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Plant Science 160 (2001) 381– 404 www.elsevier.com/locate/plantsci Review Calcium: silver bullet in signaling A.S.N. Reddy * Department of Biology and Program in Cell and Molecular Biology, Colorado State Uni6ersity, Fort Collins, CO 80523, USA Received 19 July 2000; received in revised form 5 September 2000; accepted 5 September 2000 Abstract Accumulating evidence suggests that Ca2 + serves as a messenger in many normal growth and developmental process and in plant responses to biotic and abiotic stresses. Numerous signals have been shown to induce transient elevation of [Ca2 + ]cyt in plants. Genetic, biochemical, molecular and cell biological approaches in recent years have resulted in significant progress in identifying several Ca2 + -sensing proteins in plants and in understanding the function of some of these Ca2 + -regulated proteins at the cellular and whole plant level. As more and more Ca2 + -sensing proteins are identified it is becoming apparent that plants have several unique Ca2 + -sensing proteins and that the downstream components of Ca2 + signaling in plants have novel features and regulatory mechanisms. Although the mechanisms by which Ca2 + regulates diverse biochemical and molecular processes and eventually physiological processes in response to diverse signals are beginning to be understood, recent studies have raised many interesting questions. Despite the fact that Ca2 + sensing proteins are being identified at a rapid pace, progress on the function(s) of many of them is limited. Studies on plant ‘signalome’ — the identification of all signaling components in all messengers mediated transduction pathways, analysis of their function and regulation, and cross talk among these components — should help in understanding the inner workings of plant cell responses to diverse signals. New functional genomics approaches such as reverse genetics, microarray analyses coupled with in vivo protein– protein interaction studies and proteomics should not only permit functional analysis of various components in Ca2 + signaling but also enable identification of a complex network of interactions. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Calcium; Calcium-binding proteins; Calmodulin; Protein kinase; Cell division; Calmodulin-binding proteins; Cytoplasmic streaming; Molecular motors; Kinesin; Myosin; Signal transduction; Stress; Plant defense 1. Introduction Plant growth and development is controlled by hormonal and environmental signals. Plants, unlike animals, are immobile and therefore have developed mechanisms to sense and respond to the Abbre6iations: ABA, abscisic acid; AOS, active oxygen species; [Ca2 + ]cyt, cytosolic Ca2 + ; CaM, calmodulin; CBP, calmodulin-binding protein; CBD, calmodulin-binding domain; CCaMK, Ca2 + / CaM-dependent protein kinase; CDPK, Ca2 + -dependent protein kinase; CRK, CDPK-related protein kinase; CIPK/SIPK, CBL/ SOS3-interacting protein kinase; GAD, glutamate decarboxylase; KCBP, kinesin-like calmodulin-binding protein; PDE, phosphodiesterase. * Tel.: + 1-970-4915773; fax: + 1-970-4910649. E-mail address: [email protected] (A.S.N. Reddy). biotic and abiotic stresses so that they can better adapt to their environment. How plants sense these various signals and produce an appropriate response has fascinated plant biologists over a century and has become an area of intense investigation in recent years. Research during the last two decades has clearly established that Ca2 + acts as an intracellular messenger in coupling a widerange of extracellular signals to specific responses. Although Ca2 + is implicated in regulating a number of fundamental cellular processes that are involved in cytoplasmic streaming, thigmotropism, gravitropism, cell division, cell elongation, cell differentiation, cell polarity, photomorphogenesis, plant defense and stress responses, the mechanisms 0168-9452/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 0 0 ) 0 0 3 8 6 - 1 382 A.S.N. Reddy / Plant Science 160 (2001) 381–404 by which Ca2 + controls these processes are only beginning to be understood. Because of the space limitations, my intention here is to summarize recent progress in understanding Ca2 + -mediated signal transduction pathways with emphasis on the current status of research, gaps in our knowledge and future directions. 2. Signals and cytosolic Ca2 + Improved methods to monitor free [Ca2 + ]cyt levels, especially using transgenic plants expressing Ca2 + reporter proteins, have greatly helped in demonstrating signal-induced changes in free [Ca2 + ]cyt level [1 –4]. The concentration of Ca2 + in the cytoplasm of plant cells is maintained low in the nanomolar range (100 –200 nM) [4,5]. However, Ca2 + concentration in the cell wall and in organelles is in the millimolar range (Fig. 1) [6,7]. Fig. 1. Schematic diagram illustrating the mechanisms by which plant cells elevate [Ca2 + ]cyt in response to various signals and restore Ca2 + concentration to resting level. Ca2 + channels are shown in red, whereas Ca2 + ATPases and antiporters are indicated in yellow. Arrows indicate the direction of Ca2 + flow across the plasma membrane, and into and out of cellular organelles (vacuole, plastids, mitochondria, endoplasmic reticulum and nucleus). The estimated concentration of resting levels of Ca2 + in different organelles is indicated [5,6,9,234]. Question marks indicate the lack of evidence. [Ca2 + ]cyt, cytosolic Ca2 + ; PLC, phospholipase C; R, receptor, cADPR, cyclic ADP ribose, PIP2, phosphotidyl inositol-4,5-bisphosphate, DG, diacylglycerol, PKC, protein kinase C, IP3, inositol-1,4,5-trisphosphate; ER, endoplasmic reticulum; Mt, mitochondria; Plast, plastids; PM, plasma membrane. Despite the existence of a large electrochemical gradient for Ca2 + entry into the cytoplasm, plant cells maintain their [Ca2 + ]cyt concentration at low levels, which requires active pumping of Ca2 + to the apoplast or organelles. A wide-range of signals such as light, hormones, gravity, touch, wind, cold, drought, oxidative stress and fungal elicitors have been shown to cause transient elevation of [Ca2 + ]cyt (see Table 1). A comprehensive discussion of signal-induced changes in [Ca2 + ]cyt has been published recently and will not be covered here [4,7 –9]. Current evidence indicates that 1,4,5-trisphosphate (IP3), cyclic ADP ribose (cADPR) and Ca2 + channels play an important role in elevating [Ca2 + ]cyt (Fig. 1). Ca2 + channels have been detected in the plasma membrane, vacuolar membrane, ER, chloroplast and nuclear membranes of plant cells [5]. These channels are classified based on their voltage dependence. The electrophysiological properties of all the known Ca2 + channels have been reviewed recently [5]. However, genes encoding Ca2 + channels in plants have not been identified. In plants, as in animals, phospholipase C-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) results in the production of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol. There is some indirect evidence for the involvement of heterotrimeric G proteins in promoting PIP2 hydrolysis in plants [10,11]. Phosphatidylinositol-specific phospholipase C (PI-PLC) activity and genes encoding this enzyme have been characterized from plants [12,13]. Plant PI-PLC hydrolyzes phosphatidylinositol-4,5-bisphosphate into IP3 and diacylglycerol with an absolute requirement for Ca2 + (1 mM) [12]. It was shown that one of the phospholipase C genes (AtPLC1 ) expression is induced by stresses including dehydration, salinity and low temperature [12,14]. Recently, three PI-PLC isoforms (StPLC1 to -3 ) have been isolated from guard cell enriched tissue of potato [13]. The expression pattern of the StPLC1 and -2 genes also suggest their involvement in drought stress in potato [13]. The soybean PLC showed phospholipase activity and complemented the lethal mutant phenotype of yeast lacking PLC activity. Although there is overwhelming evidence for signal-induced changes in [Ca2 + ]cyt, the information on the mechanisms by which a given signal elevates [Ca2 + ]cyt is still limited. Little A.S.N. Reddy / Plant Science 160 (2001) 381–404 383 Table 1 Effect of signals on cytosolic calcium in plants Signal Methods used to measure free cytosolic Ca2+ a Effect on [Ca2]cyt b Response References Abscisic acid Gibberellic acid Auxin NaCl 1, 2, 3 2 , ¡ Stomatal closure, gene expression a-Amylase secretion [3,20–23] [24,25] 1 1, 2, 3 [36] [27–31] Anoxia 1 Touch Wind Gravity Cold 1, 2 1, 2 1 1, 2 Drought 1, 2 Heat Shock Hypoosmotic stress Red light Ozone stress Oxidative stress (H2O2) Aluminum Pathogens and elicitors NOD factors 1, 2 2 Cell elongation and cell division Gene expression and osmolyte synthesis, K+ uptake Gene activation, adaptation to oxygen deprival Thigmomorphogenesis Morphogenesis Gravitropism COR gene expression, proline synthesis, changes in membrane lipid profile and cold acclimatization Gene expression, synthesis of osmoprotectants, and osmotolerance Thermotolerance Osmoadaptation 1 2 1, 2 Photomorphogenesis Production of AOSs Production of AOSs, HR and cell death [49] [50] [51–55] 1, 2 1, 2 , ¡ Ion imbalance Phytoalexin biosynthesis and induction of HR [56–59] [1,53,60–62] 1 Nodular formation and root hair curling [63] [32,33] [1,34–36] [37–39] [26] [40–42] [2,30,43] [44,45] [46–48] a Cytosolic free calcium levels are measured using injection of fluorescent indicator dyes (1), transgenic plants expressing aequorin (2) or cameleon (3). b Increase ( ) or decrease (¡) in cytosolic free calcium levels. COR, cold-r6 egulated; AOSs, active oxygen species; HR, hypersensitive response and [Ca2]cyt, cytosolic free calcium. is known about the mechanisms that are involved in the activation of phospholipase C and regulation of Ca2 + channels. IP3 has been shown to stimulate Ca2 + release from vacuolar store [9,15,16]. In animals, IP3 releases Ca2 + primarily from the endoplasmic reticulum (ER) whereas in plants Ca2 + is released from vacuoles. Ca2 + release from the ER elicited by IP3 has also been reported in plants [9]. IP3 − and cyclic ADP-ribose (cADPR)-gated channels that are found on the ER membrane in animals are found in the vacuolar and ER membranes in plants [9,17,18]. Recently, nicotinic acid adenine dinucleotide phosphate has been shown to release Ca2 + exclusively from plant ER [19]. These results suggest that multiple Ca2 + mobilization pathways that are regulated by different agents exist in plants. In animals, diacylglycerol, the second product of PIP2 hydrolysis, is an activator for protein kinase C. Although there are some reports indicating the presence of protein kinase C-like activity in plants, the gene encoding it has not been isolated [64]. Elevation of [Ca2 + ]cyt in response to signals could be due to influx of Ca2 + from the apoplast and/or Ca2 + release from intracellular stores (ER, vacuoles, mitochondria, chloroplasts and nucleus). Based on the type of signal or cell type internal and/or external Ca2 + stores could be involved in raising [Ca2 + ]cyt. The contribution of external Ca2 + and cellular organelles in elevating cytosolic Ca2 + in response to various signals is beginning to 384 A.S.N. Reddy / Plant Science 160 (2001) 381–404 be identified. Transgenic plants expressing a Ca2 + reporter targeted to different organelles and the use of different pharmacological agents that block or release Ca2 + from internal stores have allowed, in some cases, analysis of changes in organellar Ca2 + concentration as well as the contribution of these organelles in elevating [Ca2 + ]cyt [4,62]. These studies indicate that different signals use distinct Ca2 + stores in elevating [Ca2 + ]cyt. Cold-induced Ca2 + increase is inhibited by plasma membrane channel blockers, but is not affected by organellar channel blockers. However, wind-induced Ca2 + increase is blocked by organellar Ca2 + channel blockers whereas plasma membrane channel blockers did not have any effect [37], indicating that the extracellular Ca2 + contributes to cold-induced elevation of Ca2 + and internal Ca2 + stores contribute to wind-induced increase in [Ca2 + ]cyt. By targeting aequorin to cytoplasm and nuclear organelles, Van der Luit et al. [39] have shown that distinct cellular Ca2 + pools respond to wind and cold stimuli. Sodium chloride-induced [Ca2 + ]cyt is due to Ca2 + release from the vacuole [1,30]. Transgenic Arabidopsis seedlings in which aequorin is targeted to the cytoplasmic face of the tonoplast membrane [2] showed release of vacuolar Ca2 + in response to mannitol treatment [30]. In parsley cells, elicitor-induced sustained increase in [Ca2 + ]cyt is primarily due to the influx of extracellular Ca2 + [62]. Studies with maize suspensioncultures have shown that mitochondrial Ca2 + store contributes to anoxia induced [Ca2 + ]cyt [33]. ABA-induced changes in [Ca2 + ]cyt have been attributed to both Ca2 + release from internal stores and Ca2 + influx from external stores [65,66]. Recently, hydrogen peroxide (H2O2) has been shown to activate Ca2 + -permeable channels in the plasma membrane of Arabidopsis guard cells [55]. Furthermore, ABA induced H2O2 production in guard cells. However, H2O2-induced stomatal closing and activation of Ca2 + channels by H2O2 are disrupted in an ABA-insensitive mutant, suggesting that ABA-induced H2O2 and the H2O2-activated Ca2 + channels are important in elevating [Ca2 + ]cyt in response to ABA [55]. It is not yet known to what extent the changes in the [Ca2 + ]cyt levels are reflected in changes in free Ca2 + concentration in the nucleus. Recent studies show that there is a Ca2 + gradient between the nucleus and cytoplasm indicating the presence of regulatory mechanisms that control Ca2 + movement into and out of the nucleus [67]. ATP stimulates Ca2 + uptake into nuclei and studies implicate CaM involvement in this uptake process [67]. Currently, little is known about the participation of nuclear Ca2 + stores in increasing cytosolic Ca2 + and vice versa. Ca2 + uptake studies with isolated plant nuclei have shown that the transport of Ca2 + across nuclear membrane is an ATP-dependent process [68], suggesting that changes in cytosolic and nuclear Ca2 + may occur independently. Recently, using tobacco plants expressing Ca2 + reporter either in the cytosol or nucleus, it has been shown that changes in Ca2 + concentration in the nuclear compartment and cytosol are controlled independently [69]. These authors have also shown that the nuclear membrane is not passively permeable to Ca2 + . The signal-induced Ca2 + elevations are transient and the level of Ca2 + returns to the resting level, which requires the removal of Ca2 + from the cytosol. This requires Ca2 + transport into organelles against a concentration difference of 103 to 104 fold. Ca2 + homeostasis is achieved by high affinity Ca2 + -pumps (Ca2 + -ATPases) and low affinity Ca2 + /H+ antiporters (Fig. 1). These Ca2 + pumps and antiporters also play a crucial role in raising and restoring [Ca2 + ]cyt levels in response to various stimuli. Several Ca2 + -ATPases that belong to E6 R-type C6 a2 + -A6 TPases (ECA) and a6 utoinihibited C6 a2 + -A6 TPases (ACA) have been characterized from plants [70]. There are at least 12 Ca2 + -ATPases in plants of which eight belong to an ACA-type. In animals, autoinhibited Ca2 + -ATPases, which are regulated by Ca2 + /CaM, are exclusively in the plasma membrane whereas in plants they are primarily localized in endomembrane systems [70]. Unlike animal endomembrane Ca2 + -ATPases that lack calmodulin-binding domain, plant counterparts have a calmodulin-binding domain either at the N- or C-terminus [70 –73]. Recently, Ca2 + -ATPases that are regulated by Ca2 + /CaM have been found in the plant plasma membrane also [74,75]. However, the structural organization of plasma membranelocalized Ca2 + -ATPases in plants is different from that of animal counter parts [74,75]. The calmodulin-binding autoinhibitor region in plant plasma membrane-localized Ca2 + -ATPases is located in the N-terminus whereas it is in the C-terminal region in animal plasma membrane-localized Ca2 + -ATPases, suggesting that plant ATPases A.S.N. Reddy / Plant Science 160 (2001) 381–404 have unique structural organization. The ACAtype pumps are CaM regulated where, in the absence of activated CaM, the CaM-binding domain acts as an autoinhibitor. Binding of CaM to the autoinhibitor region causes activation of the enzyme, suggesting an important role for Ca2 + in regulating these pumps. CaM-regulated autoinhibited Ca2 + -ATPases are found in the tonoplast, ER and most recently in the plasma membrane [70 – 72,74,75]. The large number of Ca2 + -ATPases indicates complex mechanisms in regulating Ca2 + homeostasis in plants. Interestingly, the activity of the CaM-regulated ER ATPase in Arabidopsis is inhibited by a Ca2 + -dependent protein kinase, suggesting that Ca2 + both activates (through CaM) and inhibits (via CDPK) the activity of ER ATPase [76]. The phosphorylation site on the ATPase was mapped to Ser45 near the CaM-binding domain. Vacuolar localized Ca2 + /H+ antiporters (calcium ex6 changer) have been identified from Arabidopsis (CAX1 and CAX2 ) [77] and mung bean [78]. The Arabidopsis CAX1 gene functionally complements a yeast mutant defective in its antiporter activity [77]. CAX1 and CAX2 are high and low efficiency H+/Ca2 + exchangers, respectively. CAX transcripts are inducible by extracellular Ca2 + levels, Na+, K+, Ni2 + , PEG and Zn2 + but not by plant hormones. The transgenic plants expressing the CAX1 gene in sense orientation showed several abnormalities including stunted growth with poorly developed root system, necrosis of leaves and apical meristem, hypersensitivity to K+ and Mg2 + ions, and sensitivity to cold that could be reversed by Ca2 + supplementation [79]. These results indicate that the increased antiport activity of the CAX1 gene causes severe depletion in free cytosolic Ca2 + levels due to pumping of Ca2 + from cytosol to vacuole. Known Ca2 + channels, pumps and antiporters that are involved in Ca2 + homeostasis are shown in the Fig. 1. 3. Ca2 + sensors Transient Ca2 + increase in the cytoplasm in response to signals is sensed by an array of Ca2 + sensors (Ca2 + -binding proteins) which decode Ca2 + signal. Once Ca2 + sensors decode the elevated [Ca2 + ]cyt, Ca2 + efflux into the cell exterior and/or sequestration into cellular organelles such as vacuoles, ER and mitochondria restores its 385 levels to resting state. A large number of Ca2 + sensors have been characterized in plants, which can be grouped into four major classes [80,81]. These include (A) calmodulin (CaM), (B) CaMlike and other EF-hand containing Ca2 + -binding proteins, (C) Ca2 + -regulated protein kinases, and (D) Ca2 + -binding proteins without EF-hand motifs. Members of first three classes of Ca2 + sensors contain helix-loop-helix motif(s) called EF hands that bind to Ca2 + with high affinity [82]. However, different Ca2 + -binding proteins differ in the number of EF hand motifs and their affinity to Ca2 + with dissociating constants (Kds) ranging from 10 − 5 to 10 − 9 M. Binding of Ca2 + to the Ca2 + sensor results in a conformational change in the sensor resulting in modulation of its activity or its ability to interact with other proteins and modulate their function/activity. 3.1. Calmodulin CaM is a highly conserved, well-characterized and ubiquitous Ca2 + receptor in eukaryotes [82,83]. It is a small molecular weight acidic protein of 148 amino acids with four EF-hand motifs that bind to four Ca2 + ions (Fig. 2A). The crystal structure of CaM indicates that it has two globular domains, each with a pair of EF hands, connected by a central helix [84]. The binding of Ca2 + to CaM results in a conformational change in such a way that the hydrophobic pockets of CaM are exposed in each globular end which can then interact with target proteins [85,86]. In plants, there are multiple CaM genes that code for either identical proteins or contain a few conservative changes [81,83,87,88]. These small changes in amino acid composition of CaM isoforms may contribute to differential interaction of each CaM isoform with target proteins. There is considerable evidence to indicate that CaM genes are differentially expressed in response to different stimuli [81,83]. Such differential regulation is likely to be one of the mechanisms for cells to fine-tune Ca2 + signaling. Although it is preliminary, recent studies on CaM genes expression in response to different stimuli indicate that different CaM isoforms are involved in mediating a specific signal [81]. Three of the six Arabidopsis Cam genes (Cam1, -2 and -3 ) are inducible by touch stimulation [81] indicating the presence of different cis-regulatory ele- 386 A.S.N. Reddy / Plant Science 160 (2001) 381–404 ments in their promoters. The presence of multiple CaM isoforms in plants adds further complexity to the Ca2 + mediated network and points to their differential sensitivity to elevated [Ca2 + ]cyt levels in response to different stress stimuli. In potato, only one of the eight CaM isoforms (PCaM1) is inducible by touch [87]. The striking example for differential regulation of CaMs comes from the studies with soybean CaM isoforms. In soybean there are five CaM isoforms (SCaM1 to −5). SCaM1, -2 and -3 are highly conserved compared to other plant CaM isoforms including Arabidopsis CaM isoforms whereas SCaM4 and -5 are divergent and showed differences in 32 amino Fig. 2. A.S.N. Reddy / Plant Science 160 (2001) 381–404 acids with the conserved group [89]. Surprisingly, these divergent CaM isoforms are specifically induced by fungal elicitors or pathogen [90]. These results provided evidence for the differential regulation of CaM isoforms in plants. Soybean isoforms show differences in their relative abundance in vivo. The conserved isoforms are relatively abundant in their expression compared to divergent forms. All CaM isoforms activate phosphodiesterase (PDE) but differ in their activation of NAD kinase, calcineurin and nitricoxide synthase indicating Ca2 + /CaM specificity between CaM isoforms and target proteins [91]. Differential regulation of other enzymes by soybean divergent and conserved CaM isoforms has also been reported [92]. Although SCaM isoforms show similar patterns in blot overlay assays, they differ in their relative affinity in interacting with CaMbinding proteins [93]. Two divergent CaM isoforms that are found in Arabidopsis do not interact with proteins that bind to conserved CaM isoforms [94]. These studies suggest that conserved and divergent CaM isoforms may interact with different target proteins. Using agonists and antagonists of the Ca2 + signaling pathway, Ca2 + and CaM have been implicated in plant defense against pathogens [7,95]. Because the pharmacological agents have non-specific effects and because the targets of these agents are not clearly defined some of these 387 studies are not conclusive. More recent studies with transgenic plants overexpressing CaM and CaM isoforms have provided strong evidence for the involvement of CaM in plant defense responses. Transgenic plants expressing a mutated CaM with a single amino acid change (K115 to R) in the CaM sequence, which abolishes trimethylation of lysine at 115, showed increased levels of resistance to a variety of stimuli such as bacterial derived elicitor harpin, mechanical stress, and osmotic stress [96]. Furthermore, transgenic cell lines treated with cellulase or mechanical stress produced increased levels of AOSs compared to control cell lines. Production of AOSs has been shown to initiate a battery of defense responses against invading pathogens [53,60,61,97]. The enhanced resistance in transgenic plants is primarily attributed to their ability to hyperactivate NAD kinase [96]. Constitutive expression of divergent CaM isoforms SCaM4 and -5 in tobacco has also indicated a role of some CaM isoforms in plant defense against fungi and TMV. Conserved CaMs (SCaM1, -2 and -3) are not inducible by stress where as divergent CaM isoforms are normally expressed at a low level and are highly inducible in response to pathogen attack or elicitors. Transgenic plants expressing high levels of SCaM4 and -5 showed constitutive expression of salicylic acid related gene expression independent of SA Fig. 2. (A) CaM and other EF-hand motif-containing proteins from plants. AtCaM2 [98]; one of the six isoforms of CaM from Arabidopsis; At Centrin, one of the three centrins from Arabidopsis [186]; CCD-1, C-terminal centrin-like domain [235]; AtCBP22, Arabidopsis C6 alcium-B6 inding P6 rotein 22 [236]; AtTCH2 Arabidopsis T6 ouch 26 [237]; AtTCH3, Arabidopsis T6 ouch 36 [169]; PhCaM53, Petunia hybrida Calm6 odulin 53 [165]; ABI1, Arabidopsis ABA i6 nsensitive 16 [171,172], AtCBL1 to 3, Arabidopsis C6 alcineurin B6 -L6 ike-16 , -26 and -36 proteins [157]; AtCBL4, Arabidopsis CBL4 or S6 alt-O6 verly-S6 ensitive3 (SOS3) protein [31]; AtCP1, salt-induced Arabidopsis C6 alcium-Binding P6 rotein [173]; OsEFA27, rice EF hand protein responsive to A6 bscisic acid [170]; PvHRA32, bean H6 ypersensitive R6 eaction A6 ssociated protein [178], AtRBOHA, Arabidopsis R6 espiratory B6 urst O6 xidase H6 omologue A6 and BetV-4, birch pollen allergen [238]. Asterisks in CBLs indicate the myristoylation motif. Interruptions in the AtRBOHA protein are denoted by ‘//’. (B) Schematic representation of Ca2 + -regulated protein kinases and CBPs from plants. CDPK, C6 alcium-d6 ependent and calmodulin-independent p6 rotein k6 inase [194,196]; C6 RK, C6 DPK-r6 elated protein kinase [239]; CaMK (MCK1), m6 aize C6 aM-dependent protein k6 inase [104], CaMK (CB1), apple CaM-dependent p6 rotein kinase [203]; CCaMK, c6 alcium and calcium/calm6 odulin-dependent protein k6 inase [107]; SIP, one of the eight S6 OS3-i6 nteracting p6 roteins [162]; PhGAD, P6 etunia h6 ybrida g6 lutamic a6 cid d6 ecarboxylase [102]; Ca2+ ATPase (BCA1), B6 rassica C6 a2 + -A6 TPase 1 [72,112]; Ca2+ ATPase (ACA2), A6 rabidopsis C6 a2 + -A6 TPase 2 [71]; AtKCBP, Arabidopsis k6 inesin-like c6 almodulin-b6 inding p6 rotein [109]; At cNGC1, one of the six c6 yclic n6 ucleotide g6 ated c6 hannels from Arabidopsis [94,115]; HvCBT1, H6 ordeum 66 ulgare C6 aM b6 inding t6 ransporter; NtCBP4 N6 icotiana t6 abacum c6 almodulin-b6 inding p6 rotein 46 [116,117]; Zm SAUR1, Z6 ea m6 ays s6 mall a6 uxin u6 p R6 NA; PMDR1, p6 otato m6 ultidrug r6 esistance protein [119]; BjGLY I, B6 rassica j6 uncea glyoxalase I [118]; NtCB48, N6 icotiana t6 abacum c6 almodulin-b6 inding p6 rotein 48 [106]; MPCBP, m6 aize p6 ollen-specific c6 almodulin-b6 inding p6 rotein [121]. Calmodulin-binding sites that are predicted based on secondary structure but not verified experimentally are denoted with a question mark. Three other calmodulin-binding C6 a2 + -A6 TPases from plants that have been reported recently [73– 75] are not shown in this figure. (C) Putative CaM-binding myosins from Arabidopsis and their structural organization. Myosins in the Arabidopsis genome were identified by searching the database with the conserved motor domain of a myosin. The sequences that contained the motor domain of myosin were analyzed by the Simple Modular Architecture Research Tool (SMART) program. 388 A.S.N. Reddy / Plant Science 160 (2001) 381–404 throughout their life cycle and showed enhanced disease resistance to a wide spectrum of fungal pathogens and tobacco mosaic virus [90]. These studies indicate CaM isoform specificity in their target activation in Ca2 + mediated signal transduction processes in plants. Furthermore, recent studies revealed that the CaM isoforms differ in their affinity to the same target protein [91,98 – 100] highlighting the significance of the existence of multiple CaM isoforms in mediating specific Ca2 + -mediated signal transduction pathways. CaM regulates various cellular activities by modulating the activity or function of a number of proteins. In most cases, only the activated form of CaM (Ca2 + bound CaM) interacts with its target proteins. However, in some cases CaM can interact with its target proteins in the absence of Ca2 + [86]. 3.1.1. Ca 2 + -dependent CaM-binding proteins CaM is multifunctional because of its ability to interact with and control the activity of a variety of target proteins. The role of CaM in a given cell or a tissue is determined by the presence of its target proteins. In recent years efforts to dissect Ca2 + /CaM mediated signaling pathways in plants have focused on identification and characterization of CaM-binding proteins (CBPs) in plants. The amino acid sequences of the CBD in different CaM target proteins is not conserved [86]. However, CaM-binding motifs from different CaMbinding proteins form characteristic basic amphipathic a-helices with several positive residues on one side and hydrophobic residues on the other side [86]. Using biochemical assays and protein-protein interaction-based screening of expression libraries with labeled CaM : 24 proteins that interact with CaM in a Ca2 + -dependent manner have been identified from plants (Fig. 2B,C). These include NAD kinase [101], glutamate decarboxylase [102], Ca2 + /CaM kinases [103,104], elongation factor-1a [105], heat shock inducible proteins [106], CCaMK [107], transcription factor [108], kinesin-like protein [109 – 111], Ca2 + -ATPase [71,112], nuclear NTPases [113], ion transporters [94,114 –117], glyoxalase I [118], multidrug resistant protein [119], SAUR, an auxin-induced gene [120], pollen specific CBP [121] and some other proteins of unknown function [122]. The identity of the known CBPs indicate that diverse proteins implicated in a wide variety of processes including ion transport, gene regulation, cytoskeletal organization, cell division, disease resistance and stress tolerance interact with CaM (see Fig. 2B,C and Fig. 3). Interestingly, many of these CBPs have no homologs in animals. Some of these (e.g. glutamate decarboxylase, GAD) have homologues in animals but lack CaM binding motif, suggesting that they are regulated by CaM only in plants. In some cases plants and animals have similar proteins but the location of the CaM binding domain is different between plant and animal proteins (e.g. Ca2 + ATPase). Although the catalytic core in GAD, which catalyzes the conversion of glutamate into g-amino butyric acid (GABA) and CO2 is conserved across bacteria, plants and animals, the Ca2 + /CaM regulation of GAD is unique to plants [123]. GAD has been cloned and characterized from a number of plant species. Although the CBD is not conserved, all plant GADs isolated so far posses a CBD and are regulated by Ca2 + /CaM [123]. In Arabidopsis there are two GAD isoforms (GAD1 and GAD2) and the CBD of these two isoforms are different, raising the possibility for functional diversity and regulation by Ca2 + /CaM [124]. Plants produce increased levels of GABA in response to mechanical and cold stresses [125]. These environmental stresses also elevate the [Ca2 + ]cyt levels raising the possibility that elevated Ca2 + activates GAD, which in turn increases GABA levels. Transgenic tobacco plants expressing full-length GAD showed normal wild type phenotype with moderately increased GABA and reduced Glu. However, the transgenic plants expressing GAD minus CBD showed severe anatomical abnormalities in cell elongation in stem cortex and parenchyma tissues. This phenotype is shown to correlate with accumulation of extremely high levels of GABA and low levels of Glu [126]. The NAD kinase, which catalyses the conversion of NAD to NADP [101,127], is vital to living organisms especially when energy is in demand under stress conditions. Recently, it has been reported that NAD kinase plays a role in oxidative burst and in the formation of active oxygen species (AOSs) [96], which are known for their involvement in plant defense against pathogens. Several CBPs that show similarities to ion channels have been isolated from barley [114], Ara- A.S.N. Reddy / Plant Science 160 (2001) 381–404 bidopsis [94,115] and tobacco [116,117]. These proteins showed sequence similarity to cyclic nucleotide gated channels from animals and inward rectifying K+ channels from plants. These proteins have been shown to reside in the plasma membrane. Constitutive over expression of one of these proteins, NtCBP4, in sense 389 and antisense orientation in tobacco exhibited a normal phenotype under normal growth conditions. However, the NtCBP4 sense transgenic plants showed improved tolerance to Ni2 + and hypersensitivity to Pb2 + [116], suggesting a role for this CaM-binding channels in metal tolerance. Fig. 3. Ca2 + sensing proteins and their functions in plants. Four major groups of Ca2 + sensors (indicated in four boxes) have been described in plants. These include (A) Ca2 + -dependent protein kinases, (B) CaM, (C) other EF-hand motif-containing Ca2 + -binding proteins, and (D) Ca2 + -binding proteins without EF-hand motifs. Inhibition of enzyme activity is shown by ‘Þ’. CDPK, Ca2 + -d6 ependent and calmodulin-independent p6 rotein k6 inase [194,196]; Centrin from Arabidopsis [186]; AtTCH2 Arabidopsis T6 ouch 26 [237]; AtTCH3, Arabidopsis T6 ouch 36 [169]; AtCBP22, Arabidopsis C6 alcium-B6 inding P6 rotein 22 [236]; ABI1, ABA i6 nsensitive 16 [171,172]; PhCaM53, P6 etunia h6 ybrida Calm6 odulin 53 [165]; AtCBL1 to 3, Arabidopsis C6 alcineurin B6 -L6 ike-16 , -26 and -36 proteins [157]; AtCBL4, Arabidopsis CBL4 or S6 alt-O6 verly-S6 ensitive3 (SOS3) protein [31]; AtCP1, salt-induced Arabidopsis C6 alcium-Binding P6 rotein [173]; OsEFA27, rice EF hand protein responsive to A6 bscisic acid [170]; PvHRA32, bean H6 ypersensitive R6 eaction A6 ssociated [178]; CCD-1, C6 -terminal c6 entrin-like d6 omain [235]; AtRBOHA, Arabidopsis R6 espiratory B6 urst O6 xidase H6 omologue A6 [183]; BetV-4, birch pollen allergen [238]; Myosins [151,152]; s6 mall a6 uxin u6 p R6 NA [120]; EF-1 alpha, elongation factor-1 a [143]; CCaMK, c6 alcium and calcium/calm6 odulin-dependent protein k6 inase [107]; CaMK calcium/calmodulin-dependent protein kinase [104,203]; CBP1, c6 almodulin-binding p6 rotein 1 [122]; CBP5, c6 almodulin-b6 inding protein 5 [122]; MPCBP, m6 aize p6 ollen-specific c6 almodulin-b6 inding p6 rotein [121]; StCBP S6 olanum t6 uberosum c6 almodulin-b6 inding p6 rotein (Reddy, unpublished results); BjGLY I, glyoxalase I [118,138]; NAD kinase [127], NtCB48, N6 icotiana t6 abacum c6 almodulin-binding p6 rotein 48 [106]; MDR1, m6 ultidrug r6 esistance protein [119]; GAD, g6 lutamic a6 cid d6 ecarboxylase; PR proteins, p6 athogenesis r6 elated proteins [90]; cNGCs, c6 yclic n6 ucleotide g6 ated c6 hannels [94,114–117]; Ca2+ ATPase [70,71,73– 75]]; TGA, DNA binding protein [108]; NTPase, nucleoside triphosphatase [113]; KCBP, k6 inesin-like c6 almodulin-b6 inding p6 rotein [109]; PCP, p6 istil-expressed c6 alcium-binding p6 rotein. 390 A.S.N. Reddy / Plant Science 160 (2001) 381–404 A novel CaM-binding microtubule motor protein (KCBP, kinesin-like calmodulin-binding protein) was isolated from Arabidopsis and other plants [109 – 111]. This motor is unique among kinesins and kinesin-like proteins in having a CaM-binding domain adjacent to the motor domain at the C-terminus and a myosin tail homology region in the N-terminal tail [128]. KCBP binds CaM in a Ca2 + dependent manner at physiological Ca2 + concentration. KCBP binds bovine CaM and three CaM isoforms (CaM-2, -4 and -6) of Arabidopsis. However, CaM-2 showed 2-fold higher affinity to KCBP as compared with other isoforms [99]. Although, a homologue of KCBP has not been found in S. cere6isiae, C. elegans and Drosophila whose genomes have been sequenced, recently a CaM-binding C-terminal kinesin (kinesin C) was cloned from sea urchin [129]. The CBD of kinesin C shared 35% sequence identity with the CaM binding domain in KCBP. However, kinesin C differs considerably from KCBP and does not show any sequence similarity in the stalk and tail regions. The amino-terminal tail and stalk regions of KCBPs from different plant systems are highly conserved and contain myosin tail homology (MyTH4) and talin-like regions that are not present in kinesin C [130]. Further analysis of CaM-binding kinesin-like proteins from a number of phylogenetically divergent plants and animals including the primitive eukaryotes is needed to understand their origin and evolution. Motility studies with the KCBP have shown that it is a minus-end directed microtubule motor [131]. Binding of activated CaM to KCBP inhibits its interaction with microtubules [131 –133]. The CaM effect on KCBP suggests that activated CaM can act as a molecular switch to down-regulate the activity of KCBP. Among motor proteins, heavy chains of unconventional myosins also bind CaM where the inhibition of the enzymatic activity and motility by Ca2 + is similar to the effect of Ca2 + on a CaM binding KCBP, although the mechanism of inhibition is very different between these proteins [134]. Based on these studies with KCBP it is reasonable to speculate that spatial and temporal changes in free [Ca2 + ]cyt levels in response to signals are likely to regulate KCBP activity in the cell. Current evidence indicates the involvement of KCBP in trichome morphogenesis and cell division [135 – 137,240]. Glyoxalase I, which catalyses the conversion of toxic methylglyoxal to a nontoxic metabolite, expression is induced by NaCl, mannitol or abscisic acid [138]. Glyoxylase I from Brassica juncea (BjGly I) binds CaM-Sepharose and its activity is stimulated by Ca2 + /CaM [139]. Leaf discs from transgenic plants expressing BjGly I showed tolerance to methylglyoxal and salt. In contrast, the control antisense and wild type leaf discs showed loss of chlorophyll and did not survive these stresses, suggesting that BjGly I plays a role in conferring tolerance to salt stress in plants. Two nuclear proteins, a chromatin associated nucleotide triphosphatase (NTPase) [140] and a DNA binding protein (TGA3) that binds to CaM3 promoter [108], are also regulated by CaM. The binding of CaM to TGA3 enhances its binding with the promoter. In plants, Ca2 + /CaM is involved in stabilizing cortical microtubules at low Ca2 + concentrations and destabilizing the same at higher Ca2 + concentrations [141,142]. Two CBPs that show different sensitivities to Ca2 + are implicated in evoking these two opposing effects of CaM on cortical microtubules. Elongation factor1a, a CaM-binding microtubule associated protein, stabilizes microtubules and Ca2 + /CaM has been shown to inhibit elongation factor-1apromoted microtubule stabilization [143]. An auxin-induced gene product has been shown to bind CaM, suggesting the involvement of CaM in auxin action [120]. CBPs that are induced by heat shock have been isolated from tobacco [106]. One of these CBPs (NtCBP48) contains a centrally located putative transmembrane domain and a nuclear localization sequence motif [106]. Two maize proteins (CBP-1 and CBP-5) and a multidrug resistant protein also bind CaM in a Ca2 + dependent manner [119,122]. However, the functions of these are not known. A pollen specific CaM-binding protein from maize, MPCBP, [121] and CBP from developing tubers (StCBP) (Reddy, unpublished results) are also novel CBPs and have no homologs in non-plant systems. Binding of superoxide dismutase to CaM-Sepharose column suggests that it might be regulated by CaM [144]. 3.1.2. Ca 2 + -independent CaM-binding proteins Although CaM, in most cases, binds to its target proteins in the presence of Ca2 + , some CaM-binding proteins interact with CaM in the absence of Ca2 + [86]. In animals, myosins, a A.S.N. Reddy / Plant Science 160 (2001) 381–404 superfamily of actin-based motors that perform a broad array of cellular functions, bind CaM in the absence of Ca2 + [145]. The CaM-binding domain (also called ‘IQ motif’) in these myosins has a consensus sequence ‘IQXXXRGXXXR’ (I, isoleucine, Q, glutamine, R, arginine, G, glycine) which forms an a-helix [145]. Although there is compelling indirect evidence for the role of actin and actin-based motors in various transport processes in plants, little is known about plant myosins and their regulation [134]. Myosins containing IQ domains, which are typically CaM-sensitive, have been identified in plants but their interaction with, or regulation by CaM has not yet been demonstrated in these proteins [134]. The deduced amino acid sequence of plant myosins, like other known myosins, contain ‘IQ’ motifs in the neck region [146,147]. Phylogenetic analyses using the conserved motor domain grouped known myosins into 15 distinct classes [134]. Of these, published plant myosins fall into two separate groups — myosin VIII (e.g. Arabidopsis myosins ATM1 and ATM2) and myosin XI (e.g. Arabidopsis myosins MYA1 and MYA2). These two subgroups contain only plant myosins, suggesting that plants may have a unique set of myosins. By comparing the sequences in the Arabidopsis genome database with the motor domain of myosins, I have identified 17 myosin-like proteins in 60% of the sequenced genome. All Arabidopsis myosin-like proteins that are identified so far possess two to six putative CaM-binding ‘IQ’ motifs (Fig. 2C). Although the binding of CaM to any of these proteins has not been demonstrated experimentally, the presence of these motifs suggests that myosins in plants are likely to be regulated by CaM in response to changes in intracellular Ca2 + . A number of reports have shown that the cytoplasmic streaming in plant cells is regulated by the [Ca2 + ]cyt concentration [148]. Elevated levels of Ca2 + inhibits cytoplasmic streaming and the movement of organelles [149,150]. However, specific motors involved in these processes remain to be identified. The mechanisms by which Ca2 + regulates the activity of these motors is just beginning to emerge. Both CaM and CDPKs are likely to mediate Ca2 + effects on cytoplasmic streaming and organelle transport. Recently, Yokota et al. [151,152] purified two myosins from plants and demonstrated that CaM associates with the 391 purified myosin and regulates its motor activity. Ca2 + inhibits the myosin motor activity as well as myosin-activated ATPase activity in plants. The inhibition of myosin activity in the presence of Ca2 + appears to be due to dissociation of CaM from the heavy chain since Ca2 + inhibition of motor activity can be restored by exogenous addition of CaM. Some studies suggest that, in some cases, Ca2 + can effect myosins indirectly through another mechanism [153]. There is some indirect evidence in support of a role of phosphorylation catalyzed by CDPK in regulating myosin activity [154,155]. A CDPK, which has been shown to bind actin filaments, phosphorylates a putative myosin light chain in Chara [155,156]. However, the effect of such a phosphorylation is not known. 3.2. CaM-like and other EF-hand containing proteins In addition to CaM, recent studies indicate the presence of numerous CaM-like proteins in plants (Fig. 2A). However, the function of these proteins in Ca2 + signaling pathway(s) is not fully characterized as compared to that of CaM. These CaMlike proteins differ from the CaM in containing more than 148 amino acids, and one to six EFhand motifs with limited homology to CaM [83]. Hence, it is likely that these proteins are functionally distinct from CaM and are involved in controlling different Ca2 + -mediated cellular functions. Recently, a new family of Ca2 + -binding proteins, called calcineurin B-like (CBL) proteins, has been identified [31,157,158]. Studies with saltoverly-sensitive (SOS) mutants of Arabidopsis indicate a key role for Ca2 + in salt stress signaling [159 –161]. These mutants are hypersensitive to Na+ and Li+ and are unable to grow on low-K+ culture medium. These abnormal growth patterns in the presence of NaCl could be mitigated by the addition of increased levels of Ca2 + in the same medium. The SOS3 gene encodes a Ca2 + sensor protein similar to calcineurin B, a regulatory subunit of Ca2 + -dependent protein phosphatase and neural Ca2 + sensors (CNS) of animals and raises the possibility that SOS3 gene product might control the K+/Na+ transport system via a Ca2 + -regulated pathway [31]. In Arabidopsis there are at least six CBL genes encoding highly similar but functionally distinct Ca2 + -binding proteins [157]. 392 A.S.N. Reddy / Plant Science 160 (2001) 381–404 Drought, cold and wound stress signals induce AtCBL1 gene transcripts, whereas AtCBL2 and AtCBL3 are constitutively expressed [157]. Further, C6 BL-i6 nteracting p6 rotein k6 inases (CIPK1 to 4) belong to the serine/threonine class of kinases and show high homology to protein kinases SNF1 and AMPK from yeast and mammalian systems, respectively. SOS3 interacts with SOS2, which is also a member of CIPKs (also called SIPKs, S6 OS3-i6 nteracting p6 rotein k6 inases), and seven other CIPKs [162]. CIPKs interact with CBLs, but not with CaMs, in a Ca2 + -dependent manner [158]. The activity of CIPK/SIPK is activated by SOS2 (CBL4) and other CBLs in a Ca2 + -dependent manner, suggesting that CBL/SIPK complex is likely to regulate Na+ and K+ homeostasis through phosphorylation. CIPKs/SIPKs represent a novel family of Ca2 + -regulated protein kinases in plants. The interaction of calcineurin B-like (CBL) proteins with protein kinases is very surprising since the CBL proteins are known to activate a protein phosphatase in animals. In addition to Ca2 + -regulated protein kinase, there is some evidence for the role of a Ca2 + -regulated phosphatase also in salt tolerance. Ectopic expression of constitutively active yeast calcineurin has been shown to confer salt tolerance in tobacco [163,164]. Certain CaM-like proteins contain a 34 amino acid Caax-box motif (prenylation domain) at their C-termini (CTIL in PhCaM53) [165] or (CVIL in OsCaM63) [166]. Transient expression of GFP fused with the full-length CaM53 or mutated CaM53 (without CaaX-box motif) in tobacco and petunia protoplasts showed that the full-length protein localizes to plasma membrane whereas mutant CaM53 localizes to the nucleus [165,167], suggesting an important role for the prenylation domain in CaM53 localization. Ectopically expressed CaM53 with a prenylation domain in tobacco showed stunted growth and a necrotic phenotype. However, ectopic expression of neither non-prenylated form nor Caax-box motif alone showed such altered morphogenic alterations indicating the importance of the prenylation domain [165]. Arabidopsis, in addition to six CaMs, has other CaM-like genes including the TCH genes that are induced in response to various mechanical, chemical and environmental stimuli (Fig. 2A) [168]. TCH3 is 324 amino acids long and contains six EF hand motifs [169]. A cDNA sequence encoding a 27 kDa CaM-like protein (EFA27) has been isolated from ABA-treated rice seedlings [170]. It contains a single EF hand motif and the expression of EFA27 is induced in response to salt and dehydration stress, and to ABA signal. In Arabidopsis there are several EFA27 gene homologues [170], suggesting the existence of similar proteins in phylogenetically distant species. EF hand motif containing protein phosphatases were also identified in an ABA-signaling pathway (Fig. 2A) [171,172]. Another Ca2 + -binding protein, AtCP1, from Arabidopsis contains three EF hand motifs (Fig. 2A) and binds Ca2 + [173]. The AtCP1 gene transcripts are also highly inducible by NaCl treatment but not by ABA treatment indicating the specificity of this unique Ca2 + -binding protein in responding to stress factors. Several studies implicate the involvement of Ca2 + signal in plant defense responses such as phytoalexin biosynthesis, induction of defense-related genes and hypersensitive cell death [7,174,175]. Ca2 + is implicated in mediating systemin, a peptide involved wound-induced activation of defense genes in tomato [176]. Further, Flego et al. [177] showed a correlation between increased Ca2 + concentration in plants and increased resistance to bacterial pathogen Erwinia caraoto6ora. P6Hra32, a gene that is highly expressed during hypersensitive reaction in bean tissue challenged with Pseudomonas syringae, has been shown to encode a small Ca2 + -binding protein (161 aa) with four EF motifs [178]. The AOSs stimulate rapid influx of Ca2 + into the site of infection and initiate the hypersensitive response in order to develop resistance against invading pathogens [53,60]. The plasma membrane located Ca2 + channel (LEAC, l6 arge conductance e6 licitor-a6 ctivated ion c6 hannel) is likely to be involved in elevating [Ca2 + ]cyt in response to AOSs [61]. During the interaction of Arabidopsis and P. syringae, the resistance gene product, RPM1, functions immediately and elevates the Ca2 + levels [179,180]. Similar results were obtained from the C. ful6um/tomato interaction [181]. Furthermore, it was shown that the resistance gene activates a CDPK [182]. Identification and characterization of human gp91 phox (phox for phagocyte oxidase) homologues from Arabidopsis and rice (RbohA, r6 espiratory b6 urst o6 xidase h6 omologue A6 ), provided evidence for the downstream target for the ele- A.S.N. Reddy / Plant Science 160 (2001) 381–404 vated levels of Ca2 + in oxidative burst [183]. The RbohA shows high similarity to human gp91 phox, a plasma membrane bound neutrophil phagocyte oxidase that is involved in the generation of superoxide radicles via its NADPH oxidase activity. In addition to the gp91 phox region, the plant gp91 phox (RbohA) also contains two EF hand motifs which are not present in human gp91 phox suggesting that Ca2 + modulates the formation of superoxide radicles through RbohA during oxidative burst in plants [183]. Centrin (also known as caltractin) is a small (: 20 kDa) acidic protein with four Ca2 + -binding EF-hand motifs [184]. Genes encoding centrins were isolated from the salt marsh plant, Atriplex nummularia [185], Arabidopsis [186] and tobacco BY-2 cells [187]. In plants, centrins are 167 –177 amino acids long. The Arabidopsis centrin was isolated as an early-induced gene in response to bacterial inoculation. The expression of this centrin is induced when the plants are inoculated with avirulent strains of bacteria but not with virulent strains, suggesting a role for this centrin in plant defense [186]. Based on the localization of plant centrins it was suggested that centrins are involved in cell plate formation [187] and Ca2 + -regualted transport through plasmodesmata [188]. 3.3. Ca 2 + -regulated protein kinases By manipulating cellular Ca2 + levels with pharmacological agents, Ca2 + -regulated protein phosphorylation has been implicated in a variety of responses including host –pathogen interactions [182,189], cold stress [190], nuclear migration during fungal infection [97], gravitropism [80], lightregulated gene expression [191], thigmotropism [192] and hypoosmatic shock [48,193]. Studies during the last decade indicate that there are at least five types of Ca2 + -regulated protein kinases in plants: (i) a C6 a2 + -d6 ependent and CaM-independent p6 rotein k6 inases (CDPKs); (ii) C6 DPK-r6 elated k6 inases (CRKs); (iii) CaM-dependent protein k6 inases (CaMKs); (iv) C6 a2 + /CaM-dependent protein k6 inases (CCaMK); and (v) S6 OS3/C6 BLi6 nteracting p6 rotein k6 inases (SIPKs/CIPKs) (Fig. 2B). CDPKs, a new family of protein kinases, are most abundant and ubiquitous in plants [194]. Apart from plants, CDPKs are found in protozoans and algae. Ca2 + directly binds to the CDPK 393 and stimulates the kinase activity whereas CaM does not have any significant effect on the kinase activity [195]. CDPKs have a unique structural organization in which a protein kinase catalytic domain is followed by CaM-like region with four Ca2 + binding motifs (Fig. 2B). The kinase domain of CDPK shows significant homology with the mammalian Ca2 + /CaM-dependent protein kinase II (CaM K II) catalytic domain. The region that joins the kinase domain to the CaM-like region (junction region) corresponds to the autoinhibitory/CaM-binding region of CaM K II and prevents kinase activity in the absence of Ca2 + [196]. The autoinhibitory domain located in the junction region of CDPKs inhibits the kinase activity in the absence of Ca2 + . Ca2 + binding to CDPK effects confirmation of the kinase and relieves the inhibition caused by the autoinhibitory region [194]. CDPKs are found in different cellular locations. Several CDPKs have putative myristoylation sites indicating that myristoylation of CDPKs may regulate the association of CDPKs with membranes [194]. The transcripts of two Arabidopsis CDPK genes (AtCDPK1 and AtCDPK2 ) are highly inducible by drought and high salt but not by low temperature or heat stress suggesting the specificity of CDPK’s induction in response to different stress factors [197]. A CDPK from Vigna radiata is highly inducible by wounding, CaCl2, IAA and NaCl treatments [198]. There are over 40 CDPKs in the Arabidopsis genome [194] (http://plantsp.sdsc.edu/ cdpk/). CDPKs are classified into several groups based on their sequence domain organization (myristolyation, PEST and the number of EF hand motifs). Furthermore, CDPKs differ in their affinity for Ca2 + . For example, AtCDPK1 differs from AtCDPK2 in its Ca2 + stimulated activity although both of them possess four EF hand motifs. Recent studies indicate that, besides Ca2 + , lipids are involved in the regulation of CDPK activity [199,200]. A carrot CaM-like domain protein kinase, DcCPK1, resembles animal PKC in its activation by Ca2 + and certain phospholipids suggesting that lipids regulate the activity of some CDPKs and perform specific biological functions in plants [201]. Ion channels, enzymes involved in metabolism, cytoskeletal proteins, and DNA binding proteins have been identified as CDPKs substrates, suggesting their role in diverse cellular processes ranging from ion transport to 394 A.S.N. Reddy / Plant Science 160 (2001) 381–404 gene expression [194]. Sheen [202] has shown that Arabidopsis AtCDPK1 and AtCDPK1a are involved in regulating the expression of stress-inducible genes. Furthermore, phosphatases counteract these responses suggesting that involvement of Ca2 + -regulated phosphorylation is necessary for stress-induced gene expression (see Section 3.1.1 on Ca2 + and gene expression). CRKs are similar to CDPKs except that the CaM-like region is poorly conserved with degenerate EF-hands. There are at least seven CRKs in Arabidopsis. However, regulation and function of these kinases are not known [194]. A cDNA with significant sequence similarity to mammalian CaM K II has been isolated from apple [203] and maize [104] (Fig. 2B). However, the biochemical properties and regulation of these kinases are not known. A Ca2 + /CaM-dependent protein kinase (CCaMK) was characterized from lily and other plants [104,107]. Sequence analysis revealed the presence of an N-terminal catalytic domain, a centrally located CaM-binding domain and a C-terminal visinin-like domain containing only three EF hands (Fig. 2B). Biochemical studies of CCaMK established that Ca2 + /CaM stimulates CCaMK activity. In the absence of CaM, Ca2 + promotes autophosphorylation of CCaMK. The phosphorylated form of CCaMK possesses more kinase activity than the non-phosphorylated form [204]. CCaMK phosphorylates elongation factor-1a [205]. However, the physiological significance of this phosphorylation is not yet known. The affinity of CCaMK to CaM is regulated by Ca2 + stimulated autophosphorylation [206]. 3.4. Ca 2 + -binding proteins without EF-hand motifs There are several proteins that bind Ca2 + but do not contain EF-hand motifs (Fig. 3). These include annexins, calreticulin and p6 istil-expressed C6 a2 + -binding p6 rotein (PCP). Annexins are a family of proteins in plants and animals that bind phospholipid in a Ca2 + -dependent manner and contain four to eight repeats of : 70 amino acids [207]. Although the exact function of annexin is not known, plant annexins are implicated in secretory processes and some have ATPase, peroxidase or F-actin binding activities [208]. Calreticulin is a Ca2 + sequestering protein in the ER and functions as a chaperone [209]. In addition, it is implicated in Ca2 + homeostasis and other functions [210]. A 19 kDa novel Ca2 + -binding protein (PCP) that is expressed in anthers and pistil was isolated recently [211]. PCP is a high capacity (binds 20 mol of Ca2 + per mol of PCP), low affinity Ca2 + -binding protein. The pattern of PCP expression suggests a role for it in pollen-pistil interactions and/or pollen development. A cysteine class of Ca2 + -dependent protease (CDP) has been purified from Arabidopsis root cultures [212]. Its activity is specifically dependent on Ca2 + but not on other divalent cations. Structural organization of CDP is not known since the gene encoding the CDP has not been isolated. 4. Ca2 + and gene expression Although there is a great deal of information on the involvement of Ca2 + in regulating various physiological processes [213,214], the role of Ca2 + in regulating gene expression in plants is forthcoming only recently. Manipulation of [Ca2 + ]cyt by various means is shown to affect the expression of specific genes in plants. Mannitol-induced expression of RAB and AtP5CS1 genes is blocked in the presence Ca2 + channel blockers like verapamil or lanthanum or the Ca2 + chelator EGTA [30,215]. The expression of these genes in treated cultures and plants is less than that of untreated counterparts, indicating a role of Ca2 + in drought tolerance. Increased external Ca2 + or heat shock rapidly induce the expression of touch-induced CaM-related genes (TCH2, TCH3 and TCH4 ) whereas TCH1 gene, which codes for CaM, is not significantly induced [216]. Heat shock, in the presence of EGTA, a Ca2 + chelator, did not show induction of TCH genes. This EGTA effect is reversed by Ca2 + replenishment. Based on these results it was suggested that heat shock elevates [Ca2 + ]cyt levels which in turn regulates the expression of TCH2, -3 and -4 genes. Ca2 + effect on touch-induced CaM-related genes is specific since magnesium, another divalent ion, did not have any effect. Furthermore, increased Ca2 + did not effect the expression of a heat shock induced gene. Depletion of Ca2 + by Ca2 + chelator blocked ethylene-induced chitinase synthesis whereas artificial elevation of cytosolic Ca2 + with Ca2 + ionophore (ionomycin) or an inhibitor of microsomal Ca2 + ATPase induced chitinase synthesis in the absence A.S.N. Reddy / Plant Science 160 (2001) 381–404 of ethylene [217]. Accumulation of cold-induced mRNAs in alfalfa was also partially blocked by lanthanum, a Ca2 + channel blocker and a CaM inhibitor (W7) completely blocked the expression of cold-regulated genes [218]. Lam et al., [219] have shown that light induced chlorophyll a/b binding (cab) transcripts could be induced in the dark by increasing the intracellular level of Ca2 + using ionomycin. Compelling evidence for the role Ca2 + and CaM in the regulation of gene expression came from microinjection studies with phytochrome mutant (aurea) [220]. In this mutant, light-regulated genes are not expressed. Using a reporter gene fused to light-regulated promoter, it was shown that microinjection of reporter gene construct with Ca2 + or Ca2 + -activated CaM resulted in the expression of reporter gene. Partial development of chloroplasts in the aurea mutant, which requires the expression of several genes, could also be obtained by microinjection of Ca2 + and CaM into these cells [10]. In some cases, the increased levels of [Ca2 + ]cyt induced the expression of some genes and repressed others [221,222]. In tobacco, expression of one specific CaM isoform is inducible by cold and wind. By targeting aequorin to cytoplasm and nucleus, Van der Luit et al. [39] showed distinct cellular Ca2 + pools respond to wind and cold stimuli in the expression of NpCaM1 gene. Wind and cold stimuli induce [Ca2 + ]n and [Ca2 + ]cyt, respectively, suggesting that different Ca2 + transients employ distinct signal pathways in NpCaM1 gene expression [39]. Sheen [202] has elegantly demonstrated the involvement of CDPKs in stress-induced gene expression. A reporter gene (GFP/LUC) fused to a stress-inducible promoter was used in transient assays to monitor its expression under different stress conditions and in the presence of the constitutively expressed kinase domain of several CDPK isoforms. Cold, salt, dark and ABA induced the expression of the reporter gene driven by the stress-inducible promoter. The reporter gene was also expressed in the absence of stress treatments but in the presence of both Ca2 + and Ca2 + ionophore, suggesting that stress-induced gene expression is mediated by Ca2 + . Protoplasts transformed with a control construct (a reporter gene fused to stress nonresponsive promoter) did not show reporter gene expression in response to stresses or Ca2 + [202]. Since Ca2 + is able to induce the expression of a stress inducible gene, 395 Sheen tested the effect of CDPKs on the expression of stress promoter-reporter construct. The protoplasts were cotransformed with the reporter construct along with the kinase domain of eight CDPK isoforms individually. Of the eight Arabidopsis CDPKs tested, only ATCDPK1 and ATCDPK1a activated the expression of the reporter gene, indicating that specific CDPK isoforms mediate the effects of stresses on gene expression. Cotransformation of the reporter gene construct along with the ATCDPK1a and PP2C, a protein phosphatase, decreased the reporter gene expression significantly whereas cotransformation of ATCDPK1a with PP2C null mutant had no effect on the CDPK-induced reporter gene expression. These results establish an important role for specific CDPKs in stress-induced gene expression. 5. Specificity in decoding Ca2 + signal The fact that Ca2 + is a messenger in transducing a wide range of signals into diverse responses raises an important question — How does Ca2 + achieve specificity in eliciting a response to a given signal? A number of factors are likely to be involved in controlling the specificity. First, competence of an organ, a tissue or a cell type within the tissue to respond to a given stimulus. In vivo imaging of cold-induced changes in [Ca2 + ]cyt indicate that cotyledons and roots of a seedling are highly responsive whereas hypocotyls are relatively insensitive to cold shock [223]. It has also been shown that a given signal may induce a different Ca2 + signature in different cell types [224]. For example, the Ca2 + response in the endodermis and pericycle in the presence of salt and osmotic stress is different from other cell types [224]. Second, temporal and spatial changes of Ca2 + together with the extent of increase (amplitude) are likely to contribute significantly in achieving the specificity in eliciting appropriate physiological responses [225,226]. It is becoming clear that different signals cause distinct spatial and temporal changes in Ca2 + and this Ca2 + signature is likely to be important in achieving the specificity [3,6,225,227,228]. In pollen tubes, changes in cytosolic Ca2 + are limited to the tip with characteristic oscillations [226]. Some signals have been shown to cause wave-like Ca2 + increases in the cells of cotyledons. Using luminescence-imaging technol- 396 A.S.N. Reddy / Plant Science 160 (2001) 381–404 ogy, the spatial and temporal pattern of elevated [Ca2 + ]cyt in response to low temperature has been demonstrated in transgenic plants expressing aequorin [40]. These authors showed that cold-induced signal was transmitted from root to aerial tissues with a lag period of 3 min. Ozone has been shown to cause biphasic [Ca2 + ]cyt transients [50]. Using lanthanum chloride and EGTA, the activity of AOS-induced glutathione synthase was found to require a second [Ca2 + ]cyt transient peak [50]. In parsley cells, a fungal elicitor induces a biphasic Ca2 + signature and it was shown that the sustained concentrations of Ca2 + but not the rapidly induced transient peak are necessary to activate defense-associated responses [62]. The magnitude and kinetics of Ca2 + transients induced by touch, cold and fungal elicitors are also found to be different [1,37,223]. The strength of artificial wind application on transgenic aequorin seedlings correlated with the amount of [Ca2 + ]cyt levels [229]. The spatio-temporal pattern of [Ca2 + ]cyt changes could be unique to each signal [3,6,225]. Localized changes may occur in the cytoplasm or in one compartment of the cell (e.g. signal-induced changes in the cytoplasm but not in the nucleus or vice versa). Heterogeneous nature of signal-induced changes has been well documented in guard cells and in other plants cells [225]. Oscillations and waves of Ca2 + have also been reported in plants. Third, the type of Ca2 + sensors expressed in a cell and their cellular location should also contribute to the specificity. The plethora of Ca2 + sensors and their tissue specific expression and differential response to signals indicate that Ca2 + specificity is in part accomplished by the type of Ca2 + sensors and their target proteins. 6. Future directions During the last decade, significant progress has been made in demonstrating that signals not only elevate [Ca2 + ]cyt but the Ca2 + signature generated by each signal is likely to be different. Based on what is already known, it is clear that plants contain many unique Ca2 + sensing proteins with novel regulatory mechanisms that have evolved to perform plant-specific functions. It is likely that many more novel Ca2 + sensing proteins will be identified, especially as the Arabidopsis genome sequence is completed. So far : 150 proteins that are involved in Ca2 + mediated signal transduction pathways have been identified in Arabidopsis. This number is likely to reach 300 –400 in the next few years, suggesting that up to 2% of the expected number of genes in Arabidopsis encode proteins involved in Ca2 + signaling. Considering the involvement of Ca2 + in so many diverse processes it is not surprising that plants contain such a large number of proteins involved in Ca2 + signaling. Although the specific functions of most of these remain to be elucidated, a large body of indirect evidence indicates the involvement of Ca2 + sensing proteins in many cellular processes such as cytoplasmic streaming, organelle and vesicle transport, microtubule dynamics, cell division, chromosome segregation, cell elongation, tip growth and morphogenesis including some plant-specific processes. Recent studies have produced many surprises and significantly advanced our understanding of Ca2 + signaling in plants and raised many important questions. What are all the proteins involved in maintaining Ca2 + homeostasis and in elevating [Ca2 + ]cyt in response to signals? How the proteins involved in Ca2 + influx and efflux are regulated? How elevation of Ca2 + evokes different responses to different signals? What is the nature of Ca2 + signatures that are produced in response to a given signal? Are these changes confined to a subcellular compartment? What are the targets of numerous CaM-like proteins that have been identified? The next decade should bring answers to these interesting questions. The challenge now is to elucidate the role(s) of numerous Ca2 + sensing proteins and cross-talk among various components of Ca2 + signaling and other messenger mediated signaling pathways. Molecular, cell biological and genetic analysis of various proteins involved in Ca2 + signaling should permit elucidation of functional analysis of these proteins. In planta protein – protein interaction studies using new strategies such as fluorescence resonance energy transfer are necessary to verify and extend the results obtained with in vitro studies [230]. Loss-of-function experiments with individual components of Ca2 + signaling will help in understanding the function(s) of each protein involved in Ca2 + signaling. Because of the large gene families of some Ca2 + sensing proteins and likely functional redundancy or overlap with other members of the family, it will be necessary to create double or triple mutants. New A.S.N. Reddy / Plant Science 160 (2001) 381–404 technologies such as reverse genetics [231] to create knock out mutants coupled with the analysis of cell- and tissue-specific expression of individual members Ca2 + signaling pathway using microarrays [232], and non-destructive visualization of proteins in live cells using fluorescent reporters should help in gaining new insights into the function(s) of proteins involved in Ca2 + signaling and in deciphering signaling networks. Because of the involvement of Ca2 + signaling in many stress responses it is possible to generate plants that are more tolerant to biotic and abiotic stresses by manipulating one or more components of Ca2 + signaling pathway. 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