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Development 100, 181-184 (1987) Printed in Great Britain © The Company of Biologists Limited 1987 Review Article 181 Calcium: a regulation system emerges in plant cells S. GILROY, D. P. BLOWERS and A. J. TREWAVAS Botany Department, University of Edinburgh, Edinburgh EH9 3JH, UK Key words: calcium, regulation, plant cells, calmodulin, Chara, Fucus, NAD kinase, ATPases, protein kinases, cytoplasmic streaming, polarized growth Introduction Calcium occupies a pre-eminent place in the cellular control systems of animals (Campbell, 1983). Because of the cytotoxic effects of calcium, cells pay very particular attention to keeping cytoplasmic calcium levels very much lower than the normal extracellular 10~ 3 M level; usually it is in the range 1CT 8 -10~ 6 M. This is accomplished using a variety of calcium-pumping systems located both in the plasma membrane and organelles and together these operate a very efficient calcium-stat system. But, in addition, cells use the temporary elevation of cytoplasmic calcium to between 10~6 and 10~5 M that may follow plasma membrane perturbation and alteration of calcium channel activity, as signals, eliciting a variety of predetermined responses. The concentration of cytoplasmic calcium is sensed by calcium-binding proteins, most notably calmodulin, and the calcium/ calmodulin complex in turn modulates the activity of numerous enzymes and proteins. Calcium is also associated with other signalling systems such as IP3 and cyclic AMP. Calcium injected into cytoplasm has very limited mobility. This may be the consequence of efficient internal sequestration systems which may include calcium-binding proteins bound to the cytoskeleton. A localized stimulus to the plasma membrane can elicit an equally localized response in the cytoplasm. This may explain why calcium figures so strongly in the control systems involving cytoskeleton rearrangement, secretion and endocytosis, cell division, membrane electrical activity, motor and contractile activity and cell-to-cell communication. All of these processes at some stage involve a differential response between the different parts of the cytoplasm of the same cell. Calcium research in plants has only been seriously undertaken in.the last 7-8 years and this short article outlines our current understanding; greater details can be found elsewhere (Hepler & Wayne, 1985; Trewavas, 1986). Even with the limited knowledge available, however, it is evident that the control exerted in plant cells is as profound as that in animals. Is Cytoplasmic calcium regulated in plant cells? In order that changes in cytosolic calcium levels can regulate cellular processes the level of the ion itself must be closely regulated. Indirectly we know that cells must efficiently hold cytosolic calcium well below the cytotoxic concentration found in the extracellular fluid, 10~ 3 M (Hepler & Wayne, 1985), but technical problems have dogged attempts to directly measure this level in plants. However, the giant algal cells of Chara and Fucus have proved uniquely suited to microinjection of Ca2+ indicators (Williamson & Ashley, 1982) or insertion of Ca2+-sensitive microelectrodes, backed by the use of a permeant Ca 2+ indicating dye (Brownlee & Wood, 1986). These investigations have shown that lower plants maintain free, cytosolic calcium at about 10~ 7 M. Recently the development of a technique to trap Ca2+-indicating dyes in the cytoplasm of plant cell protoplasts has confirmed that a similar concentration is found in higher plant cells (Gilroy, Hughes & Trewavas, 1986). Measurements have also demonstrated the stability of the Ca2+-regulatory system that maintains this 'resting' Ca2+ level despite changes in the extracellular ionic environment, including changes in Ca 2+ concentration over the range 10~ 7 M to 10~ 3 M (Gilroy, Hughes & Trewavas, 1987). Superimposed on this stable low background, increases in Ca2+ concentration to 10~ 6 M have been observed in several plant systems. From these studies two classes of modulation have emerged: a sustained but localized ion gradient as seen in the tip-growing cells of Fucus (Brownlee & Wood, 1986) or pollen tubes (Reiss & Nobiling, 1986) and a transient increase in level as shown by lily cells undergoing mitotic progression (Keith, Raten, Maxfield, Bajer & 182 5. Gilroy, D. P. Blowers and A. J. Trewavas Selanski, 1985) or Chara cells responding to an action potential (Williamson & Ashley, 1982). Thus it has been demonstrated that plant cells precisely regulate their cytosolic Ca2+ levels. However, the molecular nature of the regulatory 'machinery' that brings this about is now only slowly being revealed. How is cytoplasmlc calcium regulated in plant cells? The low 'resting' submicromolar calcium level of the plant cell has to be maintained against a very unfavourable 10~3 M concentration of calcium found in the cell wall compartment or vacuoles/organelles. Plant cells possess a range of active transport systems which either sequester Ca 2+ into organelles or pump it back to the extracellular space. Mitochondria, chloroplasts and vacuoles accumulate Ca 2+ , often to 10~ 3 M levels, and the transporting systems of these organelles or membrane vesicle preparations generally have an affinity for Ca 2+ of less than 10~ 6 M (Moore & Akerman, 1984). In these cases, then, the organelles have relatively low affinity, but probably high capacity Ca 2+ storage sites. In contrast the Ca 2+ ATPase efflux pump at the plasma membrane and the calcium-sequestering pump of the endoplasmic reticulum have a tenfold higher affinity. It is most likely that these represent the cellular sites at which a low 'resting' Ca 2+ level is set. The plasma and vacuolar membranes may also possess Ca2+-sensitive ion channels allowing controlled influx since these membranes specifically bind Ca2+-channel antagonizing drugs in vitro (Hetherington & Trewavas, 1984; Andrejauskas, Hertel & Marme, 1986), such as verapamil and nifedipine. These same drugs are known to inhibit physiological processes in plants (Hepler & Wayne, 1985). Attempts to detect and detail the properties of calcium channels using patch/clamp technology are only at a very early stage. However clear detection of channels will require sound electrophysiological evidence; drug binding is insufficient on its own. The activity of these components of the Ca 2+ regulatory system have also been shown to be modulated by developmentally important stimuli. Active phytochrome increases Ca 2+ influx at the plasmamembrane (Roux, Wayne & Datta, 1986) whilst modulating Ca 2+ uptake by mitochondria and chloroplasts in several systems (Moore & Akerman, 1984). The activity of the plasma membrane and endoplasmic reticulum Ca2+-pumps seem to be regulated by changes in Ca 2+ level via the calcium-dependent regulatory protein, calmodulin (Moore & Akerman, 1984). Detection of a change in Intracellular free calcium by calcium-binding proteins The calcium receptor, calmodulin, has been detected and isolated from a number of different plants (Allan & Trewavas, 1985). Spinach calmodulin has been sequenced and differs by only 13 residues from bovine calmodulin (Roberts, Lukas, Harrington & Watterson, 1986). Calmodulin has been detected in both membrane and soluble plant cell fractions. Bovine and plant calmodulin are often interchangeable in activating capability. Other calcium-binding proteins have been detected in plants most notably in phloem (Sabnis & McEuen, 1986) and carrot cells (Ranjeva, Graziana, Dillenschneider, Charpentean & Boudet, 1986) and there is circumstantial evidence to support the presence of others elsewhere. Calcium- and calmodulln-dependent enzymes In plants After binding three or four calcium ions the calmodulin molecule undergoes a conformational change and then forms a ternary complex with various target proteins transducing the original calcium signal. Only a few calcium- and calmodulin-dependent enzymes have been studied in any detail in plants. The following examples are those best characterized and indicate the range of processes under control. NAD kinase (E.C.2.7.1.23) catalyses the phosphorylation of NAD to produce NADP and was the first calcium- and calmodulin-dependent enzyme to be discovered in plants (Anderson & Cormier, 1978). The capability for manipulating NAD and NADP levels argues for a pivotal role of this enzyme in the regulation of anabolic and catabolic processes. Significantly phytochrome activation by red light increases the NADP/NAD ratio suggesting a direct tie up between light, Ca 2+ , NAD kinase and metabolic regulation. Quinate:NAD oxidoreductase (QORase, E.C.I.1.1.24) catalyses the oxidation of quinate to produce dehydroquinate, an intermediate in the shikimate pathway involved in aromatic amino acid synthesis. In dark-grown carrot cells the enzyme appears to contain its own calcium-binding protein (Graziana, Dillenschneider & Ranjeva, 1984). The 'light' form of the enzyme is activated by a protein kinase and is discussed further below. Numerous adenosine triphosphatases (ATPases) have been described in plant tissues and the calciumand calmodulin-activated membrane associated types may represent active calcium pumps. Calcium- and calmodulin-activated calcium transport has been found in the plasma membrane (Dieter & Marme, Calcium regulation in plant cells 183 1980), the endoplasmic reticulum (Gross, 1982) and the tonoplast (Fukumoto & Venis, 1986). Finally, there are numerous calcium- and calmodulin-regulated protein kinases. These enzymes have the potential to amplify a weak signal and thus figure prominently in control concepts. Such enzymes have been detected in both membrane and soluble fractions and in numerous plants. However in only one or two cases have the substrates been identified. The activity of the 'light' grown form of QORase is controlled by a calcium- and calmodulin-dependent protein kinase (Graziana, Ranjeva & Boudet, 1983). In addition a plasma-membrane-associated protein kinase which autophosphorylates has also been described (Blowers, Hetherington & Trewavas, 1985). Autophosphorylation of this protein kinase alters its catalytic activity, possibly releasing it from calcium and calmodulin dependence, and thus giving prolonged activity after the transiently elevated Ca2+ concentration has returned to normal. The binding characteristics of calmodulin to enzymes have been investigated in only a few cases but presently determined binding constants span a range of 1 0 ~ 8 - 1 0 ~ 6 M as has been found for animal cells. The /CD for activation of NAD kinase by calmodulin can be 10~ 8 M although this is dependent on the source of calmodulin whereas for the plasma-membrane-bound protein kinase it is nearly 10~ 6 M. In this latter case control of enzyme activity by calmodulin availability is a real possibility. modulated (Jaffe's second rule) with Ca 2+ ionophores and Ca 2+ buffers whilst Ca2+ influxes have been blocked (Jaffe's third rule) with Ca2+-channel antagonists or by reduction of extracellular Ca 2+ . These treatments affect diverse plant processes including: cytokinin-induced bud formation in mosses; desmid morphogenesis; protoplast fusion; a multitude of phytochrome-triggered responses (notably chloroplast motility and fern spore germination); gravitropism; secretion; leaf movements; guard cell swelling and, of course, polar growth, mitosis and cytoplasmic streaming (Hepler & Wayne, 1985; Trewavas, 1986). Coupled to these studies have been observations of changes in 'membrane bound' calcium; calcium isotope fluxes and total calcium level which are thought to indicate changes in the cytoplasmic level of the ion (direct measurements have rarely proved feasible). Further evidence of Ca2+ regulation has come from the inhibitory action of anticalmodulin drugs on many of these processes (Hepler & Wayne, 1985). Though inhibition often requires high drug concentrations, which can have nonspecific effects on cell metabolism and the Ca2+-regulatory system (Gilroy, Hughes & Trewavas, 1987). Taken as a whole, although admittedly incomplete, the evidence points to Ca 2+ regulation of a wide range of physiological processes. What plant physiological processes are regulated by calcium? It should be obvious even from this brief account that much in the area of plant calcium is still based on inspired imagination and the haziest of actual knowledge rather than solid well-established fact. And yet the promise is still there; enough information has emerged to confirm what, 5-8 years ago, was then only an intuitive assessment of the importance of cytoplasmic calcium in plant growth and development. Plant calcium channels are a major area of uncertainty. Recent evidence showing that phytochrome (a plasma-membrane-located protein in certain organisms) may have protein kinase activity, could be significant here (Wong, Cheng, Walsh & Lagarias, 1986). Phosphorylation of calcium channels certainly modifies animal channel activity and could explain the tie up between red light and enhanced calcium entry. The relationship of calcium to cell growth needs detailed examination. High concentrations of calcium directly inhibit cell extension and these high concentrations probably elevate cytoplasmic calcium levels. A further gross area of ignorance concerns the substrates for calcium-regulated protein kinases in plants. We have little or no information as to their putative identity. In assessing the evidence for Ca2+ regulation of a physiological process we would do well to remember the three criteria put forward by Jaffe for the rigorous identification of such regulation: (1) the response should be accompanied by a change in intracellular [Ca 2+ ]; (2) artificial induction of such a change should stimulate the process and (3) blockage of the change should block the process. In the case of Ca2+dependent inhibition of cytoplasmic streaming (Williamson & Ashley, 1982; Hepler & Wayne, 1985) and a requirement for sustained intracellular Ca 2+ gradients for polarized growth (Brownlee & Wood, 1986; Reiss & Nobiling, 1986; Hepler & Wayne, 1985) the fragmentary data have met all of Jaffe's rules. Whilst in the case of mitosis in plant cells we lack only a direct quantification of the changes in Ca 2+ level that have been tentatively observed during mitotic progression (Keith, Raten, Maxfield, Bajer & Selanski, 1985; Hepler & Wayne, 1985). In many other processes the evidence for Ca2+ regulation is less complete but nonetheless compelling. Intracellular calcium levels have been artificially Future prospects 184 5. Gilroy, D. P. Blowers and A. J. Trewavas Probably the major impact of plant calcium work will still come in studies on polarity particularly polarized cell division, morphogenesis and cell wall secretion and patterning. Plants are organisms in which growth and development are pronounced polar phenomena, much more obviously so than in many animals. These processes in one way or another depend on cytoskeletal rearrangements and restructuring in numerous organisms, e.g. Fucus or Acetabularia. Clearly the relationship of calcium-regulated processes to cytoskeletal structure should prove exceptionally fertile ground. Since these phenomena require differential calcium levels in various parts of the same cell, the new techniques of fluorescence ratio imaging coupled with digital image processing seem ideal investigative tools (Tsien & Poenie, 1986). However, biochemical approaches to these problems are equally necessary and are likely to prove equally productive. References E. & TREWAVAS, A. J. (1985). Quantitative changes in calmodulin in NAD kinase during early cell development in the root apex of Pisum sativum L. Planta 165, 493-501. ANDERSON, J. M. & CORMIER, M. J. (1978). 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Phosphorylation of Avena Phytochrome in vitro as a probe of light induced conformational changes. /. biol. Chem. 261, 12089-12097.