the decreased photochemical efficiency for the reduction of QA(Genty et aI., 1990).

This set of data favour the interpretation of qE as an increase of the rate of Internal Conversion (or heat), which is in competition with PSII photochemistry and fluorescence where the normal LHCII antenna is present.

120

3

CI> u c: 1.2

CI>

! !i: 2 ~ 0 ::>

;:;:

~1 U

B

1.0

" ... c E ~ ... ~ ~ 0..

<l ... O.

0 100 200 300 0.1. 1.00

time (ps) I/Imax

Figure 6. (I): Fluorescence induction curve from Fo (QA fully oxidized) to Fh (QA fully reduced) obtained on excitation ofa barley leaf with intense red light (30 000 Il-E m·2 s'\The leaf was given the following pretreatments prior to exposure to the red light: A, dark adaptation; B, dark adaptation followed by 90 s irradiation with blue light (600 !l-E m·2 S·I) and 4 s dark; C, dark adaptation followed by 90 s irradiation With blue light (600 !l-E m·2 S·I) and 5 min dark. The levels and parameters associated with these fluorescene induction are reported in Table 1.

(II) Flash energy dosage response curve (I/Imax) for the fluorescence yield increase (6Ff 16Ff max) produced by single turn-over flash in pea leaves exposed to continuous light intensities of 45 (0) and 677 (0) !l-E m·2s·l . 6Ffmax is the maximum change in the flash-induced fluorecence yield above the steady-state level of fluorescence, which is produced by the non attenuated flash Imax. The curves are described by the exponential saturation function: 6Ff/6Ffmax = 1- e",(IIIm .. " (from Genty et al. 1990 Photosynthes Res, 25: 249-257.)

In leaves of higher plants lacking LHCII such as intermittent-light grown peas or chlorina f2 barley mutant, a significant qN, that resembles qE with regard to its dark relaxation kinetics (Lokstein et ai., 1994; Briantais, 1994) can be developed (however it is smaller, for the same amount of light absorbed, than in LHCII containing plants and gives rise to only a small decrease of Fo). However, at variance with leaves that contain a normal LHCII complement (Genty et ai., 1990b) it was observed (Briantais, 1994) that qN, which reduces llFlFm, did not affect the photochemical efficiency of QA photoreduction, as measured by the saturation curves of the single turnover flash-induced increase of fluorescence. This result suggests that in such LHCII deficient material, qN may involve conversion of centers to a quenching sink type. Since chloroplasts lacking LHCII have either no or very few grana stacks and display no excitonic connection between PSII units (Arntzen et ai., 1976), there is no reason for quenching sink units to influence the photochemical efficiency of active units.

Table 2. Parameters of the fluorescence induction from Fo to Fil in a barley leaf, A= dark adapted, B in the presence of non-photochemical quenching, C= after partial relaxation in the dark of the qon-photochemical quenching.

Fo Fil

t 1/2*

A 1.00 3.08 30.5

*time (Il-S) taken to reach 50% ofthe Fo to Fi j rise.

B 0.92 1.35 38.6

C 1.07 2.61 32.9

121

c) qI quenching. The origin of this component of qN (I for irreversible) is still unclear. It was characterized by its slow dark relaxation (t1/2 > 30 min) and according to Horton & Hague (1988) it becomes important at irradiances which oversaturate photosynthesis. Consequently it has been often interpreted as reflecting a photoinhibition. However,qI, as defined by its slow dark relaxation, also occurs at physiological irradiances, and its intensity dependence displays two waves with an inflexion near the saturation of photosynthesis. It has been suggest that qI may contain a contribution from a long-lasting component of qE, (a point that will be discussed by Horton, this volume).

Whatever the different mechanisms involved in the various non-photochemical quenching component, all of them, except in dim light where a decrease in PSII antenna may take place, regulate similarly PSII in the range of physiological irradiances, since a linear relationship between LlFlFm and the quantum yield of photosynthetic oxygen evolution is observed.

PSII-PSI EXCITON TRANSFER.

This exciton transfer has been extensively studied in the past in isolated thylakoids of higher plants and algae. Indeed the distribution of PSI and PSII super-complexes in the membrane varies with the cation concentration of the resuspending medium (for a review see Barber, 1982). When negative charges of proteins are shielded by cations there is a segregation ofPSII in membrane regions which become appressed with the exclusion of PSI in non appressed zones. At low cation concentration PSI and PSII are mixed and PSII to PSI exciton transfer can take place. In vivo, plants which differentiate appressed and non appressed zones do not exhibit significant changes of their functioning with the distribution of the two photo systems, which stay physically separated. Indeed the cation concentration of the stroma is always high. Therefore PSII to PSI exciton transfer may concern only a minor fraction ofPSII localized in margins and in stroma lamella.

RECOMBINATIONS

The estimation of the quantum yield of photochemical conversion depends upon if one considers the charge separation, per se, or its stabilization by the secondary donors and acceptors. Indeed the ability to extract free energy from a photo-sensitized redox reaction depends on mechanisms which control the extent of the back-reactions. Therefore the primary charge separation can have a very high yield, but depending upon the redox state of secondary donors and acceptors, the stabilization can be more or less efficient and recombinations can take place often producing delayed fluorescence.

CONCLUSION

Plants have developed photosystems, the structural organisation of which allows them to grow in dim light. But they also possess regulations of these photo systems, especially photo system II, which is up-stream of the photosynthetic process. They control, within the range of natural irradiances, the yield of photochemistry, adjusting it to the use by carbon metabolism of the products of photoconversion, reducing through these regulations the occurence of photoinhibition. These regulations are taking place at the level of the light harvesting antenna size(state transitions) and by adjustment of the various pathways of exciton deactivation. As irradiance increases the heat deactivation also increase. This adjustment of heat deactivation to changes of environmental conditions is fast (less than one minute for its relaxation).

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APPENDIX 1.

Quenching of fluorescence

In the framework of the Stem-Volmer law fluorescence quenching and the concentration of the quencher Q are related by the following equation: (FIF(Q)) -1 = K(Q) where F and F(Q) are respectively the fluorescence in the absence and the presence of the quencher Q, and K is the quenching constant.

K is the rate constant of the reaction A *+Q ~ A +Q with a non radiative dissipation of the excitation when there is a brief interaction (within the life time of the excited state A *) between A * and Q. This quenching by collision is named dynamic

In contrast, when there is a permanent link between the pigment A and the quencher, K has the meaning of the equilibrium constant of the reaction: A+Q ~ AQ. In this case the interaction between the quencher and the exciton is very much longer than the lifetime of the exciton. This quenching is named static. The Stem-Volmer equation is valid for the tWo types of quenching. If the species AQ is not fluorescent, increasing Q concentration will decrease fluorescence amplitude but no change in fluorescence life-time will occur. In contrast, in a dynamic quenching, increase in Q induces a parallel decrease of both amplitude and lifetime.

APPENDIX 2.

Photochemical (qP) and Non-Photochemical (qN) quenchings.

Referring to figure 1, qP= (Fm·-F)I(Fm·-Fo·) which measures the fraction of open (QA oxidized) PSII traps.

qN=(FmlFm.)-1 (Stem-Volmer) but often qN is calculated on the variable part of fluorescence therefore qN=[(Fm-Fo)/(Fm'­Fo.)]-l considering that the origins ofFo and of variable fluorescence are different.

Abbreviations

Fo, Fm: chlorophyll fluorescence amplitudes when all Photo system II photochemical traps are respectively open and closed; F: chlorophyll fluorescence amplitude at time t after the onset of the illumination; LHCII: light harvesting chlorophyll a-b complex associated to Photosystem II; PSI, PSIl: respectively Photosystems I and II; QA, QB: respectively primary and secondary quinonic acceptors ofPSIl.

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Protein, Reaction Center and Phtosynthetic Membranes", Brookhaven Symposia in Biology n° 28: 316-337.

Barber,1. (1982) Annu. Rev. Plant. PhysioL,33: 261-295. Bennoun, P. & Li, Y.S. (1973) Biochim. Biophys. Acta, 292: 162-168. Berry, 1.A. & Bjorkman, O. (1980) Annu. Rev. Plant. PhysioL 31: 491-543, Biggins, J. & Bruce, D. (1989) Biochim. Biophys. Acta, 973: 315-323 Bilger, W. & Schreiber, U. (1986) Photosynth. Res, 10: 303-308. Boardman, NK (1977) Annu. Rev. Plant Physiol, 28: 355-377. Bonaventura, C. & Myers, 1. (1969) Biochim. Biophys. Acta, 189: 366-389. Bradbury, M. & Baker, N.R (1981) Biochim. Biophys. Acta ,63: 542-551.

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Briantais, J-M. (1994).Photosynth Res,40:287-294. Butler, W.L.,- Visser, J.W.M. & Simons, H.L. (1973) Biochim. Biophys. Acta, 292: 140-15l. Campillo, A.J., Kollman, V.H. & Shapiro, S.L. (1976) Science, 193: 227-229 Crofts, J & Horton, P.(1991) Biochim. Biophys. Acta,1058: 187-193. Delosme, R .. Joliot, P. & Lavorel, J.(1959) C. R Acad. Sc. Paris, 249: 1409-1411. Demmig-Adams, B. (1990) Biochim. Biophys. Acta, 1020: 1-24. Demmig-Adam, B. & Adams, W.W. (1992) Planta, 171: 171-184. Falkowsky, P.G., Wyman, K., Ley, A.C. & Mauzerall, D.C.(1986) Biochim. Biophys. Acta, 249: 183-

192. Genty B, Briantais J-M & Baker N R,Biochim. Biophys. Acta, (1989) 990: 87-92. Genty, B.,Wonders, 1. & Baker, N.R. (1990a) Photosynth. Res, 26: 133-139. Genty, B., Harbinson. J., Briantais, J-M. & Baker, N.R. (1990 b) Photosynth. Res. 25: 249-257. Genty, B., Goulas, Y., Dimon, B., Peletier, G., Briantais, J-M. & Moya, I. (1992) in "Research in

Photosynthesis", (Murata Ned.) Kluwer Academic Publihers Dordrecht, Vol IV pp 603-610. Havaux, M. (1993) Plant Cell. and Environment 16: 461-467. Hodges, M., Cornic, G. & Briantais, J-M. (1989) Biochim. Biophys. Acta, 974: 289-293. Hormann, H., Neubauer, C. & Schreiber, U. (1994) Photosynth. Res., 40: 93-106. Horton, P. & Hague, A. (1988) Biochim. Biophys. Acta, 932: 107-115. Horton, P. & Lee (1985) Planta, 165: 37-42 Joliot, A. & Joliot, P. (1964) C. R Acad. Sci. Paris, 258: 4622-4625. Kim, 1.H., Glick, RE. & Melis, A. (1993) Plant Physiol., 102: 181-190. Knoetzel, J. & Simpson, D. (1991) Planta, 185: 111-123. Krieger, A. & Weis, E. (1990) in "Current Research in Photosynthesis", Vol. VI (Baltscheffsky ed.)

Kluwer Academic Publisher, Dordrecht pp. 563-566. Krieger, A., Moya, I. & Weis, E. (1992) Biochim. Biophys. Acta, 1102: 167-176. Krieger, A. & Weis, E. (1993) Photosynth. Res, 37: 117-130. Lavergne, J. & Weis, E. (1993) Photosynth. Res .. 37: 117-130 Lockstein, H., Hartel. H., Hoffmann, P. & Renger, G. (1993) 1. Photochem. Photobiol. B: BioI., 19:

217-225. Malkin, S. & Cahen, D. (1979) Photochem. Photobiol. .29: 803-813. Malkin, S .. Schreiber, U., Jansen, M., Canaani, 0., Shalgi, E., Cahen, D. (1991) Photosynth. Res., 29:

87-96. Melis, A. (1984) 1. Cell. Biochem., 24: 271-285. Paillotin, G., Swenberg, C.E., Breton, 1. & Geacintov, N.E. (1979) Biophys. 1., 25: 513-534. Ramm, D. & Hansen, U.P. (1993) Photosynth. Res., 35: 97-100. Rees, D., Noctor, G.D. & Horton, P. (1990) Photosynth., 25:.199-211. Rees, D., Noctor, G.D .. Ruban, A.V., Crofts, 1., Young. A.J. & Horton, P. (1992) Photosynth. Res., 31:

11-19 Schatz, G.H., Brock, H. & Holtzwarth, A.R (1988) Biophys. J., 54: 397-405 Schreiber, U. & Berry, 1.A. (1977) Planta, 136: 233-238. Schreiber, u., Schliva, U. & Bilger, W. (1986) Photosynth. Res., 10: 51-62. Schreiber, U. & Neubauer, C. (1987) Z. Naturforch., 42 C: 1255-1264. Sukenick, A., Bennett, 1. & Falkowsky, P. (1988) Biochim. Biophys. Acta 932: 206-215. Trissl, H.W., Gao, Y. & Wulf, K. (1993) Biophys. 1., 64: 974-988. van Grondelle, R & Amesz, 1.(1986) in "Light Emission by Plants and Bacteria" (Govindjee, Amesz

and Fork eds.) Academic Press Pub. pp 191-223. Vernotte, C., Astier, C. & Olive, 1. (1990) Photosynth. Res., 26: 203-212 Walter, R.G. & Horton. P. (1991) Photosynth. Res., 27: 121-133 Weis, E. (1982) Plant Physiol. 70: 1530-1534. Weis, E., Ball, 1.T. & Berry, 1. (1987) in "Progr. in Photosynth Res" Vol II 553-556 (Biggins 1. ed.),

Martinus Nijhoff Pub. the Nederlands. Weis, E. & Lechtenbreg, D. (1989) Philos. Trans. R Soc. London BioI. Sci. 233: 253-268.

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CHIRALLY ORGANIZED MACRODOMAINS IN THYLAKOID MEMBRANES. POSSmLE STRUCTURAL AND REGULA TORY ROLES

INTRODUCTION

Gyozo Garab

Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, Szeged, P.O.Box 521, H-6701, Hungary

In photosynthesis, the primary step in the conversion of light energy into chemical energy occurs in the reaction centers, which represents only a small fraction of the total pigment content of photosynthetic membranes. Direct excitation of the reaction centers is a very rare event even under strong sunlight. In all photosynthetic organisms the light energy is predominantly captured by antenna pigments. These pigments transfer the excitation energy to the reaction centers that convert the excited state energy to a "stable" charge separation. The reactions concurrent with the trapping of the excitation energy, e.g. fluorescence emission or dissipation of the excitation energy to heat, represent a loss in the photosynthetic utilization of light energy. In chloroplasts, on average for every reaction center, several hundred chlorophyll-a (Chl-a) and b and carotenoid molecules constitute the system of the antenna pigments.

Under "normal conditions", the antenna system minimizes quantum losses and supplies the excitation energy to the reaction centers. The efficiency of the energy migration toward the reaction centers is largely determined by the molecular architecture of the pigment system: the rate of energy transfer depends on the distance and mutual orientation of the transition dipoles of the pigment molecules (Forster, 1965; see also Borisov, this volume). The high efficiency of the energy supply to the reaction centers suggests that the structural parameters are well defined, i.e. the system is organized with sufficient rigidity not to pennit fluctuations which would lead to losses.

On the other hand, it has been demonstrated that under high light intensities, when excess energy is absorbed, controlled energy dissipation can occur in the antenna (see Chapter by P. Horton). This, by preventing photoinhibitory damage to the photosynthetic machinery, is thought to play a protective role. In general, the multilevel regulatory processes, which are capable to regulating the photosynthetic energy conversion, appear of great importance in stress physiology and plant productivity (Anderson & Andersson, 1988).

Light as an Energy Source and Information Can-;er in Plant PhYSiology Edited by Jelmings et aI., Plenum Press, New York, 1996 125

However, our understanding of the molecular architecture of the antenna system of chloroplasts is far from being complete, and relatively little is known about the molecular mechanisms leading to regulatory changes in the photophysical pathways.

In chloroplasts, the Chi molecules are bound to different pigment-protein complexes with fixed stoichiometry (see chapter by Bassi, this volume). The distanCes of the pigment molecules are determined by the binding sites on the complexes (Zuber & Brunisholz, 1991). The orientation of the transition dipoles of the pigment molecules with respect both to each other and to the protein axes are not random (Breton & Vermeglio, 1982). The pigment-protein complexes are themselves embedded in the membrane in an ordered fashion, with largely restricted precessional motion (Szito et aI. 1984). As a consequence, the Qy dipoles of the ChI-a molecules are found preferentially oriented in the plane of the thylakoid membrane. It has been suggested that this type of ordered array of the pigment dipoles facilitates long-range energy migration in a direction parallel to the membrane plane (Garab et ai, 1987).

Much data indicate that the antenna and reaction center complexes are clustered into macrocomplexes. In freeze-fracture electron micrographs of thylakoid membranes, photosystem IT (PSII) appears as 100-180 A particle. This is believed to contain a single reaction center in a core of about 80 A diameter, surrounded by a variable quantity of associated Chi alb light harvesting complex (LHCII) (Staehelin, 1976). Energy transfer interactions between particles are implied in the 'lake' models, which allow some degree of "connectivity" between photosynthetic units contained within a larger domain.

For elucidation of the structure of the antenna system and for monitoring structural rearrangements in intact thylakoid membranes non-invasive spectroscopic techniques are of special value. In this chapter, I summarize the results obtained with the aid of circular dichroism (CD) spectroscopic techniques on grana! chloroplast thylakoid membranes and aggregated LHCII. Our studies have revealed that the PSII particles in grana! thylakoid membranes and the isolated, purified LHCII readily assemble into chirally organized macrodomains. This macroorganization is sensitive to different physico-chemical factors and the composition of the membranes. The chirally organized macrodomains are proposed to constitute the structural basis for energy migration over large distances in the antenna system. This type of macroorganization also appears to play an important role in ensuring that the antenna system is capable of undergoing reversible structural rearrangements. .

PHYSICAL ORIGINS OF CD IN SYSTEMS OF DIFFERENT STRUCTURAL COMPLEXITY

Circular dichroism spectroscopy is a powerful, non-invasive technique to obtain structural information in samples of biological origin. Nearly all molecules or complexes synthesized by biological systems show optical activity, which can be measured as CD. CD is the difference in the absorbance between left (L) and right (R) circularly polarized light:

(1)

It ~ses from intra- or intermolecular asymmetry (helicity) of the molecular structure. The helicity (chirality) of structure means that it cannot be superimposed on its mirror image; this is also often called handedness. In a hierarchically organized system, the lack of symmetry can be due to different physical mechanisms which are superimposable on each other. This is demonstrated in figure 1 in the case of the pigment molecules of chloroplast thylakoid membranes.

126

"0 3.0 -;; « <l

E 2.0 .. "0 .c 1.0 u Q ~ 0.0 II "5 ~ (3 -1 .0

400 500 600 700 800

Wavelength . nm

Figure 1. Circular dichroism spectra of the pigments of pea thylakoid membranes in 80% acetonic extract ( ..• ), and in the membranes either suspended in 10 mM Tricine (PH 7.8) (-) or in the same buffer complemented with 5 mM MgClz and 300 111M sorbitol (- ). Optical pathlength, 1 cm; Chl-(a+b) content, 20 flglrnl.

Intrinsic CD of molecules

The optical activity of a monomeric chiral molecule for each electronic transition is characterized by the rotational strength of the transition, which is proportional to the area under the CD band. The rotational strength depends both on the electric dipole (Jl) and the magnetic dipole (m) ofthe transition:

iReD = Im(Jl' m) (2)

The magnetic transition dipole is a purely imaginary vector. In this equation, the Rosenfeld equation, 1m indicates that the rotational strength corresponds to the imaginary part of the scalar product of the two vectors. For a molecule to be optically active, both Jl and m must be non­zero, and m must have a component parallel to Jl. For this to occur, the molecule must have a non-zero absorbance and be asymmetric. To explain this latter condition, it is necessary to recall that the electric dipole transition moment corresponds to a linear oscillatory motion of the charge induced by the electric field of light, whereas the magnetic transition dipole can be regarded as a light-induced current loop. In asymmetric molecules, light induces a circular motion about the direction of Jl which corresponds to a helical displacement of charge. In molecules that contain a plane or center of symmetry the rotational strength vanishes (e.g. for a ring, m is perpendicular to the plane of the ring, while Jl is in the plane). This explains the correlation between the magnitude of the CD and the helicity of molecules, which facilitates the helical flow of charges (Woody, 1985).

For a single electronic transition, the intrinsic CD has the same band-shape as the absorption, and its sign is determined by the handedness of the molecule (positive or negative Cotton effect).

In monomeric solutions, the intrinsic CD of ChIs, which are nearly symmetrical, planar tetrapyrrole molecules, is very weak (Houssier & Sauer, 1970): for most transitions, the CD is about five orders of magnitude weaker than the absorbance. Carotenoids are achiral in solution (Frank & Cogdell, 1993).

Molecular complexes and small aggregates

In pigment-protein complexes, ChIs typically exhibit CD with a conservative band structure which arises from excitonic interactions (pearlstein, 1982). The intensity of these bands is generally about an order of magnitude stronger than that of the intrinsic CD of ChIs. In thylakoid membranes suspended in hypotonic, low ionic strength buffer, the CD is given rise to by the

127

particles containing the pigment -protein complexes. Thus, the CD can be considered as the weighted sum of the spectra of the complexes constituting the antenna. (Additional bands may arise from interactions between pigment molecules carried by neighboring complexes.)

For two identical pigment molecules separated by a distance vector R, if the two molecules are brought close enough to each other to interact electronically but are stilI sufficiently apart for the electrons to remain localized on each of the molecules, the absorbance band splits into two bands. The degree of separation of the bands depends on the interaction energy (Y12, cm-I) between the two dipoles (J..!.l and J..!.2):

5.04 K 1'2 V 12 = IRF

where m is in debye, R is in nm; with r = R / IRI :

(3)

(4)

The rotational strength (in units of De bye-Bohr magnetons) for the excitonic CD of a dimer is given as:

(5)

where + and - designate the lower and higher energy transitions of the two exciton states, respectively, Vo is the band center energy, and the vectors in the scalar triple product are unit

vectors, d j = I' j / II' j I ' i= 1 ,2. It is clear that the rotational strength of the dimer is independent of

the rotational strengths of the monomers. In other words, an intense CD can arise upon the interaction of molecules which possess no or little optical activity. Further, the sum of the negative and positive rotational strengths is zero, i.e. the CD of the couplet is conservative.

The theory for excitonically coupled dimers has been extended to aggregates for arbitrary size (Pearlstein, 1991). (However, it is assumed that the sizes of the aggregates are much smaller than the wavelength of the visible light). In all excitonically coupled aggregates the conservative band-structure of the CD spectra is retained, which is thus often considered as the "signature" of excitonic interactions.

Interpretation of the CD spectra of complexes in terms of exact structural parameters, although it is theoretically straightforward, is usually very difficult. The water-soluble Bchl-a­containing complex of the green photosynthetic bacterium, Prosthecocloris aesluarii, the Fenna­Matthews-Olson complex, was the first photosynthetic pigment-protein complex to be structurally characterized by atomic resolution crystallography (penna & Matthews, 1975) but only very recently has it been possible to explain its CD activity satisfactorily in terms of the known structural parameters (Lu & Pearlstein, 1993).

FOI: LHCII, in which the structure is known at 3.4 A resolution (Kuhlbrandt et al., 1994) but Chl-a and b molecules have not been identified, exact interpretation of the CD has not been presented. Recently, it has been suggested that CD of LHCII trimers originate from an array of pigment molecules in which both Chl-b-Chl-b and Chl-b-Chl-a excitonic interactions play an important role (Hemelrijk et aI., 1992). CD spectra of subchloroplast particles and complexes have also been found to be dominated by excitonically split bands (Garab et aI., 1987; Bassi et al., 1985) but in most cases no structural models are available.

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Chirally Organized Macroaggregates

Granal thylakoid membranes and macroaggregates of LHCII are characterized by non­conservative CD signals with extremely large amplitudes and long scattering tails (Garab et al., 1987; Philipson & Sauer, 1973; Gregory et al., 1980). The CD signal of these samples deviates greatly from the signal of their components: the spectra cannot be recomposed via linear combination of the component spectra (see Fig. 1). In some regions the conservative excitonic CD bands can be recognized, but the major CD bands are largely asymmetric and about an order of magnitude stronger than the CD of the monomer complexes or particles. These anomalous CD characteristics of granal chloroplasts and LHCII macroaggregates strongly resemble those in other large and highly organized systems, such as DNA aggregates, condensed chromatins, viruses etc. (Tinoco et aI., 1987). CD signals in these samples originate not solely from differential absorbance, At. -AR, but differential scattering, SL -SR, of left and right circularly polarized light also contributes to the signal of differential extinction: eL-eR=(At.-~)+(SL-SR). Systematic studies showed that these signals, although they may be accompanied by artifacts which are difficult to separate from the CD, are associated with the macroorganization of the system and provide valid and unique structural information about large chiral objects (Tinoco et al., 1987; Keller & Bustamante, 1986).

The theoretical framework for understanding the CD in large chiral assemblies has been developed extensively over the past two decades (Tinoco et al., 1987). Preferential scattering of one of the circular polarizations of the light by chiral sample has been interpreted within the framework of the CIDS theory (Circular Intensity Differential Scattering) (Bustamante et al., 1985). The theory for psi-type aggregates describes the interaction of light with large inhomogeneous molecular aggregates containing a high density of intensely interacting chromophores (psi, polymer and salt-induced). It has been shown that these chiral molecular macro structures can be imaged by differential polarization imaging, the theory of which has also been elaborated (Kim et al., 1987a; Kim et aI., 1987b).

CIDS results from interference effects of wavelets generated at different points in the object in which the point polarizable groups are helically arranged, and the pitch and the diameter of the helix are commensurate with the wavelength (d>lJlO) of the measuring light. The interference phenomenon is greatest when the wavelength of the circularly polarized light closely matches the dimensions of the macro helix (Bustamante et aI., 1985) but it is largely independent of the intensity of non-polarized scattering.

Theory predicts that CIDS:

(6)

as a function of scattering angle 0 exhibits lobes of alternating sign, the profile of which is determined by the helical parameters of the chiral macrostructure. In the first approximation, CIDS is described for non-interacting chromophores which are arranged along a macrohelix.

Psi-type CD theory (Keller & Bustamante, 1986a, 1986b) is based on the classical theory of coupled oscillators (De Voe, 1965). According to the theory of De Voe, which was elaborated for small complexes, the light induces transition dipoles in the polarizable groups of the object, and these are thought to interact as static dipoles with a distance-dependence of (3. However, in large objects, in addition to these short-range interactions long-range effects must also be considered, and in psi-type aggregates the full electrodynamic interaction among the dipoles must be considered (Keller & Bustamante, 1986b).

Psi-type CD characteristics depend largely on the size and helical parameters of the aggregate. The significance of sizes can be understood by taking into account that whereas in small aggregates, upon the interaction with the light, the entire aggregate at any instant is in the

129

same phase of the wave, in large aggregates, the sizes of which are commensurate with the wavelength of light, retardation effects also play a role. Furthennore, in large aggregates the chromophores at large distances can be coupled via radiation coupling mechanism between the dipoles (at distant points of observation the oscillating dipole can be considered as a radiating spherical wave.) The contribution of radiation coupling, which is essentially due to multiple internal scattering inside the particle, in helically organized macroaggregates packed densely with dipoles can be comparable to that of the static dipole coupling.

The electric field at any point in space x due to an oscillating electric dipole, ~, located at Xi

can be written as:

E~iPole = 47r k 2 r(x, x') ·Ili (7)

Furthennore,

(8)

which means that the electric field at any point is the superposition of the incident electric field (Eo(x» and the sum of the fields produced by all oscillating dipoles. Thus, for the general case the quantity of interest in understanding the CD is not the coupling between the individual pairs of chromophores but the coupling between any given chromophore and the rest of the chromophores in the macro domain.

The interaction tensor is given in an explicit fonn (Keller & Bustamante, 1986b):

Ikr • ikr ikr 1 r(x,X') = (3rr-l) e -(3rr-l)_1_e_+(I-rr)~--o3(r) (9)

4;r k2 r3 47r kr 2 4;rr 3 k 2

where r = x - x', r = Irl, r = r I r andk = 27r I A . The first and third tenns of the tensor, with

(3 and (\ describe the static dipole coupling and the radiation tenn, respectively. The second tenn, with (2, is called the intennediate coupling. The last term ensures that the self-interaction is zero.

It has been shown that due to static, intennediate and radiation coupling among dipoles, intense "anomalous" CD signals can arise (Keller & Bustamante, 1986a). Theory predicts that in helically organized macrodomain (Kim et a1., 1986) the magnitude of the signal is controlled by the volume, chromophore density and pitch. The shape of the spectra was shown to depend mostly on the pitch and the handedness, with sign-inverted mirror spectra for opposite handedness. Since psi-type aggregates also satisfY the criterions for CIDS, the psi-type CD is always accompanied by differential scattering.

An interesting feature of psi-type aggregates is that if the long-range coupling between the dipoles is strong, the excitation generated at one chromophore is able to delocalize over the entire aggregate. This is called collective absorbance which increases or decreases at a given wavelength, depending on how well the light is able to produce a collective excitation in the system. When the group polarizabilities are made weaker, the groups are taken further apart or the density of the chromophores inside the aggregate is diminished, the ability of an excitation created at a given position in the aggregate to transfer to a different part becomes less and less efficient. In well coupled aggregates, however, the excitation can delocalize for large distances.

Differential polarization imaging is a method which is suitable for structural investigations of large anisotropic objects. CD imaging provides information on the helical macroorganization of large molecular structures, and can resolve domains of intense chirality. The CD-image is composed of points carrying infonnation on the differential extinction of left and right circularly polarized light (Kim et a1., 1987a, 1987b).

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CHIRAL MACRODOMAINS IN THYLAKOID MEMBRANES AND LHCn

Psi-type features

It has been recognized that the anomalous features of the CD spectra of chloroplasts are associated with the ability of the granal membranes to differentially scatter left and right circularly polarized light (philipson & Sauer, 1973). Later, it was shown that scattering does not distort significantly the "true" CD bands in chloroplasts and LHCII macroaggregates (Garab et aI., 1988): in these samples magnetic CD (MCD) of Chis, which originates from inside the complexes and is therefore subjected to the same alterations (if any) as natural CD, has been shown not to be distorted significantly by differential scattering.

It has also been shown that whereas non-polarized scattering (turbidity) is largely insensitive to structural rearrangements in the membrane system, the differential polarization scattering component of the CD signal selectively responds to ultrastructural changes accompanying the formation of macroaggregates (Garab et aI., 1991). Furthermore, in chloroplast suspensIons under conditions which scatter light intensely but do not pennit the macro aggregation of particles, non-polarized scattering was shown not to distort the CD signal to any noticeable extent (Garab et aI., 1991). Thus, it was concluded that the circular differential scattering signal is superimposed to the excitonic CD signals, which carries distinct physical information on the macroorganization of the pigment system.

CIDS measurements, which were carried out at 515 nm between 0° and 170°, showed the presence of intense lobes of alternating sign, with negative and positive maxima around 15° and 70°, respectively. Modelling of the CIDS data by a simple helical array of uniaxial polarizable groups suggested that the chiral structure is left-handed with pitch and radius of the order of200-400 nm (Garab et aI., 1988).

The helically organized macrodomains were imaged in a confocal scanning differential polarization microscope (Finzi et aI., 1989,1991). The CD images displayed signals of opposite signs emerging from discrete regions with local dichroic values much larger than anticipated (Fig. 2).

It was shown, by microspectropolarimetry, that the domains with intense positive or negative CD signal exhibited broad, non-conservative bands between 650 and 700 nm (Finzi et aI., 1989). The positive and negative signals, originating from different islands, almost canceled each other out, suggesting that in a macroscopic sample the prevailing CD signals represent average values. These data rule out the interpretation of the anomalous CD of chloroplast suspensions in terms of short-range interactions. On the other hand, all available data are fully consistent with a psi-type origin of the major CD bands, i.e. an origin from chirally organized macrodomains with sizes commensurate with the wavelength of the visible light and with high density of interacting chromophores. Since in the pigment-protein complexes the chromophore density is high (about 1 Chi per nm\ and the pigment dipoles are non-randomly oriented and participate in energy transfer interactions, a macroaggregation of particles with long range order is, in fact, expected to give rise to psi-type CD if the size of aggregate reaches about one quarter of the wavelength. In accordance with the prediction of psi-type theory, it was found that the magnitude of the major CD bands in thylakoid membranes increased with the size of the macroaggregates (Barzda et aI., 1994). Size-dependency of the magnitude of the psi-type CD bands was also demonstrated with LHCII macroaggregates when the diameter of the macroaggregates was varied between about 100 nm and 5 !-Lm (Fig. 3).

Factors influencing the macrodomain organization; ultrastructure, energy migration

By measuring CD and CIDS signals in wild type and chlorina (ChI-b-Iess) mutant membranes it was shown that in wild type chloroplasts, the formation of macrodomains was

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Figure 2. High-resolution CD image of a chloroplast. The dichroic ratio, (h -10/(iL +110, varies between about -0.015 (black tones) and +0.015 (white). Bar '" 2~. (Reproduced with permission from Finzi et al., 1989.)

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Figure 3. Circular dichroism spectra ofLHCn in different aggregation states; solid line, large aggregates ('" 3-5 flm in diameter); dashes, intermediate size aggregates (1-2 mm); dots, trimeric and small aggregate «100 ~) form.

governed by interactions of LHCIIs (Garab et a1. , 1991). In the absence of major LHCn polypeptides, in the mutant, the formation of macro domains is apparently mediated by other complexes having weaker interaction capabilities.

Two external factors have been identified which regulate the macrodomain organization of thylakoid membranes (Fig. 4): (i) electrostatic screening by divalent cations under conditions that favour stacking and (ii) the osmotic pressure of the medium which probably influences the lateral interactions, i.e. the "packing" density of PSII particles. It has also been shown that these two factors govern preferentially the negative and the positive CD bands, respectively (Garab et a1., 1991).

As shown by CD measurements in juvenile wheat thylakoid membranes in which the LHCn content is under the control of a diurnal cycle, the macrodomain organization is largely facilitated by the synthesis of the peripheral complexes ofLHCII (Busheva et a1., 1991 ; Liker & Garab,

132

1994). These complexes are likely to playa role in interconnecting PSII particles. An analysis of fluorescence induction kinetics also showed that accumulation of the peripheral complexes and the increased dominance of the macrodomains were accompanied by an increase in the ability of membranes to undergo State Transitions (Busheva et ai., 1991).

It has earlier been well established that LHCII facilitates stacking of membranes (Mullet & Arntzen, 1980). The macrodomain organization is related to the high self-aggregation capability of PSII particles via adhesion of the peripheral LHCII complexes. In this way, aggregation of PSII particles can lead to the formation of nearly homogenous regions which are enriched in PSII and exclude PSI to a large extent. Both stacking, i.e. head-to-head aggregation, and "packing", i.e. lateral self-aggregation of PSII particles have been shown to occur (Garab et al., 1991; Barzda et al., 1994). This type of macroaggregate-formation can lead to the sorting and lateral separation ofPSII and PSI.

A structure-stabilizing role of the LHCII-based macrodomains is in line with the findings that purified LHCII under favorable electrostatic conditions, i.e. in the presence of cations, readily forms liquid crystalline sheets (Mullet & Arnzen, 1980; Kiihlbrandt, 1984). LHCII macroaggregates also exhibit psi-type CD bands. However, the dominant handedness in LHCII macroaggregates is different from that of the macrodomains in thylakoids (Gregory et al., 1'980) (cf Figs. 1 and 3). This is most likely the consequence of the difference between the organization ofLHCII trimers in vivo and in vitro (Kiihlbrandt et al., 1994).

100 100

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Figure 4. Dependence of the relative amplitudes of the 674 run and 688ruTI circular dichroism bands of wild type (WT) and Chl-b-Iess mutant (M) barley thylakoid membranes on the concentration ofMgCh (left) and sorbitol (right). The membranes were suspended in a buffer containing 10 ruM Tricine (PH 7.8) and 10 ruM KCI supplemented with MgCh or sorbitol. The CD intensities in WT were about 2-5 times larger than in M. (Reproduced with pennission from Garab et a1., 1991)

It is likely that chirally organized macrodomains play an important role in determining the energy migration pathways in the antenna. This structural unit could constitute the basis for the "connectivity" between PSII particles and for long-range excitation energy migration in the membrane. As pointed out above, psi-type aggregates possess the ability of delocalization of the excitation energy, which can extend virtually to the entire aggregate. It is not clear, however, whether or not in thylakoid membranes the long-range coupling between the dipoles is strong enough for such delocalization to occur. Investigations of ultrafast absorbance transients under singlet-singlet annihilation conditions on LHCII indicate that macroaggregation of LHCII is accompanied by dramatic changes in the energy migration pathways in the pigment system (Y. Barzda, G. Garab, L. Gulbinas and L. Valkunas, unpublished).

Light-induced reversible structural rearrangements; regulatory energy dissipation

The chirally organized macrodomains in chloroplasts were shown to undergo light-induced

133

reversible structural changes which could be detected in all major CD bands (Garab et al., 1988). They could be elicited either with blue or red excitation of the membranes. These reversible changes occur in the time range of lOs to 1-3 min but the rates of the changes varied from batch to batch. The eXtent of the changes varied typically between 30% and 80% of the amplitude of the major bands in the dark. These changes could be driven by either PSI or PSII, or with full chain electron transport, albeit with differences in the amplitudes and the sensitivities toward uncouplers. The light-induced ~CD could be inhibited by uncouplers and penetrating buffers (Fig. 5). This suggested the role of ~pH. However, it was also shown that a collapse of ~pH did not influence the recovery rate of the signal, thus ruling out that ~CD directly measures the transmembrane ~pH.

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Figure 5. Light-induced changes in the circular dichroism of spinach thylakoid membranes at 510 nm; the effect of2 J.tM gramicidin (gram), 4 mM NILCI, 2IJ.M nigericin (nig).and 0.5 mM imidazole (imid). Red light of6 m W /cni was turned on and off as indicated by the arrows. The thylakoid membranes were suspended in 20 ruM Tricine (pH 7.8), 0.4 M sorbitol and 5 ruM MgCh, ChI-(a+b) content, 20 mglml; optical pathlength, 1 cm. (Reproduced with pennission from Garab et aI., 1988)

Recent investigations showed that the rate of the changes varied nearly linearly with the intensity of the exciting light. More importantly, the light intensity dependence of the initial rate showed no indication for saturation even though the intensity of the beam exceeded by a factor 2-4 the saturating intensity for the rate of the photosynthetic electron transport. These data suggest that, although ~pH appears to play an important role in the structural changes, it is not caused by it. Preliminary data in thylakoid membranes strongly suggest that the changes are related to the macrodomain organization of the antenna system rather than to the operation of the photochemical apparatus per se (A. Ist6kovics and G. Garab, unpublished).

It has been shown that antimycin and myxothyazol inhibit the light-induced ~CD between 670 and 700 nm, and between 480 and 540 nm, and the turbidity changes around 510 nm (Istokovics et ai., 1992). Antimycin-sensitive turbidity changes have earlier been related to qE (Horton et al., 1991). Investigations are underway to clarifY the exact role of the structural changes, which are reflected by ~CD, in the non-photochemical quenching in the antenna. Preliminary results of a systematic investigation, which will be published elsewhere, show that most agents that affect qE also influence the light-induced ~CD. Thus, these data are in line with the hypothesis put forward by Horton et al. (1991) that the non-photochemical quenching is correlated with the aggregation state of the LHCII in the thylakoid membranes: for structural changes (~CD) to occur the macrodomains must be present. Since, however, according to our

134

results the LHCII complexes (pSII particles) appear to be highly aggregated in the dark, qE cannot be explained without invoking structural changes in the macroaggregates.

The exact physical meaning of ~CD is yet to be determined. It is clear, however, that it reflects significant changes in the macroorganization of the antenna system. In other terms, ~CD appears to indicate that the interaction of the macrodomains with light induces overall changes in the macroorganization of the antenna and the changes are not confined to some of the individual complexes.

To further clarify the role of the macrodomain organization of the antenna., systematic investigations have been carried out on LHCII. It was found that chirally organized macro domains of isolated, purified LHCII also possess the ability to undergo reversible light­induced structural rearrangements which can be detected as gross changes in the psi-type CD (Vianelli et al., 1993) (Fig. 6). Earlier, in LHCII preparations, light-induced changes in the fluorescence were reported (Jennings et al., 1991). We found that ~CD in LHCII occurs only ifit is macroaggregated. The initial rate of the ~CD was shown to be linearly proportional to the intensity of the exciting beam. It is proposed that the structural rearrangements are related to alterations in the bulk properties of the macroaggregate which may lead e.g. to proximity­changes of LHCII sheets. The physical mechanism is not known but the dynamic properties are likely to be related to the collective absorbance units occurring in psi-type aggregates.

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Figure 6. Light induced changes in the circular dichroism of purified aggregated LHCII (suspended in 10 mM Tricine, pH 7.8). Chl-(a+b) content, 20 J.!glml; optical pathlength, 1 cm; the intensity of the red actinic light was 30 mW/cm2.

CONCLUSION

Circular polarization studies have revealed the existence of chirally organized, psi-type macroaggregates in thylakoid membranes. It was shown that LHCII, which itself also readily forms chiral macrodomains, plays an important role in the adhesion of PSII particles in the membrane. It is suggested that formation of the macrodomains plays a role in the lateral separation of the two photosystems and constitute the structural basis for long-range migration and/or delocalization of the excitation energy in the granum membranes. Light-induced structural rearrangements of the macrodomains are probably involved in regulatory processes. Although the elucidation of the nature and the underlying physical mechanism of the structural reorganization requires further systematic investigations it can be concluded that psi-type macroaggregates of LHCII appear inherently flexible upon the absorbance oflight.

With LHCII the size of the aggregate can be varied over a broad range, measurements can be carried out in the visible spectral range, and our knowledge concerning the structure is far

135

more advanced than for other psi-type aggregates. Thus, LHCII appears to be well suited for systematic studies of psi-type aggregates which may have important function in different biological systems.

REFERENCES

Anderson, JM. & Andersson, B. (1988) Trends in BioI. Sci., 13: 351-355. Barzda, v., Mustardy, L. & Garab, G. (1994) Biochemistry, 33: 10837-10841. Bassi, R, Machold, O. & Simpson, D. (1985) Carlsberg Res. Commun., 50:145-162. Breton, J & Vermeglio, A (1982) in "Photosynthesis", (Govindjee, ed.), pp. 153-193, Academic Press, New York Busheva, M., Garab, G., Liker, E., T6th, Z., Szell, M. & Nagy, F. (1991) PlantPhysioI., 95: 997-1003. Bustamante, C., Maestre, M.F. & Keller, D. (1985) Biopolymers, 24 (8): 1595-1612. DeVoe, H (1965) J. Chern. Phys., 43: 3199-3208. FeIlIla, RE. & Matthews, B.W. (1975) Nature, 258: 573-574. Finzi, L., Bustamante, C., Garab, G. & Juang, C-B. (1989) Proc. Natl. Acad. Sci. USA, 86: 8748-8752. Finzi, L., Ulibarri, L. & Bustamante, C. (1991) Biophys. J., 59: 1183-1193. Forster, T. (1965) in "Modem Quantum Chemistry. Part 1lI: Action of Light and Organic Crystals", (Sinanoglu,

0., ed.), pp. 93-137, Academic Press, New York Frank, HA & Cogdell, RJ (1993) in "Carotenoids in Phoptosynthesis", (young, A & Britton, G., eels.), pp.

253-326, Chapman & Hall, London. Garab, G., Faludi-Daniel, A, Sutherland, J.C. & Hind, G. (1988) Biochemistry, 27: 2425-2430. Garab, G., Kieleczawa, J, Sutherland, J.e., Bustamante, e. & Hind, G. (1991) Photochem. PhotobioI., 54:

273-281. Garab, G., Leegood, RC., Walker, D.A., Sutherland, J.C. & Hind, G. (1988) Biochemistry, 27: 2430-2434. Garab, G., Szit6, T. & Faludi-Daniel, A (1987) in 'The Light Reactions", (Barber, J., ed.), pp. 305-339, Elsevier,

Amsterdam, New York, Oxford. Garab, G., Wells, KS., Finzi, L. & Bustamante, e. (1988) BiochemiStry, 27: 5839-5843. Gregory, RP.F., Demeter, S. & Faludi-Daniel, A (1980) Biochim. Biophys. Acta, 591: 356-360. Hernelrijk, P.w., Kwa, S.L.S., van Grondelle, R & Dekker, JP. (1992) Biochim. Biophys. Acta 1098: 159-166. Horton, P., Ruban, AV., Rees, D., Pascal, AA, Noctor, G. & Young, AI. (1991) FEBS Lett., 292 (1,2): 1-4. Houssier, C. & Sauer, K (1970) JAm. Chem. Soc., 92: 779-791. Istokovics, A, Lajk6, F., Liker, E., Barzda, v., Simidjiev, I. & Garab, G. (1992) in "Research in Photosynthesis If',

(Murata, N., ed.), pp. 631-634, Kluwer Academic Publishers, Dordrecht, Boston, London. Jennings, Re., Garlaschi, F.M. &Zucchelli, G. (1991) Photosynth. Res., 27: 57-64. Keller, D. & Bustamante, e. (1986 a) I. Chem. Phys., 84: 2961-2971. Keller, D. & Bustamante, e. (1986 b) I. Chem. Phys., 84: 2972-2979. Kim, M., Keller, D. & Bustamante, C. (1987 a) Biophys. I., 52: 911-927. Kim, M., Ulibarri, L. & Bustamante, C. (1987 b) Biophys. J., 52: 929-946. Kim, M., Ulibarri, L., Keller, D., Maestre, M.F. & Bustamante, e. (1986) I. Chern. Phys., 84: 2981-2989. Kiihlbrandt, W. (1984) Nature, 307: 478-480. Kiihlbrandt, W., Wang, D.N. & Fujiyoshi, Y. (1994) Nature, 367: 614-621. Kiihlbrandt, W., Wang, D.N. & Fujiyoshi, Y. (1994) Nature, 367: 614-621. Liker, E. & Garab, G. (1994) Physioi. Plantarum, in press, Lu, X. & Pearlstein, RM. (1993) Photochem. PhotobioI., 57: 86-91. Mullet, JE. & Arntzen, C.J. (1980) Biochirn. Biophys. Acta, 589: 100-117. Pearlstein, RM. (1982) in "PhOtOsyntllesis" (Govindjee, ed.), pp. 293-330, Academic Press, New York. Pearlstein, RM. (1991) in "Chlorophylls", (Scheer, H, ed.), pp. 1047-1078, CRC Press, Boca Raton. Philipson, KD. & Sauer, K (1973) Biochemistry, 12: 3454-3458. Staehelin, L.A. (1976) J Cell BioI., 71: 136-158. Szit6, T., Garab, G., Mustardy, L., Kiss, JG. & Faludi-Daniel, A (1984) Photobiochem. Photobiophys.,8:

239-249. Tinoco, I.J., Mickols, W., Maestre, M.F. & Bustamante, C. (1987) Aun. Rev. Biophys. Biophys. Chem., 16:

319-349. Vianelli, A, Barzda, v., Jennings, RC. & Garab, G. (1993) Book of Abstracts, 11th Internatl. Biophys. Congr.

189. (Abstract) Woody, R W. (1985) in "The Peptides", (Hraby, V.J., ed.), pp. 15-114, Acad.Press, New York Zuber, H & Biunisholz, RE. (1991) in "Chlorophylls", (Scheer, H, ed.), pp. 627-703, CRC Press, Boca Raton.

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INTERACTION OF UV RADIATION WITH THE PHOTOSYNTHETIC SYSTEMS

Janet F. Bornman

Lund University, Plant Physiology, Box 117, S-221 00 Lund, Sweden

INTRODUCTION

Over the last few decades attention has focused on the stability of the ozone layer with respect to increasing ultraviolet-B (UV-B, 290-320 nm) radiation reaching ground level. Although many of the ozone-depleting chemicals are scheduled to be phased out in the near future, in practical terms there will be large discrepancies in the rates of phasing out among countries. Chlorofluorocarbons, with lifetimes of many decades, and nitrogen oxides (N0x) are examples of compounds effective in the breakdown of stratospheric ozone (Prinn 1994). However, disturbance of ozone chemistry is also a result of interactive effects with other global changes which are occurring, the mechanisms of which are not well understood.

In this regard, interesting if alarming insight into the understanding of the Antarctic ozone "hole" has revealed that this hole seems very much dependent on a set of chemical processes initiated by formation of polar stratospheric clouds (PSCs), composed of water ice and nitric acid trihydrate, which form at night during the polar winter. These clouds act as catalytic surfaces for the release of chlorine and bromine from chemically inert molecules. When the stratosphere starts warming up again these released compounds can react with ozone molecules causing their destruction. Since the stratosphere of the Northern hemisphere is much more active (and hence temperatures are greater) than the Southern hemisphere, decreases in ozone are so far less in the North (Mahlman 1992). However, the increase in global warming may change this. With the greenhouse effect re-emission of heat energy from the earth to the upper atmosphere is decreased, which results in cooling of this atmosphere. In addition, long-term reductions in ozone will contribute to further cooling (less absorption of solar UV; Austin et al. 1992). Conditions may then become more favourable for formation of PSCs over the Arctic regions and larger areas of the Northern hemisphere.

Methane, which is much more effective in absorbing infrared radiation than C02, has also increased in recent years (now ca 1 to 1.5% year-I). This will increase the stratosphere's internal source of water vapour (oxidation of methane), also contributing to the likelihood ofPSCs forming in the Northern polar regions (Mahlman 1992). In fact recent evidence indicates a feedback mechanism whereby increases in C02 actually promote

Light as all Energy Source and Information Carrier in Plant Physiology Edited by JeIlllings et aI., Plemnn Press, New York, 1996 137

increases in methane over wetlands (Dacey et al. 1994). This is due to the fact that an increased C02 generally increases biomass allocation to below ground organs. In wetland areas this means that there is more biomass that will be decomposed anaerobically, with methane being given off as a byproduct.

In terms of the evolution of photosynthesis under a climate of intense UV radiation, different theories have been advanced for survival of primitive organisms when the oxygen content was seemingly close to zero (ca 2 x 109 years ago), and the ozone layer not yet a protective shield. Three and a half thousand million years ago the first photosynthesizing organisms, with only one photosystem, probably evolved a sulphur-based C02 fixation, and were replaced later, when atmospheric oxygen increased, by oxygen-evolving organisms with both photo systems I and II (Olson & Pierson 1986). In turn, these increases in atmospheric oxygen led to the absorption of UV radiation and gradual formation of the stratospheric ozone layer. A variety of repair and protective mechanisms evolved, indicating that UV radiation is an important environmental factor for many life forms.

Exposure of plants to both ambient and elevated UV -B radiation may induce changes in morphology, ultrastructure, physiology, biochemistry and genetics (for a review see Bornman & Teramura 1993). Often a change at one of these levels can have an effect at another level, making the distinction between different targets of UV radiation difficult. While many detrimental effects on plants by UV-B radiation have been documented, adaptive and natural photomorphogenic responses are also involved.

HOW DO PLANTS PERCEIVE UV RADIATION?

Interaction of different photoreceptors

Plants appear to have several UV photoreceptors operating at different levels for different physiological, biochemical and genetic processes. UV radiation also affects photo receptors absorbing in other wavelength ranges as well as in the Uv. Phytochrome, for instance, is interconverted between its two forms, Pfr and Pr due to absorption ofUV by the protein moiety (pratt & Butler 1970). Different photoreceptors, including UV photoreceptors, may act synergistically with other photoreceptors (Hashimoto & Yatsuhashi 1984). An example of this interaction is UV-B-induced flavonoid synthesis, which is further enhanced upon excitation of a blue light receptor (Duell-Pfaff & Wellmann 1982, Ohl et al. 1989) and can also be modified by far-red light, pointing to the involvement of phytochrome (Bruns et al. 1986).

Thus apart from evidence for distinctive, independent UV-B photoreceptors, receptors ascribed to mainly blue and UV-A regions of the spectrum also overlap in the UV-B. Both pterins and flavins have been implicated in certain responses to UV-B radiation (Galland & Senger 1988; Ensminger & Schafer 1992).

Due to their strong absorption in the UV -B region of the spectrum, other molecules which may play a part in the signalling pathway are nucleic acids, aromatic proteins and quinones (see next section). Flavonoids, pigments which increase upon exposure to UV-B radiation, and which may trigger other processes within the signalling pathway, serve several functions including the roles of effective UV filter and scavenger of oxidizing species (Takahaina et al. 1984; Husain et al. 1987; Tournaire et al. 1993). These roles offlavonoids are of particular importance for the protection of many plant processes including photosynthesis (see below) in an environment of enhanced UV-B radiation.

Response to UV-B radiation at the level of the chloroplast

Many of the mechanisms of optimization and regulation of light energy are concentrated within the photo system II (pS II) complex of the electron transport chain. It is

138

also this complex which seems to be one of the main targets of UV-B radiation (see Bornman 1989). Consequently it is to be expected that there will be a complex interaction between visible light effects and those imposed by UV-B radiation.

The synthesis, degradation and reassembly into grana thylakoid membranes of the Dl polypeptide, the apoprotein of the secondary quinone acceptor QB of the reaction centre of PS II, is responsive to several kinds of stresses, including radiation and herbicide application (see e.g. Sundby et al. 1993; Giardi et al. 1992; Draber et al. 1993). Degradation ofDl is important for the repair of PSII under photoinhibitory conditions (see Baker, this volume). Under conditions of enhanced levels of UV -B radiation the rate of degradation is increased (Greenberg et al. 1989a,b; Bornman & Sundby-Emanuelsson 1995), pointing possibly to the need to remove an increased amount of damaged Dl and/or the need for further down­regulation due to the extra burden of a high amount of energy-rich UV photons. According to work by Greenberg et al. (I989a) the degradation ofDl by UV-B radiation seems to be implemented through a quinone, the semiquinone anion radical, one of the three redox states of plastoquinone (Crofts & Wraight 1983). The Dl polypeptide thus mediates electron flow by binding and unbinding QB in its different redox states. Other photoreceptors, namely chlorophylls and carotenoids, may be responsible for triggering D 1 degradation through absorption of visible radiation. Also here there is interaction between the two photoreceptors whereby degradation rate ofDl is greater when the plant material is exposed simultaneously to both UV-B and visible radiation than if either spectral region is applied separately (Greenberg et al. 1989a; Bornman & Sundby-Emanuelsson 1995). The sensitization of the UV-B photoreceptor is followed by cleavage and the appearance of subsequent breakdown products (Greenberg et al. 1987). It has also been proposed that the process of actual degradation of D 1 by UV -B radiation differs from that by visible radiation in that UV-B-induced degradation occurs at different cleavage sites and is independent of oxygen (Friso et al. 1994b). The D2 apoprotein is also degraded by UV-B radiation. One of the main cleavage sites was identified in that region of the protein involved in binding QA, and again cleaving is apparently accomplished without the intervention of oxygen species (Friso et al. 1994a).

Another target of UV-B radiation is the chloroplast ATPase, the CFoF I-ATPase, which synthesizes ATP from ADP and phosphate using the H+ gradient, and responds to UV-B radiation in Pisum sativum with a reduction in both the activity and amount of the enzyme, with the amount showing a 35% greater reduction than the activity. Part of this discrepancy was apparently compensated for by activation of the remaining functional complexes (Zhang et al. 1994).

Due to the search for clearly defined mechanistic processes, large UV-B irradiances have been used in some of the studies discussed above and in subsequent sections; in some instances also without simultaneous exposure to visible radiation. Therefore in certain cases it remains to be shown to what extent the effect of more natural levels of UV -B radiation still will be sustained and measurable.

Response to UV-B radiation at the transcription level

Several of the processes affected by UV-B radiation have been traced back to changes in the levels of the mRNA transcripts for some of the chloroplast proteins. For example, the amounts of the 'two subunits comprising the main C02-fixing enzyme in C3 plants, ribulose 1,5-bisphosphate carboxylase (Rubisco), are reduced by UV-B radiation with the reduction being related to a decrease in the RNA transcript levels for these subunits (Jordan et al. 1992). The loss of Rubisco activity could be a direct effect on the enzyme due to UV absorption by the aromatic amino acids. Similarly, the mRNA transcripts for cab and psb A (genes encoding the Chi alb binding protein and Dl, respectively) decrease after plants are exposed to UV-B radiation (Jordan et al. 1991), as do the transcripts for the cytochrome blf complex of the electron transport chain (Zhang et al. 1994). A reduction in the transcript

139

levels for some of the polypeptides of the chloroplast ATPase complex also occurs (Zhang et al. 1994).

Some of the reductions in photosynthetic efficiency seem therefore to be a result of a decreased expression of genes coding for several of the chloroplastic proteins as well as for two of the nuclear-encoded genes, namely cab and rbc S (ChI alb binding protein and the small subunit of the Rubisco protein, respectively). This decreased gene expression could have serious consequences particularly for proteins where a rapid turnover constitutes part of their regulatory and protective function. Reductions in ChI a and b have been reported in several studies (see review by Bornman 1989, Strid & Porra 1992, Jordan et al. 1994), and may also account for decreased photosynthetic efficiency. A relationship has been proposed between the cab RNA transcript levels and the amount of Chi a and b in Pisum sativum exposed to enhanced levels ofUV-B radiation (Jordan et al. 1994). The reduction by UV-B radiation of the Chi alb binding proteins may increase in some way the degradation of the Chi molecules, possibly accounting for the decrease reported in Chi a relative to ChI b after exposure to UV-B radiation. Degradation rather than decreased biosynthesis of Chi has been suggested (Strid & Porra 1992, Jordan et al. 1994).

The superoxide dismutases (SOD), localized in the mitochondrion matrix," chloroplast stroma and thylakoid membranes, as well as in the cytosol, are efficient scavengers of superoxide radicals (02-; Hassan & Scandalios 1990). One may therefore expect that increases in SOD would occur upon exposure of plants to enhanced levels of UV-B radiation. This appears to be only partly true. In contrast to the increase in mRNA transcript levels for two other stress indicators, glutathione reductase and chalcone synthase, the transcript levels for chloroplastic SOD (Cu/Zn) of Pisum sativum declined after plants were exposed to UV -B radiation (Strid 1993). The reasons for this decline are unclear, and could be due to several mechanisms of response. Strid (1993) proposed that if increased levels of SOD during UV-B exposure were part of a signal transduction, then a decline later on is probable. Alternatively, the requirement for chIoroplastic SOD may have decreased, or in severe cases where chloroplasts are damaged by UV-B radiation, down-regulation of genes encoding chloroplastic proteins may decrease (Strid 1993). On the other hand, the amounts of mRNA for the cytosolic Cu/Zn-SOD were markedly increased in Pisum sativum after 48 h of exposure to UV-B radiation (Strid & Zilinskas 1994). It is perhaps of significance that amounts of mRNA for other chloroplast-localized proteins which were analyzed, decreased in parallel.

MEASUREMENT OF RADIA nON INTO PLANT MATERIAL

Techniques for measuring UV radiation within plant tissues (e.g. Bornman & Vogelmann 1988, Cen & Bornman 1993, Day et al. 1992, 1993) using fibre optic microprobes of quartz can be used to gauge the relative amount and quality of radiation at different tissue locations. It is also useful for making correlations between different concentrations of UV -screening compounds such as flavonoids and certain other phenolics, and the degree of protection afforded by these pigments with depth inside plant organs. In addition, changes in leaf thickness due to radiation conditions are also reflected in the light gradients within e.g. a leaf. Both these modifying factors, as well as other more tenuous ones cari alter the internal light environment for photosynthesis. This was demonstrated in a study where plants were grown under moderate levels of supplemental UV-B radiation (6.3 kJ m-2 day-l biologically effective UV-B radiation) and high photosynthetically active radiation (PAR, 400-700 nm; 1800 !lmol m-2 s_I), or the high PAR alone. Under PAR and UV-B radiation flavonoid compounds increased as did leaf thickness, while ChI fluorescence and ChI content were reduced in some of the plants studied (Bornman & Vogelmann 1991). Some of these changes were correlated to changes in PAR measured within the leaves using fibre optic microprobes (Fig. 1). Thicker leaves from the UV-B growth

140

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Figure 1. Using a quartz fibre, spectral scans of photosynthetically active radiation (pAR, 400-700 run) were taken inside leaves of Brassica campestris from plants grown either with or without supplemental UV-B radiation (6.3 kJ m-2 day-I of weighted UV irradiance using the generalized plant action spectrum of Caldwell (1971) and calculated according to Bjorn & Murphy (1985)). Normalization to 100% shows the differences in spectral quality of the different light fluxes within the leaf. The fibre optic microprobe was driven into the leaves at three different angles: 0, 30 and 1500 , in order to measure mainly collimated, forward scattered and backscattered radiation, respectively. Measurements were done with the adaxial leaf surface facing the light source. (redrawn from Bornman & Vogelmann 1991). A, control plants; B, plants grown under supplemental UV-B radiation.

conditions scattered more light, although this increase in scattering could also have been due to a decrease in ChI content found in these leaves, as well as increases in intercellular space (Bornman & Vogelmann 1991; Fagerberg & Bornman, unpubl. data). Relationships may thus exist between UV screening at different tissue locations and the degree of protection afforded the photosynthetic systems. UV-B radiation is strongly attenuated within the first few cell layers and reflects the UV treatment of the plant. An example of this was found in Brassica napus where mainly collimated or parallel 310 nm (UV-B) radiation was reduced by 95% at 60 /lm within the leaves not previously exposed to UV-B radiation compared to the same reduction already at 36 /lm for UV-B-exposed leaves (Fig. 2) (Cen & Bornman 1993). Direct comparisons of measurements of photosynthesis in rye leaves which had accumulated different amounts of flavonoids reflected increasing protection of the photosynthetic system, although the UV ~screen was apparently imperfect (Tevini et al. 1991). This non-uniform filter effect has also been noted for a variety of other

)(

~ 0 .20 r---,-------..,------,

~ bv ~ o· il 0 . 1$ prob~ C

" " i: 0 . 10

~ ~ O . O~

.. ~ 0 .00 L~::2;::= __________ __:_:_-~

o 50 .00 .SO 200 250 300

De p lh. I'm

Figure 2. Penetration of 310 run radiation in leaves of Brassica napus grown for 16 days with or without supplemental UV-B radiation (9 kJ m-2 day-I of weighted UV irradiance using the generalized plant action spectrum of Caldwell (1971) and calculated according to Bjorn & Murphy (1985)). Measurements were done using a quartz optical ,fibre, which was driven into the leaf at 00 in order to measure mainly collimated radiation. Measurements were taken with the adaxial leaf surface facing the light source. (modified and redrawn from Cen & Bornman 1993).

141

species, with the observation being that penetration of UV-B radiation was greater along anticlinal epidermal cell walls than through the protoplast ofliving cells (Day et al. 1993).

A further analysis of the influence of UV-B radiation on photosynthesis and its modification by increased levels of UV -screening pigments was done on the D 1 polypeptide of PS II (Wilson & Greenberg 1993), where it was shown that the degradation rate of D 1 was reduced in plants previously adapted to UV-B radiation before implementation of treatment protocols. This rate of degradation was positively correlated with the increased levels ofUV-B screening pigments.

INTERACTIONS OF SPECIFIC STRESS FACTORS RELATED TO PHOTOSYNTHETIC PERFORMANCE

Several studies have dealt with the potentially modifying effect of other stresses imposed on the plant together with enhanced levels ofUV .. B radiation. The net effect can be negative, positive or elicit no measurable plant response. Investigation of these multiple interactions are important in an environment constantly modified by man's activities with regard to pollution and other practices which disturb the natural environmental balance.

The role of C02

Many environmental factors imposed simultaneously with different levels of UV-B radiation have an impact on photosynthesis and other processes. Of global importance is the influence of an enhanced C02 environment. Its effect on photosynthesis is in many instances a stimulatory one, at least initially; in addition, partitioning of biomass can be affected, with allocation often increasing in plant organs below ground. This change in allocation pattern can be further modified by the interaction of UV-B and C02 (Ziska & Teramura 1992, Sullivan & Teramura 1994). A 50% increase in the rate of photosynthesis with a near doubled ambient C02 concentration was reported for rice (Ziska & Teramura 1991). Under conditions of both enhanced C02 and UV-B radiation some of the stimulatory effects from C02 alone are negated by UV-B radiation (Ziska & Teramura 1992). At the physiological level, reduction in the efficiency of some processes occur. For instance, in one of the rice cultivars tested, a C02-induced increase in the rate of C02 assimilation was eliminated by an enhanced level of UV-B radiation. Also the commonly used fluorescence ratio, FvIFm (variable fluorescence normalized to the maximum), an indicator of quantum yield or photosynthetic efficiency (Bjorkman & Demmig 1987), was similarly reduced (Ziska & Teramura 1992). A reduction in the efficiency of the electron transport chain would ultimately affect the regeneration of ribulose 1,5 bisphosphate, and thus may partially account for the decreased rate of C02 assimilation. In other instances some of the negative effects on photosynthesis by UV-B radiation are merely ameliorated by high levels of C02. However, these responses seem to be very species specific and highly dependent also on other growth conditions such as water and nutrient availability (Stewart & Hoddinott 1993).

The role of water and nutrient availability

The response of photosynthesis to enhanced UV-B radiation can also be modified by water and nutrient status of the soil. Reductions in photosynthetic activity by UV-B radiation tend to occur when water is not limiting as opposed to the influence of UV under drought conditions (Sullivan & Teramura 1990). This points to the possibility that the induction of protective mechanisms induced by another stress, e.g. water, are sufficient to also counteract the impact of a stress such as UV radiation. Alternatively, the effects of water stress mask the effect of an additional stress (Sullivan & Teramura 1990). At least in short-term studies the same trend is seen for the interactive effects of UV -B radiation and

142

nutrient supply as is observed for UV-B and water availability, namely an apparent decreased sensitivity of plants to elevated levels of UV-B radiation during concomitant mineral deficiency (Murali & Teramura 1985a,b, 1987).

PAR and UV-B radiation

One of the interesting interactions is that seen between an increased level of UV-B radiation and high amounts of PARon the photosynthetic systems (mentioned briefly above). The effect of high PAR, leading in some cases to photoinhibition (absorption oflight in excess of that which can be utilized photochemically) has been extensively studied. The additional light energy stress on the photo systems by an increased level ofUV-B radiation is manifested in the response of the D 1 polypeptide of PS II in a way in which regulatory as well as damaging effects are observed. The xanthophyll cycle, another protective mechanism for dealing with excess absorbed light energy (Demmig-Adams & Adams 1992), may also be negatively affected by enhanced UV-B radiation (Pfundel et al. 1992).

Greenberg and co-workers (1989a) have observed the increased rate ofDI degradation when Spirodela cultures are exposed to UV-B radiation and visible light as compared to exposure to visible light only. In a photoinhibition study where leaves of Brassica napus were exposed simultaneously to high PAR (1 600 Ilmol m-2 s-l) and UV-B radiation (13 kJ m-2 day-I) not only was the degradation rate of D I increased but further evidence of increased photoinhibition with UV-B radiation was reflected in higher Fo (initial fluorescence in dark-adapted material) and a decreased Fm, maximum fluorescence (Fig. 3) (Bornman & Sundby-Emanuelsson 1995). The quantum yield of PS II, estimated from the ratio FvlFm showed additional reductions in UV and PAR treated material compared to high PAR alone.

Degradation of D 1 together with other processes which respond to excess absorbed energy serve regulatory functions even though under high stress conditions damage exceeding the rate of repair may occur. The xanthophyll cycle in the thylakoid membranes helps to dissipate excess light energy non-photochemically (qNP, Demmig-Adams & Adams 1992), and this can be measured together with the light energy that is quenched photochemically (qp). The non-photochemical quenching (qNP) generally increases during photoinhibition by high PAR (Krause & Behrend 1986, Horton et al. 1988). In the Brassica study there was an even greater increase in qNP during simultaneous exposure to high PAR and UV-B radiation (Bornman & Sundby-Emanuelsson 1995), suggesting an increased turnover of the xanthophyll cycle and/or quenching by inactive PS II reaction centres (Oquist et al. 1992).

Other environmental factors

Other abiotic factors interacting in different ways with UV-B radiation for which some information exists to date, include temperature and high concentrations of metals [Caldwell 1994 (temperature), Dube & Bornman 1992 (cadmium)]. Studies of interaction of UV-B radiation with biotic factors such as fungal, viral and insect attack are still relatively few (e.g. Semeniuk & Goth 1980, Orth et al. 1990, Panagopoulos et al. 1992), and show that there is variation in the response of the plant depending in some cases on whether infection occurs before or after exposure to enhanced levels ofUV-B radiation (Orth et al. 1990). For certain of the biotic and abiotic factors part of the response by the plant may be similar and could indicate a common pathway for signal transduction.

143

., .. oi " c OJ

" ., ... .. 0 ::l ~

0.50 .--.------:---.,..-----,

0.45

0.40

0.35

0.30

<:.50

<:.<:5

2.00

1.75

1.50

1.25

1.00

0.64

0.81

0.78

0.75

0.72

0.69

0 .66

0 .63

E2Z1 HL

HL+UV

0

= HL F./Fm

~HL t:V

234 Hours of exposure

Figure 3. Monitoring of chlorophyll fluorescence parameters, F 0 ' F m and FvIF m on leaf discs of Brassica napus. During treatment conditions the leaf discs were exposed to photoinhibitory light (HL, 1600 ).tmol m-2s-1 or to HL plus 13 kJ m-2 day-I of weighted UV irradiance using the generalized plant action spectrum of Caldwell (1971) and calculated according to Bjorn & Murphy (1985). Leaf discs exposed to radiation condition were measured from 0 to 4 h. (Data extracted from Bornman & Sundby-Emanuelsson 1995).

REFERENCES

Austin, 1.. Butchart, N. & Shine, K.P. (1992) Nature, 360: 221-225. Bjorkman, O. & Demmig, B. (1987) Planta, 170: 489-504. Bjorn, L.O. & Murphy, T.M. (1985) Physio!. Veg. , 23: 555-561. Bornman, 1.F. (1989) 1. Photochem. Photobiol. , 4: 145-158. Bornman, 1.F. & Vogelmann, T.C. (1988) Physio!. Plant., 72: 699-705. Bornman, J.F. & Vogelmann, T.C. (1991) 1. Exp. Bot., 42: 547-554. Bornman, J.F. & Tera.1l1ura, A.H. (1993) in "Environmental UV Photobiology", (A.R. Young, L.O.

BjOrn, 1. Moan & W. NuItsch, eds), Plenum Pub!. Co., New York. pp. 427-471. ISBN 0-306-44443-7.

Bornman, J.F. & Sundby-E~anuelsson, C. (1995) in "Environment and Plant Metabolism", Environmental Plant Biology Series (N. Smirnoff, ed.), BIOS Sci. Pub!. , Oxford, UK. pp. 245-262.

Brnns, B. , Hahlbrock, K. & Schafer, E. (1986) Planta, 169: 393-398. Caldwell, M.M. (197 1) in" Photophysiology" (A.C. Giese, ed.), Vo!. 6, pp. 131-177. Academic Press.

New York, NY. Caldwell, R. (1994) Plant Physio!. , 104:395-399.

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Cen, y.~P. & Bornman, 1.F. (1993) Plant., 87: 249-255. Crofts, AR & Wraight. C.1. (1983) Biochim. Biophys. Acta, 726: 149-185. Dacey, J.W.H., Drake, B.G. & KIug, M.1. (1994) Nature, 370: 47-49. Day, TA, Vogelmann, TC. & DeLucia, E.H. (1992) Oecologia, 92: 513-519. Day, T.A., Martin, G. & Vogel mann, TC. (1993) Plant Cell Environ., 16: 735-741. Demmig-Adams, B. & Adams, W.W. (1992) Annu. Rev. Plant Physiol. Plant Mol. BioI., 43: 599-626. Draber, W., Tietjen, K., Kluth, 1.F. & Trebst, A (1991) Angew. Chern. Int. Ed. Engl., 30: 1621-1633. Dube, S.L. & Bornman, J.F. (1992) Plant Physiol. Biochem., 30: 761-767. Duell-Pfaff, N. & Well mann, E. (1982) Planta ,156: 213-217. Ensminger, PA & Schafer, E. (1992) Photochem. Photobiol., 55: 437-447. Friso, G., Barbato, R, Giacometti, G.M. & Barber, J. (1994a) FEBS Lett. ,339: 217-221. Friso, G., Spetea, C., Giacometti, G.M., Vass, I. & Barbato, R (1994b) Biochim. Biophys. Acta 1184,

78-84. Galland, P. & Senger, H. (1988) Photochem. Photobiol., 48: 811-820. Giardi, M.T., Rigoni, F. & Barbato, R (1992) Photosynthetica, 27: 173-182. Greenberg, B.M., Gaba, v., Mattoo, A.K. & Edelman, M. (1987) EMBO 1., 6:2865-2869. Greenberg, B.M., Gaba, V., Canaani, 0., Malkin, S .. Mattoo, A.K. & Edelman, M. (1989a) Proc. Nat.

Acad. Sci. USA 86: 6617-6620. Greenberg, B.M., Gaba, V., Mattoo, A.K. & Edelman, M. (1989b) Z. Naturforsch., 44: 450-452. Hashimoto, T. & Yatsuhashi, H. (1984) in "Blue Light Effects in Biological Systems", (H. Senger,

ed.), pp. 125- 136. Springer-Verlag, Berlin. Hassan, H.M. & Scandalios, 1.G. (1990) in "Stress Responses in Plants: Adaptation and Acclimation

Mechanisms", (R. G. Alscher & 1.R Cumming, ed.), pp. 175-199. Wiley-Liss, Inc., New York. ISBN 0-471-56810-4.

Horton, P., Oxborough, K., Rees, D. & Scholes, 1.D. (1988) Plant Physiol. Biochem., 26: 453-460. Husain, S.R, Cillard, 1. & Cillard, P. (1987) Phytochemistry, 28: 2489-2491. Jordan, B.R, Chow, W.S., Strid, A. & Anderson, 1.M. (1991) FEBS Lett., 284: 5-8. Jordan, B.R, He, 1., Chow, W.S. & Anderson, 1.M. (1992) Plant Cell Environ., 15: 91-98. Jordan, B.R, James, P.E., Strid, A. & Anthony, RG. (1994) Plant Cell Environ., 17: 45-54. Krause, G.H. & Behrend, U. (1986) FEBS Lett., 200: 298-302. Mahlman, 1.0. (1992) Nature, 360: 209-210. Murali, N.S. & Teramura, A.H. (1985a) Physiol. Plant., 63: 413-416. Murali, N.S. & Teramura, AH. (1985b) 1. Plant Nutr., 8:177-192. Murali, N.S. & Teramura, AH. (1987) 1. Plant Nutr.,IO: 501-515. Ohl, S .. Hahlbrock, K. & Schafer, E. (1989) Planta, 177: 228-236. Olson, 1.M. & Pierson, B.K. (1986) Photosyn. Res. 9: 251-259. Oquist, G., Chow, W.S. & Anderson, 1.M. (1992) Planta, 186: 450-460. Orth, AB., Teramura, AH. & Sisler, H.D. (1990) Am. J. Bot., 77: 1188-1192. Panagopoulos, I., Bornman, 1.F. & Bjorn, L.O. (1992) Physiol. Plant. 84: 140-145. Pfiindel, E.E., Pan, R-S. & Dilley, RA (1992) Plant PhysioL 98: 1372,-1380. Pratt, L.H. & Butler, W.L. (1970) Photochem. Photobiol., II: 503-509. Prinn, RG. (1994) Ambio, 23: 50-61. Semeniuk, P. & Goth, RW. (1980) Environ. Exp. Bot., 20:95-98. Stewart, J.D. & Hoddinott, 1. (1993) Physiol. Plant., 88: 493-500. Strid, A. (1993) Plant Cell Physiol. 34: 949-953. Strid, A. & Zilinskas, B.A. (1994) Abstr. 463. in "International Congress of Plant Molecular Biology",

Amsterdam. Strid, A. & Porra, R1. (1992) Plant Cell Physiol., 33: 1015-1023. Sullivan, J.H. & AH. Teramura, AH. (1990) Plant Physiology, 92: 141-146. Sullivan, 1.H. & Teramura, A.H. (1994) Plant Cell Environ., 17: 311-317. Sundby, c., Chow, W.-S. & Anderson, 1. M. 1993. Plant Physiol., 103: 105-113. Teramura, AH. 1980. Physiol. Plant., 48: 333-339. Takahama, U., Youngman, R.1., & Elstner, E.F. (1984) Photobiochem. Photobiophys., 7: 175-181. Tournaire, c., Croux, S., Maurette, M.-T, Beck, 1., Hocquaux, M., Braun, AM. & Oliveros, E. (1993)

1. Photochem. Photohiol. 19: 205-215. Tevini, M., Braun, 1. & Fieser, G. (1991) Photochem. Photobiol., 53: 329-333. Wilson, M.l. & Greenberg, B.M. (1993) Photochem. Photobiol., 57: 556-563. Zhang, 1., Hu, x., Henkow, L., Jordan, B.R & Strid, A. (1994) Biochim. Biophys. Acta, 1185: 295-

302. Ziska, L.H. & Teramura, AH. (1991) Physiol. Plant. 84: 269-276. Ziska, L.H. & Teramura, AH. (1992) C02 Plant Physiol, 99: 473-481.

145

MOLECULAR BASIS OF PHOTORECEPTION'

Francesco Lenci, Nicola Angelini, Antonella Sgarbossa

CNR Istituto Biofisica, via San Lorenzo 26, 56127 Pisa (Italy)

INTRODUCTION

Any physiological phenomenon which is driven, triggered or modulated by sunlight in living organisms (from vision to photosynthesis, from photoreactivation to vitamin D biosynthesis, from phototropism to seed germination) is the final result of a series of physical, chemical, biophysical and biochemical processes which originate from the same "elementary" event: the absorption of a photon by the photoreceptor molecule.

In order to efficiently capture light over a large part of the solar spectrum, biological photoreceptors have often high molar extinction coefficients and broad absorption bands. The spectral sensitivity of the whole photoreceptive system can be widened if different chromophores, absorbing in contiguous spectral ranges, are spatially assembled in molecular frameworks in such a way as to maximize the probability of collective interaction among them, as is the case of photosynthetic pigments (see, e.g., Borisov, Garab and Mathis, this volume).

In the plant kingdom, from higher plants to unicellular algae, light can be used both as a source of energy (photosynthesis) and as a carrier of information about the external environment (phototropism, photomorphogenesis, light-regulated intracellular redistribution of chloroplasts, photomovements of freely motile microorganisms).

Like other sensory responses to different environmental stimuli (such as temperature, pressure, chemical substances), this capability of sensing and reacting to iIIumination conditions can provide photosynthetic organisms with essential strategies for their survival (see, e.g., Presti & Delbruck, 1978; and Rudiger & Lopez-Figueroa, 1992).

In the case of photosynthesis, a complex, interconnected "hierarchy" of pigments and processes is operating: the pigments of the reaction centers are devoted to converting light energy and driving the primary photochemical reactions and those of the light-harvesting complexes are dedicated to conveying the energy of the absorbed photon to the reaction centers (see, e.g., Barber & Andersson, 1994 and the afore referred Chapters).

In photo sensory phenomena, in general, the modification of a single receptor pigment molecule induced by the absorption of one photon can be sufficient to trigger the transduction chain. The signal associated with the absorbed photon is turned into an

• Dedicated to our friend Professor Pill Soon Song on the occasion of his 60th birthday

Light as an Energy Source and Information Carrier in Plant Physiology Edited by Jelmil1gs et al., P1emll11 Press, New York, 1996 147

intracellular biochemical/biophysical signal, which is processed by the cell to eventually yield the physiological response (Lenci et al., 1991; Lipson & Horwitz, 1991).

These two different tasks of conveying and converting light energy and of detecting and transducing photic stimuli may also be accomplished by very similar chromophores. In some cases, the very diversity and specificity in photophysiological function mainly result from structural properties of the molecular environment which the chromophores are bound to (apoproteins, in most cases), rather than from a really different chemical nature of the chromophores (Schaffner et al., 1990).

Photosynthetic light-harvesting chromophores are closely spaced, bound to the antenna apoproteins so as that their dipoles have a well defined mutual orientation. In this ordered molecular structure, the photochemically not reactive light-harvesting chromophores allow smooth and efficient flow of excitation energy to the reaction center (Fig. I).

Individual chromophores of photosensing systems, on the contrary, are usually loosely bound to flexible molecular frameworks and isolated from each other. The spatial separation of the chromophores does not allow energy transfer among them and the conformational mobility of the complex gives rise, upon absorption of even a single photon by the pigment, to local rearrangements of the molecular domain around the chromophore (an interesting exception has been suggested for Euglena by several Authors, and will be discussed below). This local perturbation initiates the chain of molecular events which are at the basis of the sensory transduction process, ultimately ending in the physiological response (see, e.g., the case of phytochrome discussed in Schaffner et al., 1990 and Song, this volume) (Fig. 1).

PHOTOCOUPLING PHOTOSENSORY

light

Photosynthesis Photolransduction

Figure 1 Schematic representation of primary photomodifications in photosynthetic (left) and photosensory (right) units.

In any case, regardless of the role they play (photocoupling or photosensing), in vivo, the various photodetecting units are usually not randomly distributed, but rather assembled in ordered arrays. This molecular architecture is functional in providing living cells with efficient photoreceptor-phototransducing systems and can be carried through, for instance, if photoreceptor molecules are embedded in membranous structures (see, e.g., thylakoids in chloroplasts and rhodopsins patches in Halobacterium) or packed in quasi-crystalline subcellular organelle (see, e.g., the ParaFlagellar Body in Euglena).

148

PHOTO RECEPTORS FOR MICROORGANISMS PHOTOMOVEMENTS

Phototaxis (oriented movement toward or away from the light source) and photophobic responses (transient alterations of swimming pattern following sudden variations in fluence rate) of freely motile microorganisms are, apparently, mediated by a wide variety of photoreceptor pigments. Photosensing pigments can absorb from the near UV (flavins and pterins) to the blue (flavins, carotenoids), to the green and from the yellow-orange (rhodopsins) to the red (stentorins and blepharismins) (Fig. 2) (Lenci & Ghetti, 1989; Haupt & Haeder, 1994; Kreimer, 1994; Lenci, 1995).

In some phototactic photosynthetic bacteria, chlorophyll and some accessory pigments play the double role of driving photosynthesis and perceiving light stimuli (Raeder, this volume). In the following only, "dedicated", non-photosynthetic, photoreceptor pigments will be considered.

At present, rhodopsins in halobacteria and some flagellated algae, flavins and pterins in other green algae and hypericin-type pigments in colored ciliated protozoa have been suggested, on the basis of experimental evidence, as photopigments responsible for light­induced motile reactions in these microorganisms.

R I , N'(yO

M~N~ Flavin

o

R rfO'YYNI NH

A~ Pterin

o

~I ~ ~"~13~ CHO

~ All-trans retinal

" ~ ~

~ 11-cis retinal

CHO

Figure 2. Structure formulas of some photoreceptor chromophores

For some microorganisms, not only the structure of the photoreceptor pigment(s), but also the primary molecular events which follow the absorption of the photon, seem to be unambiguously established. In other unicells, even the nature of the molecule acting as light detector is still doubtful.

It is worth, underlining that, in most cases, considerable progress has been achieved in facing the different aspects of the problem through specific approaches, such as selection and isolation of behavioral and pigment-lacking mutants, quantitative analysis of motile responses, steady-state and time-resolved spectroscopic and microspectroscopic studies of the candidate photosensing structures.

Among photoresponsive microorganisms whose photoreceptor pigments have been extensively studied, Halobacterium halobium, Euglena gracilis and Chlamydomonas reinhardtii will be considered.

Halobacterium is a prokaryotic organism in which light is used both as an energy

149

source and as an information carrier throughout three different specialized and dedicated chromoproteins, all of them rhodopsin-like: bacteriorhodopsin and halorhodopsin for metabolic and energetic requirements, and sensory rhodopsins for perceiving and reacting to environmental light conditions (Spudich & Bogomolni, 1983 and references therein).

Among photosynthetic unicellular algae, Euglena and Chlamydomonas are case examples of special interest, on the one hand because of the amount of knowledge achieved and, on the other hand, because of the basic questions which are still unanswered.

The case of Halobacterium

In the bipolariy flagellated halophilic Halobacterium, two sensory photopigments have been identified (Sensory Rhodopsins I and II, SRI and SRII) and their photocyc1es studied in detail (Bogomolni & Spudich, 1982; Spudich & Bogomolni, 1984 and 1992; Takahashi et aI., 1985; see also Petracchi et al., 1994 and references therein).

Both sensory rhodopsins are all-trans-retinal-binding, seven helix transmembrane proteins, which undergo slow dark-reversible photocyc1es (approximately 100 times slower than bacteriorhodopsin and halorhodopsin photocyc1es). SRI exists in tW0 spectrally different forms: a thermally stable one, with absorption maximum at 587 nm, SRI587, and a long-living intermediate, with absorption maximum at 373 nm, SRI373, which thermally decays back to SRI587 in about 750 ms. SRII, with absorption maximum at 487 run, SRII487, photoconverts into a species absorbing at about 520 nm, which thermally decays back to SRII487 in about 400 ms (Fig. 3) (Spudich, 1991; Takahashi, 1991; and references therein).

SRI mediates both attractant and repellent responses: red-orange light (absorbed by SRI587) lowers the frequency of swimming reversal (attractant response), whereas blue light

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Figure 3. Schematic drawings of Sensory Rhodopsins and their photocycles (redrawn from Spudich and Bogomo1ni,1984; and Petracchi et aI., 1994).

150

(absorbed by SRI373) causes an increase of the swimming reversal frequency (repellent response). SRII (also called Phoborhodopsin) is the blue-light receptor responsible only for repellent responses (Spudich, 1991; Takahashi, 1991; and references therein).

To clarify the primary steps of signal transduction, a retinal analogue in which the a11-transl13-cis isomerization is locked was incorporated into the apoproteins of sensory rhodopsins of Halobacterium FlxSR and Flx3T mutants (blocked in retinal synthesis). It was shown that SRI 373 as well as the intermediates in the photocycle of SRII are not formed from SRIS87 and SRII487, respectively, and that all light-mediated motile responses of the cells are suppressed (Fig. 4).

These findings are clearly consistent with the hypothesis that a specific all-trans/13-cis isomerization of retinal is involved in SR's photo cycles and is required for photobehavior in Halobacterium (Yan et al., 1990b).

Selective modifications of the retinal beta-ionone ring seem not to alter the physiological function of SRI (Yan et al., 1990a). The presence of the methyl group in C-13, on the contrary, plays a key role in the interaction between retinal and SRI-apoprotein: replacement of -CH3 with -H, in fact, allows all-trans/13-cis photoisomerization but inhibits both the attractant and repellent response, thus suggesting that a steric trigger is required for signaling (Yan et aI., 1991; Spudich & Bogomolni, 1992).

A SRI- SRII

Photocycles Photo responses Photocycles Photoresponses

Responses

8 SRI

Photocycles Photoresponses Photocycles Photoresponses

------. Responses

Figure 4. Schematic representation of the effects of all-transI13-cis photoisomerization blockage (A) and C-13 dimethylation (B) on SR-I and SR-II photocyc1es and on cell photoresponses.

151

The case of Euglena

It seems established that the photoreceptor pigment of the photosynthetic flagellated alga Euglena gracilis is located in the ParaFlagellar Body (PFB), a membrane-enclosed paracrystalline structure at the base of the emergent flagellum.

Action spectra for photoaccumulation and photodispersal (Diehn, 1969; Checcucci et al., 1976) and for step-down photophobic responses (Barghigiani et ai., 1979), as well as fluorescence emission and excitation spectra of PFB in vivo (Benedetti & Lenci, 1977; Ghetti et ai., 1985), suggested flavin type chromophores as photoreceptor pigments, most likely embedded in a rigid and/or hydrophobic molecular matrix. In this physiological environment, according to the low value of fluorescence quantum yield (0.005, as compared with 0.25 offree riboflavin solutions), the first excited singlet of the pigment could undergo de-excitation pathways other than radiative decay. This low value of fluorescence quantum yield of PFB flavins is in agreement with their photoreceptor function, as no photoreceptor pigment is likely to decay from its first excited singlet state mainly through a radiative transition, but rather through reactions which can serve to trigger the first molecular steps of the photosensory transduction chain (Ghetti et ai., 1985).

Even though no unambiguous evidence for the molecular events following light absorption by flavins was provided, the possibility of a functional connection between the photoreceptor system in the PFB and the ParaFlagellar Rod, PFR (an apparently ordered structure running along the flagellum), and the role of PFR as effector were suggested (Checcucci et al., 1974a and 1974b).

Lately, the PFB of Euglena has been isolated, its morphology investigated by electron microscopy (Gualtieri et al., 1988) and its optical properties measured by means of a computer-assisted micro spectrophotometer. The structure of the optical absorption spectrum of an isolated PFB (absorption maximum around 500 nm; optical density around 1.4) indicates that rhodopsin-like molecules, rather than flavins, are present in this organelle (Gualtieri et al., 1989). The PFB fluorescence emission around 530 nm (Benedetti & Lenci, 1977) and its fluorescence excitation spectrum (Ghetti et ai., 1985) have been due to metarhodopsin (Gualtieri, 1993).

An absorption band around 500 nm, whose spectral distribution is compatible with that of a rhodopsin-like pigment, has been recently measured in the eyespot region of Euglena also by James et ai. (1992) by means of microspectroscopic technique. The intensity of this band was found to depend on the relative orientation of the plane of polarization of light and the long axis of the cell. This anisotropy was suggested to be due to the fact that rhodopsins are embedded in the quasi-crystalline structure ofPFB.

Barsanti et al. (1992 and 1993) have also reported that nicotine, which inhibits retinal biosynthesis, and hydroxylamine, which reacts with both free and opsin-bound retinal, hinder the formation ofPFB in Euglena and suppress cells photoresponsiveness, further pointing to the rhodopsin nature of the photoreceptor pigment for photomotile responses in Euglena.

As all-trans-retinal has been detected in whole Euglena cells homogenates (Gualtieri et al., 1992) and no photobleaching of Euglena rhodopsin has been observed (James et al., 1992), this retinal isomer has been suggested to be the opsin-bound chromphore and not to be released from the apoprotein upon light stimulation (Barsanti et al., 1993).

The output signal from the PFB photoreceptor could be transduced to get the final sensory· response through the ParaFlageliar rod by means of either charge fluxes or allosteric interactions (Gualtieri, 1993).

The old hypothesis of flavins as possible photoreceptor pigments in Euglena has been revived suggesting, on the basis of spectroscopic, biochemical and photobehavioral data, that in the PFB they could be combined with pterins (Schmidt et al., 1990; Brodhun & Raeder, 1990; Galland et al., 1990).

In flagellar preparations containing the PFB, fluorescence emission bands at about 520 nm and 460 nm have been detected and attributed to flavins (absorption maxima at about

152

360 and 450 run) and pterins (absorption maximum at about 350 nm), respectively (Brodhun & Raeder, 1990; Galland et aI., 1990).

The presence of pterins in the PFB is also suggested by fluorescence micrographs in vivo: the PFB spot, in fact, fluorescing around 530 nm (green) when excited at 450 nm, appears fluorescing around 450 nm (blue) when excited in the near UV (350 nm) (Brodhun & Raeder, 1990).

Convincing evidence for the contribution of both pterins and flavins to the photosensitivity of Euglena can be provided by the structure of the phototaxis action spectrum, in which the near UV band, where both pterins and flavins absorb, is significantly higher than the blue one, where only flavins absorb (Raeder & Reinecke, 1991).

Following UV-B irradiation, pterin fluorescence from PFB-extracted proteins strongly increases and Euglena cell photo responsiveness is effectively impaired (Raeder & Brodhun, 1991 and references therein). On the basis of these observations, the two chromophores have been suggested to act as photoreceptor pigments through an energy transfer mechanism. Pterins would play the role of light antenna pigments for flavins in undamaged PFB protein complexes, but would radiatively dissipate when the spoiled molecular framework did not allow efficient energy transfer to the acceptor (flavins) (Raeder & Brodhun, 1991).

The role of pterins as antenna pigments for flavins, however, seems not to be fully in agreement with the fact that excitation ofPFB, in vivo in intact cells, at about 350 nm (in the pterins absorption band) induces fluorescence in the pterin emission band (blue) rather than in the flavin emission band (green) (Brodhun & Raeder, 1990).

An even more elaborate sensing mechanism has been recently proposed for Euglena by Sineshchekov V. A. et at. (1994): a system of interacting receptor pigments would in fact funnel light energy from short-wavelength (pterins) and intermediate-wavelength (flavins) absorbing centers to a "long wavelength absorbing pigment". According to the Authors, it is not possible, at present, to conclude if this last component of the light detecting complex is the reaction center triggering the sensory response, or a further energy donor to an as yet unidentified reactive molecule, possibly a rhodopsin.

Even though not explicitly affirmed by the Authors, a direct energy transfer from pterins to the long-wavelength group has to be excluded, as the emission band of the donor and the absorption band of the acceptor do not overlap at all.

Energy transfer data, combined with fluorescence polarization results, also provide some indications about the spatial arrangements of the different pigment systems. The fact that fluorescence polarization degree is quite low after energy transfer but high within the same group of pigments (pterins or flavins or X) , is compatible with an ordered array of molecules of the same species, as could be expected from the quasi-crystalline structure of the ParaFiagellar Body (Fig. 5).

Moreover, flavin-binding as well as pterin-binding proteins have been separated and characterized by chromatographic techniques from Euglena flagella isolated with their PFB (Brodhun & Raeder, 1990 and 1995). Riboflavin binding sites have been localized both in the PFB region and along the whole flagellum of Euglena and no clear-cut explanation for this spreading of binding sites is at present available (Nebenfuehr et aI., 1991; Brodhun et ai., 1994; Neumann & Rertel, 1994).

Attempts to reconcile the rhodopsin and the flavin hypothesis either by suggesting rhodopsin as the final acceptor of a multicomponent light-harvesting and transducing system (Sineshchekov V. A. et at., 1994) or by assigning to rhodopsin the role of photoreceptor proper, and to flavins bound to flagellar proteins the function of transducing the light stimulus along the flagellum (Neumann & Rertel, 1994) seem not satisfactorily supported by experimental findings.

153

Pt

300 400

Pt

500

Wavelength (nm)

600

_ Primary

Reactions

,----, E.T. ,----, E.T. ,----, - -c

700

., o c: ., ~ l!! o

'" u::

Figure 5. (A) Absorption (--) and fluorescence (------) spectra ofpterin (Pt), flavin (FI) and unidentified (X) pbotoreceptor pigments in Euglena; (B,C) scbematic representation of energy transfer cascade in Euglena pbotoreceptor pigments (see text).

The case of Chlamydomonas

The rhodopsin nature of the photoreceptor pigment for photomotile responses in the photosynthetic flagellated alga Chlamydomonas was initially hypothesized by Foster and Smyth (1980), relating the ultrastructural and optical properties of the stigma with the structure of phototaxis action spectra previously determined (Nultsch et aJ., 1971). That a rhodopsin-like pigment is the photoreceptor for Chlamydomonas, has been subsequently confirmed by a series of behavioral studies and action spectra determination (see Ghetti & Checcucci and Marangoni et al., this volume).

Which is the opsin-bound chromophore, and which primary molecular modifications it undergoes following light absorption are, on the contrary, still open questions. As in the case of Halobacterium, the problem has been primarily investigated incorporating retinal analogues and isomers, different in their stereochemistry around their double and single bonds, into blind, retinal-lacking mutants and relating the maximum of action spectra for the photomotile responses with the absorption maximum of the added chromophore.

Foster and coworkers added several retinal analogues and short-chained compounds (including hexenal and hexanal) to FN68 mutants (almost, but not completely, blind, if grown in the dark) and measured photobehavioral responses by means of a population method. As phototaxis action spectra showed that a number of retinal analogues and even the short-chained compounds restore photoresponsiveness, it was suggested that photoisomerization of the chromophore is not required in Chlamydomonas rhodopsin for eliciting the motile response. It was proposed that a charge redistribution in the excited state of the chromophore was the primary step in rhodopsin activation. Among retinal isomers, ll-cis was found to be the most effective in restoring phototaxis in FN68 mutants (approximately three times more effective than all-trans retinal) and, consequently,

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suggested to be the natural chromophore of Chlamydomonas rhodopsin (Foster et al., 1984, 1988, 1989 and 1991; Foster & Saranak, 1988).

Quite different results were obtained by a number of Authors who used also a different, completely blind, mutant (CC-2359) and measured photomotile responses by means of cell­tracking motion analysis systems as well as population methods. No photobehavioral responses were observed after incubation with short-chained compounds and retinal analogues in which photoisomerization around C13-C14 was hindered and maximum responsiveness was found adding all-trans retinal. All these findings clearly indicate that all­trans retinal is the functionally active chromophore and that the all-trans/13-cis photoisomerization is the primary molecular reaction responsible for initiating the transduction process in Chlamydomonas (Hegemann et aI., 1991; Lawson et al., 1991; Takahashi et aI., 1991; Zacks et aI., 1993; Kroeger & Hegemann, 1994). Further support to his conclusion has recently been provided also by measurements of photoinduced electric currents in Chlamydomonas blind mutants incubated with retinal and its analogues (Sineshchekov O.A. et al., 1994).

CONCLUDING REMARKS

In photosynthesis the molecular nature and the structural organization of light harvesting and of reaction center pigments, as well as the basic mechanisms of light energy transfer and funneling, are largely known. At present, in the study of energy conversion phenomena, special attention is devoted to the early events occurring on a subnanosecond time scale, thanks also to recently developed ultrafast techniques (see Holzwarth, this volume).

In microorganisms photosensory processes, not only the primary molecular photoreactions, but also the chemical nature of the photoreceptor pigments, are, in some cases, not yet definitely clarified. The rhodopsin-like nature of Halobacterium and Chlamydomonas photoreceptor pigment, in fact, seems to be ascertained, but for Euglena both the "Flavin" and the "Rhodopsin" hypothesis sounds, per se, self-consistent and based on reliable experimental findings.

The rhodopsin hypothesis can receive firm support from evolutionary considerations: rhodopsin-like pigments, in fact, are used to sense the light environment in prokaryotes, like Halobacterium, in other flagellated unicellular algae, like Chlamydomonas, and in higher organisms. If the nature of the photoreceptor pigment is highly conserved, the rhodopsin­like nature of Euglena photoreceptor pigment is undoubtedly to be expected (Walne & Gualtieri, 1994).

The fact that rhodopsin is a seven-helix-transmembrane protein, like many other sensory receptors, seems not to be such a stringent argument in favor of the rhodopsin hypothesis for Euglena, because other photosensing receptors, like phytochrome, do not have such a conformation.

On the other hand, flavins are commonly accepted to mediate a number of sensory responses in fungi and algae induced by near-UV and blue light (Neumann & Hertel, 1994; Galland, 1992). Also in the case of photoreceptor molecules for photomotile responses of microorganisms, therefore, evolution could have advanced not only reiterating "the good solutions", but· also ramifying and compromising, ending with solutions which are not necessarily unique (or even perfect) (Block, 1992).

NOTE ADDED IN PROOF

The occurrence of a wide variety of chemical and conformational structures, for photosensing pigments, has recently been confirmed by a number of findings on the

155

Photoactive Yellow Protein (PYP), the photosensory protein for negative phototaxis of Ectothiorhodopsira halophyla. Its chromophore has been identified as p-coumaric acid (see figure below), a novel class of photosensing pigments (Hoff et aI., 1994; Baca et al., 1994), and its structure determined at high resolution to be clam-type with a.-helices and J3-sheets (McRee et aI., 1989; Borgstahl et aI., 1995).

o

HO OH

p-Coumaric acid

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PHOTO MORPHOGENIC SYSTEMS

Winslow R. Briggs, Emmanuel Liscum, Paul W. Deller, and Julie M. Palmer

Department of Plant Biology, Carnegie Institution of Washington, 290 Panama St., Stanford, CA 94305

INTRODUCTION

As sessile organisms, plants have evolved a remarkable capacity to respond to a wide range of environmental signals. An environmental factor of major importance as such a signal is light. In addition to harvesting light energy thro\lgh photosynthesis as an energy source, plants make extensive use of light to provide them with important information about their surroundings. They may respond to this information at the physiological, biochemical, or molecular levels, or some combination thereof. Indeed, virtually every phase of a plant's development is closely regulated by its light environment These collective responses are encompassed by the term photomorphogenesis. Recent reviews by Quail (1991), Furuya (1993), Kaufinan (1993), Short & Briggs (1994), and Deng (1994) and a recent comprehensive book edited by Kendrick & Kronenberg (1994) provide a broad and up-to­date coverage of the subject.

As discussed by Haupt (this volume), plants can detect and respond to four different parameters of their light environment: light spectral quality, light quantity, light duration, and light direction. After a brief consideration of some typical responses to each of these parameters in higher plants, we will discuss the use of mutants to sort out the multiplicity of roles of different photo morphogenic systems in plants. We will then focus more specifically on progress in our understanding of phototropism, a process involving detection of light direction leading to a subsequent reorientation of growth.

PLANT RESPONSES TO LIGHT QUALITY

A simple experiment demonstrates the participation of a minimum of two distinct photoreceptor systems in plant development. Pea seedlings grown in total darkness produce an extremely elongated epicotyl without a trace of chlorophyll, an apical hook that is tiny and tightly closed, and only the most vestigial of leaves. Continuous red light inhibits epicotyl growth somewhat, the hook opens, and the leaves expand somewhat, but the spindly product is hardly a normal light-grown plant. Addition of blue light to the red,

Light as an Energy Source and Information Carrier in Plant PhYSiology Edited by Jelmings et al., Plenum Press, New York, 1996 159

however, produces a normal short seedling with dark green expanded leaves and full development of photosynthetic competence. Clearly both blue and red light are required for the plant to achieve normal development. The red light effect is well-known to be mediated through the photochromic photoreceptor phytochrome while the nature of the blue light photoreceptors involved is yet to be determined (see below).

Plants grown in supplemental far red light elongate far more than their counterparts receiving equal photosynthetically active radiation but without far red, in the well known shade-avoidance response (see Smith, 1994). This response can be truly dramatic with far red-treated seedlings over twice as tall as their control counterparts without far red (Morgan, 1981). The difference in growth is related not to differences in absolute amount of light but rather to the ratio of red to far red light which in tum determines the fraction of phytochrome in the far-red absorbing form, Pfr. Inhibition of stem growth is directly related to the amount of Pfr present. In green shade, most of the red is removed from the incident light by the chlorophyll in the shading leaves, but very little of the far red, and hence most of the phytochrome is in the Pr form, and elongation growth remains rapid. Whereas in the pea experiment described above two different classes of photo receptors are involved in detecting two different spectral regions (blue and red), only a single pigment, the photo chromic photoreceptor phytochrome, is involved in the latter example (red and far red).

PLANT RESPONSES TO LIGHT QUANTITY- BOTH HIGH AND LOW FLUENCE RATES

Oxalis oregana is a species that grows deep in the redwood forests in central coastal California, and is exquisitely adapted to grow in extreme shade. However, even in the redwood forests, sunflecks are a common occurrence, and may last several minutes. Hence Oxalis leaves are occasionally exposed to an enormous step-up in light fluence rate, a step up that may be as much as 400-fold. Their response, described by Bjorkman & Powles (1981) and Powles & Bjorkman (1981), is a rapid folding ofleaflets such that the amount of intercepted light is reduced within about 5 min to about 10 % of that for the fully extended leaflets. On departure of the sunfleck, the leaflets return to their extended orientation within 20-30 min. When these workers physically prevented the leaflets from folding while simultaneously measuring photosynthesis they noted a significant reduction of carbon dioxide uptake, a reduction that required some hours at low light intensity for full recovery. This decline in photosynthetic activity is clear evidence for some sort of photodamage in the restrained leaves. Hence the folding response serves a demonstrable photoprotective function. These leaflet movements, involving rapid loss of ions and water from motor cells in the specialized organs called pulvini just at the base of the leaflets, are activated by an as yet uncharacterized blue light photoreceptor (Powles & Bjorkman, 1981).

Plants also have systems that detect extremely low fluence rates of light. Maize primary roots allowed to elongate in total darkness grow horizontally at right angles to the gravity vector (see Wilkins, 1979). A brief pulse of dim red light induces a change in orientation such that the roots now reorient their growth downward at an angle between 20 and 30 degrees (Mandoli et aI., 1984). The response is readily reversed by a brief exposure to far red light, and hence is clearly mediated by phytochrome. The response obeys the reciprocity law--as-long as the product of time and fluence rate remain constant, the response remains constant--over a period of several hours. Hence roots can perceive and respond to light fluences rates that are extremely low. Mandoli et al. (1990) have described light penetration through several different types of soils, ranging from those rich in organic matter where light penetration may be negligible to sandy soils where significant light penetration may occur through several cm. Indeed, roots growing under as much as 1.5 cm of sandy loam soil detect the extremely low levels of light penetrating from the soil surface and respond by

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downward reorientation of growth (Mandoli & Briggs, unpublished observations). This reorientation appears to be an effective adaptation against desiccation.

PLANT RESPONSES TO LIGHT DURATION

Three quarters of a century ago Garner & Allard (1920) discovered that flowering of certain species could be strictly regulated by daylength, a phenomenon known as photoperiodism. We now know that flowering induction is an extraordinarily complex process involving perception of daylength, and interaction both with temperature, and with circadian rhythms. We also know that daylength is somehow perceived by phytochrome present in young leaves or, in special cases, cotyledons. Recent reviews have focused on both physiological (Vince-Prue, 1994) and molecular-genetic (Coen, 1991; Coen & Carpenter, 1993) aspects of the flowering response. However, despite recent advances in our understanding of the developmental program for flowering at the gene level, the way in which daylength is perceived by the leaves and the information transmitted to the vegetative shoot apex still remains one of the most important unsolved problems in plant biology. the nature of the signal and the manner in which it either instructs the apex to remain vegetative or to begin the process of flower development remains elusive.

PLANT RESPONSES TO LIGHT DIRECTION

Higher plants can respond to light direction in two quite different ways. The first of these, found in species scattered over several families, is solar tracking, a phenomenon that does not involve irreversible growth changes. The second is phototropism, where true differential growth is involved. We shall deal briefly with the two phenomena here, and then discuss some recent biochemical and genetic advances in our understanding of phototropism in somewhat more detail below.

Many species of plants have leaves that track the sun, constantly adjusting their angle with respect to the petiole to keep incident solar irradiation normal to the leaf surface (or parallel to the leaf surface in an avoidance response in some cases as discussed above) as reviewed by Koller (1990). This solar tracking response is mediated by blue light, and at least for one plant there is evidence that detection of the light direction may be through dichroically oriented photoreceptors (Koller et aI., 1990). The leaf movements are driven by turgor changes in groups of motor cells in the pulvini, as is the case with the leaf folding response in Oxa/is oregana, and are fully reversible (Koller et al., 1985). Furthermore, leaves facing west at sunset will move to a "rest" position at right angles to their petioles during the night, and then, driven by a circadian rhythm, reorient to face east shortly before dawn. It is possible with a moving light source to induce leaves of one species, Lavatera cretica, to change their angle of elevation as much as 40° per hour, a rate considerably faster than the 15° per hour rate of change of the solar angle (Koller et aI., 1985). This capability is perhaps not surprising. Under cloud cover, with diffuse light, the leaf blades simply assume their rest position. Should the sun come out later in the day, they can then move rapidly into a position normal to the incident sunlight, a response that maximizes their light-harvesting capacity.

Phototropism is the directed growth of a plant organ toward (or away from) a unilateral light source, and has been the object of intensive research for over a century. The early history of phototropism is intimately associated with the early history of auxin (Went & Thimann, 1937), and the extensive physiological literature has been reviewed by lino (1990). There is still controversy over the precise role that auxin movement plays in phototropism (see the multiauthor forum in Vol. 15, No.7 of Plant, Cell & Environment, presenting a series of short commentaries by many different workers, bracketed by introductory and

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summary remarks by Trewavas, 1992a; 1992b) and there is also still controversy over the nature of the photoreceptor pigment or pigments (see below). However, some progress is being made at the biochemical and genetic levels (see Short & Briggs, 1994 and below), so some optimism is justified that both of these uncertainties will be addressed in the relatively near future. We will return to consideration of this progress following a short discussion of the use of mutants in photo morphogenesis, as mutants play an important role in much of the recent work in phototropism.

USE OF MUTANTS IN PHOTOMORPHOGENESIS

Since Koornneef et al. (1980) first described the hy mutants of Arabidopsis thaliana, mutants have played an extremely important role in elucidating the complexities of plant photomorphogenesis. Null mutants are now available both for phyA (Nagatani et aI., 1993; Parks & Quail, 1993; Whitelam et aI., 1993) and phyB (Lopez-Juez et aI., 1992; Reed et aI., 1993). Mutants of A. thaliana and several other species have been used extensively to try to sort out specific roles for the individual phytochrome species. For example, ·a mutant of Brassica napa deficient in phyB develops elongated hypocotyls in comparison to wild-type controls under continuous red or white light, indicating a role for phyB in inhibition of hypocotyl growth. However, this mutant shows normal inhibition under continuous far red light, (Devlin et aI., 1992) indicating that the so-called High Irradience Response (HIR) (see Mancinelli, 1994) can in this case be attributed to another phytochrome, probably phyA. Thus both phy A and phyB can participate in the inhibition of elongation, but in rather different ways. In another example, it has been shown that more than one phytochrome may be involved in the flowering response of A. thaliana (Halliday et aI., 1994). We have already seen above that blue light can also inhibit stem growth independent of phytochrome in pea. Hence there is an apparent functional redundancy in which several different photo receptors can contribute to the same physiological response. These may be different members of the same family of photoreceptors, e. g. phytochrome, or members of two or more groups of unrelated photoreceptors.

By and large, most of the roles described for phyA apply to etiolated seedlings. Indeed, it is well known that phyA, referred to in some earlier publications as Type I phytochrome, is rapidly degraded in its pfr form (see Furuya, 1993). Hence, not surprisingly, light-grown seedlings lacking phyA are normally indistinguishable from wild-type seedlings. However, Johnson et al. (1994) have shown a requirement for phyA for response to extended daylength in hypocotyl elongation among other things, indicating the roles for phyA are not restricted to the developmental switch from etiolated to de-etiolated growth.

Mutants in blue light responses have also been extremely useful in sorting out different photoreceptors and signal transduction chains (Liscum & Hangarter, 1994). For example, hy4mutants of A. thaliana are deficient in blue light-induced inhibition of hypocotyl elongation, but show entirely normal phototropic curvature, whereas mutants deficient in their phototropic response showed entirely normal inhibition of hypocotyl elongation (Liscum et aI., 1992). Hence these two systems, have genetically separable components. In fact it is likely that these two blue light responses are mediated by independent photoreceptors (Liscum & Briggs, 1995).

Another important class of mutants affecting photo morphogenesis have been described for which dark-grown seedlings show a phenotype with many of the properties of light­grown seedlings. This class of mutants includes the del mutants first described from Chory's laboratory (Chory et aI., 1989) and the cop mutants first described by Deng and co-workers (Deng et aI., 1991; Hou et aI., 1993). It is believed that the wild-type DET and COP gene products act as negative regulators of the de-etiolation response, and only upon photoreceptor excitation is this inhibition relieved. These mutants affecting later steps in the signal transduction pathway from the photo receptors provide potentially powerful tools to

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investigate some of the elusive steps between photoreceptor excitation and final response. Both classes of mutants apparently act downstream from both phytochrome and blue light photoreceptors (see Chory, 1993; Deng, 1994).

A detailed treatment of photo morphogenic mutants is beyond the scope of this article, but the reader should consult several recent reviews for further information: Liscum & Hangarter (1994) for mutants in blue light-regulated pathways, and Chory (1993) and Whitelam and Harberd (1994) for phytochrome mutants. Whitelam & Harberd also cover the expanding literature on the use of transgenic plants overexpressing either phy A or phyB to try to resolve the respective roles of these two photoreceptors. A good general treatment of photo morphogenic mutants has been written by Koomneef & Kendrick (1994).

RECENT STUDIES WITH MUTANTS IN THE PHOTOTROPISM PATHWAY

There is now convincing physiological and genetic evidence that a plasma membrane phosphoprotein with Mr values between 114 and 130 kD depending on the species is a ubiquitous required element in the phototropic responses of higher plants (Short & Briggs, 1994). We are currently carrying out a study of A. thaliana mutants defective in their phototropic responses (Liscum & Briggs, 1995) to try to understand the phototropism signal transduction pathway and its elements in more detail. We have isolated three different classes of mutants. The first class is defined by three independent alleles at a single locus, nphl (for ~on-phototropic fu'pocotyl). This class of mutants is deficient in or completely lacks phototropic responses, and is also deficient in or lacks the phosphoprotein. The second class is defined by two separate loci, nph2 and nph3, with a single nph2 and two nph3 alleles. These mutants show normal levels of the phosphoprotein, wild-type levels of blue light-dependent phosphorylation, and show normal gravitropism, but are deficient in or lack normal phototropic responses. The third class of mutants is defined by two alleles at a single locus, nph4. These mutants show normal levels of phosphoprotein and normal light­dependent phosphorylation but are deficient in both phototropic and gravitropic responses. Two mutants isolated in Pofi's laboratory (Khurana & Poff, 1989), strains JK224 and JK218, have been shown to be allelic to the nphl and nph3 allelic series, respectively, and have been designated nphI-2 and nph3-3 (Liscum & Briggs, 1995).

There has been considerable progress in characterizing the nph mutants with special attention to the nphl allele series, those deficient in or completely lacking the phosphoprotein (Liscum & Briggs, 1995). Save for one mutant (nph I -2) that is only deficient in first positive curvature, the most sensitive phototropic response in A. thaliana, these mutants lack all of the known phototropic responses: first positive curvature of etiolated seedlings in response to blue light; second positive curvature of etiolated seedlings in response to blue light; curvature of etiolated seedlings in response to green light (see Konjevic et aI., 1989); curvature of etiolated seedlings in response to UV -A light; and both hypocotyl and root phototropism in response to blue light in de-etiolated seedlings. These results have led to the conclusion that the phosphoprotein is a critical component in all of the known phototropic responses in A. thaliana.

We are currently using the nphl mutants in efforts to map, isolate, and characterize the gene encoding this essential protein. Preliminary genetic mapping data indicate that NPHI is located on chromosome 3, within 26 centimorgans of the glabrous (GLI) gene (Liscum & Briggs, 1995) and efforts are currently underway to isolate NPHI by using a polymerase chain-reaction (PCR) -based technique to generate fragment length polymorphisms for use as probes in cloning strategies. We will be using the same techniques in the near future (and other molecular/genetic techniques as well) to attempt to isolate the NPH2, NPH3, and NPH4 genes for detailed molecular and functional characterization.

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PURIFICATION OF THE PHOSPHOPROTEIN INVOLVED IN PHOTOTROPISM

Weare also continuing efforts to purify and characterize the NPH 1 protein itself through standard techniques, both for further biochemical characterization and for the generation of antibodies to be used as an alternate route to the gene. Although our early work was with the protein from membranes from etiolated peas, two observations prompted us to to search for another system for biochemical studies, a search that led us ultimately to choose A. thaliana. Firstly, detailed analysis of the pea phosphoprotein revealed significant heterogeneity (Oeller, unpublished observations). What was thought to be a single band on SDS-polyacrylamide electrophoresis was clearly resolved into two phosphorylated species with slightly different molecular weights. The slower-migrating species is present in low amount, and is only detected when irradiation and phosphorylation are carried out in vitro. This species is interesting in that it shows an absolute dependence on blue light for phosphorylation while the other species shows phosphorylation even in dark control preparations. The two species are further distinguished by the Ca++ requirement for their phosphorylation. The light-dependent species completely lacks Ca++ dependency, whereas the constituitively phosphorylated band shows about a 5-fold increase in phosphorylation in the presence of Ca ++.

Further attempts to purify these bands revealed yet more heterogeneity in the lower molecular weight phosphorylation-constitutive band. The band itself can be resolved into two species depending on their behavior in the presence of detergents (Triton X-IOO, Triton X-114, Thesit, Brij 35, CHAPS). One species is solubilized by detergent treatment while the other remains pelletable by centrifugation. A slight mobility difference is also discernible between the two species, with the insoluble band showing slightly higher mobility. Although blue light appears not to affect its subsequent susceptibility for phosphorylation, the band shows a clear response of another sort to blue light: Experiments with plasma membranes purified from in vivo-irradiated pea tissue indicate that this detergent-insoluble species responds to a short pulse of blue light in that it is missing from the insoluble fraction after such treatment. Whether this change represents an altered membrane association and/or blue light-induced degradation is currently in question, but antibodies should help to provide an answer.

How these three species are related to each other is currently unknown. They are probably closely related structurally, based on protein digestion experiments that produce nearly identical patterns of phosphopeptides. They could reflect degradation of a single molecule, differentially processed products of a single gene, or may be the products of different genes.

A second difficulty with the pea system is its extreme instability. Photo activity is lost rapidly on solubilization in either non-ionic or zwitterionic detergents. It has a half life best measured in minutes, making both purification and characterization studies difficult, and eliminating use of all but the fastest of techniques.

Neither the heterogeneity nor the instability of the pea system have been observed in other species analyzed to date. Hence attempts to purify this phosphoprotein have concentrated on A. thaliana. The choice to use A. thaliana resulted from several important observations. First, this species displays a single plasma-membrane-associated polypeptide that becomes heavily phosphorylated in a blue light-dependent manner and which is soluble in nonionic detergents. Second, a stainable protein band corresponding to the labeled protein band is observed on SDS polyacrylamide electrophoresis, and this protein shows a clear mobility decrease on in vitro phosphorylation. This phenomenon was first observed with pea preparations irradiated in vivo (Short et aI., 1994) but was not consistently detected if the irradiation and phosphorylation are carried out in vitro. This mobility shift is clearly and reproducibly visible after either in vivo or in vitro irradiation in A. thaliana, and appears to be dependent upon three factors: blue light, ATP, and Mg++ Third, the phosphorylation reaction in A. thaliana appears significantly more robust and stable than the pea system,

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making it far better material for purification studies, in spite of perceived difficulties related to its diminutive ·nature. A fourth characteristic that makes A. thaliana an appealing system for biochemical studies is the existence of mutants clearly lacking (or at minimum containing undetectable levels of) the protein of interest (see discussion of phototropism mutants above).

Currently detergent solubilization followed by a two step column procedure--a sizing column to remove large incompletely solubilized particles--and a Cibacron Blue column yield up to ten-fold enrichment of the phosphoprotein. Elution of the protein from SDS gels readily yields tens of micrograms, and we are optimistic that we will shortly have sufficient protein for antibody generation and microsequencing.

THE PHOTORECEPTOR PROBLEM IN BLUE LIGHT-REGULATED RESPONSES

At present, there is no conclusive evidence implicating any chemically defined photoreceptor in blue light-regulated processes in higher plants. Historically, carotenoids and flavins have been the favorite chromophore candidates, although pterins have been suggested (Galland & Senger, 1988) and most recently even retinal (Lorenzi et aI., 1994). The photoreceptor for suppression of hypocotyl growth in A. thaliana may well be the HY 4 protein, a flavin-containing DNA lyase-like molecule, recently described by Ahmad & Cashmore (1993). Mutants in the HY410cus show normal phytochrome-mediated hypocotyl suppression in red/far red regions of the spectrum, but no inhibition by blue light. The HY 4 gene was isolated with a T -DNA-tagged allele, and sequence analysis showed regions of high homology with DNA photolyases from a range of prokaryotes. These light-activated enzymes are involved in excision of thymidine dimers from damaged DNA. However, the HY 4 protein lacks a tryptophan residue specifically implicated in recognition of thymidine dimers in DNA photolyase. In addition, the HY 4 protein has a C-terminal region with some homology to tropomyosin, a domain not found in any of the known DNA lyases. Thus the protein probably does not function as a DNA lyase and may well be involved in protein­protein interactions of some kind. Although direct evidence that this interesting protein is the photoreceptor for hypocotyl inhibition is still lacking, its similarity with known photoreceptors makes it a likely candidate.

Quinones & Zeiger (I994) recently published results showing a strong correlation of phototropic sensitivity with levels of the carotenoid zeaxanthin, a component of the xanthophyll cycle discussed elsewhere in this volume (see chapter by Horton). On the strength of these correlations, they suggest that zeaxanthin may be the photoreceptor for phototropism. There are several reasons why we feel that this is not the case (Palmer et aI., 1995). First, the xanthophyll cycle--and therefore zeaxanthin--occurs in the chloroplast, whereas our evidence, both physiological and genetic, indicates that the photoreceptor resides in the plasma membrane. Second, the action spectrum for phototropism shows a strong action peak in the UV-A region of the spectrum, a peak lacking in the absorption spectrum of zeaxanthin. Finally, maize seedlings in which zeaxanthin is either reduced or completely missing either as a result of a mutation in the carotenoid biosynthesis pathway or herbicide treatment show completely normal first positive curvature, and completely normal light-induced phosphorylation of the plasma membrane-associated protein (114 kD in maize).

We currently hypothesize that the phosphoprotein is encoded by the NPH 1 locus of A. thaliana and is itself the photoreceptor for phototropism (Liscum & Briggs, 1995). Four lines of evidence support this contention, though no one is conclusive. First, solubilization of the membranes with nonionic detergents has no effect whatsoever on the quantum efficiency for photoactivation of kinase activity (Short et aI., 1993). Thus either the phosphoprotein itself is the photoreceptor moiety and some sort of autophosphorylation is involved, or the

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photoreceptor is another protein tightly associated with the phosphoprotein in a physical relationship- completely unaffected by the detergent treatment. Second, photoactivity survives electrophoresis on native gels (Warpeha & Briggs, 1993), again suggesting that if more than one protein is involved, the association must be an extremely tight one. Third, the phototropic mutant nphl-2 (Khurana et al., 1989) shows altered spectral sensitivity, with sensitivity much reduced in the blue but not in the green (Konjevic et al., 1992). It seems highly unlikely that a mutant affecting any protein but the photoreceptor could alter the spectral sensitivity of phototropism. Fourth, as mentioned above, the phosphoprotein must be involved in phototropic responses both to blue and to green light since all mutants of the nphl allele series that are insensitive to both wavelength regions lack detectable levels of the protein (Liscum & Briggs, 1995). Future studies will tell us whether this hypothesis is the correct one.

CONCLUDING REMARKS

As will have become clear from the above discussion, what is knbwn about photomorphogenic systems in higher plants varies widely depending on the system being studied. Great progress has been made in our understanding of the phytochrome family of genes and the roles of the different family members, and the cop and det mutants hold great promise as tools for probing the signal transduction pathways activated by these photo receptors. Some progress is also clearly being made in at least two blue light systems-­inhibition ofhypocotyl elongation and phototropism--though there is still much to do to gain the level of knowledge currently at hand for the phytochromes. In the near future, the combination of the powerful techniques of genetics, biochemistry, and molecular biology hold great promise for learning a great deal more about not just these systems but a wide range of other photomorphogenic systems as well.

ACKNOWLEDGEMENTS

Work from the senior author's laboratory, mentioned above, was supported by NSF Grants No. DCB 91-18392 and IBN 92-10256. The authors are grateful for this aid. This is Carnegie Institution of Washington Department of Plant Biology Publication No. 1243.

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Institution of Washington, Washington, DC, p. 59-62. Chory, J. (1993) Trends Genet., 9:167-172. Chory, J., Peto, C., Feinbaum, R., Pratt, L., & Ausubel, F. (1989) CeiL 58:991-999. Coen, E.S. (1991) Annu. Rev. Plant Physiol. Plant Mol. BioI., 41:241-279. Coen, E.S. & Carpenter, R. (1993) Plant Cell, 5:1175-1181. Deng, X.-W. (1994) Cell, 76:423-426. Deng, X.-W., Caspar, T., & Quail, P.H. (1991) Genes Dev., 5:1172-1182. Devlin, P.F., Rood, S.B., Somers, D.E., Quail, P.H., & Whitelam, G.C. (1992) Plant Physiol.,

100:1442-1447. Furuya, M. (1993) Annu. Rev. Plant Physiol. Plant Mol. BioI., 44:617-645. Galland, P. & Senger, H. (1988) Photochem. Photobiol., 48:811-820. Garner, W.W. & Allard, H.A. (1920) 1. Agri. Res., 18:553-606. Halliday, K.1., Koornneef, M., & Whitelarn, G.C. (1994) Plant Physiol., 104:1311-1315. Hou, Y., von Arnim, A.G., & Deng, X-W. (1993) Plant Cell, 5:329-339. lino, M. (1990) Plant Cell Environ., 13:633-650. Johnson, E., Bradley, M., Harberd, N.P., & Whitelam, G.c. (1994) Plant Physiol., 105:141-149. Kaufman, L.S. (1993) Plant Physiol., 102:333-337.

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Kendrick, RE. & Kronenberg, G.H.M., eds. Photomorphogenesis in Plants. 2nd ed. 1994, Kluwer Academic Publishers: Dordrecht. 828 pp.

Khurana, J.P. & Poir, K.L. (1989) Planta, 178:400-406. Khurana, J.P., Ren, Z., Steinitz, B., Parks, B., Best, T.R, & Poir, K.L. (1989) Plant Physiol., 91:685-

689. Koller, D. (1990) Plant Cell Environ., 13:615-632. Koller, D., Levitan, I., & Briggs, W.R (1985) Photochem. Photobiol., 42:717-723. Koller, D., Ritter, S., Briggs, W.R, & Shiifer, E. (1990) Planta, 181:184-190. KOIyevic, R, Khurana, J.P., & Poff, K.L. (1992) Photochem. Photobiol., 55:789-792. Konjevic, R, Steinitz, B., & Poff, K.L. (1989) Proc. Natl. Acad. Sci., U.S.A., 86:9876-9880. Koomneef, M. & Kendrick, RE. (1994 in "Photomorphogenesis in Plants", (Kendrick, RE. &

Kronenberg, G.H.M., eds.), Kluwer Academic Publishers, Dordrecht, p.601-628. Koomneef, M., Rolff, E., & Spruit, C.J.P. (1980) Heynh, Z. Pflanzenphysio!., 100:147-160. Liscum, E. & Briggs, W.R (1995) Plant Cell, 7:473-485. Liscum, E. & Hangarter, RP. (1994) Plant Cell Environ., 17:639-648. Liscum, E., Young, J.C., Poff, K.L., & Hangarter, RP. (1992) Plant Physio!., 100:267-271. Lopez-Juez, E., Nagatani, A., Tomizawa, K.-I., Deak, M., Kern, R, Kendrick, RE., & Furuya, M.

(1992) Plant Cell, 4:241-25l. Lorenzi, R, Ceccarelli, N., Lercari, B., & Gualtieri, P. (1994) Phytochem., 36:599-60l. Mancinelli, A.L. (1994) in "Photomorphogenesis in Plants", (Kendrick, R.E. & Kronenberg, G.H.M.,

eds.), Kluwer Academic Publishers, Dordrecht, p. 211-269. Mandoli, D.F., Ford, G., Waldron, L.J., Nemson, J.A., & Briggs, W.R (1990) Plant Cell Environ.,

13:287-294. Mandoli, D.F., Tepperman, J., Huala, E., & Briggs, W.R (1984) Plant Physio!., 75:359-363. Morgan, D.C. (1981) in "Plants and the Daylight Spectrum", (Smith, H., ed.), Academic Press,

London, p. 205-22l. Nagatani, A., Reed, J.W., & Chory, J. (1993) Plant Physiol., 102:269-277. Palmer, J.M., Warpheha, K.M.F., & Briggs, W.R (1995) Zeaxanthin is not the photoreceptor for

phototropism in maize coleoptiles, In prep. Parks, B.M. & Quail, P.H. (1993) Plant Cell, 5:39-48. Powles, S.B. & Bjorkman, O. (1981) Carnegie Institution of Washington Year Book 80, Carnegie

Institution of Washington, Washington, DC, p. 63-66. Quail, P.H. (1991) Phytochrome: Annu. Rev. Genet., 25:389-409. Quinones, M.A. & Zeiger, E. (1994) Science, 264:558-56l. Reed, J.W., Nagpal, P., Poole, D.S., Furuya, M., & Chory, J. (1993) Plant Cell, 5:147-157. Short, T.W. & Briggs, W.R (1994) Annu. Rev. Plant Physio!. Plant Mo!. Bio!., 45: 143-17l. Short, T.W., Porst. M., Palmer, J.M., Fernbach, E., & Briggs, W.R (1994) Plant Physio!., 104:1317-

1324. Short, T.W., Reymond, P., & Briggs, W.R (1993) Plant Physio!., 101:647-655. Smith, H. (1994) in "Photomorphogenesis in Plants", (Kendrick, RE. & Kronenberg, G.H.M., eds.),

Kluwer Academic Publishers, Dordrecht, p. 377-416. Trewavas, A.J. (1992a) Plant Cell Environ., 15:76l. Trewavas, A.J. (1992b) Plant Cell Environ., 15:793-794. Vince-Prue, D. (1994) in "Photomorphogenesis in Plants", (Kendrick, RE. & Kronenberg, G.H.M.,

eds.), Kluwer Academic Publishers, Dordrecht, p.447-490. Warpeha, K.M.F. & Briggs. W.R (1993) Aust. J. Plant Physio!., 20:393-403. Went, F.W. & Thimann, K.Y. (1937) Phytohormones, Macmillan Co., New York, 294 pp. Whitelam, G.C. & Harberd, N.P. (1994) Plant Cell Environ., 17:615-625. Whitelam, G.C., Johnson, E., Peng, J., Carol, P., Anderson, M.L., Cowl, J.S., & Harberd, N.P. (1993)

Plant Cell, 5:757-768. Wilkins, M.B. (1979) in "Encyclopedia of Plant Physiology", vo!. 7, (Haupt, W. & Feinleib, M.E.,

eds.), Springer-Verlag, Berlin, p. 601-626.

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OVERVIEW OF PHOTOSENSING IN PLANT PHYSIOLOGY

Wolfgang Haupt

Universitat Erlangen-Nurnberg Germany

INTRODUCTION

Light can be used by plants either as an energy source, as found in photosynthesis (photoharvesting), or as information about the environment (photosensing). In this latter case, a light signal starts a cascade of events that finally result in a response; and importantly, this response is energized by the plant's metabolism, with no contribution by the light energy, which usually is only needed in catalytic amounts. Thus, amplification processes are an integral part of the so-called transduction chain, which connects the light signal (or light stimulus) as the input with the terminal response as the output (cf Haupt, 1991).

There are several criteria that can be used to discriminate photosensing from photo harvesting in plants:

- if in chlorophyll-free organs or organisms a response depends on light, this cannot be based on photo harvesting. The same is true for fungi in general.

- if the action spectrum excludes chlorophylls and accessory photosynthetic pigments as being involved, the response in question is independent oflight harvesting.

- if it is evident that the energy of the input is far too low as to energize the output, this is a typical case of photosensing (e.g., the phototropic curvature of a seedling to a flash of very low fluence).

- if the pigment that absorbs the effective light is identified (e.g., by action spectroscopy) as an already known photo sensory pigment, photoharvesting is excluded as the key of this light effect; classical examples are phytochrome-mediated photoresponses (see below).

The first event in photosensing is photoperception sensu lato, which can be divided into susception and perception sensu strictu. Susception is defined as the pure physical interaction of the signal with a cell structure, whereas perception sensu strictu is the first biophysical or biochemical process following susception. This distinction has been introduced first for gravisensing (for references, cf Brauner, 1962; Larsen, 1962), where sedimentation of statoliths is termed susception, and their interaction with living cell structures denotes perception sensu strictu. In photosensing, this distinction usually is ignored; however, absorption of a photon as resulting in an excited state of the pigment corresponds to susception, and interaction of the latter with a reaction partner to perception

Light as an Energy Source and Information Carn'er in Plant Physiology Edited by Jennings et of., Plenum Press, New York, 1996 169

sensu strictu. Results of photos ensing, i.e. photoresponses in plants, comprise very different areas of

plant physiology, as will be seen from the following examples (cf Shropshire & Mohr, 1983; Kendrick & Kronenberg, 1994):

- Growth and development can be controlled by light, as e.g., etiolation and de­etiolation; control of germination of spores and seeds; orientation of growth by the light direction; control of developmental steps by the length of the daily light period.

- Biochemical and metabolic processes are under the control of light, as in, e.g., partitioning of photosynthetic products between the organs; biosynthesis or accumulation of pigments (e.g., carotenoids or anthocyanins); light control of enzyme synthesis or activation, which may be traced back to translation or transcription of genetic information as one of the most elementary effects oflight on development.

- For movement, light is one of the most important controlling factors, as, e.g., for speed, continuity and direction of locomotion in motile microorganisms; chloroplast rearrangement with respect to the light direction; opening and closing of stomata; orientation ofleaves toward the sun, ensuring an optimal position for light harvesting.

- In cell biophysics, light effects on membrane properties are known, 'resulting in control of permeability or of ion pumps.

The responses in the first two groups frequently are slow processes, taking hours or days, whereas in the latter groups fast responses are found that can start within seconds.

If we look at this variety of photoresponses, it becomes immediately obvious that for many of these responses more detailed information has to be gained, by the organism, from the light signal, beyond the simple alternative light or darkness (cf Smith, 1994). The basic parameter is light quantity, i.e., fluence rate or fluence. Besides this there are responses to light quality, which require discrimination between wave-length ranges. Moreover, light direction can be important as an orienting factor; and finally, the time pattern of light and darkness can contribute to information about the environment. The central role of light quantity also extends into the other parameters, as sensing of light quality, of vectorial properties or of time pattern of light require comparison of light quantity in wavelength ranges, in space or in time, respectively. In many cases, the full signal comprises a combination of these parameters; in photoperiodic responses, e.g., the effect of a certain time pattern can strongly depend on the light quality (cf Vince-Prue, 1994).

It is the aim of this chapter to deal with this aspect of photosensing, i.e., detection of the light signal and its parameters.

PHOTOSENSING WITH RESPECT TO THE PARAMETERS OF THE LIGHT SIGNAL.

Light Quantity as the Basic Parameter in Photosensing.

For sensing of light quantity, the number of photons has to be counted by the photo sensory pigment. This can be a time-differentiated or a time-integrated counting (cf Bjorn & Vogelmann, 1994; Smith, 1994): Time-differentiated counting allows for detecting the fluence rate at any given point in time. Time-integrated counting, instead, detects the total nufuber of photons, i.e., photon fluence, applied over the experimental period. This latter requires, of course, that the light signal has a sufficiently long after-effect. The respective memory can reside in the photosensory pigment proper, or in the transduction chain, as will be discussed below.

In a typical case of sensing of light quantity, the response must be a quantitative function of the number of photons counted, be it time-differentiated or time-integrated counting. This quantitative function between the catalytic amount of photons as the input and the quantity of the output, is an unsolved problem, and therefore, a challenge for future

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research. Besides these graded responses, all-or~none responses are occasionally found, with a threshold light quantity below which nothing will happen, but above which full response will be reached. This is interpreted, in some phytochrome-mediated responses, as a result of cooperativity in a post-sensory step (cf. Mohr & Oelze-Karow, 1976). However, this distinction between graded vs. all-or-none responses need not be an absolute one. Rather, graded responses of an organ (e.g., growth, enzyme synthesis) theoretically could be brought about by all-or-none responses at the cellular level, with the thresholds being distributed, among the cells, with a large variance. It would be interesting to ask this question for selected examples of photoresponses.

The photochemical basis of photon counting is the excitation of a pigment molecule P by absorption of a photon (cf. Lenci et aI., this volume). The resulting excited states p* relax to the ground state P, usually with a lifetime far below the millisecond range for triplet and even below the microsecond range for the singlet state (cf. Dorr, 1983), but depending also on the respective photosensory pigment. Thus, during exposure a low steady-state fraction of P*/P is established, which depends on the fluence rate. During its short lifetime, P* interacts with a reaction partner, thus starting the transduction chain. The "long-living" triplet state of flavoproteins with a lifetime in the millisecond range (Horwitz, 1994; Lenci, personal communication), instead, can also relax, via reduction, to the semiquinone form of flavin, which has a lifetime of tip to a few minutes (Hertel, 1980; Horwitz, 1994); this may then start the cascade of transduction processes. In several systems, a light-induced absorbance change (LIAC) is observed, due to reduction of a b-type cytochrome by flavin (Asard & Caubergs, 1994). However, it is doubtful whether LIAC represents a general step in the transduction chain or is only a side effect; at least it can serve as a model for post sensory processes.

The final result of this kind of photon counting, i.e. the terminal response, depends strongly on the kinetics of the subsequent reactions, and particularly on the storage of the information somewhere in the transduction chain. If there exists a store with high storage capacity, this acts as a memory; within a certain time period the total number of photons is counted. Ideally, the Bunsen-Roscoe law holds, i.e. reciprocity between fluence rate and irradiation time for a given response; this is found, e.g., in the so-called first positive phototropical curvature of oat coleoptiles (cf. Dennison, 1979; Fim, 1994). If, on the other hand, there is not much storage capacity for the information, it is the time-differentiated quantity that is counted, i.e. the photon number per time unit; consequently, the response depends on the fluence rate. These two cases, however, are theoretical extremes, as usually reciprocity holds only within some limits of time and/or fluence rate.

This can be shown, as an example, for the orientation movement of chloroplasts in duckweed, Lemna trisulca (Zurzycki et a!., 1983). If cells are irradiated by short pulses of blue light, i.e. in the range of seconds or minutes, the chloroplasts are induced to move, during a short subsequent dark period, in the direction of the appropriate arrangement without, however, completing this response. For this incomplete response, reciprocity has been found, i.e. the fluence determines the extent of the response, irrespectively of how it is composed by its factors fluence rate and time. Thus, under these experimental conditions an after-effect is involved, which decays within minutes, i.e. with kinetics not shorter than the irradiation time. The memory probably resides early in the transduction chain. If, however, the exposure is extended to longer periods, the memory becomes short-lived in comparison with the duration of the light signal, and the response now depends on the steady-state quantity, i.e. on the fluence rate, which determines the speed of movement and/or the percentage of chloroplasts participating in the response.

The phenomena discussed so far are typical of so-called photocatalysis, where one pigment can exist in its ground state and in excited states. In plant photobiology, this appears to hold for most of the blue-light absorbing photosensory pigments. More complicated is the photochrome catalysis, where a pigment can exist in two photointerconvertible ground states, besides the excited states, and is called a photochromic

171

pigment. This holds for the phytochromes, the most important family of photosensory pigments in plants. They exist in two forms, the red-light absorbing Pr with a maximum sensitivity around 660 nm, and the far-red absorbing Pfr with a maximum around 730 nm (Fig. 1). Upon absorption of a photon, the ground state Pr is excited as shown above for the blue-light pigments, but P* relaxes to the metastable Pfr (Fig. la), which can be reversed to Pr either by absorption of a photon in the far-red region (Fig. 1 b), or by a slow dark process with a half life in the range of an hour (Fig. lc); in addition, Pfr can also undergo a dark destruction with a similar time constant (cf. Mancinelli, 1994).

Pr* 6~nmj ~Ir

a/ ~ Pr Pfr

Pfr*

b ,/ \7~nm

Ifr

,/ Pfr Pr

c

dark 660nm dark

~ Pr -:;;;;:::- Pfr

~ destr

Figure 1. The phytochrome system. a. Photoconversion of Pr to Pfr via its excited state Pr* and several intermediates (summarized to Ir). b. Photoconversion (photoreversion) of Pfr to Pr via its excited state Pfr* and several intermediates (summarized to Ifr). c. Summary of all important reactions: Dark synthesis, photoreversibility, dark destruction and dark reversion.

Thus, the result of absorption of a photon by Pr is effectively stored in Pfr, and because of its long lifetime this is an excellent memory for a short light pulse. Since the emptying of the memory is very slow as compared to its filling, phytochrome usually counts the total number of photons, i.e. fluence. This is true for the so-called low fluence-rate responses, which show the classical red/far-red antagonism, as, e.g., light-dependent germination of spores or seeds. Accordingly, in these cases the Bunsen-Roscoe reciprocity law holds (cf. Kronenberg & Kendrick, 1986). Sensing of the fluence rate, on the other hand, is possible, at least theoretically, in very low fluence rates, when phototransformation Pr ~ pfr approaches the time constant of dark reversion and dark destruction of PfI:, thus avoiding saturation of pfr formation. In this case, a steady-state level of pfr is established as depending on phototransformation, dark reversion, dark destruction of Pfr, and new synthesis ofPr (cf. Mancinelli, 1994).

In conclusion, there appears to be a fundamental difference with respect to the memory: Whereas in phytochrome-mediated responses the memory is found in the photosensory pigment proper, i.e., in its physiologically active form Pfr, for blue-light responses it is postulated to reside in the transduction chain. As an intermediate case, for flavins the semiquinon form (p. 171) may serve as a short-time memory, thus also residing in the pigment.

Light Quality.

It is trivial that for each photoresponse there exists an action spectrum, which, with

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some restnctlOns, reflects the spectral absorption characteristics of the photo sensory pigment (cf. Hartmann, 1983; Schafer et al., 1983). Thus, there are spectral regions with high, low or no effectivity, but this in fact is still a kind of sensing of quantity. Instead, true sensing of light quality would mean that the organism can distinguish between different spectral regions, i.e., that different regions have qualitatively different effects, with the light quantity being of minor importance (cf. Smith, 1994).

A typical example is the so-called chromatic adaptation of some cyanobacteria. Formation of the accessory pigments of photosynthesis can be under differential control of wavelength ranges, so that phycocyanin prevails in red light, but phycoerythrin in green light. Thus, the relative amounts of these two pigments are controlled by the spectral composition of light (cf. Goldsworthy, 1991). There are good reasons to assume that in these cases the two spectral ranges with opposite effects are sensed, independently of each other, by phycocyanin and phycoerythrin. Thus, this "colour vision" requires two photo sensory pigments.

Another well-investigated example of "colour vision" by non-animal organisms is the photophobic response of Halobacterium halobium (cf. Schirnz & Hildebrand, 1991). This archaebacterium can distinguish between two spectral ranges, which have opposite effects on the time pattern offorth and back movement. This "colour vision" is again mainly based on two photosensory pigments which, however, are two interconvertible forms of one pigment species, viz. sensory rhodopsin (sR). For details, see Stavenga et al. (1991) and Lenci et al. (this volume, p. 150).

Photochromic pigments are also responsible for "colour vision" of green plants, viz. phytochromes (cf. Smith, 1994). As already mentioned above, these pigments can exist in two forms, Pr and Pfr. Particular features of phytochromes are their high sensitivity, which results in saturation of photoconversion with relatively low fluences, and overlapping of their absorption spectra. Thus, a steady-state fraction of the "active" form Pfr as related to the total amount of phytochrome Ptot is reached after a few minutes under natural light conditions. This steady-state PfrlPtot ratio = 0 depends strongly on the spectral composition of the light: With a high percentage of the red part, 0 becomes high, and vice versa (Fig. 2a). Thus, in daylight, which contains red and far-red to similar amounts (red: far-red =

1.15), a 0 value of 55 to 60% may be found (Smith, 1994). Under a leaf canopy, however, i.e. in a forest or on a field covered with broad-leaved crop plants, most of the red part is filtered out, and (besides green light) mainly far-red can reach the soil surface. This results in

a

b

R>FR Pr~Pfr

¢

R>FR Pr~Pfr

0.6 I - daylight

0.0 o

: - sugar beet crop

- 2 su or beet leaves

2 3 R:FR ratio

Figure 2. "Colour vision" by green plants: Detection of the R:FR ratio by phytochrome (R = red light, FR = far-red). a. Shift of the steady state between Pr and Pfr by the prevailing light quality. b. Quantitative dependence of0 = PfrlPtot in steady state on the R:FR ratio (Ptot = Pr + Pfr). After Smith (1994).

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a preferential photoconversion ofPfr back to Pr, and 0 may become as low as a few percent (Fig. 2b). For the human eye, this transmitted light appears green, and hence the term greenshade has been introduced; however, green light is nearly ineffective for photoconversion ofphytochromes, and thus in green shade the plant senses almost pure far­red light (Holmes, this volume). Thus, "colour" vision by phytochromes is in fact only detection of the ratio of red : far-red light. - It is important to realize once more that it is not the reduced overall effective fluence rate in the green shade that changes the behaviour, but indeed the spectral shift.

To sum up, sensing of light quality requires at least two photosensory pigments, and this theoretical requirement can be fulfilled, in practice, not only by two independent pigments, but even by a single pigment species, provided it is photochromic, thus existing in two photo-interconvertible forms. In this respect, for higher plants phytochrome(s) can be considered as "intracellular eye(s), occuring in all cells and adapting the whole organism to its light biotope" (Hartmann & Haupt, 1983).

Sensing of Vectorial Properties of Light.

There can be several reasons for a plant to respond to the direction of light. A seedling has the best chance to reach optimal light conditions for photosynthesis, if it grows toward the light source; it may then be useful to orient the leaves with their surfaces normal to the incident light. Besides these examples of phototropism, chloroplasts can rearrange themselves in the cell so as to optimize light harvesting and to minimize damage by too strong light. Similarly, free moving (motile) microorganisms or microalgae may orient their locomotion with respect to the light source (phototaxis) in order to find the optimal light environment. In these examples it is necessary for the organism or for the cell to sense the direction from which the light is coming, or in which the light traverses the organ in question.

As a basic statement, there are no cases in which the light vector itself is sensed. Rather, unidirectional light is differently absorbed in different regions of a cell or of an organ, hence the vectorial information is transformed into a gradient of light absorption, and by comparison of absorption in different regions, the final response becomes asymmetric and thus ultimately oriented with respect to the direction of the incident light (Buder, 1920).

To become effective, the spatial absorption gradient requires at least two photo sensory sites where absorption can be compared; and indeed, in most cases photo sensory pigments are found around the surface of organs or cells, as, e.g., in phototropic coleoptiles or in cells with orienting chloroplasts. In some motile unicellular algae, however, there is only one photosensory site. Here the spatial gradient is replaced by a temporal gradient, as exemplified in Chlamydomonas (on the basis of the classical hypothesis for Euglena, which, however, appears to be disproved in this particular organism; cf Haupt and Hader, 1994): By continuous rotation during locomotion, asymmetrically incident light falling on the photo sensory structure undergoes periodic modulation, and thus the vectorial information is transformed into a temporal absorption gradient. This comparison in time is sometimes called "two-instant mechanism", as compared to the "one-instant mechanism" as denoting the comparison in space (cf Haupt & Feinleib, 1979).

Thus, the main question is how an absorption gradient is established by unilateral light, be it a spatial or a temporal gradient. This can be achieved by four main principles (cf Haupt & Hader, 1994; Kraml, 1994).

Attenuation. In an etiolated seedling, unilateral light hits the proximal side with full intensity; but on its way to the distal side it is strongly attenuated by the tissues. Thus, absorption follows a gradient across the diameter of the seedling, and light-controlled growth depends on this absorption gradient. The resulting differential growth is recognized as phototropic curvature. For coleoptiles, there are several models for the causal connection

174

between the absorption gradient and the differential growth, but to date none of them is fully satisfying (cf Cosgrove, 1994).

Refraction. The classical example for a second mechanism, is the sporangiophore of Phycomyces with its phototropism. This organ is almost transparent; due to the high refractive index of the cell content it acts as a strong cylindrical collecting lens (if surrounded by air) and thus focuses unilateral light to the distal side. Since, in Phycomyces, growth is transiently stimulated by light, stronger light absorption at the distal side of the subapical growth zone results in bending toward the light.

This interpretation is based on the famous inversion experiment by Buder (1918): if a sporangiophore is immersed in paraffin oil, it will then act as a diverging lens, and accordingly phototropism becomes negative. This experiment was later extended by Shropshire (1962) and - for a completely different system - by Scholz (1976), showing quantitative dependences on the refractive index when using different oils.

Although this contribution of refraction to directional sensing is well established, there are some problems which are not yet fully understood (cf Dennison, 1979).

- It is true that absorption at the distal side is much higher than at the proximal side, but it is restricted to a narrow region of the distal half cylinder. How is the signal transduced and integrated so as to establish a preferential response of the whole distal side?

- Stimulation of growth by light is a transient effect, but for phototropic bending a long-lasting difference in growth is required and observed.

For both these problems there exist hypotheses to solve them, but they go far beyond the scope of this paper.

Focusing of light by the high refractive index of the cell can act also in a different way, depending on the cell shape and the localization of responsivity, as shown for fern protonemata (Fig.3 a; Etzold, 1965). In contrast to the subapical growth zone in the multicellular coleoptile or in the unicellular sporangiophore, growth in fern protonemata is centered at the very tip, i.e. in the apical dome. Light coming from one side is not only focused in the cross section to the distal side as in the cylindrical sporangiophore, but additionally refracted, in the apical dome, in a longitudinal direction toward the subapical region and thus outside the growth center. Accordingly, the distal part of the apical dome is bypassed by the light, only at the proximal part can light stimulate growth. In consequence, the growth center is asymmetrically activated with preference toward the light source, a positively phototropic curvature results in bulging. In this case, the important feature is not focusing, hence local increase of absorption, but bypassing, hence local decrease of absorption; in the critical region there is less light than without refraction.

tI] I

0 Q e-

O proximal distal

a b c

Figure 3. Detection of light direction according to the principle of refraction. a. Tip of the apical cell of a fern protonema with light impinging from the left. Due to refraction, the distal half of the apical dome is bypassed by the ligbt; this concerns the sensitive zone which responds to light with growth enhancement (shaded area). As a result ofthis absorption asymmetry, the growth center is shifted to the left (dotted outline). b. Cross section through a cylindrical cell, e.g. a filament of Vaucheria, with the respective light refraction. Notice the bypassed regions slightly behind the flanks. c. Chloroplast accumulation in Vaucheria (cross sections) according to the situation in b, either at the regions with the highest absorption (low fluence­rate light; top), or at those with lowest absorption (high fluence-rate light; bottom).

175

Bypassing in the cross section is used for chloroplast orientation in the coenocytic alga Vaucheria (Fig. 3b; cf Haupt & Wagner, 1984). Light refraction in the cylindrical cell results in focusing, the effect of which, however, is counteracted by some attenuation in the cell contents. Thus, there is no major difference concerning light absorption between proximal and distal regions. However, the regions at or behind the flanks are bypassed, they obtain only scattered light. The chloroplasts distribute themselves accordingly (Fig. 3c): they accumulate selectively at the proximal and distal sides or at the flanks, depending on the fluence rate. One should be aware that this is not an orientation to the light direction in its strict sense; the cell does not discriminate between the head and the tail of the vectorial arrow (cf Fig. 3c, top). By this way, the chloroplasts gather at those regions of the cell wall which are normal or parallel to the incident light, and this is precisely what they have to do in order to optimize light harvesting (Haupt & Scheuerlein, 1990).

Interference Reflection. In the hitherto unique example of the unicellular alga Chlamydomonas, there is a highly sophisticated mechanism to sense light direction (Foster & Smyth, 1980). A stack of lipid layers underlies the photo sensory structure. As these layers have a distance from each other of a quarter of a wavelength (with regard to the peak in the absorption spectrum of the photo sensory pigment), the reflected waves interfere constructively at the photosensory structure. This interference depends on the orientation of the light beam: It is maximal at normal incidence and decreases with increasing angles of incidence. In this particular case, there is only one photosensory site in the cell, and therefore directional sensing is based on a temporal gradient rather than on the usual spatial gradient (p. 174).

Dichroic Orientation. Before presenting the fourth principle of establishing an absorption gradient, the so-called action dichroism has to be introduced, i.e. the dependence of a response on the orientation of the electrical vector (E-vector) oflinearly polarized light.

In polarized light, a fern protonema grows perpendicularly to the E-vector, whereas its rhizoid grows paralIel to it (Fig. 4a; Etzold, 1965). Upon turning of the E-vector the growth direction changes accordingly; by repetitive changes all kinds of growth pattern can be produced (Fig. 4b; Wada & Sugai, 1994). This dependence of growth direction on polarized light is called polarotropism. In the protonema, the response is localized in the apical dome and can be explained on the basis of dichroic orientation of photo sensory molecules (Fig. 4c); since their transition moments are in an orientation parallel to the surface, absorption is highest in those regions, where the cell surface is parallel to the E-vector. In consequence, if the E-vector deviates from an orientation normal to the long axis of the cell, light absorption in the apical dome is asymmetric; and because of growth stimulation by light absorption, the growth center also becomes displaced, as seen in phototropism (p. 175).

Another example of action dichroism is orientation of chloroplasts to light. If Lemna is irradiated with polarized light, the anticlinal cell walls become differently occupied by the chloroplasts, depending on whether they are parallel or perpendicular to the E-vector. Again, this can be explained by surface-paralIe! orientation of photo sensory molecules, resulting in an absorption dichroism. Obviously, the orientation of photo sensory molecules is rather stable, and this requires association with cell structures, probably organelles of the cytoskeleton (cf Haupt & Wagner, 1984); this appears to hold generally for sensory molecules.

In the alga Mougeotia an action dichroism is found whose explanation is the basis for understanding directional sensing in this organism. In polarized red light, the single chloroplast turns from profile to face orientation to the light only in those cells whose long axis is normal to the E-vector, or at least forms an angle with it (cf Haupt & Scheuerlein, 1990). This is due to the dichroic orientation of phytochrome Pr. The transition moments have a prevailing orientation paralle! to the surface, with components both parallel and

176

~2

a [

b

Figure 4. Polarotropism of a fern protonema. a. Growth of a protonema in polarized light, the E-vector of which has been turned from EJ to E2 . Notice growth of the protonema cell normal to E at any time, and growth of the rhizoid parallel to E at the beginning of germination. After Etzold (1965). b. Growth pattern of five protonemata in polarized light, the E-vector of which has repeatedly been turned as required for producing each particular letter. After Wada & Sugai (1994). c. Tip of the apical cell with the surface­parallel transition moments of the photoreceptor molecules indicated by the dashes. Those molecules that can substantially absorb the polarized light (impinging from the reader to the paper with the indicated E­vector) are drawn in bold face.

start T

a b c d

®(])88 Figure 5. Dichroic photoreceptor molecules as a basis of directional sensing. Schematic cross section through a cylindrical cell with the transition moments of phytochrome Pr parallel to the surface and either parallel (dots) or normal (dashes) to the long axis of the cell. a. Starting condition. b - d. Absorption of light as indicated by bold-face dots and dashes. b. E-vector parallel to the axis: no gradient. c. E-vector normal to the axis: gradient distal and proximal vs. flanks. d. Unpolarized light: gradient qualitatively similar to c. Below: Response of the ribbon-shaped chloroplast in Mougeotia only in c and d.

normal to the long axis of the cell (Fig. 5a). If polarized light has its E-vector parallel to the axis, it finds receptor molecules or their components in the appropriate orientation all around the cell periphery, so no absorption gradient is produced, and thus no directional information is obtained; the chloroplast remains in the profile position (Fig. 5b). If, however, the light has its E-vector normal to the axis, it finds photoreceptor molecules or their components in the correct orientation only at the proximal and distal surfaces, but none at the flanks; an absorption gradient ensues, in which the edges of the chloroplast move to the sites oflowest

177

absorption, and the chloroplast eventually exposes its face to the incident light (Fig. 5c). With this basic knowledge, we can understand also the effect of unpolarized light

(Fig. 5d). This latter can be considered as comprising E-vectors having every direction. It is sufficient to consider only those components that are oriented parallel and perpendicular to the cell axis. At the proximal and distal surfaces both components are absorbed, but at the flanks, only the component with the parallel vibrating E-vector is. Thus, a gradient of absorption is generated even in unpolarized light.

This can be generalized: whenever photosensory molecules have a preferential dichroic orientation in the cell, unilateral light is differently absorbed at the flanks as compared to the distal and proximal surfaces, and thus directional information is gained. However, as for the by-passing mechanism (p. 176), in this case no discrimination can be made between "head" and "tail" of the arrow denoting the light vector.

It should be added that, in nature, dichroic orientation of photo sensory molecules does not serve to detect light polarization or its direction, but is an important means for transforming light direction into an absorption gradient. Action dichroism, as found in the laboratory, is a by-product, but as such is a valuable tool for better understanding directional sensing. .

Complications. A directional response as based on an absorption gradient requires that no spatial levelling occurs at any step in the transduction chain. Particularly two sources of levelling have to be avoided, viz. saturation and signal transmission.

Saturation. If light absorption approaches saturation, any further increase of light unavoidably decreases and finally levels the gradient. This becomes particularly obvious with phytochrome, where the primary product of light absorption is relatively stable, i.e. Pfr. If, e.g., Mougeotia is irradiated with continuous light, the less absorbing flanks should reach saturation only a few minutes later than the strongly absorbing proximal and distal sides, and thus the gradient should be levelled before a visible response can start. To overcome this problem, the cell makes use ofa particular property of phytochrome (cf. Haupt & Wagner, 1984; Kraml, 1994). The prevailing surface-parallel orientation of its transition moments, as mentioned above (p. 177, 178), is true only for the red-absorbing form Pr. The far-red absorbing Pfr, however, has a preferential orientation normal to the surface. The consequences can be seen if we consider the wavelenght-dependent steady-state level of PfrlPtot (0) in saturation (p. 173, 174, fig. 2): This 0 level depends, in addition, on the dichroic orientation, as will be shown for the Mougeotia cell (cf. Wada et aI., 1993).

Due to the different dichroic orientation of Pr and Pfr, at the proximal and distal surfaces absorption of any light by Pr is facilitated, but its absorption by Pfr is reduced (Fig.6). Thus, the photo stationary level is shifted toward Pfr. The inverse situation is found at the flanks: absorption by Pfr is facilitated, by Pr reduced, the steady state is shifted toward Pro Thus, a permanent gradient of Pfr is obtained even in saturation. - By the way, here we have an additional important function of action dichroism.

There is, at least theoretically, another possibility of overcoming the problem of saturation. For chloroplast orientation, there are several examples in which two or more independent photo sensory pigments are involved (cf. Haupt & Scheuerlein, 1990). Till now, in no case has it been proven that these pigments act in different ranges of fluence rate, so as to extend the saturation-free range by integrating over all pigments. However, for the induction' of polarity in germinating spores ofEquisetum (horse tail) or zygotes of the brown alga Fucus, two photosensory pigments have been found that indeed cover different orders of magnitude (Meyer zu Bentrup, 1964).

Signal transmission. For an oriented response, local autonomy and independence must be ensured for all transduction steps from light absorption until the terminal response, i.e., the signal must not be integrated over the whole cell, over its cross section, over its periphery or over the organ. Consistent with this requirement, for chloroplast orientation there is some evidence that several main steps are localized at the cell membrane or at the

178

1 1 1 -I-Pr Pr~Pfr

Ptr

\ - Pr~Pfr I

_1-

Figure 6. Gradient of phytochrome Pfr in saturating light, due to the different orientation ofPr and Pfr, the transition moments of which are indicated by the surface-parallel (pr) and normal (pfr) dashes. High probability of light absorption is indicated by bold-face dashes. Besides, shift of the photostationary state toward Pr or Pfr is indicated for the respective regions. After Haupt, in Haupt & Wagner (1984).

cytoskeleton. Complete lack of signal transmission, however, can cause new problems. Oriented

responses usually depend on the ratio (or difference) of absorption in different regions rather than on absolute measurements. This requires comparison between these regions and hence "exchange of information". E.g., how can a chloroplast "know" that there is another region nearby with a "preferable" level of absorption? How can a growth region in a seedling "know" whether it is more irradiated or less than the region across the diameter of the coleoptile? For the chloroplast example, an hypothesis is referred to in Wada et al. (1993); in coleoptiles, transmission of information is postulated that assists in transducing the difference (or the ratio), may be even in its amplification, yet without impairing the local autonomy (cf Firn, 1994). To date, no knowledge about this important step is available.

Unsolved problems. As an addendum, three examples will be mentioned for which the mechanism of directional sensing is hardly understood as yet. First, in some plants the leaves orient their surface normal to the sun and follow its course during the day ("sun tracking"). In some cases, the respective light sensitivity is located in the main vascular bundles of the leaf If, for simplicity, we restrict observations to deviations from normal incidence along the longitudinal axis of the leaf, it is evident that the leaf has to discriminate between tip­oriented and base-oriented angles of incidence. It has been suggested by Koller et al. (1990) that this discrimination is based on dichroic photo sensory molecules in the xylem parenchyma, and some results support this view; but in detail the respective model has not yet been proven.

Second, the orientation of the flat unicellular green alga Micrasterias denticulata superficially appears similar (Neuscheler, 1967). This alga, surrounded by water, exhibits phototaxis when gliding on a substratum; it orients its plane normal to the incident light. This orientation is possible even if the cell is resting on one of its poles. It can hardly be imagined that the small thickness of the cell could produce an effective absorption gradient; but if it does, how can the deviation from normal incidence be detected so as to result finally in asymmetric production of the jelly substance at the pole, which is responsible for locomotion or for turning toward the light?

Third, filamentous cyanobacteria like Phormidium slide back and forth on their substratum with autonomous reversals. The time pattern of these reversals can be modified by the light direction, i.e. whether light is coming from front or rear with respect to the actual direction of movement, even if movement and light direction form an angle (cf Hader, 1979). How do the organisms discriminate between these two main directions? Are intracellular or intercellular absorption gradients being formed? Which of the mechanisms presented above is being used (Haupt, 1974)?

To sum up, organisms have a variety of mechanisms to sense the direction of light;

179

these can be used also in combination. Which of them can become effective depends on several par-ameters: for the attenuation mechanism, the cell or organ must not be too transparent; for a refraction mechanism, the surrounding medium has to have a low refractive index - it functions preferentially in air; for interference reflection, an extremely ordered structure is necessary; for the dichroism mechanism, a proper orientation of photosensory molecules is required. Moreover, particular mechanisms have evolved for overcoming saturation effects. Yet there are still unsolved problems of directional sensing.

Photosensing with Respect to the Time Pattern.

Time is regularly involved in photosensing, as has been shown above with time­integrated vs. time-differentiated sensing of light quantity. Moreover, whenever a response depends quantitatively on the fluence rate, every change of the latter results in a new level of response, as, e.g., in photokinesis of filamentous cyanobacteria (cf Hader, 1979). This is trivial and is not a specific time-pattern effect. There are, however, responses that depend on the change offluence rate in a specific way, or responses that depend on a specific pattern of light-dark cycles.

Change of Fluence Rate as a Signal. For the light-growth response of the sporangiophore of Phycomyces, change of fluence rate is the signal (cf Dennison, 1979): There is a large range of fluence rates in which steady-state growth is independent of the actual fluence rate. If, however, light is suddenly increased (e.g., within a few seconds or less; i.e., "step-up") and then kept constant at the higher fluence rate, growth is transiently increased, but it returns to the former steady-state speed after 10 to 20 minutes (Fig. 7). Similarly, a "step-down" of fluence rate induces a transient decrease of growth. This light­growth response has been interpreted by a model (Delbriick & Reichardt, 1956), which is based on a few reasonable assumptions: after some time in constant fluence rate, the sensory-response system has adapted to this condition, i.e. the ambient fluence rate has become an internal standard, with which the system continuously compares the actual fluence rate. If both differ from each other, growth is accelerated or decelerated.

211 .l.._ ~~o£-___ gr_o..:::w ... th __

o 5 10 min

Figure 7. Light-growth response of the sporangiophore ofPhycomyces. Top: At zero time the fluence rate of light is increased from io to i (set up). By adaptation the internal standard (a) is set to the new fluence rate, the dotted curve represents the kinetics of adaptation. Center: the relative fluence rate with respect to the internal standard (i1a) shows a transient peak. Bottom: the growth rate (relative to that before the experiment =1) follows the i1a curve with some delay. After Delbriick & Reichardt (1956).

180

Simultaneously, the system begins to adapt to the new condition, i.e. the internal standard is changed and set t6 the new ambient fluence rate; accordingly, the growth rate returns to its former steady-state rate (Fig. 7). The molecular basis of this adaptation, however, is not yet known.

Consistently with this model, the light change has to be substantially faster than the kinetics of adaptation, otherwise, a response can hardly be expected. Finally, quantitative investigations show that the response does not depend on the absolute difference of fluence rates, I - 10 , but on the relative difference, (I - 10 ) / 10 . Thus, to obtain a given response, the change has to be greater, the stronger the starting light condition. This reminds us of the Weber-Fechner law in sensory physiology of animals.

Another response to changes in fluence rate is the photophobic respose of motile organisms. The responses in Halobacterium halobium upon crossing a light-dark border (Lenci et aI., this volume) are nothing other than a response to a step-up or step-down signal. Such photophobic responses have been investigated in more detail in filamentous cyanobacteria, and in Phormidium a model has been proposed by Hader (1979). the respective light is absorbed by phycobilins, the accessory pigments of photosynthesis; hence it acts via photo system 2 (PS 2), driving an electron transport to PS 1. During this transport, an electron pool in the chain is filled until input and output are equal; thus, under constant conditions a steady-state level is reached. Upon step-down of light, acting on PS 2, or step­up, acting on PS 1, the electron output from the pool outweighs the input; it takes some time until input and output are balanced again, and this transient lack of equilibrium appears to be the effective "product" of the photophobic signal. Its final result is premature reversal of gliding movement.

This model is strongly supported by the finding that bioelectrical effects are involved in the transduction chain from the step-down or step-up signal to the photophobic response. Moreover, it is tempting to interpret adaptation on the basis of this model. However, no generalization is possible, as adaptation can probably reside at very different sites in the transduction chain, including in the sensory system proper.

Patt~rns of Light-Dark Cycles as a Signal. Many higher plants use the daylength as a signal for timing their developmental processes, particularly to synchronize their flowering with their pollinators (cf Vince-Prue, 1994). This photoperiodism requires that these plants measure the length of the light and dark periods in the daily cycle. This is mediated by the physiological clock.

For interaction of light with the clock, at least two effects have been found, which, however, appear to concern the transduction chain rather than photosensing proper, and hence need not be discussed in detail:

- In the daily course of the clock, phases alternate with quantitatively or qualitatively different effects of light, and this is the basis for measuring the length of the day and/or of the night. This phase dependence of light effects is usually tested by "nightbreaks", i.e. by short light pulses at various times in the dark period.

- If the physiological clock is not in phase with the external light-dark cycle, the latter can reset the clock. For this resetting, the transition from darkness to light or from light to darkness is important and can be considered as the signal to be sensed. This strongly reminds us of the changes in intensity as a signal for light-growth response or for photophobic response. However, resetting of the clock by one single signal can already be a permanent effect, whereas in the examples mentioned above the response is always transient.

CONCLUDING REMARKS.

Four parameters of photosensing have been discussed in this chapter. Sensing of light quantity can be understood on the basis of general facts of photochemistry, and it depends

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mainly on the memory in the sensory-transduction system whether fluence rate as the tirne­differentiated quantity or fluence as the time-integrated quantity is measured.

Sensing of light quality, i.e. discrimination of spectral regions, requires at least two sensory pigments. These can be independent pigments, or two spectrally different forms of one photochromic system.

For sensing oflight direction the vectorial information of the light is transformed into an absorption gradient, and fluence rates are compared at different regions. Various mechanisms have evolved for establishing an absorption gradient: attenuation of light on its way through the cell or organ; refraction of light; interference reflection at highly specified structures; dichroic orientation of photosensory pigment molecules. These mechanisms can act also in combination. Moreover, in some motile organisms the spatial gradient at two or more photosensory sites can be replaced by a temporal gradient at one site, based on periodic modulation of its exposure, which can be achieved, theoretically, by any of the four above-mentioned mechanisms. Finally, attention has been drawn to a few examples in which the mechanism of directional sensing is still unsolved.

Particular steps for sensing of time pattern appear to be locked to transduction processes, but detailed knowledge is still lacking and is hence a challenge for future generations of scientists.

In the present chapter, only occasionally has attention been paid to molecular approaches. However, much of our present knowledge on photosensing is the result of molecular approaches, which had already started before this term was invented and introduced, and have recently been successfully extended to genetic approaches. Future progress will strongly depend on these approaches. However, one should always be aware that photosensing finally results in behaviour, and to understand this, one has to know the respective organisms with all of their functions. Thus, it is only the combination of molecular and organismic approaches that can promise satisfactory progress in our field.

REFERENCES.

Asard, H. & Caubergs, R (1991) in: "Biophysics of Photoreceptors and Photomovements in Microorganisms", (Lenci, F., Ghetti, F., Colombetti, G., Hader, D.-P. & Song, P.-S .. eds.), p.181, Plenum Press, New York, London.

Bjorn, L. O. & Vogelmann, T. C. (1994) in: "Photomorphogenesis in Plants", 2nd edition, (Kendrick, RE. & Kronenberg, G.H.M., eds.), p.17, Kluwer, Dordrecht.

Brauner, L. (1962) in: "Handbuch der Pflanzenphysiologie" vol. 1712, (Bunning, E., ed.), p.74, Springer, Berlin.

Buder, 1. (1918) Berichte der Deutschen Botanischen Gesellschaft, 36:104. Buder, J. (1920) Berichte der Deutschen Botanischen Gesellschaft, 38: 10. Cosgrove, D. J. (1994), in: "Photomorphogenesis in Plants", 2nd edition, (Kendrick, RE. & Kronenberg,

G.H.M., eds.), p.631, Kluwer, Dordrecht. Delbruck, M. & Reichardt, W. (1956) in: "Cellular Mechanisms in Differentiation and Growth", (Rudnick,

D., ed.), p.3; Princeton University Press. Dennison, D. S. (1979) in: "Encyclopedia of Plant Physiology", New Series, vol. 7, (Haupt. W. & Feinleib,

M. E .. , eds.), p.506, Springer, Berlin. Dorr, F. (1983) in: "Biophysics", (Hoppe, W., Lohmann, W., Markl, H. & Ziegler, H., eds.), p.265,

Springer, Berlin. Etzold, H. (1965) Planta, 64:254. Firn, R D. (1994) in: "Photomorphogenesis in Plants", 2nd edition, (Kendrick, RE. & Kronenberg,

G.H.M., eds.), p.659, Kluwer, Dordrecht. Foster, K. W. & Smyth, R D. (1980) Microbiological Review, 44:572. Goldsworthy, A. (1991) in: "Photoreceptor Evolution and Function", (Holmes, M. G., ed.), p.241, Academic

Press, London. Hader, D.-P. (1979) in: "Encyclopedia of Plant Physiology", New Series vol. 7, (Haupt, W. & Feinleib, M.

E., eds.), p.268, Springer, Berlin. Hartmann, K. M. (1983) in: "Biophysics", (Hoppe, W., Lohmann, W., Markl, H. & Ziegler. H., eds.), p.1l5,

Springer, Berlin. Hartmann, K. M. & Haupt, W. (1983) in: "Biophysics", (Hoppe, W., Lohmann, W., Markl, H. & Ziegler, H.,

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eds.), p.542, Springer, Berlin. Haupt, W. (1974) in: "Progress in Photobiology, Proceedings of the VI. International. Congress on

Photobiology Bochum", (Schenck, G. 0., ed.), 026. Haupt, W. (1991) in: "Biophysics of Photo receptors and Photomovements in Microorganisms", (Lenci, F.,

Ghetti, F., Colombetti, G., Hader, D.-P. & Song, P.-S., eds.), p.7, Plenum Press, New York, London. Haupt, W. & Feinleib, M. E. (1979) in: "Encyclopedia of Plant Physiology", New Series vol. 7, (Haupt, W.

& Feinleib, M. E., eds.), p.l, Springer, Berlin. Haupt, W. & Hader, D,-P. (1994) in "Photomorphogenesis in Plants", 2nd edition, (Kendrick, RE. &

Kronenberg, G.H.M., eds.), p.707, Kluwer, Dordrecht. Haupt, W. & Scheuerlein, R (l990) Plant Cell Envir., 13:595. Haupt, W. & Wagner, G. (l984) in: "Membranes and Sensory Transduction", (Colombetti, G. & Lenci, F.,

eds.), p.331, Plenum Press, New York, London. Hertel, R (1980) in: "Photoreception and Sensory Transduction in Aneural Organisms", (Lenci, F., &

Colombetti, G., eds.), p.89, Plenum Press, New York, London. Horwitz, B. A. (1994) in "Photomorphogenesis in Plants", 2nd edition, (Kendrick, RE. & Kronenberg,

G.H.M., eds.), p.327, Kluwer, Dordrecht. Kendrick, R. E. & Kronenberg, G. H. M., eds. (l994), "Photomorphogenesis in Plants", 2nd edition,

Kluwer, Dordrecht. Koller, D., Ritter, S., Briggs, W. R & Schafer, E. (l990) Planta, 181:184. Krarnl, M. (l994) in "Photomorphogenesis in Plants", 2nd edition, (Kendrick, RE. & Kronenberg, G.H.M.,

eds.), p.417, Kluwer, Dordrecht. Kronenberg, G. H. M. & Kendrick, R E. (1986) in "Photomorphogenesis in Plants", (Kendrick, RE. &

Kronenberg, G.H.M., eds.), p.99, Kluwer, Dordrecht. Larsen, P. (1962) in: "Handbuch der Pflanzenphysiologie" vol. 17/2, (Bunning, E., ed.), p.34, Springer,

Berlin, Mancinelli, A. L. (1994) in: "Photomorphogenesis in Plants", 2nd edition, (Kendrick, RE. & Kronenberg,

G.H.M .. eds.), p.21L Kluwer, Dordrecht. Meyer zu Bentrup, F. W. (1964) Planta, 63:356. Mohr, H. & Oelze-Karow. H. (1976) in: "Light and Plant Development", (Smith, H., ed.), p.257,

Butterworth, London,. Neuscheler. W. (1967) Zeitschr. Pflanzenphysiol., 57:46. Schafer, E., Fukshansky, L. & Shropshire, W., jr. (1983) in: "Encyclopedia of Plant Physiology", New Series

vol. 16, (Shropshire, W., jr. & Mohr, H., eds.), p.39, Springer, Berlin. Schimz, A. & Hildebrand, E. (1991) in: "Biophysics of Photo receptors and Photomovements in

Microorganisms", Lenci, F., Ghetti, F., Colombetti, G., Hader, D.-P. & Song, P.-S., eds.}, poo231, Plenum Press, New York, London,.

Scholz. A. (1976) Zeitschr. Pflanzenphysiol., 77:406. Shropshire, W. (1962) Journal of General Physiology, 45:949. Shropshire, W., jr. & Mohr, H., eds. (1983) "Encyclopedia of Plant Physiology", New Series, vol. 16,

"Photomorphogenesis", Springer, Berlin. Smith, H. (1994) in "Photomorphogenesis in Plants", 2nd edition, (Kendrick, RE. & Kronenberg, G.H.M.,

eds.), p.377, Kluwer, Dordrecht. Stavenga, D. G., Schwemer, 1. & Hellingwerf, K. J. (1991) in: "Photoreceptor Evolution and Function",

(Holmes, M. G., ed.), p.261, Academic Press. London. Vince-Pme, D. (1994) in "Photomorphogenesis in Plants", 2nd edition, (Kendrick, R.E. & Kronenberg,

G.H.M., eds.), p.447, Kluwer, Dordrecht. Wada, M .• Grolig, F. & Haupt, W. (l993) Journal of Photochemistry and Photobiology, B:Biology, 17:3. Wada, M. & Sugai, M. (1994) in "Photomorphogenesis in Plants", 2nd edition, (Kendrick, RE. &

Kronenberg, G.H.M., eds.), p.783, Kluwer, Dordrecht. Zurzycki, 1., Walczak, T., Gabrys', H. & Kajfosz, 1. (1983) Planta, 157:502.

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MECHANISMS OF PHOTORECEPTION: ENERGY AND SIGNAL TRANSDUCERS

D.-P. Hader

Friedrich-Alexander-Universitat, Institut fur Botanik and Pharmazeutische Biologie, Staudtstr. 5, D-91058 Erlangen, Germany

INTRODUCTION

The role of light for the biota is manyfold; however, basically two major purposes can be distinguished: • the energy of captured photons is utilized for energetic reasons such as in the various

forms of photosynthesis which will be covered in this NATO ASI extensively and • it serves as a key signal informing an organism about important aspects of its environment.

While the first role of light absorption is restricted to photosynthetic organisms in the widest sense, sensory mechanisms have been demonstrated in many photosynthetic and non­photosynthetic organisms (Hader, 1994) and may be an intrinsic characteristic of every living cell even though the cellular response has not yet been revealed.

Several groups of pigments have been considered as possible photo receptors in microorganisms and higher plants: carotenoids and rhodopsins, hypericin derivates, biliproteins, flavins and pterins, protoporphyrin and chlorophylls, to name a few (Lenci et aI., 1984; Nultsch & Hader, 1988). Most photoreceptor molecules of microorganisms, plants and animals are rather specialized and serve one or the other purpose listed above, but in some organisms the photoreceptors are employed for both tasks: while, e.g., phytochrome (Song, this volume) is exclusively used as a signal receiver, chlorophylls and some accessory pigments can be used both as energy transducers and as photo receptors for photo motile responses.

Almost all photoreceptor molecules consist of one (or more) chromophoric group(s) linked to a protein. However, for many sensory responses neither the nature of the chromophoric group nor the protein it is linked to has been identified. For both energetic and sensory purposes a highly ordered structure needs to be maintained by the cell, which is best achieved by membrane anchoring. Thus, it is a fair assumption that most, if not all photoreceptor molecules, are membrane bound (Hader & Lebert, 1994).

The molecular basis of photo perception leading to an excited state is covered by Lenci (this volume) and thus does not need to be repeated here. In most cases the subsequent

Light as an Energy Source and Information Carrier in Plant Physiology Edited by JelUlings et 01., Plenum Press, New York, 1996 185

photochemical reactions (e.g., photoreduction, photoconversion, photooxidation) are not known (Sol)g et ai, 1980c). The sensory transduction chain triggered by these events is also obscure for most cases; therefore the chain of events is often depicted by a black box approach (Fig. 1; Lenci et aI., 1984): the sensory transduction chain starts with the perception of a photon by a photoreceptor molecule. In some cases the absorption of a single photon may suffice to cause a response; therefore a potent energy amplification is needed (Hader, 1979; Nultsch & Hader, 1988). As the photoreceptor and the response organelle are often located in different parts of the cell, or even in different cells of a multicellular organism, a spatial transmission of the signal is needed. The final step in the transduction chain is the control of the response organelle, e.g., a change in flagellar beating or an altered morphology.

Photoperception Sensory transduction: Signal amplification Spatial transduction /

Effector response

Figure 1. Sensory transduction chain of photomovement responses in microorganisms (after Lend et aI., 1984).

The main topic of this lecture will be the discussion of systems in which pigments serve both energetic and sensory purposes. While this feature is found in many prokaryotes, some eukaryotes also seem to employ photoreceptor pigments for this dual task. Most microorganisms show three different types of photoinduced behavior: • phototaxis, which is an orientation with respect to the light direction which can be toward

the light source (positive phototaxis), away from the light source (negative) or at an angle (called diaphototaxis if perpendicular to the light beam),

• photokinesis, which describes the dependence of the steady state linear speed of movement with respect to the irradiance of the light source and

• photophobic responses, which are transient movement reactions (such as stops or turns) induced by sudden changes in the irradiance (Diehn et aI., 1977).

ENERGY CAPTURE AND PHOTOPHOBIC RESPONSES IN HALOBACTERIUM SALINARIUM

The archaebacterium Halobacterium salinarium (previously called H. halobium) and a number of other halophilic prokaryotes belong to the archaebacteria. This group is very different from all other bacteria with respect to their physiology and morphology. H. salinarium possesses a unicellular, rod-shaped cell which propels itself using a bundle of flagella arranged at one or both cell poles. Unlike E. coli, it moves equally well in either direction and changes direction of movement at regular intervals of about lOs. When the cell encounters an increase in the fluence rate of green or orange light (maximum at 587 nm) Halobacterium responds by suppressing the next endogenous reversal for a period of time. Upon a sudden decrease in the fluence rate of the same wavelength range, the cells induce a premature reversal of movement. The opposite behavior is induced by changes in blue or UV light: an increase in the fluence rate causes a sudden reversal and a decrease a prolonged swimming in the original direction. The cells respond similarly in a light gradient which results in the accumulation of bacteria in regions of optimal light conditions. The responses are similar to those of enteric bacteria in chemical gradients; therefore the reaction to an increase in green/orange light or a decrease in blue/UV has been called an attractant

186

response, and the opposite is called a repellent response. H. salinarillm synthesizes a number of bacteriorhodopsins which are similar in their

protein structure and their chromophoric group, retinal, to the rhodopsins found in eukaryotes. The most abundant of these pigments is bacteriorhodopsin (bR) which is a light­driven proton pump and carries out a primitive form of photosynthesis by generating a proton gradient across the cytoplasmic membrane exporting protons upon absorption of suitable light quanta.

Early investigations of the photobehavior resulted in the hypothesis that this pigment has a dual role in energy conversion and photo sensory processes (Nultsch & Hader, 1978; Hildebrand & Dencher, 1975). Later on, it was found that bR- mutants are still capable of sensory responses, leading to the assumption that halorhodopsin (hR), a light-driven chloride pump present in the mutants, may be the photoreceptor. This hypothesis was also proven wrong as hR- mutants showed both step-up and step-down photophobic responses. Further mutant analysis revealed a third rhodopsin present in very small amounts in the cells (Bogomolni & Spudich, 1982; Spudich, 1985; Spudich et aI., 1985). Being regarded as the photoreceptor for light-induced cellular responses, it was called sensory rhodopsin I (sR-I). As do all rhodopsins, it undergoes a pronounced photocycle after being excited by a photon (Fig. 2). The unexcited molecule absorbs at 587 nm and after excitation a long lived form, sR-I373 , is formed via several intermediates. This UVlblue absorbing form of the photochromic pigments can return to its long wavelength-absorbing form with a half life of about 750 ms (Oesterhelt & Marwan, 1990). However, upon excitation with a UVlblue photon it can return to the ground state via a different intermediate, sR-15lO. Thus, it was hypothesized that the same pigments may drive both photo sensory responses by excitation of its different photochromic states. Later on, Takahashi (1991) found an additional sensory rhodopsin (sR-II), also called phoborhodopsin, which only mediates a blue light-induced repellent response with a maximum at 480 nm. Recently the idea of a possible role for bacteriorhodopsin in photo sensory processes has seen a revival as it seems to induce similar phobic responses, which may be explained by assuming that a sudden change in the fluence rate may cause a change in the electrical or proton gradient across the cytoplasmic membrane, which in tum induces a change in flagellar activity.

As the sensory rhodopsins do not pump ions, the sensory transduction chain cannot involve membrane potential changes. In contrast, they are associated with a 97 kDa membrane-bound methyl-accepting "phototaxis" protein (MPP). Similar proteins are also involved in chemotaxis in enteric bacteria. Photosensory and chemosensory pathways seem

sR-1S87 ,

laom. ,A-I". \90 jlS

sR-l s10 1-I ,AI". ~jlS

sR-1 373

b /SR-II480,

/3ooms \

sR-II530 Intermediates

~j sR-II 350

Figure 2. Photocycle of sensory rhodopsin I and II in Halobacterium salinarium.

187

thus to converge at this point and share a common transduction chain. It is assumed that the retinal isomerization induces a conformational change in its apoprotein based on steric interaction between the chromophore and the 13-methyl group of the rhodopsin protein. Marwan and Oesterhelt (1991) have suggested that fumarate is involved in the final step of the transduction chain.

PHOTOKINESIS AND PHOBIC RESPONSES IN PHOTOSYNTHETIC BACTERIA

Many eubacteria display light-induced motor responses. None of these organisms show the true steering behavior characteristic of phototaxis, but rather depend on photokinesis and photophobic responses as their main strategies to seek and maintain a position in their habitat favorable for energy capture, growth and reproduction (Nultsch & Hader, 1988; Hader, 1988, 1989). Though not investigated to any extent, bacteria seem to utilize a number of different photoreceptor molecules, a fact which can be interpreted by stating that nature has experimented with a number of strategies and photorecepors duririg evolution from which a limited number was selected and used in higher organisms.

One example is the so-called photoactic yellow pigment (PYP) found in halophilic photosynthetic bacteria such as EctothiorhodQjpira halophila or Rhododospirillum salexigens (Meyer et aI., 1993). It is a water soluble, heat stable 14 kDa protein, the crystal structure, the amino acid and gene sequence of which are known. It has an absorption maximum at 446 nm and an emission at 495 nm. The protein is similar to a phycobilin binding protein, but the chromophore still awaits identification. Like the bacteriorhodopsins, it undergoes a photocycle with intermediates absorbing at 460 and 340 nm.

In Rhodospirillum rubrum, a photosynthetic purple bacterium, the action spectrum of photokinesis closely resembles that of photosynthesis and indicates the involvement of bacteriochlorophyll a as well as spirilloxanthin, one of the characteristic carotenoids of these organisms (Throm, 1968). The striking similarity between the spectra has early on been interpreted as an indication that the photosynthetic apparatus also serves a sensory function (Manten, 1948). There are several possible links between photosynthesis and the photoresponses. Classical inhibitors of the photosynthetic electron transport chain such as 3-(3',4'-dichlorophenyl)-l, I-dimethyl urea (DCMU) do not impair photokinesis in purple bacteria. In contrast, other inhibitors such as o-phenanthroline or atebrine drastically block the photokinetic effect. Uncouplers including dinitrophenol, imidazole and desaspidin also impair the response. These observations lead the way to the interpretation that the linkage is on the energetic level: one could hypothesize that increasing the irradiance augments the production of ATP (or proton motive force, pmf) which in turn provides more energy for the cellular engine, the flagellar motor (Valkunas et aI., 1985; Hader, 1987a). The common characteristic of these uncouplers is that they shut down the ATP production without blocking the photosynthetic electron transport. Some operate as protonophores, rendering the membrane leaky for protons. Thus, the proton gradient across the photosynthetic membranes, which in the case of purple bacteria are identical with the cytoplasmic membrane, breaks down. Let me mention in passing that most uncouplers are not specific for uncoupling the photosynthetic energy production but likewise uncouple the respiratory energy conversion. Table 1 shows the effects of several uncouplers on photokinesis in R. rubrum, indicating that in fact an increased energy production at higher irradiances is responsible for the photokinetic effect. The notion that photokinesis in purple bacteria is brought about by increasing the availability of ATP due to higher photophosphorylation was supported by the fact that externally added ATP induced an increase in the linear swimming velocity (Nultsch & Throm, 1968).

The action spectrum of photophobic responses in the purple bacterium R. rubrum also resembles the cells' absorption spectrum (Clayton, 1953a,b). However, unlike photokinesis,

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photophobic responses are inhibited by added ATP (Thomas, 1950). Inhibitors of the electron transport·· chain impair photophobic responses while uncouplers stimulate them., which has been interpreted by assuming that phobic responses are linked to the photosynthetic electron transport chain rather than to photophosphorylation. However, it is difficult to explain the rather low threshold values: in Chromatium the zero threshold of phobic responses was found at about 0.01 Ix (Schlegel, 1956) which may indicate that the photoreceptor pigments operate by a mechanism different from photosynthetic electron transport (Hader, 1987a,b). This idea is supported by the fact that aerobically grown cells which do not develop measurable amounts of bacteriochlorophyll show step-down phobic responses (Harayama & lino, 1977a). Phobic responses can also be induced by the addition of ferric ions (Harayama & lino, 1977b).

Table 1. Effects of several uncouplers on photokinesis under unaerobic conditions and on dark movement under aerobic conditions (50 % inhibition) in R. rubrum (after Throm, 1968).

anaerobic. light aerobic. dark

DNP 10-5 M

Desasidin 10-6 M

lmidazol 3xlO-5 M

10-5 M

2x10-7 M

2x10-5 M

More recently, Armitage and co-workers have investigated the sensory transduction chain oflight-induced responses in a number of purple bacteria such as Rhodopseudomonas sphaeroides (Armitage & Evans, 1985). By incorporation of potential sensitive dyes including oxanoles they could show that light-induced behavioral responses are accompanied by electrical membrane potential changes (Armitage & Evans, 1981a). An alternative method uses the electro chromic shift of membrane-bound carotenoids (Armitage & Evans, 1980, 1981b). Reaction center lacking mutants did not show a light-induced increase in swimming velocity nor a carotenoid band shift (Armitage & Evans, 1981 a,b). Photophobic responses are also accompanied by electrical potential changes: an increase in light intensity causes an increase in the membrane potential. As the membrane potential controls the direction of flagellar rotation light controls the swimming direction (Armitage & Evans, 1983).

LIGHT RESPONSES IN CYANOBACTERIA

Many cyanobacteria (formerly also called blue-green algae) are filamentous and glide when in contact with a substrate. The mechanism of movement has not yet been revealed, and there are two major models trying to explain the mode of propulsion (Hader, 1986, 1987a,b): one claims that the cells excrete slime through membrane pores pushing the filaments forward, a mechanism which has also been proposed for desmid algae (Mix, 1969). The alternative model assumes that contractile filaments in or below the cell wall layers generate undulations over the surface which allow the cells to move forward in a similar fashion to an earthworm through soil (Hader & Hoiczyk, 1992).

Cyanobacteria differ from all other photosynthetic bacteria in using chlorophyll a (but not chi b) rather than one of the bacteriochlorophylls and also in employing two photosystems (like eukaryotes) rather than one. Despite the obvious dissimilarity between the photosynthetic apparatus of cyanobacteria and that of other prokaryotes, photokinesis in

189

cyanobacteria also seems to be brought about by an increased energy production by the photosynthetic apparatus as indicated by uncoupler studies (Nultsch, 1962). However, when taking a closer look, the action spectra of different species differ in a characteristic way (Fig. 3). The Oscillatoriacean Phormidium ambiguum uses photosystem I exclusively, as indicated by the action spectrum which resembles the absorption spectrum of chlorophyll a; therefore it has been assumed that photokinesis in this species is brought about by cyclic photophosphorylation. In contrast, the Nostocacean Anabaena variabilis has an action spectrum which indicates the involvement of the accessory pigment phycocyanin, indicating the activity of non-cyclic phosphorylation (Nultsch & Hader, 1980; Nultsch, 1974; Nultsch & Hellmann, 1972).

Some cyanobacteria also show phototaxis which follows one of two different patterns. In the family of Oscillatoriaceae phototaxis is brought about by a primitive trial-and-error mechanism: filaments move along their long axis and reverse the direction of movement at a frequent but random rate. When aligned more or less parallel to the laterally impinging light rays (at low fluence rates), they tend to move more toward the light source than in the opposite direction, which, eventually, will bring the whole population closer to the light source. This behavior has been shown to be ecologically important in layered habitats such as in hot springs, where the cells move in and out of the strata depending on the irradiance (Nelson & Castenholz, 1982). At higher fluence rates the behavior is reverted, resulting in negative phototaxis. In contrast, cyanobacteria of the Nostocaceae famiIiy, have developed a true steering: when irradiated laterally the tips of filaments moving in a linear or V-shaped fashion tum toward (positive) or away (negative phototaxis) from the light source. The action spectra for these two responses in Anabaena variabilis differ in a characteristic way (Fig. 4). While the pigments responsible for positive phototaxis may be a subset of the photosynthetic pigments, those responsible for negative phototaxis have not yet been identified (Nultsch et aI., 1979). However, one interesting hypothesis assumes that the switch between positive and negative phototaxis is brought about by a sensor which measures the amount of singlet oxygen produced by excess energy absorbed by the photosynthetic apparatus (Nultsch et aI., 1983; Nultsch & Schuchart, 1985).

Photophobic responses may be the most important reactions of cyanobacteria to control their position in their microhabitat. As mentioned above the filaments glide back and forth without any visible preference. When during their random movements they leave an

6,,-------------------------,

·Ill 3 OJ c

:52

~ 2 .<: a..

\ . .:

\_./'

400

,,---'--

500 600 700

Wavelength [nm]

Figure 3. Action spectra of photokinesis in the cyanobacteria Phormidium ambiguum (dotted line) and Anabaena variabilis (unbroken line) (after Nuitsch, 1974).

190

irradiated area they often stop and reverse the direction of movement (Nultsch, 1961; Hader, 1976, 1979); this behavior is called a step-down photophobic reaction (Diehn et aI., 1977). When the organisms occupy an irradiated field they will be trapped in it (light trap). While this behavior is typical at low and intermediate fluence rates, step-up photophobic responses are observed when filaments move into fields of high fluence rates (step-up photophobic responses, Hader & Burkart, 1982). Using these two antagonistic responses (plus a less effective phototaxis and photokinesis) the organisms are able to populate areas of suitable irradiances.

The action spectrum for the step-down photophobic response strongly resembles that for photosynthetic oxygen production and also the low temperature absorption spectrum (Hader, 1974) so that there is little doubt concerning the involvement of the photosynthetic apparatus in the sensory process (Hader, 1987a,c). It could be shown with uncoupler studies that photophobic responses are independent of photophosphorylation, while inhibitors of the electron transport chain strongly suppress phobic reactions (Hader, 1974, 1975). The coupling site between the photosynthetic electron transport and photophobic responses seems to be plastoquinone, which has been confirmed by in vivo absorption spectroscopy (Hader & Poff, 1982a). In cyanobacteria the thylakoids are scattered throughout the peripheral cell plasma (chromatoplasma) and not enclosed in plastid envelopes. In the presence of light there is a vectorial proton transport into the thylakoid vesicles which causes the cytoplasma to become electrically more negative (Peschek et aI., 1985). When a filament leaves an irradiated area the proton gradient will be dissipated quickly, and the cytoplasm will becomes less hyperpolarized. Such light-induced electrical potential changes have in fact been observed in filamentous cyanobacteria (Glagolev, 1984; Hader 1978, 1979, 1981). Inhibition of the electrical potential change by the addition of membrane penetrating lipophilic cations such as triphenyl methyl phosphonium (TPMP+) affects photophobic responses. However, because of the low threshold value for photophobic responses, it is difficult to explain these reactions solely on the basis of changes in the thylakoid proton gradient (Nultsch, 1962; Nultsch & Hader, 1970). Rather, an effective amplification mechanism has to be postulated. This has indeed been found; it is based on the gating of voltage-dependent, calcium-specific membrane channels. The small electric potential change caused by the changing proton gradient across the thylakoid membrane is thought to control calcium channels in the cytoplasmic membrane. Upon a decrease in light intensity - e.g., when a filament leaves a light field - the channels are opened and this allows a massive influx

3D

·10

v~·-·-·~· (\ . I.... ..... / \j /-} '. \j \'"

·20 ~--,---,--_--, ___ -,-_--'

400 500 600 700

Wavelength [nmJ

Figure 4. Action spectra for positive (open circles) and negative (solid circles) phototaxis in Anabaena variabilis (after Nultsch et aI., 1979).

191

of calcium into the cells (Fig. 5). Inhibition of the calcium flux by adding ruthenium red, lanthanum ions or organic calcium channel blockers inhibits the photophobic response (Hader, 1982). Also, breaking down the existing calcium gradient across the cytoplasmic membrane by the application of the specific calcium ionophore, A23187 (calcimycin), drastically impaired the response (Hader, 1982; Murvanidze, 1981; Murvanidze et al., 1982). Later on, a uptake of Ca2+ through the cytoplasmic membrane during a photophobic reaction could be demonstrated using radioactively labelled calcium (Hader & Poff, 1982b). In line with these results, removal of calcium from the external medium completely inhibits photophobic reactions, while motility is not affected. Readdition of calcium restores the ability to respond (Hader & Poff, 1982b).

LIGHT-INDUCED REACTIONS IN EUKARYOTES

While most responses in eukaryotes are independent of the photosynthetic apparatus, in some cases a linkage between the two has been found. In desmids the action spectra of phototaxis, photophobic reactions and photokinesis have been found to be be similar to the absorption spectrum of the cells (Hader & Wenderoth, 1977; Wenderoth & Hader, 1979) indicating the involvement of the photosynthetic pigments. However, only photokinesis is strongly affected by inhibitors of the photosynthetic electron transport chain and by uncouplers demonstrating the link to photophosphorylation, as in the case of prokaryotes (see above). In contrast, photophobic reactions are less affected and phototaxis seems to be almost independent of the photosynthetic apparatus (Hader, 1981). This obvious enigma has not been resolved in this and a few other cases, where photosynthetic pigments seem to operate as the photoreceptor pigments, but where the rest of the photosynthetic machinery does not seem to be involved. One possible explanation could be that the photosynthetic pigments cause photodynamic reactions resulting in the production of free radicals or singlet oxygen which is sensed by the cell (Spikes, 1977; Spikes & Straight, 1981). However, this hypothesis is difficult to explain in the face of extremely low thresholds for phototaxis found

Figure 5. Model to explain photophobic reactions in Phormidium uncinatum (after Hader, 1982).

192

in some desmids (Neuscheler, 1967). In contrast, photokinesis seems to be independent of the photosynthetic apparatus in

the flagellate Euglena gracilis (Wolken & Shin, 1958; Hader & Hader, 1988). This finding has been supported by the fact that the close relative Astasia longa also shows a pronounced photokinesis, though the cells lack chloroplasts (Hader & Hader, 1989).

The role of the accessory pigments, phycobilins, in phototaxis of the photosynthetic flagellate, Cryptomonas, has also not been elucidated. The action spectrum indicates the involvement of phycoerythin, the main accessory pigment in a freshwater Cryptomonas species in phototactic orientation. In contrast, the chlorophylls a and c do not seem to playa role in photoorientation (Watanabe & Furuya, 1974).

Finally, the role of photosynthetic pigments in ciliates should be mentioned. Ciliates are non-photosynthetic organisms which show a variety of light responses, including phototaxis, photophobic responses and photokinesis (Song et aI., 1980a,b; Ghetti et aI., 1992; Ktihnel­Kratz & Hader, 1993). In some colored ciliates hypericin derivatives have been found to be the photoreceptor pigment involved (Song et a!., 1991; Ghetti, 1991). In other cases, such as the colorless Paramecium, the receptor pigments are not known (Iwatsuki & Naitoh, 1982, 1983). A number of these colorless ciliates are known to carry photosynthetic green algae as symbionts. While the aposymbiotic Climacostomu111 virens, Euplotes daidaleos and Paramecium bursaria all showed step-up photophobic responses, the presence of the symbionts has been found to add new photoresponses to the ciliates: the symbiotic forms showed step-down photophobic responses and photokinesis not found in the aposymbiotic forms (Reisser & Hader, 1984).

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Armitage, 1. P. & Evans, M. C. W. (1980) FEBS Letters 112:5. Armitage,1. P. & Evans, M. C. W. (1981a) FEMS Microbiol. Letters 11:89. Armitage, 1. P. & Evans, M. C. W (1981b) FEBS Letters 126:98. Armitage,]. P. & Evans. M. C. W. (1983) FEBS Letters 156:113. Armitage,1. P. & Evans, M. C. W. (1985) Biochim. Biophys. Acta 806:42. Bogomolni, R. A. & Spudich, J. L. (1982) Proc. Natl. Acad. Sci. USA 79:6250. Clayton, R. K. (1953a) Arch. Microbiol. 19:107. Clayton, R. K. (1953b) Arch. Microbiol. 19:125. Diehn, B., Feinleib, M., Haupt, W., Hildebrand, E .• Lenci, F. & Nultsch, W. (1977) Photochem.

Photobiol. 26:559. Ghetti, F., in "Biophysics of Photoreceptors and Photomovements in Microorganisms", (Lenci, F.,

Ghetti, F .• Colombetti, G., Hader, D.-P. & Song, P.-S., eds.), Plenum Press, New York and London, 1991, p. 257.

Ghetti, F., Checcucci, G., Lenci, F. & Heelis, P. F. (1992) 1. Photochem. Photobiol. B: Biol. 13:315. Glagolev, A. N. (1984) TIBS 9:397. Hader, D.-P. (1974) Arch. Microbiol. 96:255. Hader, D.-P. (1975) Arch. Microbiol. 103:169. Hader, D.-P. (1976) Arch. Microbiol. 110:301. Hader, D.-P. (1978) Arch. Microbiol. 118: 115. Hader, D.-P. (1979) Arch. Microbiol. 120:57. Hader, D.-P. (1981) Arch. Microbiol. 130:83. Hader, D.-P. (1982) Cell Motility 2:73. Hader, D.-P. (1986) Biochim. Biophys. Acta 864:107. Hader, D.-P. (1987a) Microbiol. Rev. 51:1. Hader, D.-P. (1987b) in "Blue Light Responses: Phenomena and Occurence in Plants and

Microorganisms", (Senger, H., ed.), eRC Press, p. 145. Hader, D.-P. (1987c) in 'The Cyanobacteria" (Fay, P. & Van Baalen, c., eds.), Elsevier, Amsterdam,

New York, Old'ord, p. 325 - 345. Hader, D.-P. (1988) 1. Photochem. Photobiol. B: Biol. 1:385. Hader, D.-P. (1989) EinfluB von UV-B-Strahlung auf die Photoorientiernng von Flagellaten, Laufener

Sem. Beitr. 3:67. Hader, D.-P. & Burkart, U. (1982) Math. Biosci. 58:1. Hader, D.-P. & Hader, M. (1988) Arch. Microbiol. 150:20.

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Hader, D.-P. & Hader, M. A (1989) Environ. Exp. Bot. 29:273. Hader, D.-P. & Hoiczyk, E. (1992) in "Algal Cell Motility" (Melkonian M., ed.), Chapman and Hall,

New York and London, p. 1. Hader, D.-P. & Lebert, M. (1994) Electrophoresis, 15:1051. Hader, D.-P. & Poff, K. L. (1982a) Arch. Microbiol., 131:347. Hader, D.-P. & Poff, K. L. (1982b) Arch. Microbiol. 132:345. Hader, D.-P. & Tevini, M. (1987) "General Photobiology" Pergamon Press, Oxford. Hader, D.-P. & Wenderoth, K. (1977) Planta 137:207. Harayama, S. & lin~, T. (1977a) 1. Bact. 131:34. Harayama, S. & lino, T. (1977b) Photochem. Photobiol. 25:571. Haupt, W. & Hader, D.-P. (1984 in "Photomorphogenesis in Plants" 4. ed., (Kendrick, R. E. &

Kronenberg, G. H. M., eds.), Kluwer Academic Publishers, Dordrecht, Boston, London, p. 707. Hildebrand, E. & Dencher, N. (1975) Nature 257:46. Iwatsuki, K. & Naitoh, Y. (1982) Experientia 38: 1453. Iwatsuki, K. & Naitoh, Y. (1983) Photochem. Photobiol. 37:415. Kiihnel-Kratz, C. & Hader, D.-P. (1993) J. Photochem. Photobiol. 19:193. Lenci, F., Hader, D.-P. & Colombetti, G. (1984) in "Membranes and Sensory Transduction",

(Colombetti, G. & Lenci, F., eds.), Plenum Press, New York, London, p. 199. Manten, A (1948) Antonie van Leeuwenhoek 14:65. Marwan, W. & Oesterhelt, D. (1991) Naturwiss. 78: 127. Meyer, T. E., Cusanovich, M. A & Tollin, G. (1993) Arch. Biochem. Biophys. 306:515. Mix, M. (1969) Arch. Microbiol. 68:306. Murvanidze, G. V. (1981) Bull. Acad. Sci. Georgian SSR 104:173. Murvanidze, G. V., Gabai, V. L. & Glagolev, A. N. (1982) 1. Gen. Microbiol. 128:1623. Nelson, D. C. & Castenholz, R. W. (1982) Arch. Microbiol. 131:146. Neuscheler, W. (1967)Z. Pflanzenphysiol. 57:151. Nultsch, W. (1961) Planta 56:632. Nultsch, W. (1962) Planta 57:613. Nultsch, W. (1974) Der EinfluB des Lichtes auf die Bewegung phototropher Mikroorganismen. I.

Photokinesis, Abhandlungen der Mbg. Gelehrten Gesellschaft 2: 143. Nultsch, W. & Hader, D.-P. (1970) Ber. Dtsche. Bot. Ges. 83:185. Nultsch, W. & Hader, D.-P. (1980) Light perception and sensory transduction in photosynthetic

prokaryotes, Structure and Bonding 41: Ill. Nultsch, W. & Hader, D.-P. (1988) Photochem. Photobiol. 47:837. Nultsch, W. & Hader, M. (1978) Ber. Dtsche. Bot. Ges. 91:441. Nultsch, W. & Hellmann, W. (1972) Arch. Microbiol. 82:76. Nultsch, W. & Schuchart, H. (1985) Arch. Microbiol. 142:180. Nultsch, W. & Throm. G. (1968) Nature 218:697. Nultsch, W., Schuchart, H. & Hohl, M. (1979) Arch. Microbiol. 122:85. Nultsch, W., Schuchart, H. & Koenig, F. (1983) Arch. Microbiol. 134:33. Oesterhelt, D. & Marwan, W. (1990) in "Biology of the Chemotactic Response", Vol. 46., (Armitage,

1. P. & Lackie, 1. M., eds.), Cambridge University Press, p. 219. Peschek, G. A, Czerny, T., Schmetterer, G. & Nitschrnann, W. H. (1985) Plant Physiol. 79:278. Reisser, W. & Hader, D.-P. (1984) Photochem. Photobiol. 39:673. Schlegel, H. G. (1956) Arch. Prot. 101:69. Song, P.-S., Hader, D.-P. & Poff, K. L. (1980a) Photochem. Photobiol. 32:781. Song, P.-S., Hader, D.-P. & Poff, K. L. (1980b) Arch. Microbiol. 126:181. Song, P.-S., Suzuki, S., Kim, I.-D. & Kim, 1. H. (1991) in "Biophysics of Photoreceptors and

Photomovements in Microorganisms", (Lenci, F., Ghetti, F., Colombetti, G., Hader, D.-P. & Song, P.-S., eds.), Plenum Press, New York, London, p. 21.

Song, P.-S., Walker, E. B. & Yoon, M. 1. (1980c) in "Photoreception and Sensory Transduction in Aneural Organisms", (Lenci, F. & Colombetti, G., eds.), Plenum Press, New York, London, p. 241.

Spikes, 1. D. (1977) in "The Science of Photobiology", (Smith, K. C., ed.), Plenum Press, New York, p. 81.

Spikes, 1. D. & Straight, R. (1981) in "Oxygen and Oxy-radicals in Chemistry and Biology", (Rodgers, M. A J. & Powers, E. L., eds.), Academic Press, New York, p. 421.

Spudich,1. (1985) in "Sensory Perception and Transduction in Aneural Organisms", (Colombetti, G., Lenci, F. & Song, P.-S., eds.), Plenum Press, New York, London, p. 113.

Sundberg, S. A, Bogomolni, R. A & Spudich, J. L. (1985) 1. Bact. 164:282. Takahashi, T. (1991) in "Biophysics of Photoreceptors and Photomovements in Microorganisms",

(Lenci, F., Ghetti, F., Colombetti, G., Hader, D.-P. & Song, P.-S., eds.), Plenum Press, New York, London, p. 249.

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Thomas,1. B. (1950) Biochim. Biophys. Acta 5:186. Throm, G. (1968) Arch. Prot. 110:313. Valkunas. L., Razjivin, A. & Trinkunas, G. (1985) Photobiochem. Photobiophys. 9: 139. Watanabe, M. & Furuya. M. (1974) Plant Cell Physiol. 15:413. Wenderoth, K. & Hader, D.-P. (1979) Planta 145: 1. Wolken,1. 1. & Shin, E. (1958) J. Protozool. 5:39.

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LIGHT SIGNAL TRANSDUCTION MEDIA TED BY PHYTOCHROMES

Debbie Sommer and Pill-Soon Song

Department of Chemistry University of Nebraska-Lincoln Lincoln, Nebraska USA 68588

INTRODUCTION

Light signal transduction in plants is mediated by the concerted interaction of UV-B, blue/uV-A, and red-light photoreceptors. The physiological effects of the activation of these photoreceptors are dramatically illustrated by comparing the morphologies of dark grown seedlings to those exposed to ambient light (Fig. 1). Dark-grown (etiolated) seedlings are typically white in color, have elongated stems, and lack leaves and flowers. Exposure to light induces photomorphogenesis, which is characterized by a rapid inhibition of stem elongation and the development of leaves and photosynthetically competent chloroplasts, which results in greening (de-etiolation).

The major photoreceptors that elicit these responses are the phytochromes (for recent reviews, see Quail, 1991; Furuya, 1993). In addition to promoting de-etiolation of dark­grown plants, this family of five structurally homologous proteins, designated phytochromes A, B, C, D, and E, transduce red light signals into a diverse array of developmental and morphogenic responses throughout the life cycle of plants, from seed to germination to senescence, including the regulation of stem elongation, pigment accumulation, flowering, and gravitropism. These photoreceptors exist in two interconvertible forms, Pr and Pfr, each of which has a distinct absorbance spectrum. In the dark, phytochrome is synthesized as the red-light absorbing Pr form, which has a major absorbance maxima at 666~668 nm. Upon absorption of red light, the Pr form of phytochrome undergoes rapid photochemical and conformational changes to the Pfr form, which has a spectral wavelength maximum near 730 nm. Pfr, which is conventionally thought to be the biologically active photoisomer, alters the expression of several genes by up to 100X the levels expressed in the dark within minutes (Tobin & Silveithorne, 1985; Kaufman et aI., 1986; Thompson & White, 1991). pfr can then be converted back to the Pr form by far-red (~730 nm) irradiation, thus serving as a light­regulated switch, according to the scheme shown:

Light as an Energy Source and [nlmmation Carrier in Plant Physiology Edited by Jelmings et ai., Plenum Press, New York, 1996 197

/red~ Precursor ... Pr Plant Cell Pfr ... Signal CJscade ...

~ far-red

RegWatioo of gene txpreSSim

In this review, the current state of the relationships between the phytochromes and the mechanisms of the red-light signal transduction cascade will be reviewed with emphasis on the recent literature.

Figure 1. Morphological changes resulting from light signal transduction. Pea plants were grown in either the dark (right) or under ambient light (left) for seven days. The dark grown plants, in which the phytochromes are in the "inactive" Pr forms, have elongated stems, unopened leaves and a white (etiolated) appearance. The conversion of the phytochromes to the Pfr forms by red/white light induces photomorphogenesis, characterized by the inhibition of stem length, the opening of leaves and the accumulation of chlorophyll (de-etiolation). Photograph by Lily Deforce.

THE PHYTOCHROME GENE FAMILY

Cloning of all five phytochrome genes from Arabidopsis has revealed that the phytochromes are a diverse but structurally conserved family of photo receptors (Clack et aI., 1994). The recently adopted nomenclature for the phytochrome genes and their products is outlined in Table 1 (Quail et aI., 1994). All five gene products are predicted to be soluble cytoplasmic proteins of similar size (approximately 1100 amino acids). The extent of homology is highest between phytochromes Band D, which are 80% identical. The identities between the other phytochromes range from 46-56%. Despite their lack of absolute identities, however, there are no large non-homologous domains present in any of the five proteins except for 10-40 amino acid extensions of the N- and C- termini of the larger phytochromes Band D.

Northern blot analyses of the phytochrome mRNA levels indicate that all five phytochromes are expressed in the roots, leaves, stems, and flowers of green plants.

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Table 1. Phytochrome Nomenclature

Designation/Symbol

phytochrome or phy

Pr Ptf Pr A, PrB, etc.

PtfA, PtfB, etc.

PHYmRNA

PHY

phy

Description

Fully assembled phytochrome apoprotein containing the covalently attached chromophore (haloprotein). The haloprotein gene products encoded by the five phytochrome genes are designated phyA, phyB, etc. Symbol for the red-light absorbing form of phytochrome. Symbol for the far-red-light absorbing form of phytochrome. Red-light absorbing form of phytochrome A, phytochrome B, etc. Far -red-light absorbing form of phytochrome A, phytochrome B, etc. Symbol for the mature transcript of a phytochrome gene. Transcripts of the various subfamilies are designated PHYA mRNA, PHYB mRNA, etc. Symbol for a wild-type phytochrome gene. The genes for the subfamilies are designated PHYA, PHYB, etc. In cases where very closely related wild-type genes exist within one subfamily, the designations used are PHYAl, PHYA2, etc. Designates a mutant phytochrome gene. Mutant alleles are designatedphyA-l, PhyB-l, etc.

Although phytochrome A expression decreases by a factor of 200 upon exposure to light (Somers et aI., 1991) and its expression further decreases in light-grown plants as they approach the flowering stage, the expression of the other four phytochrome genes appears not to be light regulated and remain uniform regardless of the developmental stage of the plant or its exposure to light (Clack et aI., 1994). In addition, phytochrome A, unlike the other phytochromes, is rapidly degraded after conversion to the Pfr form and is therefore referred to as "light labile" (reviewed by Quail, 1991; Furuya, 1993).

Transgenic and photomorphogenic mutant plants are currently being used to assign specific physiological roles to the protein products of the five phytochrome genes (reviewed by Reed et aI., 1992; Whitelam & Harberd, 1994). The overexpression of mono cot phytochromes A and B in transgenic dicot species has indicated that, although the amino acid homology between monocot and dicot phytochromes ranges from 63-73% (Sharrock et aI., 1986; Dehesh et aI., 1991), the structural motifs that are responsible for the incorporation of the chromophore, the activation of the light-mediated signal transduction pathway, and the proteolytic degradation of phytochrome A ptf are conserved between mono cot and dicot phytochromes (Boylan & Quail, 1989; Kay et aI, 1989; Keller et aI., 1989; Boylan & Quail, 1991; Cherry et aI., 1991; McCormac et al., 1991; Wagner et aI, 1991; Whitelam et aI., 1992). Analyses of transgenic overexpressers and mutants deficient in phytochrome A, B, or both A and B, have indicated that the roles and interactions of these phytochromes in the regulation of plant development are complex. Plants that are deficient in phytochrome A display no gross morphological differences compared to wild-type controls when grown under continuous white or red light, although they do display elongated hypocotyls and defects in seed germination when grown under far-red light (Nagatani et aI., 1993; Parks & Quail, 1993; Whitelam et aI., 1993). Phytochrome B deficient mutants, on the other hand, display a number of morphological abnormalities when grown under red or white light, but not far-red, including elongated hypocotyls, stems, and root hairs, increased apical dominance, and a reduction in chlorophyll accumulation Adamse et al. 1987; Childs et aI., 1991; Childs et aI., 1992; Devlin et aI., 1992; Lopez-Juez et aI.,

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1992; Nagatani et aI., 1991b; Reed et aI., 1993; Somers et aI., 1991). It appears that although phytochrome B plays major roles in plant development, the roles of phytochrome A are less pronounced, often complementing or antagonizing the actions of phytochrome B. For example, phytochromes A and B appear to have opposing functions in the regulation of seed germination, as studies using phytochrome A, B, and AB null mutants indicated that under far-red light, phytochrome A promotes germination, while phytochrome B inhibits it (Johnson et aI., 1994; Reed et aI., 1994). The antagonistic actions ofphytochromes A and B have also been demonstrated with respect to flowering: while phytochrome A promotes flowering, phytochrome B apparently inhibits flowering (Goto et aI. 1991; Reed et aI, 1993; Reed et aI., 1994). On the other hand, the overexpression of either phytochrome A or Bin transgenic plants results in light-grown dwarfed plants, indicating that phytochromes A and B may play complementary roles (Boylan & Quail, 1991; Nagatani et aI., 1991a; Wagner et aI, 1991; Whitelam et aI., 1992; McCormac et aI., 1993). It has been demonstrated that both phytochromes control certain aspects of de-etiolation, particularly the regulation of hypocotyl growth under red light or white light applied with light and dark cycles (Johnson et aI., 1994; Reed et aI., 1994). Furthermore, both phytochrome A or B can induce chlorophyll accumulation after a pulse of red light (Reed et aI., 1994).

Mutational analyses have also revealed that phytochrome B, in conjunction with one or more of the other members of the phytochrome family, elicits shade-avoidance responses in plants, which promote stem elongation in response to low ratios of red/far-red light encountered under canopies. End-of-day far-red responses, which allow plants to detect and respond to changes in the red/far-red ratio of light as evening approaches, are also mediated by phytochrome B (Nagatani et aI., 1991 b; Whitelam & Smith, 1991; Smith et aI., 1992; Robson et aI., 1993). In addition, the Pr form of phytochrome B mediates gravitropism (Liscum & Hangarter, 1993). Phytochrome A is apparently the photoreceptor that perceives daylength in light-grown seedlings (Johnson et aI., 1994; Reed et aI., 1994).

In summary, it appears that phytochromes A and B are active in both dark- and light­grown plants and that they perform distinct as well as over-lapping functions, some synergistic, others antagonistic, in light signal transduction. Although they are not yet available, the analyses of transgenic and mutant plants overexpressing or deficient in phytochromes C, D, and E will further clarify the complex roles and interactions of the phytochrome gene family.

Photo morphogenic mutants with lesions in genes encoding proteins other than the phytochromes are also being used to dissect the phytochrome-mediated signal transduction pathway(s). These mutants include those that exhibit anomalous responses to red or far-red light that are not attributable to the phytochromes and those that display a light-grown phenotype when grown in complete darkness (Chory, 1992; Chory, 1993; Deng, 1994; Whitelam & Harberd, 1994). So far, several Arabidopsis and one pea loci have been reported that, when mutated, result in dark-grown plants that, despite the absence of a light signal, display characteristics consistent with exposure to light. These characteristics include the expression of light-inducible genes, the inhibition of hypocotyl elongation, anthocyanin accumulation, and leaf and chloroplast development (see references cited by McNellis et aI., 1994). The protein products of these genes, designated DET I, 2, and 3 (DE-eTiolated), COP I, 2, 3, 4, 8, 9, 10, and 11 (COnstitutively Photo morphogenic), and LIPI (Light Independent Photomorphogenesis) function downstream of the phytochromes, possibly by repressing photomorphogenesis in the absence of light in wild-type plants (Chory et aI., 1989; Chory et aI., 1991; Deng et aI., 1991; Frances et aI., 1992; Cabreray Poch, et aI., 1993; Hou et aI., 1993; Wei & Deng, 1992; Wei et aI., 1994a). Three of these genes, as well as a fourth whose protein product appears to function in a network of signal transduction processes that includes the phytochromes, have been cloned (Deng et aI., 1992; Castle et aI., 1994; Pepper et aI., 1994; Wei et aI., 1994b). Remarkably, in general, these proteins, DETl, COPl, COP9, and FUS6/COP11, bear no strong over-all homologies to any known proteins, which suggests that at least part of the phytochrome signal transduction chain in

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plants utilizes proteins not present, or not yet diScovered, in other organisms. DETl encodes a protein of ca. 62 kDa that is localized to the nucleus and may indirectly repress transcription (pepper et al., 1994), while COPI is a 74 kDa protein that bears some homology to the B subunits of heterotrimeric G proteins (Deng et al., 1992). COP9 has a predicted molecular mass of 22.5 kDa and appears to form a large complex with other proteins in the dark (Wei et aI., 1994b), while FUS6/COPll encodes a protein with a predicted molecular mass of50.5 kDa that contains a putative ATP/GTP binding site (Castle et aI., 1994).

THE PHYTOCHROME PROTEIN

Thus far, only phytochromes A and B have been purified. Of the two, phytochrome A from oat is the most extensively characterized and will be used here as a model for discussing the structure of the phytochrome molecules. Phytochrome is a globular dimeric molecule that contains one linear tetrapYI70le chromophore per monomer, which is covalently bound via a thioether linkage to cysteine 321 (Lagarias & Rapoport, 1980; Hershey et aI., 1985; Jones & Quail, 1986). Biochemical and genetic analyses have indicated that phytochrome monomers posses discrete domains: a N-terminal domain of approximately 74 kilodaltons that contains the chromophore and a C­terminal domain responsible for dimerization. These two regions are linked by a proteolytically vulnerable "hinge region" (reviewed by Quail, 1991). Electron microscopy suggests that dimerization of the phytochrome monomers forms a V-shaped structure (Jones & Erickson, 1989). Within the amino and carboxy domains, there appear to be several structurally and functionally significant subdomains, including the hydrophilic 6-10 kDa N terminus (Fig. 2). Spectroscopic data indicate that this region interacts with the Pfr chromophore, since its loss is associated with a 8-nm shift in the absorbance maximum ofPfr (Jones et aI., 1985) and with a decrease in the a-helical folding of phytochrome (Chai et al., 1987; Deforce et aI., 1994; Sommer & Song, 1990), as described later. It has been reported that the removal of residues 7-69 alters the biological activity of phytochrome A expressed in light-grown transgenic tobacco plants (Cherry et aI., 1992). It should be noted, however, that these results have not been duplicated in transgenic Arabidopsis, although it does appear that the removal of this 10 kDa fragment results in the inactivation of phytochrome in plants that are grown under far-red light (Boylan et al., 1994; Discussed in more detail below). Overexpression of phytochromes A and B in bacteria, yeast, and transgenic plants has revealed that apophytochrome apparently catalyzes the covalent attachment of its own chromophore and that the region responsible for this enzymatic activity lies between amino acids 70 and 398 (Lagarias & Lagarias, 1989; Deforce et aI., 1991; Cherry et aI., 1992; Cherry et al., 1993; Deforce et al., 1993; Kunkel et al., 1993). Although phytochrome truncated at residue 398 can incorporate the chromophore and is photoreversible, the absorbance spectra are blue-shifted, indicating that the region between amino acids 399 and 672 interact with the chromophore and are necessary for spectral integrity. Phytochrome truncated to the C-terminal side of amino acid 672 retains the spectral characteristics of native phytochrome (Cherry et aI., 1993). A histidine-to-tyrosine mutation at His-283, which is conserved in all five of the Arabidopsis phytochromes, produces a mutant phenotype in Arabidopsis, which indicates that this residue is critical for phytochrome action in vivo (Reed et aI., 1993). Dimerization within the C-terminal domain apparently involves complex interactions between several regions, including regions defined by amino acids 623-673 and 991-1093 (Deforce et aI., 1991; Cherry et al., 1993; Edgerton & Jones, 1992; 1993). It appears that the C-terminal domain is essential for phytochrome signal transduction, as removal of as few as 35 amino acids from the C-terminus inactivates oat phytochrome in vivo (Cherry et al., 1993; Boylan et al., 1994). Sites for the ubiquitination of the pfr form of

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Alpha Helix

n Lyase Activity Dimerization

I I I • •• • •• •

N c

100 200 300 .00 500 800 700 800 IlOO 1000 1100

U U Spectral Integrity LPfr Degradation.J

I I Receptor Binding Site Receptor Activation/Release

Figure 2. Structural features of the phytochrome molecule. As a result of the Pr to Pfr phototransformation, the a-helical folding of the N-terminus of the phytochrome molecule increases as it interacts with the bilin­like chromophore, which is attached to cysteine 321 (Lagarias & Rapoport, 1980; Chai et aI., 1987; Sommer & Song, 1990; Deforce et al., 1994). This region has been shown to be, in some cases, essential for the biological activity of phytochrome (Cherry et al., 1992; Boylan et aI., 1994). The N-terminal two-thirds of the phytochrome molecule contains the structural features that are responsible for the autocatalytic covalent attachment of the chromophore and spectral integrity (Deforce et al., 1991; Cherry et al ., 1992; Cherry et al., 1993). In addition, the site at which phytochrome binds to its as-yet-unidentified receptor may also lie in this region (Boylan et aI., 1994). The C-terminal portion of the phytochrome molecule contains the sequences responsible for dimerization and for the ubiquitination of phytochrome A Pfr, which leads to its degradation in vivo (Shanklin et al., 1989; Deforce et al., 1991; Cerry et al ., 1993; Edgerton & Jones, 1993; Vierstra, 1993). Point mutations demonstrated to alter the biological activity of phytochrome are indicated by the circles (reviewed by Xu et aI., 1995).

phytochrome A, which results in its rapid destruction, also reside in the C terminus (Shanklin et aI., 1989; Vierstra, 1993).

From the evidence presented above, it is clear that phytochrome-mediated signal transduction requires the involvement of both the amino and carboxyl domains. Using results obtained from a set of experiments that investigated the dominant negative effects of oat phytochrome A mutants on the photo morphogenesis of Arabidopsis seedlings, Quail and colleagues have proposed that there are three regulatory subdomains involved in phytochrome signal transduction (Boylan et aI., 1994). Their assay system used oat phytochrome constructs that lacked 52 amino acids from the N-terminus, the entire C­terminal domain, or a deletion of residues 617-687. When grown under continuous far-red high irradiance conditions, all three mutant phytochromes were dysfunctional, as determined by comparing hypocotyl length to that of Arabidopsis over-expressing wild-type oat phytochrome. According to their interpretation, these observations suggest that the mutant phytochromes were bound to phytochrome's reaction partner, thus blocking the ability of ArabidojJsis phytochrome A to bind to it and initiate signal transduction. Since these plants displayed mutant phenotypes, however, it was suggested that despite the ability of the mutant phytochromes to bind to the reaction partner, they were unable to activate it. When the mutants were grown under continuous white or red light, however, the N-terminal mutant exhibited normal phytochrome regulatory properties, while the C-terminal mutants remained dysfunctional. Based on these observations, the following hypothesis was proposed: (1) The site at which phytochrome binds to its reaction partner in response to light lies between amino acid residues 52 and 617, (2) The site that activates the reaction

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partner and/or stimulates the release of the reaction partner so that it may transduce the light signal lies in the C-terminal domain of phytochrome, and (3) The N-terminal 52 amino acids have a role in phytochrome-mediated signal transduction only under high-irradiance far-red light. It was proposed that under these conditions the N-terminus participates in the activation of the reaction partner and/or modulates its release. Taken together, these results suggest that the N-terminus is required only for seedling development and is not necessary for white- or red-light photoregulation.

THE PHYTOCHROME PHOTOTRANSFORMA nON

Phytochrome transduces light signals through small but significant photoreversible changes in the structures of its chromophore and polypeptide. When Pr is activated by red light, a cis-trans isomerization at the C 15 double bond between the C and D rings of the chromophore occurs within a few picoseconds, partially accounting for the spectral differences between the two forms of phytochrome (Rudiger et a!., 1983; Thummler & Rudiger, 1983; Savikhin et aI, 1993). The most recent laser photolysis data of native oat phytochrome suggest that this ZZZ to ZZE isomerization involves five kinetic intermediates, rather than four (Zhang et a!., 1992). As a consequence of the photoisomerization of the chromophore, the D ring of the chromophore twists slightly out of plane (Rospendowski et a!., 1989).

A variety of biochemical probes and spectroscopic techniques have been used to detect conformational changes in the phytochrome polypeptide. Experiments using limited digestion by prot eases revealed differences in digestion patterns between the two forms of phytochrome and have demonstrated that the N-terminus of Pr is more susceptible to cleavage than is the N-terminus ofPfr (Lagarias & Mercurio, 1985; Vierstra & Quail, 1986). Conformational changes have also been detected with monoclonal antibodies that bind preferentially with either Pr or Pfr, but not both (Thomas et a!., 1984; Cordonnier et a!., 1985), and with kinases, which differentially phosphorylate Pr and Pfr (Wong et a!., 1986). Spectroscopic techniques have demonstrated that the Pfr chromophore is preferentially bleached by a variety of chemical agents, which suggests that the Pr to Pfr phototransformation results in increased exposure of the Pfr chromophore (reviewed by Song et a!., 1991).

It appears that the conformational changes that result from the phytochrome phototransformation occur primarily at the amino terminus of the phytochrome molecule. It has been shown that upon Pr to Pfr phototransformation, the distance between the chromophore and the N-terminus decreases by 12 A (Farrens et a!., 1992). CD data indicate that a-helical folding of native phytochrome increases 3-5% upon photo conversion of phytochrome from the Pr to the Pfr form (Chai et a!., 1987; Sommer & Song, 1990; Deforce et a!., 1994). However, there is no detectable photoreversible change observed with phytochrome lacking the 6 kDa N-terminus or in the presence of an antibody that binds near the N-terminus (Vierstra et aI., 1987; Chai et aI., 1987). This increase in a-helical folding occurs within milliseconds of irradiation (Bjorling et aI., 1992; Chen et aI., 1993).

A model incorporating the data outlined above has been proposed. According to this model, the Pr' chromophore resides within a hydrophobic pocket, which is relatively inaccessible to chemical agents. Upon phototransformation to the Pfr form, the chromophore reorients, increasing its interaction with the N-terminus of the polypeptide, which results in an increase in alpha-helical folding. This model accounts for the increased susceptibility of the N-terminus to proteolytic attack in the Pr form, as well as the increased susceptibility of the chromophore to modification by chemical agents (Song et aI., 1991).

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THE PHYTOCHROME SIGNAL TRANSDUCTION PATHWAY

As outlined above, phytochrome is synthesized as Pr and is converted to the conformationaIly distinct Pfr form by red/white light. This change in the conformation of phytochrome produces dramatic changes in the biochemistry and morphology of the plant. The major unresolved question in phytochrome research is "How does phytochrome exert its action?". It is generally accepted that phytochrome does not directly interact with the genes it regulates and that all of the components of the signal transduction chain are present in the cell before the conversion of Pr to Pfr (Quail, 1991). Therefore, it is assumed that phytochrome exerts its action through a second messenger. But what is this second messenger? Currently, there are three major hypotheses on how phytochrome's "message" might be transduced. These are through: (A) calcium-calmodulin, (B) phosphorylation, and (C) the currently most promising pathway, G-proteins.

A. Calcium Ions

The hypothesis that calcium ions may play an essential role in phytochrome-mediated signal transduction was first proposed in 1976 on a theoretical basis (reviewed by Roux, 1984; Roux, 1992). In the years that have foIlowed, there have been a large number of reports, often contradictory, on the role of calcium ions in ihytochrome signal transduction. Some of the more salient aspects of the role of Ca + ions in phytochrome signal transduction are outlined below.

Investigations off ern spore germination, leaf movements, and protoplast swelling, all of which are phytochrome-mediated responses, have supported the hypothesis that calcium ions play an important role in phytochrome signal transduction (reviewed by Tretyn et aI., 1991). It has been demonstrated that red-light and calcium ions stimulate fern spore germination. Germination does occur in darkness, however, in the presence of Ca2+ ions and the ionophore A23187. On the other hand, calcium channel blockers such as La3+ and C02+, as weIl as calmodulin inhibitors, prevent the induction of germination by light (Wayne & Hepler, 1984). In addition, red light mediated leaf unrolling and dark-induced leaflet closure have both been shown to be inhibited by EGT A, a calcium chelator (Viner et aI., 1988; Roblin et aI., 1990; Tretyn & Kendrick, 1990). The sweIling of dark adapted protoplasts in response to red light, a photoreversible phenomenon that has frequently been used to investigate phytochrome-mediated signal transduction, has also been shown to be calcium ion dependent. It has been demonstrated that Ca2+ and A23187 can mimic the effects of red light on protoplast swelling in the dark, while calcium chelators and channel blockers inhibit red-light induced swelling (Bossen et aI., 1988; Tretyn et aI., 1990a; Bossen etaI., 1991;UetaL, 1991). .

There have been numerous reports of the modulation of Ca2+ ion fluxes and intraceIlular calcium ion concentrations by phytochrome. The direction of these fluxes have differed, however. While it has been reported that phytochrome promotes calcium uptake and/or increases intraceIlular concentrations in Mougeotia ceIls, fern spores, and protoplasts from various species (Dreyer & Weisenseel, 1979; Das & Sopory, 1985; Wayne & Hepler, 1985; Tretyn, 1987; Berkelman et aI., 1990; Chae et aI., 1990), there have also been several reports of calcium ion efflux from some of these same systems (Hale & Roux, 1980; Roux, 1984; Takagi & Nagai, 1988; Scheuerlein et aI., 1991). It is likely that these differences are the result of the method of measurement (Tretyn et aI., 1991). Recent results using laser scanning confocal microscopy in conjunction with caged Ca2+ ions strongly support the hypothesis that red-light initiated signal transduction is mediated, at least in part, by cytosolic calcium concentrations (Shacklock et aI., 1992). These authors reported that the release of calcium ions from the caged forms resulted in protoplast swelling in the dark. It appears that the phytochrome-mediated response is preceded by a transient increase in free calcium ions, which is then foIlowed by a net efflux. It was concluded that although red-light

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promotes both an increase in cytosolic free calcium concentrations, as well as a net efllux of Ca2+ ions, calcium ion efllux is not involved in the swelling of the protoplasts.

The calcium hypothesis of phytochrome signal transduction is also supported by the observation that the activities of several enzymes are modulated by both red light and calcium. In addition, gibberellin synthesis and peroxidase secretion are also mediated, in part, by red light and calcium ions (reviewed by Roux, 1984; Tretyn et aI., 1991; Roux, 1992).

The phytochrome signal transduction chain apparently involves the activation of calcium-dependent protein kinases, as there have been reports of red-light, calcium­dependent phosphorylation of nuclear proteins from pea and from oat protoplasts (Datta et aI., 1985; Park & Chae, 1989; Park & Chae, 1990; Roux, 1992). The phosphorylation of these proteins was inhibited by calcium chelators, and by protein kinase C inhibitors, respectively. The role of protein phosphorylation in phytochrome-mediated signal transduction is discussed in more detail below.

Recent investigations of the role of calcium ions in the modulation of phytochrome­regulated gene expression has lent considerable credibility to the suggestive data outlined above. It has been shown that W -7, a calmodulin antagonist, blocks the red-light dependent expression of the light-harvesting chlorophyll alb-binding (cab) proteins in soybean suspension cultures, while calcium ionophores induce low levels of cab mRNA accumulation in the dark (Lam et aI., 1989). Similar results were also reported for soybean cultures that were treated with bacterial toxins, which resulted in the expression of cab genes in the dark. The subsequent addition ofW7 prevented the expression of these genes in the dark (Romero & Lam, 1993). In a particularly interesting set of experiments, Chua and colleagues have used the aurea tomato mutant, which lacks photodetectable phytochrome A, to dissect the phytochrome signal transduction pathway (Neuhaus et aI., 1993; Bowler et aI., 1994a). By microinjecting mutant protoplasts with various compounds and assaying for phytochrome­mediated gene expression and chloroplast development, they initially identified two distinct pathways in the red light signal pathway. One of these pathways requires calcium and calmodulin and regulates cab gene expression and the synthesis of some of the proteins of the photosynthetic complexes. These results will be discussed in detail below.

Although the calcium hypothesis of phytochrome action has many merits and it is difficult to argue that calcium does not play a role in red-light transduction processes, this hypothesis has not been able to fully explain the mechanism by which phytochrome exerts its action. Therefore, it can be concluded that calcium ions playa major, but not causal role in phytochrome signal transduction. In this context, we will next review the literature on another apparently important process in phytochrome signal transduction, protein phosphorylation.

B. Protein PhosphorylationlDephosphorylation

Phytochrome researchers have attempted to answer three basic questions regarding the potential role of reversible protein phosphorylation in the red-light mediated signal transduction chain: (a) Is protein phosphorylation/dephosphorylation involved in phytochrome signal transduction?, (b) Is the phytochrome molecule itself a substrate for light-dependent phosphorylation in vivo and, if so, what is the significance? and, (c) Is phytochrome a light regulated protein kinase? The answers to these questions, as determined thus far, will be discussed below. Unfortunately, only the first question can presently be answered with certainty.

(a) Is protein phosphorylation/dephosphorylation involved in phytochrome signal transduction? A number of investigations using light-treated homogenates, protoplasts, and isolated nuclei have indicated that the phosphorylation states of several proteins are reversibly modified by red and far-red light (reviewed by Singh & Song, 1990;. Table 2).

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Table 2. Reports of PhosphorylationlDephosphorylationlKinases in Phytochrome-Mediated Signal Transduction

Year Observation

1978 Phytochrome is a phosphoprotein in vivo (Quail et al. 1978) 1980 118 kDa phytochrome from A vena contains one mol of phosphate per

monomer. (Hunt & Pratt 1980) 1985 Red light mediates increases in acid & alkaline phosphatases in

Sorghum. (Rajasekhar & Sopory 1985) Red-light enhancement of phosphorylation of pea nuclear proteins of Mr 77,64, and 47 kDa enhanced by calcium. Reversed by calmodulin inhibitors, calcium chelators. (Datta et al. 1985)

1986 Polycation-dependent protein kinase activity is associated with purified oat phytochrome. Pr is the preferred substrate for calcium. and cyclic nucleotide-dependent kinases in vitro. Pr was phosphorylated near the N-terminus, while the Pfr phosphorylation site was in the C-terminus. (Wong et al. 1986)

1988 Pulse of red light promoted the rapid phosphorylation of a 30 kDa protein and the dephosphorylation of 32 & 29 kDa proteins in oat coleoptile tips. (Otto & Schafer 1988)

1989 Oat phytochrome contains nucleotriphosphate binding sites. (Wong & Lagarias 1989) Characterization of the phytochrome-associated kinase. (Wong et al. 1989) Highly purified phytochrome lacks kinase activity. (Grimm et al. 1989; Kim et al. 1989) 32 and 27 kDa proteins from oat protoplast phosphorylated in red­light. Labeling reduced with calcium chelator. (Park & Chae, 1989) Binding of AT-l to promoters of light-regulated genes is dependent upon phosphorylation. (Datta & Cashmore 1989)

1990 Red-light induced phosphorylation of 32 & 27 kDa protoplast proteins involves protein kinase C. (Park & Chae 1990) Phosphorylation of Pr in vitro by cAMP-dependent kinase occurs at Ser 17. Pfr is phosphorylated at Ser 598. (McMichael & Lagaris 1990) Protein kinase C inhibitor inhibits red-light induced swelling of wheat protoplasts. (Bossen et al 1990)

1991 45 & 17 kDa proteins were phosphorylated in response to red light in barley nuclei. 17 kDa protein was identified as H3 histone. (Grimm et al1991) The phosphorylation & dephosphorylation of 75 and 60 and 10 and 68 kDa proteins in isolated oat nuclei is modulated by red/far-red light. The reactions are blocked by cholera toxin. 75 & 24 kDa proteins detected with anti-G protein antibody. (Romero et al. 1991;a and b) Functional & structural homologies noted between phytochrome & bacterial sensors. (Schneider-Poetsch & Braun 1991; Schneider­Poetsch et al. 1991)

1992 Phosphorylation of 3AF3, a nuclear binding protein, is required for rbcS-3A expression. (Sarokin & Chua 1992)

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Age-dependent red light phosphorylation of 76-55 kDa proteins and dephosphorylation of a 94 kDa protein in soluble Sorghum extracts. (Doshi et al. 1992) cDNA of 145 kDa moss phytochrome indicated presenc of a raflmos kinase homologous domain. (Thiimmler et al. 1992)

1993 145 kDa moss phytochrome with a putative kinase domain undergoes red-light-dependent calcium-sensitive autophosphorylation. (A1garra et al 1993) Red-light phosphorylation of 70 & 60 kDa proteins in wheat protoplasts by caged calcium. Caged inositol triphosphate increased labeling of70 kDa protein. (Fallon et a11993) 70 kDa nuclear protein CA-1 is a phosphoprotein. May function as a transcriptional repressor of a cab gene in Arabidopsis.

(Sun et a11993) 1994 Autophosphorylation of a 60 kDa calcium/red light sensitive protein

may be a link in both the red and blue light signal transduction pathways. (Fallon & Trewavas 1994) A 40 kDa protein was red-light phosphorylated in fractions of purified parsley cytosol. (Harter et aI. 1994a) Phosphorylation of a 92 kDa protein from pea microsomal fractions was stimulated by red-light. Phosphorylation was reversed in the presence of pertussis toxin. (Hasunuma et al. 1994) Light-dependent tyrosine phosphorylation of a 88 kDa protein from cyanobacteria. (Warner & Bullerjahn 1994) Kinase activity immunoprecipitated with maize phytochrome. Phosphorylation was reduced with red-light irradiation. (Biermann et al 1994) FUS6, with a predicted molecular mass of 50.5 kDa, which may act downstream of phytochrome, contains a potential protein kinase C phosphorylation site. (Castle & Meinke 1994) COP9, a protein with predicted molecular mass of 22.5 kDa that is apparently involved in phytochrome signal transduction, contains putative protein kinase C and cAMP-dependent protein kinase phosphorylation sites. (Wei et al. 1994b) A tyrosine kinase inhibitor blocked the light-regulated expression of chalcone synthase in soybean cell cultures, while a non-specific kinase inhibitor blocked the expression of cab genes. (Bowler et al. 1994b) Phosphorylation/dephosphorylation regulates the translocation of cytosolic proteins into the nucleus, which participate in the regulation oflight-regulated genes. (Harter et al 1994b)

Although early studies often failed to identify the specific intracellular locations or identities of the proteins involved, they provided initial evidence that the dynamic processes of phosphorylation/dephosphorylation are indeed components of the phytochrome signal transduction chain. It is now apparent that phosphorylation of both cytosolic and nuclear proteins are involved in the transmission of red-light signals. While Harter et al. (Harter et aI., 1994a) have demonstrated that a cytosolic protein is phosphorylated within seconds of red-light irradiation, several nuclear proteins have also been observed to undergo reversible phosphorylation in response to red light (Datta et aI., 1985; Grimm et aI., 1991; Romero et aI., 1991a). The binding of certain nuclear proteins to the promoters oflight regulated genes has also been shown to be dependent on the phosphorylation states of the proteins (Datta & Cashmore, 1989; Sarokin & Chua, 1992; Sun et aI., 1993), as is the translocation of a

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cytosolic protein into the nucleus that binds to the promoter elements of light-regulated genes (Harter et aI., 1994b). Furthermore, at least two of the gene products involved in the transmission of red-light signals downstream of phytochrome, COP9 and FUS6 (previously discussed) contain putative protein kinase C phosphorylation sites. In addition, COP9 also possesses a putative site for phosphorylation by cAMP-dependent protein kinases (Castle et aI., 1994; Wei et aI., 1994b).

The importance of tyrosine phosphorylation in the transduction of signals in animal systems is well documented (Fantl et aI., 1993). Although there is currently little evidence for or against the involvement of tyrosine phosphorylation in light-mediated signal transduction in plants, this possibility may be worthy of investigation in view of a recent report that light-mediated signal transduction in cyanobacteria involves tyrosine phosphorylation of a 88 kDa protein (Warner & BulleIjahn, 1994) and the observation that the expression of a phytochrome-regulated gene is inhibited by tyrosine kinase inhibitors (Bowler et aI., 1994b).

(b) Is the phytochrome molecule itself a substrate for light-dependent phosphorylation in vivo and, if so, what is the significance? The question of whether or not phytochrome undergoes light-dependent phosphorylation is currently unresolved. Early ill vivo labeling experiments and biochemical characterization of the phytochrome molecule indicated the presence of one phosphate moiety per phytochrome monomer (Quail et aI., 1978; Hunt & Pratt, 1980). In later studies employing phosphorylation as a probe of red-light induced conformational changes in the phytochrome molecule, Lagarias and co-workers reported that phytochrome is a substrate for mammalian cyclic AMP- and GMP-dependent kinases, a calcium-activated phospholipid dependent kinase, and a polycation-dependent protein kinase associated with purified phytochrome in vitro (Wong et aI., 1986). They found that the phosphorylation of phytochrome by these kinases depended on the conformation of the phytochrome molecule. While the Pr form of phytochrome was phosphorylated by all four kinases, the Pfr form was significantly phosphorylated only by cAMP-dependent kinase. The specific amino acid residues phosphorylated by these kinases were also conformation­dependent; while Pr was phosphorylated on the N-terminus at Ser 17, Ser 598 was phosphorylated in the Pfr form (McMichael & Lagaris, 1990). These differences may be accounted for by changes in the secondary structure of the N-terminus between the Pr and pfr forms of phytochrome (Chai et aI., 1987).

Despite these observations, however, there is currently no concrete evidence, positive or negative, that phosphorylation/dephosphorylation of phytochrome occurs in vivo and is responsible for red-light signal transduction. Although many possible mechanisms could be involved, it is interesting to note, however, that the deletiOn/replacement of potentially phosphorylable serine residues in the N-terminus of phytochrome over-expressed in transgenic tobacco results in phenotypic alterations compared to over-expression of wild­type phytochrome (Cherry et aI., 1992; Stockhaus et aI., 1992). In one of these studies, the biological activity of phytochrome increased when ten N-terminal serine residues were replaced with alanine residues (Stockhaus et aI., 1992). The authors suggested that one possible interpretation was that after the photoconversion of wild-type phytochrome to the pfr form, phytochrome is inactivated by serine phosphorylation. Removal of this site, therefore, results in a prolonging of the active state and an enhancement in the biological activity ofPfr. Alternatively, the serine-to-alanine replacements may stabilize and/or increase the amphiphilic a-helical folding of the N-terminus (Parker et aI., 1991), increasing its biological activity.

(c) Is phytochrome a light regulated protein kinase? A considerable amount of controversy has arisen since it was suggested that phytochrome may possess intrinsic kinase activity in 1986 (Wong et aI., 1986). Subsequent reports from other laboratories indicated that highly purified phytochrome possesses no endogenous kinase activity and suggested

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that the activity previously reported (Wong et aI., 1986; Wong et aI., 1989) was the result of the presence of a co-purifYing contaminant (Kim et aI., 1989; Grimm et aI., 1989). It has since been suggested that the discrepancies between these reports may be attributable to differences in purification protocols (i.e., whether phytochrome was isolated in the Pr vs. the Pfr form) or to protein denaturation during purification. The issue has recently been re­opened with the observation that Pr is specifically phosphorylated in salt- and detergent­washed anti-phytochrome immunoprecipitates and that the kinase activity is either intrinsic to phytochrome or tightly associated with phytochrome (Biermann et aI., 1994).

Although there is limited experimental evidence that phytochrome from higher plants functions as a protein kinase, cDNA cloning and preliminary biochemical assays indicate that phytochrome from the moss Ceratodon pll1pureus may function in this capacity (Thiimmler et aI., 1992; Algarra et aI., 1993). The phytochrome gene from this organism encodes a polypeptide with a predicted molecular mass of 145 kDa. While the N-terminal portion of the protein is homologous to other known phytochromes, particularly phytochrome B, the C-terminal domain displays limited homology to protein-tyrosine kinases and to the RaflMos family of serine/threonine kinases. Initial biochemical characterization of extracts indicates that this novel phytochrome undergoes autophosphorylation in response to red lIght. However, these kinase motifs are apparently not present in phytochromes from other species, including mosses related to C. purpureus, which suggests that the kinase motif is unique to this particular moss.

On the basis of amino acid homologies, Schneider-Poetsch has proposed the hypothesis that phytochrome may function as a tyrosine kinase in a two-component system analogous to those used by bacterial sensor proteins (Schneider-Poetsch et aI., 1991; Schneider­Poetsch & Braun, 1991; Schneider-Poetsch, 1992). In response to environmental stimuli, these proteins undergo conformational changes in their N-terminal regions, which stimulates autophosphorylation at a conserved C-terminal histidine. The phosphate is then transferred to a regulator molecule, which ultimately affects gene transcription/enzyme activity. In addition to the sequence homologies between phytochrome and these proteins, the authors supported their hypothesis by noting that; (1) phytochromes and the bacterial proteins share similar hydropathy profiles, (2) a conserved tyrosine residue in phytochrome sequences corresponds to the conserved phosphorylated histidine in the bacterial proteins, and (3) both phytochromes and the bacterial transmitters are dimeric molecules. This hypothesis also proposes a means to reconcile the discrepancies discussed above regarding the detection of phytochrome kinase activity. According to this hypothesis, phytochrome may possess kinase activity only in the presence of a specific "receiver" molecule that may be lost during some purification procedures. Although interesting, this hypothesis lacks experimental verification, however.

As with the calcium hypothesis of red-light signal transduction, the evidence unequivocally suggests that protein phosphorylation plays a major role in phytochrome signal transduction. However, there is no evidence that suggests. that the phosphorylation/dephosphorylation of phytochrome or any other protein is the initial step in the signal transduction pathway.

C. GTP-Binding Protein Mediated Signal Transduction

A common theme that has emerged during the investigation of signal transduction processes in animals is that guanine nucleotides and the proteins they bind to play essential roles. Two major classes of signal-transducing GTP-binding proteins have been identified in animals: the heterotrimeric G proteins and the low molecular weight ras-like proteins (for reviews, see Gilman, 1987; Simon et aI., 1991; Lowy & Willumsen, 1993). Both classes of proteins cycle between a GDP bound "off' state and a GTP bound "on" state in response to specific chemical or physical stimuli. The heterotrimeric G proteins are composed of three subunits: an a. subunit of ca. 40-50 kDa that binds GDP or GTP, a f3 subunit of ca. 38 kDa,

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and a 8-10 kDa y subunit. In the absence of a stimulus, the GDP-bound a subunit and the /3 and y subunits associate with their particular receptor. Activation of the receptor by the appropriate stimulus results in an exchange of GDP for GTP, which stimulates the dissociation of the three subunits and promotes the binding of the GTP-bound a subunit to a specific effector. The heterotrimeric G-protein receptors identified so far include those that respond to hormones, neurotransmitters, odorants, and light. The G protein coupled effectors include ion channels and enzymes such as adenylate cyclase, guanylate cyclase, phosphodiesterase, and phospholipases. The ras-like GTP-binding proteins, on the other hand, are monomeric proteins of 20-30 kDa that form complexes with other cytosoliclmembrane-bound proteins. Although the functions of most of these proteins have not yet been determined, it is apparent that they transduce a diverse array of physiological signals, including those that regulate growth, development, proliferation, secretion, and intracellular transport. The best characterized ras receptors described thus far are the tyrosine kinase growth factor receptors that transduce signals primarily by initiating a phosphorylation cascade.

Recent evidence suggests that phytochrome-mediated red-light responses are transduced through both heterotrimeric as well as ras-like GTP-binding proteins. Initial clues that GTP-binding proteins are involved in red-light signal transduction came from experiments using protoplasts and crude extracts of etiolated seedlings. Bossen et al. (Bossen et aI., 1990; Bossen et aI., 1991) reported that the introduction ofGDP-/3-S, a G­protein inhibitor, by electroporation prevented the red light-induced swelling of protoplasts. On the other hand, GTPyS, a G-protein activator, induced swelling of the protoplasts in the dark, mimicking the effects of red light. Investigations of the binding of GTPyS to crude pea and oat extracts revealed that red light stimulated the binding of GTPyS, compared to unirradiated controls (Hasunuma et aI., 1987; Romero et aI., 1991b).

The use of bacterial toxins, which regulate the activity of GTP-binding proteins, has also indicated that these proteins are involved in the phytochrome signal transduction pathway. The a subunits of the G-proteins that stimulate adenylate cyclase (Gs), are ADP­ribosylated by cholera toxin at a conserved arginine residue, which results in an enhancement in the activity of Gs. On the other hand, pertussis toxin ADP-ribosylates the inhibitory G a­subunit, Gi (i.e., those that inhibit adenylate cyclase), thus preventing the activation of Gi. Romero et al (Romero et aI., 1991a) demonstrated that incubation of etiolated oat seedlings with cholera toxin (which activates Gs-like G proteins) mimicked the effect of red light on the expression of phytochrome and cab genes (ie., phytochrome was down regulated, while cab gene expression was up-regulated). Similar results were also obtained with soybean suspension cultures that were incubated in the dark with either cholera or pertussis toxin (Romero & Lam, 1993). These results suggest that phytochrome may activate a Gs-like G protein or inhibit a Gi-like G protein. A possible mechanism of phytochrome action suggested from these experiments is that in the dark an inhibitory Gi-like G protein is active. Red light transformation of phytochrome then activates a Gs-like protein, overcoming the inhibition of the inhibitory G protein. As we will see below, however, there are alternative explanations.

Suggestive evidence that low molecular weight GTP-binding proteins may playa role in phytochrome signal transduction was initially obtained from Western blot analyses of crude plant extracts. Romero et ai. (Romero et aI., 1991b) reported the presence of a 24 kDa protein that was recognized by an antibody raised against a conserved region of animal G proteins. Further experiments revealed that a 24 kDa protein preferentially bound GTP. This evidence suggested that a ras-like GTP-binding protein is involved in phytochrome signal transduction. There have also been reports that the binding of GTP to low molecular weight proteins in pea nuclei is regulated by phytochrome (Clark et aI., 1993). A further correlation between phytochrome and ras-like GTP binding proteins has been established at the transcriptional level. The expression of two ras-like proteins, pra 2 and pra 3, parallels that

210

of phytochrome; the mRNA levels of all three are down-regulated by red light (Yoshida et ai., 1993). Although the significance of these observations is not yet clear, it may be that certain ras-like proteins are more active in the dark (by virtue of their greater abundance) and that their activity then decreases as a result of down-regulation by phytochrome.

Suggestive evidence that heterotrimeric G-proteins may play a role in phytochrome signal transduction has also been obtained from Western blot analyses. Muschietti et al. (Muschietti et ai., 1993) detected the presence of a 43 kDa protein in crude membrane preparations from alfalfa seedlings that was recognized by anti-transduciniGi antibody. A 43 kDa protein was also photoaffinity labeled with a GTP analog and was ADP-ribosylated by cholera toxin.

To date, the most convincing data concerning the role of GTP-binding proteins in phytochrome-mediated signal transduction has come from the laboratory of Nam-Hai Chua. As outlined above, this laboratory has developed an assay system employing protoplasts from the aurea tomato mutant, which is deficient in photodetectable phytochrome A. In an initial set of experiments, Neuhaus et ai. (1993) reported that microinjection of oat phytochrome A or GTPyS restored wild-type characteristics (i.e., anthocyanin accumulation and chloroplast maturation). The introduction oflow concentrations ofGTPyS (which failed to elicit a response) with cholera toxin, also mimicked the effect of injection of phytochrome. Co-injection of phytochrome and GDP-/3-S, or phytochrome and pertussis toxin, however, failed to restore the wild-type characteristics. Furthermore, it was also shown that although microinjection of calcium ions could trigger the production of PS 11, LHCI/II, ATP synthetase, and RUBISCO, it did not stimulate the production of cytochrome b6f and the photosystem I core components. Therefore, maturation of the chloroplasts did not occur. These results suggested that while calcium ions could trigger some phytochrome­mediated responses, it cannot fully substitute for phytochrome.

In an extension of this work, Bowler et ai. (Bowler et ai., 1994a) have recently reported that microinjection of cyclic GMP triggered the production of anthocyanins, injection of calcium ions triggered the activation of cab gene expression, and that co­injection of cyclic GMP with calcium ions resulted in the development of fully mature chloroplasts. That is, cyclic GMP and calcium ions apparently take the place of phytochrome in rescuing the aurea mutant. [It should be noted, however, that the aurea phenotype is not considered to be due to a phytochrome A deficiency (reviewed by Whitelam & Harberd, 1994) and that the validity of some of these results has been questioned.]

Based on experiments using various activators/inhibitors of signal transduction components, Bowler et al. (Bowler et aI., 1994b) have concluded that phytochrome signal transduction involves three separate pathways. One of these pathways, apparently utilizes G­protein activation of guanylyl cyclase and at least one down-stream tyrosine kinase to activate anthocyanin biosynthesis, while a second pathway utilizes a G-protein, calcium, calmodulin, and protein kinases to activate cab gene expression. The third pathway utilizes both cGMP and calcium/calmodulin to activate the gene encoding ferredoxin NADP+ oxidoreductase, a component of PSI. Although the activation of these pathways results in the development of mature chloroplasts, it appears that considerable "cross talk" exists between these pathways. For example, a component(s) ofthe pathway that induces cab gene expression apparently can negatively regulate anthocyanin biosynthesis and vice versa (Bowler et ai., .1994b).

There are many possible interpretations and conclusions that can be drawn from the results described in the proceeding pages. The simplest is that a transducin-like protein transduces phytochrome responses. This hypothesis would account for most of the data reported by Neuhaus et al. (Neuhaus et aI., 1993) and Bowler et al. (Bowler et al., 1994a): transducin is activated by GTP and is inhibited by GDP, it is ADP-ribosylated by both cholera and pertussis toxins, and in vertebrates, it activates cGMP phosphodiesterase.

However, that are other possible hypotheses that could be drawn. One is that a Gi-Gs­ras signal transduction cascade is involved in phytochrome transduction. This hypothesis

211

could potentially account for the observation that pertussis toxin regulates the expression of phytochrome and cab genes in soybean cultures in the absence of light (Romero & Lam, 1993). The inhibitory G proteins (Gi) are uncoupled from their receptors by pertussis toxin. Therefore, if a Gi-like protein, that represses cab gene expression in the dark is modified, cab gene expression could be activated in the absence of light. It has recently become apparent that Gi-like proteins are activators of ras. For example, it has been shown that adrenergic agonists acting on a2A receptors and carbachol acting on muscarinic m2 receptors, which are both Gi receptors, activate ras-like proteins, which then initiate phosphorylation cascades (Alblas et ai., 1993; Winitz et al., 1993). In fact, many of the activities once prescribed to the Gi proteins are now being reassigned to ras activation via Gj. (Although there are not yet any reports of cholera toxin modulating ras, a similar scenario could be possible with ras activation/inactivation by Gs). Therefore, the results of Romero & Lam (1993) as well as reports of phytochrome activation of ras, could be accounted for by the participation of a Gi-like protein in phytochrome signal transduction. In the dark, Gj activates ras-like protein(s). The activation of ras results in the accelerated hypocotyl growth characteristic of etiolated seedlings, which in analogous to the uncontrolled growth in animals that occurs when ras becomes "hyperactivated" . The conversion of phytochrome to the pfr form could uncouple the Gi-ras pathway/activate a Gs-like protein.

A major inconsistency in proposing that phytochrome signal transduction involves GTP-binding proteins is the fact that all known heterotrimeric G protein receptors (of which there are more than 100 known) are characterized by seven hydrophobic stretches of amino acids that form transmembrane a-helices (Strader et ai., 1994). Furthermore, these receptors are characteristically glycosylated and/or palmitylated. The ras receptors characterized so far are primarily membrane-bound tyrosine kinases. Phytochrome, on the other hand, is primarily a cytosolic protein [although there have been several reports that it associates with membranes/liposomes (Kim et ai., 1983; Napier & Smith, 1987; Singh et al., 1989; Lamparter et al., 1992; Terry et ai., 1992)]. Furthermore, sequence analysis fails to indicate motifs characteristic of membrane proteins (Hershey et ai., 1985; Sharrock et al., 1986; Sharrock & Quail, 1989) and the glycosylation/palmitylation of phytochrome has not been substantiated. Although the phytochromes may represent a novel class of G-protein receptors, this possibility seems remote in view of the dissimilarities between the phytochromes and the G-protein receptors thus far characterized. Despite the lack of experimental evidence to support such a hypothesis, it could be proposed that the phytochromes activate a membrane receptor(s) in a manner analogous to the activation of the maltose chemotaxis transducer by the maltose binding protein following ligand binding in certain bacteria (reviewed by Manson, 1992). By analogy to the bacterial signal transduction pathway, the conformational change that results from the absorption of red light by phytochrome may stimulate the binding of phytochrome to a membrane-bound receptor, which in tum activates the G-protein(s).

The hypothesis that GTP-binding proteins are involved in red light signal transduction provides a unifying framework in .which to view research that has previously seemed disconnected. For example, there are several reports that phytochrome-mediated signal transduction results in alterations of potassium, sodium, and calcium fluxes (Brownlee & Kendrick~ 1979; Roth-Berjerano & Nejidat, 1987; Blum et ai., 1992; discussed above) and in alterations in the levels and/or activities of phosphoinositides (Guron et al., 1992; Mehta et al., 1993), diacylglycerol (Morse et al., 1989; Park & Chae, 1990), protein kinase C (park & Chae, 1990; Chandok & Sopory, 1992) and phospholipase C (Melin et ai., 1987). Furthermore, it appears that some red-light induced effects can be mimicked by cAMP (Kim et al., 1986; Avalos & Vicente, 1987; Chung et al., 1988; Mateos et al., 1993), acetylcholine (reviewed by Bossen et ai., 1989; Kim et ai., 1990; Tretyn et ai., 1990b; Tretyn et al., 1990c), and that hormones, including auxins (Shinkle & Briggs, 1984; Yahalom et ai., 1988; Kuhn & Galston, 1992), gibberellins (see ref cited by Chung et ai., 1988; Nick & Furuya,

212

GTP GOP

/ '\..

8JJ I I

Tyrosine

Kineses EJ I

8 /

Ser/Thr Klnases

Tyrosine

Klnas98

Transcriptional Repression (DETjCOP?)

(.)

Pr Red Light

Pfr (Inhibits?)

Far-Red Light

~ GTP GOP GTP GOP

/ '\.. / '\..

8cP ~-8 cP 1 I

~G EJLC---e I ~ I I GTP cGMP ~

j H'

I I PIP IP+OAG

(+):@GMP2j,(.)@ (+) ~ ()

I Kina D Gl 1 I--~·· POE

l----I---~ ~ : 1

I : 8 1- - - - - - - -1- - - - -I 1

Ser/Thr Klnases

Gene Transcription (Ie., Chalcone Synthase)

SerfThr Klnases

Gene Transcription Gene Transcription (Ie., CAB)

Figure 3. Possible components of the phytochrome-mediated signal transduction pathways. In the dark, Pr may activate an inhibitory Gi-like protein a-subunit, which could potentially activate raf-like kinases and ras-like GTP-binding proteins. Phospholipases (PLC) and protein kinase C (PKC) may be activated by the p and y subunits of the heterotrimeric G protein. The activation of these molecules could initiate a phosphorylation cascade that could ultimately repress the transcription of light-regulated genes, possibly through the DET and COP gene products. Upon photoconversion of phytochrome to the Pfr form, transducin-andlor Gs-like GTP-binding proteins may be activated and stimulate the activities of guanylyl cyclase (GC), phospholipases (pLC), and ras proteins. These regulatory molecules may ultimately up­regulate gene transcription through pathways that activate calcium channels and mediate its release from intracellular stores and utilize inositol triphosphates (lP3), diacylglycerol (DAG), calmodulin (CaM), and various protein kinases.

1993; Toyomasu et ai, 1994a; Weller et aI., 1994), ethylene (Suzuki & Taylorson, 1981; Vangronsveld et aI., 1988), abscisic acid (Bossen et aI., 1991; Toyomasu et ai, 1994b) and cytokinin (Tong et aI., 1983; Cohen et aI., 1988) interact with phytochrome to regulate plant growth and development. It is well established that in animals all of these processes are regulated by the heterotrimeric and/or ras like GTP-binding proteins. Although there have not yet been functional relationships established between phytochromes and specific GTP­binding proteins, several heterotrimeric and ras-like GTP-binding proteins have been cloned from plants (Ma et al. 1990; Terryn et aI., 1993; Verma et aI., 1994) and at least one low molecular weight GTP-binding protein has been isolated and partiaIly characterized from oats (Sommer & Song, 1990).

CONCLUSIONS

As we have seen, evidence has accumulated indicating that calcium ions, protein phosphorylation and GTP-binding proteins play important roles in the phytochrome signal transduction chain. Based on this evidence, which is admittedly, in part, circumstantial, it

213

appears that the phytochromes transduce light signals through the interaction of these three regulators, as well as possibly more mechanisms, that have not yet been identified. Based on the evidence outlined above, as well as on what is known about signal transduction in animal systems, the model depicted in Fig. 3 is proposed. This model, which is largely speculative, predicts that in the dark, Pr activates inhibitory G proteins, possibly in conjunction with a ras-like protein. The activation of these proteins triggers a signal transduction cascade that results in the repression of the transcription of light-regulated genes, possibly through the protein products of the COP and/or DET genes. Photoconversion of Pr to pfr may then activate transducin- or Gs-like proteins, which in tum may modulate the activities of guanylyl cyclase, calcium, channels, phospholipases, and/or protein kinases, which ultimately regulate gene transcription.

The possibility that plants transduce red-light signals via GTP-binding proteins raises many intriguing questions. For example, do the phytochromes interact with membrane­bound G-protein receptors and how many phytochrome receptors exist? Do each of the five phytochromes share the same receptors or do they each interact with one or more distinct molecules? Furthermore, how many "effectors" do each receptor interact with, and what level of "cross talk" exists between the components of the transduction chain(s). It is likely that there will be no simplistic answers to these questions. The phytochromes therefore represent one of the most challenging puzzles remaining in the field of signal transduction today.

Acknowledgement.

This work was supported by NIH grant GM36956.

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218

LIGHT PENETRATION INTO THE CANOPY OF TERRESTRIAL ECOSYSTEMS

M. G. Holmes

Department of Plant Sciences University of Cambridge Cambridge CB2 3EA United Kingdom

INTRODUCTION

The description of the penetration of radiation into a vegetation canopy would appear to be a simple task. In essence, radiation approaches an object and is variously reflected, absorbed, and transmitted. Although these are concepts in photophysics which it should be possible to describe in simple and easily quantifiable terms, natural radiation is a complex phenomenon which varies in quantity, quality, and time. The vegetation canopy is also a complex object which varies not only in the spectral characteristics of its elements, but also spatially and temporally.

As well as bearing in mind the dynamic nature of the natural radiation environment, it is important not to forget that a description of the radiation at the Earth's surface and within plant canopies is part of a complex chain in events and is not necessarily a description of the important radiation for plant photobiology, which is the radiation which arrives at the photoreceptor. The radiation at the photoreceptor has passed through many potential modifications from the sun via the atmosphere via vegetation canopies via water in aquatic environments via tissue, before eventually arriving at the photoreceptor.

THE NATURAL RADIATION ENVIRONMENT

When primitive life formed, the solar energy arriving at Earth was very similar to that of today with the exception that there was a much higher level ofultraviolet-B (UV-B; 280-320nm) radiation. As aquatic oxygen-producing organisms multiplied and had their effect on the atmosphere, a substantial proportion of the oxygen was converted into ozone in the stratosphere with the result that much of the harmful UV-B radiation was absorbed before it reached the Earth. This provided the conditions in which terrestrial vegetation could exist and resulted in the solar spectrum which now arrives at ground and sea level.

Solar radiation has four important characteristics for plants: one, it provides heat, primarily from the infra-red wavelengths; two, it provides a source of energy

Light as an Energy Source and Information Carrier in Plant Physiology Edited by Jennings et al., Plenum Press, New York, 1996 219

(photosynthesis, ca. 400-700nm; PAR): three, it provides information (e.g. phototropism, ca. 350-5QOnm; photomorphogenesis, ca. 600-750nm); four, it is a source of damage (e.g. DNA dimer formation, ca. 290-350nm).

THE CANOPY RADIATION ENVIRONMENT

When solar radiation penetrates a vegetation canopy it is selectively attenuated by scattering and absorption. This results in a change in both the quantity and quality of the radiation within the canopy. This basic phenomenon has been known since the earliest measurements by Zederbauer (1908) and Knuchel (1914). Somewhat more modern data are depicted in Figure 1. From these spectra it can be seen that there is a reduction in the blue waveband (400-500nm), a reduction in PAR, and a reduction in the red/far-red (RlFR) ratio. The RIFR ratio is a convenient measure of the radiation which determines the photoequilibrium state of the photoreceptor phytochrome (Holmes & Smith, 1975). The fundamental causes for the various spectral and quantitative changes are described in the chapter titled 'Interception of Light and Light Penetration in Plant Tissues' in this volume.

8.---------------------------------~

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400 soo 600 700 800

Wavelength (nm)

Figure 1. Spectral quality of radiation under clear skies within a wheat canopy at ground level (lowest curve), 0~8m above ground (middle curve), and above the canopy (top curve). (After Holmes & Smith, 1977).

It is important to note at this stage that most radiation measurements within canopies have been made with a cosine-corrected receiver which is not the optimal sensor for such measurements. The optimal sensor does not exist; it would have diffusing surfaces and the same physical dimensions as the plant(s) which grow in the radiation being measured. A horizontal cosine-corrected sensor is probably the best compromise for a canopy of

220

horizontally oriented leaves. As Holmes and Attridge (1987) pointed out on the basis of studies on Sinapis"alba, vertically oriented organs such as hypocotyls can be more sensitive to radiation from the side than from above; they also showed that radiation changes below canopies showed more dramatic changes in light quantity and quality when the measurements were made with a sideways pointing sensor than with a vertically positioned sensor. Reduced tillering in grasses grown near a green canopy (Casal et aI, 1986) led Ballan~ et al (1987) to support the conclusion that radiation received from the side can be more important than radiation from above. Sattin et al (1994) compared the quality and quantity of vertically and horizontally propagating radiation within maize, soybean and wheat canopies. The measurements confirmed the markedly reduced R:FR ratio of the horizontal component. Another important aspect of this study was that it demonstrated the individuality of different canopy types. The main differences were that the seasonal changes in the spectral quality of radiation varied at different rates between canopies and that the extent to which the R:FR ratio was decreased also depended on canopy type.

SKY CONDITION

Daylight consists of two components; these are direct solar radiation and indirect sky radiation (diffuse radiation). Direct solar radiation is effectively a point source with an angular diameter of about 0.5°; it arrives as parallel radiation at the canopy. Diffuse radiation arrives at the canopy from all parts of the sky and it is rarely uniformly distributed; the radiance at the zenith is typically 2.1 to 2.4 times the radiance at the horizon for an overcast sky (e.g. Monteith & Unsworth, 1990).

The relative proportions of direct and diffuse radiation obviously depend mainly on sky conditions (i.e. the amount and density of cloud cover) and on solar elevation (see below). Readers requiring more detail are referred to Jones (1992) for a review of natural radiation for the non-specialist. In essence, the important point to note about sky condition is that the extent of cloud cover varies enormously according to location and season. In the UK., for example, the proportion of diffuse radiation ranges from about 55% in summer to about 70% in winter. In arid regions, the average proportion of diffuse radiation is more typically half, or less, of these values. Under clear skies, the proportion of direct solar radiation contributing to the total global radiation increases with increasing solar elevation; at solar elevations above 30°, direct solar radiation accounts for at least 80% of the total under clear skies (Robinson, 1966).

Apart from a brief flurry of work in the 1960s (Vezina & Pech, 1964; Vezina & Grandtner, 1965; Federer & Tanner, 1966; Vezina & Boulter, 1966), the effect of cloud cover on light quality below vegetation canopies has been largely ignored. However, sky conditions can have a substantial effect on the vegetation canopy radiation regime (e.g. Holmes & Smith, 1977; Figure 2). The comparison shown in Figure 2 was measured at the same location within a wheat canopy under either clear or totally overcast conditions, but with the same solar elevations. The main effect is that a cloudy sky can cause a less marked reduction in the R:FR ratio than that observed when the sky is clear (Figure 3).

SOLAR ELEVATION

Solar elevation affects both the quantity and quality of radiation within canopies. In broad terms, the effect of solar elevation on radiation quantity is straightforward because the quantity within the canopy increases in approximate proportion to the quantity arriving at the surface of the canopy; as solar elevation decreases, radiation penetration into the canopy also decreases. Modifications to this relationship are caused mainly by canopy architecture

221

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2.5

0.5

O+-------~------r_------r_----~ 400 500 600

Wavelength (nm) 700 800

Figure 2. Comparison of the relative spectral distribution of radiation within a wheat canopy on a clear (broken curve) and on a totally overcast (continuous curve) day. Both spectra were measured at the same position in the canopy and at similar solar elevations and growth stages. (After Holmes & Smith, 1977).

and the relative contributions of direct and diffuse radiation. Consideration of solar elevation effects automatically involves the influence of leaf

orientation on the attenuation of the incident radiation. The penetration of direct solar radiation into planophile canopies (with predominantly horizontal leaves) tends to be relatively unaffected by solar elevation (Figure 4); by contrast, the penetration of direct radiation into erectophile canopies (with predominantly vertical leaves) is strongly dependent on solar elevation, with penetration becoming greater as the sun approaches the zenith. An intermediate situation occurs if we consider a spherical leaf distribution. Here the leaves are considered as being theoretically arranged on the surface of a sphere and therefore have a probability of being oriented at all possible angles. Natural canopies cannot normally be classified as purely planophile or erectophile. Soybean, for example, lies approximately between planophile and the example shown for spherical leaf distribution. Maize or wheat, on the other hand, lie approximately between the erectophile and spherical curves.

The effect of solar elevation on light quality within a canopy is slightly more complex. As with light quantity, light quality also depends on canopy architecture because the pathlength of the direct beam varies greatly according to solar elevation. In addition, there are daily differences in the proportions of direct and diffuse radiation penetrating the canopy. For example, at low solar elevations much lower RFR ratios prevail in a maize canopy (Sinclair & Lemon, 1973) than in a wheat canopy (Holmes & Smith, 1977). This is because at low solar elevation canopy architecture and planting density results in direct radiation being a greater contributor to the total radiation within the maize canopy than in a wheat canopy. At high solar elevations, however, the reverse is true, with lower R:FR ratios being found in wheat than in maize, even when identical leaf area indices are compared (Sattin et

222

1.5,-------------------,

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o .~

/'

............................. -~-~-/ 0.5

O.O+-----,----,----r-----r--~

0.0 0.2 0.4 0.6 0.8 1.0

Height above ground (m)

Figure 3. The effect of sky condition on the relationship between the ratio of R:FR radiation and depth within a wheat canopy. The solar elevation was 43-47° and 42-48° under the clear and overcast skies respectively. Open symbols, clear skies; closed symbols, overcast skies. (After Holmes & Smith, 1977).

ai, 1994). In the situation shown in Figure 5 for a wheat cariopy, it can be seen that the R:FR ratio is lowest at both low and high solar elevations with the highest R:FR values being observed at a solar elevation of about 40°. This situation arises from the changing contributions of direct and diffuse radiation as solar elevation increases, and from the marked changes in direct radiation attenuation with changing solar elevation which affect primarily erectophile canopies, such as wheat.

SUNFLECKS

Sun flecks are areas within a vegetation canopy which receive direct solar radiation. Several attempts have been made to quantifY sunflecks. Most of these attempts have been empirical, and much of this work relates to the two decades of studies started by Evans in 1939. The commonest methods of study have been canopy photography, direct observation, timing, and direct measurement of the quantity and quality of radiation. Despite these efforts, and attempts to model them mathematically (e.g. Monteith, 1965), sunflecks are very difficult to quantifY because they vary in size and duration. Under calm conditions, the duration for which an area of ground or vegetation is exposed to the sunfleck (sometimes called time flecks) depends on the size of the fleck; the larger the fleck, the longer the exposure time. As a rule of thumb, it can be said that sunfIecks have a size of the order of mm, and a duration of only a few minutes; this generalisation should be compared with the discussion of gaps below.

223

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Figure 4. Relationship between the extinction coefficient for direct radiation and solar elevation for various leaf orientations. H = horizontal distribution (planophile); S = spherical distribution; V = vertical distribution (erectophile). (After Jones, 1992).

Representative sunflecks are shown in Figure 6. The main characteristics to note are that, in comparison to shade light, sunflecks are depleted more in the B waveband and less in the R waveband. As a result, the R:FR ratio is not as much reduced as in shade light, but it is lower than in the incident radiation above the canopy. Although these features are common to all canopies, the extent to which the R:FR ratio is altered appears to depend on canopy structure: a study on wheat provided an average sunfleck R:FR value of 1.00, and a range from 0.76 to 1.15 (Holmes & Smith, 1977); in a similar study on sugar beet, sunfleck R:FR values as low as 0.36 were observed (Holmes, 1981).

A common feature of sunflecks which can be seen in the data presented here (Figure 6) is that the quantity of FR radiation can be greater per unit area in the sunfleck than that incident at the top of the canopy. This results from a funnelling effect in which FR radiation is reflected from surrounding leaves and is concentrated onto the sunfleck. Similar observations of high FR levels to these within a wheat canopy have been made near the top of maize canopies (Sinclair & Lemon, 1973). In conjunction with the high FR irradiances which can be created within the receiving plant tissue (see 'Interception of Light and Light Penetration in Plant Tissues' in this volume) it is possible to predict very high FR radiation levels in the epidermal cells of sub-canopy species.

Movement of the canopy by wind can cause oscillations in time and space and these are sometimes referred to as wind flecks. The duration of these oscillations is typically between 5 and 20 cycles per second and therefore provides extremely fast and large variations in both the quality and quantity of radiation received by any vegetation in their path. The spectral characteristics of wind flecks are similar to sunflecks.

224

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Figure 5. The effect of solar elevation on the ratio of R:FR radiation at various depths within a wheat canopy. (After Holmes & Smith, 1977).

In contrast to sunflecks, canopy gaps are larger areas of direct solar radiation with diameters of up to a few meters. Canopy gaps provide a wide range of environmental changes which are of significance for the growth and development of plants (e.g. Pickett & White, 1985). Although gaps obviously increase the quantity of light penetrating into the canopy, the radiation has always been difficult to quantifY; this is partially because there are diurnal and - depending on latitude - seasonal changes in light quantity. It is therefore very difficult to obtain adequate experimental data. Furthermore, the radiation quantity also depends on the size of the gap and on the height and the density (especially with regard to chlorophyll) of the surrounding vegetation. Canham (1984) is one of several authors who have attempted to provide an analytical description of radiation quantity in and around canopy gaps.

As pointed out earlier, the spatial orientation if the detector is important because some plants are more sensitive to radiation from the side than above (Holmes & Attridge, 1987). Although measurements with vertically oriented cosine-corrected sensors may be appropriate for assessment of PAR, detector orientation and design are probably very important in canopy gaps because the spectral variation in sideways propagating radiation (which is primarily reflected radiation) can be much greater than in the radiation received from above (e.g. Sattin et ai, 1994). Indirect evidence for substantial spectral changes in the proximity of vegetation comes from studies of the physiological responses of plants (e.g. Kasperbauer et ai, 1984; Casal et ai, 1986) and the effects of radiation from the adjacent vegetation on the photoconversion of phytochrome (Smith et ai, 1990).

225

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6

4

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I • I

I " I "

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Wavelength (nm)

700 800

Figure 6. Spectral photon flux distribution of sunflecks at various heights in a wheat canopy. A, above canopy (R:FR = 1.17); B, 0.8m (R:FR = 0.90); C, 0.6m (R:FR = 0.96); D, O.4m (R:FR = 0.93); E, 0.2m above ground level (R:FR = 0.93). (After Holmes & Smith, 1977).

TEMPORAL CHANGES IN THE LIGHT ENVIRONMENT

Temporal changes in the light environment are mainly cyclic. The major cyclic changes are of the order of, 1) years to thousands of years (ranging from 11 year sunspot cycles to the multi-millennial cyclic changes in the eccentricity of the Earth's orbit), 2) annual (i.e. seasonal, depending on the position of Earth's axis relative to sun), 3) daily (which depend on the Earth's rotation on its axis), and 4) minutes (resulting from solar activity). Non-cyclic changes of relevance range from several years (e.g. stratospheric ozone layer changes) through days or hours (primarily cloud cover) to hours and minutes (e.g. aspect and shading).

Seasonal variations in the quantity of available radiation are most noticeable as one moves away from the equator and the annual variation in daylength increases. In addition, the annual variation in the quantity of radiation also increases. As a partial consequence, there is also a marked variation in the growth pattern of vegetation. The annual changes in light quality below vegetation canopies are less well documented and we can consider an early example of such a study which describes the phenomena and also shows the possible ecological consequences of these spectral changes. Figure 7 shows the annual variation in both the quantity (described by PAR) and the quality (described by the R:FR ratio) beneath a wheat canopy. There is no reason to suppose that the situation is different within other annual canopies except that the duration of shading and the absolute values of PAR and R:FR ratio may vary.

It can be seen in the Figure that when the wheat grows above the sensor (i.e. above any potentially competitive seed or plant) the amount of PAR and the R:FR ratio decrease. This

226

1.0

0.8

0.6 0 .::: .. .... ~

"'"' ~ 0.4

0.2

O.O-t-------r----.,------,...---------\ 110 140 170 200 230

Day of year

Figure 7. Seasonal changes in PAR (closed circles) and the ratio ofR:FR radiation (open symbols) Within a wheat canopy in 1973. The mature crop was harvested on 13/8/73. Values are normalised to 1.0 to aid comparison. (After Holmes, 1981)

change in both quantity and quality continues until senescence occurs - in this case in late June - at which time the quality of radiation starts to return to that of normal daylight. The change results primarily from chlorophyll destruction during leaf senescence. By contrast, however, the change in light quantity is negligible. The clear inference of these different phenomena is that any seed or plant exploiting phytochrome as an indicator of shade would have a marked advantage over a seed or plant using light quantity as an indicator of it's surroundings; this is because using quality would initiate germination or promote growth earlier and thereby take advantage of the detection of the imminent death of its senescing competitors. Similar data do not appear to be available for other crops. However, it is clear from a comparison study of maize, soybean and wheat that the seasonal changes in the R:FR ratio differ substantially between canopy types (Sattin et ai, 1994).

PENETRATION OF UV-B RADIATION

There has been an increased interest in UV-B (280-320nm) radiation in recent years as a result of fears that depletion of the stratospheric ozone layer by pollutants may be increasing the amount of ambient UV -B at ground level. There has been very little research on the penetration of UV-B radiation into vegetation canopies, partly because accurately calibrated instruments are rare and expensive, and partly because it is assumed that the attenuation of UV -B will be strong and the fraction of UV -B penetrating will therefore be insignificant.

However, UV -B levels below vegetation canopies have been calculated (Allen et aI,

227

1975) and measured previously (DeLucia et aI, 1991: Grant, 1991; Lee & Downum, 1991; Yang et al; 1993; Brown et al, 1994). The studies were made on different canopy types and the amount of transmitted UV-B varied enormously, ranging from virtually zero in a sUbtropical canopy (possible instrument limitations; Lee & Downum, 1991) to more than 80% penetration in a com canopy (Grant, 1991), thereby indicating major dependence on canopy type. Brown et al (1994) measured both UV-B and PAR transmittance in forests and found a significantly greater penetration ofUV-B than PAR in temperate mixed deciduous, dry subtropical and moist tropical forests. Underneath a Cotoneaster canopy, the UV­B:PAR ratio increases by about 50% in sunflecks and by over 300% in shade (Table 1).

A decrease in PAR is important because the damaging effectiveness ofUV-B radiation tends to be greater when the amount of PAR available to a plant is low (e.g. Mirecki & Teramura, 1984; Flint et aI., 1985; Cen & Bornman, 1990; Deckmyn et aI, 1994). Although photosynthesis is probably directly involved (Adamse & Britz, 1992), a definitive explanation is not yet available.

Decreasing the R:FR ratio can result in reduced tolerance to UV-B radiation. The only experiments available using polychromatic light to simulate natural conditions indicate that simulating canopy shade by reducing the R:FR ratio reduces the ability of Brassica napus seedlings to maintain healthy growth when compared with plants receiving daylight (Table 2). Adding UV -B to daylight inhibited dry weight accumulation by 18%; if the R:FR ratio was reduced (by addition of FR) but PAR remained the same, UV -B inhibited dry weight accumulation by 41%. Clearly, more research directed at trying to understand canopy radiation environments in the UV-B, PAR and photomorphogenetic wavebands is required; because all these wavelengths influence plant growth and development, we need to know the relative roles played by all three of them.

Table 1. PAR, UV-B, R:FR ratio and UV-B:PAR ratio in sunflecks and shade below a Cotoneaster canopy. The data are normalised to 1.00 in the daylight outside the canopy (M G Holmes, unpublished data).

Daylight Sunfleck Shade PAR 1.00 0.57 0.09 UV-B 1.00 0.84 0.28 R:FR ratio 1.00 0.72 .032 UV-B:PAR ratio 1.00 1.47 3.11

Table 2. Final dry weights (normalised to 1.0 for daylight) of Brassica napus plants exposed to various polychromatic irradiation treatments for three weeks. Experiments were run in a glasshouse which absorbs most UV-B radiation, so all plants received background UV-B (0.2 Wm-2; Philips TLl12 filtered through cellulose acetate). Additional UV-B (also with Philips TLl12 filtered through cellulose acetate) was 2.0 Wm-2 for 6h dai1, centred at noon. The FR treatment (radiation from 500W projectors filtered through red and blue plastic and heat absorbing glass; 8h dai1, centred at noon) produced a range ofR:FR ratios which depended on solar angle and cloud cover. Experiments are from April to June 1992 (M G Holmes, unpublished data).

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Daylight Daylight + UV-B Daylight + UV-B + FR Daylight + FR

Relative dry weight 1.00 0.82 0.59 1.02

R:FR ratio 1.09 - 1.19 1.09 - 1.18 0.24 - 0.86 0.26 - 0.79

REFERENCES

Adamse, P. & Britz, S.1. (1992) Photochem. Photobiol. 56: 645-650. Allen, L.H., Jr., Gausman, H.W. & Allen, W.A. (1975) J. Env. Qual. 4: 285-294. Balian!, e.L., Sanchez, RA, Scopel, AL., Casal, 1.1. & Ghersa, e.M. (1987) Plant Cell Environ. 10: 551-

557. Baraldi, R, Rossi, F .. FacinL 0., Fasolo, F., Magli, M. & Nerozzi, F. (1994) Physiol. Plant. 91: 339-345. Brown, M.J., Parker, G.G. & Posner, N.E. (1994) 1. Ecol. 82: 843-854. Canham, C.D. (1988) Ecology 69: 1634-1638. Casal, 1.1., Sanchez, RA & Deregibus, V.A. (1986) Exp. Environ. Bot. 26: 365-371. Cen, Y-P. & Bornman, J.F. (1990) 1. Exp. Bot. 41: 1489-1495. Deckrnyn, G., Martens, C. & Impens, I. (1994) Plant, Cell Environ. 17: 295-301. DeLucia, E.H., Day, T.A. & Vogelmann, T.e. (1991) Current Topics in Plant Biochemistry and Physiology

10: 32-48. Evans, G.C. (1939) J. Ecol. 27: 436-482. Federer, C.A. & Tanner, C.B. (1966) Ecology 47: 555-560. Flint, S.D., Jordan, P.W. & Caldwell, M.M. (1985) Photochem. Photobiol. 41: 95-99. Grant, RH. (1991) Agron. J. 83: 391-396. Holmes, M.G. (1981) in "Plants and the Daylight Spectrum (H. Smith, ed.). Academic Press, London. Holmes, M.G. & Attridge, T.H. (1987) Proc. Symp. Plant Photomorphogenesis, p.26, (Mitrakos, K,

Georghiou, K & Thanos. C.A, eds.). Holmes, M.G. & Smith, H. (1977) Photochem. Photobiol. 25: 539-545. Jones, H.G. (1992) Plants and Microclimate, pp. 9-45. Cambridge University Press. Kasperbauer, M.J., Hunt, P.G. & Sojka, RE. (1984) Physiol. Plant. 61: 549-554. Knuchel, H. (1914) Zentralanstalt Forst!. Versuchswesen 11: 1-94. Lee, D.W. & Downum, KR (1991) Int. J. Biometeorol. 35: 48-54. Mirecki, RM. & Teramura. AH. (1984) Plant Physiol. 74: 475-480. Monteith, 1.L. (1965) Ann. Bot. 29: 17-37. Monteith, J.L. & Unsworth, M.H. (1990) Principles of Environmental Physics. Edward Arnold, London. Pickett, S.T.A. & White, P.S. (1985) The ecology of natural disturbance and patch dynamics. Academic

Press, Orlando, Florida. USA. Robinson, N. (1966) Solar radiation. Elsevier. Sattin, M., Zuin, M.C. & Sartorato, I. (1994) Plant. 91: 322-328. Sinclair, T.R & Lemon, E.R (1973) Solar Energy 15: 89-97. Smith. H., Casal, J.J. & Jackson, G.M. (1990) Plant Cell Environ. 13: 73-78. Vezina, P.E. & Boulter, D.WK (1966) Can. J. Bot. 44: 1267-1284. Vezina. P.E. & Grandtner. M.M. (1965) Ecology 46: 869-872. Vezina, P.E. & Pech, G.Y. (1964) Forest Sci. 10: 443-451. Yang, x., Miller, D.R & Montgomery, M.E. (1993) Agric. and Forest Meteorol. 67: 129-146. Zederbauer, C. (1908) Forest. Quart. 6: 255-262.

229

LIGHT PENETRATION AND EFFECTS ON AQUATIC ECOSYSTEMS

D.-P. Hader

F riedrich-Alexander -U niversitat, Institut fur Botanik und Pharmazeutische Biologie, Staudtstr. 5, D-91058 Erlangen, Fed. Rep. Germany

BIOMASS PRODUCTIVITY IN THE OCEANS

About 50% of the biomass productivity on this planet depends on aquatic ecosystems (Houghton & Woodwell, 1989). Since freshwater habitats occupy only 0.5% of the total water surface the major share is represented by marine systems. In these the main biomass producers are (mostly unicellular) phytoplankton organisms. However, also macroalgae -with a few exceptions - limited to coastal areas, have a large share in productivity. Almost all primary and secondary consumers in the intricate aquatic ecosystems depend on these primary producers.

The total production of biomass in the oceans can be deduced from the fact that about 100 gigatons (Gt) of carbon (in the form of carbon dioxide) are incorporated annually by these organisms (Fig. 1). This is on the same order of magnitude as all terrestrial ecosystems taken together. Thus, a total of about 200 Gt of carbon are cycled annually, which is a considerable share of the total reservoir of735 Gt in the atmosphere. In comparison, carbon release from anthropogenic sources into the atmosphere amounts to about 5 Gt from fossil fuel burning and about 2 Gt from (mainly tropical) deforestation, respectively. However, only 3 of the total 7 Gt are found to accumulate in the atmosphere annually, and there is a current debate on the fate of the missing 4 Gt.

On a global scale, biomass productivity is not uniform: in the oceans most of the productivity is concentrated in the subpolar regions and higher latitudes while the concentration of phytoplankton in the tropics and subtropics are a factor of 10 to 100 smaller (Ehrendorfer, 1991). One exception to this rule are the upwelling areas on the continental shelves (Fig. 2). This uneven distribution of primary productivity - and as a consequence that of primary and secondary consumers - has been exclusively attributed to a scarcity of nutrients and to non-permissive surface temperatures at low latitudes. However, recently, excessive short wavelength ultraviolet radiation (UV-B, 280 - 315 nm) has been considered as another limiting factor as its irradiance is about seven times higher in the tropics than at mid latitudes (Hader et aI., 1989, 1991; Helbling et aI., 1992). The hypothesis is supported by the fact that the large phytoplankton blooms in the oceans and lakes occur in

Light as an Energy Source and Information Carrier in Plant Physiology Edited by Jennings et al., Plenum Press, New York, 1996 231

Atmosphere: 735

1 I 2

104 100

~ • ~ Terrestrial: 1500 -- --

Oceans: 36000 / Fossil energy: 7500

20° ,-----t---~----_h----~

10° 10°

- -. 0° _L

0° '..J ,

20° 20° -,

30° 30°

40°

40°

50°

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60°

Figure 2. Distribution of biomass in the Southern Atlantic Ocean (after Ehrendorfer, 1993).

they also contribute to the attenuation of light in the water column. Depending on the absorbing properties of the water column, Ierlov (1950, 1970) has classified marine waters into several types ranging from oceanic waters type I (clearest waters) through III followed by coastal waters types 1 through 9 (very turbid).

The best way to describe the ultimate depth to which photosynthetic organisms can perform positive net photosynthesis is by defining the euphotic zone, in which the surface irradiance is attenuated to 0.1%. This is the irradiance at which very low light-adapted organisms such as some cyanobacteria or cryptophyceae can still perform photosynthesis. In physical terms the depth of the euphotic zone can range from a few decimeters in turbid coastal waters to several dozens of meters in clear open oceanic waters (Siebeck & Bohm, 1987; Giskes and Kraay, 1990; Smith et aI., 1992). One easy way of estimating the absorption is to lower a Secchi disk into the water column until it moves out of sight. A Secchi disk is a circular plate with a white, highly reflective coating and a diameter of about 25 cm. It carries several holes for better contrast during visual estimation.

The attenuation in the water column is spectrally dependent: long wavelengths (red and far red) are absorbed more than intermediate wavelengths (blue-green). At the other end of

233

the spectrum, the shorter the wavelength, the more attenuated is the radiation. In the past, this fact has led to the assumption that ultraviolet radiation, especially the detrimental short wavelength UV -B radiation does not penetrate much into the water column. This notion was further nourished by lack of appropriate instrumentation (Smith & Baker, 1978; Baker & Smith, 1982; Smith & Tylor, 1976).

The technical problem in these measurements is that a very small fluence rate of ultraviolet radiation needs to be measured against a large background of visible radiation. UV -B represents only about 0.15% of the total solar energy received at the earth's surface and UV-A amounts to about 4% (Hader & Tevini, 1987). Especially at the short wavelength band of solar energy transmitted through the atmosphere, a steep logarithmic drop in irradiance occurs over a small wavelength range. Measurements performed with a spectroradiometer equipped with a single monochromator fail especially in this region as the internal scattering of these instruments is in the range of 1%. To use a practical example, if the background solar radiation in the visible range amounts to about 1 W m-2 nm-I, the instrument will show a signal of 10 mW m-2 nm-I, regardless of which wavelength has been chosen; it will show this value also in the UV-C region (below 280 nm) even though these wavelengths do not penetrate through the terrestrial atmosphere. The solution to this measurement problem is to use a double monochromator which limits the scattered radiation to 0.01%. Thus, a double monochromator spectroradiometer (such as Bentham or Optronic) with an ultralow-scatter holographic grating provides useful readings down to 10-5 W m-2

nm- I .

A recent development for underwater light measurements uses a different concept: the LUVSS (light and ultraviolet submersible spectroradiometer) instrument separates the short and long wavelength ranges with a dichroic mirror, and each spectral range is evaluated with a separate monochromator. Using this instrument, Smith and coworkers (1992) were able to accurately determine the penetration of solar radiation into clear oceanic waters in the Antarctic (Fig. 3). In contrast to older assumptions UV-B was found to penetrate to 35 m (1% transmission). This indicates that an increase in solar UV-B irradiation may be of major concern for aquatic ecosystems.

10' .,,------------,

Om

10m

E c:

10~

300 400 500 600 700

Wavelength [nm]

Figure 3. Penetration of solar radiation into clear Antarctic water measured at increasing depth (after Smith et aI., 1992).

234

Due to multiple (Rayleigh) scattering in the water the amount of direct radiation decreases with depth in favor of indirect (scattered) radiation. This effect increases with shorter wavelengths according to the Rayleigh law: scattering increases with the inverse of the fourth power of the wavelength. Thus, a considerable component of solar radiation even comes from below, which is not reflected from the bottom. Since an organism does not discriminate between direct and scattered radiation, measurements with a cosine corrected receiver are of no use under these circumstances. Therefore, we have developed a 41t receiver for a double monochromator spectroradiometer (Optronic 742 and 752). At one end of a 10-m waterproof quartz cable there is an air-filled internally frosted quartz sphere 40 mm in diameter (Fig. 4a). The other end of the quartz cable shows a rectangular cross section of 18 x 0.25 mm which fits exactly onto the entrance slit of the monochromator in order to transmit all collected light. However, this device still has a considerable forward characteristics because of the limited opening angle of the quartz light guide. In a subsequently developed device, a 20-m quartz light guide is used, which consists of 72 individual fibers. The polished ends of these were threaded into equidistant holes in a saltwater resistant metal sphere from the inside, and cast in resin, so that the fibers point in different directions covering the whole 41t space (Fig. 4b). Some of the light direction·s can be shielded by blinds to block out radiation impinging from above, below or the equator.

Using these devices, measurements of solar UV irradiation and penetration into the water column were made in the Baltic Sea, the North Sea and the Mediterranean. Figure 5 shows spectra of solar UV irradiation at the sea surface and in the water column for cloudless sky and 348 Dobson units (DU; 1 DU equals about 0.01 mm total ozone column) (Piazena & Hader, 1994). Using the action spectrum for inhibition of motility in the flagellate Euglena gracilis (Hader & Liu, 1990), the spectral efficiency was calculated for these data (Fig. 5b), which shows a peak at about 310 nm.

The irradiance spectra recorded at different depths of natural waters show significant decreases, especially in the UV-B range. Figure 6 shows the penetration of solar irradiance into the water column integrated both over the ranges 290 - 320 nm and 290 - 400 nm for

• •

•

a b

Figure 4. Device to measure spectra1light distribution in the water column, which consists of a quartz cable connected to an Optronic double monochromator spectroradiometer. The receiver for solar radiation is either a hollow, internally frosted, quartz sphere (a or the 72 fibers of the quartz cable are inserted into equidistant holes so that the tips point into different directions covering the whole 41t space (b).

235

10 • 10.1

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10·) >-., u g e .. .. ;g '6

10·" 10·'

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10-1

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Wavelength Inm) Wavelength Inm)

Figure 5. Underwater spectra of solar radiation at the surface and penetrating into the water column in turbid coastal waters in the Baltic Sea near the island of Hidden see (a) and convolution of these spectra with the action spectrum for inhibition of motility in the flagellate Euglena by UV-B irradiation (b).

10 •

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e

.~

.~ 10-' ~1

e .. ~ 1>2

10-1

10-'

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Depth 1m)

Figure 6. Penetration of solar irradiance in the ranges of 290 - 320 and 290 - 400, respectively, into turbid waters near Hiddensee (Baltic Sea) and clear waters near Sardinia (Mediterranean).

the turbid waters off the island of Hidden see (Baltic Sea) and for the clear waters in the near shore zone off the island of Sardinia (Mediterranean Sea). Due to strong mixing and negligible stratification of temperature and salinity in the upper water column, the vertical gradients of transmission differ only little from a linear slope on a semi-logarithmic scale. In order to quantifY the light penetration, attenuation coefficients can be estimated for specific wavelengths, such as, e.g., 310 nm (C3JO) which can be used to determine the critical depth dc which is defined by the decrease of the solar irradiance to 1 % in a water column. While the transmission of Mediterranean waters is close to that of distilled water (c3lO= 1.8-2.3 m- I ,

dc = 2.0-2.4 m) attenuation coefficients for coastal lagoons are much higher (C3lO = 14.0 m-I, dc = 0.33 m). Note, that due to multiple reflection, radiation penetrates much deeper into the water column than can be judged from the absorption coefficient (Piazena & Hader, 1994).

VERTICAL DISTRIBUTION OF PRIMARY PRODUCERS IN THE WATER COLUMN

Being sessile plants, macroalgae are known to follow well established patterns of

236

vertical distribution down the water column. Few algae are adapted to the spray water area above the tidal zone (supralittoral) where they are exposed to unfiltered solar radiation. Many more species are found in the eulittoral, defined by the tidal range. Below this is the sublittoral which falls dry only under extreme conditions. Light sensitive algae may be limited to the understory of kelp forests in the sublittoral, or to caves and crevices where they are exposed to less than 1 or 10% of the surface irradiance. The record lowest occurrence was found in some red algae at a depth of 268 m in the Bahamas (0.001 % of the surface light), but reportedly, growth in these organisms is extremely slow, being of the order of a few cells per year (Luning, 1985).

In contrast, phytoplankton are free to move up and down in the water column and can be passively distributed in the mixing layer, which extends down to the pycnoclin by the action of wind and waves (Smith, 1989; Ignatites, 1990). However, phytoplankton are not equally distributed within the water column, but rather, use highly sophisticated strategies to move to a specific depth with optimal light conditions for growth and reproduction.

These organisms are faced with a dilemma: on the one hand they have to find a position close to the surface in order to receive sufficient solar radiation for photosynthesis, and on the other hand they cannot tolerate the high irradiance of unfiltered solar radiation (Raven, 1991). Most of the phytoplankton are found within the euphotic zone. A fair number of plankton are capable of active movement, such as flagellates or ciliates. However, not actively motile organisms are also known to move up and down in the water column by changing their buoyancy. Cyanobacteria produce gas vacuoles, to decrease their specific weight, and diatoms employ oil droplets for the same purpose (Walsby, 1987; Walsby et aI., 1992). Some of these organisms form a neuston at the surface of a body of water, and are thus fully exposed to solar radiation (Kol, 1929; Pringsheim, 1956; Gerber & Hader, 1994).

Many phytoplankton organisms are known to undergo substantial vertical migrations in the water column (Yentsch et aI., 1964; Taylor et aI., 1966; Tyler & Seliger, 1978, 1981; Bums and Rosa, 1980) to optimize their position with respect to the incident irradiance. As the light intensity changes with solar elevation and is altered by a varying cloud cover, the organisms have to constantly readjust their positions in the water column. Several dinoflagellate species have been observed to move up to 15 m up and down each day. Many cells move to the surface during daytime and to lower layers at night (Estrada et aI., 1987; Holmes et aI., 1967), guided by light (Hader, 1987, 1988a, 1991a; Ekelund & Hader, 1988; Nultsch & Hader, 1988; Hader, 1991b) and gravity (Bean, 1985; Hader, 1987; Hader et aI., 1990). At noon, some species have been shown to avoid the layer immediately at the surface and to return later in the afternoon (Eggersdorfer & Hader, 1991a,b). Upward movement is usually guided by negative gravitaxis (Hader, 1987; Rhiel et aI., 1988b; Tirlapur et aI., 1993), often supported by positive phototaxis at low irradiance (Hader et aI., 1981; Liu et aI., 1990; Watanabe & Furuya, 1974). In many organisms, upward movement is compensated by an antagonistic downward swimming, controlled by, e.g., negative phototaxis elicited by high irradiance. Other organisms switch to a movement perpendicular to the incident light (diaphototaxis) to stay at a suitable depth (Liu et aI., 1990; Rhiel et aI., 1988a,b).

Several techniques have been developed to determine the vertical distribution of phytoplankton in the water column. One approach took the concept of the water column literally and com.sisted of aim long Plexiglas column lowered into the water (Hader & Griebenow, 1988). 18 outlets were spaced at even distances along the length of the column and connected by silicon hoses to a peristaltic pump capable of handling 18 samples in parallel. A 3 m long column was used for marine applications (Eggersdorfer & Hader, 1991a,b). A disadvantage of this technique is that only small samples can be drawn in order not to deplete the volume in the column substantially. Consequently, dense populations need to be used in order to obtain statistically significant numbers for cell counting. In addition, the cells may behave abnormally when confined to a column 10 cm wide.

Therefore, the technique to determine the vertical distribution of phytoplankton in

237

marine ha~itats was changed by using 20 submergible pumps, which are lowered into the water column at equidistant intervals (Fig. 7) (Hader, 1994). Samples of 1 I each are drawn from the sea (which takes about 1 min) and later concentrated in the laboratory by centrifugation or tangential flow filtration. The number of motile and non motile organisms is counted using a computer-based automatic image analysis system developed for this purpose (Hader & Griebenow, 1987; Hader & Vogel, 1991; Hader, 1988b, 1992). The image analysis system is capable of discriminating between cells and seston particles by use of size thresholds. In addition, fluorescence microscopy is employed to identify life cells: the samples are stained with acridine orange, which interacts with DNA and RNA by intercalation or electrostatic attraction. The resulting fluorescence emission (at around 620 nm) is recorded by the CCD camera of the image analysis system. In contrast to DAPI (4',6-diamino-2-phenylindole dihydrochloride) staining, the cells do not need to be chemically fixed and are even motile after staining.

Figure 8a shows a typical example of a vertical distribution during a dinoflagellate (mostly Ceratium species) bloom in the North Sea during calm weather at around 11 a.m. (Hader, 1994). A few hours later the cells moved down in the water column. On the following day there were high winds and waves which caused the organisms to be more or

'I

Figure 7. 20 pumps are used to draw 1 liter samples each to determine the vertical distribution of phytoplankton in the water column.

238

Cells per ml Cells per ml

o 500 1000 1500 2000 2500 3000 o 500 1000 1500 2000 2500 3000

o

2 2

I I ~ 3 ~ 3 .. Q. C-Ol 01 0 0

4 4

5 5

6 a 6 b

Figure 8. Vertical distribution of a phytoplankton bloom (mainly CeratiulIl species) near Helgoland (North Sea) during calm weather (a) and during high winds (b).

less equally distributed throughout the water column (Fig. 8b). Similar distributions were also found inthe Baltic Sea, while in the Mediterranean the distribution extended over greater depth due to much clearer water and a resulting deeper euphotic zone (see above). These observations indicate that the cells adjust to the transparency of the water column and move closer to the surface in turbid waters. Thus, also in habitats of low transparency they may be exposed to inhibitory levels of solar UV irradiation

EFFECTS OF HIGH IRRADIANCES ON ORGANISMS IN THE WATER COLUMN

High fluence rates of visible and specifically short-wavelength UV radiation may cause damage to macroalgae and phytoplankton such as changes of the DNA structure (Karentz et aI., 1991), photosynthetic pigment bleaching (Ekelund & Hader, 1988; Hader et al., 1988; Nultsch & Agel, 1986), inhibition of motility in swimming organisms (Hader et aI., 1989, 1991; Hader, 1993a,b) and disturbance of orientation (Hader & Hader, 1988a,b, 1989, 1990; 1991). Photosynthetic oxygen generation and biomass production is impaired (Smith et aI., 1992; Haberlein & Hader, 1992; Ziindorf & Hader, 1991), and changes in enzyme activity and in nitrogen incorporation have been reported (Dahler, 1985; Dahler et aI., 1986, 1987).

Even short exposure to unfiltered solar radiation impairs the percentage of motile cells in many phytoplankton species and decreases the swimming velocity (Worrest & Hader, 1989; Hader & Worrest, 1991). This reduces the ability of the organisms to adjust their position in the water column to the changing light climate. Using UV cut-off filters, which block short wavelength radiation, or a layer of artificially produced ozone indicates that UV­B has a large effect on motility, even though also UV-A and visible radiation may impair motility in some organisms (Tirlapur et aI., 1993; Sebastian et aI., 1994). Simultaneously, the ability to respond to external stimuli is affected by solar UV irradiation. Phototaxis as well as

239

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Figure 9. Oxygen production of Nodularia spumigena at various depths in the water column

gravitaxis decrease in precision until the (at that time still motile) cells move into random directions. This also reduces the chances of a population to move to and stay at a depth with suitable irradiances.

The next question is how photosynthesis is affected by the irradiances (UV-B, UV-A and visible) at the depth the organisms a located. Earlier experiments have shown that the photosynthetic pigments are bleached within minutes or hours when exposed to unfiltered solar radiation (Hader et aI., 1989, 1991). Also the fluorescence emission from the photosynthetic apparatus changes dramatically, indicating that the energy transfer from the accessory pigments to the reaction centers is affected (Gerber & Hader, 1993). This notion is confirmed by PAM (pulse amplitude modulation) fluorescence measurements (Hanelt, 1992; Hanelt et aI., 1992, 1993; Herrmann et aI., 1994), indicating that the organisms enter the state of photoinhibition when exposed to strong solar radiation (Kolbowski et aI., 1990; Krause & Weis, 1991; Trebst, 1991).

Because these measurements are carried out under artificial conditions a device has been developed which allows the measurement of photosynthetic oxygen production of macroalgae or phytoplankton above and in the water column under solar irradiation (Hader & Schafer, 1994a,b). Measurements are based on determination of oxygen exchange using a Clark electrode (Dubinsky et aI., 1987). The organisms are located in a cuvette in the top lid made from UV-B transmitting Plexiglas, and the medium is agitated with a magnetic stirrer from below. The signal from the electrode is amplified and transmitted to an analog/digital (AID) card housed in the extension box of a notebook computer. Two additional integrated sensors monitor light intensity and temperature; all values are constantly displayed and stored for future calculations. The device can be used above or below water; in the latter case it is lowered into the water column from a buoy or from a research vessel.

The oxygen production shows a pronounced dependence on the available light intensity and can be negative even at a depth of a few meters in some species (Fig. 9). The photosynthetic performance is closely related to the irradiance and the transparency of the water column as examples from coastal lagoons of the Baltic Sea, the North Sea and the Mediterranean show. There are significant differences between species adapted to surface waters or to different depths in the water column. When exposed to strong solar irradiation many species show reversible photoinhibition or even photodamage, which is caused in part by visible radiation and to a larger extent by UV.

240

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Fischer, Stuttgart, Jena, New York, p.892. Ekelund, N. & Hader, D.-P. (1988) Plant Cell Phvsiol. 29: 1109. Estrada, M., A1cataz, M., & Marrase (1987) Inv.' Pesq. 51:443. Gerber, S. & Hader, D.-P .• 1993, Env. Exp. BioI. 33:515. Gerber, S. & Hader, D.-P. (1994) FEMS Microbiol. Ecol. 13:177. Gieskes, W. C. & Kraay, G. W. (1990) Biomass Newsletter 12: 12. Green. A. E. S. (1983) Physiol. Plant. 58:351. Haberiein, A. & Hader, D.-P. (1992) Acta Protozool. 31 :85. Hader, D.-P. (1987) Arch. Microbiol. 147:179. Hader, D.-P. (1988a) 1. Photochem. Photobiol. B: BioI. 1:385. Hader, D.-P. (1988b) Proc. Indian Acad. Sci. (Plant Sci.) 98:227. Hader, D.-P. (1991a) in "Photobiology. The Science and its Applications" (Riklis, E., ed.), Plenum

Press. New York and London. p. 329. Hader, D.-P. (l991b) in "Biophysics of Photo receptors and Photomovements in Microorganisms"

(Lenci, F .. Ghetti, F., Colombetti, G., Hader, D.-P., & Song, P.-S., eds.), Plenum Press, New York and London, p. 203.

Hader, D.-P. (1992) Acta Protozool. 30:135. Hader, D.-P. (1993a) in "Progress in Phycological Research" (Round, F.E. & Chapman, D.J., eds.),

vol. 9, p. 1. Hader, D.-P. (1993b) in "UV-B Radiation and Ozone Depletion. Effects on Humans, Animals, Plants,

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Hader, D.-P. (1994) Vertical distribution of marine phytoplankton in the water column, in press. Hader, D.-P., Colombetti, G., Lenci, F., & Quaglia, M. (1981) Arch. Microbiol. 130:78. Hader, D.-P. & Griebenow, K. (1987) EDVMed. BioI. 18:37. Hader, D.-P. & Griebenow, K. (1988) FEMS Microbiol. Ecol. 53:159. Hader, D.-P. & Hader, M. (1988a) Current Microbiol. 17:215. Hader, D.-P. & Hader. M. (1988b) Arch. Microbiol. 150:20. Hader, D.-P. & Hader, M. A. (1989) Environ. Exp. Bot. 29:273. Hader, D.-P. & Hader, M. (1990) 1. Photochem. Photobiol. B: BioI. 5: 105. Hader, D.-P. & Hader, M. (1991) Env. Exp. Bot. 31:33. Hader, D.-P. & Liu, S.-M. (1990) Curr. Microbiol. 2I:l61. Hader, D.-P. & Schafer, J. (1994a) 1. Plant Physiol. 144:293. Hader, D.-P. & Schafer, J. (l994b) Environm. Monitor. Assessm., 32:259. Hader, D.-P. & Tevini, M. (1987) "General Photobiology", Pergamon Press, London. Hader, D.-P. & Worrest, R. C. (1991) Photochem. Photobiol. 53:717. Hader, D.-P. & Vogel, K. (1991) J. Math. BioI. 30:63. Hader, D.-P., Vogel, K. & Schafer, 1. (1990) Microgravity sci.technol. III, IlO. Hader, D.-P., Worrest, R. C. & Kumar, H. D. (1989) Aquatic ecosystems, UNEP Environmental

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Effects Panel Report 33. Hader, D.-P., Rhiel,E. & Wehrmeyer, W. (1988) FEMS Microbiol. Ecol. 53:9. Hanelt, D. (1992) Mar. Ecol. Progr. Ser. 82:199. Hanelt, D., Hupperts, K. & Nultsch, W. (1992) Bot. Acta 105:278. Hanelt, D., Hupperts, K. & Nultsch, W. (1993) Mar. Ecol. Progr. Ser. 97:31. Helbling, E. W., Villafane, V., Ferrario, M. & Holm-Hansen, O. (1992) Marine Ecology Progress

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Series 80:89. Herrmann, H., Ghetti, F., Scheuerlein, R & Hader, D.-P. (1994) 1. Plant Physiol., 145:221. Holmes R W., Williams, P. M. & Eppley, R W. (1967) Limnol. Oceanogr. 12:503. Houghton, R. A. & Woodwell, G. M. (1989) Scientific American 260:18. Ignatiades, L. (1990) 1. Plankton Res. 12:851 Jerlov, N. G. (1950) Nature 166:. Jerlov, N. G. (1970) in "Marine Ecology" (Kinne, D., ed.), vol. 1, p. 95. Karentz, D., Cleaver, J. E., & Mitchell, D. L. (1991) 1. Phycol. 27:326. Kerr, R A. (1989) Science 262:501. Kol, E. (1929) "Wasserbliite" der Sodateiche auf der Nagy Magyar Alfold (Grofien Ungarischen

Tiefebene), I, Arch. Protistenk. 66:515. Kolbowski, 1., Reising, H. & Schreiber, U. (1990) Photosynth. Res. 25:309. Krause, G. H. & Weis, E. (1991) Ann. Rev. Plant. Physiol. Plant Mol. BioI. 42:313. Liu, S.-M., Hader, D.-P. & Ullrich, W. (1990) FEMS Microbiol. Ecol. 73:91. Liining, K. (1985) "Meeresbotanik" Thieme, Stuttgart. Madronich, S., Bjorn, L. D., Ilyas, M. & Caldwell, M. M. (1991) Changes in biologically active

ultraviolet radiation reaching the earth's surface, United Nations Environmental Program, 1. Nultsch, W. & Agel, G. (1986) Arch. Microbiol. 144:268. Nultsch, W. & Hader, D.-P. (1988) Photochem. Photobiol. 47:837. Piazena, H. (1990) Z. Physiother. 42:357. Piazena, H. (1991) Z. Meteorol. 41, 273. Piazena, H. & Hader, D.-P. (1994) Photoche. Photobiol. 60:463 Pringsheim. E. G. (1956) Nova Acta Leopoldina 125:5. Raven, 1. A. (1991) J. Photochem. Photobiol., B: BioI. 9: 239. Rhiel, E., Hader, D.-P. & Wehrmeyer, W. (1988a) 1. Photochem. Photobiol. B: Biol. 2: 123. Rhiel, E., Hader, D.-P. & Wehrmeyer, W. (1988b) Plant Cell Physiol. 29:755. Sebastian, C., Scheuerlein, R & Hader, D.-P. (1994) 1. Exp. Marine BioI. Ecol., 182:251. Siebeck, D. & Bohm, U. (1987) Untersuchungen zur Wirkung der UV-B-Strahlung aufkleine

Wassertiere, BPT Bericht, Gesellschaft fur Strahlen- und Umweltforschung, Miinchen, p. 84. Smith, R. (1989) Photochem. Photobiol. 50:459. Smith, R C. & Baker, K. S. (1978) Photochem. Photobiol. 29:311. Smith, R C. & Tyler, 1. E. (1976) in "Photochemical and Photophysical Reviews" (Smith, R C., ed.),

vol. 1, 117. Plenum Press, London, New York. Smith, R C., Prezelin, B. B., Baker, K. S., Bidigare, R R, Boucher, N. P., Coley, T., Karentz, D.,

Macintyre, S., Matlick, H. A., Menzies, D., Dndrusek, M., Wan, Z., & Waters, K. 1. (1992) Science 255: 952.

Taylor, W. R, Seliger, H. H., Fastie, W. G., & McElroy, W. D. (1966) J. Mar. Res. 24:28. Tirlapur, U., Scheuerlein, R, & Hader, D.-P. (1993) FEMS Microbiol. Ecol. 102:167. Trebst, A. (1991) Z. Naturf. 46:557. Tyler, M. A. & Seliger, H. H. (1978) Limnol. Oceanogr. 23:227. Tyler, M. A. & Seliger, H. H. (1981) Limnol. Dceanogr. 26:310. Walsby, A. E. (1987) in "The Cyanobacteria" (Fay, P. & Van Baalen, C., eds.), Elsevier Science

Publishers, p. 385. Walsby, A. E., Kinsman, R, & George, K. I. (1992) J. Microbiol. Meth. 15:293. Watanabe, M. & Furuya, M. (1974) Plant Cell Physiol. 15:413. Worrest, R C. & Hader, D.-P. (1989) Environmental Conservation 16:261 Yentsch C. S., Backus, R H. & Wing, A. (1964) Limnol. Dceanogr. 9:519. Ziindorf, 1. & Hader, D.-P. (1991) Arch. Microbiol. 156:405.

242

INTERCEPTION OF LIGHT AND LIGHT PENETRATION IN PLANT TISSUES

M. G. Holmes

Department of Plant Sciences University of Cambridge Cambridge CB2 3EA United Kingdom

INTRODUCTION

There are three potential fates for a photon arriving at a tissue: it can be reflected, absorbed, or transmitted. All three factors are inter-related. The probability of one of these fates depends on the wavelength and the angle of incidence of the radiation, and on several characteristics of the tissue. The important characteristics of the tissue are the size and distribution of the elements with different refractive indices, and the concentration, distribution and absorption characteristics of absorbing particles. The amount of radiation transmitted is a function of all the above parameters. The complex interaction of reflectance, absorptance and scattering is important for most photo responses in plants, whether they are involved in obtaining energy (e.g. photosynthesis), information (e.g. photomorphogenesis) or responding to the destructive effects oflight (e.g. DNA dimer formation).

To understand the radiation environment of a photoreceptor, we have to have knowledge about the fate of radiation at the air/tissue interface, and important phenomena within plant tissue such as absorptance and radiation scattering. All of these combine to determine the quantity and quality of the tissue radiation environment. In this approach, we consider the phenomena which control the entrance of radiation, and then move on to consider the parameters which control the fate of radiation within the tissue. It is essential that the student understands the terminology of irradiance, fluence rate, and other units of light measurement; their importance, and the relevance of different methods of radiation measurement cannot be exaggerated.

CONTROLLING THE ENTRANCE OF LIGHT

Plants which depend on light as a source of energy can run the risk of receiving too much radiation. Apart from temperature effects, the two most serious effects of excessive radiation are nucleic acid lesions caused by UV-B (ultraviolet-B; 280-320nm) radiation, and photodestruction of chlorophyll by excited singlet state oxygen. Although plants have

Light as an Energy Source and Information Carrier in Plant Physiology Edited by Jemlings et al., Plenum Press, New York, 1996 243

evolved photoreceptors which function to repair or prevent the damaging consequences of excessive r~diation, it is of obvious adaptive value if unwanted radiation can either be reflected away from the tissue, or can be absorbed before it reaches sensitive targets. Epicuticular wax and leaf hairs are commonly used to reflect excessive radiation of all wavelengths, while pigmentation in leaf hairs and epidermal layers are only suited for absorbing shorter wavelengths. In some instances it is necessary to capture as much radiation as possible and plants appear to have evolved structural modifications to enhance penetration of radiation.

ANATOMY

Leaves tend to absorb well in the PAR (photosynthetically active radiation; 400-700nm) and the far infra-red (above about 1400nm), but poorly in the near infra-red (ca. 700-1400nm) waveband. This is largely a function of their poor reflectance in the PAR and far infra-red and high reflectance in the near infra-red wavebands. For the shorter wavelengths, it is possible to generalise that conifers have low reflectance (ca.· 10%), and that crop plants and deciduous plants have a fairly high reflectance (ca. 30%); the reflectance of leaves of pubescent and species with waxy cuticles tends to be very high with values approaching 40% (Linacre, 1969: Gates, 1980; Stanhill, 1981; Monteith and Unsworth, 1990). A study by Mooney et al (1977) on Atriplex hymellelytra which grows in Death Valley in California showed a seasonal variation in leaf reflectivity at 550nm. These leaves exhibited low reflectance in winter and high reflectance in summer; this was a function of leaf water content, with low water content resulting in high reflectivity.

WAXES

The main purpose of the waxy epicuticular layer is to reduce water loss (Cutler et ai, 1980; Juniper & Jeffiee, 1982). It is known that UV-B radiation can induce the formation of wax (Basiouny et ai, 1978). Cen and Bornman (1993) recorded that UV-B treated leaves of Brassica napus formed a dense epicuticular wax layer which may have increased UV-B reflectance. Steinmiiller and Tevini (1985) found that UV-B caused an increase in epicuticular wax in all the species tested (barley, bean, and cucumber). They described the biochemical changes in the wax components in a subsequent publication (Tevini & Steinmiiller, 1987).

The significance of the changes in epidermal wax is not clear, especially when it is borne in mind that several environmental factors regulate wax production (e.g. Hull et ai, 1975). The absorptance ofUV-B radiation by wax increases very strongly with wavelengths below about 300nm and this increase in absorptance is much more marked than with supposed screening pigments (e.g. Bornman & Vogel mann, 1988). However, not enough is known about the relative absorbance of waxes compared to screening pigments in order to state the relative importance of waxes. Rough epidermal cells can increase absorption of radiation because they increase internal reflections (Bernhard et aI, 1968); whether or not increased wax deposition can modulate such effects is unknown. More knowledge about the reflective properties of the different waxes, their refractive indices, and their effect on cuticle morphology in relation to light will help us understand the relative importance of wax in regulating radiation penetration into plant tissues.

LEAF HAIRS

Many plants use pubescence to reduce transpiration (e.g. Ehleringer et ai, 1976).

244

However, there is_evidence that some species use a pubescent layer to reduce either the UV­B or longer wavelength radiation penetrating into the tissue. Observations with Espeletias (Goldstein et ai, 1989) and Encelia farinosa (Ehleringer & Bjorkman, 1978) have shown that a significant role of hairs in these species is to reduce the amount of visible light entering the mesophyll. The PAR reflectance of the pubescent E. farinosa is about 50% greater than the glabrous coastal species E. californica (Ehlenringer, 1980; Fig. 1). One characteristic of leaf hairs is that they can have a high refractive index. In soybean, for example, the leaf hairs have a refractive index of about 1.48; this compares to a value of about 1.41 for the epidermal cell walls (e.g. Woolley, 1975). Dry cell walls have an even higher refractive index. This high refractive index step increases the potential for reflecting potentially harmful radiation. It is possible that hairs on the lower epidermis can also cause increased reflectance from the upper epidermis (Eller, 1977).

1.0

0.8

~ 0.6 .............. ...

~ ~

0.4

0.2

.. ........

O.O..L,----,-----....------,.------.-l 400 500 600 700 800

Wavelength (nm)

Figure 1. Absorption spectra of the glabrous coastal Encelia cali/arnica (upper curve), E. virginiensis (second curve), and the pubescent desert species E. !arinasa (lower three curves). The plants were gmwn in an aridity gradient, with dryness increasing from the top to the bottom curve. (After Ehleringer, 1980).

The high "refractive index of plant hairs is not their only characteristic which affords protective reflectance to the plant. Karabouniotis et al (1992) found that the pubescent layers of Olea europea and Olea chrysophylla exhibited considerable absorbance in the UV­B region, with a maximum near 310nm. By contrast, absorbance in the PAR region was negligible. Both Olea species, and a range of other pubescent species contained phenolics with a high flavonoid content. It is probable that the pigmentation caused the relative differences in absorbance in the UV-B and the PAR regions. It is noteworthy that only the young leaves of Olea carry hairs. As the leaves mature and the hairs are lost, the epidermis -

245

which contains high concentrations ofUV-B absorbing pigmentation - appears to provide a substantial <UV-B screen. Lovelock et al (1992) found that the amount of epidermal UV absorbing compounds varied between species in their study of tropical mangroves. Sun leaves tended to have higher contents of these compounds, but several other factors were also involved.

On a larger scale, spines on cactus can increase leaf reflectance (Gates et aI, 1965; Gates, 1980). In the cactus Opllntia bllgelonii, the orientation of the spines results in a reduction of the quantity of the radiation at the epidermis at high solar elevations, but they appear to provide an increase in the irradiance at low elevations (Nobel, 1983). Whether or not there is an equivalent situation with leaf pubescence is unknown.

LENS EFFECTS (FOCUSING)

The radiation regime within tissue is initially controlled by the air/tissue interface. At this stage the extent to which radiation penetrates is determined primarily by the differences in refractive index and this in turn depends on the angle of incidence of the radiation. For a perfectly flat surface, Snell's Law determines the quantity of radiation entering the tissue. In Nature, however, perfectly flat tissue surfaces rarely exist. On a relatively large scale, such as leaves, waxy cuticles can smooth surfaces, but the overall topography is dominated by the natural way in which cells tend to produce a non-planar surface. The result is an array of structures (i.e. cells) which can act as convex lenses, each producing what is commonly termed as a lens effect. On a smaller scale, many unicellular structures with cylindrical or spherical shapes can act as lenses.

Lens effects were first studied in detail by Haberlandt (1914) who used ray-tracing diagrams to demonstrate that individual cells in the epidermis of various leaves could focus light. He showed that differently shaped cells would focus the incoming light at different areas of those cells. Much more recently, Bone et al (1985) reported strong lens effects in the leaves of plants in tropical understories. These plants tend to receive very low levels of PAR and often exist close to the photosynthetic compensation point of C3 plants. Bone et al calculated that the lens effect of epidermal cells can produce a ten-fold concentration of PAR onto the chloroplasts within the cells.

It is important to note that the lens effect will only occur in direct light. This has been demonstrated in the temperate species Oxalis ellropea where Poulson and Vogelmann (1990) used image analysis to measure the uniformity of the radiation field below a stripped epidermis. They found that diffuse incident radiation resulted in a uniform radiation field but that collimated light, which is equivalent to direct solar radiation, produced a highly non­uniform radiation field in which radiation was concentrated in areas where the irradiance was twice that of the incident.

The evidence for whether or not epidermal focusing is of ecological advantage is unresolved. It has been suggested that the lens effect may help in very low light conditions, but as it is only truly functional in direct solar (sunfleck) radiation it is questionable whether it is of help because sunflecks typically have a higher fluence rate than diffuse canopy light (see chapter on 'Light Penetration into the Canopy of Terrestrial Ecosystems'). Further evidence against epidermal focusing being of ecological advantage is that in plants growing in open habitats it may cause photoinhibition and damage the photosynthetic system, although the effect may be moderated by chloroplast avoidance movements (Gabry~ & Walczak, 1980; Zurzycki, 1961).

In addition to the above arguments, several species have developed an elegant avoidance reaction to excessive levels of light in which high irradiances of blue light, such as those found in sunflecks, cause leaflets to close rapidly. As Vogelmann (1994) points out, focusing may be a secondary effect. The most likely explanation is that the convex epidermal

246

cells function to decrease specular reflection and therefore cause absorption of more of the diffuse light which predominates within vegetation canopies.

Most experimental work on lens effects have centred on cylindrical tissues because they are relatively simple to work with. One of the best documented instances is that of the sporangiophore of Phycomyces. The sporangiophore is phototropic; when irradiated from one side it bends towards the light. The lens effect occurs when light passes from a medium with low refractive index (i.e. air) into a medium with high refractive index (i.e. plant tissue); what is happening here is that the fluence rate on the distal side of the sporangiophore is approximately double that on the proximal side (Fig. 2), and the sporangiophore bends towards the light. If the refractive index surrounding the sporangiophore is changed by submersing it in oil, it bends away rather than towards the light because the fluence rate is now greater at the proximal side because the refractive index step which is required to produce a lens effect has been removed.

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Degrees

Figure 2. Light gradients hum (solid curve) and lOmm (broken curve) below the sporangium of a Phycomyces sporangiophore. J is the amount of radiation relative to the incident radiation (10). The degree values refer to the azimuth angle, where 90° is directly opposite the irradiated surface. (After Dennison & Vogel mann, 1989).

Under normal conditions, the fluence rate difference across an object such as a sporangiophore depends on three main factors, these being the angle of collimation, the angle of incidence, and the degree of scattering within the tissue. Although refractive index differences are the over-riding factor, refractive index tends to be fairly similar between tissue types, with values between about l.3 and 1.45 being typical. It is worth noting that similar lens effects have been observed in the rhizoids of Marchantia and Lunularia, but the physiological effect is that they bend away from the light under natural conditions. If they

247

are immersed in mineral oil they bend towards the light because the refractive index change has been abolished (Humphrey, 1966).

The phototropic curvature of Avena coleoptiles is the best studied example of possible lens effects in higher plants. When exposed to low light levels, coleoptiles bend towards the light (first positive curvature); when the amount oflight is increased, they start to bend away from the light (negative curvature). If the coleoptiles are immersed in paraffin oil, which has a similar refractive index to the tissue, the response to unidirectional light is reversed so that low fluence rates produce small, but negative curvature and high fluence rates now result in positive curvature (Humphry, 1966; Meyer, 1969).

Although these experiments provide some evidence for the elimination of the lens effect by altering refractive indices, there is a real possibility that anoxia resulting from immersion in oil may have affected the response. Shropshire (1974) attempted to circumvent this problem by comparing the effects of convergent and divergent light on Avena coleoptile phototropism. He found that convergent light produced the expected positive phototropism, but that divergent light resulted in negative curvature. Although technical difficulties with this approach have been pointed out (Iino, 1990; Vogelmann, 1994), Shropshire's experiments do add to the circumstantial evidence for lens effects in higher plants. Nevertheless, it has to be borne in mind that extremely steep blue light gradients exist across coleoptiles (Vogelmann & Haupt, 1985); each cell responds to the amount of light it receives, so the overall phototropic response may be a product of the individual cell responses.

The study oflens effects in cylindrical higher plant organs is even more complex than in simple structures such as the Phycomyces sporangiophore. The first problem is that multiple scattering occurs and this is difficult to account for. The optical pathlength is also long and complex, although theoretical consideration of a maize mesocotyl does predict that focusing can occur across the width of the organ (Steinhart 1991). What is not understood is the possible role offocusing by individual epidermal cells of coleoptiles and mesocotyls, because a situation analogous to that described by Haberlandt (1914) in individual leaf epidermal cells will presumably exist. Another major problem is that, as mentioned above, the response (positive or negative curvature) depends on the fluence rate.

PHOTOPROTECTION BY PIGMENTS

Several pigments can act as efficient absorbers of potentially damaging excessive levels of UV-B radiation, but the flavonoids are generally considered to be the main screening pigment which acts to protect plants (e.g. Caldwell et ai, 1983; Hahlbrock et al, 1982; McClure, 1975; Shibata, 1915). The main evidence for flavonoids as protective screens derives from their strong absorptance in the UV-B waveband, and the fact that they are primarily found in the vacuoles of the epidermal cells (e.g. Beerhues et ai, 1988; Jahnen & Hahlbrock, 1988; Robberecht & Caldwell, 1978; Schmelzer et ai, 1988; Schnabl et ai, 1986) where they can provide maximum protection to lower cell layers and in the vacuoles of the upper mesophyll tissues (Jahnen & Hahlbrock, 1988). A contributory and compelling argument for their role as protectors is that flavonoid biosynthesis is induced by UV-B radiation in many of the systems which have been studied (Bruns et ai, 1986; Chappell & Hahlbrock, 1984; Hahlbrock et ai, 1976; Tevini et ai, 1983; Wellmann, 1975).

In practice, UV-B absorbing pigments are traditionally measured using a methanolic extract (Mirecki & Teramura, 1984) for which the absorptance at 300nm is recorded. This provides an overall view of flavonoid derived absorbers but suffers from the potential problem that it describes the gross absorptance of the entire tissue extract. As described in a later section, the spatial distribution of absorbers is extremely important in determining the extent to which radiation is attenuated. In addition, the absorptance of an extract does not necessarily represent the absorbing potential of the intact tissue (c.f. Fig. 3).

248

1.00..,..L-----I....----..L---------'~

0.80

0.60 ., u .§ ~ '" ,J:l · < 0.40 · · · · · · · · · · · 9 · · · · · · 0.20 · · · · b

0.00 400 500 600 700

Wavelength (nm)

Figure 3. The spectral absorptance of an entire tobacco leaf (square symbols), an infiltrated leaf (closed circles), and a pigment extract of equivalent pigment concentration. (After Moss and Loomis, 1952).

One of the most striking spectral characteristics of pl~nt epidermal tissue is that it tends to allow the penetration ofa much larger percentage of PAR than UV-B (e.g. Gausman et aI, 1975). Looking at a range of species, Robberecht and Caldwell (1978) found that epidermal peels tended to transmit less than 10% of the incident UV-B radiation; this contrasts with the PAR waveband of which more than 80% is typically transmitted. They concluded that much of the UV-B absorptance (between 20 and 57%) was probably caused by pigmentation. Pigmentation of floral bodies also appears to protect the potentially sensitive pollen from damage by UV-B radiation (Brehm & Krell, 1975; Flint & Caldwell, 1983). Ontogenetically, UV-B damage is most likely to occur when the pollen penetrates the stigmatic tissues (Flint & Caldwell, 1983, 1985).

In a study of 22 plant species, Day et al (1992) found that UV -B radiation penetrated deepest into the tissue of herbaceous dicotyledons, followed by woody dicotyledons and grasses, and attenuation was greatest in conifers. This and other studies (e.g. Bornman & Vogelmann, 1988; Cen and Bornman, 1993; Bornman and Teramura, 1993) using fibre optic micro probes in intact tissue show that the epidermal layer provides extremely strong attenuation. N~vertheless, physiological experiments show that UV-B can penetrate deep enough to cause significant damage. Whether this response is the result of a small increase in UV-B above a normally tolerable level, or whether screening is not distributed with adequate uniformity across the epidermis is not known

Studying accumulation of UV-absorbing flavonoids induced by UV-B radiation in Arabidopsis thaliana L., Lois (1994) found that younger leaves are more readily damaged by UV-B than older leaves, and that the amount of damage correlated closely with flavonoid content. An Arabidopsis mutant which was very sensitive to UV -B radiation was deficient in a derivative of the flavonol kaemferol (Lois & Buchanan, 1994). Similarly, Stapleton and Walbot (1994) showed that the DNA in maize plants which contained flavonoids was

249

protected from damage by UV -B radiation compared to the damage in flavonoid deficient mutants.

On the basis of the new work with flavonoid deficient mutants and the various forms of evidence cited above, it would appear that flavonoids are the main screening pigments which plants have adopted to regulate shorter wavelengths penetrating their tissue. However, new physiological (Britz, pers. comm.) and mutant studies (Last, pers. comm.) which have not yet been published suggest that flavonoids are not always the most effective protective compounds.

The formation of anthocyanin in response to UV -B is not generally considered to be a significant contributor to the protection of plant cells against UV -B radiation. There is very little evidence for anthocyanin acting as other than a by-product ofUV-B induced stress in which anthocyanin is produced in parallel with other flavonoids which absorb more strongly in the UV-B waveband. However, a study by Takahashi et al (1991) does suggest that anthocyanin can reduce UV -B damage. They induced controlled amounts of anthocyanin in Centaurea cyanus L. cell suspension cultures using UV -containing white light and then determined the sensitivity (i.e. survival) of the cells, and the pyrimidine dimer ccmtent of the cells, in response to UV-B light. They found that both the sensitivity of the cells, and the formation of pyrimidine dimers in response to UV-B, decreased with increasing anthocyanin content. It has to be noted, however, that the absorption spectra of the tissues indicated a shoulder at 330nm which may have indicated the presence of another flavonoid or phenyl propanoid compound.

CONTROLLING THE PENETRA nON OF LIGHT

Anatomy

Radiation absorptance by leaves tends to increase with leaf thickness and density. The example (Fig. 4) of the relationship between absorptance and specific leaf weight shown here was measured by Osborne and Raven (1986) and is for eight different species. Sheehy (1975) has demonstrated a parallel decrease in leaf transmittance with specific leaf weight (data not shown). Exceptions to this relationship do exist. One cause can be the effect of pubescence on the leaf reflective properties which can result in decreased absorptance with increasing specific leaf weight in Encelia farinosa.

It is always important to bear in mind that the absorptive properties can change during natural development (Causton & Venus, 1981) and as a result of several environmental factors (Evans, 1972). Although variations in absorptance can probably be described in terms of pigment content (g m-2), the phenomenological optical constant for scattering (m-I)

and tissue thickness (m), the relative importance of these factors can vary greatly. Working with seaweeds, Ramus (1978) has shown that light absorption can be independent of pigment content in fronds of Codium fragile, whereas absorptance is strongly dependent on pigment content in the thallus of the green alga Viva lactllca. Ramus suggests that the different absorbing characteristics of C. fragile may be caused by air spaces in the fronds acting as light-guiding structures.

Scattering

Scattering may be defined as any phenomenon which causes a photon to be deflected from its original direction of propagation. Scattering is only poorly understood and much of our knowledge is still based on the pioneering work of Mie (1908). The subject is complex, and although the situation in whole plants is unresolved, it is worth summarising what is known about single particles (independent scattering) and about situations in which scattering mainly results from radiation already deflected by other particles (multiple scattering).

250

0.9

0.8

0.7

0.6

o 2 3 4 5

Specific leaf weight (g ffi-2 x 10)

Figure 4. The relationship between specific leaf weight and absorptance for several plant species. (After Osborne & Raven, 1986).

In independent scattering, every particle is considered individually. To include other particles, the total number of particles is multiplied by the effect of radiation on one particle. The size of the particle is very important. For particles much smaller than the wavelength, Rayleigh scattering applies; scattering is inversely proportional to the fourth power of the wavelength; this type of scattering only occurs at the molecular level. It is important to note the wavelength dependency. Scattering increases with particle size. For particles greater than about 0.2 !!m, the increase in scattering with particle size is less than that predicted by Rayleigh (e.g. Penndorf, 1962), and the wavelength dependency begins to be lost.

Backward scattering predominates with particles less than about 1.0 !!m in diameter and greater than about 20!!m (see van de Hulst, 1957 and references therein). For particles in the range 1.0 to 20.0!!m (which includes many plastids and cells), forward scattering predominates very strongly. Most of this scattering is caused by diffraction, interference and related phenomena (e.g. Hodgkinson & Greenleaves, 1963; Latimer, Moore & Bryant, 1968). Extrapolating to whole tissues, it follows that the ratio of forward to backward scattering depends on the size distribution of cellular components. About 85% of radiation is scattered forwar-ds in biological tissues (Diffey, 1983; Latimer & Noh, 1987). Scattering is strongly dependent on the refractive index of the particles and backscatter increases with increasing refractive index (van de Hulst, 1957).

With multiple scattering, photons are scattered by more than one particle and the chance of absorption is greater than with single particles. Multiple scattering leads to loss of directionality of the radiation, with the result that equal amounts of radiation may propagate in all directions (e.g. Woodward, 1964; Billmeyer & Richards, 1973). However, the size of plant cells and their components results in predominantly forward scattering in tissues. The relatively large size of these components also means that scattering is independent of

251

wavelength (Mie scattering). The wavelength dependent attenuation of radiation within tissue is mainly caused by absorption by pigments. As far as is known, molecular scattering (which is wavelength dependent) plays a very minor role. The main cause of scattering in plants is intercellular air spaces which account for about one third of their volume (Fig. 5). This type of scattering is caused by refractive index differences. Further scattering is caused by particles of various sizes.

§ M M \0

Cd 1'1 .9 ., ., ·s ., iii ~

100

80

60

40

20

0+-----.-----.----.-----.-----4 o 2 3

Deptb(mm)

4 5

Figure 5. The influence of intercellular air spaces on the transmission of red light through different depths of sunflower hypocoty\s. Infiltration of the air spaces with oil markedly reduces the attenuation of light because the similar refractive index of oil reduces light scattering. (After Parsons et aI, 1984).

Theoretical analyses

Almost all radiation is scattered to some extent in plant tissues. This results in an internal light regime in which radiation travels in all directions. Even if collimated light is used, Kortum (1969) has demonstrated that the radiation becomes predominantly diffuse after between two and eight scattering events. Beer's Law cannot be used in theoretical approacp.es to light trapping because (a) pigments are not homogeneously distributed, and because (b) the pathlength is increased by scattering. For many years, analysis of the propagation of scattering radiation has been based on the Kubelka-Munk (K-M) theory (Kubelka, 1948, 1954). The approach considers radiation travelling in both forward and reverse directions. The theory has been modified and elaborated by Kortum (1969), Fukshansky (1981) and Seyfried and Fukshansky (1983). The analysis is too complex to review here, and the reader is referred to the above citations; alternatively, simplified versions have been described variously by Francis and Clydesdale (1975), Judd and Wyszecki (1975), Osborne and Raven (1986) and Seyfried (1989). Only a brief summary of

252

the Kubelka-Munk Theory is given in the following section.

Kubelka-Munk Theory

The basic ingredients of the Kubelka-Munk (K-M) theory (after Kubelka, 1948) are: (a) the tissue is plane parallel and is infinite, (b) two radiation fluxes are involved, I and J, and they travel in opposite directions, (c) for each flux, photons travel within a solid angle of 21, (d) there is no refractive index difference between the medium and its surroundings, (e) the medium is macrohomogeneous, (f) the incident radiation is diffuse, and infinite.

The K-M theory is often used under invalid conditions. The commonest error is that collimated light is usually used in experiments. This may not be a serious problem because much of the radiation is diffused by the tissue (Kortum, 1969). The other main abuse is that the refractive index of tissue is typically much higher than that of air (ca. 1.4 vs. 1.0). The result is that the tissue acts as a light trap (e.g. Vogelmann & Bjorn, 1984; Seyfried & Schafer, 1985) and there are also effects on the radiation gradients within the tissue (e.g. Seyfried & Fukshansky, 1983). This problem has been partially overcome by accounting for surface reflection (Seyfried, Fukshansky & Schafer, 1983) and by using a more complex approach (Fukshansky-Karazinova et ai, 1986).

It is informative to look at one example of the application of the K-M theory to plant tissues in order to emphasise a few characteristics of the results obtained. Seyfried and Fukshansky (1983) and Seyfried and Schafer (1983) calculated light gradients at three wavelengths for dark-grown (Fig. 6a) and green, light-grown (Fig. 6b) Cucurbita pepo cotyledons. The first point to note is that in the virtual absence of absorption there is an approximately linear decrease in fluence rate with depth (at 730 and 660nm in dark-grown, and at 730nm in green, light-grown tissue). When absorbing pigments are present, there is an exponential decrease in fluence rate with depth (41 Onm in dark-grown and 660 and 410nm in light-grown tissue).

The second point to note from the theoretical predictions from the K-M theory is that, in the absence of significant absorption, the internal fluence rate (I) can be substantially higher than in the incident fluence rate (10). Even with absorption, the irradiated epidermal layer can have an IlIa value greater than 1.0 (Fig. 7). Experimental measurements of phytochrome photo conversion (Seyfried and Schafer, 1985) and of light penetration (Knapp et ai., 1988) have confirmed empirically the fairly good accuracy of the K-M theory in predicting the characteristics of plant tissue internal light regimes.

Radiative transfer theory

Models which include both diffuse and collimated radiation propagating in both directions have existed for a long time. Details are beyond this review, but the interested reader is directed to Schuster (1905), Volz (1962, 1964), Mudgett and Richards (1971) and Latimer and Noh (1987). The important characteristic of these models is that they avoid the criticism of the K-M theory about it applying only to diffuse radiation. To do this, they introduce collimated light in addition to diffuse radiation.

The radiative transfer equation (Chandrasekhar, 1960) describes losses by absorption and scattering of radiation passing through a specific point. The equation considers angular effects of the incident radiation. Generally speaking, the radiative transfer equation requires input parameters which are not all readily measurable. Readers interested in the theory should consult Chandrasekhar (1960), van Gernert and Star (1987), Mudgett and Richards (1971) and Meador and Weaver (1979).

The radiative transfer theory has gained increasing popularity with plant photobiologists because of its applicability to collimated radiation in a highly scattering medium such as plant tissue. However, it does have its limitations. One of the greatest is that the theory requires some knowledge of the scattering phase function (essentially, the angle

253

3

2 •••••••••••

'. •••••••• ••• ~.60nm

'. '. ' . ......

(a) etiolated

". '. '. '. '. '. '. '. '. '"

O+--------r------~r==-----~ 0.0 0.5 1.0

Depth (mm)

1.5

3

2

(b) green

0.5

Depth (mm)

1.0

Figure 6. Light gradients in etiolated (a) and green (b) Cucurbita pepo cotyledons calculated using the Kubelka-Munk theory. Blue light absorbing carotenoids are present in both etiolated and green cotyledons and cause exponential blue light gradients. Red and blue light absorbing chlorophyll is only present in the green cotyledons, so an exponential red light gradient is only found in the green cotyledons. (After Kazarinova-Fnkshansky et ai, 1985).

of scattering) within the tissue. This is difficult to determine. A rigorous application of the theory requires knowledge of the size and distribution of both the absorbing and the scattering bodies within the tissue; none of these characteristics are simple to define. Several of these limitations have been studied by Asimov and Fukshansky (pers. comm.) who have now developed equations for stochastic radiative transfer. Much of their approach is based on work evolved in theoretical fluid dynamics (Monin & Yaglom, 1965) using Keller and Friedmann's 1924 study.

Tissue anatomy and the alteration of light scattering

Plants act as excellent light traps for two reasons. The main reason is that the jump in refractive index at the air/cuticle interface creates a reflecting surface. As a consequence, light is reflected back and forth within the tissue. The second reason is that internal scattering increases the pathlength and therefore increases the duration for which radiation is within the tissue. The result is that there is effectively a build-up of radiation before it is either absorbed or transmitted by the tissue. This phenomenon is seen in the build-up immediately below the epidermis of long wavelengths (>700nm) which are not substantially absorbed, and the lesser fluence rate of shorter wavelengths.

The concept of radiation amplification (or light trapping) is elegantly discussed by Vogelmann and Bjorn (1984). In essence, they describe the fact that a particle will receive I amount of radiation from a point source; if a perfect mirror is placed on the opposite side, it will receive 2 x I amount of radiation; if the particle is placed in an integrating sphere into

254

5~---------------------------,

4

3

o S

2

O~~-----r-----r----~----,--J o 2 3 4

Deptb(mm)

Figure 7. Red (660nm) and far-red (730nm) light gradients in a green Phaseo/us vulgaris hypocotyl. (After Vogelmanll, 1994).

which the light source enters, then it will receive up .to lOx I amount of radiation (Wendlandt & Hecht, 1966). Methods of radiation measurement must be understood to comprehend this subject area.

Multiple scattering also greatly increases the quantity of radiation which is absorbed by tissues because the increases in pathlength increase the probability of absorption. One of the earliest demonstrations of this phenomenon was by Moss and Loomis (1952) who showed that elimination of air spaces in a leaf by infiltration with a liquid of similar refractive index reduced the amount of PAR absorbed by the tissue (Fig. 3). It is also noteworthy that the absorptance of the whole leaf is markedly greater than that of a pigment extract of equivalent concentration.

When the amount of available light is limited, it is obvious that light trapping can be advantageous for photosynthesis. Similarly, amplification may be of advantage in photomorphogenesis, although no substantive evidence is available and photo morphogenetic responses tend to have very high sensitivity (Holmes & Wagner, 1980). Disadvantages may occur in photosynthesis where excessive light can be damaging. In the case of UV-B radiation, trapping is probably of negligible importance for the whole leaf because penetration is limited. Conversely, it would certainly be beneficial for leaf tissue to concentrate UV-B absorbing pigments in the upper epidermis because not only does this cell layer receive the highest levels of incident solar UV -B radiation, but it also receives the highest fluence rate as a result of reflection from the upper epidermal cell distal walls.

255

Fluorescence

Chlorophyll a fluorescence at 688nm may affect PfrlPtot ratios. It is difficult to measure this experimentally, but Lork and Fukshansky (1985) have calculated that the effect is likely to be less than about 15% in a leaf under natural daylight. Although it is unlikely to be of significance in the natural environment, fluorescence of protochlorophyll and chlorophyll can affect the accuracy of laboratory photomorphogenesis studies. The problem mainly arises when chlorophyll containing plants are irradiated with blue light; although the radiation source may be very pure, some of this radiation can be fluoresced in the red waveband and initiate responses through phytochrome rather than a blue absorbing pigment. For example, Holmes and Schafer (1981) noted that fluorescence in the hypocotyls of Sinapis alba in response to monochromatic blue light produced enough red radiation to elicit an independent response through phytochrome to account for about half of the total response which was ostensibly caused by blue light.

Light piping

Light entering the end of a transparent cylinder will be internally reflected by the cylinder walls as long as there is a refractive index difference between the cylinder and its surroundings. As a result, radiation can propagate along the inside of cylinders. The distance it can travel is determined by the attenuation (scattering plus absorption) within the cylinder. The angle at which light is able to enter the cylinder (the acceptance angle) is limited (typically in the approximate range 45 to 60°). This angle is related to the angles at which radiation can travel through the cylinder; oblique angles are internally reflected, but steeper angles (more perpendicular to the longitudinal wall) are transmitted, and therefore escape. Vogelmann (1994) has pointed out that cylinders of plant tissue can be considered to be analogous to man-made optical waveguides because (a) light guiding occurs even when the organ is bent, (b) acceptance angles are similar in both (typically 45-60°), (c) light is transmitted with some degree of coherency.

Mandoli and Briggs (1982a, b) have demonstrated that mung bean hypocotyls and oat coleoptiles can act as light guides and that they can transmit radiation to a distance of up to about 45mm. However, it is not surprising that plant cells are relatively inefficient transmitters oflight (ca. 1-2% efficiency) when compared to man-made optical fibres. Each cylindrical cell contains scatterers and absorbers. The radiation also has to pass through two cell walls as it transfers from one cell to the next; in addition, there is usually a large vacuole within the cell and an air space (with a consequent refractive index jump) between the cells. It has been demonstrated by infiltrating tissue with oil or with water that light passes through vacuoles and cytoplasm and is reflected by the outer walls where there is a major change in refractive index. However, this relatively poor efficiency is probably unimportant for photomorphogenesis because the responses are sensitive to levels of radiation which are many orders of magnitude lower than those found in incident daylight (Holmes & Wagner, 1980).

The internal light regime and photomorphogenesis

The internal radiation regime of tissues can have substantial effects on plant responses. For example, inhibition of hypocotyl elongation in Phaseo/us is caused by red light converting phytochrome in the Pr form into Pfr. In etiolated tissue the action maximum is near 660nm which is close to the absorption maximum of Pfr. In green plants, however, the action maximum for the hypocotyl response is near 620nm instead of 660nm (Jose & Schafer, 1978). This is probably because 620nm radiation is less strongly attenuated by chlorophyll than 660nm and is therefore proportionately more effective.

Direct evidence that chlorophyll causes action maximum shifts comes from the work of

256

Beggs et al (1980). They observed an action maximum for inhibition of green Sinapis alba seedling hypocoty1s at 640nm. When chlorophyll was removed artificially (by treating with Norflurazon, which inhibits carotenoid synthesis and thereby allows chlorophyll photodestruction) they found that the action maximum returned to 677nm, near the absorption maximum ofPr. It is important to point out that the shorter wavelength peak may not be due entirely to chlorophyll screening; Pr also has an electronic excitation band around 610nm (Song et ai, 1979) and this may partially account for two action maxima being recorded in the red waveband (Holmes & Schafer, 1981).

Kazarinova-Fukshansky et al (1985) give a detailed analysis of distortions of action spectra in photomorphogenesis which are caused by light gradients in plant tissue. They calculated that action spectra in etiolated tissue should bear a close resemblance to absorption spectrum of phytochrome in etiolated tissue, but that action spectra in green tissue would show negligible action in the blue waveband and a shift of the red peak towards longer wavelengths. The authors also considered the effects of light gradients. on phytochrome cycling rates. The resultant action spectra showed wavelength shifts which depended on the depth of tissue which was considered. Clearly, the location of the photoreceptor is very important because both the quality and the quantity of radiation varies with position in the tissue.

Photomorphogenesis in aquatic plants

Reductions in the red:far-red (R:FR) ratio cause in vivo reductions in phytochrome photoequilibrium (i.e. PfrlPtot ratio) and these reductions correlate well with a range of photomorphogenetic responses (Holmes & Smith 1975). An important feature of the relationship between R:FR ratio and PfrlPtot ratio is that phytochrome is most sensitive to the range of R:FR values which are found in terrestrial environments; these range between about 1.15 in natural daylight and about 0.05 in dense canopy shade (Holmes & Smith, 1977). The question therefore arises of whether or not phytochrome can function in aquatic plants because the range of R:FR ratios found in aquatic environments tends to be upwards of a value of about 1.2 and is therefore outside the R:FR range to which phytochrome is sensitive (Smith & Holmes 1977).

The UV and PAR wavebands are poorly attenuated by pure water and wavelengths above about 700nm are more strongly absorbed (Curcio & Petty, 1951). Dissolved inorganic material accentuates the absorption oflonger wavelengths (Kasha, 1948) and further reduces the R:FR ratio. Phytochrome modulated responses do occur in aquatic environments, so it is necessary to explain how they can occur if the radiation environment is outside the R:FR range ratio to which phytochrome can respond. The answer appears to lie in the fact that a chlorophyll-containing tissue modifies its own radiation regime in such a way that phytochrome can be photoconverted using the prevailing aquatic radiation environment.

Holmes (1988) showed that aquatic plants probably act as 'mini-canopies'. Using a combination of data he showed that phytochrome uses the modified radiation within a tissue to establish the nature of the surrounding radiation environment (Fig. 8). The exercise confirms that major changes in phytochrome photoequilibrium in etiolated tissue occur within the relatively narrow R:FR range of 0.05 to 1.15 in etiolated tissue; within green leaves, the range of R:FR ratios which produce equivalent changes in phytochrome photoequilibrium is extended up to approximately 5.0. Using very thick leaves, it is can be seen that a R:FR ratio of 15 or greater will produce the same pfrlPtot value as that produced by a ratio of 1.5 in etiolated tissue. This approach begs many questions: the main problems are analysis of the angle of incidence of the radiation and the fact that refractive index changes are different. Nevertheless, the important issue is that aquatic plants appear to modifY phytochrome photoequilibrium by selective absorption in a manner that enables phytochrome to function.

257

Etiolated Phaseolus

0.75

] .8 0.5

~ Il.

0.25

O~r------.------.------.------~ o 5 10 15 20

R:FR ratio

Figure 8. Calculated phytochrome photoequilibria within etiolated and green Phaseo/us vulgaris and Crassula leaves. (After Holmes, 1988).

The sieve effect

Beer's Law states that absorptance is directly proportional to pigment concentration. However, Beer's Law applies only when pigments are in relatively low concentrations and are uniformly distributed, such as in solutions under laboratory conditions. In plant tissues, pigments are often in high concentration and their distribution is rarely uniform because they are concentrated in organelles such as chloroplasts. The error involved in misusing Beer' s Law can be envisaged using the simple schematic description Fig. 9 which was adapted from Vogelmann (1994). In this hypothetical situation a cuvette contains a pigment which transmits 50% of the incident light (Fig. 9a). When the same amount of pigment occupies only half of the cuvette, one half transmits 100% and the other half transmits 25% of the incident light, giving a mean transmittance value of62.5% instead of the expected 50% (Fig. 9b). If the same amount of pigment is restricted to an even smaller volume of the cuvette, the mean transmittance value increases even further above the expected value (Fig. 9c). This is a simplified summary of the sieve effect and it describes the non-uniform distribution of absorbers. As can be seen in Fig. 9, its overall effect is to lead to an underestimation of pigment content.

The term "sieve effect" was originally proposed by Rabinowitch (1956) on the basis that radiation appeared to pass through gaps between particles in a suspension. Duysens (1956) demonstrated the role of self-shading by particles in reducing absorption efficiency and the tendency of the sieve effect to flatten the absorption spectra. Several workers have made subsequent contributions of our knowledge of the sieve effect - also called the "package effect" (Kirk, 1983) - and the reader interested in greater detail is referred to

258

111111 !!llll l!!!!l al· .. ·~·· '. ' .. ":1 b l r~::.~':<1 ci 1'(/1

~ ~ ~ ~ ~ 100% 25% 100% 12.5%

SO%T =62.5% T =78.2% T

Figure 9. Schematic description of the sieve effect. For details, see text. (After Vogelmann, 1994)

Fukshansky (1987). The consequence of the sieve effect is the same in both cell suspensions and in entire

tissues: the accumulation of pigments into organelles results in lower absorptance and this is largely caused by self-shading. Experimental study of the sieve effect is not always straightforward because it is often dynamic. Chloroplast orientation and positioning can change with time as a result of directly induced light avoidance or as a function of an endogenous circadian rhythm (Haupt, 1973; Britz, 1979; Haupt, 1982). One of the most striking examples of the sieve effect has been demonstrated in Ulva. In this green alga, the transmission of blue and red wavelengths through the thallus varies with a circadian rhythm which parallels the circadian rhythm in the migration of the chloroplasts between the face and profile, or side walls of the cells (Britz et ai, 1976). Transmittance of blue and red radiation was greatest (i.e. absorptance was lowest) when the chloroplasts were near the side walls, and least when they were near the face walls (Britz & Briggs, 1987).

Although chloroplast migration or orientation, abs6rptance, and photosynthesis are related, our understanding of the relationship is not complete. For example, the change in photosynthesis in leaves as a result of chloroplast movement is much greater than the change in absorptance (Zurzycki, 1961; Lechowski, 1974). Also, on the basis of data from seaweeds, it is known that the arrangements of photosynthetic pigments and their activity do not always run in parallel (Nultsch et ai, 1981). One factor which may account for apparent discrepancies is light scattering. As we have seen earlier, scattering increases the optical pathlength and leads to increased probability of absorption. Work on leaves has shown that scattering can cancel out the sieve effect (Inoue & Shibata, 1974).

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427-471, Plenum, New York. Bone, RA, Lee, D.W. & Norman, J.M. (1985) Appl. Optics 24: 1408-1412.

259

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PHOTOSENSORY TRANSDUCTION IN FLAGELLATED ALGAE

R. Marangoni, E. Lorenzini and G. Colombetti

Istituto di Biofisica CNR 156127 Pisa, Italy

FOREWORD

Solar energy practically provides all the heat and light that our Planet receives and is therefore at the basis of every vital process. It is generally accepted that life, as we know it, has been shaped by the physical properties of our sun, a G spectral class star, and that all living organisms have evolved under the influence of the solar radiation filtered by the Earth's atmosphere.

It is a problem for exobiologists and perhaps science-fiction writers to imagine what kind of life would evolve under a different sun, but some general rules could probably apply for life to exist in a form not too different from that we know: y- and X-rays, high-energy particles, UV-C and probably UV-B should be cut-off by the planet's atmosphere (and magnetic field), the sun emission should not vary too much or too rapidly in time, the average temperature of the planet should not exceed certain limits. With regard to this, it may be interesting to recall that the Gaia hypothesis proposed by Lovelock (I979) suggests that life, once evolved, has been able to interact with the planet atmosphere, modifying it and making the successive development of life possible.

At any rate, our planet is continuously bathed in a flux of photons of wavelengths ranging from the near-UV to the infrared, and life has naturally evolved under this continuous presence. Photons carry energy and also information. Photon energy is directly utilised in metabolic processes such as photosynthesis or ATP-synthesis in Halobacteria. Energy is easy to measure and for a monochromatic radiation is given by the well known formula E = h ciA, where h = 6.63 10-34 Js-4, c = 2.99 108 ms-1 and A is the wavelength in meters.

The information carried by photons is more difficult to quantify; living organisms are able to extract information on the external environment usually by measuring and comparing the number of photons received in different instants of time (more now than before) or in different spatial locations (more here than there) and sometimes use this information to tell the direction of the incoming light; there are also organisms that can discriminate the polarisation state of a beam of light and use this information to discriminate directions (dichroic receptors) and organisms that can distinguish between the different wavelengths of

• Dedicated to our friend Professor Pill-Soon Song on the occasion of his sixtieth birthday.

Light as an Energy Source and Information Carrier in Plant Physiology Edited by Jemlings et 01., Plemun Press, New York, 1996 263

light (colors) (Colombetti & Petracchi, 1989), possessing more than one type of visual pigments. The amount of data obtainable is, of course, much higher when the organism evolves the capability of perceiving images of the world outside; in this case it becomes possible to classify the objects around, to recognise them (this also requires a certain degree of memory) and to use this information, for example, to catch food or to avoid predators. There are also other processes where light is utilised as a regulatory signal in biological processes such as gene expression, cell reproduction, enzyme activation, DNA repair a.s.o. (Kreimer, 1994 and references therein).

In order to be able to exploit luminous information, the organisms had to select molecules capable of absorbing photons of the appropriate wavelength thus generating signals to be used as input for subsequent metabolic processes. Light perception is just one of the more general class of biological processes known as cell responses to external signals or stimuli; all these processes share general features indicating that nature has followed a quite limited number of "sensory" pathways; cells have usually slightly modified one chain of metabolic events to adapt it to some other purpose. It has recently been· suggested (Gualtieri, 1993) that "visual" pigment may also derive from a common ancestor and belong to the same class of molecules, rhodopsins, found ubiquitously, from man to archaebacteria. Of course, this does not exclude that other systems, as seems to be the case for some coloured ciliates, may have evolved and utilised another (or more than one other) class of pigments. The main steps of the photo sensory transduction chain in some of these systems, such as Blepharisma japoniclIm and Stentor coentiells, seem, however, to be similar to those of systems using rhodopsin pigments (Fabczak et al., 1993b; Fabczak et al., 1993d).

INTRODUCTION

Freely swimming organisms can react to light stimuli by altering their swimming behaviour. There are several different types of so called "photomotile behaviour" or simply "photobehaviour". Here we will only briefly recall the main types of photo movements that will be mentioned in the following (the reader is referred to the literature for a detailed description of the appropriate terminology, Diehn et aI., 1977):

i) photokinesis - cell speed depends on light intensity and wavelength; it is defined positive if the cell speed is higher in the light than in the dark, negative ifit is lower.

ii) step-up and step-down photophobic responses - when exposed to a sudden increase or decrease of illumination the cells stop their forward swimming , then swim backwards or tumble on the spot for a time interval that depends on the intensity of the stimulus (Hegemann & Bruck, 1989).

iii) phototaxis - this is a movement oriented according to the direction of the incoming light; it is defined as positive if the cells move towards the light source, and as negative if they move away from it. Phototaxis is certainly the most interesting and intriguing among the different types of photomotile responses, one of the reasons probably being the structural complexity required from apparently simple organisms to detect and track a signal such as light direction. There is, nowadays, general consensus that phototactic systems are usually able to follow light direction even in the absence of spatial gradients of light (Colombetti et al., 1992). The relationship between structure and function in phototaxis has been dealt with in some detail by Foster and Smyth (1980), and recently by Kreimer (1994). The main conclusion is that cells are able to track light direction by modulating the light signal falling on their photoreceptors. This modulation can be achieved by combining shielding/screening devices or reflecting mirrors with a photoreceptor unit, in what is known as a directional antenna. The absolute modulation depth (the difference between maximum and minimum signal, or contrast) is important in this model and we will examine later a case in which optimum contrast is achieved when the directional antenna includes a reflecting mirror.

A common problem for all the types of photomotile reactions mentioned above is how

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light perception is linked to the mechanisms controlling cell motility, which in freely swimming organisms, is usually brought about by cilia and flagella (Colombetti & Marangoni, 1991). At present, not much is really known about how light signals, processed by the photo sensory transduction chain, are able to affect flagellar or ciliary beating. There is evidence (Harz et aI., 1992; Sineshchekov, 1991a; Fabczak et al., 1993a; Fabczak et al., 1993c) that in some flagellates and ciliates light can alter membrane conductances, opening or closing ion channels, ultimately leading to a Ca-ions (Ca++) influx in the ciliary or flagellar region, with the final result of an alteration in the beating pattern of the motor organelle.

In this paper we will focus our attention on the primary processes following light absorption in two flagellated algae Chlamydomonas reinhardtii and Haematococcus pluvialis.

PHOTORECEPTOR PIGMENTS

Several studies carried out in the last decade (Sineshchekov, 1991a,b; Zacks et al., 1993; Kroger & Hegemann, 1994; Sineshchekov et aI., 1994) give strong evidence in favour of all-trans retinal rhodopsin as the photoreceptor pigment both in C. reinhardtii and H. pluvialis. While the results obtained for H. pluvialis are mainly based on action spectrum determinations, those for C. reinhardtii are mainly based on studies of carotenoid less mutants. A more detailed discussion of some of the experimental results leading to this conclusion can be found in the chapters by Lenci et al. and by Ghetti and Checcucci in this volume.

The largest body of information on the events following light absorption in C. reinhardtii and also in H. pluvialis derives from measurements of membrane potentials or currents in single cells. Some previous attempts had been made using conventional intracellular microelectrode penetration (Nichols & Rikmenspoel, 1978), but the cell damage was so high that it was not possible to obtain really meaningful information on the status of the cell membrane potential and of its variation upon illumjnation. Significant progress in the understanding of the electric processes linked to the photomotile reactions was obtained by measuring membrane potential differences or currents using the suction pipette method (Ristori et al., 1981; Sineshchekov, 1991a,b; Harz & Hegemann, 1991, Harz et al., 1992). The suction pipette system is similar to the whole-cell patch clamp (Hille, 1992), the major difference being that the input resistance is of the order of 200-250 MQ for C. reinhardtii and apparently of the order of some tens of MO for H. pluvialis. This resistance is far from those necessary for a good patch clamp, which are of the order of some tens of GO. It is, therefore, not possible to use this configuration to measure single channel currents and properties. Some problems derive from the presence of the cell wall in C. reinhardtii, which makes it very difficult to perform this kind of measurements in the wild type that, in fact, are carried out on the wall-less mutant CW2. H. pluvialis is easier to deal with, thanks to its greater dimensions and elastic cell wall (Harz et aI., 1992; Sineshchekov, 1991a,b).

The suction pipette technique, combined with the use of microbeams of light, showed that the photoinduced signals are generated only when the stigma region is illuminated, thus indicating that the photoreceptor of these algae is located in that region (Ristori et al., 1981; Sineshchekov, 1991a; Harz et al., 1992). It has also allowed the researchers to measure i) the signals generated in a cell as a function of the relative position of the light source and of the photoreceptor and ii) the action spectrum for signal generation. A short summary of these experiments is presented below:

i) the cells, held by the suction pipette, can be oriented according to the stimulus direction and the relative position of the photoreceptor region and of the light beam, which can be varied at will. It is thus possible to measure light-induced electric signals in different configurations, determining, for instance, the back-to-front contrast (defined as the

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photoinduced signal obtained with the receptor facing the light source, minus the one with the photoreceptor pointing away from it); it is possible, in this way, to have a quantitative estimate of how efficient a light directional antenna may be (Foster & Smyth, 1980). The data from H. pluvialis show that the contrast measured is about three; the same kind of measurement on C. reinhardtii (Harz et aI., 1992) gives a contrast factor of about eight; this is much more than expected on the basis of a simple screening mechanism (the cell overall absorbance can only explain a factor of 2) and thus gives support to the idea that the stigma acts as a quarter-wave stack reflector (Harz et al., 1992). The difference in the contrast factors between C. reinhardtii and H. pilivialis may be explained by taking into account the different structures of their eyespots, a multilayered structure acting as an efficient quarter wave stack in C. reinhardtii and a single layered stigma acting as a less efficient quarter wave reflector in H. pluvialis (Sineshchekov, 1991a).

ii) The efficiency of a quarter-wave reflector depends also on the angle of incidence of the actinic light, besides the obvious dependence on wavelength. Therefore, Harz et al. (1992) have determined an action spectrum for single cells by keeping them with the stigma facing the light source (Harz et aI., 1992). The resulting action spectrum is a rhodopsin-like spectrum with no fine structure. This is different from the results of Sineshchekov (1991a), who found that the action spectrum has an overall resemblance to a rhodopsin-like spectrum, but shows a detailed fine structure, that the author partly ascribes to a high spatial rigidity of the chromophore in the protein moiety, and partly to the possible presence of more than one receptor pigment.

SENSORY TRANSDUCTION

From the evidence reported above, it is at present accepted that the photoreceptor pigment driving the cell motor response to light in both C. reinhardtii and H. pilivialis is a rhodopsin-like molecule. The question remains open as to how the signal absorbed by the photoreceptor is transduced into an alteration of flagellar motility. In higher organisms the light signal starts a complex chain of enzymatic reactions, eventually leading to the opening or closing of ion channels. In these cases, the depolarisation or hyperpolarisation of the photoreceptor membrane is the input for the subsequent signal elaboration and transmission to the central nervous system. In unicells, on the other hand, the electric signal in itself may be able to regulate and control the beating of the motor organelle (Machemer, 1986). We will discuss below in some detail the characteristics of the electric signals that are generated in these systems under the action of blue-green light. Not much is known on the nature of the biophysical and biochemical events leading from the excited rhodopsin to the alteration of ion channel conductance. In higher organisms (and perhaps also in a colored ciliate, Fabczak et aI., 1993b) G-proteins play a major role in signal transduction (Rayer et aI., 1990). There is also evidence that G-proteins are present in C. reinhardtii, where eyespot fractions have been shown to contain at least three types of G-proteins, homologous to vertebrate G-proteins (f..- and /3-subunits (Korolkov et aI., 1990). There is, however, to the best of our knowledge, no further evidence indicating a possible role for G-protein­controlled biochemical events in microorganisms similar to those observed in higher organisms. With regard to this, it may be interesting to recall that the transducer in the photoserisory transduction chain of the photomotile responses of Halobacterium halobium is probably not a G-protein (Spudich, 1993).

There is at present consensus that Ca-ions (Ca++) playa major role in photoresponses of C. reinhardtii and H. pluvialis. The experimental evidence derives mainly from measurements of the photo motile reactions or of the photoinduced electric currents in the presence of different levels of external Ca ++ (Hegemann et aI., 1990 and references therein) and of different Ca++channel inhibitors (Nultsch et aI., 1986, Hegemann et ai., 1990). In brief, both phototaxis and photophobic response are gradually abolished in the presence of

266

Ca++ channel inhibitors such as ro-conotoxin GVIA (ro-CgTx) and pimozide (Hegemann et aI., 1990), which inhibit selectively two different types (N or L) of Ca++ channels. There seems to be, moreover, another type of Ca++ channel, which is involved in phototaxis, but not in the photophobic response, and is blocked by Verapamil (Hegemann et aI., 1990). The electric signal recorded from single cells depends on the external Ca ++ concentration, even if it is not possible to inhibit it completely in either system (Sineshchekov, 1991 b; Harz & Hegemann, 1991). Though the involvement of Ca++ in the sensory transduction chain of both cell species is clear, there is no precise idea of how the system works, except for the general concept that calcium can regulate the beating of cilia and flagella (Kreimer, 1994). There is experimental evidence in the literature that this is indeed the case for isolated flagella and in detergent treated cells (Hyams & Borisy, 1978; Kamiya & Witman, 1984). More recently, Korngreen & Priel (1994) have been able to measure, simultaneously, ciliary beating frequency and intracellular free calcium in rabbit tracheal cells, showing that these two quantities do not fully correlate.

As briefly mentioned above, C. reinhardtii and H. pluvialis show photoinduced membrane potentials or currents, and it is possible to measure them by using the suction pipette technique. There are three main types of electric signals in H. pluvialis (Fig. 1): one is known as Primary Potential Difference (PPD) and is usually accompanied by a late potential difference (LPD); if the PPD is over a certain threshold it is followed by a regenerative response (RR) (Sineshchekov, 1991a). The situation is quite similar in C. reinhardtii except for the absence of the LPD (Harz et aI., 1992). A Photoreceptor Current (PRC) is accompanied, when over this threshold, by a fast flagellar current (FFC) usually followed by a slow flagellar current (SFC) (Fig. 2 and Fig. 3). The FFC shape and amplitude are almost independent of the light fluence rate, whereas its delay time with respect to light stimulation decreases with increasing light intensity, as show in Fig. 3 (Harz & Hegemann, 1991).

In H. piuvialis, the PPD is biphasic and one of the components has a very fast rising time (less than 30 I-1-s) that does not depend on external conditions such as light intensity and temperature. This fast component has been attributed to some not better defined charge movements in the photoreceptor molecules, similar in a way to the early receptor potential in vision (Cone, 1972). The physiological role of this signal, if any, is not clear at the moment. The second component of the photoreceptor current has a delay time that depends on light intensity and decreases from 400l-1-s to 120~IS with increasing light intensity as shown in Fig. 4 (Sineshchekov et aI., 1990). The fast signal saturates only at high photon exposures (Irradiance x time) of the order of 1020 photons/m2 (Fig. 5 and Fig. 6) (Harz et aI., 1992; Sineshchekov 1991a). The difference between the two curves derives probably from the fact that in H. pluvialis the curve is restricted to a higher range of photon exposures, and its initial (sigmoidal?) part is not shown. Also, the signal recorded from C. reinhardtii corresponds probably to the slow component of the PPD in H. pluvialis, due to the much lower time resolution of the experimental apparatus used by Harz et al. (1992), which is of the order of 500 I-1-S.

The photophobic step-up reactions of H. pluvialis cells, observed directly under the microscope, show a strict correlation with the flagellar RR (Sineshchekov, 1991a), while in C. reinhardtii the slow flagellar current has a time kinetics closely resembling that of the period of backward swimming during a step-up photophobic response. It is possible that the fast flagellar current corresponds to the stop reaction which initiates the photophobic step­up response.

The sign of the light-induced electric signals depends on how a cell is sucked into the micropipette; in particular, the signal varies with the relative position of the stigma region and of the flagella, with respect to the inner side of the micropipette. Several different configurations are possible, stigma and flagella inside the pipette, stigma inside and flagella outside, stigma outside and flagella inside, stigma and flagella outside. There is also a report in the literature (Beck & Uhl, 1994) of a case in which the stigma and one flagellum are

267

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Figure 1. Light-induced electrical responses in If. pluvialis. PPD = primary potential difference, LPD = late potential difference, RR = regenerative response (redrawn from Sineshchekov, 1991a).

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50ms

1 2PA

Figure 2. C. reinhardtii (strain CW2): correlation between position of flagella and eyespot in the suction pipette and photoinduced current: 1) eyespot in and flagella out; 2) flagella in and eyespot out; 3) flagella and eyespot out; FFC = Fast Flagellar Currents; SFC = Slow Flagellar Currents. A) Flash stimulation (redrawn from Harz et aI., 1992).

268

1 10ms

2 PpA 3

4

5

A

Figure 3. C. reinhardtii: electrical responses as a function oflight intensity: 1= 5%; 2 = 10%; 3 = 25%; 4 = 50%; 5 = 100% that corresponds to 2x1019 photons/m2 The recordings were performed when flagella and eyespot were out of the suction pipette and the eyespot was towards the actinic light. A) Flash stimulation (redrawn from Harz and Hegemann, 1991).

0.2 ms

~l

Figure 4. The delay of the second component of the photoreceptor current decreases with increasing intensity of light flash (0.1 11m2 for upper curve and 18% of this value for lower curve). The light flash (arrow) was in the plane of the H. pluvialis cell (redrawn from Sineshchekov et aI., 1990).

outside and the second flagellum inside the pipette. Some typical signals are reported in Fig. 2 and Fig. 7.

As we have previously mentioned, all the above described photoinduced electrical signals are mediated by calcium ions. In particular, as shown in Fig. 8 and Fig. 9, the fast flagellar currents (or the RR signal) are abolished almost completely in the presence of calcium chelators or calcium channel blockers, whereas the PRe (or the PPD) are present even in the absence of calcium ions (Harz et aI., 1992; Sineshchekov, 1991b). Recently Beck

269

~

::I ~ E Q) 0.5 ... ... ::J U

.::.: ('II Q) a.

o ...! .. d ••• • ..... • ••• lIuI. •• ...... I

10'• 10'9 1020 1021

photon fluence

Figure 5. C. reinhardtii (strain CW2): light intensity saturation of photoreceptor peak current normalised to the maximum experimental value. Photon fluence is in photonim2 (redrawn from Harz et aI., 1992).

2.5

2.0 ....... > E

"'"' 1.5 >

~ - 1.0 c III -0 a..

0.5

0.0 0 2 3 4

Photon fluence. E (photons/m2 )( 10-20)

Figure 6. H. pluvialis: computer fit of stimulus-response data for primary potential current (redrawn from Sineshchekov 199Ia).

and Uhl (1994) have shown that there are voltage-sensitive calcium channels in the flagellar membrane of C. reinhardtii. An important point to consider is that the photocurrents discussed above are those due to high intensity stimulation and are, in fact, more directly connected to the photophobic step­up response than to phototaxis. At lower fluences, where phototaxis takes place, the PRe and the flagellar currents are too small to be presently measurable.

In H. pluvialis measurements of the flagellar beating frequency show that the two flagella have different sensitivity to light stimulation (Sineshchekov, 1991 b). By simulating the condition of a periodic illumination of the photoreceptor region, it was shown that the

270

A 2

c

2

Figure 7. H. pluvialis: correlation between position of flagella and stigma in the suction pipette and photoinduced courrent: A) flagella and stigma out; B) flagella in and stigma out; C) flagella out and stigma in; D) flagella and stigma in. 1 = primary potential difference; 2 = regenerative response. The arrow indicates the flash stimulus (redrawn from Sineshchekov, 1991 a).

a

c

A

b

d

B

10ms

1 2PA

Figure 8. Photoresponse of two different (A and B) cells ofe. reinhardtii (strain CW2) in the presence of different extracellular calcium concentrations: a) 1.0 f.lM Ca++ b) 0.1 f.lM Ca++ c) 0.1 f.lM Ca++ d) 0.01 f.lM Ca++ (redrawn from Harz et a!., 1992).

cis-flagellum (the one closer to the stigma) beats more frequently and the trans-flagellum less frequently wh,en light falls upon the stigma region, whilst the trans-flagellum increases its beating frequency when the stigma and the cell body shade the presumed photoreceptor, as shown in Fig. 10 (Sineshchekov, 1991a); in a swimming cell this would result in an orientation of the cells away from the light source. The general principle of an asymmetry of the two flagella seems to hold also in C. reinhardtU, where, on the other hand, the two flagella seem to show different amplitudes of beating upon light stimulation (NuItsch & RUffer, 1994), rather than different beating frequencies.

271

60

a

40

20

o

o 10 20 30 40

Time, ms

Figure 9. Effect of external calcium concentration in removing PPD and RR in H. p!uvialis: a) 500 f.tM Ca++b) with 2 mM EGTA (redrawn from Sineshchekov, 199Ib).

1 off

1 on

1 off

300 ms

1 on

1 off

30

20

>-U I: G.I tT G.I ... ... III .5 ... cu G.I ,g

... ~ G.I III cu ... N %

> E or "C :::l ::: ii E C(

Figure 10. Opposit photoresponses of the cis- and trans-flagella of a H. pluvialis cell. Light on and off are indicated by arrows (redrawn from Sineshchekov, 1991a).

For both types of cells the integral of the PPD (or of the PRC) necessary to induce the RR response (flagellar current) is almost constant at different light intensities. This means that a well defined amount of charge must enter the cell to activate the voltage-dependent calcium channels, corresponding to a membrane depolarisation of several tens of m V (Sineshchekov, 1991b, Harz et al., 1992). In particular in C. reinhardtii, the membrane depolarisation can be estimated by assuming that the total photoreceptor current entering a cell is of the order of 40 pA and that it lasts for about 4 ms, corresponding to a total charge

272

of about 106 elementary charges. Assuming a spherical cell with a specific capacitance of 1 /..lF/cm2 (Hille, 1992) and a radius of8 /..lm, its total capacitance amounts to 2 pF (4 1t r2 ~ 200 /..lm2) and we obtain

I1V = Q/C = 160 x 10-15 12 x 10-12 = 80 x 10-3 V (1)

This should be more than enough to activate voltage-dependent calcium channels; in fact, a membrane depolarisation of the order of some m V is enough to activate this type of channel in ciliates (Machemer, 1986). We will now estimate how many channels should be activated in order to account for the measured photo current of 40 pA. Let G be the unitary calcium channels conductance (1-25 pS), let V = 100 mV be the steady membrane potential and let us assume ohmic conductance: from Ohm's Law (S is the total conductance, expressed in mhos or siemens)

V = if S = if nG (2)

where n is the total number of channels (provided that they are all open at the same time, which is very unlikely). Inserting in (2) the value of I1V from (1) and the photo current measured, we have

0.1 x n x G = 40 x 10-12 A (3)

that is

n x G = 40 x 10-12 I 10-1 = 400 x 10-12 S (4)

From (4) we obtain for G = 1 n = 400 and for G=25 n = 16 This means that the measured photocurrent can be generated by the opening of a

number of channels ranging from 16 to 400, depending on the unitary Ca++ channel conductance.

It can be estimated that under illumination conditions giving rise to half maximum photoreceptor current, about 12% of the rhodopsin molecules would be activated, that is about 1200 molecules; with a 1: 1 ratio rhodopsin to activated channels, a unitary conductance of only 0.3 pS would suffice to account for the measured photoreceptor current.

Even if it is taken into account that these calculations are only estimates of orders of magnitude, they indicate that, in principle, there is no need for signal amplification in the sensory transduction chain of the wall-less mutant CW2 of C. reinhardtii. It has been suggested (Harz et aI., 1992) that this might indicate that rhodopsin and the photoreceptor channel form one protein complex.

This is in apparent contradiction to the conclusions of Sineshchekov (1991a), who indicates that in H. pluvialis the system needs a large amplification: in H. pluvialis the photoreceptor current is of the order of 107 unit charges/s, the number of excited rhodopsin is of the order of 103, therefore one can conclude that one rhodopsin must move about 104 charges, and this is taken as the necessary amplification factor. On the other hand, if one takes into account that in ohmic membranes the amount of current flowing depends on both the number of channels open and on their conductance, the data reported by Sineshchekov (1991a) are in reasonable agreement (within an order of magnitude) with those of Harz et aI. (1992). Moreover, one should define, in advance, what amplification means in these systems. In vision, one usually defines as amplification factor (about 106) the ratio of the number of cGl\1P molecules activated to the number of excited rhodopsins. In vision, the stoichiometry between the number of cGl\1P molecules and that of closed channels is about 1. If we assume that also in microorganisms amplification means the ratio between number of

273

channels open (or closed) and number of rhodopsins excited, then there is no need for amplification in H. pluvialis as well.

REFERENCES

Beck, C. & Uhl, R (1994) 1. of Cell BioI. 125, 1119-1125. Colombetti, G., Braucker, R & Macheme,r H. (1992) J. Photochem. Photobiol. B: BioI. 15, 253-257. Colombetti, G. & Marangoni, R (1991) in "Biophysics of photoreceptors and photomovements in

microorganisms", (Lenci F., Ghetti F., Colombetti G., Hader D.P. & Song P.S. Eds.) Plenum Press, New York, 53-71.

Colombetti, G. & Petracchi, D. (1989) Crit. Rev. Plant Sci. 8, 309-355. Cone, RA. (1972) Nature New BioI. 236, 39-43. Diehn, B., Feinleib, M.E., Haupt, W., Hildebrand, E., Lenci, F. & Nultsch W. (1977) Photochem. Photobiol.

26, 559-560. Fabczak, H., Park, P.B., Fabczak, S., Tao, N. & Song P.S. (1993a) Photochem. Photobiol. 57,696-701. Fabczak, H., Park, P.B., Fabczak, S. & Song P.S. (1993b) Photochem. Photobiol. 57, 702-706. Fabczak, H., Tao,"N., Fabczak, S. & Song P.S. (1993c) Photochem. Photobiol. 57,872-876. Fabczak H., Tao N., Fabczak S., Song P.S. (1993d) Photochem. Photobiol. 57, 889-892. Foster, K.W. & Smyth, RD. (1980) Microbiol. Rev. 44, 572-630. Gualtieri, P. (1993) J. Photochem. Photobiol. B (BioI.) 19,3-14. Harz, H. & Hegemann, P. (1991) Nature 351,489-491. Harz, H., Nonnengasser, C.& Hegemann, P. (1992) Phil. Trans. R Soc. Lond. B 338, 39-52. Hegemann, P. & Bruck, B. (1989) Cell Mot. and the Cytosk. 14,501-515. Hegemann, P., Neumeier, K., Hegemann, U. & Kuehnle E. (1990) Photochem. Photobiol. 52,575-578. Hille, B. (1992) Ionic channels of excitable membranes. (Second Edition) Sinauer Associates Inc.,

Publishers Sunderland, Massachusetts. Hyams, 1.S. & Borisy G.G. (1978) 1. Cell Sci. 33, 235-253. Kamiya, R & Witman G.B. (1984) 1. Cell BioI. 98, 97-107. Korngreen, A & Priel Z. (1994) Biophys. J. 67,377-380. Korolkov, S.N., Garnovskaya, M.N., Basov, AS., Chunaev, AS. & Dumler, LL. (1990) FEBS Letters 270,

132-134. Kreimer, G. (1994) lnt. Rev. Cytol. 148,229-311. Kroger, P. & Hegemann, P. (1994) FEBS Letters 341, 5-9. Lovelock, J.E. (1979) "Gaia". Oxford University Press, Oxford and New York. Machemer, H. (1986) in Membrane Control of Cellular Activity. (Liittgau H.C. ed.), (Fortschr. Zool./Prog.

Zool., 33), 205-250. Nichols, K.M. & Rikmenspoel, R (1978) J. Cell Sci. 29, 233-247. Nultsch, W. & Riiffer, U. (1994) Naturwissenschaften. 81, 164-174. Nultsch , W., Pfau, J. & Dolle, R (1986) Arch. Microbiol. 144, 393-397. Rayer, B., Naynert, M. & Stieve, H. (1990) J. Photochem. Photobiol., B: BioI. 7, 107-148. Ristori, T., AscoJi, C., Banchetti, R, Parrini ,Po & Petracchi D. (1981) Proc. 6th. Int. Congr. Protozool.,

Warsaw, 314. Sineshchekov, O.A. (199Ia) in "Light in Biology and Medicine" (Douglas RH., Moan 1., Ront6 G. Eds.)

Plenum Press, New York, 2,523-532. Sineshchekov, O.A (1991b) in "Biophysics of photoreceptors and photomovements in microorganisms",

(Lenci F., Ghetti F., Colombetti G., Hader D.P. & Song P.S. Eds.) Plenum Press, New York, 191-202. Sineshchekov, O.A., Litvin F.F., Keszthelyi L. (1990) Biophys. J. 57, 33-39. Sineshchekov O.A., Govuronova, E.G., Der, A., Keszthelyi, L. & Nultsch W. (1994) Biophys. J. 66, 2073-

2084. Spudich, J.L. (1993). J. Bacteriol. 175,7755-7761. Zacks, D.N., Derguini, F., Nakanishi, K. & Spudich L. (1993) Biophys. J. 65, 508-518.

274

ACTION SPECTROSCOPY'

Francesco Ghetti and Giovanni Checcucci

CNR Istituto di Biofisica, via San Lorenzo 26, 56127 Pisa, Italy

INTRODUCTION

Action spectroscopy is a non-destructive technique for studying in vivo the absorption properties and, in some cases, the primary reactions of photoreceptors involved in triggering biological photoresponses. Action spectroscopy can be used in the investigation of any Iight­dependent phenomenon for which a standard response can be defined. It consists in the quantitative analysis of the response of the system as a function of the wavelength of the stimulating light and the outcome of this analysis, the action spectrum, is a measure of the relative efficiency of light of different wavelengths in inducing a defined effect on the examined biological system.

The determination of action spectra has provided much valuable information and several reviews dealing with action spectroscopy have been published in the past (Shropshire, 1972; Colombetti & Lenci, 1980; Hartmann, 1983; Schafer et at., 1983; Galland, 1987; Lipson, 1991; Coohill, 1991; Coohill, 1992).

The aim of action spectroscopy is the identification of the pigment responsible for the observed photoresponse. In fact, provided some necessary conditions are met (see below) the structure of the action spectrum will be similar to that of the pigment absorption spectrum.

As stressed by some authors (Colombetti & Lenci, 1980; Gualtieri, 1993), however, action spectroscopy does not allow in general a precise identification of the pigment responsible for the photoresponse. This can be due to various reasons: for example, most of the published action spectra lack good wavelength resolution, many pigments have similar absorption spectra in the visible range and, moreover, the absorption properties of a pigment in vivo can be strongly influenced by its molecular environment, making the comparison between action and absorption spectra difficult.

Nevertheless, when the isolation and the biochemical characterisation of the photoreceptor are not possible, several techniques are available for the identification of the photoreceptor pigment (e.g.: micro spectrophotometry, microspectrofluorometry or use of specific quenchers of the excited states of the photoreceptor), but only action spectroscopy

• Dedicated to our friend Professor Pill-Soon Song on the occasion of his sixtieth birthday.

Light as an Energy Source and Infonnation Canier in Plant PhYSiology Edited by Jelmings et a/., Plenmn Press, New York, 1996 275

allows us to establish a direct link between the functional absorption of photons by the presumed photoreceptor and the observed response.

ACTION SPECTROSCOPY

A prerequisite for the determination of an action spectrum is the definition of a physical or numerical unit for quantifying the photoresponse. In the literature it can vary from the length or the weight of a plant to the concentration of a gas or of an enzyme, from the angle of bending of a sporangiophore to the number of photomotile microorganisms accumulating in a light trap, etc ..

An action spectrum is the plot of the effectiveness of the light in inducing the observed photoresponse as a function of wavelength. The stimulating light should be, therefore, as much monochromatic as possible. Selecting the different wavelengths by means of interference filters, band-widths between 5 and 10 nm can normally be obtained. On the other hand, the use of gratings allows narrower bands but, in conventional monochromators, they strongly reduce the size of the incident beam. Tuneable dye-lasers, pumped with a continuous light source, such as, for example, an Argon laser, can provide highly monochromatic light with power of the order of O. 1 Wand could represent a good alternative to conventional light sources.

Absorption spectra of biological photo receptors molecules at room temperature show, in general, several nanometer wide bands and therefore excitation bandwidths narrower than about 5 nm could be unnecessary (Hartmann, 1983). On the other hand, these photoreceptors are often rigidly embedded in spatially ordered receptive units and their absorption spectra can show ;n vivo a relatively resolved vibrational structure, thus justifying the use of narrower excitation bandwidths.

The use of highly monochromatic light, however, is only a necessary condition to solve the above-mentioned problem of the low wavelength resolution of action spectra. In order to reveal shoulders or narrow peaks, the interval between the used wavelengths should be as small as possible, compatibly with the bandwidths of the stimulating light (the intervals should be not lower than half of the excitation bandwidth (Hartmann, 1983)).

The effectiveness of the light in inducing the photoresponse can be quantified, for example, as the amount of light that is required by the system in order to produce a fixed level of response. Due to the stoichiometric relationship between the number of photons absorbed by the photoreceptor and the number of photoreceptor molecules producing the primary reaction initiating the phototransduction chain, the "strength" of the stimulus should be expressed as a fluence rate (number of photons or moles of photons (einstein) per square meter per second).

The basic assumption of action spectroscopy is that at each wavelength the photoresponse depends on the stimulating light only through the photoreceptor primary reaction triggering the transduction chain or, in other words, at equal levels of response at different wavelengths correspond equal rate constants of primary reaction (Schafer et a!., 1983; Galland, 1987). If the dependence of the response on the strength of the stimulus is monotonous, this relationship becomes bijective:

(1)

where kAn and RAn are the rate constant and the response at wavelength An, respectively. Being kA = FAosA°<\l, (FA is the fluence rate, expressed in einsteinom-2os-1, SA is the molar extinction coefficient of the photoreceptor and <\l is the quantum efficiency of the primary reaction), it is possible to deduce the fundamental relationship of action spectroscopy:

(2)

276

It states that, when equal responses are measured, the molar extinction coefficients of the photoreceptor are proportional to the reciprocal of the fluence rates at different wavelengths. This means that in a hypothetical experiment, where at each examined wavelength the same level of response is obtained, the plot of the reciprocal of the fluence rates used versus the wavelength should have the same shape as the absorption spectrum of the photoreceptor in its physiological environment.

In relationship (1) the response R is related to the rate of the primary reaction. An equivalent relationship could be written linking the response to the total quantity of product and relationship (2) can be extended to fluences NA (NA = FA·At) if the exposure time At is constant at each wavelength:

RAI = RA2 => EAl : EA2 = IfFAI : IfFA2 = lI(FAl·At) : lI(FA..2·At) = IINAI : IINA2

If the examined system obeys the law of reciprocity (Shropshire, 1972), i.e. the response depends on the total dose, it is possible to use different irradiation times at different wavelengths.

In deducing relationship (2), the quantum efficiency of the primary reaction, <1>, has been assumed independent of wavelength. If this condition is not met, the action spectrum will result distorted relative to the absorption spectrum (Schafer et al., 1983). On the other hand, if in the photoreceptor molecule the primary reaction originates from the first singlet excited state or from the first triplet state, and the rates of the various radiationless transitions have the usual values of a normal organic molecule, the assumption of the independence of <I> from wavelength can be considered true (Galland, 1987).

Other sources of wavelength-dependent distortion of the action spectrum can be found in the environment where the pigment is embedded in vivo. In fact, the presence in the cell of other pigments can cause screening or reflection effects, in such a way that the effectiveness of a particular spectral range on the photoreceptor could be reduced or enhanced, respectively (see Fig. 1). Moreover, in a cellular environment with a high number of scattering centers, the efficiency in eliciting the photoresponse of the shorter wavelengths can be apparently decreased, because of the dependence of the diffusion on the inverse of the wavelength (see Fig. 1). The sample should also not absorb too much light at the tested wavelengths, so that, at least ideally, every photoreceptor involved in the photoprocess could be exposed approximately to the same amount of photons and, consequently, have the same probability of responding (Coohill, 1991). Finally, as mentioned above, it has to be taken into account that the molecular environment can drastically change the absorption characteristics of the photoreceptor pigment, thus making difficult its identification.

From relationship (2) it follows that a simple procedure to determine an action spectrum is to choose a standard level of response and to measure at each wavelength the fluence rate (or the dose) inducing that response. However, since a necessary prerequisite is that the magnitude of the response is proportional to the intensity of the stimulus, a succession of graded responses should be measured, for each wavelength, in order to construct a fluence rate-response curve and to verity that the chosen standard level of response fallon the linear part of the curve.

An important criterion to determine if the fluence rate-response curves at the different tested wavelengths are indicative of the same mechanism of action is to check if they can be reasonably superimposed, by multiplying each of them by an appropriate fluence factor, thus showing that they have the same "shape" (Coohill, 1991). In other words, given any pair of fluence rate-response curves, the ratio of two fluence rates corresponding to the same level of response should be approximately a constant for any level of response and, consequently, the difference of their logarithms should be also a constant. This implies that the curves plotted on a logarithmic abscissa should appear parallel.

If a fluence rate-response curve has been determined with a sufficient number of data points, it can be fitted with an appropriate mathematical function. This, besides allowing the

277

A Screening pigment

B Reflecliog pigment

C Scaltcring

: I ~ I I I \. I ! I I

, I , ,

I I I I I i I I

l I Ii Ii 1 j U II f:'~~~t

I II. I

I ~ ~ I I I

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j •• :~ ' .) •• ' ~:.'.: • ;.<' • . . . . .. ' . , I "

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Wavelength

Wavelength

Wavelength

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E-o .,

.D <

~ l! ..: .. '"

.. c '§ li VI

Wavelength

il . ~ ~ I.I.l

Wavelength

~ " > '£ ~ Ul

Wavelength

Figure 1. Sources of wavelength-dependent distortion of an action spectrum. A: In the presence of a screening pigment, the overlapping of a band in the absorption spectrum of the photoreceptor pigment (continuous line) with a band in the absorption spectrum of the screening pigment (dashed line) results in a depression of the corresponding band in the action spectrum (effectiveness). B: In the presence ofa reflecting pigment, the overlapping of a band in the absorption spectrum of the photoreceptor pigment (continuous line) with the reflection band of the screening pigment (dashed line) results in an enhancement ofthe corresponding band in the action spectrum (effectiveness). C: The presence of scattering results in a depression of the short-wavelength part of the action spectrum (effectiveness).

calculation of the fluence rates for any standard level of response, makes it possible to check more precisely if the curves have the same shape.

The kinetic properties of the photoreceptor system determine the shape of the fluence rate-response curves, i.e. the mathematical function that best fits the data points could, in principle, give information on the kinetic mechanism of the primary step of the phototransduction process. Two general kinetic models have been discussed illustrating two important types of photoreactions, in which a single, non-photochromic photoreceptor is involved: photoproduct formation and photocatalysis (Hartmann, 1983; Galland, 1987). Biological systems and relative kinetic models based on photochromic photoreceptors, i.e. on photo receptors existing in two ground state forms reverting to each other by effect of light (e.g. phytochrome), are discussed in detail in Hartmann (1983) and in Schafer et al. (1983).

In the kinetic model for photoproduct formation, the photoreceptor P from its excited state P* is converted, through a one-step or a multi-step dark reaction, into a product A; the photoresponse R is proportional to the concentration of A. It can be demonstrated (Hartmann, 1983) that the relative photoresponse Rrel is an exponential function of the product of the fluence rate by the exposure time t with El '<!> as parameter:

278

or, as a function of the fluence (NA = FA·t):

As Rrel depends on the fluence, the reciprocity law should apply. Examples of photobiological processes following this kinetic mechanism are the light­

dependent synthesis of chlorophyll in higher plants, the photoinactivation by UV light of enzymes and viruses and the UV photodamage of bacteria and protozoa (Hartmann, 1983).

In the kinetic model for photocatalysis the light-produced active state P A of the photoreceptor P functions as a catalyst regulating the photoresponse R. P A reverts in the dark to P with a rate constant kA It can be demonstrated (Hartmann, 1983) that the photoresponse R is a linear function of the exposure time t multiplied by a hyperbolic function of the fluence rate with the ratio kAiI>A·<I> as parameter:

As R depends linearly on the exposure time and hyperbolically on the fluence rate, the reciprocity law is not valid. If the exposure time is kept constant for all the experiments the relative photoresponse Rrel becomes:

Examples of photobiological processes following this kinetic mechanism are the photosynthesis and the photoactivation of enzymes, such as the cytochrome oxidase or the nitrate reductase (Hartmann, 1983). Hyperbolic functions have been used also to fit the fluence-response curves for various photoprocesses occurring in Phycomyces, such as photo morphogenic responses and photogravitropic equilibrium of sporangiophores (Lipson, 1991 ).

ACTION SPECTROSCOPY OF ALGAL PHOTOTAXIS

Action spectroscopy has been widely used in the study of photomovements of microorganisms, mainly with the aim of identifYing the photoreceptor molecules involved in the process or, at least, of classifYing them in a particular biochemical class (carotenoids, flavins, etc.). In the following, an overview is presented of experimental works in which action spectroscopy was applied with this purpose to the study of motile reactions of uniceIIular algae, with some emphasis on more recent publications. For more complete information on this subject that takes into account results obtained also with other techniques than action spectroscopy, the reader can refer to some general reviews on this topic (Colombetti & Lenci, 1980; Nultsch & Hader, 1989; Colombetti & Petracchi, 1989; Lenci & Ghetti, 1989; Song & Poff, 1989; Kuznicki et al., 1990; Lenci et al., 1991; Kreimer, 1994). As discussed above, in fact, action spectra can provide only a first hint about the nature of the photoreceptor involved, and other experimental approaches should be adopted in order to enrich the information obtained from action spectroscopy.

279

For phototactic algae, where the interpretation of photomovement action spectra is complicated by the presence of screening pigments or reflecting organelles (stigma or eyespot), Foster and Smyth (1980) proposed, in order to identifY the photoreceptor, the determination of an action spectrum obtained by plotting against wavelength the reciprocal of the fluence rate at which the maximum response occurs, showing how this value can be obtained from the fluence rate-response curves. Under some conditions (Foster & Smyth, 1980), this spectrum should approximate the photoreceptor absorption spectrum, without any distortion due to screening or reflecting pigments.

Foster and Smyth, reinterpreting on the basis of this model previous data by Nultsch et af. (1971), produced an action spectrum suggesting a rhodopsin-like photoreceptor for the phototactic response of the flagellated alga Chlamydomonas reinhardtii (Foster & Smyth, 1980). Later, this hypothesis was confirmed using a blind mutant, which exhibits photoresponse only at very high fluence rates: incorporating analogues of the retinal chromophore into the mutant cells, it was shown, in fact, that the photoresponse was restored also at lower fluence rates and that the maxima of the action spectra were shifted consistently with the absorption maxima of the different incorporated retinal analogues (Foster et af., 1984).

By means of action spectroscopy measurements, performed at the Okazaki Large Spectrograph (Watanabe et af., 1982; Watanabe, 1991), the photophobic and phototactic responses of DUl/abella salina were showed to be triggered by two different photoreceptors, which in both cases, were suggested to be a rhodopsin or a carotenoprotein (Wayne et ai., 1991).

In the case of the flagellated alga Euglena gracilis, action spectra for the phototactic and the photophobic response, based either on population or on single cell measurements, indicated that flavin molecules may be responsible for photobehaviour (Diehn, 1969; Checcucci et aJ., 1976; Barghigiani et aJ., 1979). More recently, phototaxis action spectra based on computer-assisted tracking of individual cells showed, both for green and dark bleached E. gracilis, a good agreement with the hypothesis that, besides flavins, pterin molecules also act as photoreceptors (Hader & Reinecke, 1991). On the basis of action spectroscopy, a flavin-type photoreceptor was also proposed for the photophobic response of the euglenoid flagellate Astasiajritschii (Mikolajczyk & Walne, 1990).

For the photoorientation of the dinoflagellate Peridinium gatunense an action spectrum was determined showing a marked effect in the spectral range 550-720 nm; the involvement of photosynthetic or accessory pigments was excluded on the basis of experiments with inhibitors of photosynthetic electron transport chain and of the lack of action bands in the blue-green spectral range, and no hypothesis on the nature of this photoreceptor was put forward (Liu et al., 1990).

In brown algae, for the phototaxis of the gametes of EctocaTpUS siliculoslls and of the zoospores of Pseudochorda gracilis, action spectra in the range 400-500 nm, with two bands at about 420 and 460 nm, were determined (Kawai et af., 1990, 1991). The combined action of a free and of a protein-bound flavin (8-hydroxy-5-deazaflavin) was suggested to explain the 420 nm and the 460 nm bands, respectively (Kawai et aJ., 1991).

ACTION SPECTROSCOPY OF ULTRAVIOLET EFFECTIVENESS

Action spectroscopy can also provide significant information in the field of ecophysiological plant research and, more generally, in the field of environmental photobiology. In fact, action spectroscopy is a useful tool for investigating the biological effects of ultraviolet radiation, and, in particular, of the UV-B wavelengths (280-315 nm), on terrestrial plants and phytoplankton organisms (Bornman, 1989; Coohill, 1989; Coohill, 1991; Coohill, 1992; Bornman & Teramura, 1993; Holm-Hansen et al., 1993; Hader, 1993; Bornman, this volume).

280

A 1.00

0.75

.~ ::l

~ 0.50 ·0

'" <i ~

0.25

1.e+21 l.e+22

Fluence [photons· m 2]

8 1.0

III

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Wavelength [nmJ

Figure 2. Dose-response curves (A) and actiou spectrum (B) of the effectiveness ofUV-B wavelengths in inhibiting the photosynthetic ox)'gen production in the unicellular flagellate alga Dunaliella salina. The irradiation source was a 1 kW Xenon lamp filtered with 10 nm band (BWHM) interference filters. Photosynthetic oxygen production was determined at room temperature by means of a Clark-type electrode inserted in a cuvette for liquid-phase measurements. The dose-response curves were obtained by fitting the data points Witll the Hill functiony = a·xn / (xl1 + bn), following the procedure indicated by Ensminger et al. (1990).

In this field, more accurate and reliable predictions can be obtained from action spectra measured under polychromatic irradiation, in order to expose the investigated organisms not only to the UV-B wavelengths, but also to UV-A (315-400 nm) and visible radiation, that can activate enzymatic repair mechanisms. This experimental technique can be realized by using a broad band radiation source that simulates the solar spectral irradiance and progressively reducing the irradiation spectral band by means of cut-off filters. Assuming additivity of different wavelengths in producing the observed biological response, the contribution of a particular irradiation band can be estimated by calculating the differences in irradiance obtained, using pairs of successive cut-off filters and the corresponding differences in effectiveness in order to identifY the acting wavelengths and quantifY the resulting response, respectively (Rundel, 1983).

In order to asses the potential effects of increasing levels of UV -B radiation due to the depletion of the atmospheric ozone layer, valuable information can be provided by the

281

1.00E+OO

1.00E-OI

1.00E-03

Action spectrum

, , " : , . , ; " " ,; "

SUD simulator r •••

, I SUD

specttal irradiances

Weighting functions

1.00E·04 +--",-~--r-+'--",-~-",-~-",-~--.

275 280 285 290 295 300 305 310 315 320 325 330

Wavelength [nm]

Figure 3. Biological weighting functions obtained by combining the action spectrum for UV-B inhibition of photosynthetic oxygen production in the unicellular flagellate alga Dunaliella salina (see Fig, 2) with a real solar spectrum and with the spectrum ofa solar simulator (Setkmeyer & Payer, 1993), with enhanced emission in the UV-B range, The action spectrum is normalized to 1.0 at 280 nm, whereas the solar spectrum (measured at Tramariglio, about 40°36' N, 8°11' E, Alghero, Sardinia, Italy, May 1993) and the sun simulator spectrum is normalized to 1.0 at 330 nm.

determination the "biological weighting function", i.e., the spectral efficiency of a particular artificial or natural broad-band irradiation source in producing the observed photoresponse. This is obtained by multiplying the action spectrum for the observed photo response by the spectral irradiance of that source, measured under a specific irradiation condition. This could be, for instance, the estimated solar spectrum at ground level at different percentages of depletion in stratospheric ozone (Coohill, 1989; Coohill, 1991; Cullen et aI., 1992),

As a case example, in Fig. 2 the dose-response curves and action spectrum are shown of the effectiveness ofUV-B wavelengths in inhibiting the photosynthetic oxygen production in the unicellular flagellate alga Dunaliella salina, The action spectum was determined under monochromatic irradiation, using a 1 kW Xenon lamp as light source and narrow-band interference filters. Fig. 3 shows two different biological weighting functions obtained by combining this action spectrum with the solar spectrum and with the spectrum of a solar simulator (Seckrneyer & Payer, 1993), with an emission showing an increase in irradiance in the UV-B range comparable to those observed under the ozone hole (Holm-Hansen et al., 1993),

282

Acknowledgements

This work has been supported by a grant from the European Community Research Project 'Environment' (Contract EV5V-CT91-0026).

REFERENCES

Barghigiani, C., G. Colombetti, B. Franchini & F. Lenci (1979) Photochem. Photobiol. 29,1015-1019. Bornman, J. F. (1989) J. Photochem. Photobiol., B: BioI. 4, 145-158. Bornman, J. F. & A. H. Teramura (1993) in "Environmental UV Photobiology", (A. R Young, L. O. Bjorn,

J. Moan & W. Nultsch, eds.), Plenum Press, New York, pp. 379 - 425. Checcucci, A., G. Colombetti, R Ferrara & F. Lenci (1976) Photochem. Photobiol. 23, 51-54. Colombetti, G. & F. Lenci (1980) in "Photoreception and Sensory Transduction in Aneural Organisms" (F.

Lenci & G. Colombetti, eds.), New York, Plenum Press, pp. 173-188. Colombetti, G. & D. Petracchi (1989) Critical Reviews in Plant Sciences 8, 309-355. Coohill, T. P. (1989) Photochem. Photobiol. 50, 451-457. Coohill, T. P. (1991) Photochem. Photobiol. 54, 859-870. Coohill, T. P. (1992) J. Photochem. Photobiol., B: BioI. 13,95-98. Cullen, J. c., P. J. Neale & M. P. Lesser (1992) Science 258, 646-650. Diehn, B. (1969) Biochim. Biophys. Acta. 177, 136-143. Ensminger, P. A., X. Chen & E. D. Lipson (1990) Photochem. Photobiol. 51,681-687. Foster, K W. & RD. Smyth (1980) Microbiol. Rev. 44,572-630. Foster, KW., J. Saranak, N. Patel, G. Zarilli, M. Okabe, T. Kline & K Nakanishi (1984) Nature 311, 756-

759. Galland, P. A. (1987) in "Blue Light Responses: Phenomena and Occurrence in Plants and Microorganisms"

(H. Senger, ed.), Boca Raton, CRC Press, pp. 37-52. Gualtieri, P. (1993) J. Photochem. Photobiol., B: BioI. 19,3-14. Hader, D-P. (1993) in "Progress in Phycological Research 9", (F.E. Round & D.J. Chapman, eds.), Biopress

Ltd., pp. 1 - 45. Hader, D.-P. & E. Reinecke (1991) Acta Protozool. 30, 13-18. Hartmann, K M. (1983) in "Biophysics" (W. Hoppe, W. Lohmann, H. Markl & H. Ziegler, eds.), Berlin,

Springer-Verlag, pp. 115-144. Holm-Hansen, 0., D. Lubin & E.W. Helbling (1993) in "Environmental UV Photobiology", (A. R Young,

L. O. Bjorn, J. Moan & W. Nultsch, eds.), Plenum Press, New York, pp. 379 - 425. Kawai, H., D. G. Muller, E. FoIster & D.-P. Wider (1990) Planta 182,292-297. Kawai, H., M. Kubota, T. Kondo & M. Watanabe (1991) Protoplasma 161, 17-22. Kreimer, G. (1994) Int. Rev. Cytol. 148,229-310. Kuznicki, L., E. Mikolajczyk & P. L. Walne (1990) Critical Reviews in Plant Sciences 9, 343-369. Lenci, F. & F. Ghetti (1989) J. Photochem. Photobiol., B: BioI. 3, 1-16. Lenci, F., F. Ghetti, G. Colombetti, D.-P. Hader & P.-S. Song (eds.) (1991) Biophysics of photo receptors and

photomovements of microorganisms, NATO ASI Series A211, New York, Plenum Press. Lipson, E. D. (1991) in "Biophysics of photoreceptors and photomovements of microorganisms" (F. Lenci,

F. Ghetti, G. Colombetti, D.-P. Hader & P.-S. Song, cds.), NATO ASI Series A211, New York, Plenum Press, pp. 293-309.

Liu, S. M., D.-P. Hader & W. Ullrich (1990) FEMS Microbiol. Ecol. 73, 91-102. Mikolajczyk, E. & P. L. Walne (1990) 1. Photochem. Photobiol., B: BioI. 6,275-282. Nultsch, W. & D.-P. Hader (1989) Photochem. Photobiol. 47, 837-869. Nultsch, W., G. Throm & 1. von Rimscha (1971) Arch. Mikrobiol. 80, 351-369. Rundel, RD. (1983) Physiol. Plant. 58,360-366. Schafer, E., L. Fukshansky & W. Shropshire, Jr., (1983) in "Encyclopaedia of Plant Physiology", NS, Vol.

16. Photomorphogenesis (W. Shropshire, Jr., & H. Mohr, eds.), Berlin, Springer-Verlag, pp. 39-68. Seckmeyer, G. & H.-D. Payer (1993) J. Photochem. Photobiol., B: BioI. 21, 175-181. Shropshire, W., Jf. (1972) in "Ph}10chrome" (K. Mitrakos & W. Shropshire, Jr., eds.), London, Academic

Press, pp. 161-181. Song, P.-S. & K L. Poff (1989) in "The science of photobiology", 2nd ed., (K. C. Smith, ed.), New York,

Plenum Publishing Corp., pp. 305-346. Watanabe, M. (1991) in "Biophysics of photoreceptors and photomovements of microorganisms" (F. Lenci,

F. Ghetti, G. Colombetti, D.-P. Hader & P.-S. Song, eds.), NATO ASI Series A211, New York, Plenum Press, pp. 327-337.

Watanabe, M., M. Furuya, Y. Miyoshi, Y. Inoue, 1. Iwahashi & K Matsumoto (1982) Photochem. Photobiol. 36, 491-498.

Wayne, R., A. Kadota, M. Watanabe & M. Furuya (1991) Planta 184,515-524.

283

PHOTOREGULA TION OF FUNGAL GENE EXPRESSION

Enrique Cerda-Olmedo and Luis M. Corrochano

Departamento de Genetica, Universidad de Sevilla, Apartado 1095, E-41080 Sevilla, Spain.

INTRODUCTION

Photoresponses do not have to be mediated by fresh gene expression. Some responses are so fast that they do not allow enough time for synthesis, maturation, and transport first of the mRNA and then of the polypeptide, and for protein activity. This is the case for phototropism towards blue light, a phenomenon that has been particularly well described in the fungus Phycomyces and in some plants (Galland, 1990).

Other photoresponses, such as photomorphogenesis and photocarotenogenesis, are comparatively slow and leave ample time for the modulation of gene expression. Red, blue, and near ultraviolet radiations influence developmental processes in the fungi and in plants. The elements of the signalling pathway and the involvement of gene expression are under active investigation in various plants (Chory, 1993; Quail, 1994; Short & Briggs, 1994). There are many examples of photo morphogenesis in the fungi (Kumagai, 1988; Corrochano and Cerda-Olmedo, 1991). The biosynthesis of carotenoids is almost universally regulated by light (Avalos et aI., 1993).

PHOTOMORPHOGENESIS IN THE FUNGI

Blue light, together with many other signals from the environment, defines the onset of some developmental pathways (e.g., sporulation) and the choice between alternative developmental pathways (e.g., different kinds of reproductive structures).

In the dark, Phycomyces blakesleeanus produces two kinds of vegetative reproductive structures, called macrophores and microphores. Blue light increases the production of macrophores and decreases that of microphores. These two responses exhibit a two-step dependence on exposure. Each response has one component with a threshold at about 10-4

This review IS dedicated to Prof. Charles Yanofsky on the occasion of his 70th birthday.

Light as an Energy Source and In/onnation Can'ier in Plant Physiology Edited by JelU1ings et al., Plenum Press, New York, 1996 285

J m-2 and another with a threshold at about 1 J m-2, which suggests the existence of two photosystems. The effect of light depends on the product of its intensity and the exposure time, as in photography. This reciprocity holds for exposures of 12 s to 3 h, at least, and implies that the cell "counts" and "remembers" the photons received over a long time interval before taking developmental decisions. The absolute threshold is very low: the cell responds to about ten photons per 11m2, and these may be given at a rate of one per 11m2 each 20 min. The response requires the previous development of competence to light, which is maximal in 48-h-old cultures under certain conditions (Corrochano & Cerda-Olmedo, 1992).

In Neurospora crassa blue light promotes the formation of the vegetative and sexual reproductive structures (conidia and protoperithecia). Only intense light induces a response: the threshold for the protoperithecia is about 4 J m-2 (Degli-Innocenti & Russo, 1984). Additionally, conidiation is subject to an endogenous circadian rhythm: bands of conidia stand out on the hyphal mats at constant distances that correspond to the mycelial expansion in 21.5 h. This phenomenon, which is particularly clear in the bd ("band") mutant, can be phase-shifted by blue light (Sargent & Briggs, 1967). The circadian rhythms of such distant relatives as Neurospora and Drosophila are surprisingly related, as shown by the similarity of the genes responsible for them (Dunlap, 1993).

In Aspergillus (Emericella) nidulans conidiation is induced by red light (Mooney & Yager, 1990). The exposure must last for at least 15-30 min and the threshold is very high, about 200 J m-2 at the most effective wavelength (680 nm). A far-red exposure (730 nm) abolishes the induction of conidiation by a previous red one. This is reminiscent of phytochrome in plants. Other fungi that respond to red light include Conidiobolus and Pi/obolus (Page, 1962).

PHOTOCAROTENOGENESIS

Among the many examples of photoinduced carotenogenesis we may cite the increased accumulation of neurosporaxanthin in Fusarium aquaeductuum (Bindl et ai., 1970), Neurospora crassa (Zalokar, 1955), and Gibberella jujikuroi (Avalos & Cerda-Olmedo, 1987), of torulene in Rhodotorula minuta (Tad a & Shiroishi, 1982), and of 13-carotene in Phycomyces blakesleeanus.

Photocarotenogenesis in Phycomyces is the sum of two components; one has a very low threshold (10-) J m-2) and a weak response; the other is less sensitive (threshold, 100 J m-2), but produces large increases in carotene accumulation with no signs of saturation even at 2 x 106 J m-2 (Bejarano et ai., 1991). The competence period for "photocarotenogenesis" is the same as that for photomorphogenesis when tested under the same culture conditions. A requirement for new gene expression was suggested by the inhibition with cycloheximide (Jayaram et ai., 1979) and experiments in vitro (Salgado et ai., 1991).

Photocarotenogenesis is a conspicuous blue light effect in Neurospora because mycelia are white when grown in the dark and orange when grown in the light. A requirement for new protein synthesis was suggested by experiments with cycloheximide (Rau et ai., 1968). The response has two components, with thresholds at about 10 and 200 J m-2, respectively. To activate the second component the exposure m.ust extend itself over at least 15 min (Schrott, 1980). Conidia always contain carotenoids, even ifformed in the dark.

The biological role of photocarotenogenesis is obscure. The ability to make carotenes does not influence the fitness of Phycomyces when exposed to various radiations, including blue light in the presence of photo sensitizers, hydrogen peroxide, and other damaging agents (Martin-Rojas, 1994).

286

PHOTOINDUCffiLE GENES

The first evidence of photoinduced accumulation of mRNAs was obtained with Fusarium aqllaedllctuum and later extended to Neurospora. Illumination led to the appearance of new mRNAs, which could be translated to specific polypeptides in vitro (Schrott & Rau, 1977; Mitzka-Schnabel et al. 1984; Chambers et aI., 1985). The details of the process were obtained after the isolation and characterization of genes whose transcription is induced by light.

Genes for photocarotenogenesis in Neurospora

The albino mutants of Neurospora, which produce white mycelia when grown in the light, involve three al genes, numbered in inverse order with respect to their action in the pathway (Harding & Turner, 1981; Morelli et al. 1993). All three al genes have been isolated by complementation with the corresponding mutants and characterized in detail.

Gene al-3, responsible for geranylgeranyl pyrophosphate synthetase (Sandmann et aI., 1993), was isolated first (Nelson et aI., 1989). Its mRNA becomes detectable shortly after a brief illumination, reaches its maximum concentration in about 15 min, and is then rapidly degraded (Baima et aI., 1991). Induction adapts to continuous illumination in the sense that the mRNA level decreases after 30 min and eventually disappears. This pattern of immediate induction and adaptation is characteristic of many other photoinduced genes.

Light regulates in a similar way the expression of gene al-2 (Schmidhauser et aI., 1994), responsible for phytoene synthetase, which converts geranylgeranyl pyrophosphate into the first carotene, and gene al-I (Schmidhauser et aI., 1990), responsible for phytoene dehydrogenase and the manufacture of coloured carotenes.

Genes for photomorphogenesis in Neurospora

The COil genes were isolated because their mRNAs are expressed upon conidiation (Berlin and Yanofsky, 1985; Springer, 1993) and the ccg genes, because they are under the control of the circadian clock (flock-font rolled genes) (Loros et aI., 1989). Some of these genes turned out to be regulated by light (Lauter & Russo, 1991; Lauter & Yanofsky, 1993; Arpaia et aI., 1993). The same genes and possibly others could be isolated as bli genes on the basis of their !2lue-light inducible expression (Sommer et aI., 1989). With one exception, the precise function of these genes is unknown.

The exception is the gene isolated independently as ccg-2 and bli-7, isolates which turned out be identical (Lauter et aI., 1992; Bell-Pedersen et aI., 1992) to gene eas, already detected because of the "easily wettable" phenotype of a mutant. The product is a hydrophobin, a small and very hydrophobic protein of the cell wall of the conidia, where it forms bundles of rod lets visible to the electron microscope (Beever & Dempsey, 1978).

Some genes (con-5, con-6, con-JO, bli-3, and bli-4) are immediately and transiently expressed after a brief illumination, very much like the al genes (Sommer et al. 1989; Lauter & Russo, 1991; Lauter & Yanofsky, 1993). The mRNA of two other genes (eas and bli-I3) is present at low level in the dark, take much longer to be photoinduced, and remain detectable for several hours (Sommer et aI., 1989; Lauter et aI., 1992; Arpaia et aI., 1993). Photoinduction was observed in submerged cultures, where no conidiation occurs, and in mutants defective in different conidiation steps (Sommer et aI., 1989; Lauter & Russo, 1991; Lauter & Yanofsky, 1993).

In addition to eas (ccg-2), con-6 and con-lO are expressed rhythmically at a low level in the dark under the control of the circadian clock (Loros et aI., 1989; Arpaia et aI., 1993; Lauter & Yanofsky, 1993). They remain photoinducible in strains with an accentuated cycle (bd) and in the absence of an operative clock (in thejrequency mutantjrq9). In these cases the kinetics of photoinduction are modified.

287

Genes of the sensory transduction pathway in Neurospora

The wc ("white collar") mutations were identified as regulators of the photocarotenogenesis in Neurospora (Harding & Turner, 1981), and it was later found that all the blue-light responses of Neurospora required functional wc-J and wc-2 genes (Degli­Innocenti & Russo, 1984).

Photoinduction of translatable mRNA requires the presence of the wild type alleles of wc-J and wc-2 in all the blue light-induced genes investigated (Nelson et al. 1989; Sommer et al. 1989, Schmidhauser et al. 1990; Lauter & Russo 1991; Arpaia et al. 1993; Schmidhauser et al. 1994). The products of the wc genes mediate common, and presumably early, steps in the signalling pathways for blue-light effects in Neurospora.

Gene wc-J has been recently isolated by G. Macino and his coworkers. The sequence suggests a Zn-finger protein with the ability to bind the so called GAT A sequence present in many promoters. In fact, the protein, expressed in Escherichia, binds a GAT A sequence in the al-3 promoter (G. Macino, personal communication). Transcription factors that bind GAT A sequences are known in many organisms and playa role in the photoregulation of plant genes (Gilmartin et aI., 1990).

The hydrophobins of Schizophyllum

Three hydrophobins accumulate in the cell walls of Schizophyllum commune. Like eas, the corresponding genes are photoinducible in the mycelia six hours after the beginning of illumination with far ultraviolet radiation (Yli-Mattila et aI., 1989; Wessels et aI., 1991).

Photoinducible genes of Aspergillus

The detailed genetic dissection of conidiation in Aspergillus nidulans (Timberlake, 1990; 1993) led to the isolation of several genes that are expressed both in the course of conidiation and upon illumination. This is the case of the regulatory genes brlA and abaA (Mooney & Yager, 1990). It is not yet clear whether this is a direct effect or an indirect consequence of the induction of conidiation by light.

The amino-end of the brlA gene product has some similarities with plant phytochromes (Griffith et aI., 1994) that may be of biological significance given the red/far-red reversibility exhibited by conidiation in Aspergillus and the developmental effects of phytochrome in plants.

Gene rodA, responsible for the hydrophobin in the rodlets of the conidial cell wall (Stringer et aI., 1991), is homologous to the Neurospora gene eas.

A carotenogenic gene from Phycomyces

The extreme sensitivity and complexity of the photoresponses of this organism have not been duly explored at the molecular level because of the primitiveness of Phycomyces gene cloning techniques. Gene carB, responsible for the four dehydrogenations needed to convert phytoene into lycopene (Aragon et aI., 1976) has been isolated recently by A. P. Eslava and his coworkers (unpublished results). Induction by blue light is particularly fast, because it reaches' its maximum in 5 min, and quantitatively modest, about fivefold. This value is consistent with the fact that Phycomyces contains appreciable amounts qf J3-carotene and expresses the carB gene when grown in the dark.

Structure of blue light-inducible promoters

The promoters of many genes must respond to signals originated by blue light, developmental transitions, and the circadian clock. It is particularly worth knowing whether

288

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Figure 1. Expression of con-ID'-'lacZ. Top: the construct consists of the intact conoiD upstream region and a con-ID'-'lacZ translational fusion that contains the first 40 codons of con-JO fused in phase to the 8th codon of E. coli lacZ. The transcription initiation site (+ I ) and the coding regions are indicated. Bottom: ~-galactosidase activity (nmollminlmg protein) in mycelia and conidia. The mycelia were exposed to white light that contained 6 W m-2 blue light. From Corrochano et al. (J 995).

potential site

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• • Figure 2_ Structure of the conoID gene and its promoter. The transcription initiation site (+ I) and the coding regions are indicated. E, enhancers, one of them located in an intron. CRS-B, a region that binds a mycelial protein. There are two binding sites for putative factors that cause repression in the dark, two other for activation of transcription during conidiation, and another for repression in the absence of conidiation. From Corrochano et aI. (1995).

the different signals act on the same or different DNA segments in the blue-light inducible promoters.

A few homologies and coincidences have been noticed in the sequence of various promoters: a highly conserved 13 bp sequence is present at different locations in the promoters of genes al-J, al-2, and al-3 (Schmidhauser et aI., 1994). A sequence called CRS-

289

B is present in the promoters of several genes that are expressed during conidiation, such as ai-I, con-6; con-l0, and eas; this sequence is recognized by a protein present in the mycelia but not in the conidia (White & Yanofsky, 1993). The promoter of gene al-3, composed of the 226 bases that precede the transcription start, is surprisingly small, given the complicated regulation of its expression; photoinduction is determined by two short sequences within that promoter: one, called APE, finds similarities in the promoters of ai-I, con-l0, and eas; the other belongs to the CCAAT family of regulatory elements (Carattoli et aI., 1994).

There is a similar segment in the promoter of eas and the promoter of gene grg-l (Eberle & Russo, 1992), isolated as a glucose repressible gene (McNally & Free, 1988) and identical to ccg-l (Loros et aI., 1989), which is induced 5-10 fold during the circadian cycle. This segment is different from the segment required for the repression by glucose (Wang et aI., 1994) and could be involved in circadian expression and photoinduction. It is not known whether grg-l is photoinducible. Three DNA segments in the eas promoter are involved in photoinduction, and none of them coincides with the segment shared with grg-l (KaIdenhoff & Russo, 1993).

The regions of the con-l0 promoter responsible for light and developmental induction have been investigated recently by measuring j3-galactosidase activity in a set 'of con-l0'­'lacZ fusions (Corrochano et aI., 1995). Photoinduction of lacZ by the con-l0 promoter is a two-step response. A brief exposure (5 s) induced a transient activation that reached a maximum after 1 min. A period of 15 min in the dark or light was then required before the appearance of an additional activation (Fig. 1). This is similar, down to quantitative detail, to the two steps ofphotocarotenogenesis (Schrott, 1980), and suggests that similar molecular mechanisms might be involved.

Deletions within the promoter of con-l0 identified distinct sequences that govern the conidiation-specific and the mycelial-specific expression of the gene (Fig. 2). The regulation by light seems independent of mycelial repression and appears as a release of repression in the dark. A repeated 17-bp sequence acted as a transcriptional enhancer required for maximal mycelial and conidial expression. Two CRS-B sequences may be conidiation-activation sites, since their deletion markedly reduced expression in the conidia. One of the dark-repression sites contains several tandem repeats of a GATA sequence (Corrochano et aI., 1995). These results seem at odds with the view of the wc-l gene product as a GAT A-specific activator and the CRS-B-binding protein as a repressor. The role of these proteins in the regulation of the con-JO promoter remains to be elucidated.

Rapid progress in the characterization of photoinducible genes is now to be expected, not only in the filamentous fungi, but in plants and other organisms as well.

NOTE ADDED IN PROOF

The following relevant papers appeared after this review was submitted: • Photoregulation of genes for carotenoid byosynthesia in Neurospora (Arpaia et aI.

1995; Li & Schmidhauser, 1995) • A new method to isolate blue light regulation mutants of Neurospora (Carattoli et aI.,

1995) • Effects of light on the circadian clock of Neurospora (Arpaia et aI., 1995;

Crosthwaite et aI., 1995).

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799. White, B. T. & Yanofsky, C. (1993) Dev. BioI. 160:254-264. Yli-Mattila, T., Ruiters, M. H. 1. & Wessels, J. G. H. (1989) Curf. Microbiol. 18:289-295. Zalokar, M. (1955) Arch. Biochem. Biophys. 56:318-325.

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PHOTOTROPISM IN PHYCOMYCES·

Enrique Cerda-Olmedo and Virginia Martin-Rojas

Departamento de Genetica, Universidad de Sevilla, Apartado 1095, E-41080 Sevilla, Spain

INTRODUCTION

Blue light is used as a source of information on the environment by all kind of living beings, from microbes to animals. The Zygomycete fungus Phycomyces blakesleeanus is notorious for the variety of responses to blue light, various invisible radiations, and other stimuli. The copious literature on the behaviour of Phycomyces accumulated over more than a century was critically examined in Bergman et al. (1969) and Cerda-Olmedo and Lipson (1987). Galland (1990) updated and compared knowledge on Phycomyces and plant phototropism. We will discuss here only the aspects that have progressed in the last five years.

The vegetative reproductive structures of this fungus, called sporangiophores, are large coenocytes, not divided into cells, and come in two very different sizes. The giant ones, or macrophores, grow several centimeters long guided by many stimuli. The dwarf ones, or microphores, are about 1 mm long.

Many stimuli modify the growth of the macrophores. They cause transient changes in growth velocity (mecisms) when applied symmetrically around the macrophore axis and modify the direction of growth (tropisms) when applied asymmetrically. The most studied stimulus is blue light, and the most studied response, tropism, but similar responses are caused by wind, gravity, pressure, and the presence of obstacles. Other major effects of blue light are the increased production of macrophores, the decreased production of microphores, and the increased accumulation of J3-carotene, a yellow pigment.

Phototropism, photomorphogenesis, and photocarotenogenesis occur in many fungi and plants, with biophysical similarities that suggest common underlying mechanisms. Most organisms are much less sensitive than Phycomyces to light, as indicated by their thresholds. Many fungi and plants can see full moonlight. Phycomyces compares favourably with the human eye: we and Phycomyces can see the stars.

The various tropisms and photomorphogenesis presumably improve the dispersion of Phycomyces spores, allowing the macrophores to grow into open air, where spores can stick to or be eaten by passing animals. Microphores, much less onerous to build than

• This review is dedicated to Prof. Horst Senger on his 65th birthday.

Light as an Energy Source and Information Carrier in Plant Physiology Edited by JeIUlings et af., Plenum Press, New York, 1996 293

macrophores, will be formed when there are few chances for wide spore dispersal and the fungus resigns itself to leaving the spores in situ.

TROPISM TO BLUE LIGHT

The direction of growth

The growing zone of the macrophore is a transparent cylinder about 100 /.lm in diameter and about 3 mm long. The photoreceptors are bound to the outside plasma membrane of the cell, as shown by the different effect of light with different polarization planes (Jesaitis, 1974) and other results (Steinhardt et al., 1989). Blue light stimulates growth, as shown by the transient increase in growth velocity that follows a transient or sustained increase in the fluence rate. Macrophores grow towards a lateral blue . light beam (positive phototropism). The stimulation of growth and positive phototropism seem contradictory results: absorption and scattering, although very modest for visible light in wild-type macrophores, ensure that the total fluence is larger for the side towards the light beam, the proximal side, than on the distal one. The proximal side would be expected to grow faster, and this would provoke a negative phototropism.

Lateral illumination produces sharp focusing bands on the distal side. The distribution, and not the total fluence, on the distal side, is a critical factor for the production of positive phototropism. This hypothesis was proven by immersion of the macrophores in fluids of high refractive index. The macrophores act then as divergent lenses and grow away from the light.

New examples of negative phototropism to blue light have been described, and they stress the role of macrophore optics in phototropism. Piloboloides pi/) mutants are characterized by the continuous widening of the growing zone of their macrophores, which may reach 500 /.lm in diameter. Phototropism is positive when the maximum diameter of the growing zone is smaller than 210 /.lID, and negative when it is larger (Koga et aI., 1984; Ootaki & Tsuru, 1993).

Phototropism never brings Phycomyces to grow straight into the light beam. When vertical macrophores are illuminated horizontally, the final angle is 71° from the vertical or 19° from the light beam. This result was traditionally attributed to a compromise between positive phototropism and gravitropism, which tell the macrophore to grow towards different directions. This explanation was proven false by changing the relative orientation of light and gravity. The final angle is 13° from the light beam for horizontal macrophores illuminated vertically and 16° when gravity is removed by the use of a c1inostat. Phototropism does not reach the exact direction of the light beam because of a negative phototropism caused by light shining at a small angle with the macrophore axis. Gravitropism plays a marginal role, except in a supergravitropic mutant (Ootaki et al., 1991a).

Consideration of macro ph ore optics (Tsuru et aI., 1988; Ootaki et al., 1988; Ootaki et al., 1991a) attributed these two cases of negative phototropism to increased attenuation due to the increased length of the light path. Increased attenuation could also be caused by increased absorption, for example, in a mutant with a very high carotene content (Ootaki et aI., 1988; Ootaki et aI., 1991b). However, the proposition that positive tropism occurs when the maximal fluence rate on the distal side (the focusing peaks) is larger than the one on the proximal side (Tsuru et al., 1988) cannot be accepted, because there are examples to the contrary (Fukshansky, 1993; Martin-Rojas et aI., 1995).

Precise measurements and calculations have provided detailed knowledge of the distribution of light fluence around the macrophore under a vanety of circumstances and have estimated the distribution of photoreceptor excitation (Dennison & Vogelmann, 1989; Fukshansky & Richter, 1990; Fukshansky, 1993). The important conclusion is that the

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direction of phototropism is determined by the energy fluence in a narrow central strip of the distal side. Bright focusing bands in this strip cause positive phototropism. Negative phototropism is observed even in the presence of focusing bands, if they lie laterally on the distal side.

Wild-type action spectra

Refined quantitative descriptions of phototropism, photo mecism, photo morphogenesis, and photocarotenogenesis can be extended over enormous fluence ranges. In each case, the results exhibit a two-step dependence on exposure, which suggests the existence of two photosystems. The stimulus-response relationships present great similarities (Corrochano et aI., 1988), except for high-fluence photocarotenogenesis (Bejarano et al., 1991).

Action spectroscopy has not helped to identifY the photoreceptors beyond a general suggestion that they may contain riboflavin or carotenoids as chromophores. Part of the difficulty is that the action spectra for the growth responses of Phycomyces depend very much on the actual experimental conditions. The variety has been enriched by n:cent research: the action spectrum for photomecism depends on the wavelength and intensity of the reference light (Ensminger et aI., 1991). The action spectra for the growth responses are not determined exclusively by the photo receptors, but are influenced by the geometry and the optics of the macrophore; dichroism and variations in the distribution offluence around the macrophore must be taken into account (Fukshansky, 1993).

Geometrical factors should be averaged out in the six action spectra for photomorphogenesis and photocarotenogenesis (Corrochano et aI., 1988; Bejarano et aI., 1991). These spectra present overall similarities, but also small significant differences which suggest variations in the structure or the operation of the respective photoreceptors.

Genetics of phototropism

Genetic analysis identifies the elements in the different signal pathways and informs about their relationships. Mutants were isolated on the basis of defects in phototropism (the mad mutants), ultraviolet tropisms (uvi), photocarotenogenesis (Pic), and "photomorphogenesis" (pmg). Biochemical mutants of interest include those with modifications in carotene biosynthesis (car), riboflavin biosynthesis (rib) and riboflavin permeation (dar). A mutant isolated for other reasons exhibits a very fast gravitropism (geo) and another one has a defect in phosphodiesterase (pde).

The mad mutants define ten unlinked genes, named in alphabetical order of discovery, the latest additions being mad! and madJ (Campuzano et aI., 1990, and in press). Mutants in some of the genes (madA, B, C, and 1) are "night blind". Their stimulus-response curves are similar to those of the wild type, but shifted to higher light intensities, and they possess normal tropisms to stimuli other than light. "Night-blind" madA and made mutants are defective not only in phototropism, but in photomorphogenesis and photocarotenogenesis as well.

Mutants in the other genes, called "stiff", are defective in the tropisms to other stimuli and must therefore be involved in the regulation of cell-wall growth. This conclusion is supported by the analysis of the fine fluctuations of growth rate under constant conditions of illumination (Ensminger & Lipson, 1992).

Many mutations are highly pleiotropic. The genetic analysis can be summarized in a chart of sensory pathways, similar to the usual biochemical pathways with the difference that information, and not necessarily chemicals, flows along them. The elements of the sensory pathways (sensors, effectors, and intermediate elements) are identified by the genes that control them. The sensory pathway chart conveys a first impression of parsimony. For example, blue-light receptors depending on the madA and madB gene functions combine with different effectors to carry out various photoresponses, such as tropism,

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carotenogenesis, and morphogenesis. The growth regulation mediated by the genes defined by "stiff' mutants carries out the responses to blue light, gravity, and obstacles.

The small and transient variation in growth velocities called photomecism is unlikely to playa biological role by itself and is considered just an expression of the basic mechanisms that cause phototropism. In this view, phototropism would be the result of photomecism and additional spatial and genetic factors. This is supported by the observation that most mad mutants are simultaneously defective in both phototropism and photomecism. The additional gene functions that together with photomecism cause phototropism may be represented by gene madH, since a mutant in this gene has a normal action spectrum for photomecism (Ensminger & Lipson, 1991).

Adaptation

The Phycomyces growth responses depend more on changes and asymmetries in illumination than on the actual fluence. Macrophores easily adapt to fluence changes, and much faster to increases than to decreases (review by Galland, 1989). Adaptation is easly shown by shifting macrophores grown under a bright symmetrical illumination to a dim lateral illumination: tropism occurs after a delay that increases with the logarithm of the ratio between the light fluences (Delbruck & Reichardt, 1956). The time constant is about 6 min.

If macrophores are shifted from a bright symmetrical illumination to darkness for a certain time and then to a test unilateral illumination, the kinetics of adaptation can be followed by monitoring the onset of phototropism. In this case the kinetics of adaptation are more complicated (Galland et aI., 1989b), initially fast (time constant about 1 min) and then slower (time constant about 11 min). The adaptation is accelerated by substituting very dim illumination (called subliminal because it produces no tropism by itself) for the period in the dark (Galland et aI., 1989a). The highest sensitivity occurs at 485 nm, with a threshold of about 10-11 W m-2, but visible light up to 680 nm in wavelength is quite effective (Chen et aI., 1993). There are two extraordinary aspects to this response; its extremely low threshold, about 100 times lower than the threshold for phototropism, and the effectiveness of the green to red spectral range. This response and an inhibitory effect of red light on the blue-light response (Loser & Schafer, 1986) contrast with all the other photoresponses, which cut off abruptly just above 500 nm.

TROPISM TO ULTRA VIOLET RADIA nON

The macrophores grow away from ultraviolet sources. This response was believed to be mediated by the blue-light pathway, since blue-light receptors are expected to absorb ultraviolet radiation as well. The changed direction was attributed to the presence in the macrophores of abundant gallic acid, whose high ultraviolet absorption would effectively shelter the distal side from stimulation (Delbruck & Shropshire, 1960). In keeping with this view, mutants isolated because of defective tropism to ultraviolet radiation exhibited defective tropisms to blue light and were affected in known mad genes (Campuzano et aI., 1994).

In fact, Phycomyces has a separate sensory system responsive to ultraviolet C radiation; but not to blue light. This is supported by the isolation of lIvi mutants with normal blue tropism and defective ultraviolet tropism (Martin-Rojas et aI., 1995), and by mutants with low gallic acid content (isolated in this laboratory by D. Weinkove). The lIvi mutant and the wild type respond equally to blue light. The mutant responds more slowly than the wild type to ultraviolet radiances (Fig. 1).

The mad mutants isolated because of their defective tropism towards blue light are more or less defective in their response to ultraviolet radiation, but this defect is only slight in the "stiff' mutants tested (A.P. Eslava, pers. comm.). This result may mean that an

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important pathway for ultraviolet tropism does not use the same growth regulation as other tropisms.

SENSORY TRANSDUCERS

Photo receptors

It is possible, but unproven, that many or all of the blue-light receptors in distantly related organisms are homologous and derive from a very early ancestor. The blue-light sensory transduction pathways of Phycomyces share some basic elements, since all the blue light responses depend on genes madA and madB. Genes madB, madC, and mad! (Ensminger et aI., 1990) are likely to be responsible for the photoreceptor for phototropism: the corresponding mutations not only render phototropism less effective than in the wild type, but change the shape of the action spectra.

The nature of the chromophore for blue photoreception has been the subject of long controversies with no conclusive results. The modification of the action spectrum in the presence of roseoflavin (Otto et aI., 1981), a riboflavin analogue, is a strong, but not decisive indication that riboflavin is part of the photoreceptor. The madA mutants have very low flavin content, but an increased riboflavin supply did not improve their defective phototropism (HoW et aI., 1992a).

The pterins are also excellent candidates for chromophores in phototropism. The mad! mutants have a consistent loss of an unidentified pterin (Hohl et aI., 1992b).

Although f3-carotene could be a blue-light receptor because of the similarities between its absorption spectrum and many action spectra, there have been many doubts as to its capacity for photochemical activity. Colour mutants, due to structural and regulatory mutations in carotene biosynthesis, reveal that f3-carotene is not required for photocarotenogenesis (Bejarano et aI., 1991) or phototropism (Bergman et aI., 1973; Presti et aI., 1977), but is essential for photomorphogenesis (Corrochano & Cerda-Olmedo, 1990). Since plant mutants deprived of carotenoids are phototropic (Galland, 1990), carotenoids are unlikely to act as photoreceptors in plant phototropism, but they seem to improve the response, probably by enhancing the contrast between the proximal and the distal sides of the coleoptile (Vierstra & Poff, 1981). Circumstancial evidence (Quinones & Zeiger, 1994) implicates zeaxanthin in phototropism in Zea mays coleoptiles (see Briggs, this volume).

Mediators and effectors

The players in the transduction of the signals may include the GTP-binding proteins and the H+ -ATPase present in the plasma membranes of Phycomyces (Rausch et aI., 1988; Hasunuma et aI., 1993; Ashktorab and Cohen, 1994).

Blue light causes a hyperpolarization of the mycelial hyphae, probably mediated by the action of a H+-ATPase. The result is an acidification of the cell (by 0.3 pH units). The observation that mutants in genes madA, madB and madC are defective in this response suggests that hyperpolarization is an early process leading to phototropism (Weiss & Weisenseel,1990).

Phototropism depends on the regulation of chitin biosynthesis and breakdown, as shown by the in vitro activation of chitin synthesis by blue light and by the study of some "stiff' mutants (Herrera-Estrella & Ruiz-Herrera, 1983). The regulation of growth and the activation of chitin synthetase depends on calcium ions and calmodulin (Ruiz-Herrera et al., 1990). Adaptation to the dark is slightly accelerated by calcium ions (Sineshchekov & Lipson, 1992), while trifluoperazine, an inhibitor of calmodulin, inhibits growth and phototropism (Valenzuela & Ruiz-Herrera, 1989). Calcium ions are localised in the endoplasmic reticulum, vacuoles, and mitochondria of the macrophores (Morales &

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-100 A

..... -80 Cl c»

:2-C) -60 c ;; c c» -40 III -0 c» -20 'EI c

"" 0

20 0 5 10 15 20

Time [min)

B

Figure 1. A. Negative tropism of a wild type and an uvi mutant macrophore during unilateral ultraviolet irradiation (277 nm, 3xlO·3 W m·2) . The mutant shows a longer latency than the wild type. B. Responses of wild type (left) and mutant (right) sporangiophores to visible and ultraviolet radiations. A culture with mature sporangiophores grown under zenithal white light was exposed successively to visible light for 140 min and to ultraviolet radiation (277 om) for 30 min, both coming from the left of the figure.

Ruiz-Herrera, 1989), but the concentrations in the plasma membrane or the cell wall, sites of the photoreceptors and the chitin fibers, respectively, were inappreciable. Detailed electron micrograhs of the macrophores failed to detect specialised structures that might be involved in sensory transduction (Morales & Ruiz-Herrera, 1993). The distribution of microvesic1es(chitosomes) in the growing zones of young bending macrophores is asymmetrical: there are more on the proximal side, the one that grows the least (Morales & Ruiz-Herrera, 1990). The increased demand for chitosomes posed by phototropism seems to be met bY'a faster consumption, rather than an increased production, of chitosomes.

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22l. Sineshchekov, AV. & Lipson, E.D. (1992). Photochem. Photobiol. 56: 667-675. Steinhardt, AR, Popescu, T. & Fukshansky, L. (1989). Photochem. Photobio!. 49: 79-87. Tsuru, T., Koga, K., Aoyama, H. & Ootaki, T. (1988). E,,'P. Mycol. 12: 302-312. Valenzuela, C. & Ruiz-Herrera, 1. (1989). Current Microbiol. 18: 11-14. Vierstra, RD. & Poff, K.L. (1981). Plant Physio!. 68: 798-801. Weiss, 1. & Weisenseel, M.H. (1990). 1. Plant Physiol. 136: 78-85.

299

WHAT CAN ERRORS CONTRIBUTE TO SCIENTIFIC PROGRESS?

Wolfgang Haupt

Universitat Erlangen-Niimberg, Germany

Although scientific progress depends on precise and correct experiments, occasionally errors in experiments can also be constructive and contribute to success. Examples of this apparent paradox will be given in this paper, using light control of fern-spore germination as the respective system.

Spores of Dryopteris filix-mas have an absolute requirement for light, and it was already established by Mohr (1956) that the sensory pigment is phytochrome with its low fluence-rate response. Thus, the first step after light absorption is transformation of the red­light absorbing form Pr of phytochrome into the far-red absorbing form Pfr. Since the latter is known as the physiologically active form, it can be considered as an internal signal which eventually controls germination. If the transduction of this signal to the terminal response is to be analyzed, it is necessary to separate, to identify and to characterize certain steps in this transduction chain, and this was the scientific question, underlying the investigations of the present paper. Isolation of steps in the transduction chain requires that the respective phases do not overlap too much in the population of spores, i.e. that the developmental processes are sufficiently synchronized, and this was the initial difficulty.

Far-red reversible phytochrome effects usually require a single light pulse in the range of seconds or minutes only (cf. Mancinelli, 1994). However, in his investigation on fern­spore germination, Mohr (1956) had to apply red light continuously during 24 hours. Obviously, these spores were poorly synchronized, the long irradiation time was necessary to meet all spores in their sensitive phase. In the present investigation, we succeded, with appropriate pretreatments, in inducing germination by a single pulse of light in the range of seconds or minutes. However, these results were extremely unreliable, with scattering between 20 and 60 percent germination for identical treatments (Haupt, 1985). This weak point could be traced back to shortcomings in the method, as will be shown.

The experiments had been started with the method of Mohr: Spores were sown on agar in small petri dishes (3 cm 0), the agar was dissolved in quartz-distilled water without any additions After preliminary experiments the number of petri dishes had to be increased. Thus, one stock of dishes had repeatedly been used and cleaned after each experiment, but the new stock had been thoroughly cleaned only once or twice. By good luck they could be distinguished even in retrospect, and it was found that the high germinations were correlated with the new dishes and the low germinations with the old ones (Tab. 1 ). It was concluded that the glass vessels contained traces of substances that were leached out after repeated experiments and washings, and germination was improved by these traces.

Light as an Energy Source and Information Carrier in Plant Physiology Edited by Jelmings et af., Plenum Press, New York, 1996 301

Table 1. Light induction of spore germination in Dryopteris filix-mas: initial results on the dependence on inorganic ions. 30 h after sowing on agar, red light (R) is applied. Percent germination is evaluated 5 days after sowing. Agar is dissolved in pure water or in an inorganic nutrient solution (Haupt, 1985).

Light duration Water agar Water agar nutrient agar

in old petri dishes in new petri dishes

R 12 h 70 85 96

R2min 19 58 93

Dark 2 18 71

As a consequence, in the next experiment the pure water was replaced by an inorganic nutrient solution which was expected to contain the necessary substances. As a result, high percentages of germination were obtained and could reliably be reproduced (Tab. 1). Moreover, this reliably high germination could still be found if the nutrient solution was diluted to one hundredth. Thus, traces of ions in the submiIlimolar range were already effective (Haupt, 1985). This finding called for investigation of the nature of the substances in question. A few results of Scheuerlein and his group will be mentioned below.

This first example can be summarized as follows. Uncritical use of a method from the literature without testing its suitability resulted in the initial failure of reliable results. However, disentangling the source of error ended in the discovery of the requirements of inorganic ions for germination; a new scientific problem was born from a shortcoming in designing the early experiments. Without this error, no attempt would have been made to ask for a requirement of inorganic ions.

The second error followed immediately. The experiments on water agar confirmed the early results by Mohr (1956) that almost no germination can be obtained in complete darkness. On nutrient agar, however, a significant level of dark germination was observed (Tab. 1 ), and still worse, this level was extremely unstable, ranging from a few percent up to 70%. As light effects cannot be investigated without reliable dark controls, the source of a possible error had to be searched for. Two possibilities could be considered: i) Part of the spores do not require Pfr at all. ii) The spores contain already Pfr, even if no light pulse has been applied, whatever the reason.

To test this alternative, the spores were given a saturating far-red pulse shortly after sowing, thus reverting all Pfr which might have been present in the spores. The result was a perfect zero germination (Tab.2). Thus, the spores cannot germinate without Pfr, and this is proof for the second alternative (Haupt, 1985). The question therefore arose where the spores had acquired their Pfr from. Again, two possibilities could be envisaged: i) During spore development on the mother plant, Pr was phototransformed to Pfr; some of this was conserved during the drying period, and thus was still found in the rehydrating spore after sowing. ii) The spores obtained light after sowing, which had been overlooked so far.

To test this alternative, the method had to be critically reconsidered. So far, the spores were always sown in day-light and put into darkness after between a few minutes and half an hour. This appeared adequate, as, according to Mohr (1956), red light becomes effective only if applied after a preinduction period of about 24 hours following sowing. However, this late acquirement of light sensitivity had been reported for spores sown on pure water agar. Could this be transferred to the nutrient agar? This question has been tested with two experiments on nutrient agar (Tab.2):

302

- Spores were sown under green safe light and then put into darkness: No germination was observed.

- Spores were sown very fast in dim day-light and put into darkness between 15 and 30 seconds thereafter: Again no germination was obtained. However, substantial germination was observed after 5 min of this "sowing light" (Haupt, 1985) or sometimes even less (psaras and Haupt, 1989).

Table 2. Light induction of spore germination in Dryopteris filix-mas: Germination of "dark controls" on nutrient agar under various conditions. After Haupt (1985).

Sowing condition Daylight 15-30 min Daylight 15-30 min Daylight 15-30 min Daylight 0.5 min Green safelight 15 min

Far red (10 min), Time after sowing no

after 6 h after 18 h

no no

Germination (%) 71 o o o o

Thus, inorganic substances as contained in the nutrient solution make the spores sensitive to light within minutes after sowing. However, the important thing is: this early light signal is fully reversible by far-red until 18 to 24 h after sowing (cf Tab.2). Thus, the information of the light signal is still stored in pfr after that time, and this Pfr has not yet acted. Obviously, the cell is not yet prepared to accept and to process this internal signal Pfr. Development of the cell's competence to react to pfr requires about 24 hours; this development is started by sowing and proceeds in the preinduction phase independent of whether or not Pfr is already present (Haupt and Psaras, 1989).

Thus, the second error resulted in drawing the attention to the distinction between sensitivity of phytochrome to light and to competence of the cell to respond to Pfr. This distinction is a well-established fact in other systems (Mohr, 1983). In fern spores, the former is acquired within minutes after sowing and hence is probably a pure physical effect, due to hydration of phytochrome, whereas development of competence to Pfr requires many hours and is assumed to be a biochemical effect, locked to dark processes that start with sowing. Both, the physical and the biochemical processes occur in the apparently dry spore, which does not show any sign of swelling even after days in darkness. Yet, there must be a limited water uptake, just enough to hydrate the phytochrome and those cell compartments in which competence to Pfr develops, i.e., where factors are provided that are required for the action ofPfr.

In conclusion, hydration appears to proceed in two steps. The first step is independent of light, it is strongly limited and therefore probably restricted to the outermost regions of the spore. The second step requires a light signal and results in full hydration of all organelles and compartments. In addition, the sensitization of phytochrome and the development of competence to pfr in a spore hydrated only superficially is one more piece of evidence for phytochrome and its primary reaction partner(s) being localized close to the cell surface, at least in fern spores.

There is an additional conclusion that can be drawn from the results. It has been shown that Pfr, generated by red light shortly after sowing, remains remarkably stable during a period of about 20 to 24 hours until the cell has become competent, i.e., during the preinduction phase; the half time of its disappearance by dark destruction or dark reversion amounts to many hours (Haupt and Filler, 1988), i.e., much longer than in phytochrome I, the phytochrome species of etiolated plants. Accordingly, the spore phytochrome (as far as control of germination is concerned) belongs to the stable phytochrome, the phytochrome II family (cf Quail, 1994).

303

On this basis, characterization of early processes after sowing can be started, viz., the preinduction phase and the "coupling phase", when pfr starts the transduction chain (called "coupling ofPfr to the transduction chain"). This coupling phase can be quantified by far-red reversibility kinetics: After a red-light pulse, Pfr is allowed to act for various times, after which it is withdrawn by applying a far-red pulse.

In a first experiment, these kinetics are run 24 h after sowing. Fig.l (second curve) shows that until about 4 h the effect of red light is fully reversible, Pfr has not yet coupled in any of the spores. Afterwards increasing percentages of spores escape far-red reversibility, and far-red becomes completely ineffective after about 20 h (not shown in the Figure), coupling of Pfr is now completed, and the transduction chain has escaped Pfr control in the whole population. If the red-light pulse is given 48 h after sowing, the "lag phase" of 4 h, i.e. the coupling time of the "fastest" spores, is the same, although beyond 4 h the slope of the curve is steeper (Fig. 1, upper curve). Thus, the preinduction phase is close to saturation after 24 h. If, in contrast, the preinduction phase is shortened, the coupling kinetics are delayed accordingly (Fig. 1, lower curves). This confirms the conclusion that a certain state of competence has to be reached before coupling can begin.

100

~ c 0 50 ~ c E Ii; 0)

0 4 6 8 10 12 14 16

llt [hl

Figure 1. Coupling curves (escape curves) of Dryopteris filix-mas as depending on the time of irradiation after sowing. The protocol is indicated in the inset, with Rand FR denoting red and far-red light, respectively. The controls without FR amount to between 80 and 90 percent. Partly after Haupt & Psaras (1989).

With these results we have obtained kinetics of the preinduction phase, in which the conditions required for coupling are established, and of the coupling phase, in which pfr interacts with its reaction partner. All subsequent processes are preliminarily summarized as postcoupling phase.

For further characterization, an attempt can be made to find external factors that affect these phases differently. According to Mohr (1956), a modest elevation of the ambient temperature can strongly inhibit the light-controlled germination without damaging the spores irreversibly. We now ask whether this inhibition is phase specific. The respective experiments are easy to design, but again liable to errors, as will be shown.

For technical reasons, the experiments had been done with the closely related species Dryopteris paleacea (Haupt, 1990). The spores were induced by light under optimal conditions, i.e. after a 48 h preinduction phase has generated full competence to Pfr. 22 h after the red pulse, far-red is given to terminate the presence ofPfr; under these conditions, pfr must have coupled in all spores. Besides of the standard temperature of 22°C, the whole experiment runs also at 27 or 32°C. As Fig.2 (left column of data, bottom) shows, germination is almost completely blocked, confirming the earlier results of Mohr. If the temperature is raised only during one of the three phases, the temperature effect is clearly restricted to the coupling phase, in the preinduction and postcoupling phases no effect is found (Fig.2).

304

germination ("I -"'9 FR Opcl .. otftl Dldlx·mos ,

27 'C ~ 2rc1 92 71

~ 87 44

~ 15 40

~~ 91 71

2TC 7 9 22"C

Figure 2. Phase-specific inhibition by elevated temperatures on light-induced spore germination according to the protocols at the left side. 48 h after sowing, spores were given a red pulse (R), and the Pfr action terminated, after 22 h, by far-red (FR). The temperature is indicated along the time axis. Hatched areas denote presence ofPfr. In the data columns, inhibited germinations are indicated by italics.

When the experiment was repeated with Dryopteris filix-mas, a puzzling result was obtained (Haupt, 1991): The well-defined restriction of the effect to the coupling phase disappeared, the preinduction phase proved to be sensitive to temperature as well (Fig.2, right column). As it appeared unlikely that this should be a fundamental species-specific difference, it was assumed that once more an error was involved.

This assumption has been tested on a broader experimental basis. The coupling phase has been extended or shortened so as to obtain coupling kinetics, and the temperature has been raised during a 48 h preinduction phase. In both, D.filix-mas and D.paleacea the coupling kinetics are strongly affected by the previous temperature as long as the coupling time is sufficiently below saturation (Fig.3). Qualitatively the pairs of coupling curves with and without temperature pretreatment are similar in both species; there is only a quantitative difference: The effectiveness of the pretreatment is lost earlier in D.paleacea than in D.filix­mas. In the initial experiment, by chance a coupling time had been chosen for which the difference between both species is most spectacular and thus was erroneously suggested to be qualitative.

sowing R FR , , ", , 22'C

~22'C 100

~ .-~~: c .~ / <i;'C 0 :g 50 c , ,

D.filix-mas E ~ /

Ol

/ ---- --. a t--.

100 6t[hl 9 16 24 CD

~ '-.>~F' ..... -_ ... -

c 22'C /3ZC 0 :g 50

, c ,

D.pa/eacea E ,

~ , Ol , ,

a _ • ____ .1

I--,

Figure 3. The effect of a temperature pretreatment on the coupling curve. Protocol on top, with Rand FR denoting red and far-red. R was given 48 h after sowing. Partly after Haupt (1991).

305

sowing 22·C n·c

dlWelopmenl

01 compe h!nCe

rOP'r

l 32·C

T - RED-J n ·c

coopling deloy

I Figure 4. Schematic presentation of the early phases after sowing and their interpretation along a time axis from above to below. The increasing widths of the diagrams after sowing indicate development of competence, which is completed at the arrowheads. Coupling is indicated by bold lines. In the right diagram, the temperature pretreatment for, e. g., 24 h (shaded) results in partial loss of competence; reestablishing of full competence causes delay in the start of coupling. Left diagram: control without temperature pretreatment.

When in a preinduction phase of 48 or 72 h the temperature treatment was restricted to 24 h, inhibition was found only for an elevated temperature during the terminal 24 h. It was concluded that the temperature reversibly inactivates a factor that is produced in the preinduction phase and later on required for coupling (Haupt, 1991). The nature of this factor remains to be elucidated.

It can now be interpreted why this inhibition depends on the coupling time (Fig.4): After the temperature treatment has inactivated (or reduced) the essential factor, this latter has to be reestablished, i.e. the preinduction phase has to be prolonged, its processes extend now into the time after the red-light pulse (right scheme in Fig.4). Thus, under this condition coupling can start only with a delay, just as in those experiments where the preinduction phase was too short (cf Fig. I ). This delay becomes obvious if we don't allow for coupling sufficiently long. If, however, coupling is possible for a fully saturating time, any after-effect on the coupling kinetics remains hidden, and this was the set-up of the original experiment, which gave the erroneous result.

To summarize the error once more: use of saturating coupling times resembles an end­point determination, which neglects any possible influence on the kinetics and thus causes a serious loss of important information. Accordingly, the temperature effect on the preinduction phase could be found only with comparative coupling kinetics.

After this message had been learned, it appeared advisable to re-examine the obvious insensitivity of the postcoupling phase to elevated temperatures, i.e. to extend also these experiments to protocols with non-saturating coupling periods. Thus, coupling curves were run, and the temperature was raised after the far-red pulse (Haupt, 1992). Similarly as by the temperature pretreatment, the coupling curve is shifted by the aftertreatment, too (Fig.S). However, what has in fact been shifted cannot be the coupling process proper, as it is hardly conceivable that escape from far-red reversibility can be abolished in retrospect.

Before the result and the source of error can be interpreted, the terminology of the phases has to be reconsidered. Till now, the coupling phase was defined on the basis of the whole population; it was taken as terminated when the last spore had completed its coupling. However, full understanding requires us to consider the individual spore, as schematically shown in Fig.6. Four to six hours after the red pulse, a low percentage of spores has definitely coupled, they germinate after withdrawal ofPfr and thus have escaped pfr control.

306

R FR I 'C32"C

--4-~-22"C fit

o 6 9 12

fl.t [hI

Figure 5. Apparent shift of the coupling curve by a temperature treatment after the action ofPfr. The protocol is indicated in the inset, with R and FR denoting red and far-red, respectively. The "control" denotes a preparation without FR. After Haupt (1992).

RED oj 01 01 01 01

4h ,J coupling

12h postcoupling

tim~ processes

16hl 20hl

fast medium slow

Figure 6. Schematic presentation of individual spores, covering the range from the extremely fast to the extremely slow couplers, which require about 4 and 20 h, respectively, for coupling (bold lines).

For all others, the information is still contained in Pfr, as proven by the far-red reversibility. With increasing time, more and more spores terminate their coupling period and start the subsequent processes. Finally, 20 h after red, nearly all spores have coupled. At this late stage the earliest spores have already entered their postcoupling phase 14 to 16 hours before. Thus, at any time after the red-light pulse, we always have a mixture of stages; i.e., coupling, early and late postcoupling phases are shifted to each other in the population.

On this basis, the new results are easy to explain. The shift of the coupling curve as an effect of the temperature treatment after coupling means that during a limited period in the early postcoupling phase there is still sensitivity to temperature. All those spores are inhibited which are in this early part of the postcoupling phase at the time of temperature treatment. As the curve is shifted by 4 to 5 hours, this early period should last about that time. Consistently, the effect is found as well, if the temperature is raised, after far-red, only for three to six hours; but if the onset of temperature treatment is delayed to 6 h after far­red, it remains ineffective (Haupt, 1992).

Thus, for each spore there is a period with an average of about 5 hours after coupling, in which it is still sensitive to temperature. Whenever the time between red and far-red was below saturation for the population, there were always subpopulations that coupled as late as in the period of 5 hours immediately preceding the far-red pulse, which hence had not yet passed the first 5 hours of the postcoupling phase, in which they were affected by the temperature treatment.

To sum up, correcting the erroneous conclusion about insensitivity in the postcoupling phase demonstrated the necessity of distinguishing more carefully between statements for

307

the whole population and those for the individual spore. It has been shown that the so-called coupling phase of the population in fact consists of a mixture of phases on the basis of the individual spores, the time courses of which are spread over at least 14 hours. Moreover, it resulted in a more detailed knowledge about the postcoupling phase: The coupling phase (of the individual spore) is followed first by a 5-hours "post coupling phase A", which is sensitive to temperature, and then by a "postcoupling phase B", which no longer is affected by elevated temperatures.

The improved scheme derived from these results and interpretations is shown in Tab.3, supplemented by a few results of Scheuerlein and his group. Without external calcium in submillimolar concentrations, there is hardly any germination possible. Interestingly, however, the competence of the individual spore to the calcium effect is strongly limited in time. This time window of only one hour or even less is located about 20 h after coupling (Durr and Scheuerlein, 1990). Thus, the temperature-insensitive part of the postcoupling phase can now further be subdivided into one (B) preceding the calcium phase (C) and one following it (D). In this latter, parameters of germination very soon become detectable, and the reaction chain leading to them may be branched, as concluded from additional effects of external factors, including requirement of additional light and/or calcium signals (asterisk in Tab.3; Scheuerlein, personal communication).

Another phase-specific (and thus phase-characterizing) factor is nitrate, again effective in the submillimolar range. Although there is no absolute requirement, nitrate can strongly enhance a non-saturating light effect during the coupling phase (of the whole population), and to some degree a few hours before and thereafter (Haas and Scheuerlein, 1990, 1991). Thus, it is tempting to correlate the time courses of nitrate and temperature effects with each other, but more detailed comparative experiments are still needed.

CONCLUDING REMARKS.

Four examples have been presented, showing erroneously designed experiments, which nevertheless turned into constructive tools in solving scientific problems, or which have established new problems for future research. These examples can be summarized as follows:

- Working with an inappropriate medium for sowing resulted in the discovery of the effects of the nutrient solution, and opened the door for the presently ongoing efforts to analyze the action mechanism of calcium and nitrate.

- Lack of care during sowing drew the attention to the distinction between fast acquirement of light sensitivity and slow development of competence for the action of the resulting internal signal Pfr; it furthermore led to the knowledge about biphasic hydration of the spore.

- Uncritical use of saturation in order to keep one factor constant caused rethinking about the role of kinetics, when phase specificities are to be analyzed.

- Unawareness of the difference between the coupling phase of the population and that of the individual spore eventually improved the understanding of problems in a population with an all-or-none response. This together with the previous error improved the theoretical background for future research.

This 'latter point may stimulate, in addition, rethinking about the difference between graded and all-or-none responses. This difference might not always be as fundamental as usually thought: theoretically, a graded response to Pfr in a tissue or in an organ could be the result of all-or-none responses of the individual cells, whose sensitivity to Pfr or its time course would be scattered over a wide range as in a population of spores. It is a challenge to find an example of such a theoretically predicted "pseudograded" response.

308

Table 3. Schematic model of the sequence of phases in the light-induced spore germination ofDryopteris filix"mas. Pr, Pfr: red and far-red absorbing form of phytochrome, respectively. +L\T: increase of temperature by 5 or lOoC. *: additional light and/or calcium required.

SOWING Phytochrome Pr is insensitive to light

.J.. 5 min hydration ofPr

.J.. Phytochrome Pr has become sensitive to light

.J.. preinduction phase 24 h

.J..

development of competence

Spores have become competent to respond to Pfr; the stage of competence is sensitive to elevated temperature (+ L\ T)

LIGHT PULSE (minutes, seconds or milliseconds)

.J.. coupling phase 4 to 20 h +L\ T inhibits, nitrate enhances

(the time requirement depends on the individual spore)

.J.. Pfr has started the transduction chain,

a temperature-sensitive product ofPfr action has been formed .J..

postcoupling phase A 5h .J..

sensitive to +L\T

The first product of Pfr action has acted and can no longer control the subsequent cascade of events

.J.. postcoupling phase B 15 h

.J.. not affected by +L\T

Spores require external calcium

postcoupling phase C .J.. 1 h .J..

calcium phase

spores have lost competence to external calcium; if calcium has not been available, further development stops

.J.. postcoupling phase D hours and days branching of transduction chain

.J.. The first signs of germination become visible, e.g.

chlorophyll formation rhizoid initiation neoformation ofplastids* rhizoid growth*

309

REFERENCES.

Diirr, S. & Scheuerlein, R. (1990) Photochem. Photobiol., 52:73. Haas, C. 1. & Scheuerlein, R. (1990) Photochem. Photobiol., 52:67. Haas, C. 1. & Scheuerlein, R. (1991) 1. Plant Physiol., 138:350. Haupt, W. (1985) Planta, 164:63. Haupt, W. (1990) Photochem. Photobiol., 52:57. Haupt, W. (1991) Photochem. Photobiol., 54:811. Haupt, W. (1992) 1. Plant Physioi., 140:575. Haupt, W. & Filler, E. (1988) Bot. Acta, 101:160. Haupt, W., & Psaras, G. K. (1989) 1. Plant Physiol., 135:31. Mancinelli, A. L. (1994) in: "Photomorphogenesis in Plants" 2nd edition, (Kendrick, R.E. & Kronenberg,

G.H.M., eds.). p.21I, Kluwer, Dordrecht Mohr, H. (1956) Planta, 46:534. Mohr, H. (1983) in: "Encyclopedia of Plant Physiology" New Series vol. 16, (Shropshire, W. jr.& Mohr, H.

eds.), p. 336, Springer, Berlin,. Psaras, G. K. & Haupt, W. (1989) Bot. Acta, 102:222. Quail, P. H. (1994) in: "Photomorphogenesis in Plants" 2nd edition, (Kendrick, R.E. & Kronenberg, G.H.M.

eds.), p.71, Kluwer, Dordrecht,.

310

INDEX

Action spectroscopy, 275, 279, 280, basic assumption, 276, foundamental relationship, 276

Action spectrum 275-278, 280, 282, distortion, 277

Action spectrum, 188, 190, 191, 193,295,297 Adaptation, 296, 297 Algae, 237 Aquatic ecosystems, 231, 232, 234 ATP synthase, 17

Bacteriopheophytin, 78 Bacteriorhodopsin, 187, 188 f3-carotene, 286, 288, 293, 297 Biomass productivity, 231, 232 Blue light, 285, 286, 288, 293,294, 296, 297

photoreceptor, 160

Ca ++ channel inhibitors, 266 Calcium, 308, 309 Calcium ions, 204, 205, 211, 213 Calmodulin, 204, 205, 206, 211, 213 Canopy architecture, 221, 222 Canopy gaps, 225 Carotenoids, 149 CD

molecular complexes and small aggregates, 127

molecules, 127 Chi rally organized macroaggregates, 129 Chlorophyll fluorescence, 99, 113 Chloroplast, 197,200,205,211 CIDS (Circular Intensity Differential Scattering),

129 Circular dichroism spectroscopy, 126 Conidiation, 286, 287, 288, 289, 290 Coupling factor, 24 CP24,44,46,47,49, 51, 53, 54, 56,60

structure 51 CP26,44, 46, 47, 49, 51, 53,54,60

structure 51 CP29,44,46, 47, 49, 51,54,60

structure 51 CP43, 43, 44 CP47, 43, 44 Critical distance, 37 Crystallography, 77 Cyanobacteria, 189, 190, 191

Cycloheximide, 286 C)iochrome bf complex, 24 Cytochrome b559, 84

Cytochrome c, 80

~pH importance in qE, 101

D1 protein degradation and repair, 93

DCMU,188 Deep funnel, 69, 72 Delocalized proton pools, 10 Dephosphorylation, 205, 206, 207, 208, 209 Dichroism, 295 Dinoflagellate, 237, 238 Dipole-dipole interaction, 32 Dipole approximation, 33

Electron transport, 1,5-10, 12-15 Electron-phonon coupling, 66 Emerson effect, 5 Energy equilibration, 65 Energy transducers, 185 Energy transduction, 17 Errors, 301, 304 Evolution

of the chlorophyll alb antenna proteins, 54

Fern spore, 204, 303 Flavins, 149, 152, 153, 155 Fluorescence, 256 Focusing, 246, 248 Forster theory, 32 Fungi, 285, 286,290

Gprotein, 201, 206, 209, 210, 212, 213, 214, 266 Gene expression, 285, 286 Germination, 301, 302, 303, 304, 305, 308, 309 Gravitaxis. 237, 240 Gravitropism, 294, 295 GTP-binding protein, 209, 210, 211, 212, 213,

297

H+-ATPase,297 High irradiance, 237,239 Homogeneous broadening, 66, 69 Hydrophobin, 287, 288

311

lnductiveresonance,32 Inhomogeneous broadening, 66, 67 Interaction energy, 32, 33

Kubelka-Munk theory, 253, 254

Leafhairs, 244, 245 Leaflet movements, 160 Lense.ffec~,246,247,248 LHCI, 41, 42, 44, 47, 54 LHCII, 42, 43, 45, 46, 47, 48, 49, 50, 51, 53, 54,

59,60 structure, 49

Light gradien~, 247, 248, 253-255, 257 Light penetration, 232, 236, 253 Light piping, 256 Light-induced reversible structural

rearrangements, 133 Localized proton pools, 10

Macroalgae, 231, 232, 236, 239, 240 Macrophore, 293-298 Mad mutan~, 295, 296 Marcus theory, 77, 79 Mecisms, 293 Microphore, 293 Molecules mutual orientation, 34 Motile reactions, 279 Motility, 235, 236, 239 mRNA, 285, 287, 288 Mutan~

in photomorphogencsis, 162 in the phototrophism pathway, 163

Nitrate, 308, 309 Nonphotochemical quenching, 47, 48, 51, 99,

113 Non-hemic iron, Fe2+, 82 Nuclear genome, 41, 54

Oat, 201-203, 205, 206, 210, 211, 213 Overlap integral, 33 Ozone, 232, 235, 239 Ozone layer, 137, 138

P-700,85 dimer,78 triplet state, 81

P680 dimer,83 dry matter production, 95 electron donor to P680+ ,83 in the natural environment, 94 monomer, 83 photoinhibition, 89 triplet state, 83, 92

PPUR, 220, 225, 226, 227,228 Pfr, 197-199,201-204,206,208,209,212-214,

301-309 Phoborhodopsin, 187 Phosphorylation, 204-210, 212, 213 Phosphorylation, 24 Photoacoustic, 114

312

Photocarotenogenesis, 285, 286, 288, 290, 293, 295,297

Photocatalysis kinetic model, 279

Photochemical quenching, 113 Photoinduced behavior, 186 Photoinduced electric signals, 265, 266, 269 Photoinducible genes, 287, 290 Photoinhibition, 15 Photokinesis, 186, 188, 189, 190-193,264 Photomecism, 295, 296 Photomorphogenesis, 197, 198,200,202,243,

255-257,285-287,293,295,297 Photomovemen~, 147, 149, 186,264 Photomovements, 279 Photophobic responses, 152, 186-191, 193,264 Photophosphorylation, 8, 10 Photoproduct formation

kinetic model, 278 Photoprotection by pigments, 248 Photoreception pigments, 265 Photoreception, 147, 185,297 Photoreceptor pigments, 149, 152-155 Photoreceptor, 197, 198,200 Photoresponses, 285, 288 Photosensing ,148, 149, 155 Photosensory transduction, 152 Photosinthetic unit, 2, 5 Photosynthesis, 233, 237, 240

light response, 89 quantum efficiency, 90

Photosynthetic bacteria, 188, 189 Photosynthetic electron transfer, 17 Photosynthetic oxygen production, 239, 240 Photosynthetic pigment, 239, 240 PhQtosystem I, 5-8, 10-13,23,41,42,44,45,60,

65,72,73,84 core complex, 42 primary electron donor, 76 reaction center, 23

PSI-LHCI, 44 Photosystem II, 5-8, 10-15,41-47,49,56,59,60,

65, 66,69-73 complex, 20, 91 connectivity 116 core complex, 41, 42, 43, 46, 49, 60 photodamage, 84, 91 photoinactivation ofthe acceptor side, 92 photoinactivation of the donor side, 92 polypeptides, 82 reaction center, 20, 47, 81 regulatory phenomena, 13

Phototaxis, 149, 153, 154, 186-188, 190-193, 237,239,264

Phototropism, 161,293-297 phosphoprotein involved, 164

Phytochrome 160, 197-200,301,303,309 Phytochrome

gene family, 198,200 phototransformation, 203 protein, 201

Ph)10plankton, 231, 232, 237, 239, 240 Pigment binding protein, 41, 56

Pigments, 185-187, 189, 190, 192, 193 Pi10boloides motants, 294 Plastid genome, 41 Pr, 197-200,202-204,206,208,209,214 301

302,309 ' , Primary photoreactions, 147 Promoter, 288, 289, 290 Protein kinase, 205-209, 211-214 Proton electrochemical potential, 23 Protoplasts, 204-207, 210, 211 Pterins, 149, 152, 153, 297 Purple bacteria, 76

QA

Os

electron transfer, 79 properties, 79

electron transfer, 79 properties, 79 reaction center, 75

qE,100 sites of, 102

qI, 100 qT,IOO

R:FR ratio, 221-224, 226-228 Radiative transfer theory, 253 Ras, 209-214 Reciprocity, 171, 172 Refraction, 175, 176, 180, 182 Refractive coefficient, 36 Regulation of energy distribution, 11 Response

blue light regulated, 165 folding, 160 high fluence, 160 to light direction ,161 to light duration, 161 low fluence, 160

Retinal, 265 ~odops.in, 148-150, 152-155, 185, 187, 188,265 Riboflavlll, 295, 297

Scattering, 243, 247, 248, 250-256, 259 Sensors, 225 Sensory rhodopsin, 150, 151, 187 Sensory transduction, 148, 264-266, 288, 297 Seston, 232, 238 Shallow funnel 69, 71-73 Sieve effect, 258, 259 Signal transducers, 185 Signal transduction, 197-205,207-214 Singlet ox)'gen, 92 Sky condition, 221, 223 Slow trapping

physiological role, 73 in PSII, 72

Solar elevation, 221-225 Solar radiation, 232, 234-237, 239, 240 Solar tracking 161 Spatial absorption gradient, 174 Spectral broadening, 66 Spectral heterogeneity 65

Spectroradiometer, 234, 235 Sporangiophores, 293, 298 Spore, 301-303 State transitions, 100, 116 Stokes shift, 34 Sun tracking, 179 Sunflecks, 160, 223-226,228

Temporal absorption gradient, 174 Temporal changes, 226 Thermal radiometry, 114 Thylakoid membrane

lipid composition 19 Tissue anatomy, 254 Tomato aurea mutant, 205, 211 Torulene, 286 Toxin, 205-207, 210-212 Transduction chain, 169-172, 178, 181 Trifluoperazine, 297 Tropism, 293, 295, 296

UV radiation, 137, 138, 142,239,296,298 within plant tissue, 140

UV PAR,143 photoreceptors, 138 role of C02, 142 role of nutrient availability, 142 role of water, 142

UV-A, 232, 234, 239, 240 UV-B, 219,227,228,231,232,234-236,239,

240 action spectrum, 281 degradation ofDl, 139, 143 dose-response curves, 281 at the ·trascription level, 139

Variations of antenna size, 115 Vertical distribution, 232, 237, 238, 239

Water column, 232-240 Waxes, 244

Xanthophyll cycle, 104

Zeaxanthin, 90, 104,297

313