[advances in botanical research] volume 22 || calcium ions as intracellular second messengers in...

Calcium Ions as Intracellular Second Messengers in Higher Plants

ALEX A . R . WEBB. MARTIN R . McAINSH. JANE E . TAYLOR and ALISTAIR M . HETHERINGTON

Division of Biological Sciences. University of Lancaster. Lancaster LA1 4YQ. UK

I . Introduction ............................................................................... 45

Paradigm .............................................................................. 47 A . Calcium Ions as Second Messengers - the Animal Cell

I1 . Calcium Ions as Second Messen ers in Plant Cells ........................... 49 Methods for Measuring [Ca I i in Single Cells ........................... 49

B . Measurement of [Ca2+Ii .......................................................... 60

111 . Measurements of Stimulus-Induced Changes in [Ca2+Ii in Plants ........ 68

IV . The Calcium Homeostatic Apparatus ............................................. 69 A . Mechanisms of Generating Increases in [CazfIi ........................... 69 B . Ca2+-ATPases ...................................................................... 83

A . B+

V . The Problem of Specificity ........................................................... 84 A . Other Second Messengers ........................................................ 84 B . The Calcium Signature - a Stimulus-Specific Calcium Signal ........ 85 C . Calcium Signatures in Plant Cells ............................................. 86

Acknowledgements ...................................................................... 88 VI . Future Prospects ......................................................................... 87

References ................................................................................. 88

I . INTRODUCTION

Over the past decade evidence has been rapidly accumulating that points to the importance of the free calcium ion in the control of a diverse array of plant

Advances in Botanical Research Vol . 22 incorporating Advances in Plant Pathology ISBN 0-12-005922-3

Copyright 0 1996 Academic Press Limited All rights of reproduction in any form reserved

46 A. A. R. WEBB el al.

processes (Bush, 1993; Poovaiah and Reddy, 1993; Bowler and Chua, 1994; Gilroy and Trewavas, 1994). The common role that calcium plays in all these processes is to act as an intracellular second messenger. This implies that alterations (usually increases) in the concentration of free calcium ions in the cytosol ([Ca2'Ii) act as an intermediary in the coupling of an extracellular stimulus to its characteristic intracellular response. An important corollary is that cytosolic calcium ion homeostasis is under strict control. In this review we have chosen to use the animal cell paradigm of calcium-based second messenger systems as a comparator for examining the evidence that plant cells employ a similar mechanism for stimulus-response coupling. Clearly, 'there are inherent dangers in this approach and many pitfalls await the unwary if such a path is followed too closely. However, on the basis of the available evidence it would seem that, provided that one is cautious and is careful not to discard too readily data that do not apparently fit the model, there is much to be learnt from this approach.

We start this review with an overview of the workings of calcium-based second messenger systems in animal cells. We then move to plant cells and first consider the methodology that is currently available for studying free calcium in single cells. We believe that an understanding of the technology and its limitations is vital to assessing the subsequent material on the (increasing) range of stimuli which appear to use calcium ions as second messengers in higher plants. As will become apparent the subject of the involvement of calcium ions in signal transduction is a very large field and a detailed discussion of all its facets would be far beyond the scope of this review. Accordingly, we have chosen to concentrate on certain areas that are currently attracting a great deal of interest. One of these areas is concerned with mechanisms of encoding specificity in the calcium messenger system. To examine this in more detail requires a thorough discussion of the calcium homeostatic machinery. Finally, we close with our personal perspective on the direction research in this field may follow for the next few years.

It is necessary to mention those areas which, although of great importance to plant cell signalling, will not be covered here. Obviously the lines of demar- cation between these various fields are entirely artificial and only serve to delimit the areas for reviewers' convenience. One major area that will not be discussed is other second messengers. In this review we concentrate on the calcium ion but there is evidence that changes in cytosolic pH may also be important on transducing certain signals and it would appear from the literature that the time is ripe for re-evaluation of the role of CAMP and other cyclic nucleotides. Additionally, there is evidence, albeit fragmentary at present, that lipid-derived second messengers such as lyso lipids and diacylglycerol, may also have a signalling role. It seems likely that there will be other second messengers, yet to be discovered, that may play a critical role in stimulus-response coupling. Within calcium signalling one area that we will not be discussing in any great detail is the field of downstream processing of

CALCIUM IONS AS INTRACELLULAR SECOND MESSENGERS 47

the calcium signal. Obviously this is of great importance to the generation of the final response and many of the primary and secondary effectors involved in these downstream events are now being identified and characterized. This subject is covered in detail in other reviews (Bush, 1993; Poovaiah and Reddy, 1993; Bowler and Chua, 1994; Gilroy and Trewavas, 1994). Finally, it is important to note that the area of calcium signalling in plants has been the subject of number of recent useful reviews (Johannes et al., 1991; Bush, 1993; Poovaiah and Reddy, 1993; Bowler and Chua, 1994; Gilroy and Trewavas, 1994) and it is recommended that the reader should consult these to complete the picture of calcium-based stimulus-response coupling pathways in plants.

A. CALCIUM IONS AS SECOND MESSENGERS - THE ANIMAL

CELL PARADIGM

Intracellular second messengers are used to couple extracellular stimuli such as hormones to their characteristic intracellular responses. The interaction of the extracellular stimulus with its receptor sets in motion a train of events which results in an alteration (most frequently an increase) in the concentration of another molecule in the cytosol. This alteration then triggers the cell's internal machinery to produce the response (such as secretion, contraction or alterations to gene expression) that is typical of the primary stimulus. As the field of stimulus-response coupling had its origins in mammalian physiology where the primary stimulus is itself frequently secreted from an endocrine gland and is known as a first messenger, then it follows that the cytosolic molecules that link the first messenger with the final response should be called intracellular second messengers.

In terms of second-messenger based signal transduction pathways, mammalian cells are the best understood. cAMP was the first second messenger to be described (Robinson etal., 1971) and since then the list has expanded to include GMP, cytosolic free calcium ions ( [Ca2'Ii), diacylglycerol, inositol( 1,4,5)trisphosphate (Ins( 1,4,5)P,) and cADP ribose (cADPR) and it seems likely that others will be identified as our understanding of signalling pathways increases. Next to cAMP the best studied of these second messengers is [Ca2+Ii. In mammalian cells an array of first messengers make use of calcium-based signal transduction pathways in their mechanism of action. Interestingly, these range from simple molecules such as neurotrans- mitters to the relatively complex peptide growth factors. Perhaps equally striking is the breadth of the responses that are elicited by these first messengers. These include physiological examples such as muscle contraction, through calcium-mediated regulation of metabolic pathways such as in the control of glycogen metabolism in hepatocytes, to responses that must ultimately reflect alterations to gene expression as in the control of cell proliferation (Berridge, 1993a; Tsunoda, 1993). Finally, the fact that the calcium messenger

48 A. A. R. WEBB ef at.

system has been employed and is retained in such a diverse array of cells would suggest that it is both effective and flexible. This latter point is important as it implies that there is an inherent capacity for encoding specificity in the system which allows it to couple faithfully with the appropriate intracellular responses.

Having introduced the concept of calcium ions as second messengers it is now time to consider the events that lead from first messenger reception to the generation of the increase in [Ca2+Ii - the calcium signal. Although we deal with these steps in greater depth later as the detailed comparison of plant and animal cell proceeds, it is worth looking at the process in overview. The calcium signal may be fuelled by two possible sources. In animal cells the concentration of calcium outside the cell is at least 1000-fold greater than the cytosolic concentration. Clearly, one source of calcium is from the cell’s exterior, and animal cells are indeed equipped with a range of calcium channels the operation of which allows the selective and controllable entry of calcium down its electrochemical gradient into the cytosol (Tsien and Tsien, 1990; Hofmann et al., 1994). Voltage operated calcium channels (VOC) are regulated by the membrane potential, which in turn can be influenced by a number of first messengers. Other calcium channels are linked to receptors (receptor-operated channels [ROC]) and, as they are regulated by receptor occupancy, provide a direct link between first messengers and the influx of calcium. A third class of calcium channel in the plasma membrane is regulated by intracellular second messengers (second messenger-operated channels [SMOC]) and gives the cell the potential to utilize feedback to either amplify or abolish the calcium signal. Clearly, the machinery exists at the plasma membrane to allow calcium entry and through the regulation of these channels to exert a high level of control over the process. When these factors are coupled to regulation of the spatial distribution of these channels across the surface of the cell the possibilities for generating controlled, localized calcium entry and generating a high degree of specificity in the calcium messenger system are obvious.

The other possible sources of free calcium used to generate the calcium signal are the internal stores. Release of calcium from these sites is indirect and usually relies on the participation of other second messengers. Of these mechanisms, perhaps the best understood is the coupling process which proceeds through the operation of the phosphoinositide cycle. In this system, the binding of an extracelldar first messenger to a receptor belonging to the seven membrane pass or serpentine superfamily of membrane receptors generates a signal, which results in the activation of the membrane-bound enzyme phospholipase C (PLC). Transmission of the signal to the PLC from the receptor proceeds through the involvement of heterotrimeric G proteins. Once the PLC is activated it hydrolyses the membrane lipid phosphatidyl inositol(4,5)bisphosphate [PtdIns(4,5)P2 ] to yield diacylglycerol (DAG) and Ins(1 ,4,5)P3. The DAG produced is involved in the activation of


the pleiotropic enzyme protein kinase C while the Ins( 1 ,4,5)P3, being water soluble, diffuses into the cytosol. Once in the cytosol it is available for binding to its receptor located on the surface of intracellular membranes. Binding of the ligand to its receptor results in opening of outwardly directed calcium channels and causes calcium to be released into the cytosol. Importantly, these membranes also contain receptors for cADP-ribose, which is another second messenger involved in the generation of the calcium signal (Galione, 1993). Once generated, the increase in calcium can become autocatalytic and from a local site can generate global increases in calcium which can take a variety of forms such as oscillations, spikes and waves depending on the agonist and cell type. Importantly, it has been suggested that the calcium signal contains information that may contribute to the specificity of the response (Berridge, 1990). Finally, calcium 'is a somewhat paradoxical messenger in that if its concentration is allowed to increase unchecked it can act as a cytotoxin. To prevent this, under physiological conditions Ca2+-ATPases are responsible for dissipating the calcium signal and returning the concentration of free calcium to its resting level by either pumping the calcium out of the cell or back into intracellular stores.

11. CALCIUM IONS AS SECOND MESSENGERS IN PLANT CELLS

In the following sections we review the evidence that indicates that calcium ions behave as second messengers in the cells of higher plants. Central to the study of calcium ions as second messengers is the ability to measure accurately the concentration of free calcium ions in the cytosol of single cells and to detect changes that occur after the application of an appropriate stimulus. For this reason, we begin this section with an account of the different techniques that can be used to address this question and indicate what information each of the protocols reveals about cytosolic calcium dynamics.

A. METHODS FOR MEASURING [Ca2+]i IN SINGLE CELLS

Several techniques are available for measuring [Ca2+li in single cells, most of which were originally developed for use with animal cells. These include the use of ion-selective electrodes, luminescent proteins and Ca2+-sensitive dyes. Over the past 10 years the number of reports of stimulus-induced changes in [Ca2+Ii in plants has increased markedly (Table I). In the majority of these studies fluorescent Ca2+-sensitive dyes have been used for the measurement of [Ca"], . Recombinant aequorin techniques have also been developed for monitoring [Ca2+Ii in plants (Knight eta/ . , 1991, 1992, 1993).

TABLE I Measurements of stimulus-induced changes in plant cell [Ca2+ Ji using fluorescent Ca2+-sensitive dyes and

aeguorin-based techniques

Stimuli Cell type

ABA Guard cells

Coleoptiles Hypocot yls Roots Aleurone protoplasts Guard cell protoplasts

Injected “caged” ABA Guard cells Auxin Coleoptiles

Hypocotyls Roots Root hairs

(3‘43

H2 0 2

Cytokinin

Methyl viologen Red light

Far-red light

UVA (pulses) Salt stress Heat stress Mechanical stimuli

(touch & wind) Cold shock

Aleurone protoplasts

Protonema Guard cells Aequorin-transformed seedlings Guard cells Leaf protoplasts

Leaf protoplasts Leaf protoplasts Alga Root protoplasts Mesophyll protoplasts Aequorin-transformed seedlings

Aequorin-transformed seedlings

Ca2+ response Reference

Increase

Increase Increase Increase Decrease Increase Increase Decrease Decrease Decrease Variable (increases &

Increase decreases)

Increase Increase Increase Increase Increase

Increase Decrease Increase Increase Increase Increase

Increase

McAinsh eta/. (1990, 1992); Gilroy et a/. (1991); Irving et a/. (1992)

Gehring et al. (1 990a) Gehring et a/. ( 1990a) Gehring et al. (1 990a) Wang eta/. (1991) Schroeder & Hagiwara (1990) Allan et al. (1994) Gehring et al. (1990a) Gehring et al. (1990a) Gehring et a/. (1 990a)

Ayling et at. (1 994) Bush & Jones (1988); Gilroy

& Jones (1992) Hahm & Saunders (1991) McAinsh (1994) Price et al. (1 994) McAinsh ( I 994) Chae eta/. (1990); Shacklock

eta/. (1992); Volotovski e ta / . (1993a,b)

Volotovski e ta / . (1993a,b) Chae et al. (1990) Russ eta/. (1991) Lynch et al. (1989) Biyaseheva et al. (1 993) Knight eta/. (1991, 1992,

Knight e ta / . (1991, 1992, 1993)

1993)

Fungal elicitors

IP, Injected “caged” Ins( 1 ,4,5)P3 Injected GTPyS Mitosis (anaphase) a-Factor Incompatible stigmatic

S-glycoprotein Electric fields Fertilizationlegg

activation Cell polarization [Ca2 I,

Injected “caged” Ca2+

Injected Ca2+ A23187 + Ca2+ Br-A23 187

Aequorin-transformed seedlings Increase Suspension culture cell protoplasts Increase

Guard cells Leaf protoplasts Stamen hairs Endosperm Yeast Pollen tubes

Pollen tubes Fucus

Fucus Guard cells

Leaf protoplasts Aleurone protoplasts Rhizoid Pollen tubes

Moss protonema Spores Alga Isolated mitochondria Guard cells

Leaf protoplasts Pollen tubes

Stamen hairs Rhizoid Pollen tubes Suspension culture cell protoplasts

Increase Increase Increase Increase Increase Increase

Increase Increase

Increase Increase

Increase Increase Increase Increase


Increase Increase

Increase Increase Increase Increase

Knight ef a/. (1991) Messiaen et a/. , (1993);

Messiaen & Van Cutsem ( 1994)

Gilroy et a/. (1990) Shacklock eta/. (1992) Zhang eta/. (1990) Keith eta/. (1985) Iida eta/. (1990) Franklin-Tong et a/. (1993)

Malho eta/. (1994) Robinson (1990); Roberts

Berger & Brownlee (1993) Gilroy et a/. (1991); McAinsh

Volotovski eta/. (1993a,b) Bush & Jones (1987) Hodick eta/. (1991) Obermeyer & Weisenseel

Hahm & Saunders (1991) Scheuerlein eta/. (1991) Russ eta/. (1991) Zottini & Zannoni (1993) Gilroy et a/. (1 990); McAinsh

Shacklock eta/. (1992) Franklin-Tong eta/. (1993);

Malho et a/. (1994) Zhang eta/. (1990) Brownlee & Wood (1986) Franklin-Tong et a/. (1993) Messiaen & Van Cutsem

et a/. (1994)

et a/. (1995)

(1991)

etaf. (1995)

(1 994)

TABLE I continued

Ionomycin + CaZ+ Injected Caz+-saturated

Verapamil

~ a ~ ’

Calmodulin antagonists

Vanadate

EGTA

Br,-BAPTA

(C4/80, W-7)

Injected EGTA A23187 + EGTA

Br2-BAPTA2+ Injected Ca -free

Br,-BAPTA Injected C1- K +

Decrease in K f

NaN, M-N-ethyl maleimide Iontophoretic

&NO,

microinjection

Isolated mitochondria Stamen hairs

Rhizoid Root hairs Pollen tubes

Pollen tubes

Pollen tubes

“Seedling” (cotyledon & hypocot yl) protoplasts

Guard cells Root protoplasts Spores Alga Stamen hairs Embryogenic suspension culture

Pollen tubes Stamen hairs

cell

Stamen hairs Rhizoids Suspension culture cell protoplasts

Guard cells Suspension culture cell protoplasts Suspension culture cell protoplasts Root hairs Pollen tubes

Increase Increase

Decrease Decrease Decrease

Decrease

Decrease

Decrease

Decrease Decrease Decrease Decrease Decrease Decrease

Decrease Decrease

Increase Increase Increase


Zottini & Zannoni (1993) Zhang et al. (1990)

Brownlee & Pulsford (1988) Clarkson et al. (1988) Obermeyer & Weisenseel

Obermeyer & Weisenseel

Obermeyer & Weisenseel

(1991)

(1991)

(1991)

Elliot & Petkoff (1990)

Gilroy et al. (1991) Gilroy et a/. (1 986) Scheuerlein et ul. (1 99 1) Russ eta/. (1991) Zhang et at. (1 990) Timmers et al. (1991)

Miller eta/. (1992) Zhang et al. (1990)

Zhang eta/. (1990) Hodick etal. (1991) Messiaen & Van Cutsem

Gilroy eta/. (1991) Gilroy et al. (1 989) Gilroy et a/. (1989) Clarkson eta/. (1988) Malho et a/. (1994)

(1 994)

ns(1,4,5) ABA, abscisic acid; GA3, gibberellic acid; UVA, ultra-violet A; Ins(1,4,5)P3, inositol trisphosphate; GTPyS, guanosine S‘-O-(3 ’- thiotrisphosphate); BAPTA, bis-(O-aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid; EGTA, ethyleneglycol-bir-(~-aminoethyl)-N,N,N’,N‘- tetraacetic acid.


TABLE I1 Non-ratiometric and ratiometric (dual-excitation and dual-emission) fluorescenl

Ca + -sensitive dyes

Non-ratiornetric dyes F~uo-3 Rhod-2 Calcium Green ( - I , -2 and

Calcium Orange ( - 5N) Calcium Crimson

- 5N)

Ratiometric dyes Dual-excitation dyes

Fura-2 Quin-2 Fura Red BTC

Indo- 1 Dual-emission dyes

Structurally linked dyes Calcium Green-1 and Texas Red 70000 Dextran

Excitation wavelengths (nm)

Emission wavelengths (nrn)

490 550

490 550 590

340 and 380 330 and 350 420 and 480 400 and 480

360

490 and 570

530 580

530 590 610

510 490 660 540

405 and 480

530

I . Fluorescent Ca2+-sensitive dyes Several fluorescent Ca 2+-sensitive dyes, and their conjugates, are commercially available (Table 11). These can be broadly grouped into two categories: non-ratiometric or single-wavelength dyes, and ratiometric or dual-wavelength dyes.

Non-ratiometric dyes. The most commonly used non-ratiometric dyes are fluo-3 and Calcium Green. These exhibit an increase in fluorescence across the whole of the emission spectra on binding Ca2+ (Fig. 1A). Therefore, at a given wavelength [Ca2+Ii is proportional to the intensity of fluorescence. Typically, the emission maximum, approx. 530 nm for fluo-3 and Calcium Green (490 nm excitation), is used. However, the amount of fluorescence measured in a cell is not simply dependent on [Ca2+Ii but also on the amount of dye present, which will vary from cell to cell, the distribution of the dye within the cell, and the degree of dye loss and photobleaching that occurs during measurements of [Ca2+Ii. These factors complicate the quantification of [Ca2+Ii using non-ratiometric dyes.

Ratiometric dyes. Ratiometric dyes exhibit a shift in either their excitation or emission spectra when they bind Ca2+. Consequently, they are subdivided

54 A. A. R. WEBB et at.

I z 43 3 CM trae ca2* .- c v)

Q) - c - - C

m U X w

.- c

+ .-

500 5% so0 650 2 50 300 350 400

Wavelength (nm) Wavelength (nm)

C Ex = 355 nm

4w 450 553

Wavelength (nm)

Em = 530 nm 39.8 IIM free Ca2'

D

I 450 500 550 600 650

Excitation wavelength (nm)

i0

Fig. 1. Emission spectra for (A) fluo-3 (a non-ratiometric dye); (B) fura-2 (a dual- excitation ratiometric dye); (C) indo-1 (a dual-emission ratiometric dye); and (D) Calcium Green-1 and Texas Red dextran (a 70 000 MW dextran conjugate of both Calcium Green and Texas Red which can be used as a dual-excitation ratiometric dye). Redrawn from Haugland (1992) with permission.

into: (i) dual-excitation dyes, the most widely used of which is fura-2 (Fig. 1B); and (ii) dual-emission dyes, the most commonly used of which is indo-1 (Fig. 1C). This shift is utilized in the measurement of [Ca2+Ii. For example, fura-2 (a dual-excitation dye) exhibits a shift in its excitation maximum (510nm emission) on binding CaZ+, such that the fluorescence at shorter wavelengths, typically 340 nm, increases with increasing [Ca2+Ji whereas the fluorescence at longer wavelengths, typically 380 nm, decreases with increasing [Ca2+li (Fig. 1C). By calculating the ratio of the fluorescence intensities at


these two wavelengths a measurement is obtained that is proportional to [Ca2+Ii but independent of the amount of dye present in the cell. The same principle applies to dual-emission dyes except that it is the shift in the emission maximum that is utilized for calculating the Ca2+-dependent ratio. This technique eliminates potential artefacts that may be associated with cell-to-cell variations in dye loading, dye distribution and dye loss (through leakage or photobleaching). In addition, it allows accurate calibration of the fluorescence ratio (see Tsien and Rink, 1980; Grynkiewicz e ta / . , 1985).

The majority of ratiometric measurements of [Ca2+Ii have been made using dyes that are excited by short wavelength ultraviolet light (UV), for example fura-2, 340/380 nm excitation maxima; indo-1, 360 nm excitation maximum. However, in general, UV-excitable dyes cannot be employed in studies requiring the release of active compounds from their inactive "caged" forms by UV-photolysis (McCray and Trentham, 1989) or in imaging studies using confocal scanning laser microscopy (CSLM) (White et al., 1987; Shotton, 1989). The latter would require a laser capable of emitting lines in the UV range. Advanced confocal microscopes equipped with this facility have only recently become commercially available. Therefore, these studies have relied heavily upon non-ratiometric dyes with excitation maxima at longer wavelengths.

Recently, long wavelength ratiometric dyes have become available. These include Fura Red, which may act as a ratiometric dye, using excitation of 490nm for the Ca2+-sensitive signal and of 440nm for the Ca'+-insensitive signal (535 nm emission), and BTC, a coumarin benzothiazole-based indicator, which exhibits an excitation maximum shift from 480 nrn to 400 nm on binding Ca2+ (540 nm emission). In addition, cells can be coloaded with two separate non-ratiometric dyes, the spectral properties of which allow a ratio to be calculated that is independent of dye concentration, for example with fluo-3 and Fura Red. This technique assumes that both dyes adopt equivalent distribution patterns within the cell, which may not always occur. A 70000MW dextran conjugate of both Calcium Green and Texas Red (Calcium Green-1 and Texas Red dextran) has been developed to avoid this problem. This uses excitations of 490 nm (Calcium Green-1) for the Ca2+-sensitive signal and 570 nm (Texas Red) for the Ca2+-insensitive signal (530nm emission) (Fig. 1D). Since both dyes are structurally linked an identical distribution is assured. However, many of these dye combinations have still to be applied successfully to the measurement of [Ca2+li in plant cells.

The majority of fluorescent Ca*+-sensitive dyes are produced in both the free acid and the acetoxymethyl esterified (AM) form. Many of them are also available conjugated to specific biomolecules. The most common conjugates are those in which the dye is attached to a high molecular weight (MW) water-soluble dextran (typically with molecular weights of 10 000, 70 000 or 500 000). However, Molecular Probes, Inc. (Eugene, Oregon, USA) have developed an amine-reactive fura that can be covalently attached to any

56 A. A. R. WEBB et al.

suitable biomolecule. Fura-BSA is one example of such a conjugate. In addition, site-selective conjugates of certain Ca2+-sensitive dyes are available. These include NuCaGreen, a 70 000 MW dextran conjugate of Calcium Green and a nuclear localization peptide, which can be used specifically to monitor changes in nuclear [Ca2+Ii. Other site-selective dyes include rhod-2 AM, which is the only cell-permeant dye that has a net positive charge causing its sequestration into mitochondria in some cells, and fura-C,, and fura-indoline -CIS, which are lipophilic Ca2+ indicators that preferentially associate with cell membranes allowing changes in [Ca2+Ii occurring close to membranes to be studied (Etter et al., 1994). With the exception of fura-indoline-CI8, which exhibits a shift in its excitation maximum from approx. 575nm to approx. 510 nm upon binding Ca2+, making it a potential longer-wavelength ratiometric dye, the site-selective forms of the dyes and their conjugates possess similar fluorescence characteristics to the parent dyes.

Ca2+-sensitive dyes have also been developed which possess different affinities for Caz+ making them suitable for determining [Ca2+Ii across a wide range of concentrations. For example, there are three types of Calcium Green commercially available (see Table 11); Calcium Green-1 has the highest affinity for Ca2+ (Kd - 189nM), making it best suited for monitoring small changes in [Ca2+Ii (Haugland, 1992), while Calcium Green-5N (and Calcium Orange-5N) has a low affinity for Ca2+ (Kd - 3.3 p ~ ) making it suitable for measuring unusually large pulses of [Ca2+Ii (e.g. during Ca2+-induced Ca2+ release) (Kuba and Takeshita, 1981; Goldbeter et al., 1990; Berridge, 1993b). Calcium Green-2 has an intermediate affinity for Ca2+. The magnesium indicators (mag-fura-2 and -5, mag-fura red, mag-indo-1, mag-quin-1 and -2, Magnesium Green), also have a moderate affinity for Ca2+ (e.g. Magnesium Green: Kd for Ca2+ - 4.8 pM) and may therefore also be useful in detecting large "spikes" in [Ca2+Ii up to 1 p~ (Collins etal., 1991; Konishi et al., 1991; Hurley et al., 1992).

2. Dye loading techniques A range of techniques have been developed for introducing Ca2+-sensitive dyes into single cells. Several of these have been used to load plant cells, including: microinjection, low pH loading, ester loading, electroporation and digitonin permeabilization. These have met with varying degrees of success. The reasons for this remain unclear.

Microinjection. This technique has been widely applied to the study of [Ca2+Ii in plant cells, and has met with a high degree of success (Brownlee and Pulsford, 1988; Clarkson etal., 1988; Gilroy etal., 1990, 1991; McAinsh etal., 1990, 1992, 1995; Zhang etal., 1990; Hodick etal., 1991; Miller etal., 1992; Thiel et al., 1993; Allan et al., 1994; Ayling et al., 1994; Franklin-Tong etal., 1993; Malh6 etal., 1994; Roberts etal., 1994). There are both advantages and drawbacks associated with microinjection. The major advantage is


that it allows a wide range of both charged and uncharged compounds to be rapidly introduced directly into the cytoplasm of a cell enabling the intracellular environment to be manipulated and the effects monitored. The major drawback is that it is an invasive, and potentially disruptive technique requiring the impalement of a cell with a microelectrode or micropipette with the associated structural damage this may cause. Therefore, it is essential to estab- lish the viability of a cell following microinjection. A range of parameters have been used to assess cell viability following microinjection (see Blatt et al., 1990; McAinsh et al., 1990,1992,1995; Gilroyet al., 1991). Microinjection techniques fall into two groups: iontophoretic injection and pressure injection (Callaham and Hepler, 1991). In addition, patch clamp techniques have also been used to introduce compounds into protoplasts held in the whole-cell configuration in a manner analogous to microinjection (Schroeder and Hagiwara, 1990).

Iontophoretic microinjection is a technique that relies on the movement of charged molecules, for example dyes with a net negative or positive charge, driven by an electrical current. Under such conditions currents are carried by the ions in the solution rather than by the transfer of electrons resulting in ionic movements without any accompanying change in cell pressure or cell volume (Callaham and Hepler, 1991). This makes the iontophoretic microinjection ideally suited for use in plants, particular in studies on turgor regulating cells, for example stomatal guard cells (Gilroy etal., 1990, 1991; McAinsh et al., 1990, 1992, 1995; Allan et al., 1994). Both continuous current and current pulses have been used for the iontophoretic microinjection of plant cells. The latter avoids the protracted hyperpolarization of the plasma membrane which occurs when passing negative currents (depolarization when using positive currents) and reduces the effect of charging cells by allowing time for the cell to discharge in between successive current pulses.

Although cell loading by iontophoretic microinjection can be controlled by regulating the iontophoretic current, additional factors also influence the delivery of a dye from the electrode. These include competing ions, differences in characteristics between individual electrodes (e.g. tip resistance) and cell type. However, in practice, the same quantity of a dye can be repeatably injected into cells although the absolute amount present may be difficult to calculate (see Purves, 1981 for additional details regarding microelectrode techniques).

At Lancaster we use microelectrodes (< 0.25 pm tip diameter), pulled from 0.68-mm filamented electrode glass, to impale stomatal guard cells in detached epidermis. The tip diameter of the electrode is dictated by the size of cell to be impaled and the molecular weight of the compound to be injected. External diameters which have been used for studies in plants range from 0.1 to 1 .O pm (see Read et al., 1992). Negatively charged compounds, for example fura-2, are iontophoretically injected into the cytosol of the cells using negative current pulses (1 .O nA, 2 Hz, 200 ms duration) for 1 min (McAinsh et al., 1990, 1992, 1995) using an isolated stimulator and an intracellular amplifier (World


Precision Instruments [WPI], Sarasota, Florida, USA). Positively charged compounds are injected by positive current, while uncharged compounds may be co-injected into cells together with a high K + buffer. Currents used for the iontophoretic microinjection of plant cells range from <0.1 to 10nA passed for 10s to 10min (see Read etal., 1992). The tips of the electrodes are filled with the Ca’+-sensitive dye at a concentration of 10mM and back- filled with 3 M KCI for use with WPI electrode holders containing a sintered Ag-AgC1 pellet to make the connection with the external circuit. Concentra- tions of dyes used to fill electrodes range from 5 0 p ~ to 10mM (see Read etal., 1992). Using apparatus described above it is also possible to monitor membrane potential as an additional indicator of cell viability (Brownlee and Pulsford, 1988; Clarkson et al., 1988; Callaham and Hepler, 1991; McAinsh et a/. , 1 992).

Pressure microinjection uses pressure to inject uncharged compounds or proteins that are not sufficiently mobile in an electrical current to allow successful iontophoretic microinjection. In addition, it can be used to inject mixtures of either charged and/or uncharged compounds. Iontophoretic injection of mixtures will tend to preferentially inject individual components on the basis of the different charges they carry. Pressure microinjection, however, will maintain the integrity of the mixture during injection, ensuring that the composition of the mixture entering the cell is the same as that in the pipette.

Injection of compounds into the cytosol of cells using pressure is, in general, harder to achieve than iontophoretic microinjection due to the large pipette tip diameter required. This is a particular problem with walled cells (rather than protoplasts) when the requirement for a sharp pipette to penetrate the wall conflicts with the need to have a large, open tip. Bevelling of the micropipettes, giving them a profile similar to that of a hypodermic needle, may help to overcome this probIem by increasing the internal tip diameter while ensuring that the pipette remains sharp (Kaila and Voipio, 1985). In addition, pressure microinjection is complicated by the high pressure that must be applied inside the pipette in order to overcome the back pressure exerted by the cell’s turgor. For example, the turgor inside the guard cells of open stomata is approximately 60 bars (Weyers and Meidner, 1990). This is at the working limit for most commercial injection systems. Pressure microinjection has been successfully used in such diverse cell types as fungal hyphae (Money, 1990) and the eggs and rhizoids of Fucusserratus (Berger and Brownlee, 1993; Roberts et al., 1994).

Acid loading. The free acid form of the Ca2+-sensitive dyes dissociate at physiological pHs into the anionic state rendering them cell impermeant. However, at mildly acid pHs this is prevented, allowing them to enter cells in the uncharged, undissociated form. Once inside the cell the dyes encounter the higher pH of the cytosol and dissociate into the cell impermeant anionic state and become trapped inside the cell. This phenomenon, referred to as


“ion trapping”, can be used to load cells using the acid-loading technique (Callaham and Hepler, 1991).

This technique was first described by Gilroy etal. (1986) as a method for loading root protoplasts with quin-2 and has subsequently been used to load protoplasts from a range of different tissues (see Table I); for example protoplasts from barley aleurone layers (Bush and Jones, 1987, 1988), oat (Volotovski el al., 1993a), maize root tips (Lynch et al., 1989; Kiss et al., 1991) and pea mesophyll (Biyaseheva etal., 1993). There are few reports of this method working with walled cells (e.g. Halachmi and Eilam, 1989; Callaham and Hepler, 1991; Hahm and Saunders, 1991; Russ etal. , 1991). In addition, the extreme conditions employed during acid loading protocols can have a marked affect on cell viability following loading. Typically, cells are maintained at pH 4.5 for > 2 h at dye concentrations > 5 O p M (Callaham and Hepler, 1991). These factors, therefore, severely limit the potential usefulness of this technique for studying [Ca2+Ii in plant cells.

Ester loading. The acetoxymethyl (AM) esters of the Ca’+-sensitive dyes are highly lipophilic and therefore are supposed to pass freely through the plasma membrane into the cell where they are hydrolysed by intracellular esterases. This traps the dye inside the cell, within the cytoplasm (Tsien, 1981). The AM esters of the dyes are insensitive to Ca2+ and some, for example flU0-3 AM, are also non-fluorescent. Therefore, hydrolysis releases the active form of the dyes.

Ester loading is commonly used to introduce dyes into animal cells. Inclu- sion of compounds such as Pluronic F-127 (Molecular Probes, Inc., Eugene, Oregon, USA), a low-toxicity detergent, and foetal calf serum in the loading solution have also been reported to increase the success of loading (Haugland, 1992; Graziana et al., 1993). A wide range of conditions (20min to 24 h incubations in 1-50 pM of the AM esters at 4-30’ C) (see Read et al . , 1992) have been used in an attempt to load plant cells with the AM esters of the CaZ+-sensitive dyes with varying degrees of success. These include studies of [Ca2+Ii in both individual plant cells (Keith etal. , 1985; Nobiling and Reiss, 1987; Chae et al . , 1990; Elliot and Petkoff, 1990) and tissue sections (Gehring eta/ . , 1990a,b; Williams et al., 1990) (see Table I).

The success of ester loading varies markedly from cell type to cell type (ester loading appears to be most successful in non-green tissues) and with the dye used. This variability may at least be partially the result of cell wall associated esterases which cleave the dye before it enters the cell (Callaham and Hepler, 1991; Cork, 1986). In addition, dyes may become rapidly compartmentalized on entering the cell, rather than remaining in the cytosol, through the same mechanism as ester loading due to the presence of extracytoplasmic esterases in subcellular compartments such as the endoplasmic reticulum and vacuole (Callaham and Hepler, 1991).

Electroporation (electroperrneabilization). This technique is a general


method that allows nearly any compound to be directly introduced into cells. It is best suited for use with wall-less cell suspensions - that is, protoplasts (Callaham and Hepler, 1991) - but can also be applied to walled cells growing on suitable support media (e.g. Jackson and Heath, 1990). High voltage (1.1-7.5 kV cm-') electrical pulses (10-6-10-3 s duration) are used to cause a transient permeabilization of the plasma membrane (Tsong, 1991). The conditions required to achieve this vary with each type of cell and the compound to be loaded (see Read etal., 1992 for a review of conditions used). A range of protoplasts have been loaded with Ca2+-sensitive dyes using this technique, including barley (Gilroy et al., 1986), wheat (Shacklock et af., 1992), yeast (Iida et af., 1990) and fern (Scheuerlein etal., 1991) (see Table I). There are two major drawbacks with electroporation: (i) the enzymatic isolation of protoplasts may alter the physiology of the cell or destroy vital surface proteins; and (ii) the transient permeabilization of the plasma membrane may allow the leakage of vital compounds and metabolites out of the cell as well as facilitating the loading of dyes. Therefore, the usefulness of this technique as a tool for studying [Ca2+Ii in plants is questionable.

Digitonin permeabilization. Permeabilization of plant cells, allowing the entry of the free acid forms of the Ca2+-sensitive dyes, may also be achieved using digitonin. A concentration of 0.1070 digitonin has been shown to enable the loading of fluo-3 into the cytosol of embryonic cells of Daucus carota (Timmers et al., 1991). Although fluo-3-loaded Daucus embryos have been reported to continue their development, this technique still remains to be shown to be a viable method of dye loading in plants.

B. MEASUREMENT OF [Ca2+]i

Quantification of the fluorescence signal from cells loaded with Ca2+-sensitive dyes can be achieved using either photometric or imaging with both conventional microscopy and confocal scanning laser microscopy techniques. Each of these provides a different level of information regarding the quantitative, temporal and spatial aspects of changes in [Ca2+Ii within cells. The apparatus required for each of these techniques differs markedly although they all have four components in common. These are: (i) an excitation light source (allowing the selection of excitation wavelengths); (ii) specimen holder (for the immobilization and maintenance of cells in order to facilitate dye loading, often by microinjection, and repeated measurements of fluorescence signals); (iii) detector (enabling the emission wavelengths to be specified); and (iv) signal processing (for amplification, recording and analysis of data). Many of the practical aspects of the measurement of [Ca2+], have been reviewed previously (e.g. see Cobbold and Rink, 1987; Tsien and Hartoonian, 1990; Callaham and Hepler, 1991; Read et al., 1992).


I . Fluorescence ratio photometry This technique provides information about both quantitative and temporal aspects of the [Ca”], dynamics of cells, and is characterized by a very high degree of temporal resolution (but no spatial resolution: measurements of [Ca2+], obtained are an average value for the whole of the cell). Conse- quently, ratio photometry has a low dependence on complicated hardware and computer software.

Figure 2 shows a schematic representation of the apparatus required for ratio photometry. Typically, excitation light is provided by a xenon light source. This provides a more uniform spectral output than a mercury lamp. The excitation wavelength is selected using narrow band-pass filters. For dual- excitation photometry, the excitation wavelength is alternated using a rapid filter changer or spinning rotor (a monochromator can also be used in combination with a “Chopping” device). Cells are viewed using an inverted epifluorescence microscope and quartz optics. This allows a high degree of flexibility with respect to the perfusion and microinjection of cells. A dichroic mirror is used to reflect short wavelength excitation light onto cells while passing the longer wavelength emission light to the detector. Fluorescence emissions are detected using a photomultiplier tube (PMT). When a dual- emission dye is used two PMTs are required. These are mounted at 90” to each other and a second dichroic mirror is used to split the two emission wavelengths; the shorter are reflected into one PMT and the longer are passed to the other. Specific wavelengths are selected using narrow band-pass filters. Commonly, shutters are used to protect both cells and the detector from stray illumination. Excitation of cells and data acquisition are synchronized by computer. The data, in the form of the analogue output from the PMTs are subsequently digitized and fed into the host computer for storage and ratio analysis.

2, Fluorescence ratio imaging Imaging techniques provide information about the spatial distribution of [Ca”], , in addition to quantitative and temporal changes, generating a “Ca2+-map” of the cell. This adds an additional layer of complexity to both the hardware and software required for these measurements.

Excitation of cells is achieved as with ratio photometry (Fig. 2). However, fluorescence emissions are recorded using a video camera. Typically, a low light level CCD (charge coupled device) camera is employed for imaging [Caz+Ii in plant cells. At Lancaster we use a cooled Extended ISIS-M intensified CCD camera (Photonic Science, Kent, UK). This contains two inten- sifier screens in series and is typically operated at -5°C to reduce the dark current (or “thermal noise”) generated by the detector in the absence of any light input. A rapid filter changer or spinning rotor is required for use in conjunction with dual-emission dyes to enable images at the two emission wavelengths to be captured. Excitation of cells and image acquisition are

I I XENONLIGHT SOURCE

-1 + FILTERS

I o I I liquid light guide 1 z

I I I variableaperture 1 d 81-1 dicroic mirror

EPIFLUORESCENCE MICROSCOPE

DETECTOR CHANGER

FRAMESTORE + IMAGE PROCESSOR VIDEO CAMERA

AMPLl FlER

HOST COMPUTER

AMPLIFIER

Fig. 2. A schematic representation of the apparatus used at Lancaster for the measurement of [Caz+Ii using fluorescent Ca*+-sensitive dyes. Microscope system: Systems are based around a Nikon (Telford, UK) Diaphot TMD inverted epifluorescence microscope. Excitation light is provided by a Nikon 75/100 W xenon light source. Excitation wavelengths and intensities are selected by a spinning rotor holding six narrow band-pass filters, in combination with metal gauze neutral density filters, and transmitted to the microscope via a liquid light guide (Cairn Research Ltd, UK). A Nikon CF Fluor DL 40x, oil immersion lens (aperture 1.30) and non-fluorescent immersion oil (Sigma, UK) are used for all measurements. Excitation and emission wavelengths are split using an appropriate combination of filters and dichroic mirror (Nikon). Photometry: Photometric measurements are made using a Cairn Spectrophotometer system (Cairn Research Ltd, Kent, UK). The system consists of: two 9924B photomultiplier tubes (Thorn EMI, UK) and amplifiers, together with an analogue-digital converter located in the host computer (Cairn Research Ltd). Emission wavelengths are specified using narrow band-pass filters (NikonICairn Research Ltd) and split into the two photomultiplier tubes for dual emission measurements using an appropriate dichroic mirror (Cairn Research Ltd). Data are acquired and processed using Cairn C32 software (Cairn Research Ltd). Imaging: A cooled Extended ISIS-M intensified CCD camera (Photonic Science, Kent, UK) is used to record images. Images are acquired using an ARGUS-50 image analysis system (Hamamatsu Photonic UK Ltd, Middlesex, UK), consisting of an ARGUS-50 image processor and video monitor, together with a frame store located in an 80486 host computer (Viglen, USA) running ARGUS-50 Ca2+ concentration analysis software (Hamamatsu Photonic UK Ltd). Emission wavelengths are specified using narrow band-pass filters (Nikon). Images are subsequently processed using either ARGUS-50 control software or ARGUS-50 Ca2+ concentration analysis software (Hamamatsu Photonic UK Ltd). SimuItaneous photometry and imaging: The two photomultiplier tubes and imaging camera are mounted on a purpose built detector changer enabling rapid switching between Ca2+ measurement systems. This is connected to the side camera port of the microscope via a variable aperture PFX shutter system (Nikon).


synchronized by computer. The output of the camera is digitized, typically up to a maximum resolution of 512 x 512 pixels, sent to the host computer for storage and analysis. Fluorescent images are normally displayed in 256 grey level intensities. Ratio images, calculated on a pixel by pixel basis, are coded as different pseudocolours on the basis of [Ca2+Ii.

There are two important factors that must be taken into account when using fluorescent dyes to measure [Ca2+Ii in plants: (i) cell autofluorescence; and (ii) the signal-to-noise ratio. Plant cells are highly autofluorescent at the excitation wavelengths of many of the Ca2+-sensitive dyes. It is essential to correct for the contribution this makes to the total fluorescence signal at both excitation/emission wavelengths. In photometric studies autofluorescence is normally subtracted on-line using a mean value determined for each cell prior to loading with dye (e.g. see McAinsh etal., 1990, 1992, 1995). However, in imaging studies cell movements complicate this processes, making it impos- sible to subtract the initial image of the unloaded cell from all subsequent images. Therefore, a calculated mean autofluorescence is subtracted from images, pixel by pixel, at the end of each experiment (e.g. see Gilroy etal., 1990). Following autofluorescence correction the signal from dye-loaded cells is often very low making measurements extremely noisy. The noise can be reduced by integrating successive fluorescence measurements, increasing the signal-to-noise ratio.

At Lancaster we have systems for making both photometric and imaging measurements of [Ca’’], using either non-ratiometric or ratiometric Caz+- sensitive dyes. We have also developed a system that enables us to make simultaneous photometric and imaging measurements (see Fig. 2). The major advantage of this is that it combines the high level of temporal resolution obtained with photometry with the spatial resolution of imaging. In addition, photometry provides a Ca2+-dependent ratio “on-line” so that changes in [Ca2+Ii can be monitored throughout the course of the experiment. This allows image capture to be optimized, images being obtained at the crucial points during changes in [Ca2’], . Sophisticated imaging systems (using either conventional microscopy or CSLM) have improved temporal resolution. How- ever, this is still significantly less than that obtainable by photometry. They are also capable of calculating ratio images on-line, although they do this at the expense of temporal resolution. The large amount of computer memory required for storing the images obtained during rapid, on-line ratio imaging is also prohibitive, limiting the time course of experiments substantially.

3. Images obtained by conventional microscopy are prone to out of focus blur due to light from both above and below the plain of focus. This may distort the image of the part of the cell which is in the focal plain, reducing the spatial resolution of the technique. CSLM allows optically thin sections of cells to be examined producing images that do not suffer from blur due to out of focus

Confocal scanning laser microscopy (CSLM)


light (White et al., 1987; Shotton, 1989). Sophisticated hardware and software are both required to capture these images. However, the high resolution images produced should increase the level of spatial information obtained.

Confocal microscopes illuminate a very small, precisely defined spot on the cell with the image of a pin hole. The fluorescence is viewed through a second pin hole at an identical image plane. Fluorescent light from out of focus regions of the cell is out of focus when it reaches the observation pin hole, and as such is almost totally excluded from entering the detector. Pin point illumination is achieved using a laser while fluorescence emissions are detected using a single PMT. An image is built up by scanning the confocal pin holes in two dimensions over the cell. In addition, the scan can be moved vertically through the thickness of the cell allowing a series of sequential, optically pure images to be obtained. These can be reconstructed to produce a three- dimensional representation of the distribution of [Ca2+Ii throughout the cell.

There are several disadvantages associated with CSLM, the major one of which is the restricted number of lines emitted by lasers which can be used for exciting Ca2+-sensitive dyes. Until recently only the non-ratiometric dyes with long wavelength excitation maxima were suitable for use with confocal microscopes. In addition, although most of the commercial confocal microscopes are capable of achieving a high degree of spatial resolution they have a very low level of temporal resolution. Photobleaching of dyes and cell damage, resulting from the intensity of laser illumination and localized heating effects, may also limit the number of applications for which CSLM is suited.

4. Aequorin Aequorin is a photoprotein from the coelenterate Aequorin victoria consisting of apoaequorin, a 22 000 MW polypeptide, and coelenterazine, a hydrophobic luminophore. The photoprotein has three Ca2+-binding sites. Upon binding Ca2+ the luminophore is discharged and emits a finite amount of blue light (470nm). Consequently, aequorin has been used to measure [Ca2+Ii in both animal (see Cobbold and Lee, 1991) and plant cells (Williamson and Ashley, 1982; Gilroy etal., 1989).

There are both advantages and disadvantages associated with the use of aequorin for measuring [Ca2+Ii in single cells as compared to fluorescent Caz+-sensitive dyes (see Cobbold and Lee, 1991). Advantages include:

1. Measurements of [Ca2+Ii using aequorin require less complicated hard-

2. The technique involves the quantification of blue light emissions, therefore,

3. There is no intracellular compartmentalization or leakage of the photo-

4. There is no buffering of [Ca2+li by the photoprotein.

ware and software.

there is no autofluorescence since no excitation light is used.

protein due to its high molecular weight and negative charge.


5 . Aequorin has no toxic effects within cells.

Disadvantages included:

1 . In general, photoproteins have to be introduced into cells by pressure microinjection due to their high molecular weight. This requires the use of micropipettes with a large tip diameter increasing the potential for cell damage (similar problems may also be encountered with microinjection of the high molecular weight dextran conjugates of the fluorescent Ca2+-sensitive dyes into certain smaller cell types).

2. The signal recorded from aequorin-loaded cells is normally too low to allow imaging of changes in [Ca2+Ii using this technique.

Therefore aequorin and fluorescent Ca2+-sensitive dyes can be viewed as complementary techniques for measuring [Ca2+Ii, providing different approaches to study similar questions.

5. Recombinant aequorin Recently, a new technique has been developed for the measurement of [Ca2+Ii in plants using aequorin. This employs recombinant DNA technology. Tobacco plants have been transformed with a chimeric gene construct consisting of the 35s cauliflower mosaic virus promoter fused to the apoaequorin-coding region from complementary DNA (cDNA), so that they constitutively express apoaequorin (Knight et af . , 1991). Treatment of transformed seedlings with coelenterazine results in the reconstitution of functional aequorin inside cells. Consequently, the luminescence of these plants should provide a direct measure of [Ca2+Ii within cells. Using this technique a wide range of stimuli have been shown to stimulate an increase in [Ca2+ji in intact plants. These include wind, touch, cold and fungal elicitors (Knight e taf . , 1991, 1992, 1993; also see Fig. 3).

In addition to reporting “whole-plant” [Ca”], , it is now possible to measure [Ca2+Ii in specific subcellular organelles using recombinant aequorin techniques. This is achieved by fusing the apoaequorin cDNA to an organelle specific target sequence. In animals, a chimeric mitochondria1 apoaequorin cDNA has been used to transform bovine endothelial cells, specifically tar- geting the apoaequorin to mitochondria (Rizzuto et af . , 1992). These cells have been used to demonstrate ATP-induced increases in mitochondrial [Ca2+Ii (Rizzuto etaf . , 1992). Work is currently in progress to specifically target the apoaequorin to different cell types, subcellular organelles and membrane- bound proteins in plants.

As with the use of aequorin microinjected into cells there are both advantages and disadvantages associated with the recombinant aequorin technique for measuring [Ca2+Ii. The major advantage is that it provides a non-invasive method for introducing a Ca2+-sensitive indicator directly into the cytosol of cells. This eliminates potential cell damage (although it prevents manipulations


30

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Fig. 3. Changes in the cytosolic Ca 2+-dependent luminescence of aequorin- transformed tobacco seedlings, recorded using a chemilurninometer. Traces show the effects of (a) touch (seedlings were touched once every minute [arrows] for four successive minutes with a fine wire, after which water at 0°C was added); (b) temperature shock (seedlings were rapidly transferred from 20°C to between 0 and 50°C by the addition of water a t the appropriate temperature); (c) fungal elicitors (seedlings were treated with [ 11 untreated yeast elicitor; [2] trifluoroacetic acid- hydrolysed yeast elicitor; [3] void volume from a Sephadex G-25 column of yeast elicitor preparation; [4] proteinase K digested yeast elicitor; and [ 5 ] untreated Glio- cfudiurn deliquescens elicitor); (d) wind (seedlings were subjected to wind forces of 1-12N). Data redrawn from Knight era/. (1991, 1992) with permission.

of the intracellular environment during the course of experiments). In addition, measurements can also be made on intact seedlings rather than isolated tissues or cells. The major disadvantage is that it is not clear whether all cells or only certain cell types exhibit a change in [Ca2+li (Poovaiah and Reddy, 1993). This problem is compounded by the lack of data regarding the unifor- mity of expression of apoaequorin throughout transformed plants. The first


of these problems may be answered using a sensitive photon counting camera to image aequorin luminescence at the single cell level (Read etal., 1992). Recently, Knight el al. (1993) have imaged changes in the Ca2+-dependent luminescence of aequorin-transformed tobacco seedlings; in the cotyledons, hypocotyls and roots in response to cold shock, and in the cotyledons in response to mechanical stimuli (wounding and touch). However, the second problem may prove a little harder to address. Other disadvantages include:

1 . Only a finite amount of functional aequorin is reconstituted within cells. Once this has been discharged the transformed plants are no longer capable of reporting changes in [Ca2+Ii. This problem is compounded by the relatively low levels of expression which have so far been achieved in these plants. This may be overcome by the use of a stronger promoter in the apoaequorin chimeric gene construct or the use of coelentrazine analogues, with different biological activities, to reconstitute functional aequorin within plants (Knight el al., 1993).

2. At present, the lack of both tissue and cell specificity severely limits the number of applications for recombinant aequorin techniques. Single cell studies, for example stomata1 guard cells, will not be possible until the advent of suitable cell-specific promoters for the apoaequorin chimeric gene constructs used in plant transformations.

3. The kinetics (i.e. rapid, transient increases) of all the changes in [Ca2+Ii determined by recombinant aequorin techniques vary markedly from those measured using fluorescent Ca2+-sensitive dyes (see Fig. 3). This makes comparisons between data difficult.

111. MEASUREMENT OF STIMULUS-INDUCED CHANGES IN [Ca"], IN PLANTS

There are an increasing number of reports of stimulus-induced changes in plant cell [Ca2+Ii determined using fluorescent Ca2+-sensitive dyes or aequorin- based techniques. Studies have been conducted in a variety of different species on a range of different cell types. These have revealed both increases and decreases in [Ca2+li in response to a diverse array of treatments. Table I provides a list of the studies conducted to date, including the type of cell under investigation, the stimuli used and the change in [Ca2+Ii reported. It is apparent that calcium ions act as second messengers in coupling a diverse range of important stimuli to an equally broad array of physiological responses and that this occurs in a number of different cell types. What is even more striking is that if we look at the most intensely investigated cell (the guard cell) it is apparent that stimuli which bring about opposite effects on guard cell turgor, namely auxins and abscisic acid, both stimulate increases in [Ca2+Ii. We discuss the implications of this apparently paradoxical result later. However,


before addressing this point it is necessary to consider the processes that contribute to calcium homeostasis in plant cells. As we have discussed already, a great deal more is known about the processes that regulate calcium influx and efflux in animal cells and for this reason we relate what is known of calcium homeostasis in the plant cell to the picture that has emerged from studies with animal cells.

IV. THE CALCIUM HOMEOSTATIC APPARATUS

A. MECHANISMS OF GENERATING INCREASES IN [Ca2+Ii

Agonist-induced elevations in [CaZ+Ii are brought about via a number of complex and interconnecting pathways. In plant cells these pathways are beginning to be elucidated. Studies of vertebrate and invertebrate cells have described elegant systems that are capable of generating waves or agonist- specific oscillations and spikes in [Ca2+Ii. In order to describe what has been learnt about the mechanisms by which plant cells elevate [Ca2+Ii, it will be necessary to summarize the pertinent components of animal cell signalling pathways (Fig. 4). For more detailed information regarding animal signalling pathways the reader is referred to excellent reviews of the subject by Berridge and Dupont (1994), Fasolato eta/ . (1994), Berridge (1993a,b), Fewtrell(1993), Galione (1993) and Tsien and Tsie (1990).

1. Ca2+ may enter the cytosol either from the extracellular space via an influx across the plasma membrane or via release from intracellular compartments which act as Ca” stores. Release of Ca2+ from intracellular stores requires the involvement of diffusible messengers in indirect pathways. These are described later. Influx of calcium across the plasma membrane is due to the movement of the ion, down the electrochemical gradient, through membrane- spanning pore-forming proteins. Agonist-sensitive opening or closing of these Ca’+-permeable channels can be via an indirect pathway involving diffusible second messengers. Such channels are known as second messenger-operated channels (SMOC). Alternatively, channels such as receptor-operated channels (ROC) and voltage-operated channels (VOC) are regulated directly.

The most extensively studied calcium influx pathways are those mediated by the VOC (Tsien and Tsien, 1990). The Ca2+-permeable VOC have been characterized on the basis of their pharmacology, electrophysiology and cellular distribution into four main types, L, T, N and P. These definitions may require adjustment in the light of more recent molecular investigations (Tsien and Tsien, 1990). The characteristic features of these VOC are that they are activated by membrane depolarization, exhibit high selectivity for

Direct pathways for elevating [Ca2+],

Fig. 4. A schematic representation of the mechanisms for increasing [Ca2+Ii in mammalian cells. Details and abbreviations can be found in the text. Solid lines indicate pathways resulting in an increase in [Ca”];. Dashed lines represent feedback and regulatory mechanisms. Ins(1,4,5)P3 is represented by 4 and Ins(1,3,4,5)P4 by s.


Ca2+ over other ions as a result of high affinity Ca2+-binding sites on the protein, are selectively modulated by a number of agonists and their subcellular distribution is heterogeneous. The L,-type VOC are found in both excitable and non-excitable cells. The L-type VOC are unique among the Ca'+-permeable VOC in that they are blocked by the 1,4-dihydropyridines (Tsien and Tsien, 1990).

Channel activity can be studied using patch-clamp analysis, in which changes in electrical current across the plasma membrane are monitored by sealing a very small diameter glass electrode to the membrane (Satter and Moran, 1988). Such analysis has demonstrated that the L-type VOC are activated by high voltages. This is also the case with N and P but not T-type VOC (Tsien and Tsien, 1990).

A number of cDNAs encoding subunits of the mammalian L-type channels have been cloned and sequenced providing structural data. Microinjection of mRNA encoding sense and antisense for subunits of the L-type VOC into Xenopus oocytes, followed by expression and subsequent patch-clamp analysis, has provided information regarding the role of the individual subunits of the channel protein in pore formation and gating (Tsien and Tsien, 1990).

Higher plant channels which allow the flux of CaZ+ into the cytosol across the plasma membrane were first identified in guard cells (Schroeder and Hagiwara, 1990; Cosgrove and Hedrich, 1991). Schroeder and Hagiwara (1990), using the whole cell patch clamp configuration, identified a relatively non-selective plasma membrane channel which was permeable to Ca2+. This channel showed repetitive and transient activation by abscisic acid (ABA), which was accompanied by concomitant increases in [Caz+li . The process by which ABA gates this channel is unknown but Schroeder and Hagiwara (1990) suggested that the repetitive nature of the activation of the channel makes it unlikely that it is an ROC, and is probably an SMOC. Given the apparent importance of this channel in contributing to ABA-induced increases of [Ca2+Ii, it is clear that further study of the regulation of this channel is required. The stretch activated channel reported by Cosgrove and Hedrich (1991) is discussed later.

Ca2+-influx into carrot protoplasts can be inhibited by pharmacological agents such as the phenylalkylamines (verapamil, D600, D800), the diphenyl- butylpiperidines (fluspirilene, R66204) and bepridil which are all believed to bind and block L-type channels in mammals (Graziana etal., 1988; Hether- ington etal. , 1992). Furthermore, binding sites for these agents have been located on the plasma membrane of carrot protoplasts (Graziana et al., 1988). In the study of Graziana el al. (1988), binding of 1,4 dihydropyridines (nitren- dipine, nifedipine, (+)PN200-110) was not detected and these compounds had little effect on 4sCa2+ uptake, even though the 1,4,-dihydropyridines are thought also to antagonize L-type VOC. Further analysis of Ca2+-channel antagonist binding in carrot protoplasts resulted in the purification of a


75 kDa plasma membrane protein which bound an azido derivative of phenylalkylamines ([3H]LU49888) (Thuleau et al., 1990, 1993; Hetherington et al., 1992). Verapamil-binding proteins also have been purified from maize plasma membranes (Harvey et al., 1989).

Reconstitution of the [3H]LU49888-binding protein in liposomes resulted in the formation of a Ca2+-permeable channel (Thuleau etal., 1993). Patch- clamp studies of this channel identified that it was short-lived. Following its disappearance the channel was replaced by one that was permeable to both CaZ+ and C1-. This second channel could be blocked by the addition of bepridil and verapamil providing the first direct measurement of block of a plant Caz+-channel (Thuleau et al., 1993).

The above findings suggested the presence of Ca2+-channels in the plasma membranes of plant cells which on the basis of pharmacology at least showed some similarity to those present in mammalian cells. Recent major advances suggest that Ca2+-permeable VOC present in the plasma membrane of plant cells differ significantly from those in mammalian cells.

A Ca2+-permeable VOC in the plasma membrane of carrot cell protoplasts has been reported by Thuleau etal. (1994). This channel was activated by depolarization, with a permeability sequence as follows Ba2+ > Ca2+ > Mg2+. The channel was selective for Ca2+ over K + but a permeability to K+ was not entirely excluded.

Uptake of 45Ca2+ into plasma membrane vesicles and whole protoplasts was used to identify a voltage-dependent calcium transporter in roots of Zea mays (Marshall etal., 1994). Maximum 4sCa2+ uptake was activated at - 80 mV suggesting that in vivo, this Ca2+ current is activated by depolarization of the membrane from resting (estimated to be - 150 mV). A comprehen- sive study of the pharmacology of the 45Ca2+ uptake was undertaken. This described differences between plant plasma membrane Ca2+-permeable VOC and their mammalian equivalents. The maize Caz+-flux was insensitive to verapamil, nifedipine and diltiazem, cadmium and Cu2+. Ruthenium red (which is usually considered an inhibitor of endomembrane Ca2+ channel activity) was inhibitory. 45Ca2+ uptake was inhibited by 20-30070 by the following ions: Ba2+, Cs+, Ni2+, Zn*+, MgZ+, Gd2+. Whereas, La3+, Nd3+, Mn2+ inhibited the influx by approximately 70%. It was demonstrated that Z . mays root cells possess two Ca2+ uptake pathways, the first is lanthanum- sensitive and dominates, accounting for approximately 70% of uptake with maximal uptake occurring at - 80 mV, the second is Gd3+-sensitive, accounts for 30% of uptake and peaks at -60mV. 45Ca2+ uptake was almost completely inhibited by the presence of 1 mol m-3 of each La3+ and Gd2+ (Marshall et al., 1994).

Pineros and Tester (1993) reported a Ca2+-permeable channel in plasma membrane vesicles of root cells of wheat. This VOC also was activated by depolarization from the resting potential. Verapamil inhibited the channel open time whereas AIC13 ( 7 0 ~ ~ ) completely blocked the channel. These


authors suggested a role for the root plasma membrane Ca2+-permeable VOC in aluminium toxicity. The wheat Ca2+-permeable VOC and the voltage-dependent Ca2+-fluxes across maize root cell plasma membranes differed in their sensitivity to verapamil. This suggests that the activities of different Ca2+channels were being observed during the studies of Pineros and Tester (1993) and Marshall etal. (1994). Whether these channels were activated by dissimilar conditions or reside in different root cells or species remains to be established.

It is not known whether any of the voltage-dependent Ca2+-influx activities described in carrot protoplasts, maize root protoplasts or plasma membrane vesicles of wheat root are involved in signal transduction processes. A number of investigations have attempted to identify the role of plasma membrane Ca2+-channels in physiological responses by testing the sensitivity of these responses to agents known to antagonize mammalian Ca2+- permeable VOC (e.g. McAinsh ef al., 1991; Knight et al., 1992). However, the Ca2+-permeable VOC in plant plasma membranes show very different sensitivity to inhibitory agents compared to that shown by mammalian channels (Marshall et al., 1994). Therefore, some caution should be employed before agents whose pharmacology has been determined from studies of mammalian cells are used in plant-based studies. Additionally it is clear that plant plasma membrane Ca2+-permeable VOC show altered sensitivity to certain agents in different cells and conditions (Pineros and Tester, 1993; Thuleau et al . , 1993, 1994; Marshall et al., 1994). Verapamil, bepridil, Gd3+, La3+ and certain 1-4 dihydropyridines have all been demonstrated to inhibit directly the flux through outward rectifying K + channels, further complicating the use of these compounds in assessing the importance of Ca 2+-channels in contributing to agonist-induced increases in plant [Ca2+Ii (Terry et al., 1992).

ROCs open in response to the binding of an external ligand. The activation is direct and occurs without the intervention of a diffusable messenger, though such messengers may be involved in modulation of the channel activity. ROC may [e.g. the N-methyl-D-aspartate (NMDA) receptor] or may not (e.g. the ATP receptor) be additionally gated by voltage (Tsien and Tsien, 1990). As far as we are aware there are no reports of Ca2+-permeable ROC activity in the plasma membrane of plant cells.

A considerable inward Ca2+ conductance in smooth and skeletal muscle cells is carried by stretch activated channels (SAC) (Tsien and Tsien, 1990). Cosgrove and Hedrich (1991) have identified Ca2+-permeable SAC in the plasma membrane of guard cell protoplasts of V. faba, which when activated allowed Ca2+-influx. The role of the SAC Ca2+-permeable channel in the signal transduction network of guard cells remains to be determined. In addition, SAC permeable to K + and anions were also located in the guard cell plasma membrane, these may also be important in regulating stomata1 movements. Another Ca2+-permeable SAC has been identified by patch-clamp analysis of the plasma membrane of epidermal cells of onion bulbs (Ding and


Pickard, 1993a,b). The onion Ca2+-permeable SAC exhibited a voltage- dependent component to its gating by mechanostimulation. This channel showed a biphasic response to Gd3+ and La3+. At low concentrations Gd3+ (< 1 p ~ ) and La3+ (< 25 pM) stimulated channel activity in response to mechanoactivation, whereas at higher concentrations both Gd3+ (> 1 p ~ )

and La3+ (> 4 0 ~ ~ ) were inhibitory. The effects of Gd3+ and La3+ could have been a result of blockage and/or interference with the mechanosensor mechanism. The tension-dependent activity of the onion Ca2+-selective SAC increased as temperature was dropped from 25°C to 6°C (Ding and Pickard, 1993b). Ding and Pickard (1993b) proposed that temperature-sensitive plasma membrane Ca2+-selective SAC may be involved in thermonastic responses, such as the closure and reopening of petals and thermotropic responses.

Communication between some animal cells can involve the spread of waves of Ca2+ (or another diffusable messenger such as Ins(1,4,5)P3) via gap junc- tions (Tsien and Tsien, 1990; Berridge, 1993a). Whether plant cells com- municate with each other in a similar manner via plasmodesmata requires investigation. It is worthy of note that the most well understood plant signalling pathways are found in the guard cell, which lack such connections (Willmer, 1983).

2. In many animal cells binding of an agonist to a cell surface G-protein linked receptor or tyrosine kinase receptor results in the accumulation of a diffusible messenger such as Ins(1,4,5)P3 (or possible cADPR) which in turn elevates cytosolic free calcium via release from internal stores or via influx through SMOC (Fig. 4).

Indirect pathways for generating an increase in [Ca2+li

Ins(l,4,5)P3-sensitive Ca2+ release. Hydrophilic Ins(1 ,4,5)P3 (as well as another messenger, DAG) is generated by the action of PLC upon PtdIns(4,5)P2 which is found in the inner leaflet of the plasma membrane. PtdIns(4,5)P2 is a phospholipid formed via the successive phosphoryia- tions of phosphatidylinositol (PtdIns) to phosphatidylinositol 4-phosphate (Ptd(4)InsP) and to PtdIns(4,5)P2 (Berridge and Irvine, 1989).

Activation of a G-protein linked receptor results in a conformational change in loops I1 and 111 of the receptor which causes the G protein to dissociate. The cy subunit exchanges guanosine diphosphate (GDP) for guanosine triphosphate (GTP) and activates PLC-01 and the GPy subunits activate PLC- p, both of which generate Ins(1 ,4,5)P3. Agonists such as platelet-derived growth factor cause the dimerization of tyrosine kinase receptors. Dimeriza- tion allows the kinase domains of these receptors to autophosphorylate specific tyrosine residues, thereby exposing docking sites for SH2 domains on PLC-yl. Phosphorylation of this enzyme results in the generation of Ins( 1 ,4,5)P3 (Berridge, 1993a).

Ins(1 ,4,5)P3 diffuses into the cytosol where it binds the Ins(1,4,5)P3-


receptor (IP3R) resulting in release of Ca2+ from Ins( 1 ,4,5)P3-sensitive stores, thereby elevating [Ca”], (Berridge, 1993a; Mikoshiba et al., 1993). IP3R is a tetrameric transmembrane protein which contains the Ins( 1,4,5)P, binding site at the N-terminal end located in the cytoplasm. The C-terminal contains a membrane-spanning region which forms a Ca2+ channel. Binding of Ins( 1 ,4,S)P3 results in a conformational change which is probably associated with channel opening. IP3R are found on the membranes of rough and smooth ER, therefore these are believed to be the Ins(1 ,4,5)P3-sensitive stores (Berridge, 1993a).

Ins( 1 ,4,5)P3-induced Ca2+-release via the IP3R has been demonstrated via the challenge of permeabilized cells with Ins(1,4,5)P3 (Hill et al., 1988), microinjection of Ins( 1,4,5)P, into single cells (Payne et al., 1988), photolysis of caged-Ins( 1 ,4,5)P3 in Xenopus oocytes (Parker and Ivorra, 1990), challenge of intact membrane vesicles (Galione et al., 1993b), patch-clamp recordings of purified IP3R reconstituted in planar lipids (Ehrlich and Watras, 1988) and transfection of L-fibroblasts with IP3R-encoding cDNA (Mikoshiba etal., 1993). Such studies have demonstrated that the IP3R has a bell shape response to CaZ+, with this ion being stimulatory in the 100-300nM region (Berridge and Dupont, 1994). The role of Ca2+ in regulating the IP3R will be discussed later. IP3R is inhibited by heparin, caffeine and ethanol. The use of heparin in whole cell studies is complicated by the inhibition of Ins( 1 ,4,5)P3 generation by this agent (Berridge, 1993a).

Cloning of the IP3R has identified at least four families, all of which share significant homology (Berridge, 1993a; Mikoshiba et al., 1993). The primary sequence of the IP3R shows no homology with those of plasma membrane VOC Ca2+ channels (Mikoshiba etal., 1993). A highly conserved region in the free C-terminal domain appears to be involved in channel opening because antibodies directed against this region can either enhance or inhibit Ins( 1 ,4,5)P3-induced Ca2+-release (Berridge, 1993a,b). Deletion of any fraction of another highly conserved 650 amino-acid sequence at the N-terminal prevents Ins( 1,4,5)P3 binding (Mikoshiba et al., 1993).

Each of the four Ins(l,4,5)P3-binding sites of the tetrameric channel are available, though there is some debate as to whether the binding of the four molecules of Ins( 1 ,4,5)P3 act co-operatively or not, to open the channel (Ber- ridge, 1993a). Ins(1,4,5)P3 can release only a proportion of the Ca2+ present in Ins(1 ,4,5)P3-sensitive stores. This “quanta1 calcium release” is probably a result of variations in receptor sensitivity. These variations in sensitivity may be due to receptor modulation by a sensor which increases IP3R sensitivity as the store fills and/or due to receptor heterogeneity as a result of transcrip- tion from different genes or post-translational modification (Berridge, 1993a).

In addition to its role of releasing intracellular Ca2+ , Ins(1,4,5)P3 can generate an influx of Ca2+ across the plasma membrane via activation of an SMOC which is an inositol phosphate receptor (IPR). The IPR is more sensitive than the IP3R t o inositol 1,3,4,54etrakisphosphate, a phosphorylated


15000

- E n

= 10000

U

UJ

- n I- - 0 c 0. U J 0 c a u Q)

Q) - - a rn 5000 -

I

P N

c.

0

3500

GroPl Gr"P'P

3000

2500

z n U

0

v) 0 c

-2000 = c

-1500 ';

-1000 m=

7 L

t

t GroPlPZ

10 20 30 40 50

fraction

Fig. 5. High performance liquid chromatography of deacylated phospholipids from [32P]-labelled guard cell protoplasts of Commelina communis $0 ), chromatographed with mammalian [3H]-glycerophosphatidylinositol (GroPI), [ HI- glycerophosphatidylinositol phosphate (GroPIP) and [3H]-glycerophosphatidylinositol bisphosphate (GroPIP,). Tritiated compounds (0). Webb, A. A. R., Drobak, B. K. and Hetherington, A. M. (unpublished observations).

derivative of Ins(1 ,4,5)P3. Therefore it appears that both these inositol phosphates can mobilize an influx of calcium (Berridge, 1993a,b; Fasolato et al., 1994).

Plant cells possess PtdIns, PtdIns(4)P and PtdIns(4,5)P2, along with other inositol-containing phospholipids and the appropriate enzymes for their inter- conversion (Drobak, 1992, 1993; Hetherington and Drobak, 1992; Cote and Crain, 1993; Parmar and Brearly, 1993). However, the relative abundance of PtdIns(4,5)P2 is very low in plant ceIls compared to that in animaI cells (Fig. 5 ; and see Hetherington and Drobak, 1992).


Despite the presence of the inositol-containing phospholipids and PLC activity in plant plasma membranes, a clear identification of Ins( 1 ,4,5)P3 in a higher plant cell has yet to be achieved (Hetherington and Drobak, 1992; Cote and Crain, 1993; Drobak, 1993; Drobak e taf . , 1994). However, plant cells are competent to respond to Ins( 1 ,4,5)P3. Release of Ins( 1 ,4,5)P3 in the cytosol of guard cells resulted in an increase in [Ca2+Ii, inactivation of the plasma membrane K+ -influx channel, activation of the depolarizing "leak" current and subsequent stomata1 closure (Blatt et af . , 1990; Gilroy et af . , 1990). The Ins( 1 ,4,5)P3-sensitive Ca2+-store in guard cells remains to be determined. We describe the progress that has been made in identifying Ins( 1 ,4,5)P3-sensitive stores and receptors in other higher plant cells.

On the basis of the current evidence plant cells possess an IP3R that is similar to that identified in mammalian cells. However, the plant IP3R is on the tonoplast and does not appear to be located on the endoplasmic reticulum (ER) (Canut e taf . , 1993). Ins(1,4,5)P3 has been reported to release Ca2+ from isolated vacuoles (Drobak and Ferguson, 1985; Ranjeva et al., 1988; Alexandre et af . , 1990) and tonoplast vesicles or microsomes (Schumaker and Sze, 1987; Brosnan and Sanders, 1990; Canut etaf . , 1993). Patch-clamp analysis of whole vacuoles isolated from red beet has demonstrated that Ins(] ,4,5)P3 releases Cat+ from the vacuole via a Ca2+-permeable channel (Alexandre et a f . , 1990).

Ins( 1 ,4,5)P3-induced Ca2+-release measured either by 4sCa2+ flux from tonoplast vesicles and whole vacuoles or by patch-clamp analysis, has a number of features in common with Ins(1 ,4,5)P3-induced calcium release via the IP3R in mammalian cells. Ca2+-release by the vacuole is specific for Ins( 1 ,4,5)P3 over other inositol phosphates, this specificity is similar to that demonstrated by the mammalian IP3R (Johannes et a f . , 1992b). The vacuolar Ins( 1 ,4,5)P3-sensitive Ca2+-release is inhibited by heparin and this inhibition appears to be due to competition with Ins(1,4,5)P3 for binding sites, rather than due to blockage of the channel (Johannes e taf . , 1992b; Brosnan and Sanders, 1990, 1993). The estimated Kd for Ins( 1 ,4,5)P3-induced Cat+- release from the higher plant vacuole is in the range 0.2-1 pM which compares well with the Kd for release via the mammalian IP3R (0.1-1 p ~ ) (Alexandre and Lassalles, 1992). Ins(1,4,5)P3 is unable to release all the Caz+ taken up by tonoplast vesicles (Brosnan and Sanders, 1990; Canut et af . , 1993). Whether this quanta1 release by the vacuole is a result of modification of receptor sensitivity as appears to be the case with the IP3R in animals, or is a result of the presence of both Ins( 1 ,4,5)P3-sensitive and -insensitive vacuolar stores is not known. However, an Ins( 1 ,4,5)P3-independent release pathway is present at the tonoplast (see below).

A striking difference between the vacuolar 1ns(l,4,5)P3-sensitive Ca2+-release and the mammalian IP3R is the lack of inhibition of the vacuolar system by extravacuolar Cat+. Alexandre etaf . (1990) were able to detect activity of the vacuolar Ins(1 ,4,5)P3-sensitive Ca2+-channel in the


presence of millimolar Ca2+ at the cytoplasmic face of the tonoplast. “Cyto- plasmic” Ca2+ concentrations of this order result in the inhibition of the mammalian IP3R (Berridge, 1993a; Berridge and Dupont, 1994).

The mammalian IP3R consists of both a receptor and channel. It is not known whether the Ins(1 ,4,5)P3-receptor in plants and the tonoplast Ins( 1 ,4,5)P3-sensitive Caz+-channel reside on the same protein. However, Brosnan and Sanders (1993) describe the identification of high affinity binding sites for Ins(1,4,5)P3 on the tonoplasts of red beet. Ca2+-release by red beet vacuoles has previously been shown to be Ins( 1 ,4,5)P3-sensitive (Alexandre et al., 1990; Brosnan and Sanders, 1990). The Ins(1 ,4,5)P3-specific binding site appears to be proteinaceous. Furthermore, the binding of Ins(1,4,5)P3 is inhibited by competition with heparin, has an estimated Kd of 0.1 p ~ , and is specific for Ins(l,4,5)P3 over other inositol phosphates. The above data suggest that the red beet tonoplast Ins( 1 ,4,5)P3-specific binding site may be involved in Ins( 1,4,5)P3-induced Ca2+-release from the vacuole (Brosnan and Sanders, 1993).

Binding of Ins( 1 ,4,5)P3 to the red beet tonoplast Ins( 1 ,4,5)P3-binding site was inhibited by sulphydryl reagents (Brosnan and Sanders, 1993). Preincuba- tion of red beet microsomes with Ins(1,4,5)P3 protected the binding site from sulphydryl reagents. These results are very similar to those obtained with the mammalian IP3R. It is believed in the case of the mammalian IP3R that sulphydryl reagents act upon cysteine residues in the Ins( 1 ,4,5)P3-binding site. It has yet to be confirmed that these reagents act in a similar manner on the plant Ins( 1 ,4,5)P3 receptor.

Ryanodine receptors and cADPR-sensitive release. Another principle intracellular Ca2+-release channel is the ryanodine receptor (RYR) (Tsien and Tsien, 1990; Berridge, 1993a,b; Fewtrell, 1993; Berridge and Dupont, 1994). The RYR shows many structural and functional homologies with the IP3R suggesting common evolutionary origins, though the RYR is larger. There are three families of RYR. RYRl is found in skeletal muscle, RYR2 in cardiac muscle and RYR3 in non-muscle cells. The RYR are tetrameric and show considerable sequence homology between families and a certain amount with the IP3R, especially in the C-terminal membrane-spanning region (Berridge, 1993a,b). At nanomolar concentrations the plant alkaloid ryanodine results in channel opening and release of Ca2+ from stores, whereas at concentrations greater than micromolar, ryanodine inhibits RYR. Caffeine activates RYR, unlike the IP3R (Berridge, 1993a). RYRl can also be activated by the cell surface dihydropyridine receptor which appears to be in physical contact with the channel protein (Berridge, 1993a; Fewtrell, 1993).

Recently, advances have been made in understanding the in vivo regulation of RYR. Caz+ can be released from ryanodine-sensitive stores by the messenger cADPR (Galione, 1993; Galione et d. , 1993a,b; Thorn et al., 1994). In sea urchin eggs cADPR synthesis from P-NAD+ (P-nicotinamide adenine


dinucleotide) is activated by the action of 3 ' ,5 '-cyclic guanosine mono- phosphate (cGMP) on the synthetic enzyme ADP-ribosyl cyclase and it has been proposed that cADPR may transduce signals generated by cell surface receptors linked to cGMP production (Galione etal., 1993b). However, it is unlikely that cGMP will regulate cADPR production in all cell types (Thorn et al. , 1994).

Recently, Allen et al. (1995) have demonstrated that cADPR can release Ca2+ from plant vacuoles via a voltage-dependent pathway.

IP3R and R YR and calcium-induced calcium release. Both ryanodine- and Ins(l,4,5)P3-sensitive stores can reside in the same cells. Not all animal cells possess both types of stores. In sea urchin eggs the presence of both types of stores appears to represent redundant calcium-release pathways (Galione et al., 1993a), whereas in pancreatic acinar cells the operation of both release pathways appears to be required for the generation of the full Ca2+ response (Thorn et al., 1994).

The common sensitivity of RYR and IP3R to Ca2+ has led to the suggestion that both these stores are involved in calcium-induced calcium release (CICR) (Berridge, 1993a,b; Berridge and Dupont, 1994). CICR is a feed- forward process by which small elevations in [Ca2+], stimulate large releases of Ca2+ from both ryanodine and Ins( 1 ,4,5)P3-sensitive stores. The CICR properties of the IP3R result in an all-or-none response in which a sudden and near maximal release occurs as Ins(1,4,5)P3 concentrations rise. This is a con- sequence of released Ca2+ inducing further Ca2+ release (Berridge, 1993a).

Berridge and his co-authors believe that an important role of cADPR and Ins(1,4,5)P3 is to sensitize RYR and IP3R respectively to Ca2+ in order to induce CICR (Berridge 1993a,b; Berridge and Dupont, 1994). Other researchers place greater emphasis on the ability of Ins(1 ,4,5)P3 to evoke release, while acknowledging the role of Ca2+ in sensitizing the IP3R (Berridge, 1993a; Fewtrell, 1993 and references therein).

Agonist-induced oscillations and waves of increase of [Ca2+Ii across the cell have been observed in many animal cells types (Berridge, 1993b; Fewtrell, 1993; Miyazaki and Shirakawa, 1993). Release of Ca2+ from intracellular stores can form an important component in the generation and maintenance of waves and oscillations. A number of models have been proposed which describe how the above mechanisms interact to generate complex [Ca2+Ii signals. Fewtrell (1993) classified these models into three main classes; those where the abundance of Ins(1 ,4,5)P3 oscillates, those where Ins(1 ,4,5)P3 increases but does not oscillate and those where no increase of Ins(1 ,4,5)P3 is required to generate the Ca2+ signal. Whether such models will require modification in the light of recent discoveries concerning the role of cADPR as a messenger remains to be determined. The reader is referred to the following reviews where models of spatial and temporal variations in [Ca2+Ii are discussed in detail: Berridge and Dupont (1994), Fewtrell (1993), Berridge


(1993a,b), Tsien and Tsien (1990). An interesting recent finding suggests that the accumulation of Ca2+-stores in restricted regions of the cell may also be important in defining subcellular [Ca2+Ii gradients (Stendahl et al., 1994).

Ca2+ -release pathways have been described in plant cells which appear to be distinct from the Ins (1 ,4,5)P3 ryanodine/cADPR-sensitive systems described above (Johannes et al., 1992a,b; Gelli and Blumwald, 1993; Allen and Sanders, 1994; Ward and Schroeder, 1994). The Ins( 1,4,5)P3-insensitive channels are located in the tonoplast of the vacuole, though it is not known yet whether this represents the same Ca2+-store as the Ins(l,4,5)P3-sensitive store. One of the Ins(l,4,5)P3-insensitive influx pathways at the tonoplast may be involved in CICR (Ward and Schroeder, 1994).

In describing fluxes across the tonoplast we shall adopt the conventions of Bertl etal. (1992) in which the voltage across the tonoplast (V,) is defined as follows:

Vrn = Vcytosol - Vvacuole

Positive or outward currents, therefore, represent the flux of cations out of the cytosol (i.e. into the vacuolar lumen). Therefore, under resting conditions the cytoplasm is negative relative to the vacuole due to the action of H+-translocating ATPases and pyrophosphatases in the tonoplast (Rea and Poole, 1993). Not all the articles referred to here have used the convention proposed by Bertl etal. (1992). For clarity such data have been redefined accordingly.

Ward and Schroeder (1994) proposed that in plant cells CICR requires the co-ordinated action of at least two distinct classes of tonoplastic channel, a voltage-independent Ca*'-sensitive K+-influx channel (VK) and a very high conductance, voltage- and Ca2+-gated cationic channel which has previously been designated the slow vacuolar (SV) channel. The VK channels identified by Ward and Schroeder (1994) in the tonoplast of guard cells of V. faba allowed K+-influx from the vacuole and were activated by increases in [Ca2+li; however, they were not permeable to Caz+. Alkalinization of the cytosol resulted in a reduction of VK activity. The SV channel, unlike the VK channel, was demonstrated to be capable of permitting Ca2+-influx from the vacuole to the cytosol (Ward and Schroeder, 1994). SV-type channels have been described in the tonoplasts of a number of plant cells; they are characterized by high conductances, activation at positive membrane potentials and permeability to cations, though so far only in the guard cell have they been demonstrated to be Ca2+-permeable (Ward and Schroeder, 1994 and references therein).

The SV channel is open at positive membrane potentials, which are unlikely to occur in resting cells. This has meant that it has proved difficult to ascribe a physiological role for the SV channel, but Ward and Schroeder (1994) pro- pose that it is via this channel that CICR may occur in plant cells.


The guard cell VK is activated by elevations in [Ca2+], . Therefore, Ward and Schroeder (1994) proposed that this channel is activated by an initial rise in [Ca2+Ii in response to a stimulus which promotes stomata1 closure, such as ABA (McAinsh etal., 1990, 1992; Schroeder and Hagiwara, 1990). Opening of the VK results in an influx of K + which in turn makes the tonoplast potential more positive. At positive potentials SV is activated by increases in [Ca2'Ii and therefore in the model of Ward and Schroeder (1994) this channel will allow influx of Ca2+, which will serve to both further elevate [Ca2+Ii and make the membrane potential even more positive. The activation of SV by increased [Ca2+Ii, in barley aleurone cells, appears to involve the action of calmodulin (Bethke and Jones, 1994). K+-influx across the tonoplast cannot be maintained at potentials positive of about 100 mV. Ward and Schroeder (1994) proposed that the activity of H+-translocating pyrophosphatases and ATPases in the tonoplast maintains a potential negative enough to allow continued influx of K + into the cytosol from the vacuole.

The Ward and Schroeder model is very intriguing and it proposes an elegant mechanism for CICR which is dissimilar to those evolved by animal cells. It is perhaps surprising that plant cells possess an Ins( 1 ,4,S)P3-sensitive Ca2+-channel which on the basis of current data appears to be similar to the IP3R, but have evolved an alternative pathway for CICR.

At least three other Ins( 1 ,4,5)P3-insensitive Ca2+-permeable influx channels have been identified in the tonoplast (Johannes eta/. , 1992a,b; Gelli and Blumwald, 1993; Allen and Sanders, 1994). The first of these was described by Johannes etal. (1992a,b) in sugar beet tap roots. This voltage-gated Ca2+-influx channel is very different from the SV described above. It is open at negative potentials and has a much lower conductance (12pS), which saturates at -40mV. The channel is inhibited by Zn2+ and Gd3+ and appears to be insensitive to Ins(1 ,4,5)P3, heparin, ryanodine and [Ca2+Ii. A very similar channel has been identified in the tonoplast of V. faba guard cells alongside another Ins( 1 ,4,5)P3-insensitive Ca2+-permeable channel (Allen and Sanders, 1994). This second channel shows some similarities with the one described above. It is active at negative membrane potentials and is inhibited by Gd3+. They differ in that the second channel has a higher conductance (27pS), is inhibited by nifedipine and is gated open by high vacuolar Ca2+ concentrations (Allen and Sanders, 1994). The physiological significance of the presence of two similar tonoplastic Ca2+-influx channels is not known. Allen and Sanders proposed that the two channels are involved in transducing different signals or may contribute to different types of elevations in [Ca2+Ii.

Gelli and Blumwald (1993) also identified an Ins(1 ,4,5)P3-insensitive Ca2+-influx channel in the tonoplast which is active at negative membrane potentials. Unlike those channels identified by Sanders and his coworkers, this channel is regulated by increasing [Ca2+Ii. The Ca2+ channels in sugar beet


tonoplasts described by Gelli and Blumwald (1993) were inhibited by nifedipine, verapamil and La3+.

It has proved difficult to identify physiological roles for the tonoplast Ins( 1 ,4,5)P3-insensitive Ca2+-influx channels which are open at negative membrane potentials (i.e. all those described above except the SV channel). This is because their voltage dependencies are such that at physiological membrane potentials the channels would always be active, resulting in uncontrolled Ca2+-influx. However, Allen and Sanders (1994) report that the two Ca2+-influx channels, active at negative membrane potentials in the guard cell tonoplast, are also regulated by vacuolar pH. Assuming a vacuolar pH of 5 .O, these channels would not normally be open. Therefore, it is possible that tonoplastic CaZ+ channels which are open at negative membrane potentials may be gated by other unidentified agents. If these putative regulators are identified, it may prove possible to define a role for these channels in regulating increases in [ca2+l i .

It is clear that a number of distinct pathways exist in the tonoplast to regulate Ca2+-influx into the cytosol. Whether these pathways contribute to the elevations in [Ca2+Ii in response to different stimuli or whether they are involved in generating a variety of patterns of increase in [Ca2+Ii remains to be determined.

Another Ca2+-permeable channel has been identified in the tonoplast of sugar beet cells. The channel described by Pantoja etaf . (1992) was outward- rectifying and active at positive membrane potentials. Pantoja et af. (1992) proposed that this channel is involved in the uptake of Ca2+ from the cytosol following CICR. However, it is unlikely that under normal conditions Ca2+], would reach the millimolar concentration required for this channel's activity. It is interesting that the model proposed by Ward and Schroeder (1994) for CICR would result in the positive membrane potentials required for this Ca2+-efflux channel to open. Other Ca2+-uptake mechanisms, i.e. the Ca2+-ATPases, are discussed later.

Capacitative Ca2+ entry. Depletion of Ca2+-containing stores by a number of agents stimulates an influx of Ca2+ across the plasma membrane (Putney and Bird, 1993; Fasolato etaf . , 1994). The process has been terr-ied kapaci- tative entry". Recently evidence has accumulated that depletion of intracellular stores results in the release of a diffusible messenger, Ca2+-influx factor (CIF) (Parekh e taf . , 1993; Randriamampita and Tsien, 1993). CIF has not been fully characterized but it is known to be a small (< 500 Da), lipophilic, phosphate-containing anion that can induce Ca2+-influx in a number of cells. It is found in the cytosol of resting cells and the organelles of active cells (Randriamampita and Tsien, 1993; Fasolato e taf . , 1994). A small G protein may also be involved in capacitative Ca2+ entry (Putney and Bird, 1993; Fasolato et af., 1994).


CIF probably acts upon a Ca2+-release activated Ca2+-channel (CRAC) present in the plasma membrane. This influx channel is activated by agents which deplete intracellular stores and is inhibited by high [Ca2+Ii (Fasolato et al. , 1 994).

The presence of a Ca2+ chelator outside the guard cell can deplete intracellular [Ca2+Ii (Gilroy etal. , 1991; Webb etal., In Press). It has not been investigated whether such a depletion results in an influx of Ca2+ across the guard cell plasma membrane. Furthermore, it has not been demonstrated that an extracellular Ca2+-chelator depletes intracellular Ca2+-stores stores in addition to cytosolic Ca2+ in guard cells.

B. Ca2+-ATPases

In mammalian tissues, [Ca2+Ii is returned to and maintained at resting levels by the action of Ca'+-translocating ATPases in the plasma membrane (PM Ca2+-ATPase) and the membranes of the ER and sarcoplasmic reticulum (SR). These Ca2+-ATPases remove Ca2+ from the cytosol, and in the case of the ER/SR Ca2+-ATPases consequently bring about loading of intracellular Ca2+-stores (Tsien and Tsien, 1990; Berridge, 1993a). The PM Ca2+- ATPases are directly activated by calmodulin and show little sequence similarity with ER/SR Ca2+-ATPases which are indirectly activated by calmodulin.

The current literature concerning Ca2+-ATPase activity in plant cells has recently been reviewed by Evans (1994). To avoid repetition we shall summarize the major conclusions of that excellent article, to which the reader is referred. In plant cells, calmodulin-stimulated Ca2+-ATPase activity is associated with the plasma membrane and intracellular membranes. Mem- brane purification, protein purification and reconstituted and immunological studies have demonstrated that the vast majority of calmodulin-stimulated Ca2+-ATPase activity is associated with the intracellular membranes, most likely the ER. This is totally unlike the situation in mammalian cells. Plants also possess a Ca2+-ATPase activity which is not stimulated by calmodulin and molecular studies have identified plant homologues of a mammalian SR Ca2+-ATPase.

The complexity of plant Ca2+-ATPases means that further work is required before the role of each type of pump in signalling can be assessed. However, in the case of gibberellic acid (GA)-induced secretion of a-amylase by barley aleurone, a physiological role for the calmodulin-stimulated ER Ca2+-ATPase has been described. Calmodulin-stimulated calcium transport into the ER is associated with secretion of a-amylase. It is believed that Ca2+-binding stabilises a-amylase in vivo. Furthermore GA also stimulates increased calmodulin levels in this tissue (Evans, 1994).

84 A. A. R . WEBB et al.

V. THE PROBLEM OF SPECIFICITY

It is clear from the data presented in Table I that calcium ions are used as second messengers to couple a diverse array of stimuli to their characteristic responses. On the basis of this evidence it would seem likely that this list will expand in the next few years as the techniques for measuring [Ca2+Ii are applied to other cell types. Given the apparent ubiquity of the calcium messenger system the question of specificity arises. Clearly, there is every reason to believe that specificity can be built into signal transduction systems at the level of the receptor and indeed there has been progress on the isolation of molecules with properties which would suggest that they may well serve this purpose (Chang etal., 1993; Jones, 1994). Based on work on the animal heterotrimeric G proteins it also seems possible that there is the capacity for building in specificity in terms of receptor43 protein interactions where specific G-protein subunits can interact with specific effector molecules. However, difficulties begin to manifest themselves at the level of the calcium signal. One example, which has been alluded to already, should illustrate the problem. This is the action of auxins and ABA on stomata1 guard cells. Although these compounds bring about opposite physiological effects they both cause an increase in [Ca*'], . The question, then, is how can an increase in calcium control the fluxes of ions responsible for both an increase or decrease in turgor?

A. OTHER SECOND MESSENGERS

One possible solution to this problem relies on the participation of other second messengers in the generation of the final response. Is there any evidence to suggest that this may be the case in plant cells? Again guard cells provide some of the most relevant evidence. It has now been shown convinc- ingly that the elevation of free calcium induced by ABA is accompanied by an increase in cytoplasmic alkalinization while in the case of auxins the pH of the cytoplasm drops (Irving etal., 1992; Blatt and Thiel, 1993). Although it is not clear whether protons are behaving as second messengers and their source has yet to be accounted for, the ability to modulate pH concurrently with calcium begins to present an attractive mechanism for the differential control (in this specific example) of the ion channel activity responsible for mediating the changes in guard cell turgor. Again, space prevents a full discussion of the possibilities for fine control using this approach but for a very useful and detailed account the reader should consult Blatt and Thiel (1993). In addition to protons/hydroxyl ions there is evidence for the involvement of other putative second messengers in plant cells. Again much of the most recent evidence has come from guard cells where it is possible to use patch clamp analysis to study the effect of putative second messengers on the ion channels


responsible for bringing about turgor changes. Such studies have generated data which suggest that guard cells are capable of responding to CAMP, DAG and Ins(1,4,5)P3 (Assmann, 1993; Li e ta / . , 1994). In addition to these data are the results from the Chua laboratory which strongly support the involvement of cGMP in the phytochrome stimulus-response coupling pathway (Bowler etal. , 1994).

B. THE CALCIUM SIGNATURE - A STIMULUS-SPECIFIC CALCIUM SIGNAL

Another possibility for generating specificity which would not necessarily rely on other second messengers would be to encrypt information into the calcium signal itself. Such an idea has generated much interest in animal cell biology. Whittaker and colleagues (Galione et a/. , 1993b) have coined the term “calcium signature” for a stimulus-specific calcium signal. There are two obvious (but not necessarily separate) ways of encoding information in the calcium signal. The first is to generate increases in calcium which occur in localized subregions of the cell that are close to the primary and secondary effector molecules required to generate a particular response. Such a mechanism would require the localization of either plasma membrane calcium channels or subcellular release sites to an area overlying the effector molecules. In the case of the plasma membrane channels this might be fairly simple and depend on receptor clustering. However, the situation with the subcellular sites is much more complex as it would also require the participation of other second messengers. In animal cells there is evidence these strategies are utilized. In the case of pancreatic acinar cell it has been shown that application of acetylcholine or cholecystokinin at physiological concentrations results in a localized increase of free calcium and this correlates in spatial terms with the cellular distribution of the a-amylase-containing zymogen granules (Kasai et al. , 1993; Thorn etal., 1993).

Temporal aspects of the calcium signature may also be very important, especially in situations where an agonist induces intracellular calcium to oscillate. This phenomenon has been recorded in many cells in response to a variety of agonists. In overview, it has been found that the pattern of oscillations depends on both the type and strength of the agonist. These observations led to the suggestion that the frequency and amplitude of the oscillations may encode signalling information. The frequency of the oscillations varies from approximately 10 s up to several minutes (Tsunoda, 1993). Superimposed on these temporal elements are spatial considerations. It is clear that in certain cells the oscillations can propagate as a wave which traverses the cell. At the moment the precise physiological significance of waves and standing oscillations is a matter of some debate (Tsien and Tsien, 1990; Amundson and Clapham, 1993). However, their characterization and in particular the mechanism of their generation is much better understood (Berridge, 1993a,b;

86 A. A. R. WEBB er al.

Berridge and Dupont, 1994; Bootman, 1994). Here there is good evidence that the delicate interplay of influx across the plasma membrane in combination with the release from intracellular stores is capable of generating the subtle but highly reproducible pattern observed in a number of cell types. When oscillations and localized increases are superimposed on an asymmetric distribution of effector molecules then it is obvious that there is great scope for generating specificity.

C. CALCIUM SIGNATURES IN PLANT CELLS

Is there any evidence for a calcium signature in plant cells? Again the guard cell is probably the model in which to address this question. Imaging studies (Gilroy etal. , 1991; McAinsh etal., 1992; Shacklock etal., 1992) have certainly revealed that there is a markedly heterogeneous distribution of calcium in single guard cells and other cell types. Interestingly, it is apparent that after the addition of ABA certain areas in the guard cell remain calcium quiescent while others demonstrate marked increases in calcium (McAinsh et al., 1992). However, we do not yet know whether it is possible to correlate these localized “hot spots” with the distribution of ion channels. Asymmetric distribution of calcium is also observed in tip growth, where there is a localized calcium gradient towards the tip (and growing region) of both Fucus rhizoids (Brownlee and Wood, 1986) and pollen tubes (Miller etal., 1992). In these cases it is possible that the elevated calcium is associated with the control of tip growth (Battey and Blackbourn, 1993).

Oscillations in cytosolic free calcium have also attracted attention in higher plants, although many of the earlier reports have been discounted on the basis of imaging artefacts (Read etal., 1993). Recently, however, we have found good evidence that [Ca”], in the guard cell oscillates in response to elevated external free calcium and that the pattern of oscillations is absolutely dependent on the strength of the external stimulus (McAinsh et al., 1995). This result has added significance as it is also possible to correlate both the pattern of the oscillations and the strength of the external stimulus with the extent of the physiological response. We have investigated the origin of the calcium used to generate the oscillations and have found evidence to suggest that there is both calcium influx across the plasma membrane and calcium release from internal stores. The next key question to address is whether this pattern of oscillations can be altered by the addition of another external stimulus. The second feature that these results highlight is the importance of measuring the concentration of free calcium in the apoplast as we have shown that external calcium above and including 100pm induced oscillations in guard cell [Ca2+Ii. The possibility that cytosolic free calcium in these cells may be in a natural state of oscillation requires further investigation.

In summary, although the evidence is as yet fragmentary it seems likely that,


as in animals, plant cells will rely on the interaction of different signalling pathways and different forms of agonist-induced signal to define specificity. When this is combined with differential localization of receptors and effectors there would seem to be ample opportunity for encoding specificity of the calcium signal.

VI. FUTURE PROSPECTS

Given the large number of physiological stimuli which are already documented to use calcium ions as second messengers in plants it seems likely that this list will continue to expand in the near future. This process will certainly be aided by the new technological approaches such as recombinant aequorin and long wavelength indicators which will make the quantification of cytosolic free calcium much easier. However, there is still the wider question of the overall control of calcium homeostasis to consider. For example, there is still much work to be done in the characterization of the intracellular stores. In particular, although research has tended to focus on the vacuole, is there a major role for the ER and do other organelles contribute to the maintenance of cytosolic calcium levels? In this review we have not discussed the role of the family of calcium-binding proteins including calmodulin. These proteins could have a major role to play in the control of calcium homeostasis and this will require detailed investigation.

Recently, there have been some exciting developments in terms of defining plant calcium-permeable channels (Thuleau etal., 1993, 1994). It can be anticipated that the next few years will result in the opening up of this field through expression of the cloned genes in heterologous systems combined with patch clamp analysis. This type of experiment is likely to reveal some of the complexities and subtleties associated with the regulation of these channels, Similarly, isolation of the genes for signal transduction components will be important for a number of reasons. Over-expression studies in heterologous systems will be important for defining in biochemical terms the activity of the encoded protein. The access to purified proteins will certainly help to shed some light on specificity through the accurate documentation of substrates and activation optima. Over-expression is also the first step in the series of steps leading to a full molecular characterization of the protein either by NMR or X-ray crystallography. Here solution structures may be of great value in working out the molecular basis of interactions with other components of signal transduction components. Access to signalling genes and their promoters will also be of use in studies of the physiological function of the gene product through transgenic strategies. Already this work is bearing fruit (reviewed by Taylor et al., 1994).

However, one of the greatest challenges will be to disentangle the complexities of interacting signal transduction pathways which combine to generate

88 A. A. R. WEBB et at.

a single response. Here the picture is getting more complex with the appearance of additional putative second messengers such as cADPR, protons, DAG, CAMP and cGMP. Teasing apart the fundamentals of how these individual messengers are orchestrated to produce the final response will be one of the greatest challenges.

ACKNOWLEDGEMENTS

AARW, MRM, JET and AMH are all grateful to the BBSRC for research grant support (Biological Adaptation to Global Environment Change Pro- gramme and Intracellular Signalling Programme), the results of which are described in this review. In addition MRM is grateful to the Royal Society for the award of a University Research Fellowship.

REFERENCES

Allan, A. C., Fricker, M. D., Ward, J . L., Beale, M. H. and Trewavas, A. J. (1994). Two transduction pathways mediate rapid effects of abscisic acid in Commelina guard cells. The Plant Cell 6, 1319-1328.

Allen, G. J. and Sanders, D. (1994). Two voltage-gated, calcium release channels core- side in the vacuolar membrane of broad bean guard cells. The Plant Cell 6,

Allen, G . J., Muir, S. R. and Sanders D. (1995). Release of CaZ+ from individual plant vacuoles by both InsP, and cyclic ADP-ribose. Science 268, 735-737.

Alexandre, J. and Lassalles, J. P. (1992). Intracellular CaZ+ release by InsP3 in plants and effect of buffers on Ca2+ diffusion. Philosophical Transactions of the Royal Society of London B, 338, 53-61.

Alexandre, J., Lassalles, J. P. and Kado, R. T. (1990). Opening of Ca2+ channels in isolated red beet root vacuole membrane by inositol 1,4,5-triphosphate. Nature

Amundson, J. and Clapham, D. (1993). Calcium waves. Current Opinion in Neuro- biology 3, 375-382.

Assmann, S. M. (1993). Signal tranduction in guard cells. Annual Review of Cell Biology 9, 345-375.

Ayling, S. M., Brownlee, C. and Clarkson, D. T. (1994). The cytoplasmic streaming response of tomato root hairs to auxin; observations of cytosolic calcium levels. Journal of Plant Physiology 143, 184-188.

Battey, N. H. and Blackbourn, H. D. (1993). The control of endocyclosis in plant cells. New Phytologist 128, 307-338.

Berger, F. and Brownlee, C. (1993). Ratio confocal imaging of free cytoplasmic calcium, gradients in polarising and polarised Fucus zygotes. Zygote 1, 9-15.

Berridge, M. J. (1990). Calcium oscillations. Journal of Biological Chemistry 265,

Berridge, M. J. (1993a). Inositol trisphosphate and calcium signalling. Nature 361,

Berridge, M. J. (1993b). Ca2+-induced and Ca2+ release and inositol trisphosphate,

Berridge, M. J . and Dupont, G. (1994). Spatial and temporal signalling by calcium.

685-694.

343, 567-570.

9583-9586.

315-325.

intracellular channels and calcium spiking. Biomedical Research 14, 21-29.

Current Opinion in Cell Biology 6, 261-274.

CALCIUM IONS AS LNTRACELLULAR SECOND MESSENGERS 89

Berridge, M. J. and Irvine, R. F. (1989). Inositol phosphates and cell signalling. Nature

Bertl, A., Blumwald, E., Coronado, R., Eisenberg, R., Findlay, G., Gradman, D., Hille, B., Kohler, K., Kolb. H.-A., MacRobbie, E., Meissner, G., Miller, C., Neher, E. and Palade, P. (1992). Electrical measurements on endomembranes. Science 258, 873.

Bethke, P. C. and Jones, R. L. (1994). Ca2+ calmodulin modulates ion channel activity in storage protein vacuoles of barley aleurone cells. The Plant Ce// 6,

Biyaseheva, A. E., Molotkovskii, Y. G. and Mamonov, L. K. (1993). Increase of free Ca2+ in the cytosol of plant protoplasts in response to heat stress as related to CaZ+ homeostasis. Russian Plant Physiology 40, 613-618.

Blatt, M. R. and Thiel, G. (1993). Hormonal control of ion channel gating. Annual Review of Plant Physiology and Plant Molecular Biology 44, 543-567.

Blatt, M. R., Thiel, G. and Trentham, D. R. (1990). Reversible inactivation of K + channels of Vicia stomata1 guard cells following the photolysis of caged inositol 1,4,5-trisphosphate. Nature 346, 766-769.

Bootman, M. (1994). Questions about quanta1 Ca2+ release. Current Biology 4,

Bowler, C. and Chua, N. H. (1994). Emerging themes of plant signal transduction. Plant Cell 6, 1529-1541.

Bowler, C., Neuhaus, G., Yamagata, H. and Chua, N. H. (1994). Cyclic-GMP and calcium mediate phytochrome phototransduction. Cell 77, 73-81.

Brosnan, J. M. and Sanders, D. (1990). Inositol trisphosphate-mediated Ca2+ release in beet microsomes is inhibited by heparin. FEES Letters 260, 70-72.

Brosnan, J . M. and Sanders, D. (1993). Identification and characterization of high- affinity binding sites for inositol trisphosphate in red beet. The Plant Cell 5 ,

Brownlee, C. and Pulsford, A. L. (1988). Visualization of the cytoplasmic Ca2+ gradient in Fucus serratus rhizoids: correlation with cell ultrastructure and polarity. Journal of Cell Science 91, 249-256.

Brownlee, C. and Wood, J . W. (1986). A gradient of cytoplasmic free calcium in growing rhizoid cells of Fucus serratus. Nature 320, 624-626.

Bush, D. S. (1993). Regulation of cytosolic calcium in plants. Plant Physiology 103,

Bush, D. S. and Jones, R. L. (1987). Measurement of cytoplasmic calcium in aleurone protoplasts using INDO-I and FURA-2. Cell Calcium 8, 455-472.

Bush, D.S. and Jones, R. L. (1988). Cytoplasmic calcium and alpha amylase secretion from barley aleurone protoplasts. European Journal of Cell Biology. 46,466-469.

Callaham, D. A. and Hepler, P. K. (1991). Measurement of free calcium in plant cells. In ''Cellular Calcium. A Practical Approach" (J. G. McCormack and P. H. Cobbold, eds), pp. 383-410. Oxford University Press, New York.

Canut, H., Carrasco, A., Rossignol, M. and Ranjeva, R. (1993). Is vacuole the richest store of IP,-mobilizable calcium in plant cells? Plant Science 90, 135-143.

Chae, Q., Park, H. J. and Hong, S. D. (1990). Loading of quin-2 into the oat proto- plast and measurement of cytosolic calcium ion concentration changes by phytochrome action. Biochimica et Biophysica Acta 1051, 115-122.

Chang, C., Kwok, S. F., Bleecker, A. B. and Meyerowitz, E. M. (1993). Arabidopsis ethylene-response gene ETRI-similarity of product to 2-component regulators. Science 262, 539-544.

Clarkson, D. T., Brownlee, C. and Ayling, S. M. (1988). Cytoplasmic calcium measurements in intact higher plant cells: results from fluorescence ratio imaging of fura-2. Journal of Cell Science 91, 71-80.

Cobbold, P. H. and Lee, J . A. C. (1991). Aequorin measurements of cytoplasmic free

341, 197-205.

277-285.

169-172.

93 1-940.

7-13.


calcium. In "Cellular Calcium. A Practical Approach" (J. G. McCormack and P. H. Cobbold, eds), pp. 55-82. Oxford University Press, New York.

Cobbold, P. H. and Rink, T. J . (1987). Fluorescence and bioluminescence measurement of cytoplasmic free calcium. Journal of Biochemistry 248, 3 13-328.

Collins, J. H., Theibert, J. L., Francois, J.-M., Ashley, C. C. and Potter, J. D. (1991). Amino acid sequences and Ca*+-binding properties of two isoforms of barnacle troponin C. Biochemistry 30, 702-707.

Cork, R. J. (1986). Problems with the application of quin-2-AM to measuring cytoplasmic free calcium. Plant, Cell and Environment 9, 157-161.

Cosgrove, D. J. and Hedrich, R. (1991). Stretch-activated chloride, potassium, and calcium channels coexisting in plasma membranes of guard cells of Vicia faba L. Planta 186, 143-153.

Cote, G. G. and Crain, R. C. (1993). Biochemistry of Phosphoinositides. Annual Review of Plant Physiology and Plant Molecular Biology 44, 333-356.

Ding, J. P. and Pickard, B. G. (1993a). Mechanosensory calcium-selective cation channels in epidermal cells. The Plant Journal 3, 83-110.

Ding, J. P. and Pickard, B. G. (1993b). Modulation of mechanosensitive calcium- selective cation channels by temperature. The Plant Journal 3, 713-720.

Drobak, B. K. (1992). The plant phosphoinositide system. Biochemical Journal 288,

Drobak, B. K. (1993). Plant phosphoinositides and intracellular signalling. Plant Physiology 102, 705-709.

Drobak, B. K. and Ferguson, I. B. (1985). Release of Ca2+ from plant hypocotyl microsomes by inositol-l,4,5-trisphosphate. Biochemical and Biophysical Research Communications 130, 1241-1246.

Drobak, B. K.. Watkins, P. A. C., Valenta, R., Dove, S. K., Lloyd, C. W. and Staiger, C. J. (1994). Inhibition of plant plasma membrane phosphoinositide phospholipase C by the actin-binding protein, profilin. The Plant Journal 6 ,

Ehrlich, B. E. and Watras, J. (1988). Inositol 1,4,5-trisphosphate activates a channel from smooth muscle sarcoplasmic reticulum. Nature 336, 583-586.

Elliott, D. C. and Petkoff, H. S. (1990). Measurement of cytoplasmic free calcium in plant protoplasts. Plant Science 67, 125-131.

Etter, E.F., Kuhn, M.A. and Fay, F.S. (1994). Detection of changes in near- membrane Ca2+ concentration using a novel membrane-associated Ca2+ indicator. Journal of Biological Chemistry 269, 10141-10149.

Evans, D. E. (1994). PM-type calcium pumps are associated with higher plant cell intracellular membranes. Cell Calcium 15, 241-246.

Fasolato, C., Innocenti, B. and Pozzan, T. (1994). Receptor-activated Ca2+ influx: how many mechanisms for how many channels? Trends in Pharmacological Sciences 15, 71-83.

Fewtrell, C. (1993). Ca2+ Oscillations in non-excitable cells. Annual Review of Physiology 55, 427-454.

Franklin-Tong, V.E., Ride, J. P., Read, N, D., Trewavas, A. J. and Franklin, F. C. H. (1993). The self-incompatibility response in Papaver rhoeas is mediated by cytosolic free calcium. The Plant Journal 4, 163-177.

Galione, A. (1993). Cyclic ADP-ribose: a new way to control calcium. Science 259,

Galione, A., White, A., Willmott, N., Turner, M., Potter, B. V. L. and Watson, S. P. (1993a). cGMP mobilizes intracellular Ca2+ in sea urchin eggs by stimulating cyclic ADP-ribose synthesis. Nature 365, 456-459.

Galione, A., McDougall, A., Busa, W. B., Willmott, N., Gillot, I. and Whitaker, M. (1993b). Redundant mechanisms of calcium-induced calcium release underlying

697-712.

3 89-400.

325-326.


calcium waves during fertilization of sea urchin eggs. Science 261, 348-352. Gehring, C. A., Irving, H. R. and Parish, R. W. (1990a). Effects of auxin and abscisic

acid on cytosolic calcium and pH in plant cells. Proceedings of the National Academy of Sciences USA 87, 9645-9649.

Gehring, C. A., Williams, D. A., Cody, S. H. and Parish, R. W. (1990b). Photo- tropism and geotropism in maize coleoptiles are spatially correlated with increases in cytosolic free calcium. Nature 345, 528-530.

Gelli, A. and Blumwald, E. (1993). Calcium retrieval from vacuolar pools. Charac- terization of a vacuolar calcium channel. Plant Physio/ogy 102, 1139-1 146.

Gilroy, S. and Jones, R. L. (1992). Gibberellic acid and abscisic acid coordinately regulate cytoplasmic calcium and secretory activity in barley aleurone protoplasts. Proceedings of the National Academy of Sciences USA 89, 3591-3595.

Gilroy, S. and Trewavas, A. J. (1994). A decade of plant signals. BioEssays 16,

Gilroy, S . , Hughes, W. A. and Trewavas, A. J. (1986). The measurement of intracellular calcium levels in protoplasts from higher plant cells. FEBS Letters

Gilroy, S., Hughes, W. A. and Trewavas, A. J . (1989). A comparison between Quin-2 and Aequorin as indicators of cytoplasmic calcium levels in higher plant cell protoplasts. Plant Physiology 90, 482-491.

Gilroy, S., Read, N. D. and Trewavas, A. J . (1990). Elevation of cytoplasmic calcium by caged calcium or caged inositol trisphosphate initiates stomata1 closure. Nature 346, 769-771.

Gilroy, S., Fricker, M. D., Read, N. D. and Trewavas, A. J. (1991). Role of calcium in signal transduction of Comrnelina guard cells. The Plant Cell 3, 335-344.

Goldbeter, A., Dupont, G. and Berridge, M. J. (1990). Minimal model for signal- induced Ca2+ oscillations and for their frequency encoding through protein phosphorylation. Proceedings of the National Academy of Sciences USA 87,

Graziana, A., Fosset, M., Ranjeva, R., Hetherington, A.M. and Lazdunski, M. (1988). Ca2+ channel inhibitors that bind to plant cell membranes block Ca2+ entry into protoplasts. Biochemistry 27, 764-768.

Graziana, A., Bono, J-J. and Ranjeva, R. (1993). Measurements of cytoplasmic calcium by optical flourescence in plant systems. Plant Physiology and Biochemistry 31, 277-281.

Grynkiewicz, G., Poenie, M. and Tsien, R. Y. (1985). A new generation of Ca2+ indicators with greatly improved fluorescence characteristics. Journal of Biological Chemistry 260, 3440-3450.

Hahm, S. H. and Saunders, M. J. (1991). Cytokinin increases intracellular Ca2+ in funaria: detection with Indo-1. Cell Calcium 12, 675-681.

Halachmi, D. and Eilam, Y. (1989). Cytosolic and vacuolar Ca2+ concentrations in yeast cells measured with the Ca2+-sensitive fluorescence dye indo-I. FEBS Letters 256, 55-61.

Harvey, H. J., Venis, M. A. and Trewavas, A. J. (1989). Partial purification of a protein from maize (Zea mays) coleoptile membranes binding the calcium channel antagonist verapamil. Biochemical Journal 257, 95-100.

Haugland, R. P. (1992). “Molecular Probes. Handbook of Fluorescent Probes and Research Chemicals”. Molecular Probes, Inc., Eugene, Or.

Hetherington, A. M. and Drobak, B. K. (1992). Inositol-containing lipids in higher plants. Progress in Lipid Research 31, 53-63.

Hetherington, A. M., Graziana, A., Mazars, C., Thuleau, P. and Ranjeva, R. (1992). The biochemistry and pharmacology of plasmamembrane calcium channels in plants. Philosophical Transactions of the Royal Society of London B 338,91-96.

677-682.

199, 217-221.

146 I - 1465.


Hill, T. D., Dean, N. M. and Boynton, A. L. (1988). Inositol 1,3,4,5-tetrakisphosphate induces Ca2+ sequestration in rat liver cells. Science 242, 1176-1 178.

Hodick, D., Gilroy, S., Fricker, M. D. and Trewavas, A. J. (1991). Cytosolic Ca2+ -concentrations and distributions in rhizoids of Chara fragilis Desv. determined by ratio analysis of the fluorescent probe Indo-1. Botanica Acta 104,

Hofmann, F., Biel, M. and Flockerzi, V. (1994). Molecular basis for Ca2+ channel diversity. Annual Review of Neuroscience 17, 399-418.

Hurley, T. W., Ryan, M. P. , and Brinck, R. W. (1992). Changes of cytoplasmic Ca2+ interfere with measurements of cytosolic Mg2+ using mag-fura-2. American Journal of Physiology 263, 300-307.

Iida, H., Yagawa, Y . and Anraku, Y. (1990). Essential role for induced Ca2+ influx followed by [Ca2+Ii rise in maintaining viability of yeast cells late in the mating pheromone response pathway. A study of [Ca2+Ii in single Saccharomyces cerevisiae cells with imaging of FURA-2. The Journal of Biological Chemistry

Irving, H. R., Gehring, C.A. and Parish, R. W. (1992). Changes in cytosolic pH and calcium of guard cells precede stomata1 movements. Proceedings of the National Academy of Sciences USA 89, 1790-1794.

Jackson, S. L. and Heath, I. B. (1990). Roles of calcium ions in hyphal tip growth. Microbiological Reviews 57, 367-382.

Johannes, E., Brosnan, J. M. and Sanders, D. (1991). Calcium channels and signal transduction in plants cells. BioEssays 13, 33 1-336.

Johannes, E., Brosnan, J .M. and Sanders, D. (1992a). Parallel pathways for intracellular Ca2+ release from the vacuole of higher plants. The Plant Journal

Johannes, E., Brosnan, J. M. and Sanders, D. (1992b). Calcium channels in the vacuolar membrane of plants: multiple pathways for intracellular calcium mobilization. Philosophical Transactions of the Royal Sociely of London B 338,

Jones, A. M. (1994). Auxin binding proteins. Annual Review of Plant Physiology and Plant Molecular Biology 45, 393-420,

Kaila, K. and Voipio, J. (1985). A simple method for dry bevelling of micropipettes used in the construction of ion-selective micro-electrodes, The Journal of Physiology 369, 8.

Kasai, H., Li, Y. X. and Miyashita Y. (1993). Subcellular distribution of Ca2+ release channels underlying Ca '+ waves and oscillations in exocrine pancreas. Cell 74, 669-677.

Keith, C. H., Ratan, R., Maxfield, F. R., Bajer, A. and Shelanski, M. L. (1985). Local cytoplasmic calcium gradients in living mitotic cells. Nature 316, 848-850.

Kiss, H. G., Evans, M. L. and Johnson, J. D. (1991). Cytoplasmic calcium levels in protoplasts from the cap and elongation zone of maize roots. Protoplasma 163,

Knight, M. R., Campbell, A. K., Smith, S. M. and Trewavas, A. J. (1991). Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytoplasmic calcium. Nature 352, 524-526.

Knight, M. R., Smith, S. M. and Trewavas, A. J. (1992). Wind-induced pIant motion immediately increases cytosolic calcium. Proceedings of the National Academy of Sciences USA 89, 4967-4971.

Knight, M. R., Read, N. D., Campbell, A. K. and Trewavas, A. J. (1993). Imaging calcium dynamics in living plants using semi-synthetic recombinant aequorins. The Journal of Cell Biology 121, 83-90.

Konishi, M., Hollingworth, S., Harkins, A. B. and Baylor, S. M. (1991). Myoplasmic

222-228.

265, 1 3 39 1 - 1 3399.

2, 97-102.

105-1 12.

181-188.


calcium transients in intact frog skeletal muscle fibers monitored with the fluorescent indicator furaptra. Journal of General Physiology 97, 271-301.

Kuba,Y. and Takeshita, S. (1981). Stimulation of intracellular Ca2+ oscillations in sympathetic neurone. Journal of Theoretical Biology 93, 1009-1031.

Li, W. W., Luan, S., Schreiber, S. L. and Assmann, S. M. (1994). Cyclic-AMP stimulates K + channel activity in mesophyll cells of Vicia faba. Plant

Lynch, J., Polito, V. S. and Lauchli, A. (1989). Salinity stress increases cytoplasmic Ca activity in maize root protoplasts. Plant Physiology 90, 1271-1274.

Malho, R., Read, N. D., Pais, M. S. and Trewavas, A. J. (1994). Role of cytosolic free calcium in the reorientation of pollen tube growth. The Plant Journal5,331-341.

Marshall, J., Corzo, A., Leigh, R. A. and Sanders, D. (1994). Membrane potential- dependent calcium transport in right-side-out plasma membrane vesicles from Zea mays L. roots. The Plant Journal 5, 683-694.

McAinsh, M. R. (1994). Effects of oxidative stress on stomatal behaviour and guard cell cytosolic free Ca2+. Plant Physiology S105, 101.

McAinsh, M. R., Brownlee, C. and Hetherington, A. M. (1990). Abscisic acid-induced elevation of guard cell cytosolic Ca2+ precedes stomatal closure. Nature 343,

McAinsh, M. R., Brownlee, C. and Hetherington, A. M. (1991). Partial inhibition of ABA-induced stomatal closure by calcium-channel blockers. Proceedings of the Royal Society of London B-Biological Sciences 243, 195-201.

McAinsh, M. R., Brownlee, C. and Hetherington A. M. (1992). Visualizing changes in cytosolic-free C a Z + during the response of stomatal guard cells to abscisic acid. The PIant Cell 4, 1113-1122.

McAinsh, M. R., Webb, A. A. R., Taylor, J. E. and Hetherington, A . M . (1995). Stimulus-induced oscillations in guard cell cytosolic free calcium. The Plant Cell

McCray, J. A. and Trentham, D. R. (1989). Properties and uses of photoreactive caged compounds. Annual Review of Biophysics and Biophysical Chemistry 18,

Messiaen, J. and Van Cutsem, P. (1994). Pectic signal transduction in carrot cells: membrane, cytosolic and nuclear responses induced by oligogalacturonides. Plant Cell Physiology 35, 677-689.

Messiaen, J., Read, N. D., Van Cutsem, P. and Trewavas, A. J . (1993). Cell wall oligogalacturonides increase cytosolic free calcium in carrot protoplasts. Journal of Cell Science 104, 365-371.

Mikoshiba, K., Furuichi, T., Miyawaki, A., Yoshikawa, S., Nakagawa, T., Yamada, N., Hamanaka, Y., Fujino, I., Michikawa, T., Ryo, Y., Okano, H., Fujii, S. and Nakade, S. (1993). Inositol trisphosphate receptor and Ca2+ signalling. Philosophical Transactions of the Royal Society of London B-Biological Sciences

Miller, D. D., Callaham, D. A., Gross, D.J. and Hepler, P. K. (1992). Free Ca2+ gradient in growing pollen tubes of Lillum. Journal of Cell Science 101, 7-12.

Miyazaki, S. and Shirakawa, H. (1993). Ca2+-induced Ca2+ release and inositol trisphosphate. Ca2+-induced Ca2+ release mediated by the IP, receptor is responsible for C a 2 + waves and C a 2 + oscillations at fertilization of mammalian eggs. Biomedical Research 14, 35-39.

Money, N. P. (1990). Measurement of hyphal turgor. Experimental Mycology 14,

Nobiling, R. and Reiss, H.-D. (1987). Quantitative analysis of calcium gradients and activity in growing pollen tubes of Lilium longiflorum. Protoplasma 139, 20-24.

Obermeyer, G. and Weisenseel, M. H. (1991). Calcium channel blocker and calmodulin

Physiology 106, 957-961.

186-1 88.

7 1207- 12 19.

239-270.

340, 345-349.

4 16-425.


antagonists affect the gradient of free calcium ions in lily pollen tubes. European Journal of Cell Biology 56, 319-327.

Pantoja, O., Gelli, A, and Blumwald, E. (1992). Voltage-dependent calcium channels in plant vacuoles. Science 258, 1567- 1570.

Parekh, A. B., Terlau, H. and Stuhmer, W. (1993). Depletion of InsP, stores activates a Ca2+ and K + current by means of a phosphatase and a diffusible messenger. Nature 364, 814-818.

Parker, I. and Ivorra, I. (1990). Inhibition by Ca2+ of inositol trisphosphate- mediated Ca2+ liberation: A possible mechanism for oscillatory release of [Ca2+Ii. Proceedings of the National Academy of Sciences USA 87, 260-264.

Parmar, P. N. and Brearley, C. A. (1993). Identification of 3- and 4-phosphorylated phosphoinositides and inositol phosphates in stomata1 guard cells. The Plant Journal 4, 255-263.

Payne, R., Walz, B., Lery, S. and Fein, A. (1988). The localization of calcium release by inositol trisphosphate in Limulus photoreceptors and its control by negative feedback. Philosophical Transactions of the Royal Society of London B 320, 359-379.

Pineros, M. and Tester, M. (1993). Plasma membrane Ca2+ channels in roots of higher plants and their role in aluminium toxicity. Plant and Soil 155/156,

Poovaiah, B. W. and Reddy, A. S. N. (1993). Calcium and signal transduction in plants. Critical Reviews in Plant Sciences 12, 185-21 1.

Price, A. H., Taylor, A., Ripley, S. J., Griffiths, A., Trewavas, A. J. and Knight, M. R. (1994). Oxidative signals in tobacco increase cytosolic calcium. The Plant Cell 6 , 1301-1310.

Purves, R. D. (1981). “Microelectrode. Methods for Intracellular Recording and Ionophoresis”. Academic Press, London.

Putney, J. W. and Bird G. St J.(1993). The signal for capacitative calcium entry. Cell

Randriamampita, C. and Tsien, R. Y. (1993). Emptying of intracellular Ca2+ stores releases a novel small messenger that stimulates Ca2+ influx. Nature 364,

Ranjeva, R., Carrasco A. and Boudet, A. M. (1988). Inositol trisphosphate stimulates the release of calcium from intact vacuoles isolated from Acer cells. FEBS Letters

Rea, P. A. and Poole, R. J. (1993). Vacuolar H+-translocating pyrophosphatase. Annual Review of Plant Physiology and Plant Molecular Biology 44, 157-180.

Read, N. D., Shacklock, P. S., Knight, M. R. and Trewavas, A. J. (1993). Imaging calcium dynamics in living plant cells and tissues. Cell Biology International 17,

Read, N. D., Allan, W. T. G., Knight, H., Knight, M. R., Malho, R., Russell, A., Shacklock, P. S. and Trewavas, A. J. (1992). Imaging and measurement of cytosolic free calcium in plant and fungal cells. Journal of Microscopy 166,57-86.

Rizzuto, R., Simpson, A. W. M., Brini, M. and Pozzan, T. (1992). Rapid changes of mitochondria1 Ca2+ revealed by specifically targeted recombinant aequorin. Nature 358, 325-327.

Roberts, S. K.. Gillot, I. and Brownlee, C. (1994). Cytoplasmic calcium and Fucus egg activation. Development 120, 155-163.

Robinson, G. A., Butcher, R. W. and Sutherland, E. W. (1971). “Cyclic AMP”. Academic Press, New York.

Robinson, K. R. (1990). Temporal and spatial changes in Ca2+ during plant development. In “Calcium in Plant Growth and Development” (R. T. Leonard and P. K. Hepler, eds), pp. 11 1-1 19. American Society of Plant Physiologists Symposium Series, Vol 4.

119-122.

75, 199-201.

809-8 14.

230, 137-141.

1 11-125.


Russ, U., Grolig, F. and Wagner, G. (1991). Changes of cytoplasmic free Ca2+ in the green alga Mougeotia scalaris as monitored with Indo- 1 , and their effects on the velocity of chloroplast movements. Planta 184, 105-12.

Satter, R. L. and Moran, N. (1988). Ionic channels in plant cell membranes. Physiologia Plantarum 72, 816-820.

Scheuerlein, R., Schmidt, K., Poenie, M. and Roux, S. J. (1991). Determination of cytoplasmic calcium concentration in Dryopteris spores. A developmentally non- disruptive technique for loading of the calcium indicator fura-2. Planta 184,

Schroeder, J. I. and Hagiwara, S. (1990). Repetitive increases in cytosolic Ca2+ of guard cells by abscisic acid activation of nonselective Ca2+ permeable channels. Proceedings of the National Academy of Sciences USA 87, 9305-9309.

Schumaker, K. S . and Sze, H. (1987). Inositol 1,4,5-trisphosphate releases Ca2+ from vacuolar membrane vesicles of oat roots. Journal of Biological Chemistry 262,

Shacklock P. S., Read, N. D. and Trewavas, A. J. (1992). Cytosolic free calcium mediates red light-induced photomorphogenesis. Nature 358 753-755.

Shotton, D. (1989). Confocal scanning optical microscopy and its applications for biological specimens. Journal of CeN Science 89, 175-206.

Stendahl, O., Krause, K-H., Krischer, J., Jerstrom, P., Theler, J-M., Clark, A., Carpentier, J-L. and Lew, D. P. (1994). Redistribution of intracellular Ca2+ stores during phagocytosis in human neutrophils. Science 265,

Taylor, J. E., McAinsh, M. R., Montgomery, L., Renwick, K. F., Webb, A. A . R. and Hetherington, A. M. (1994). The use of transgenesis to investigate signal transduction pathways. Biochemical Society Transactions 22, 949-952.

Terry, B. R., Findlay, G. P. and Tyerman, S . D. (1992). Direct effects of Caz+-channel blockers on plasma membrane cation channels of Amaranthus tricolor protoplasts. Journal of Experimental Botany 43, 1457-1473.

Thiel, G., Blatt, M. R., Fricker, M. D., White, I. R. and Millner, P. (1993). Modula- tion of K + channels in Vicia stomata1 guard cells by peptide homologs to the auxin-binding protein C terminus. Proceedings of the National Academy of Sciences USA 90, 11493-11497.

Thorn, P., Gerasimenko, 0. and Petersen, 0. H. (1994). Cyclic ADP-ribose regulation of ryanodine receptors involved in agonist evoked cytosolic C a 2 + oscillations in pancreatic acinar cells. The EMBO Journal 13, 2038-2043.

Thorn, P., Lawrie, A. M., Smith, P. M., Gallacher, D. V. and Petersen, 0. H. (1993). Local and global cytosolic Ca2+ oscillations in exocrine cells evoked by agonists and inositol trisphosphate. Cell 74, 661-668.

Thuleau, P., Graziana, A., Canut, H. and Ranjeva, R. (1990). A75-kDa polypeptide, located primarily a t the plasma membrane of carrot cell-suspension cultures, is photoaffinity labeled by the calcium channel blocker LU 49888. Proceedings of the National Academy of Sciences USA 87, 1oooO-1OOO4.

Thuleau, P., Graziana, A., Ranjeva, R. and Schroeder, J. I. (1993). Solubilized proteins from carrot (Daucus carota L.) membranes bind calcium channel blockers and form calcium-permeable ion channels. Proceedings of the National Academy of Sciences USA 90, 765-769.

Thuleau, P., Ward, J. M., Ranjeva, R. and Schroeder, J. I. (1994). Voltage-dependent calcium-permeable channels in the plasma membrane of a higher plant cell. The EMBO Journal 13, 2970-2975.

Timmers, A. C. J . , Reiss, H.-D. and Schel, J . H. N. (1991). Digitonin-aided loading of Fluo-3 into embryogenic plant cells. Cell Calcium 12, 515-521.

Tsien, R. Y. (1981). A non-destructive technique for loading calcium buffers and indicators into cells. Nature 290. 527-528.

166- 174.

3944-3946.

1439-1441.


Tsien, R. Y. and Hartoonian A. T. (1990). Practical design criteria for a dynamic ratio imaging system. Cell Calcium 11, 93-109.

Tsien, R. Y. and Rink, T. J . (1980). Neutral carrier ion selective microelectrodes for measurement of intracellular free calcium. Biochimica et Biophysica Acta 599,

Tsien, R. Y. and Tsien, R. W. (1990). Calcium channels, stores and oscillations.

Tsong, T. Y. (1991). Electroporation of cell membranes. Biophysical Journal 60,

Tsunoda, Y. (1993). Receptor operated C a 2 + signalling and cross-talk in stimulus secretion coupling. Biochemica et Biophysica Act! 1154, 105-156.

Volotovski, I. D., Sokolovski, S. G., Nikiforov, E. L. and Zinchenko, V. P. (1993a). Influence of CaZ+ transport inhibitors on light-induced Ca2+ oscillations in plant cell suspension. Photosynthetica 29, 169- 176.

Volotovski, I. D., Sokolovsky, S. G., Nikiforov, E. L. and Zinchenko, V. P. (1993b). Calcium oscillations in plant cell cytoplasm induced by red and far-red light irradiation. Journal of Photochemistry and Photobiology B-Biology 20, 95-100.

Wang, M., Van Duijn, B. and Schram, A. W. (1991). Abscisic acid induces a cytosolic calcium decrease in barley aleurone protoplasts. FEBS Letters 278, 69-74.

Ward, J . M. and Schroeder, J . I. (1994). Calcium-activated K + channels and calcium- induced calcium release by slow vacuolar ion channels in guard cell vacuoles implicated in the control of stomatal closure. The Plant Cell 6 , 669-683.

Weyers, J . and Meidner, H. (1990). “Methods in stornatal physiology”. Longman, London.

White, J . G., Amos, W. B. and Fordham, M. (1987). An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy. Journal of Cell Biology 105, 41-48.

Williams, D. A., Cody, S. H., Gehring, C. A., Parish, R. W. and Harris, P. J . (1990). Confocal imaging of ionised calcium in living plant cells. Cell Calcium 11,

Williamson, R. E. and Ashley, C. C. (1982). Free CaZ+ and cytoplasmic streaming in

Willmer, C. M. (1983). “Stomata”. Longman, London. Zhang, D. H., Callaham, D. A. and Hepler, P. K . (1990). Regulation of anaphase

chromosome motion in Tradescantia stamen hair cells by calcium and related signaling agents. The Journal of Cell Biology 111, 171-182.

Zottini, M. and Zannoni, D. (1993). The use of Fura-2 fluorescence to monitor the movement of free calcium ions into the matrix of plant mitochondria (Pisum sati- vum and Helianthus tuberosus). Plant Physiology 102, 573-578.

623-638.

Annual Review of Cell Biology 6 , 715-760.

296-334.

291-297.

the alga, Chara. Nature 296, 647-651.

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