chlorophyll: a symptom and a regulator of plastid development

New Phytol. (1997), 136, 163-181

Tansley Review No. 92Chlorophyll: a symptom and a regulator ofplastid development

BY HOWARD THOMAS

Cell Biology Department, Institute of Grassland and Environmental Research, PlasGogerddan, Aberystwyth, Ceredigion SY23 3EB, UK

{Received 2 December 1996)

CONTENTSSummary'IntroductionI.

II.III.

IV.

V.

VI.

VII.

Chlorophyll and related tetrapyrrolesChlorophyll-protein complexes1. Light-harvesting and core complexes2. Resolution of the molecular structure of

LHCChlorophyll biosynthesis1. The C5 pathway2. Degradation of C5 pathway

intermediates3. Genetic perturbation of chlorophyll

biosynthesis4. Early light-inducible proteinsNADPH-protochlorophyllideoxidoreductase1. General features of POR regulation2. PORA and PORB3. POR and plastid differentiation4. POR degradation5. Chlorophyll synthesis in the dark6. POR in relation to expression and

turnover of other plastid proteinsAssembling chlorophyll-proteins1. Integration of LHCP2. Role of chlorophyll in stabilizing

nascent LHCPChlorophyll turnover at the steady state

163164164164164

165166166

166

167167

168168168169170171

171171171

171172

1. Metabolism of labelled chlorophyll2. DI protein turnover3. Sun-shade acclimation4. Phaeophorbide in PS II

VIII. The plastid family treeIX. The pathway of chlorophyll catabolism

1. The first three reactions2. Phaeophorbide a oxygenase3. Gene expression in chlorophyll

catabolism4. Subcellular organization of chlorophyll

catabolism5. Interconversion of chlorophylls a and b

X. Chlorophyll and associated proteins insenescence1. Thylakoid disassembly in gerontoplasts2. Mobilization of chlorophyll-proteins in

senescence3. Chlorophyll breakdown: catabolism or

detoxification ?XI. How does chlorophyll stabilize associated

proteins ?1. Protein conformation2. C^^tochrome/3. Protease inhibition by tetrapyrroles?

XII. ConclusionAcknowledgementsReferences

172172172173173173173173

174

174175

175175

175

176

176176176177177177177

SUMMARY

The metabolism of chlorophylls and related tetrapyrroles directly infiuences, and is mfiuenced by, the protems andcell structures with which they are associated. During net accumulation, de-greening and at the steady state,chlorophyll and its derivatives are important elements in the post-translational regulation of the expression ofgenes for chloroplast proteins. At the same time, they represent potential photodynamic hazards against whichgreen cells need to have protective mechanisms. This review deals with genetic, chemical and environmental

AXvS^r^ll!tS.^nlvuHnate;CC,corecon.plex; ELIP eaH.cataboHte; LHC, light-harvestmg complex; LHCP, Iight-harvestmg chlorophyll-bindmg protem; LSD, arge s u b u n ^ N C C non-fluorescent chlorophyll cataboHte; Nfl, Norflurazon; PhaO, Phaeophorbide a oxygenase; PLB, prolame lar body, POR, iNADPH-protochlorophylliie oxidoreductase; Pr/Pfr, red/far-red absorbing forms of phytochrome; PS, Photosystem; Rubisco, ribulose-1,.-bisphosphate carboxylase/oxygenase; SSU, small subunit; WRL, weak red hght.

164 H. Thomas

perturbations of chlorophyll biosynthesis that impact on protein stability, membrane organization andsusceptibility to photodamage. NADPH-protochlorophyllide oxidoreductase is considered in detail as a pigment—protein regulating, and regulated by, chlorophyll metabolism. The question of the extent and significance ofchlorophyll turnover at the steady state is addressed, with particular emphasis on the dynamics of the photosystemn reaction centre. The pathway of chlorophyll catabolism is described, along with its interrelationship withprotein mobilization in chloroplast senescence. Finally, the structural basis of pigment-protein interaction andstability is examined, and the discussion ends by expressing some general thoughts about the control of proteinlifetimes in the living cell.

Key words: Chlorophyll a and b, tetrapyrroles, chlorophyll-protein complexes, chlorophyll biosynthesis, turnoverand catabolism, plastids.

I. INTRODUCTION

Chlorophyll is familiar. The light absorbed andrefiected by green plant tissues closely matches thelight sensitivities of the eyes of Old-World primates,including Homo sapiens. There is good evidence thatthe L-M (red—green) cone system has evolvedthrough spectral tuning by leaf reflectance, a domi-nant feature of the visual environment (Osorio &Bossomaier, 1992). Its very familiarity makes it easyto overlook chlorophyll and to underestimate itssignificance. Of course, it is universally appreciatedthat chlorophyll is part of photosynthesis, inter-cepting light energy that ultimately powers theassimilation of COg. But chlorophyll is far frombeing merely a passive colouring agent. The messageof this discussion is that it plays a crucial part incorrectly building the photosynthetic apparatus andin the controlled deconstruction of plastids duringsenescence.

II. CHLOROPHYLL AND RELATED

TETRAPYRROLES

Figure 1 summarizes the chemical constitutionsof chlorophylls a and b and related compoundsfound in green plants. Some features of these struc-tures are relevant to the subject of the regulatoryfunctions of macrocyclic tetrapyrroles. Chlorophyllsand their proto- and phaeo- derivatives have moreor less extensive conjugated double-bond systemsand thus absorb strongly at visible (particularly redand blue) as well as ultraviolet A wavelengths.

Absorption of light quanta by chlorophyll andrelated tetrapyrroles results in excitation to thesinglet state (^Chl). If the energy of absorbed quantacannot be dissipated through photosynthetic electrontransport or some other quenching mechanism, themolecule can undergo spin-inversion intersystem-crossing to produce the much longer-lived triplet(^Chl) state. Triplet-state tetrapyrroles are highlyactive photosensitizers. Photodynamic damage canbe propagated in cells either directly by reactionbetween ^Chl and substrates such as lipid fatty acidsto produce a free radical cascade (type I mechanism)or by a type II mechanism in which reaction with Ogproduces toxic singlet oxygen (Spikes & Bommer,1991).

Chlorophyll, its biosynthetic precursors and itsbreakdown products are potentially destructive whenilluminated. This poses special problems for pigmentmetabolism during net assembly and dismantling ofthe photosynthetic apparatus, as well as at the steadystate. In the viable green cell, the structural role ofchlorophyll and the need to hold its photodynamictendencies in check are inseparable.

III. CHLOROPHYLL-PROTEIN COMPLEXES

1. Light-harvesting and core complexes

Free chlorophyll is readily isolated from green tissuesby lipophilic solvents. Gentler extraction pro-cedures, using subcellular fractionation followed byincubation of green membranes with surfactants,remove pigments as complexes with specific proteins(Ogawa, Obata & Shibata, 1966; Thornber, Smith &

CH,-<^ —CH2CH3

CH3 -7\J<^ J^)>~CH^

t-Hj

CO2R

R = H ChlorophyllideR = phytol Chlorophyll

CH,-

CH,,?

-CH2CH3

- C H ,

= CH3 Chlorophyll(ide) a

CH2 CO2CH3CO2R

R = H PhaeophorbideR = phytol Phaeophytini^ = CHo Phaeo a

= CHO Chlorophyll(ide) b * = CHO Phaeo b

Figure 1. Structures of chlorophyll and related tetrapyrroles.

CH-r

Haem

Chlorophyll and plastid development 165

stroma

Figure 2. Schematic diagram of LHCP II in relation to chlorophylls and orientation in the thylakoidmembrane. Significant amino acids are indicated by single-letter codes and sequential positions in thepolypeptide. Chlorophylls are labelled al-al and bl-h6. After Kuhlbrandt, Wang & Fujiyoshi (1994).

Bailey, 1966). The best modern procedures yield adozen or more separable complexes and little or nofree pigment. For example Photosystem (PS) I frombarley has been resolved by electrophoresis into acore complex (CC I) and at least two light-harvestingchlorophyll-protein complexes (LHC Ia, Ib). CC Iaccounts for c. 20 % of the total chlorophyll of themembrane and is composed of a major polypeptideof c. 58 kDa and chlorophyll a but not h. The LHCI complexes are made of chlorophylls a and h withapoproteins in the range 20-25 kDa (Dreyfuss &Thornber, 1994). Barley PS II gives a morecomplicated pattern, with at least three CC II andfour LHC II complexes resolved by electrophoresis.LHC l ib is the major pigment-protein complex inthe membrane, accounting for 40% of the totalchlorophyll and is composed of apoproteins of25-30 kDa and chlorophylls a and h (Peter &Thornber, 1991).

2. Resolution of the molecular structure of LHC

The nature of the association between pigments andproteins has been established in atomic detail forseveral complexes of prokaryotic origin (Papiz et al.,1996). Of the pigment proteins of angiosperms, thebest characterized is the light-harvesting complex ofangiosperm PS II, which has been resolved to

0-34 nm by Kuhlbrandt, Wang & Fujiyoshi (1994).On gentle isolation from thylakoids, LHC II isobtained as a trimer. Fach monomer comprises asingle LHCP molecule {c. 230 amino acids, relativemolecular mass 25 kDa), 12-15 chlorophylls {a and bin approx. equal molar ratio) and two carotenoids(usually lutein-but see below). Based on hydropathyplots of the derived amino acid sequence of LHCP,it had been anticipated that the protein has threemembrane-spanning helices; and studies of mem-brane orientation had shown that the C and Ntermini of the polypeptide are luminal and stromalrespectively. Flectron crystallography confirmedthese features of LHC II structure and enabled 12chlorophyll molecules and the two carotenoids to beaccurately positioned within the molecular structureof the monomer unit (Kuhlbrandt et al., 1994).

The schematic in Figure 2 shows an N terminalsegment of 54 amino acids leading to the Bmembrane-spanning helix (amino acids 55—89), anon-helical luminal loop, the C helix from residue123 to 143, a stromal loop, then the A helix (170-199)and finally the C-terminal segment on the lumenside, including a small run of helix from 205 to 214.The two-fold symmetry that gives a distinctive X-shape to spans A and B is braced by two luteins, onerunning from about residue 197 (luminal end of helixA) to 160 (stromal loop between helices A and C) and

166 H. Thomas

the other from about 48 (N terminal stromal and POR is probably constitutive. Because proto-segment) to around 100 (luminal loop between chlorophyllides do not normally build up in dark-helices B and C). incubated tissue, either ALA supply must be limiting

Distinguishing chlorophyll a from b is beyond the or products downstream of ALA are diverted tolevelof resolution of electron crystallography. Never- other fates. Studies with the tigrina series of barleytheless, spectroscopic and other evidence allows each mutants reveal the existence of at least four genesof the twelve chlorophyll molecules within the crystal concerned with regulating the flow of metabolitesstructure tentatively to be assigned an identity, between ALA and protochlorophyllide. FeedbackThese are shown in Figure 2, together with their inhibition is a possibility, since both haem andpresumptive ligands within the helices. Histidine divinyl-protochlorophyllide have been observed to(H) is the commonest ligand in other tetrapyrrole- inhibit ALA formation in vitro (Castelfranco &polypeptide complexes; here only two chlorophylls Beale, 1983). Genetic manipulation has to some{a5 and b3) are coordinated in this way. Glutamine degree simulated in light-grown plants the sup-(Q-aJ, 66), glutamate (E-fli, fl4, &5) and asparagine pressed state of the ALA to protochlorophyllide(N~a2) are other identifiable ligands (Figure 2). The pathway characteristic of etiolated tissue: transgenicdistances betw^een the individual chlorophylls and tobacco plants expressing antisense genes for thebetween chlorophylls and carotenoids are consistent ALA-forming enzymes glutamate-tRNA synthasewith known energy transfer rates. (Andersen, 1992) and glutamate-1-semialdehyde

The functions of the lutein molecules are of aminotransferase (Hofgen et al., 1994) are pale orinterest. A structural role is suggested by obser- variegated. Light causes elevated steady-state levelsvations that in vitro assembly of the complex from of niRNAs encoding some enzymes in the tetra-the separate components is absolutely dependent on pyrrole biosynthetic pathway, for example ferro-the presence of stoichiometric amounts of the chelatase (Smith et al., 1994), but depresses otherscarotenoid (Plumley & Schmidt, 1987). A light- such as the Mg chelatase component OLfVEharvesting function is considered less likely, but (Hudson et al., 1993) and POR (see Section V).special significance is attached to the efficiency with Another facet of the regulation of chlorophyllwhich carotenoids quench chlorophyll triplets. Al- biosynthesis that merits study in higher plants wasthough lutein normally does these important jobs described by Johanningmeier (1988), who foundwithin LHC II, it is not essential for photosynthesis, evidence that intermediates in the pathway influencePogson et al. (1996) isolated lutein-less mutants of the expression of nuclear genes in Chlamydomonas.Arabidopsis that have perfectly normal levels of It has been proposed that tetrapyrroles are importantchlorophylls and apparently fully functional light- elements in the signalling mechanism that co-harvesting structures. Lutein is replaced by vio- ordinates nuclear gene expression and plastid dif-laxanthin in these mutants, though this carotenoid is ferentiation (Reinbothe et al., 1996)less effective than lutein when used for in vitro

assembly (Plumley & Schmidt, 1987). The part ^ ^ , • r r ,, J ; , , 1 11 • I T T TT>^ TT • ^- Degradation of Co pathway intermediates

played by chlorophylls m assembling LHC 11 isdiscussed in Section XI. It is reasonable to conclude that substrate-level

regulation by feedback loops is essential for matchingthe supply of photodynamic precursors and end-

IV. CHLOROPHYLL BIOSYNTHESIS t-i^ J f . . ^ , .products to the capacity oi quenching mechanisms to

1 T j /-.r 1 conduct them safely into stable complexes. Implicit1. Ihe C5 pathway . .' . .

m such a scheme is a requirement for intermediatesEarly in mesophyll cell development, chlorophyll— upstream of an inhibited step to be redirected intoprotein complexes are put together. Chlorophyll catabolism, but the biochemistry of these branch-biosynthesis has a direct influence on expression of lines in the metabolic network is rather poorlygenes for plastid proteins, particularly at the post- defined. Oxidative enzymic activities able to bleachtranscriptional level. An outline of the C5 pathway of chlorophyll and precursors have been detected insidetetrapyrrole synthesis in green plants is shown in and outside immature plastids and chloroplastsFigure 3. (Hougen, Meller & Gassman; 1982; Luthy et al..

At all stages of leaf and plastid development, from 1984; Whyte & Castelfranco, 1993 ; Jacobs & Jacobs,etiolated (von Wettstein, Gough & Kannangara, 1993; Shioi et al., 1995; see Fig. 4). We may1995) through to senescent (Hukmani & Tripathy, speculate that they represent built-in escape routes1994), externally-supplied 5-aminolaevulinate that render potentially phototoxic porphyrin pre-(ALA) is metabolized in the dark to protochloro- cursors harmless. Some measurements of the turn-phyllides, the immediate precursors of the light- over of chlorophyll and precursors in etiolated andrequiring step at NADPH-protochlorophyllide oxi- greening tissues suggest that these catabolic systemsdoreductase (POR). This tells us that the section of are surprisingly active (Stobart & Hendry, 1984;the biosynthetic pathway between ALA dehydratase Hendry & Stobart, 1986).


Glutamate Glu-tRNA

Glutamate-1-semialdehyde

ligase

Glu-tRNAreductase

GSA

Glutamyl-tRNA

COOH

COOH

HOOC

HOOC

Porphobiiinogen

COOH

aminotransferase

ALAdehydratase

Porphobiiinogendeaminase

UroporphyrinogenIII synthase

ALA

• Hydroxymethylbllane COOH

CH.,

COOHUroporphyrinogen Uroporphyrinogen Coproprphyrinogen

COOH COOH

CH CH,

/// decarboxylase ||i

CoproporphyrinogenIII oxidase

Protoporphyrinogen Protoporphyrinogen ProtoporphyrinIX IX oxidase

Ferrochelatase

:ooH

CH,

COOH COOH

COOH COOH

CH,

Haem Mg chelatase/Me transferase/cyclase

COOH COOH

CH,

DivinylcHaProtochlorophyllide a

8-vinyl MonovinylProtochlorophyllide

Chlorophyllide a Chlorophvll svnthetase

• C H ,

CO2CH3COOH

Chlorophyll a oxygenase

Figure 3. The C5 pathway of chlorophyll biosynthesis. Structures of photodynamic intermediates are shown,as is the origin of haem.

3. Genetic perturbation of chlorophyll biosynthesis

The regulatory capacities of the tetrapyrrole bio-synthetic network are limited, however, and may beeasily overridden chemically or genetically. Thetetrapyrrole intermediates accumulated when ALAis fed in the dark are inadequately served by thenormal in vivo quenching mechanisms so that whenthe tissue is illuminated, there is often fatal photo-damage. Mock et al. (1995) and Kruse, Mock &Grimm (1995) rendered tobacco plants highly photo-sensitive by means of antisense down-regulation ofuroporphyrinogen decarboxylase and coproporphy-rinogen oxidase, respectively. Chlorophyll Z?-less mu-tants are known in a number of plant species.Recently it has been shown that many of these do notrepresent biochemical lesions in the chlorophyll ajb

interconversion but rather have leaky mutations ofearlier steps in the common pathway (Falbel &Staehelin, 1996). A threshold level of chlorophyll aseems to be required before chlorophyll b biosyn-thesis can commence. Thus less extreme disturbanceof the pathway than dark-feeding precursors orantisense knockout is expressed in 6-less mutants bysuppressing chlorophyll b synthesis and the associ-ated assembly of light-harvesting complexes.

4. Early light-inducible proteins

Within a few hours of exposing etiolated seedlings tolight, distinctive mRNAs appear and increase tomaximal levels (Meyer & Kloppstech, 1984; Grimm& Kloppstech, 1987). These messages encode earlylight-inducible proteins (EL IPs) which becom^e

168 H. Thomas

C5 pathway Protoporphyrins

Haem ProtochlorophyliidesGassman/

Whyte enzyme

132-HO-Chl a

Lipoxygenase Peroxidase Chlorophyll1 I X , - . oxidase

Chlorophyll ib

S^ Mg 13dechelatase

Oxidases,peroxidases,

glycosyltransferases...

Chlorophyllase

Chlorophyllide

NCC

Figure 4. Degradation of chlorophyll and related com-pounds in green cells of angiosperms. Biosynthesis isrepresented by filled arrows, breakdown by open arrows.The products of (per)oxidation are unidentified, except for132-HO-chlorophyll a, shown by Schoch et al. (1984) to beamongst the reaction products of chlorophyll oxidase.

integrated into the thylakoid membrane. It is nowknown that FLIPs are induced at all stages of leafdevelopment, even into senescence, in response tohigh light (photoinhibitory) stress (Potter & Klopps-tech, 1993; Humbeck, Kloppstech & Krupinska,1994). Gene sequence comparisons establish FLIPsto be members of the LHCP multigene families(Green & Pichersky, 1994). That FLIPs are likely tobe pigment—proteins may be inferred not only fromtheir LHC-like structures (Green & Kuhlbrandt,1995) but also from their homology with algalcarotenoid-binding proteins (Levy et al., 1993).FLIPs are considered to be important factors incontrolling photodynamic damage when the inten-sity of illumination threatens to overrun the quench-ing capacity of the photosynthetic apparatus. Bybinding photoconvertible xanthophylls, they can actas hyper-stable LHCs under light stress (Krol et al.,1995). FLIP mRNA is short-lived and, duringrecovery from light-stress, the protein is rapidlydegraded (Adamska, Kloppstech & Ohad, 1993),perhaps by a thylakoid-associated serine protease

(Adamska et al.., 1996). There is no evidence eitherway, but one may speculate that, like other pigment-associated proteins, alteration in the nature of theassociation between apoprotein and chromophoremay trigger proteolysis.

V. NADPH-PROTOCHLOROPHYLLIDEOXIDOREDUCTASE

1. General features of FOR regulation

Fach of the steps in chlorophyll biosynthesis inwhich macrocyclic tetrapyrroles are substrates orproducts has implications relevant to the subject ofthe present review, but one invites particular at-tention. POR represents an interesting model forchlorophyll—proteins in general, illustrating twomajor regulatory themes: light-mediated changes intranscription; and post-translational control viadifferential proteolytic susceptibility. POR is animportant enzyme in the control of chloroplastassembly. It is the point at which the chlorophyllbiosynthetic pathway is light-dependent (Griffiths,1978). The enzyme uses light as a kind of substrate.It is abundant in dark-grown tissues, as is its mRNA,but on exposure to light, amounts decrease (Maple-ston & Griffiths, 1980; Forreiter et al., 1990). PORexpression is modulated by the phytochrome systemat the level of repression of nuclear gene transcription(Mosinger et al., 1985). It is regulated post-translationally via turnover, resulting in the declineof the protein in the light (Kay & Griffiths, 1983). Inthe absence of light, POR exists as a stable enzyme-substrate complex between apoprotein, NADPHand protochlorophyllide (Oliver & Griffiths, 1982).On illumination, there is conversion to an enzyme-product complex which is highly susceptible toproteolytic attack (Reinbothe et al., 1995c).

2. PORA and PORB

The POR story just presented is now known to applyto PORA, one of two light-dependent POR enzymesfound in angiosperms. The highly photoregulatedPORA characteristic of greening etioplasts is presentat vanishingly small levels in green tissues, and yetthere is abundant evidence that such tissues canphotoreduce protochlorophyllide. The enzyme re-sponsible is referred to as PORB. Holtdorf et al.(1995) have described just such a form of POR: it isclosely related in protein and gene structure toPORA but is not expressed in a light- or phyto-chrome-sensitive manner. This form of POR alsodiffers from PORA in the mechanism whereby itsprecursor is imported into the plastid from the site ofsynthesis in the cytosol.

PORA can be expressed as the unprocessedprecursor pPORA in E coli transformed with cDNAencoding the barley enzyme (Schulz et al., 1989).

Chlorophyll and plastid development

Table 1. Abundances of plastid types in primary leaves of wheat treatedwith water, 0-1 mm GA^ and/or 1 mM gabaculine in darkness for up to 48 hafter germination {data of Younis, Ryberg & Sundqvist, 1995)

Hours

36

42

48

Treatment

ControlGA3GabaculineGA3 + gabaculine

ControlGA3GabaculineGA3 -4- gabaculineControlGA3GabaculineGA3 + gabaculine

Amyloplasts

49158489

262

7281

60

4265

Proplastids

187

136

120

1610

640

23

Young + matureetioplasts

337835

6198129

88965812

Radioactively-labelled pPORA is taken up by iso- for 42 h with 1 mM gabaculine, synthesis of non-lated etioplasts and processed to the mature size, phototransformable protochlorophyllide was de-Reinbothe et al. (1995 (i) showed that import is creased by c. 80%. Etioplast differentiation wasdependent on the availability of protochlorophyllide, correspondingly slowed, so that even at 48 h onlywith which pPORA associates before removal of the just over half the plastids were etioplasts (Table 1).transit peptide. There is evidence that docking Early processes in chloroplast differentiation havebetween pPORA and protochlorophyllide at the also been studied along the age-gradient of ex-etiopiast envelope is facilitated by chaperonin(s) and panding grass leaves. Here the youngest cells at theprevented by externally-supplied protochlorophyl- base of the lamina are enclosed by the sheaths oflide in a mutually antagonistic way. If pPORA is previous leaves and contain organelles with charac-complexed with chlorophyllide it cannot be imported teristics intermediate between etioplasts and pro-or processed (Reinbothe et al., 1995c). Association plastids. Chloroplasts become fully differentiated aswith (proto)chlorophyllide clearly has profound cells emerge from the sheath into full light. Theinfluences on (p)PORA conformation, with impli- expression pattern of PORA along the age gradientcations for the enzyme's subcellular localization and of cells in the fourth leaf of Lolium temulentummetabolism. By contrast, PORB import to plastids seedlings is similar to that observed during greeningdoes not require protochlorophyllide, though newly- of etiolated whole leaves (Davies et al., 1989;imported PORB forms a stable ternary complex with Ougham & Davies, 1990). The proplastids of youngprotochlorophyllide and NADPH (Reinbothe et al., leaf cells of Lolium treated with gabaculine lack any1995 6). internal membrane structure, and assembly of the

thylakoids in maturing chloroplasts is severely. . disrupted. This is accompanied by a marked in-

3. FOR and plastid differentiation 1 -i • • r 1 i u n J T -LJ/ T^ TT r^ -'-' hibition of chlorophyll and LHCP II formation

Etioplasts contain prolamellar bodies (PLBs), para- (Davies et al., 1990a). Schunmann, Ougham & Turkcrystalline structures largely made of lipids and (19946) and Schunmann & Ougham (1996) alsoordered arrays of PORA in its photosensitive ternary exploited the developmental gradient along theenzyme-substrate complex form. Illumination dis- expanding leaf in a study of slender, a barleyperses the PLB as PORA is degraded and the plastid overgrowth mutant with much decreased sensitivitydevelops into a chloroplast. PORA has a structural to low temperature constraints on cell extensionrole in PLB assembly, and the supply of proto- (Schunmann, Harrison & Ougham, 1994a). Theychlorophyllide is decisive for this role. For example, isolated by differential screening a cDNA corre-Table 1 presents data of Younis, Ryberg & Sundqvist sponding to an mRNA that is abundant in young(1995) on the proportions of different plastid types in expanding tissue of wild-type leaves but very muchdark-grown wheat seedlings over a period of intense less so in the mutant. Sequencing revealed thisetioplast differentiation. Between 36 h and 48 h of cDNA to be identical with PORA. Expression ofseedling development etioplasts increased from 33 to POR along a gradient of cell age in barley leaves was88 % of the total plastid population. similar to that observed in L. temulentum. Altered

Gabaculine is an inhibitor of tetrapyrrole bio- expression in slender implies that the growth of thesynthesis in plants. In etiolated wheat tissue treated ceil within which plastids are differentiating also

170 H. Thomas

Nuclear genome

por cab rbcS

Pfr down-regulates Pfr up-regulates

Plastid genome

rbcL psbA

TRANSCRIPTION

TRANSLATION

pPOR pLHCP/

pSSU

IMPORT & PROCESSING

^Pchlide

POR LHCP II SSU LSU

POR-PchI LHCII rubisco DI

Figure 5. General features of transcriptional and post-transcriptional regulation of gene expression for someimportant plastid proteins, emphasising post-translational protein: chlorophyll and protein: protein inte-ractions. Light exerts its influence through the phytochrome Pr/Pfr system (encoded by nuclear gene phy) andalso directly modifies the stability of POR (nuclear gene por) and the DI protein of PS II (plastid gene psbA).Chlorophyll supply determines the lability of LHCP (nuclear gene cab). Stoichiometric association of the large(LSU) and small (SSU) subunits (genes rbcL, rbcS) is necessary for Rubisco function and survival. Fast andSlow refer to the susceptibility of different forms of each protein to proteolytic attack.

exerts a regulatory influence over POR, though themechanism is not clear. The etiolated leaf stimulatedto green by transfer from darkness to full illumi-nation is a very convenient experimental subject thathas revealed much about POR and its controls.Nevertheless, there are special features of PORduring plastid differentiation in growing cells underless extreme light regimes that are better studied inthese more 'natural' developmental systems.

An intriguing aspect of plastid differentiationraising further questions about POR metabolismconcerns the reversal of senescence. In some plants,notably Nicotiana, the plastids of yellow leaves(gerontoplasts-see Section VIII) can be induced toredifferentiate into chloroplasts. We found thatPORA is immunologically undetectable in miaturegreen and senescent yellow leaves of intact N. rusticaplants. But if the yellow leaf is stimulated to regreenby cutting off the shoot above it, treating withcytokinin and maintaining the plant in dim light, theresumption of chlorophyll biosynthesis is accom-panied by strong induction of POR (H. Zavaleta, B.J. Thomas, I. M. Scott & H. Thomas, unpublished).This implies that POR expression, import, assemblyand turnover systems, like those working in thegreening etioplast, probably also operate during thegerontoplast-chloroplast transition.

4. POR degradation

The ternary complexes of both PORA and PORBwith protochlorophyllide and NADPH are stable inthe dark. By contrast, the POR-chlorophyllideassociation is susceptible to proteolysis. In vitro it isvery difficult to dislodge chlorophyllide from PORAand so it might be that each POR molecule survivesonly one turn of the reaction cycle, making it a trulysuicidal enzyme. On the other hand there is evidencefrom studies with mutants that PORA in vivo is notstrictly a one-shot enzyme and that, during greeningof etiolated tissue, a given molecule of PORA can goround the catalysis cycle several times before it isdegraded (Reinbothe et al, 1996). Proteolytic ac-tivities have been detected within etioplasts andproposed to be responsible for attacking susceptibleforms of PORA (Hammp & De Filippis, 1980;Walker & Griffiths, 1986; Honda, Tanaka & Tsuji,1994). A light-induced nuclear-encoded ATP-de-pendent pH 6"5 protease that increases to maximalactivity as the etioplast-to-chloroplast transitionreaches completion might be responsible for regu-lating PORB turnover (Reinbothe, Apel & Rein-bothe, 1995fl; Reinbothe et al, 1995 b). Althoughthe mechanisms of suicidal light-dependent turnoverof POR are becoming clearer, the reasons for such an


apparently inefficient and wasteful molecular life- but instead to focus on the regulatory role ofstyle are less obvious. Photoactive chromophore- pigments. The abundance of LHC lib makes it aassociated proteins often carry out high-risk jobs in favoured subject for investigation. Precursors of thethe cell and may be designed to be disposable (the LHC II apoproteins (pLHCP) w hen presented toDI protein is another example-see Section VII). It isolated plastids are taken up by an energy-requiring,has been suggested that proteolytic fragments of post-translational mechanism (Waegemann, PaulsenPOR have a function in conveying newly made & Soil, 1990). Soon after uptake, LHCP can bechlorophyllide to the developing thylakoid mem- detected in LHC lib within the thylakoids. Experi-brane (Reinbothe et al., 1996). In any case, notions ments with mutagenized pLHCP (Kohorn & Tobin,of materials-efficiency and energy-efficiency do not 1987) and with LHCP fused with the Rubisco transitnecessary have the same significance for plants that peptide (Lamppa, 1988) have established that maturethey do for animals and other heterotrophs (Thomas, LHCP contains all the necessary information for1994). correct targetting and assembly within the chloro-

plast. Integration of apoproteins into the thylakoid^ _ , , , „ 7 • • 7 7 7 membrane requires Mg-ATP and at least two5. Chlorophyll synthesis in the dark , . ,., r /n <? r^t-

chaperonm-like stroma factors (rayan oc Clme,There is reason to believe that forms of POR other 1991). It is thought that chlorophyll {b in particular)than A and B exist in higher plants. Based on careful is also necessary for stable integration (Kohorn &quantifications of pigments and patterns of radio- Auchincloss, 1991), and studies in vitro show thatlabelling in barley, Tradescantia and Zostera, Adam- apoproteins and pigments in the correct ratio have ason and co-workers have produced persuasive evi- strong tendency towards self-assembly (Plumley &dence that angiosperms can synthesize chlorophyll Schmidt, 1987; Paulsen, Finkenzeller & Kiihlein,in the dark (Adamson, Hiller & Vesk, 1980; 1993). Initial integration of (p)LHCP occurs largelyAdamson, Packer & Gregory, 1985; Walmsley & in the intergranal thylakoid membranes, followed byAdamson, 1995). Light-independent chlorophyll migration into the stacked region (Kohorn & Yakir,biosynthesis is well established in green algae, 1990).pteridophytes and gymnosperms. In these plants thechloroplastic genes chlL, chlN and chlB (structurally ^ ^ ^ / , j chlorophyll in stabilizing nascent LHCPunrelated to PORA/B) encode a protochlorophyllidereduction pathway that can work in darkness (Suzuki Many different lines of evidence show that light-Si Bauer, 1992; Li, Goldschmidt-Clermont & harvesting proteins uncomplexed to chlorophyll areTimko, 1993). These genes have been lost from the subject to fast degradation. For example, chloro-angiosperm plastid genome. There are no reports of phyll 6-Iess mutants express genes for LHCs, andanything like them in the nuclear genome either, so make LHC proteins, at essentially normal rates, butit might be that light-independent chlorophyll without chlorophyll 6 to form complexes with correctsynthesis in angiosperms occurs via yet another form stoichiometries, the proteins are turned over rapidlyof POR. At present, the regulatory role of pig- (White & Green, 1987; Harrison, Nemson & Melis,ment-protein interactions in these emerging areas of 1993). Exposing etiolated tissue to intermittent lightPOR metabolism can only be guessed at. also disturbs the balance between protem and

chlorophyll supply during complex assembly, re-sulting in differential turnover (Bennett, 1981;

6. POR in relation to expression and turnover of ^^^.^^ ^ Green, 1988; Tanaka, Tanaka & Tsuji,other plastid proteins 1992). Dahlin & Timko (1994) used an in vitroIn plastid assembly we see two major regulatory integration system to show the relationship betweenthemes- light-mediated changes in transcription; pigment availability and stability of apoproteinsand post-translational control via differential pro- during assembly of complexes. When pea plantsteolytic susceptibility. Figure 5 is a picture of plastid were grown in weak red light (WRL), their leavesassembly looked at m this way, showing a direct contained c. 29% of the chlorophyll and 66 /o of theassociation between POR metabolism and construe- carotenoids of high-light leaves. In plants exposed totion of light-harvesting and other chlorophyll- WRL plus the carotenoid synthesis inhibitor Nor-protein complexes. fl^razon (Nfl), pigments were further decreased to

14% and 5% respectively. When pLHCP wasincubated with thylakoids plus plastid lysate it was

VI. ASSEMBLING CHLOKOPHYLL-PROTEINS integrated into the membrane. Compared with high-

light controls, with plastid preparations from WRL1. Integration of LHCP plants only c 30 % of the integrated protein wasThe mechanism of chlorophyll-protem complex stable to treatment with the protease the^rmolysin,assembly has been much studied and it is not my whilst for WRL + Nfi the figure was only D /o. Thusintention to review this subject comprehensively, the resistance of newly-mtegrated apoprotem to

172 H. Thomas

proteolysis is nicely in step with the chlorophyll integration of newly synthesized Dl (Mullet,status of the tissue from, which the plastid prep- Gamble-Klein & Klein, 1990). A study with radio-aration was made. Reciprocal incubations with labelled ALA by Feierabend & Dehne (1996)lysates and membranes from different sources and provided evidence that Dl turnover is probablyparallel experiments with the Rieske FeS protein, accompanied by chlorophyll turnover in vivo, underwhich does not complex with pigments in vivo, moderate light as well as under photoinhibitorysupported the notion that chlorophyll regulates the conditions. Clearly the supply of new chlorophyll isintegration of its associated proteins, except at met by the operation of the whole C5 pathway andabnormally low carotenoid levels. not only from pools of immediate precursors or

Conversely, antisense transgenic tobacco plants in chlorophylls transferred from other complexes,which LHCP II transcripts have been decreased to Raskin, Fleminger & Marder (1995) pulse-labelledalmost undetectable levels make essentially normal barley leaves with [^*C]ALA and found that, over theamounts of properly integrated and functional LHC short term, the specific radioactivity of reaction(Flachmann & Kiihlbrandt, 1995). All in all, the centre chlorophyll was much lower than that ofstory is consistent: chlorophyll is necessary for thylakoids as a whole or of a grana-enriched fraction,stabilizing complexes against attack by proteases in On the other hand during a cold chase following avivo and its rate of supply sets the pace for the rate *C feed, reaction centres lost label faster than didof appearance of functional, integrated light-harves- thylakoids and grana. The authors suggest that theseting and reaction-centre units. data are consistent with the existence of different

pools of newly synthesized chlorophyll with fast,slow or intermediate turnover kinetics, to which the

VII. CHLOROPHYLL TURNOVER AT THE STEADY ,.^ . . , . , . ,diiierent pigment—protein complexes withm the

STATE r- o r- r

membrane have access.. , , J ,. r 7 7 77 7 71 7 77 The degradative side of the chlorophyll turnover1. Metabolism of labelled chlorophyll . ... . , , , T I

equation seems likely to be photodynamic. It almost

We have seen that the chlorophyll biosynthesis certainly does not occur through the catabolicpathway seems to remain in place in leaf tissue that pathway that operates when green tissue turns yellowhas completed net accumulation of the pigment and in senescence, since a mutant of Festuca pratensis inpassed into the steady state, and even into early which one step of this pathway is disabled (Vicentini,senescence. If the capacity to make chlorophyll is Iten & Matile, 1995) turns over Dl in the lightpresent at and beyond chlorophyll steady state in leaf perfectly normally (Hilditch, Thomas & Rogers,cell development, is there a balancing degradative 1986). Protein turnover in the core complex of PS IIcapacity too ? In other words, does chlorophyll turn is not the same in photoinhibition as under non-over at a significant rate ? This question remains photoinhibitory conditions; the process when photo-generally unresolved. One early radiolabelling study inhibition is exerted through donor-side limitation isshowed considerable differences between dicots and different from that of acceptor-side photoinhibitionmonocots (Perkins & Roberts,1983). Incorporation (Barber & Andersson, 1992). But a common featureof precursor into chlorophyll was high in mature of all modes of Dl turnover might be the dislodgingdicot leaves and negligible in comparable monocot of chlorophyll leaving a proteolytically-susceptibletissue. Differences in radiolabelling between the apoprotein.chlorophylls of different complexes within the samethylakoid membrane have also been reported ^ r, 7 7 7-, „ * 1 o T <,->-, r\ ^- >jun—shade acclimation

(Brown, Acker & Duranton, 1975).Leaf and chloroplast structural and photosynthetic

^ ^^ . characteristics are sensitive to the light intensity2. Dl protein turnover u- u ^u ^ 1 T-U 1 J 1 •

under which they develop, ihus leaves developingA process with which continuous replacement of in shade have a higher ratio of light-harvestingchlorophyll might be expected to be associated is (relatively chlorophyll 6-rich) to reaction centre (a-turnover of the Dl protein of PS II. Dl has a short rich) complexes than those exposed to brighter lighthalf-life in light and a high rate of degradation is (Anderson, Chow & Goodchild, 1988). Maturematched by an equally high rate of synthesis (Mattoo leaves are also capable of acclimation to decreasedet al., 1984). Under photoinhibitory conditions, Dl light levels. Hidema et M. (1991) showed thatdegradation in isolated PS II particles is simul- exposing fully-expanded leaves of rice to lowtaneous with, or even anticipated by, destruction of irradiance strongly retarded the post-maturity loss ofthe reaction centre chlorophyll PggQ (Telfer et al., total chlorophyll during senescence while the ratio of1994). Dl may be regarded as a chlorophyll-binding bto a appreciably increased. Similar responses wereprotein and therefore might be expected to become observed in Lolium temulentum leaves (Mae et al.,susceptible to proteolysis when dissociated from the 1993). Hidema et al. (1992) confirmed that thepigment. Chlorophyll is certainly required for stable diminished rate of chlorophyll loss and the increase

Chlorophyll and plastid development

mb-.a ratio in shaded rice leaves were associated function in the life of the leaf. The gerontoplast is

with retention of LHC II; furthermore, ^^N- not a dead organelle, it is a plastid with the definedlabellmg revealed that LHC II protein turned over and controlled task of salvaging material for useat a negligible rate under these conditions. The elsewhere. The visible symptom of senescence isevidence for contmued synthesis and breakdown of yellowing, which tells us that chlorophyll is beingnon-reaction-centre chlorophyll under such steady- removed from the thylakoid membrane. Is thestate conditions is at best contradictory. In the dismantling of thylakoid complexes during yellowingabsence of much de novo synthesis, an increase in the the converse, inverse or reverse of assembly duringh:a ratio could be explained by conversion of the greening of expanding tissue ?chlorophyll a to b during acclimation to shade. Itmay be that this in turn is part of a metabolic cycleregulating the balance of b to a, and hence that of ^^- "^^^ PATHWAY OF CHLOROPHYLL

light-harvesting to reaction centre. The evidence for, CATABOLISM

and wider significance of, a reaction scheme runningfrom chlorophyll b to a are discussed in Section IX ^- '^^^ fi""'^ ^hree reactions

^ • The breakdown of chlorophyll into phytol, Mg " anda primary cleavage product of the porphyrin moiety

4. Phaeophorbide in PS ff °^^^^^ ^^ ^^^^^ consecutive reactions (Matile &Krautler, 1995 ; Matile et al, 1996; Fig. 4). The first

A minor but intriguing aspect of chlorophyll metab- step in chlorophyll catabolism is removal of theolism during assembly and turnover of the PS II phytol tail of chlorophyll a to produce chloro-reaction centre concerns phaeophorbide. Spectro- phyllide. Chlorophyllase, the enzyme that carries outscopic evidence has established that the D1/D2 this reaction, is associated with plastid membranescomplex is associated with phaeophorbide a as well and is probably constitutive but latent throughoutas chlorophyll a. The origin and structural sig- much of the development of green tissues (Amir-nificance of this chlorophyll derivative (which re- Shapira, Goldschmidt & Altman, 1987; Rodriguez,presents no more than 0-1 % of total chlorophyll ) is Gonzalez & Linares, 1987; Brandis, Vainstein &not very clear, but some experiments using high- Goldschmidt, 1996). Phytol seems to be quite stablesensitivity luminescence analyses of greening mem- during leaf senescence and persists largely in esteri-branes and tissues (Ignatov & Litvin, 1994) point to fled form (Peisker et al, 1989). Although Mg fallsthe chlorophyllide product of POR as its immediate out of chlorophyll all too easily in vitro, particularlyprecursor. It was suggested that chlorophyllide to if the pH is on the acid side, conditions in the cell do(presumably) phaeophorbide is catalysed by Mg not ordinarily favour this and an activity called Mgchelatase running in reverse, and that the complete dechelatase is required. Mg dechelatase was orig-sequence on through phytylation involves several inally identified in green algae (Ziegler et al, 1988)steps and is light-regulated. There was evidence that and has been detected in higher plants by assayingassembly of the functional PS II core complex the conversion of chlorophyllide into phaeophorbideproceeds via an association between Dl, D2, chloro- or dechelation of chlorophyllin (Langmeier, Gins-phyll a and phaeophorbide a, lacking Pgso- Radio- burg & Matile, 1993 ; Vicentini e a/., 1995; Shioi eilabelling studies are also consistent with biosyn- al., 1996). Here again, the activity appears to bethesis, rather than in situ chlorophyll dechelation, as constitutive and latent. Phaeophorbide a oxygenasethe origin of phaeophytin (Raskin et al, 1995). (PhaO), the third step, is most significant for thePhaeophytin biosynthesis might turn out to be a yellowing of senescent leaves because opening thesignificant feature of PS II turnover; if so, it will be porphyrin macrocycle is associated with the loss ofimportant to understand how the process is regulated green colour (Hortensteiner, Vicentini & Matile,(Ignatov & Litvin, 1994). 1995).

VIII. THE PLASTID FAMILY TREE 2. Phaeophorbide a oxygenase

We have seen that net assembly of complexes is The first stable product of PhaO is a linearcharacteristic of the differentiation of chloroplasts tetrapyrrole. The chemical structure of the fluores-from etio-granal, pro-granal or pre-granal plastids. cent chlorophyll catabolite (FCC) made by the PhaOEqually dramatic changes occur in other parts of the of rape cotyledons (FCC-2) has been determinedplastid developmental network (Thomson & (Fig. 6 b). The reaction that produces FCC-2Whatley, 1980). Senescence is the period of chloro- requires O^ and involves Fe which operates in aplast to gerontoplast transition. Not everyone likes redox-cycle driven by reduced ferredoxin (Matile etthe term gerontoplast (coined by Sitte, 1977), but I al, 1996). PhaO is specific for phaeophorbide a asdo because it emphasizes that maturity to senescence substrate; phaeophorbide 6 is a competitive inhibitorrepresents not a loss or deterioration but a change of of FCC production in vitro (Hortensteiner et al.

174 H. Thomas

Me

o'(a) Reaction intermediate

Me Me

Me

O

NCC-1 R = COCHjCOO-NCC-2 R = CgH.,.,05 (yg-glucose)NCC-3 R = H

Figure 6. Structures of chlorophyll eatabolites fromBrassica napus. (a) First detectable intermediate in themacrocycle-opening phaeophorbide a oxidase reaction, (b)Fluorescent product of the PhaO reaction, (c) Three non-fluorescent terminal eatabolites (Matile et al., 1996;Miihlecker & Krautler, 1996).

1995). The production of primary FCC fromphaeophorbide a requires the presence of a fer-redoxin-dependent reductase, a soluble stroma pro-tein responsible for the reduction of a double bond inthe pyrrole system following the action of the ring-opening oxygenase. The transient intermediate inthe PhaO reaction, a red bilin, is chemically identicalto a terminal chlorophyll catabolite excreted byChlorella (Gossauer, 1994; Hortensteiner, Krautler& Matile, unpublished; Fig. 6a).

3. Gene expression in chlorophyll catabolism

Experiments with inhibitors of protein synthesisindicate that the activation process unmaskingchlorophyllase and Mg dechelatase during sene-scence requires new translation (Thomas et al,1989), which in turn might mean that one or moreimportant senescence-regulating genes are turnedon. PhaO is not latent but its activation is sensitive totranslation inhibitors, which suggests that it is madede novo in senescence (Schellenberg, Matile &Thomas, 1990). Mutants in three species {Festueapratensis, Phaseolus vulgaris, Pisum sativum) displaynormal activation of chlorophyllase and dechelataseduring senescence, but PhaO is not induced and leaftissue remains green (Bachmann et al, 1994;Vicentini et al, 1995; Thomas et al, 1996). Thecoordinated action of several gene products might benecessary for invoking the chlorophyll catabolismpathway in senescence. One of these products (eitherencoded by the PhaO structural gene itself, or by aregulator of it) is missing or damaged in stay-greenmutants, giving rise to the non-yellowing phenotype.In the case of Festuea, it was shown that LHC IIcomplexes from plastids of the stay-green mutantcomprise LHCP plus polar (phytol-less) chlorophyllderivatives (Thomas et al, 1989). Schoch & Brown(1986) observed that treating LHC II with chloro-phyllase in vitro resulted in the formation of a stablechlorophyllid—protein complex. It is probable, there-fore, that chlorophyll and its downstream eatabolitesremain complexed with protein right up until PhaOopens the macrocycle. At this stage the protein isliberated and becomes vulnerable to attack byproteases. Thus the principle of turnover-levelregulation maintaining pigment—protein stoichi-ometry applies not only during complex formationbut also when pigment—proteins are dismantledduring gerontoplast development.

4. Subcellular organization of chlorophyll catabolism

A surprising recent finding is that much of themachinery of chlorophyll catabolism is located in theplastid envelope. Essentially all of the chlorophyllaseand PhaO activity is associated with this membrane(Matile & Schellenberg, 1996), and the gerontoplastenvelope also appears to be equipped with a carrier

Ghlorophyll and plastid development 175

that is responsible for exporting newly produced (ribulose-l,5-bisphosphate carboxylase/oxygenase)catabolites into the cytosol, since FCC is released is the single most important source of remobilizableinto the medium if ATP is provided at the cytosolic protein N in the leaf and its degradation, accom-face of the intact organelle (Matile et al, 1996). How panied by loss of COg fixation activity, is a prominentdoes chlorophyll get from the thylakoid to the feature of senescence (Makino, Mae & Ohira, 1984;envelope? Are there specialized carrier proteins? Or Crafts-Brandner, Salvucci & Egli, 1990). The ultra-do the pigment-binding proteins of thylakoid com- structureof senescing chloroplasts (that is, differenti-plexes become mobile ? Might there be a role for the ating gerontoplasts) reveals a loss of stroma materialplastoglobuli (Tevini & Steinmuller, 1985) or similar as Rubisco disappears, but the most dramaticbodies (Picher et al, 1993; Ghosh et al, 1994), changes concern thylakoid membranes. Contactproviding hydrophobic, antioxidant-rich vehicles for between the lamellae in grana stacks is loosened andmoving chlorophyll between membranes? These the membranes disperse as the number and size ofquestions cannot be answered yet, but the impli- plastoglobuli increase (Tevini & Steinmuller, 1985).cations for the interlinking of the fates of pigments It is significant that although stroma and granaand proteins are clear. components are greatly altered, the plastid envelope

retains integrity to the end of senescence (Butler &, , , , „ J , Simon, 1971; Thomas, 1977; Thomson & Whatley,

5. Interconversion of chlorophylls a and o i n o n \ r> • • r ^ u i i - j * ^•' ^ -^ 1980). Reorganization of thylakoid structure is

A notable feature of the chemistry of the catabolites accompanied by changes in photosynthetic lightso far characterized is that they are all derivatives of reactions and electron transport. The functions andchlorophyll fl (Muhlecker & Krautler, 1996). protein complements of PS II and the cyto-Phaeophorbide & is not a substrate for PhaO. And chrome 6//complexes are particularly labile duringyet chlorophyll 6 is clearly broken down in sen- senescence (Ben-David, Nelson & Gepstein, 1983;escence: how? Recently Japanese workers have Holloway, Maclean & Scott, 1983; Woolhouse &described the conversion of chlorophyll 6 to a in Jenkins 1983; Roberts e/f a/., 1987) but under normaldeveloping plastids (Itoh e a/., 1994; Itoh, Ohtsuka circumstances components of different complexes& Tanaka, 1996; Ohtsuka, Itoh & Tanaka, 1997). are lost in a more-or-less coordinated fashionSchumann et al. (1996) also found that etioplasts (Schmidt, 1988).could convert Zn-phaeophorbide b into Zn-phaeo-phytin a. Could this sequence or something similar ^ Mobilization of chlorophyll-proteins in senescencebe active in gerontoplasts ? It might be relevant inthis connection that the soybean stay-green variant I have argued that when proteins form complexescytG degrades chlorophyll a but chlorophyll b (and with their pigment chromophores they lock intolight-harvesting complexes) are relatively stable stable structures. It follows that proteolyticremobili-(Guiamet etal, 1991). Is cytG a mutation of a gene zation during senescence requires the chlorophyll-operating in the b to a pathway? If so, it offers a protein complexes to be dissociated. Disassembly oftempting target for molecular characterization of this pigment-protein complexes is potentially hazardousstep, since cj ^G is a cytoplasmic (presumably plastid because it separates chlorophyll from the variousDNA) gene. mechanisms which, in the intact thylakoid, prevent

Figure 4 presents the interconversion of a and b as photodynamic damage. We can now see how efTec-a cycle with implications not only for chlorophyll tively metabolism via PhaO solves the problem. Bycatabolism but also for steady-state adjustments, opening the macrocycle and destroymg the residualsuch as those occurring during light acclimation (see conjugated bond system m two virtually simul-VII 3 ) It would not be surprising to find that the taneous reactions, phaeophorbide is rendered photo-operation of the b-a cycle is accompanied by, and dynamically impotent and the pigment-bmdmgeven the instigator of, dynamic changes in the protein can be released for recyclmg. Even so, as mproteins of thylakoid complexes. pigment-protein assembly, it is possible to invoke

photodynamic damage during complex mobilizationby excessive illumination or chemical interference

X. CHLOROPHYLL AND ASSOCIATED PROTEINS (-fhomas & Matile, 1987; Kar et al, 1993).IN SENESCENCE The interdependence of the degradation of chloro-

phyll and of the proteins that bind it is clearly1. Thylakoid disassembly in gerontoplasts exemplified in stay-green senescence mutants. Re-CO, assimilation decreases during leaf senescence, tention of pigment has been shown to be associatedand it is well established that the limiting process with immobilization of thylakoid protems m non-determining photosynthetic rate over the period of yellowing variants of -veral species (Thomas &decline is sited within the plastid and not, for Smart, 1993; Bachmann . . al, 1994; Guamet &example, at the level of stomatal resistance (Fnednch Giannibelli, 1994). This has important implications& HufTaker, 1980; Gay & Thomas, 1996). Rubisco for the internal mtrogen economy of such plants.

176 H. Thomas

Pigment-protein complexes in the thylakoids mightaccount for over 30 % of the total salvagable proteinof chloroplasts. If chlorophyll degradation is im-paired, much of the N of thylakoids is unavailable forrecycling. It follows that, when N supply is limited,stay-green plants pay a significant penalty comparedwith normally yellowing types in terms of rates ofgrowth and development, because their internal Nrelations are compromised by a large pool ofinaccessible protein in senescent leaves (Bakken etal., 1996; Hauck, 1996). In this sense, it is not anexaggeration to consider chlorophyll catabolism as apace-setting process with implications for whole-plant development and ecological fitness.

3. Chlorophyll breakdown: catabolism ordetoxification ?

Conversion of chlorophyll to FCC is a delicateoperation upon which controlled redistribution ofthe N from pigment-binding proteins is absolutelydependent. The terminal products of chlorophyllbreakdown are non-fluorescent tetrapyrroles (NCCs-Fig. 6 c) which accumulate in the cell vacuole. Thekinetics of FCC and NCC appearance, and acomparison of their molecular structures, suggest aprecursor-product relationship (Matile & Krautler,1995; Matile et al, 1996). Radiolabelling hasrevealed that carbon from the pyrroles of chlorophyllis neither lost as COg nor exported from senescentleaves to other parts of the plant but remains asterminal catabolites (Peisker et al., 1990; Matile etal, 1996). Moreover, Curty & Engel (1996) haverecently shown that the total chlorophyll a + b isconverted mole-for-mole into a single a-type NCCin autumn leaves of Cercidiphyllum japonicum(further support for a 6 to a pathway). It may beconcluded that plants catabolize chlorophyll intowater-soluble porphyrin derivatives which accumu-late in mesophyll cell vacuoles during foliar sen-escence. The N and C that get biosynthesized intothe chlorophyll of a green cell are in that cell forgood. In other words, the raw material of chloro-phyll, unlike that of proteins and other constituents,is not salvaged during senescence for use elsewhere.It seems that this is the price to be paid for gainingaccess to the N of thylakoid proteins. Furtherfeatures of the later stages of chlorophyll breakdownlead to an unusual conclusion.

Amongst the chemical structures of catabolites sofar described are two conjugates. The NCC-2 ofBrassica napus is a ^-glucoside and NCC-1 ismalonated (Muhlecker & Krautler, 1996-Fig. 6 c).All NCCs are highly oxidized and hydroxylated.Recently the tonoplastic ATP-dependent trans-porter that delivers NCCs to the vacuole has beencharacterized (Hinder et al, 1996; Matile et al,1996). Conjugation/hydroxylation and ATP-driventransfer to the vacuole are characteristic fates of

xenobiotics in plant cells. Considering the con-versions by which the intact macrocycle of chloro-phyll is rendered photodynamically safe, and also thesequestration of breakdown products in the vacuoleas terminal metabolites, it is difficult to escape theconclusion that this is a detoxification sequencerather than a catabolic pathway. The highly photo-destructive properties of chlorophyll mean that itmust be handled like a toxic compound by the cell inorder to gain access to the considerable store of Ninvested in the chlorophyll-binding proteins.

XI. HOW DOES CHLOROPHYLL STABILIZE

ASSOCIATED PROTEINS?

1. Protein conformation

It is clear that plant cells are equipped with themeans to test apoproteins for the correctness of theirassociations with chlorophyll (or chlorophyllide, orprotochlorophyllide, or phaeophorbide) and to de-stroy proteins that fail this test. What is it about sucha chromophore that protects the complex fromproteolysis? One possibility is that association be-tween the pigment and its apoprotein moves hydro-phobic zones, or refractory peptide bonds, to theoutside of the complex, thus frustrating a waitingprotease. Paulsen et al. (1993) found that folding ofthe light-harvesting protein is facilitated by chloro-phyll. They overexpressed LHCP II in E. coli andreconstituted complexes with chlorophyll. Circulardichroism measurement indicated that the pro-portion of alpha helix increased from 20 to 60%when protein was renatured with chlorophyll. Therenatured complex was resistant to attack by trypsin,in contrast to the protein alone. As well as wild-typeLHCP, they overexpressed a form of the protein inwhich valine-229, in the 4th position from the Cterminus (Fig. 2), was replaced with cysteine by site-directed mutagenesis. Where it is accessible to thereagent, cysteine in a polypetide chain is modified bythe thiol label eosin maleimide. Paulsen et al. (1993)showed that renaturation with chlorophyll makes acysteine residue in the membrane-spanning B helixinaccessible (which is not too surprising), but alsothe substitute cysteine at position 229. This residueis well beyond the D helix and hydropathy modelswould place it outside the hydrophobic core of themolecule, in a proteolytically-exposed position (Fig.2). It seems that the stabilizing influence of thenearby chlorophyll (b3) extends to this part of thecomplex. The consequence of this for LHCP in vivowould be to limit proteolytic attack from the lumendirection. A possibly related observation here con-cerns the behaviour of cytochrome / .

2. Cytochromef

Cytochrome/ is significantly more stable in stay-greens than in yellowing genotypes (Davies et al.

Chlorophyll and plastid development -t nn

1990 ; Bachmann et al, 1994). The protein has a play m survivmg the proteolytic pressures of cyto-smgle membrane-spanning region, with most of the plasmic existence. General rules relating proteinpolypeptide Cham exposed within the lumen. The structure and half-life have been proposed from timedecreased lability of cytochrome/is not explained by to time (see, for example, Ferreira & Davies, 1986;the thylakoid lumen's being a no-go area for Chiang & Dice, 1988; Hersko & Ciechanover, 1992;proteolysis m stay-greens, because in Festuea the Varshavsky, 1992), though, as in subcellular tar-extrinisic, lumen-facing PS II protein OEC33 is getting, higher-order conformation is likely to be atdegraded normally (Hilditch et al, 1989). We least as decisive as amino acid composition orsuggest that the lummal portion of cytochrome/is sequence. The exquisite poising of metabolismstabilized by its association with haem, just as the C within the viable cell can be accounted for in part byterminus of LHCP II is made inaccessible by the equilibrium between, on the one hand, stablechlorophyll. Note the similarity between the struc- protein conformation (for example, adequate sub-tures of haem and chlorophyllide (Fig. 1). Does this strate supply ensuring continuous occupation of anmean that at least some of the haem is catabolized via enzyme's active site, locking the entire protein into athe PhaO route and that disabling this pathway in refractory configuration) and, on the other, a con-stay-greens limits loss of this chromophore and stantly probing and testing proteolytic milieu. Wedegradation of the associated protein during sen- see a clear demonstration of such a regulatory systemescence ? It seems unlikely, but not impossible. in the post-translational turnover behaviour of POR

in light and dark. I have argued throughout this

3. Protease inhibition by tetrapyrrolesl ""^""^^^ ^^^^^ through their influence on proteinconformation, chlorophyll and its derivatives play

Facilitated folding into resistant conformations looks crucial, and often pace-setting, roles in the regulationto be the probable mechanism by which chlorophyll of gene expression in the broad sense. The prop-and other tetrapyrroles confer stability on the osition could be extended to any and all metabolitesproteins with which they are complexed, but there that dock shape-specifically with proteins and multi-are other, additional, possibilities. One that needs protein complexes. If a protein is gainfully employedfurther investigation is that tetrapyrroles are protease in a structural or catalytic task, it is relatively safe. Ifinhibitors. Vierstra & Sullivan (1988), investigating not, it will be culled. What better way of attuningphytochrome turnover, found that haemin could gene expression to the needs of metabolism andinhibit proteolysis by the ubiquitin pathway. Bind- physiology ?ing haemin stabilizes part of the bovine serumalbumin molecule against proteolysis, perhaps at, . 1 J- 1 T • • • 1 1 • 1 A C K N O W L E D G E M E N T S

least m part by directly limiting peptidolytic attack(Shin, Yamashita & Hirose, 1994). The hydrophilic I thank Kate Griffiths, Sandy Cowan, Iain Donnison,macrocyclic derivatives of chlorophyll should be Barbara Hauck and Helen Ougham for (mostly con-examined for similar regulatory effects on catabolic structive) criticism and discussion, and Philippe Matile forf^nvirTT /ic collaboration over the years. The author's research on

matters green is supported by, amongst other organ-izations, the UK Biotechnology and Biological Sciences

XII. CONCLUSION Research Council and Unilever pic.

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chlorophyll: a symptom and a regulator of plastid development

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