Review Calcium: silver bullet in signalingarquivo.ufv.br/dbv/pgfvg/BVE684/htms/pdfs_revisao... ·...

Plant Science 160 (2001) 381–404

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 inplant responses to biotic and abiotic stresses. Numerous signals have been shown to induce transient elevation of [Ca2+]cyt inplants. Genetic, biochemical, molecular and cell biological approaches in recent years have resulted in significant progress inidentifying several Ca2+-sensing proteins in plants and in understanding the function of some of these Ca2+-regulated proteinsat the cellular and whole plant level. As more and more Ca2+-sensing proteins are identified it is becoming apparent that plantshave several unique Ca2+-sensing proteins and that the downstream components of Ca2+ signaling in plants have novel featuresand regulatory mechanisms. Although the mechanisms by which Ca2+ regulates diverse biochemical and molecular processes andeventually physiological processes in response to diverse signals are beginning to be understood, recent studies have raised manyinteresting 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 messengersmediated transduction pathways, analysis of their function and regulation, and cross talk among these components — should helpin understanding the inner workings of plant cell responses to diverse signals. New functional genomics approaches such as reversegenetics, microarray analyses coupled with in vivo protein–protein interaction studies and proteomics should not only permitfunctional 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

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1. Introduction

Plant growth and development is controlled byhormonal and environmental signals. Plants, un-like animals, are immobile and therefore havedeveloped mechanisms to sense and respond to the

biotic and abiotic stresses so that they can betteradapt to their environment. How plants sensethese various signals and produce an appropriateresponse has fascinated plant biologists over acentury and has become an area of intense investi-gation in recent years. Research during the lasttwo decades has clearly established that Ca2+ actsas an intracellular messenger in coupling a wide-range of extracellular signals to specific responses.Although Ca2+ is implicated in regulating a num-ber of fundamental cellular processes that areinvolved in cytoplasmic streaming, thigmotropism,gravitropism, cell division, cell elongation, cell dif-ferentiation, cell polarity, photomorphogenesis,plant defense and stress responses, the mechanisms

Abbre6iations: ABA, abscisic acid; AOS, active oxygen species;[Ca2+]cyt, cytosolic Ca2+; CaM, calmodulin; CBP, calmodulin-bind-ing protein; CBD, calmodulin-binding domain; CCaMK, Ca2+/CaM-dependent protein kinase; CDPK, Ca2+-dependent proteinkinase; CRK, CDPK-related protein kinase; CIPK/SIPK, CBL/SOS3-interacting protein kinase; GAD, glutamate decarboxylase;KCBP, kinesin-like calmodulin-binding protein; PDE, phosphodi-esterase.

* Tel.: +1-970-4915773; fax: +1-970-4910649.E-mail address: [email protected] (A.S.N. Reddy).

0168-9452/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.

PII: S 0 1 68 -9452 (00 )00386 -1

A.S.N. Reddy / Plant Science 160 (2001) 381–404382

by which Ca2+ controls these processes are onlybeginning to be understood. Because of the spacelimitations, my intention here is to summarizerecent progress in understanding Ca2+-mediatedsignal transduction pathways with emphasis onthe current status of research, gaps in our knowl-edge and future directions.

2. Signals and cytosolic Ca2+

Improved methods to monitor free [Ca2+]cyt

levels, especially using transgenic plants expressingCa2+ reporter proteins, have greatly helped indemonstrating signal-induced changes in free[Ca2+]cyt level [1–4]. The concentration of Ca2+

in the cytoplasm of plant cells is maintained low inthe nanomolar range (100–200 nM) [4,5]. How-ever, Ca2+ concentration in the cell wall and inorganelles is in the millimolar range (Fig. 1) [6,7].

Despite the existence of a large electrochemicalgradient for Ca2+ entry into the cytoplasm, plantcells maintain their [Ca2+]cyt concentration at lowlevels, which requires active pumping of Ca2+ tothe apoplast or organelles.

A wide-range of signals such as light, hormones,gravity, touch, wind, cold, drought, oxidativestress and fungal elicitors have been shown tocause transient elevation of [Ca2+]cyt (see Table 1).A comprehensive discussion of signal-inducedchanges in [Ca2+]cyt has been published recentlyand will not be covered here [4,7–9]. Currentevidence indicates that 1,4,5-trisphosphate (IP3),cyclic ADP ribose (cADPR) and Ca2+ channelsplay an important role in elevating [Ca2+]cyt (Fig.1). Ca2+ channels have been detected in theplasma membrane, vacuolar membrane, ER,chloroplast and nuclear membranes of plant cells[5]. These channels are classified based on theirvoltage dependence. The electrophysiologicalproperties of all the known Ca2+ channels havebeen reviewed recently [5]. However, genes encod-ing Ca2+ channels in plants have not beenidentified.

In plants, as in animals, phospholipase C-medi-ated hydrolysis of phosphatidylinositol-4,5-bispho-sphate (PIP2) results in the production ofinositol-1,4,5-trisphosphate (IP3) and diacylglyc-erol. There is some indirect evidence for the in-volvement of heterotrimeric G proteins inpromoting PIP2 hydrolysis in plants [10,11]. Phos-phatidylinositol-specific phospholipase C (PI-PLC)activity and genes encoding this enzyme have beencharacterized from plants [12,13]. Plant PI-PLChydrolyzes phosphatidylinositol-4,5-bisphosphateinto IP3 and diacylglycerol with an absolute re-quirement for Ca2+ (1 mM) [12]. It was shownthat one of the phospholipase C genes (AtPLC1)expression is induced by stresses including dehy-dration, salinity and low temperature [12,14]. Re-cently, three PI-PLC isoforms (StPLC1 to -3)have been isolated from guard cell enriched tissueof potato [13]. The expression pattern of theStPLC1 and -2 genes also suggest their involve-ment in drought stress in potato [13]. The soybeanPLC showed phospholipase activity and comple-mented the lethal mutant phenotype of yeast lack-ing PLC activity. Although there is overwhelmingevidence for signal-induced changes in [Ca2+]cyt,the information on the mechanisms by which agiven signal elevates [Ca2+]cyt is still limited. Little

Fig. 1. Schematic diagram illustrating the mechanisms bywhich plant cells elevate [Ca2+]cyt in response to varioussignals and restore Ca2+ concentration to resting level. Ca2+

channels are shown in red, whereas Ca2+ ATPases andantiporters are indicated in yellow. Arrows indicate the direc-tion of Ca2+ flow across the plasma membrane, and into andout of cellular organelles (vacuole, plastids, mitochondria,endoplasmic reticulum and nucleus). The estimated concen-tration of resting levels of Ca2+ in different organelles isindicated [5,6,9,234]. Question marks indicate the lack ofevidence. [Ca2+]cyt, cytosolic Ca2+; PLC, phospholipase C;R, receptor, cADPR, cyclic ADP ribose, PIP2, phosphotidylinositol-4,5-bisphosphate, DG, diacylglycerol, PKC, proteinkinase C, IP3, inositol-1,4,5-trisphosphate; ER, endoplasmicreticulum; Mt, mitochondria; Plast, plastids; PM, plasmamembrane.

A.S.N. Reddy / Plant Science 160 (2001) 381–404 383

Table 1Effect of signals on cytosolic calcium in plants

ReferencesEffect onMethods used to measure free ResponseSignalcytosolic Ca2+ a [Ca2]cyt

b

Abscisic acid , ¡1, 2, 3 Stomatal closure, gene expression [3,20–23]2 a-Amylase secretionGibberellic [24,25]

acid Auxin Cell elongation and cell division1 [36] Gene expression and osmolyte synthesis,1, 2, 3NaCl [27–31]

K+ uptake Gene activation, adaptation to oxygen1Anoxia [32,33]

deprival Touch Thigmomorphogenesis1, 2 [1,34–36] Morphogenesis1, 2 [37–39]Wind

Gravity 1 Gravitropism [26] COR gene expression, proline synthesis,1, 2 [40–42]Cold

changes in membrane lipid profile andcold acclimatization

Drought 1, 2 Gene expression, synthesis of [2,30,43]osmoprotectants, and osmotolerance

Thermotolerance [44,45]Heat Shock 1, 22 OsmoadaptationHypoosmotic [46–48]

stress Red light Photomorphogenesis1 [49] Production of AOSs2 [50]Ozone stress

[51–55]Oxidative 1, 2 Production of AOSs, HR and cell deathstress(H2O2)

, ¡Aluminum Ion imbalance1, 2 [56–59][1,53,60–62]Pathogens 1, 2 Phytoalexin biosynthesis and induction of

HRandelicitors

Nodular formation and root hair curlingNOD factors [63]1

a Cytosolic free calcium levels are measured using injection of fluorescent indicator dyes (1), transgenic plants expressingaequorin (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 involvedin the activation of phospholipase C and regula-tion of Ca2+ channels.

IP3 has been shown to stimulate Ca2+ releasefrom vacuolar store [9,15,16]. In animals, IP3 re-leases Ca2+ primarily from the endoplasmicreticulum (ER) whereas in plants Ca2+ is releasedfrom vacuoles. Ca2+ release from the ER elicitedby IP3 has also been reported in plants [9]. IP3−

and cyclic ADP-ribose (cADPR)-gated channelsthat are found on the ER membrane in animalsare found in the vacuolar and ER membranes inplants [9,17,18]. Recently, nicotinic acid adeninedinucleotide phosphate has been shown to releaseCa2+ exclusively from plant ER [19]. These resultssuggest that multiple Ca2+ mobilization pathways

that are regulated by different agents exist inplants. In animals, diacylglycerol, the secondproduct of PIP2 hydrolysis, is an activator forprotein kinase C. Although there are some reportsindicating the presence of protein kinase C-likeactivity in plants, the gene encoding it has notbeen isolated [64].

Elevation of [Ca2+]cyt in response to signalscould be due to influx of Ca2+ from the apoplastand/or Ca2+ release from intracellular stores (ER,vacuoles, mitochondria, chloroplasts and nucleus).Based on the type of signal or cell type internaland/or external Ca2+ stores could be involved inraising [Ca2+]cyt. The contribution of externalCa2+ and cellular organelles in elevating cytosolicCa2+ in response to various signals is beginning to


be identified. Transgenic plants expressing a Ca2+

reporter targeted to different organelles and theuse of different pharmacological agents that blockor release Ca2+ from internal stores have allowed,in some cases, analysis of changes in organellarCa2+ concentration as well as the contribution ofthese organelles in elevating [Ca2+]cyt [4,62]. Thesestudies indicate that different signals use distinctCa2+ stores in elevating [Ca2+]cyt. Cold-inducedCa2+ increase is inhibited by plasma membranechannel blockers, but is not affected by organellarchannel blockers. However, wind-induced Ca2+

increase is blocked by organellar Ca2+ channelblockers whereas plasma membrane channelblockers did not have any effect [37], indicatingthat the extracellular Ca2+ contributes to cold-in-duced elevation of Ca2+ and internal Ca2+ storescontribute to wind-induced increase in [Ca2+]cyt.By targeting aequorin to cytoplasm and nuclearorganelles, Van der Luit et al. [39] have shownthat distinct cellular Ca2+ pools respond to windand cold stimuli. Sodium chloride-induced[Ca2+]cyt is due to Ca2+ release from the vacuole[1,30]. Transgenic Arabidopsis seedlings in whichaequorin is targeted to the cytoplasmic face of thetonoplast membrane [2] showed release of vacuo-lar Ca2+ in response to mannitol treatment [30].In parsley cells, elicitor-induced sustained increasein [Ca2+]cyt is primarily due to the influx of extra-cellular Ca2+ [62]. Studies with maize suspension-cultures have shown that mitochondrial Ca2+

store contributes to anoxia induced [Ca2+]cyt [33].ABA-induced changes in [Ca2+]cyt have been at-tributed to both Ca2+ release from internal storesand Ca2+ influx from external stores [65,66]. Re-cently, hydrogen peroxide (H2O2) has been shownto activate Ca2+-permeable channels in theplasma membrane of Arabidopsis guard cells [55].Furthermore, ABA induced H2O2 production inguard cells. However, H2O2-induced stomatal clos-ing and activation of Ca2+ channels by H2O2 aredisrupted in an ABA-insensitive mutant, suggest-ing that ABA-induced H2O2 and the H2O2-acti-vated Ca2+ channels are important in elevating[Ca2+]cyt in response to ABA [55].

It is not yet known to what extent the changesin the [Ca2+]cyt levels are reflected in changes infree Ca2+ concentration in the nucleus. Recentstudies show that there is a Ca2+ gradient betweenthe nucleus and cytoplasm indicating the presenceof regulatory mechanisms that control Ca2+

movement into and out of the nucleus [67]. ATPstimulates Ca2+ uptake into nuclei and studiesimplicate CaM involvement in this uptake process[67]. Currently, little is known about the participa-tion of nuclear Ca2+ stores in increasing cytosolicCa2+ and vice versa. Ca2+ uptake studies withisolated plant nuclei have shown that the transportof Ca2+ across nuclear membrane is an ATP-de-pendent process [68], suggesting that changes incytosolic and nuclear Ca2+ may occur indepen-dently. Recently, using tobacco plants expressingCa2+ reporter either in the cytosol or nucleus, ithas been shown that changes in Ca2+ concentra-tion in the nuclear compartment and cytosol arecontrolled independently [69]. These authors havealso shown that the nuclear membrane is notpassively permeable to Ca2+.

The signal-induced Ca2+ elevations are tran-sient and the level of Ca2+ returns to the restinglevel, which requires the removal of Ca2+ fromthe cytosol. This requires Ca2+ transport intoorganelles against a concentration difference of103 to 104 fold. Ca2+ homeostasis is achieved byhigh affinity Ca2+-pumps (Ca2+-ATPases) andlow affinity Ca2+/H+ antiporters (Fig. 1). TheseCa2+ pumps and antiporters also play a crucialrole in raising and restoring [Ca2+]cyt levels inresponse to various stimuli. Several Ca2+-AT-Pases 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]. Thereare at least 12 Ca2+-ATPases in plants of whicheight belong to an ACA-type. In animals, autoin-hibited Ca2+-ATPases, which are regulated byCa2+/CaM, are exclusively in the plasma mem-brane whereas in plants they are primarily local-ized in endomembrane systems [70]. Unlike animalendomembrane Ca2+-ATPases that lack calmod-ulin-binding domain, plant counterparts have acalmodulin-binding domain either at the N- orC-terminus [70–73]. Recently, Ca2+-ATPases thatare regulated by Ca2+/CaM have been found inthe plant plasma membrane also [74,75]. However,the structural organization of plasma membrane-localized Ca2+-ATPases in plants is different fromthat of animal counter parts [74,75]. The calmod-ulin-binding autoinhibitor region in plant plasmamembrane-localized Ca2+-ATPases is located inthe N-terminus whereas it is in the C-terminalregion in animal plasma membrane-localizedCa2+-ATPases, suggesting that plant ATPases


have unique structural organization. The ACA-type pumps are CaM regulated where, in theabsence of activated CaM, the CaM-binding do-main acts as an autoinhibitor. Binding of CaM tothe autoinhibitor region causes activation of theenzyme, suggesting an important role for Ca2+ inregulating these pumps. CaM-regulated autoinhib-ited Ca2+-ATPases are found in the tonoplast, ERand most recently in the plasma membrane [70–72,74,75]. The large number of Ca2+-ATPasesindicates complex mechanisms in regulating Ca2+

homeostasis in plants. Interestingly, the activity ofthe CaM-regulated ER ATPase in Arabidopsis isinhibited by a Ca2+-dependent protein kinase,suggesting that Ca2+ both activates (throughCaM) and inhibits (via CDPK) the activity of ERATPase [76]. The phosphorylation site on the AT-Pase was mapped to Ser45 near the CaM-bindingdomain. Vacuolar localized Ca2+/H+ antiporters(calcium ex6 changer) have been identified fromArabidopsis (CAX1 and CAX2) [77] and mungbean [78]. The Arabidopsis CAX1 gene function-ally complements a yeast mutant defective in itsantiporter activity [77]. CAX1 and CAX2 are highand low efficiency H+/Ca2+ exchangers, respec-tively. CAX transcripts are inducible by extracellu-lar Ca2+ levels, Na+, K+, Ni2+, PEG and Zn2+

but not by plant hormones. The transgenic plantsexpressing the CAX1 gene in sense orientationshowed several abnormalities including stuntedgrowth with poorly developed root system, necro-sis of leaves and apical meristem, hypersensitivityto K+ and Mg2+ ions, and sensitivity to cold thatcould be reversed by Ca2+ supplementation [79].These results indicate that the increased antiportactivity of the CAX1 gene causes severe depletionin free cytosolic Ca2+ levels due to pumping ofCa2+ from cytosol to vacuole. Known Ca2+

channels, pumps and antiporters that are involvedin Ca2+ homeostasis are shown in the Fig. 1.

3. Ca2+ sensors

Transient Ca2+ increase in the cytoplasm inresponse to signals is sensed by an array of Ca2+-sensors (Ca2+-binding proteins) which decodeCa2+ signal. Once Ca2+ sensors decode the ele-vated [Ca2+]cyt, Ca2+ efflux into the cell exteriorand/or sequestration into cellular organelles suchas vacuoles, ER and mitochondria restores its

levels to resting state. A large number of Ca2+

sensors have been characterized in plants, whichcan be grouped into four major classes [80,81].These include (A) calmodulin (CaM), (B) CaM-like and other EF-hand containing Ca2+-bindingproteins, (C) Ca2+-regulated protein kinases, and(D) Ca2+-binding proteins without EF-hand mo-tifs. Members of first three classes of Ca2+ sensorscontain helix-loop-helix motif(s) called EF handsthat bind to Ca2+ with high affinity [82]. How-ever, different Ca2+-binding proteins differ in thenumber of EF hand motifs and their affinity toCa2+ with dissociating constants (Kds) rangingfrom 10−5 to 10−9 M. Binding of Ca2+ to theCa2+ sensor results in a conformational change inthe sensor resulting in modulation of its activity orits ability to interact with other proteins and mod-ulate their function/activity.

3.1. Calmodulin

CaM is a highly conserved, well-characterizedand ubiquitous Ca2+ receptor in eukaryotes[82,83]. It is a small molecular weight acidicprotein of 148 amino acids with four EF-handmotifs that bind to four Ca2+ ions (Fig. 2A). Thecrystal structure of CaM indicates that it has twoglobular domains, each with a pair of EF hands,connected by a central helix [84]. The binding ofCa2+ to CaM results in a conformational changein such a way that the hydrophobic pockets ofCaM are exposed in each globular end which canthen interact with target proteins [85,86]. In plants,there are multiple CaM genes that code for eitheridentical proteins or contain a few conservativechanges [81,83,87,88]. These small changes inamino acid composition of CaM isoforms maycontribute to differential interaction of each CaMisoform with target proteins. There is considerableevidence to indicate that CaM genes are differen-tially expressed in response to different stimuli[81,83]. Such differential regulation is likely to beone of the mechanisms for cells to fine-tune Ca2+

signaling.Although it is preliminary, recent studies on

CaM genes expression in response to differentstimuli indicate that different CaM isoforms areinvolved in mediating a specific signal [81]. Threeof the six Arabidopsis Cam genes (Cam1, -2 and-3) are inducible by touch stimulation [81] indicat-ing the presence of different cis-regulatory ele-


ments in their promoters. The presence of multipleCaM isoforms in plants adds further complexity tothe Ca2+ mediated network and points to theirdifferential sensitivity to elevated [Ca2+]cyt levelsin response to different stress stimuli. In potato,only one of the eight CaM isoforms (PCaM1) isinducible by touch [87]. The striking example for

differential regulation of CaMs comes from thestudies with soybean CaM isoforms. In soybeanthere are five CaM isoforms (SCaM1 to −5).SCaM1, -2 and -3 are highly conserved comparedto other plant CaM isoforms including Arabidop-sis CaM isoforms whereas SCaM4 and -5 aredivergent and showed differences in 32 amino

Fig. 2.


acids with the conserved group [89]. Surprisingly,these divergent CaM isoforms are specifically in-duced by fungal elicitors or pathogen [90]. Theseresults provided evidence for the differential regu-lation of CaM isoforms in plants. Soybean iso-forms show differences in their relative abundancein vivo. The conserved isoforms are relativelyabundant in their expression compared to diver-gent forms. All CaM isoforms activate phosphodi-esterase (PDE) but differ in their activation ofNAD kinase, calcineurin and nitricoxide synthaseindicating Ca2+/CaM specificity between CaMisoforms and target proteins [91]. Differential reg-ulation of other enzymes by soybean divergentand conserved CaM isoforms has also been re-ported [92]. Although SCaM isoforms show simi-lar patterns in blot overlay assays, they differ intheir relative affinity in interacting with CaM-binding proteins [93]. Two divergent CaM iso-forms that are found in Arabidopsis do notinteract with proteins that bind to conserved CaMisoforms [94]. These studies suggest that conservedand divergent CaM isoforms may interact withdifferent target proteins.

Using agonists and antagonists of the Ca2+

signaling pathway, Ca2+ and CaM have beenimplicated in plant defense against pathogens[7,95]. Because the pharmacological agents havenon-specific effects and because the targets ofthese agents are not clearly defined some of these

studies are not conclusive. More recent studieswith transgenic plants overexpressing CaM andCaM isoforms have provided strong evidence forthe involvement of CaM in plant defense re-sponses. Transgenic plants expressing a mutatedCaM with a single amino acid change (K115 to R)in the CaM sequence, which abolishes trimethyla-tion of lysine at 115, showed increased levels ofresistance to a variety of stimuli such as bacterialderived elicitor harpin, mechanical stress, and os-motic stress [96]. Furthermore, transgenic cell linestreated with cellulase or mechanical stress pro-duced increased levels of AOSs compared to con-trol cell lines. Production of AOSs has been shownto initiate a battery of defense responses againstinvading pathogens [53,60,61,97]. The enhancedresistance in transgenic plants is primarily at-tributed to their ability to hyperactivate NADkinase [96].

Constitutive expression of divergent CaM iso-forms SCaM4 and -5 in tobacco has also indicateda role of some CaM isoforms in plant defenseagainst fungi and TMV. Conserved CaMs(SCaM1, -2 and -3) are not inducible by stresswhere as divergent CaM isoforms are normallyexpressed at a low level and are highly inducible inresponse to pathogen attack or elicitors. Trans-genic plants expressing high levels of SCaM4 and-5 showed constitutive expression of salicylic acidrelated 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 fromArabidopsis; 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, ArabidopsisC6 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 xidaseH6 omologue A6 and BetV-4, birch pollen allergen [238]. Asterisks in CBLs indicate the myristoylation motif. Interruptions in theAtRBOHA 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, oneof 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 otatom6 ultidrug r6 esistance protein [119]; BjGLY I, B6 rassica j6 uncea glyoxalase I [118]; NtCB48, N6 icotiana t6 abacum c6 almodulin-b6 indingp6 rotein 48 [106]; MPCBP, m6 aize p6 ollen-specific c6 almodulin-b6 inding p6 rotein [121]. Calmodulin-binding sites that are predictedbased on secondary structure but not verified experimentally are denoted with a question mark. Three other calmodulin-bindingC6 a2+-A6 TPases from plants that have been reported recently [73–75] are not shown in this figure. (C) Putative CaM-bindingmyosins from Arabidopsis and their structural organization. Myosins in the Arabidopsis genome were identified by searching thedatabase with the conserved motor domain of a myosin. The sequences that contained the motor domain of myosin were analyzedby the Simple Modular Architecture Research Tool (SMART) program.


throughout their life cycle and showed enhanceddisease resistance to a wide spectrum of fungalpathogens and tobacco mosaic virus [90]. Thesestudies indicate CaM isoform specificity in theirtarget activation in Ca2+ mediated signal trans-duction processes in plants. Furthermore, recentstudies revealed that the CaM isoforms differ intheir affinity to the same target protein [91,98–100] highlighting the significance of the existenceof multiple CaM isoforms in mediating specificCa2+-mediated signal transduction pathways.

CaM regulates various cellular activities bymodulating the activity or function of a number ofproteins. In most cases, only the activated form ofCaM (Ca2+ bound CaM) interacts with its targetproteins. However, in some cases CaM can inter-act with its target proteins in the absence of Ca2+

[86].

3.1.1. Ca2+-dependent CaM-binding proteinsCaM is multifunctional because of its ability to

interact with and control the activity of a varietyof target proteins. The role of CaM in a given cellor a tissue is determined by the presence of itstarget proteins. In recent years efforts to dissectCa2+/CaM mediated signaling pathways in plantshave focused on identification and characteriza-tion of CaM-binding proteins (CBPs) in plants.The amino acid sequences of the CBD in differentCaM target proteins is not conserved [86]. How-ever, CaM-binding motifs from different CaM-binding proteins form characteristic basicamphipathic a-helices with several positiveresidues on one side and hydrophobic residues onthe other side [86].

Using biochemical assays and protein-proteininteraction-based screening of expression librarieswith labeled CaM :24 proteins that interact withCaM in a Ca2+-dependent manner have beenidentified from plants (Fig. 2B,C). These includeNAD 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-likeprotein [109–111], Ca2+-ATPase [71,112], nuclearNTPases [113], ion transporters [94,114–117], gly-oxalase I [118], multidrug resistant protein [119],SAUR, an auxin-induced gene [120], pollen spe-cific CBP [121] and some other proteins of un-known function [122].

The identity of the known CBPs indicate thatdiverse proteins implicated in a wide variety ofprocesses including ion transport, gene regulation,cytoskeletal organization, cell division, disease re-sistance and stress tolerance interact with CaM(see Fig. 2B,C and Fig. 3). Interestingly, many ofthese CBPs have no homologs in animals. Some ofthese (e.g. glutamate decarboxylase, GAD) havehomologues in animals but lack CaM bindingmotif, suggesting that they are regulated by CaMonly in plants. In some cases plants and animalshave similar proteins but the location of the CaMbinding domain is different between plant andanimal proteins (e.g. Ca2+ ATPase).

Although the catalytic core in GAD, whichcatalyzes the conversion of glutamate into g-aminobutyric acid (GABA) and CO2 is conserved acrossbacteria, plants and animals, the Ca2+/CaM regu-lation of GAD is unique to plants [123]. GAD hasbeen cloned and characterized from a number ofplant species. Although the CBD is not conserved,all plant GADs isolated so far posses a CBD andare regulated by Ca2+/CaM [123]. In Arabidopsisthere are two GAD isoforms (GAD1 and GAD2)and the CBD of these two isoforms are different,raising the possibility for functional diversity andregulation by Ca2+/CaM [124]. Plants produceincreased levels of GABA in response to mechani-cal and cold stresses [125]. These environmentalstresses also elevate the [Ca2+]cyt levels raising thepossibility that elevated Ca2+ activates GAD,which in turn increases GABA levels. Transgenictobacco plants expressing full-length GAD showednormal wild type phenotype with moderately in-creased GABA and reduced Glu. However, thetransgenic plants expressing GAD minus CBDshowed severe anatomical abnormalities in cellelongation in stem cortex and parenchyma tissues.This phenotype is shown to correlate with accu-mulation of extremely high levels of GABA andlow levels of Glu [126].

The NAD kinase, which catalyses the conver-sion of NAD to NADP [101,127], is vital to livingorganisms especially when energy is in demandunder stress conditions. Recently, it has been re-ported that NAD kinase plays a role in oxidativeburst and in the formation of active oxygen spe-cies (AOSs) [96], which are known for their in-volvement in plant defense against pathogens.

Several CBPs that show similarities to ion chan-nels have been isolated from barley [114], Ara-


bidopsis [94,115] and tobacco [116,117]. Theseproteins showed sequence similarity to cyclicnucleotide gated channels from animals andinward rectifying K+ channels from plants.These proteins have been shown to reside in theplasma membrane. Constitutive over expres-sion of one of these proteins, NtCBP4, in sense

and antisense orientation in tobacco exhibited anormal phenotype under normal growth condi-tions. However, the NtCBP4 sense transgenicplants showed improved tolerance to Ni2+ andhypersensitivity to Pb2+ [116], suggesting a rolefor this CaM-binding channels in metal toler-ance.

Fig. 3. Ca2+ sensing proteins and their functions in plants. Four major groups of Ca2+ sensors (indicated in four boxes) havebeen described in plants. These include (A) Ca2+-dependent protein kinases, (B) CaM, (C) other EF-hand motif-containingCa2+-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]; AtTCH2Arabidopsis 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 , -26and -36 proteins [157]; AtCBL4, Arabidopsis CBL4 or S6 alt-O6 verly-S6 ensitive3 (SOS3) protein [31]; AtCP1, salt-induced ArabidopsisC6 alcium-Binding P6 rotein [173]; OsEFA27, rice EF hand protein responsive to A6 bscisic acid [170]; PvHRA32, bean H6 ypersensitiveR6 eaction A6 ssociated [178]; CCD-1, C6 -terminal c6 entrin-like d6 omain [235]; AtRBOHA, Arabidopsis R6 espiratory B6 urst O6 xidaseH6 omologue A6 [183]; BetV-4, birch pollen allergen [238]; Myosins [151,152]; s6 mall a6 uxin u6 p R6 NA [120]; EF-1 alpha, elongationfactor-1 a [143]; CCaMK, c6 alcium and calcium/calm6 odulin-dependent protein k6 inase [107]; CaMK calcium/calmodulin-dependentprotein kinase [104,203]; CBP1, c6 almodulin-binding p6 rotein 1 [122]; CBP5, c6 almodulin-b6 inding protein 5 [122]; MPCBP, m6 aizep6 ollen-specific c6 almodulin-b6 inding p6 rotein [121]; StCBP S6 olanum t6 uberosum c6 almodulin-b6 inding p6 rotein (Reddy, unpublishedresults); 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-bindingp6 rotein.


A novel CaM-binding microtubule motorprotein (KCBP, kinesin-like calmodulin-bindingprotein) was isolated from Arabidopsis and otherplants [109–111]. This motor is unique amongkinesins and kinesin-like proteins in having aCaM-binding domain adjacent to the motor do-main at the C-terminus and a myosin tail homol-ogy region in the N-terminal tail [128]. KCBPbinds CaM in a Ca2+ dependent manner at phys-iological Ca2+ concentration. KCBP binds bovineCaM and three CaM isoforms (CaM-2, -4 and -6)of Arabidopsis. However, CaM-2 showed 2-foldhigher affinity to KCBP as compared with otherisoforms [99]. Although, a homologue of KCBPhas not been found in S. cere6isiae, C. elegans andDrosophila whose genomes have been sequenced,recently a CaM-binding C-terminal kinesin (ki-nesin C) was cloned from sea urchin [129]. TheCBD of kinesin C shared 35% sequence identitywith the CaM binding domain in KCBP. How-ever, kinesin C differs considerably from KCBPand does not show any sequence similarity in thestalk and tail regions. The amino-terminal tail andstalk regions of KCBPs from different plant sys-tems are highly conserved and contain myosin tailhomology (MyTH4) and talin-like regions that arenot present in kinesin C [130]. Further analysis ofCaM-binding kinesin-like proteins from a numberof phylogenetically divergent plants and animalsincluding the primitive eukaryotes is needed tounderstand their origin and evolution.

Motility studies with the KCBP have shownthat it is a minus-end directed microtubule motor[131]. Binding of activated CaM to KCBP inhibitsits interaction with microtubules [131–133]. TheCaM effect on KCBP suggests that activated CaMcan act as a molecular switch to down-regulate theactivity of KCBP. Among motor proteins, heavychains of unconventional myosins also bind CaMwhere the inhibition of the enzymatic activity andmotility by Ca2+ is similar to the effect of Ca2+

on a CaM binding KCBP, although the mecha-nism of inhibition is very different between theseproteins [134]. Based on these studies with KCBPit is reasonable to speculate that spatial and tem-poral changes in free [Ca2+]cyt levels in response tosignals are likely to regulate KCBP activity in thecell. Current evidence indicates the involvement ofKCBP in trichome morphogenesis and cell divi-sion [135–137,240].

Glyoxalase I, which catalyses the conversion oftoxic methylglyoxal to a nontoxic metabolite, ex-pression is induced by NaCl, mannitol or abscisicacid [138]. Glyoxylase I from Brassica juncea(BjGly I) binds CaM-Sepharose and its activity isstimulated by Ca2+/CaM [139]. Leaf discs fromtransgenic plants expressing BjGly I showed toler-ance to methylglyoxal and salt. In contrast, thecontrol antisense and wild type leaf discs showedloss of chlorophyll and did not survive thesestresses, suggesting that BjGly I plays a role inconferring tolerance to salt stress in plants.

Two nuclear proteins, a chromatin associatednucleotide triphosphatase (NTPase) [140] and aDNA binding protein (TGA3) that binds to CaM-3 promoter [108], are also regulated by CaM. Thebinding of CaM to TGA3 enhances its bindingwith the promoter. In plants, Ca2+/CaM is in-volved in stabilizing cortical microtubules at lowCa2+ concentrations and destabilizing the same athigher Ca2+ concentrations [141,142]. Two CBPsthat show different sensitivities to Ca2+ are impli-cated in evoking these two opposing effects ofCaM on cortical microtubules. Elongation factor-1a, a CaM-binding microtubule associatedprotein, stabilizes microtubules and Ca2+/CaMhas been shown to inhibit elongation factor-1a-promoted microtubule stabilization [143]. Anauxin-induced gene product has been shown tobind CaM, suggesting the involvement of CaM inauxin action [120]. CBPs that are induced by heatshock have been isolated from tobacco [106]. Oneof these CBPs (NtCBP48) contains a centrallylocated putative transmembrane domain and anuclear localization sequence motif [106]. Twomaize proteins (CBP-1 and CBP-5) and a multi-drug resistant protein also bind CaM in a Ca2+-dependent manner [119,122]. However, thefunctions of these are not known. A pollen specificCaM-binding protein from maize, MPCBP, [121]and CBP from developing tubers (StCBP) (Reddy,unpublished results) are also novel CBPs and haveno homologs in non-plant systems. Binding ofsuperoxide dismutase to CaM-Sepharose columnsuggests that it might be regulated by CaM [144].

3.1.2. Ca2+-independent CaM-binding proteinsAlthough CaM, in most cases, binds to its

target proteins in the presence of Ca2+, someCaM-binding proteins interact with CaM in theabsence of Ca2+ [86]. In animals, myosins, a


superfamily of actin-based motors that perform abroad array of cellular functions, bind CaM in theabsence of Ca2+ [145]. The CaM-binding domain(also called ‘IQ motif’) in these myosins has aconsensus sequence ‘IQXXXRGXXXR’ (I,isoleucine, Q, glutamine, R, arginine, G, glycine)which forms an a-helix [145]. Although there iscompelling indirect evidence for the role of actinand actin-based motors in various transport pro-cesses in plants, little is known about plantmyosins and their regulation [134]. Myosins con-taining IQ domains, which are typically CaM-sen-sitive, have been identified in plants but theirinteraction with, or regulation by CaM has not yetbeen demonstrated in these proteins [134]. Thededuced amino acid sequence of plant myosins,like other known myosins, contain ‘IQ’ motifs inthe neck region [146,147]. Phylogenetic analysesusing the conserved motor domain groupedknown myosins into 15 distinct classes [134]. Ofthese, published plant myosins fall into two sepa-rate groups — myosin VIII (e.g. Arabidopsismyosins ATM1 and ATM2) and myosin XI (e.g.Arabidopsis myosins MYA1 and MYA2). Thesetwo subgroups contain only plant myosins, sug-gesting that plants may have a unique set ofmyosins. By comparing the sequences in the Ara-bidopsis genome database with the motor domainof myosins, I have identified 17 myosin-likeproteins in 60% of the sequenced genome. AllArabidopsis myosin-like proteins that are iden-tified so far possess two to six putative CaM-bind-ing ‘IQ’ motifs (Fig. 2C). Although the binding ofCaM to any of these proteins has not been demon-strated experimentally, the presence of these mo-tifs suggests that myosins in plants are likely to beregulated by CaM in response to changes in intra-cellular Ca2+.

A number of reports have shown that the cyto-plasmic streaming in plant cells is regulated by the[Ca2+]cyt concentration [148]. Elevated levels ofCa2+ inhibits cytoplasmic streaming and themovement of organelles [149,150]. However, spe-cific motors involved in these processes remain tobe identified. The mechanisms by which Ca2+

regulates the activity of these motors is just begin-ning to emerge. Both CaM and CDPKs are likelyto mediate Ca2+ effects on cytoplasmic streamingand organelle transport. Recently, Yokota et al.[151,152] purified two myosins from plants anddemonstrated that CaM associates with the

purified myosin and regulates its motor activity.Ca2+ inhibits the myosin motor activity as well asmyosin-activated ATPase activity in plants. Theinhibition of myosin activity in the presence ofCa2+ appears to be due to dissociation of CaMfrom the heavy chain since Ca2+ inhibition ofmotor activity can be restored by exogenous addi-tion of CaM. Some studies suggest that, in somecases, Ca2+ can effect myosins indirectly throughanother mechanism [153]. There is some indirectevidence in support of a role of phosphorylationcatalyzed by CDPK in regulating myosin activity[154,155]. A CDPK, which has been shown tobind actin filaments, phosphorylates a putativemyosin light chain in Chara [155,156]. However,the effect of such a phosphorylation is not known.

3.2. CaM-like and other EF-hand containingproteins

In addition to CaM, recent studies indicate thepresence of numerous CaM-like proteins in plants(Fig. 2A). However, the function of these proteinsin Ca2+ signaling pathway(s) is not fully charac-terized as compared to that of CaM. These CaM-like proteins differ from the CaM in containingmore than 148 amino acids, and one to six EF-hand motifs with limited homology to CaM [83].Hence, it is likely that these proteins are function-ally distinct from CaM and are involved in con-trolling different Ca2+-mediated cellularfunctions.

Recently, a new family of Ca2+-bindingproteins, called calcineurin B-like (CBL) proteins,has been identified [31,157,158]. Studies with salt-overly-sensitive (SOS) mutants of Arabidopsis in-dicate a key role for Ca2+ in salt stress signaling[159–161]. These mutants are hypersensitive toNa+ and Li+ and are unable to grow on low-K+

culture medium. These abnormal growth patternsin the presence of NaCl could be mitigated by theaddition of increased levels of Ca2+ in the samemedium. The SOS3 gene encodes a Ca2+ sensorprotein similar to calcineurin B, a regulatory sub-unit of Ca2+-dependent protein phosphatase andneural Ca2+ sensors (CNS) of animals and raisesthe possibility that SOS3 gene product might con-trol the K+/Na+ transport system via a Ca2+-reg-ulated pathway [31]. In Arabidopsis there are atleast six CBL genes encoding highly similar butfunctionally distinct Ca2+-binding proteins [157].


Drought, cold and wound stress signals induceAtCBL1 gene transcripts, whereas AtCBL2 andAtCBL3 are constitutively expressed [157]. Fur-ther, C6 BL-i6 nteracting p6 rotein k6 inases (CIPK1 to4) belong to the serine/threonine class of kinasesand show high homology to protein kinases SNF1and AMPK from yeast and mammalian systems,respectively. SOS3 interacts with SOS2, which isalso a member of CIPKs (also called SIPKs,S6 OS3-i6 nteracting p6 rotein k6 inases), and seven otherCIPKs [162]. CIPKs interact with CBLs, but notwith CaMs, in a Ca2+-dependent manner [158].The activity of CIPK/SIPK is activated by SOS2(CBL4) and other CBLs in a Ca2+-dependentmanner, suggesting that CBL/SIPK complex islikely to regulate Na+ and K+ homeostasisthrough phosphorylation. CIPKs/SIPKs representa novel family of Ca2+-regulated protein kinasesin plants. The interaction of calcineurin B-like(CBL) proteins with protein kinases is very sur-prising since the CBL proteins are known to acti-vate a protein phosphatase in animals. In additionto Ca2+-regulated protein kinase, there is someevidence for the role of a Ca2+-regulated phos-phatase also in salt tolerance. Ectopic expressionof constitutively active yeast calcineurin has beenshown to confer salt tolerance in tobacco[163,164].

Certain CaM-like proteins contain a 34 aminoacid Caax-box motif (prenylation domain) at theirC-termini (CTIL in PhCaM53) [165] or (CVIL inOsCaM63) [166]. Transient expression of GFPfused with the full-length CaM53 or mutatedCaM53 (without CaaX-box motif) in tobacco andpetunia protoplasts showed that the full-lengthprotein localizes to plasma membrane whereasmutant CaM53 localizes to the nucleus [165,167],suggesting an important role for the prenylationdomain in CaM53 localization. Ectopically ex-pressed CaM53 with a prenylation domain in to-bacco showed stunted growth and a necroticphenotype. However, ectopic expression of neithernon-prenylated form nor Caax-box motif aloneshowed such altered morphogenic alterations indi-cating the importance of the prenylation domain[165].

Arabidopsis, in addition to six CaMs, has otherCaM-like genes including the TCH genes that areinduced in response to various mechanical, chemi-cal and environmental stimuli (Fig. 2A) [168].TCH3 is 324 amino acids long and contains six EF

hand motifs [169]. A cDNA sequence encoding a27 kDa CaM-like protein (EFA27) has been iso-lated from ABA-treated rice seedlings [170]. Itcontains a single EF hand motif and the expres-sion of EFA27 is induced in response to salt anddehydration stress, and to ABA signal. In Ara-bidopsis there are several EFA27 gene homologues[170], suggesting the existence of similar proteinsin phylogenetically distant species. EF hand motifcontaining protein phosphatases were also iden-tified 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 genetranscripts are also highly inducible by NaCl treat-ment but not by ABA treatment indicating thespecificity of this unique Ca2+-binding protein inresponding to stress factors.

Several studies implicate the involvement ofCa2+signal in plant defense responses such asphytoalexin biosynthesis, induction of defense-re-lated genes and hypersensitive cell death[7,174,175]. Ca2+ is implicated in mediating sys-temin, a peptide involved wound-induced activa-tion of defense genes in tomato [176]. Further,Flego et al. [177] showed a correlation betweenincreased Ca2+ concentration in plants and in-creased resistance to bacterial pathogen Erwiniacaraoto6ora. P6Hra32, a gene that is highly ex-pressed during hypersensitive reaction in bean tis-sue challenged with Pseudomonas syringae, hasbeen shown to encode a small Ca2+-bindingprotein (161 aa) with four EF motifs [178]. TheAOSs stimulate rapid influx of Ca2+ into the siteof infection and initiate the hypersensitive re-sponse in order to develop resistance against in-vading pathogens [53,60]. The plasma membranelocated Ca2+ channel (LEAC, l6 arge conductancee6 licitor-a6 ctivated ion c6 hannel) is likely to be in-volved in elevating [Ca2+]cyt in response to AOSs[61]. During the interaction of Arabidopsis and P.syringae, the resistance gene product, RPM1, func-tions immediately and elevates the Ca2+ levels[179,180]. Similar results were obtained from theC. ful6um/tomato interaction [181]. Furthermore,it was shown that the resistance gene activates aCDPK [182]. Identification and characterization ofhuman gp91phox (phox for phagocyte oxidase) ho-mologues from Arabidopsis and rice (RbohA,r6 espiratory b6 urst o6 xidase h6 omologue A6 ), providedevidence for the downstream target for the ele-


vated levels of Ca2+ in oxidative burst [183]. TheRbohA shows high similarity to human gp91phox, aplasma membrane bound neutrophil phagocyteoxidase that is involved in the generation of super-oxide radicles via its NADPH oxidase activity. Inaddition to the gp91phox region, the plant gp91phox

(RbohA) also contains two EF hand motifs whichare not present in human gp91phox suggesting thatCa2+ modulates the formation of superoxide radi-cles through RbohA during oxidative burst inplants [183].

Centrin (also known as caltractin) is a small(:20 kDa) acidic protein with four Ca2+-bindingEF-hand motifs [184]. Genes encoding centrinswere isolated from the salt marsh plant, Atriplexnummularia [185], Arabidopsis [186] and tobaccoBY-2 cells [187]. In plants, centrins are 167–177amino acids long. The Arabidopsis centrin wasisolated as an early-induced gene in response tobacterial inoculation. The expression of this cen-trin is induced when the plants are inoculated withavirulent strains of bacteria but not with virulentstrains, suggesting a role for this centrin in plantdefense [186]. Based on the localization of plantcentrins it was suggested that centrins are involvedin cell plate formation [187] and Ca2+-regualtedtransport through plasmodesmata [188].

3.3. Ca2+-regulated protein kinases

By manipulating cellular Ca2+ levels with phar-macological agents, Ca2+-regulated protein phos-phorylation has been implicated in a variety ofresponses including host–pathogen interactions[182,189], cold stress [190], nuclear migration dur-ing fungal infection [97], gravitropism [80], light-regulated gene expression [191], thigmotropism[192] and hypoosmatic shock [48,193]. Studies dur-ing the last decade indicate that there are at leastfive types of Ca2+-regulated protein kinases inplants: (i) a C6 a2+-d6 ependent and CaM-indepen-dent p6 rotein k6 inases (CDPKs); (ii) C6 DPK-r6 elatedk6 inases (CRKs); (iii) CaM-dependent proteink6 inases (CaMKs); (iv) C6 a2+/CaM-dependentprotein k6 inases (CCaMK); and (v) S6 OS3/C6 BL-i6 nteracting p6 rotein k6 inases (SIPKs/CIPKs) (Fig.2B).

CDPKs, a new family of protein kinases, aremost abundant and ubiquitous in plants [194].Apart from plants, CDPKs are found in proto-zoans and algae. Ca2+ directly binds to the CDPK

and stimulates the kinase activity whereas CaMdoes not have any significant effect on the kinaseactivity [195]. CDPKs have a unique structuralorganization in which a protein kinase catalyticdomain is followed by CaM-like region with fourCa2+ binding motifs (Fig. 2B). The kinase domainof CDPK shows significant homology with themammalian Ca2+/CaM-dependent protein kinaseII (CaM K II) catalytic domain. The region thatjoins the kinase domain to the CaM-like region(junction region) corresponds to the autoin-hibitory/CaM-binding region of CaM K II andprevents kinase activity in the absence of Ca2+

[196]. The autoinhibitory domain located in thejunction region of CDPKs inhibits the kinase ac-tivity in the absence of Ca2+. Ca2+ binding toCDPK effects confirmation of the kinase and re-lieves the inhibition caused by the autoinhibitoryregion [194]. CDPKs are found in different cellularlocations. Several CDPKs have putative myristoy-lation sites indicating that myristoylation ofCDPKs may regulate the association of CDPKswith membranes [194].

The transcripts of two Arabidopsis CDPK genes(AtCDPK1 and AtCDPK2) are highly inducible bydrought and high salt but not by low temperatureor heat stress suggesting the specificity of CDPK’sinduction in response to different stress factors[197]. A CDPK from Vigna radiata is highly in-ducible by wounding, CaCl2, IAA and NaCl treat-ments [198]. There are over 40 CDPKs in theArabidopsis genome [194] (http://plantsp.sdsc.edu/cdpk/). CDPKs are classified into several groupsbased on their sequence domain organization(myristolyation, PEST and the number of EFhand motifs). Furthermore, CDPKs differ in theiraffinity for Ca2+. For example, AtCDPK1 differsfrom AtCDPK2 in its Ca2+ stimulated activityalthough both of them possess four EF handmotifs. Recent studies indicate that, besides Ca2+,lipids are involved in the regulation of CDPKactivity [199,200]. A carrot CaM-like domainprotein kinase, DcCPK1, resembles animal PKCin its activation by Ca2+ and certain phospho-lipids suggesting that lipids regulate the activity ofsome CDPKs and perform specific biological func-tions in plants [201]. Ion channels, enzymes in-volved in metabolism, cytoskeletal proteins, andDNA binding proteins have been identified asCDPKs substrates, suggesting their role in diversecellular processes ranging from ion transport to


gene expression [194]. Sheen [202] has shown thatArabidopsis AtCDPK1 and AtCDPK1a are in-volved in regulating the expression of stress-in-ducible genes. Furthermore, phosphatasescounteract these responses suggesting that involve-ment of Ca2+-regulated phosphorylation is neces-sary for stress-induced gene expression (seeSection 3.1.1 on Ca2+ and gene expression).

CRKs are similar to CDPKs except that theCaM-like region is poorly conserved with degener-ate EF-hands. There are at least seven CRKs inArabidopsis. However, regulation and function ofthese kinases are not known [194]. A cDNA withsignificant sequence similarity to mammalian CaMK II has been isolated from apple [203] and maize[104] (Fig. 2B). However, the biochemical proper-ties 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 presenceof an N-terminal catalytic domain, a centrallylocated CaM-binding domain and a C-terminalvisinin-like domain containing only three EFhands (Fig. 2B). Biochemical studies of CCaMKestablished that Ca2+/CaM stimulates CCaMKactivity. In the absence of CaM, Ca2+ promotesautophosphorylation of CCaMK. The phosphory-lated form of CCaMK possesses more kinase ac-tivity than the non-phosphorylated form [204].CCaMK phosphorylates elongation factor-1a[205]. However, the physiological significance ofthis phosphorylation is not yet known. Theaffinity of CCaMK to CaM is regulated by Ca2+-stimulated autophosphorylation [206].

3.4. Ca2+-binding proteins without EF-handmotifs

There are several proteins that bind Ca2+ butdo not contain EF-hand motifs (Fig. 3). Theseinclude annexins, calreticulin and p6 istil-expressedC6 a2+-binding p6 rotein (PCP). Annexins are a fam-ily of proteins in plants and animals that bindphospholipid in a Ca2+-dependent manner andcontain four to eight repeats of :70 amino acids[207]. Although the exact function of annexin isnot known, plant annexins are implicated in secre-tory processes and some have ATPase, peroxidaseor F-actin binding activities [208]. Calreticulin is aCa2+ sequestering protein in the ER and functionsas a chaperone [209]. In addition, it is implicated

in Ca2+ homeostasis and other functions [210]. A19 kDa novel Ca2+-binding protein (PCP) that isexpressed in anthers and pistil was isolated re-cently [211]. PCP is a high capacity (binds 20 molof Ca2+ per mol of PCP), low affinity Ca2+-bind-ing protein. The pattern of PCP expression sug-gests a role for it in pollen-pistil interactionsand/or pollen development. A cysteine class ofCa2+-dependent protease (CDP) has been purifiedfrom Arabidopsis root cultures [212]. Its activity isspecifically dependent on Ca2+ but not on otherdivalent cations. Structural organization of CDP isnot known since the gene encoding the CDP hasnot been isolated.

4. Ca2+ and gene expression

Although there is a great deal of information onthe involvement of Ca2+ in regulating variousphysiological processes [213,214], the role of Ca2+

in regulating gene expression in plants is forth-coming only recently. Manipulation of [Ca2+]cyt

by various means is shown to affect the expressionof specific genes in plants. Mannitol-induced ex-pression of RAB and AtP5CS1 genes is blocked inthe presence Ca2+ channel blockers like verapamilor lanthanum or the Ca2+ chelator EGTA[30,215]. The expression of these genes in treatedcultures and plants is less than that of untreatedcounterparts, indicating a role of Ca2+ in droughttolerance. Increased external Ca2+ or heat shockrapidly induce the expression of touch-inducedCaM-related genes (TCH2, TCH3 and TCH4)whereas TCH1 gene, which codes for CaM, is notsignificantly induced [216]. Heat shock, in thepresence of EGTA, a Ca2+ chelator, did not showinduction of TCH genes. This EGTA effect isreversed by Ca2+ replenishment. Based on theseresults it was suggested that heat shock elevates[Ca2+]cyt levels which in turn regulates the expres-sion of TCH2, -3 and -4 genes. Ca2+ effect ontouch-induced CaM-related genes is specific sincemagnesium, another divalent ion, did not have anyeffect. Furthermore, increased Ca2+ did not effectthe expression of a heat shock induced gene. De-pletion of Ca2+ by Ca2+ chelator blocked ethyl-ene-induced chitinase synthesis whereas artificialelevation of cytosolic Ca2+ with Ca2+ ionophore(ionomycin) or an inhibitor of microsomal Ca2+-ATPase induced chitinase synthesis in the absence


of ethylene [217]. Accumulation of cold-inducedmRNAs in alfalfa was also partially blocked bylanthanum, a Ca2+ channel blocker and a CaMinhibitor (W7) completely blocked the expressionof cold-regulated genes [218]. Lam et al., [219]have shown that light induced chlorophyll a/bbinding (cab) transcripts could be induced in thedark by increasing the intracellular level of Ca2+

using ionomycin. Compelling evidence for the roleCa2+ and CaM in the regulation of gene expres-sion came from microinjection studies with phy-tochrome mutant (aurea) [220]. In this mutant,light-regulated genes are not expressed. Using areporter gene fused to light-regulated promoter, itwas shown that microinjection of reporter geneconstruct with Ca2+ or Ca2+-activated CaM re-sulted in the expression of reporter gene. Partialdevelopment 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, theincreased levels of [Ca2+]cyt induced the expressionof some genes and repressed others [221,222]. Intobacco, expression of one specific CaM isoform isinducible by cold and wind. By targeting aequorinto cytoplasm and nucleus, Van der Luit et al. [39]showed distinct cellular Ca2+ pools respond towind and cold stimuli in the expression of Np-CaM1 gene. Wind and cold stimuli induce [Ca2+]nand [Ca2+]cyt, respectively, suggesting that differ-ent Ca2+ transients employ distinct signal path-ways in NpCaM1 gene expression [39].

Sheen [202] has elegantly demonstrated the in-volvement of CDPKs in stress-induced gene ex-pression. A reporter gene (GFP/LUC) fused to astress-inducible promoter was used in transientassays to monitor its expression under differentstress conditions and in the presence of the consti-tutively expressed kinase domain of several CDPKisoforms. Cold, salt, dark and ABA induced theexpression of the reporter gene driven by thestress-inducible promoter. The reporter gene wasalso expressed in the absence of stress treatmentsbut in the presence of both Ca2+ and Ca2+

ionophore, suggesting that stress-induced gene ex-pression is mediated by Ca2+. Protoplasts trans-formed with a control construct (a reporter genefused to stress nonresponsive promoter) did notshow reporter gene expression in response tostresses or Ca2+ [202]. Since Ca2+ is able toinduce the expression of a stress inducible gene,

Sheen tested the effect of CDPKs on the expres-sion of stress promoter-reporter construct. Theprotoplasts were cotransformed with the reporterconstruct along with the kinase domain of eightCDPK isoforms individually. Of the eight Ara-bidopsis CDPKs tested, only ATCDPK1 andATCDPK1a activated the expression of the re-porter gene, indicating that specific CDPK iso-forms mediate the effects of stresses on geneexpression. Cotransformation of the reporter geneconstruct along with the ATCDPK1a and PP2C, aprotein phosphatase, decreased the reporter geneexpression significantly whereas cotransformationof ATCDPK1a with PP2C null mutant had noeffect on the CDPK-induced reporter gene expres-sion. These results establish an important role forspecific CDPKs in stress-induced gene expression.

5. Specificity in decoding Ca2+ signal

The fact that Ca2+ is a messenger in transduc-ing a wide range of signals into diverse responsesraises an important question — How does Ca2+

achieve specificity in eliciting a response to a givensignal? A number of factors are likely to be in-volved in controlling the specificity. First, compe-tence of an organ, a tissue or a cell type within thetissue to respond to a given stimulus. In vivoimaging of cold-induced changes in [Ca2+]cyt indi-cate that cotyledons and roots of a seedling arehighly responsive whereas hypocotyls are relativelyinsensitive to cold shock [223]. It has also beenshown that a given signal may induce a differentCa2+ signature in different cell types [224]. Forexample, the Ca2+ response in the endodermisand pericycle in the presence of salt and osmoticstress is different from other cell types [224]. Sec-ond, temporal and spatial changes of Ca2+ to-gether with the extent of increase (amplitude) arelikely to contribute significantly in achieving thespecificity in eliciting appropriate physiological re-sponses [225,226]. It is becoming clear that differ-ent signals cause distinct spatial and temporalchanges in Ca2+ and this Ca2+ signature is likelyto be important in achieving the specificity[3,6,225,227,228]. In pollen tubes, changes in cyto-solic Ca2+ are limited to the tip with characteristicoscillations [226]. Some signals have been shownto cause wave-like Ca2+ increases in the cells ofcotyledons. Using luminescence-imaging technol-


ogy, the spatial and temporal pattern of elevated[Ca2+]cyt in response to low temperature has beendemonstrated in transgenic plants expressing ae-quorin [40]. These authors showed that cold-in-duced signal was transmitted from root to aerialtissues with a lag period of 3 min. Ozone has beenshown to cause biphasic [Ca2+]cyt transients [50].Using lanthanum chloride and EGTA, the activityof AOS-induced glutathione synthase was foundto require a second [Ca2+]cyt transient peak [50].In parsley cells, a fungal elicitor induces a biphasicCa2+ signature and it was shown that the sus-tained concentrations of Ca2+ but not the rapidlyinduced transient peak are necessary to activatedefense-associated responses [62]. The magnitudeand kinetics of Ca2+ transients induced by touch,cold and fungal elicitors are also found to bedifferent [1,37,223]. The strength of artificial windapplication on transgenic aequorin seedlings corre-lated with the amount of [Ca2+]cyt levels [229]. Thespatio-temporal pattern of [Ca2+]cyt changes couldbe unique to each signal [3,6,225]. Localizedchanges may occur in the cytoplasm or in onecompartment of the cell (e.g. signal-inducedchanges in the cytoplasm but not in the nucleus orvice versa). Heterogeneous nature of signal-in-duced changes has been well documented in guardcells and in other plants cells [225]. Oscillationsand waves of Ca2+ have also been reported inplants. Third, the type of Ca2+ sensors expressedin a cell and their cellular location should alsocontribute to the specificity. The plethora of Ca2+

sensors and their tissue specific expression anddifferential response to signals indicate that Ca2+

specificity is in part accomplished by the type ofCa2+ sensors and their target proteins.

6. Future directions

During the last decade, significant progress hasbeen made in demonstrating that signals not onlyelevate [Ca2+]cyt but the Ca2+ signature generatedby each signal is likely to be different. Based onwhat is already known, it is clear that plantscontain many unique Ca2+ sensing proteins withnovel regulatory mechanisms that have evolved toperform plant-specific functions. It is likely thatmany more novel Ca2+ sensing proteins will beidentified, especially as the Arabidopsis genomesequence is completed. So far :150 proteins that

are involved in Ca2+ mediated signal transductionpathways have been identified in Arabidopsis. Thisnumber is likely to reach 300–400 in the next fewyears, suggesting that up to 2% of the expectednumber of genes in Arabidopsis encode proteinsinvolved in Ca2+ signaling. Considering the in-volvement of Ca2+ in so many diverse processes itis not surprising that plants contain such a largenumber of proteins involved in Ca2+ signaling.Although the specific functions of most of theseremain to be elucidated, a large body of indirectevidence indicates the involvement of Ca2+ sens-ing proteins in many cellular processes such ascytoplasmic streaming, organelle and vesicle trans-port, microtubule dynamics, cell division, chromo-some segregation, cell elongation, tip growth andmorphogenesis including some plant-specific pro-cesses. Recent studies have produced many sur-prises and significantly advanced ourunderstanding of Ca2+ signaling in plants andraised many important questions. What are all theproteins involved in maintaining Ca2+ homeosta-sis and in elevating [Ca2+]cyt in response to sig-nals? How the proteins involved in Ca2+ influxand efflux are regulated? How elevation of Ca2+

evokes different responses to different signals?What is the nature of Ca2+ signatures that areproduced in response to a given signal? Are thesechanges confined to a subcellular compartment?What are the targets of numerous CaM-likeproteins that have been identified? The nextdecade should bring answers to these interestingquestions. The challenge now is to elucidate therole(s) of numerous Ca2+ sensing proteins andcross-talk among various components of Ca2+

signaling and other messenger mediated signalingpathways. Molecular, cell biological and geneticanalysis of various proteins involved in Ca2+ sig-naling should permit elucidation of functionalanalysis of these proteins. In planta protein–protein interaction studies using new strategiessuch as fluorescence resonance energy transfer arenecessary to verify and extend the results obtainedwith in vitro studies [230]. Loss-of-function experi-ments with individual components of Ca2+ signal-ing will help in understanding the function(s) ofeach protein involved in Ca2+ signaling. Becauseof the large gene families of some Ca2+ sensingproteins and likely functional redundancy or over-lap with other members of the family, it will benecessary to create double or triple mutants. New


technologies such as reverse genetics [231] to cre-ate knock out mutants coupled with the analysisof cell- and tissue-specific expression of individualmembers Ca2+ signaling pathway using microar-rays [232], and non-destructive visualization ofproteins in live cells using fluorescent reportersshould help in gaining new insights into the func-tion(s) of proteins involved in Ca2+ signaling andin deciphering signaling networks. Because of theinvolvement of Ca2+ signaling in many stressresponses it is possible to generate plants that aremore tolerant to biotic and abiotic stresses bymanipulating one or more components of Ca2+

signaling pathway. Although some progress hasalready been made in this area [90,96,116,233],there is a great potential in producing crop plantswith desirable traits by engineering Ca2+ signalingpathway.

Acknowledgements

I would like to thank Dr Irene Day and DrVaka Reddy for critically reading the manuscript;Bryan Criswell for his help in preparing thefigures. My apologies to those colleagues in thefield whose work was not mentioned due to spacelimitations. Research on Ca2+ signaling in mylaboratory is supported by grants from NSF,Agricultural Experiment Station and NASA.

References

[1] M.R. Knight, A.K. Campbell, S.M. Smith, A.J. Tre-wavas, Transgenic plant aequorin reports the effects oftouch and cold-shock and elicitors on cytoplasmic cal-cium, Nature 352 (1991) 524–526.

[2] H. Knight, A.J. Trewavas, M.R. Knight, Cold calciumsignaling in Arabidopsis involves two cellular pools anda change in calcium signature after acclimation, PlantCell. 8 (1996) 489–503.

[3] G.A. Allen, J.M. Kwak, S.P. Chu, J. Llopis, R.Y.Tsien, J.F. Harper, J.I. Schroeder, Cameleon calciumindicator reports cytoplasmic calcium dynamics in Ara-bidopsis guard cells, Plant J. 19 (1999) 735–747.

[4] H. Knight, Calcium signaling during abiotic stress inplants, Int. Rev. Cytol. 195 (2000) 269–324.

[5] P.J. White, Calcium channels in higher plants, Biochim.Biophys. Acta 1465 (2000) 171–189.

[6] A.J. Trewavas, R. Malho, Ca2+ signaling in plant cells:the big network!, Curr. Opin. Plant Biol. 1 (1998)428–433.

[7] A.S.N. Reddy, V. Reddy, Calcium as a messenger instress signal transduction, in: M. Pessarakali (Ed.),Handbook of Plant and Crop Physiology, MercelDekker, New York, 2000, in press.

[8] D.S. Bush, Calcium regulation in plant cells and its rolein signaling, Ann. Rev. Plant Physiol. Plant Mol. Biol.46 (1995) 95–122.

[9] D. Sander, C. Brownlee, J. Harper, Communicatingwith calcium, Plant Cell. 11 (1999) 691–706.

[10] C. Bowler, G. Neuhaus, H. Yamagata, N.-H. Chua,Cyclic GMP and calcium mediate phytochrome photo-transduction, Cell 77 (1994) 73–81.

[11] C. Bowler, N.-H. Chua, Emerging themes of plantsignal transduction, Plant Cell. 6 (1994) 1529–1541.

[12] T. Hirayama, C. Ohto, T. Mizoguchi, K. Shinozaki, Agene encoding a phosphatidylinositol-specific phospho-lipase C is induced by dehydration and salt stress inArabidopsis thaliana, Proc. Natl. Acad. Sci. USA 92(1995) 3903–3907.

[13] J. Kopka, C. Pical, J.E. Gray, B. Muller-Rober, Molec-ular and enzymatic characterization of three phospho-inositide-specific phospholipase C isoforms frompotato, Plant Physiol. 116 (1998) 239–250.

[14] T. Hirayama, N. Mitsukawa, D. Shibata, K. Shinozaki,AtPLC2, a gene encoding phosphoinositide-specificphospholipase C, is constitutively expressed in vegeta-tive and floral tissues in Arabidopsis thaliana, PlantMol. Biol. 34 (1997) 175–180.

[15] I.Y. Perera, I. Heilmann, W.F. Boss, Transient andsustained increases in inositol 1,4,5–trisphosphate pre-cede the differential growth response in gravistimulatedmaize pulvini, Proc. Natl. Acad. Sci. USA 96 (1999)5838–5843.

[16] C. Pical, T. Westergren, S.K. Dove, C. Larsson, M.Sommarin, Salinity and hyperosmotic stress inducerapid increases in phosphatidylinositol 4,5-bisphos-phate, diacylglycerol pyrophosphate, and phosphatidyl-choline in Arabidopsis thaliana cells, J. Biol. Chem. 274(1999) 38232–38240.

[17] G.J. Allen, S.R. Muir, D. Sanders, Release of Ca2+

from individual plant vacuoles by both InsP3 and cyclicADP-ribose, Science 268 (1995) 735–737.

[18] Y. Wu, J. Kuzma, E. Marechal, R. Graeff, H.C. Lee,R. Foster, N.H. Chua, Abscisic acid signaling throughcyclic ADP-ribose in plants, Science 278 (1997) 2126–2130.

[19] L. Navazio, M.A. Bewell, A. Siddiqua, G.D. Dickin-son, A. Galione, D. Sanders, Calcium release from theendoplasmic reticulum of higher plants elicited by theNADP metabolite nicotinic acid adenine dinucleotidephosphate, Proc. Natl. Acad. Sci. USA 97 (2000)8693–8698.

[20] M.R. McAinsh, C. Brownlee, A.M. Hetherington, Ab-scisic acid -induced elevation of guard cell cytosolicCa2+ precedes stomatal closure, Nature 343 (1990)186–188.

[21] M.R. McAinsh, C. Brownlee, A.M. Hetherington, Vi-sualizing changes in cytosolic Ca2+ during the responseof stomatal guard cells to abscisic acid, Plant Cell. 4(1992) 1113–1122.


[22] S. Gilroy, M.D. Fricker, N.D. Read, A.J. Trewavas,Role of calcium in signal transduction of Commelinaguard cells, Plant Cell. 3 (1991) 333–364.

[23] M. Wang, B. Van Duijn, A.W. Schram, Abscisic acidinduces a cytosolic calcium decreases in barley aleuroneprotoplasts, FEBS Lett. 278 (1991) 69–74.

[24] S. Gilroy, R.L. Jones, Gibberellic acid and abscisic acidcoordinately regulate cytoplasmic calcium and secre-tory activity in barley aleurone protoplasts, Proc. Natl.Acad. Sci. USA 89 (1992) 3591–3595.

[25] D.S. Bush, R.L. Jones, Cytoplasmic calcium and a-amylase secretion from barley aleurone protoplasts,Eur. J. Cell Biol. 46 (1988) 466–469.

[26] C.A. Gehring, D.A. Williams, S.H. Cody, R.W. Parish,Phototropism and geotropism in maize coleoptiles arespatially correlated with increases in cytosolic free cal-cium, Nature 345 (1990) 528–530.

[27] J. Lynch, A. Lauchli, Salinity affects intracellular cal-cium in corn root protoplasts, Plant Physiol. 87 (1988)351–356.

[28] J. Lynch, V.W. Polito, A. Lauchli, Salinity stress in-creases cytoplasmic Ca activity in maize root proto-plasts, Plant Physiol. 90 (1989) 1271–1274.

[29] D.S. Bush, Effects of gibberellic acid and environmen-tal factors on cytosolic calcium in wheat aleurone cells,Planta 199 (1996) 566–574.

[30] H. Knight, A.J. Trewavas, M.R. Knight, Calcium sig-nalling in Arabidopsis thaliana responding to droughtand salinity, Plant J. 12 (1997) 1067–1078.

[31] J. Liu, J.K. Zhu, A calcium sensor homolog requiredfor plant salt tolerance, Science 280 (1998) 1943–1945.

[32] C.C. Subbaiah, D.S. Bush, M.M. Sachs, Elevation ofcytosolic calcium precedes anoxic gene expression inmaize suspension-cultured cells, Plant Cell. 6 (1994)1747–1762.

[33] C.C. Subbaiah, D.S. Bush, M.M. Sachs, Mitochondrialcontribution to the anoxic Ca2+ signal in maize sus-pension-cultured cells, Plant Physiol. 118 (1998) 759–771.

[34] A.J. Russell, M.R. Knight, D.J. Cove, C.D. Knight,A.J. Trewavas, T.L. Wang, The moss, Physcomitrellapatens, transformed with apoaequorin cDNA respondsto cold shock, mechanical perturbation and pH withtransient increases in cytoplasmic calcium, Transgen.Res. 5 (1996) 167–170.

[35] D.H. Polisensky, J. Braam, Cold-shock regulation ofthe Arabidopsis TCH genes and the effects of modulat-ing intracellular calcium levels, Plant Physiol. 111(1996) 1271–1279.

[36] V. Legue, E. Blancaflor, C. Wymer, G. Perbal, D.Fantin, S. Gilroy, Cytoplasmic free Ca2+ in Arabidop-sis roots changes in response to touch but not gravity,Plant Physiol. 114 (1997) 789–800.

[37] M.R. Knight, S.M. Smith, A.J. Trewavas, Wind-in-duced plant motion immediately increases cytosoliccalcium, Proc. Natl. Acad. Sci. 89 (1992) 4967–4971.

[38] C. Thonat, N. Boyer, C. Penel, J.C. Courduroux, T.Gaspar, Effects of mechanical stimulation on localiza-tion of annexin-like proteins in Bryonia dioica in-ternodes, Protoplasma 176 (1993) 133–137.

[39] A.H. van Der Luit, C. Olivari, A. Haley, M.R. Knight,A.J. Trewavas, Distinct calcium signaling pathwaysregulate calmodulin gene expression in tobacco, PlantPhysiol. 121 (1999) 705–714.

[40] A.K. Campbell, A.J. Trewavas, M.R. Knight, Calciumimaging shows differential sensitivity to cooling andcommunication in luminous transgenic plants, CellCalc. 19 (1996) 211–218.

[41] M.F. Thomashow, Role of cold-responsive genes inplant freezing tolerance, Plant Physiol. 118 (1998) 1–8.

[42] H. Knight, E.L. Veale, G.J. Warren, M.R. Knight, Thesfr6 mutation in Arabidopsis suppresses low-tempera-ture induction of genes dependent on the CRT/DREsequence motif, Plant Cell. 11 (1999) 875–886.

[43] I. Johansson, C. Larsson, B. Ek, P. Kjellbom, Themajor integral proteins of spinach leaf plasma mem-branes are putative aquaporins and are phosphorylatedin response to Ca2+ and apoplastic water potential,Plant Cell. 8 (1996) 1181–1191.

[44] A.E. Biyaseheva, Y.G. Molotkovskii, L.K. Mamonov,Increase of free Ca2+ in the cytosol of plant protoplastin response to heat stress as related to Ca2+ homeosta-sis, Rus. Plant Physiol. 40 (1993) 540–544.

[45] M. Gong, A.H. van der Luit, M.R. Knight, A.J. Tre-wavas, Heat-shock-induced changes in intracellularCa2+ level in tobacco seedlings in relation to thermo-tolerance, Plant Physiol. 116 (1998) 429–437.

[46] A.R. Taylor, N.F.H. Manison, C. Fernandez, J. Wood,C. Brownlee, Spatial organization of calcium signalinginvolved in cell volume control in the Fucus rhizoid,Plant Cell. 8 (1996) 2015–2031.

[47] A.R. Taylor, N.F.H. Manison, C. Brownlee, Regula-tion of channel activity underlying cell volume andpolarity signals in Fucus, J. Exp. Bot. 48 (1997) 579–588.

[48] K. Takahashi, M. Isobe, S. Muto, An increase incytosolic calcium ion concentration precedes hypoos-motic shock-induced activation of protein kinases intobacco suspension culture cells, FEBS Lett. 401 (1997)202–206.

[49] P.S. Shacklock, N.D. Read, A.J. Trewavas, Cytosolicfree calcium mediates red-light induced photomorpho-genesis, Nature 358 (1992) 753–755.

[50] H. Clayton, M.R. Knight, H. Knight, M.R. McAinsh,A.M. Hetherington, Dissection of the ozone-inducedcalcium signature, Plant J. 17 (1999) 575–579.

[51] A.H. Price, A. Taylor, S.J. Ripley, A. Griffiths, A.J.Trewavas, M.R. Knight, Oxidative signals in tobaccoincrease cytosolic calcium, Plant Cell. 6 (1994) 1301–1310.

[52] A. Levine, R. Tenhaken, R. Dixon, C. Lamb, H2O2

from the oxidative burst orchestrates the plant hyper-sensitive disease resistance response, Cell 79 (1994)583–593.

[53] A. Levine, R.I. Pennell, M.E. Alvarez, R. Palmer, C.Lamb, Calcium-mediated apoptosis in a plant hyper-sensitive disease resistance response, Curr. Biol. 6(1996) 427–437.

[54] M.R. McAinsh, H. Clayton, T.A. Mansfield, A.M.Hetherington, Changes in stomatal behavior and guardcell cytoplasmic free calcium in response to oxidativestress, Plant Physiol. 111 (1996) 1031–1042.


[55] Z.-M. Pel, Y. Murata, G. Benning, S. Thomine, B.Kluesener, G.J. Allen, E. Grill, J.I. Schroeder, Calciumchannels activated by hydrogen peroxide mediate ab-scisic acid signaling in guard cells, Nature 406 (2000)731–734.

[56] S. Lindberg, H. Strid, Aluminium induces rapidchanges in cytosolic pH and free calcium and potas-sium concentrations in root protoplasts of wheat(Triticum aesti6um), Physiol. Plant 99 (1997) 405–414.

[57] D.L. Jones, L.V. Kochian, S. Gilory, Aluminium in-duced a decrease in cytosolic calcium concentration inBY-2 tobacco cell cultures, Plant Physiol. 116 (1998)81–89.

[58] D.L. Jones, S. Gilroy, P.B. Larsen, S.H. Howell, L.V.Kochian, Effect of aluminum on cytoplasmic Ca2+

homeostasis in root hairs of Arabidopsis thaliana (L.),Planta 206 (1998) 378–387.

[59] C. Plieth, B. Sattelmacher, U.P. Hansen, M.R. Knight,Low-pH-mediated elevations in cytosolic calcium areinhibited by aluminium: a potential mechanism foraluminium toxicity, Plant J. 18 (1999) 643–650.

[60] C.J. Lamb, R.A. Dixon, The oxidative burst in plantdisease resistance, Ann. Rev. Plant Physiol. Plant Mol.Biol. 48 (1997) 251–275.

[61] S. Zimmermann, T. Nurnberger, J.-M. Frachisse, W.Wirtz, J. Guren, R. Hedrich, D. Scheel, Receptor-medi-ated activation of a plant Ca2+-permiable ion channelinvolved in pathogen defense, Proc. Natl. Acad. Sci.USA 94 (1997) 2751–2755.

[62] B. Blume, T. Nurnberger, N. Nass, D. Scheel, Recep-tor-mediated increase in cytoplasmic free calcium re-quired for activation of pathogen defense in parsley,Plant Cell. 12 (2000) 1425–1440.

[63] D.W. Ehrhardt, R. Wais, S.R. Long, Calcium spikingin plant root hairs responding to Rhizobium nodula-tion signals, Cell 85 (1996) 673–681.

[64] S.K. Sopory, M. Munshi, Protein kinases and phos-phatases and their role in cellular signalling in plants,CRC Crit. Rev. Plant Sci. 17 (1998) 245–318.

[65] M.R. McAinsh, C. Brownlee, A.M. Hetherington, Vi-sualising changes in cytosolic Ca2+ during the responseto stomatal guard cells to abscisic acid, Plant Cell. 4(1992) 1113–1122.

[66] J.I. Schroeder, S. Hagiwara, Repetitive increases incytosolic Ca2+ of guard cells by abscisic acid activationof nonselective Ca2+-permeable channels, Proc. Natl.Acad. Sci. USA 87 (1990) 9305–9309.

[67] P. Nicotera, D.J. McConkey, D.P. Jones, S. Orrenius,ATP stimulates Ca2+ uptake and increases the freeCa2+ concentration in isolated rate liver nuclei, Proc.Natl. Acad. Sci. USA. 86 (1989) 453–457.

[68] T.D. Bunney, P.J. Shaw, P.A. Watkins, J.P. Taylor,A.F. Beven, B. Wells, G.M. Calder, B.K. Drobak,ATP-dependent regulation of nuclear Ca(2+ ) levels inplant cells, FEBS Lett. 476 (2000) 145–149.

[69] N. Pauly, M.R. Knight, P. Thuleau, A.H. van der Luit,M. Moreau, A.J. Trewavas, R. Ranjeva, C. Mazars,Control of free calcium in plant cell nuclei, Nature 405(2000) 754–755.

[70] M. Geisler, K.B. Axelsen, J.F. Harper, M.G. Palmgren,Molecular aspects of higher plant P-type Ca(2+ )-AT-Pases, Biochim. Biophys. Acta 1465 (2000) 52–78.

[71] J.F. Harper, B. Hong, I. Hwang, H.Q. Guo, R. Stod-dard, J.F. Huang, M.G. Palmgren, H. Sze, A novelcalmodulin-regulated Ca2+ -ATPase (ACA2) fromArabidopsis with an N-terminal autoinhibitory do-main, J. Biol. Chem. 273 (1998) 1099–1106.

[72] S. Malmstrom, H.E. Akerlund, P. Askerlund, Regula-tory role of the N terminus of the vacuolar calcium-ATPase in cauliflower, Plant Physiol. 122 (2000)517–526.

[73] C.C. Subbaiah, M.M. Sachs, Maize cap1 encodes anovel SERCA-type calcium-ATPase with a calmodulin-binding domain, J. Biol. Chem. 275 (2000) 21678–21687.

[74] M.C. Bonza, P. Morandini, L. Luoni, M. Geisler,M.G. Palmgren, M.I. De Michelis, At-ACA8 encodes aplasma membrane-localized calcium-ATPase of ara-bidopsis with a calmodulin-binding domain at the Nterminus, Plant Physiol. 123 (2000) 1495–1506 in Pro-cess Citation.

[75] W.S. Chung, S.H. Lee, J.C. Kim, W. Do Heo, M.C.Kim, C.Y. Park, H.C. Park, C.O. Lim, W.B. Kim, J.F.Harper, M.J. Cho, Identification of a calmodulin-regu-lated soybean Ca(2)+ -ATPase (SCA1) that is locatedin the plasma membrane, Plant Cell. 12 (2000) 1393–1408.

[76] I. Hwang, H. Sze, J.F. Harper, A calcium-dependentprotein kinase can inhibit a calmodulin-stimulatedCa2+ pump (ACA2) located in the endoplasmicreticulum of Arabidopsis, Proc. Natl. Acad. Sci. USA97 (2000) 6224–6229.

[77] K.D. Hirschi, R.G. Zhen, K.W. Cunningham, P.A.Rea, G.R. Fink, CAX1, an H+/Ca2+ antiporter fromArabidopsis, Proc. Natl. Acad. Sci. USA 93 (1996)8782–8786.

[78] H. Ueoka-Nakanishi, T. Tsuchiya, M. Sasaki, Y.Nakanishi, K.W. Cunningham, M. Maeshima, Func-tional expression of mung bean Ca2+/H+ antiporterin yeast and its intracellular localization in thehypocotyl and tobacco cells, Eur. J. Biochem. 267(2000) 3090–3098.

[79] K.D. Hirschi, Expression of Arabidopsis CAX1 in to-bacco: altered calcium homeostasis and increased stresssensitivity, Plant Cell. 11 (1999) 2113–2122.

[80] B.W. Poovaiah, A.S.N. Reddy, Calcium and signaltransduction in plants, CRC Crit. Rev. Plant Sci. 12(1993) 185–211.

[81] R.E. Zielinski, Calmodulin and calmodulin-bindingproteins in plants, Ann. Rev. Plant Physiol. Plant Mol.Biol. 49 (1998) 697–725.

[82] D.M. Roberts, A.C. Harmon, Calcium modulatedproteins: targets of intracellular calcium signals inhigher plants, Ann. Rev. Plant Physiol. Plant Mol.Biol. 43 (1992) 375–414.

[83] W.A. Snedden, H. Fromm, Calmodulin, calmodulin-re-lated proteins and plant responses to the environment,Trends Plant Sci. 3 (1998) 299–304.

[84] Y.S. Babu, C.E. Bugg, W.J. Cook, Structure ofcalmodulin refined at 2.2 A, resolution, J. Mol. Biol.204 (1988) 191–204.

[85] K.T. O’Neil, W.F. DeGrado, How calmodulin binds itstargets: sequence-independent recognition of am-


phiphilic a-helices, Trends Biochem. Sci. 15 (1990) 59–64.

[86] A.R. Rhoads, F. Friedberg, Sequence motifs forcalmodulin recognition, FASEB J. 11 (1997) 331–340.

[87] D. Takezawa, Z.H. Liu, G. An, B.W. Poovaiah,Calmodulin gene family in potato: developmental andtouch-induced expression of the mRNA encoding anovel isoform, Plant Mol. Biol. 27 (1995) 693–703.

[88] T. Yang, G. Segal, S. Abbo, M. Feldman, H. Fromm,Characterization of the gene family in wheat: structure,chromosomal location, and evolutionary aspects, Mol.Gene Genet. 252 (1996) 684–694.

[89] S.H. Lee, J.C. Kim, M.S. Lee, W.D. Heo, H.Y. Seo,H.W. Yoon, J.C. Hong, S.Y. Lee, J.D. Bahk, I.Hwang, J. Cho, Identification of a novel divergentcalmodulin isoform from soybean which has differen-tial ability to activate calmodulin-dependent enzymes,J. Biol. Chem. 270 (1995) 21806–21812.

[90] W.D. Heo, S.H. Lee, M.C. Kim, J.C. Kim, W.S.Chung, H.J. Chun, K.J. Lee, C.Y. Park, H.C. Park,J.Y. Choi, M.J. Cho, Involvement of specific calmod-ulin isoforms in salicylic acid-independent activation ofplant disease resistance responses, Proc. Natl. Acad.Sci. USA 19 (1999) 766–771.

[91] S.H. Lee, H.Y. Seo, J.C. Kim, W.D. Heo, W.S. Chung,K.J. Lee, M.C. Kim, Y.H. Cheong, J.Y. Choi, C.O.Lim, M.J. Cho, Differential activation of NAD kinaseby plant calmodulin isoforms. The critical role of do-main I, J. Biol. Chem. 272 (1997) 9252–9259.

[92] S.H. Lee, J.D. Johnson, M.P. Walsh, J.E. Van Lierop,C. Sutherland, A. Xu, W.A. Snedden, D. Kosk-Kosicka, H. Fromm, N. Narayanan, M.J. Cho, Differ-ential regulation of Ca2+/calmodulin-dependentenzymes by plant calmodulin isoforms and free Ca2+

concentration, Biochem. J. 350 (2000) 299–306.[93] S.H. Lee, M.C. Kim, W.D. Heo, J.C. Kim, W.S.

Chung, C.Y. Park, H.C. Park, Y.H. Cheong, C.Y.Kim, S.H. Lee, K.J. Lee, J.D. Bahk, S.Y. Lee, M.J.Cho, Competetive binding of calmodulin isoforms tocalmodulin-binding proteins: implications for the func-tion of calmodulin isoforms in plants, Biochim. Bio-phys. Acta 1433 (1999) 56–67.

[94] C. Kohler, G. Neuhaus, Characterisation of calmodulinbinding to cyclic nucleotide-gated ion channels fromArabiodpsis thaliana, FEBS Lett. 4710 (2000) 133–136.

[95] E. Blumwald, G.S. Aharon, B.C. Lam, Early signaltransduction pathways in plant-pathogen interactions,Trends Plant Sci. 3 (1998) 342–346.

[96] S.A. Harding, S.H. Oh, D.M. Roberts, Transgenictobacco expressing a foreign calmodulin gene shows anenhanced production of active oxygen species, EMBOJ. 17 (1997) 1137–1144.

[97] H. Xu, M.C. Heath, Role of calcium in signal transduc-tion during the hypersensitive response caused by ba-sidiospore-derived infection of the cowpea rust fungus,Plant Cell. 10 (1998) 585–598.

[98] B. Liao, M.C. Gawienowski, R.E. Zielinski, Differen-tial stimulation of NAD kinase and binding of peptidesubstrates by wild-type and mutant plant calmodulinisoforms, Arch. Biochem. Biophys. 327 (1996) 53–60.

[99] V. Reddy, F. Safadi, R.E. Zielinski, A.S.N. Reddy,Interaction of a kinesin-like protein with calmodulinisoforms from Arabidopsis, J. Biol. Chem. 274 (1999)31727–31733.

[100] Z. Liu, M. Xia, B.W. Poovaiah, Chimeric calcium/calmodulin-dependent protein kinase in tobacco: differ-ential regulation by calmodulin isoforms, Plant Mol.Biol. 38 (1998) 889–897.

[101] S. Muto, S. Miyachi, Properties of a protein activatorof NAD kinase from plants, Plant Physiol. 59 (1977)55–60.

[102] G. Baum, Y. Chen, T. Arazi, H. Takatsuji, H. Fromm,A plant glutamate decarboxylase containing a calmod-ulin binding domain: Cloning, sequence and functionalanalysis, J. Biol. Chem. 268 (1993) 19610–19617.

[103] B. Watillion, R. Kettmann, P. Boxus, A. Burny, Acalcium/calmodulin-binding serine/threonine proteinkinase homologue to the mammalian type II calcium/calmodulin-dependent protein kinase is expressed inplant cells, Plant Physiol. 101 (1993) 1381–1384.

[104] Y.-T. Lu, H. Hidaka, L.J. Feldman, Characterizationof a calcium/calmodulin-dependent protein kinase ho-molog from maize roots showing light-regulated gravit-ropism, Planta 199 (1996) 18–24.

[105] N.A. Durso, R.J. Cyr, A calmodulin-sensitive interac-tion between microtubules and a higher plant homologof elongation factor-1 alpha, Plant Cell. 6 (1994) 893–905.

[106] Y.-T. Lu, M.A.N. Dharmasiri, H.M. Harrington,Characterization of a cDNA encoding a novel heat-shock protein that binds to calmodulin, Plant Physiol.108 (1995) 1197–1202.

[107] S. Patil, D. Takezawa, B.W. Poovaiah, Chimeric plantcalcium/calmodulin-dependent protein kinase gene witha neural visinin-like calcium-binding domain, Proc.Natl. Acad. Sci. 92 (1995) 4897–4901.

[108] D.B. Szymanski, B. Liao, R.E. Zielinski, Calmodulinisoforms differentially enhance the binding ofcauliflower nuclear proteins and recombinant TGA3 toa region derived from the Arabidopsis Cam-3 pro-moter, Plant Cell. 8 (1996) 1069–1077.

[109] A.S.N. Reddy, F. Safadi, S.B. Narasimhulu, M.Golovkin, X. Hu, A novel plant calmodulin-bindingprotein with a kinesin heavy chain motor domain, J.Biol. Chem. 271 (1996) 7052–7060.

[110] A.S.N. Reddy, S.B. Narasimhulu, F. Safadi, M.Golovkin, A plant kinesin heavy chain-like protein is acalmodulin-binding protein, Plant J. 10 (1996) 9–21.

[111] W. Wang, D. Takezawa, S.B. Narasimhulu, A.S.N.Reddy, B.W. Poovaiah, A novel kinesin-like proteinwith a calmodulin-binding domain, Plant Mol. Biol. 31(1996) 87–100.

[112] S. Malmstrom, P. Askerlund, M.G. Palmgren, Acalmodulin-stimulated Ca2+-ATPase from plant vac-uolar membranes with a putative regulatory domain atits N-terminus, FEBS Lett. 400 (1997) 324–328.

[113] H.L. Hsieh, C.G. Tong, C. Thomas, S.J. Roux, Light-modulated abundance of an mRNA encoding acalmodulin-regulated, chromatin-associated NTPase inpea, Plant Mol. Biol. 30 (1996) 135–147.


[114] R.C. Schuurink, S.F. Shartzer, A. Fath, R.L. Jones,Characterization of a calmodulin-binding transporterfrom the plasma membrane of barley aleurone, Proc.Natl. Acad. Sci. USA 95 (1998) 1944–1949.

[115] C. Kohler, T. Merkle, G. Neuhaus, Characterization ofa novel gene family of putative cyclic nucleotide- andcalmodulin-regulated ion channels in Arabidopsisthaliana, Plant J. 18 (1999) 97–104.

[116] T. Arazi, R. Sunkar, B. Kaplan, H. Fromm, A tobaccoplasma membrane calmodulin-binding transporter con-fers Ni2+ tolerance and Pb2+ hypersensitivity in trans-genic plants, Plant J. 20 (1999) 171–182.

[117] T. Arazi, B. Kaplan, H. Fromm, A high-affinitycalmodulin-binding site in a tobacco plasma-membranechannel protein coincides with a characteristic elementof cyclic nucleotide-binding domains, Plant Mol. Biol.42 (2000) 591–601.

[118] V.S. Veena, S.K. Reddy, A. Sopory, Glyoxalase I fromBrassica juncea : molecular cloning, regulation and itsover-expression confer tolerance in transgenic tobaccounder stress, Plant J. 17 (1999) 385–395.

[119] W. Wang, D. Takezawa, B.W. Poovaiah, A potatocDNA encoding a homologue of mammalian multidrugresistant P-glycoprotein, Plant Mol. Biol. 31 (1996)683–687.

[120] T. Yang, B.W. Poovaiah, Molecular and biochemicalevidence for the involvement of calcium/calmodulin inauxin action, J. Biol. Chem. 275 (2000) 3137–3143.

[121] F. Safadi, V. Reddy, A.S.N. Reddy, A pollen-specificnovel calmodulin-binding protein with tetratricopeptiderepeats, J. Biol. Chem. 275 (2000) 35457–35470.

[122] A.S.N. Reddy, D. Takezawa, H. Fromm, B.W. Poova-iah, Isolation and characterization of two cDNAs thatencode for calmodulin-binding proteins from corn roottips, Plant Sci. 94 (1993) 109–117.

[123] W.A. Snedden, N. Koutsia, G. Baum, H. Fromm,Activation of a recombinant petunia glutamate decar-boxylase by calcium/calmodulin or by a monoclonalantibody which recognizes the calmodulin binding do-main, J. Biol. Chem. 271 (1996) 4148–4153.

[124] M. Zik, T. Arazi, W.A. Snedden, H. Fromm, Twoisoforms of glutamate decarboxylase in Arabidopsis areregulated by calcium/calmodulin and differ in organdistribution, Plant Mol. Biol. 37 (1998) 967–975.

[125] A.I. Ramputh, A.W. Bown, Rapid g-aminobutyric acidsynthesis and the inhibition of the growth and develop-ment of oblique banded leaf-roller larvae, Plant Phys-iol. 111 (1996) 1349–1352.

[126] G. Baum, S. Lev-Yadun, Y. Fridmann, T. Arazi, H.Katsnelson, M. Zik, H. Fromm, Calmodulin binding toglutamate decarboxylase is required for regulation ofglutamate and GABA metabolism and normal develop-ment in plants, EMBO J. 15 (1996) 2988–2996.

[127] J.M. Anderson, H. Charbonneau, H.P. Jones, R.O.McCann, M.J. Cormier, Characterization of the plantnicotinamide adenine dinucleotide kinase activatorprotein and its identification as calmodulin, Biochem-istry 19 (1980) 3113–3120.

[128] V. Reddy, A.S.N. Reddy, A plant calmodulin-bindingmotor is part kinesin and part myosin, Bioinformatics15 (1999) 1055–1057.

[129] G.C. Rogers, C.L. Hart, K.P. Wedman, J.M. Scholey,Identification of kinesin-C, a calmodulin-binding car-boxy-terminal kinesin in animal (Strongylocentrotuspurpuratus) cells, J. Mol. Biol. 294 (1999) 1–8.

[130] S.E. Abdel-Ghany, A.S.N. Reddy, A novel calcium/calmodulin-regulated kinesin-like protein is highly con-served between monocots and dicots, DNA Cell. Biol.19 (2000) 567–578.

[131] H. Song, M. Golovkin, A.S.N. Reddy, S.A. Endow, Invitro motility of AtKCBP, a calmodulin-binding ki-nesin-like protein of Arabidopsis, Proc. Natl. Acad.Sci. USA. 94 (1997) 322–327.

[132] B.E. Deavours, A.S.N. Reddy, R.A. Walker, Ca2+/calmodulin regulation of the Arabidopsis kinesin-likecalmodulin- binding protein, Cell. Motil. Cytoskel. 40(1998) 408–416.

[133] S.B. Narasimhulu, A.S.N. Reddy, Characterization ofmicrotubule binding domains in the Arabidopsis ki-nesin-like calmodulin-binding protein, Plant Cell. 10(1998) 957–965.

[134] A.S.N. Reddy, Molecular motors and their functions inplants, Intl. Rev. Cytol. (2001) (in press).

[135] J. Bowser, A.S.N. Reddy, Localization of a kinesin-likecalmodulin-binding protein in dividing cells of Ara-bidopsis and tobacco, Plant J. 12 (1997) 1429–1438.

[136] D.G. Oppenheimer, M.A. Pollock, J. Vacik, D.B. Szy-manski, B. Ericson, K. Feldmann, D. Marks, Essentialrole of a kinesin-like protein in Arabidopsis trichomemorphogenesis, Proc. Natl. Acad. Sci. USA 94 (1997)6261–6266.

[137] J.W. Vos, F. Safadi, A.S. Reddy, P.K. Hepler, TheKinesin-like calmodulin binding protein is differentiallyinvolved in cell division, Plant Cell. 12 (2000) 979–990.

[138] J. Espartero, I. Sanchez-Aguayo, J.M. Pardo, Molecu-lar characterization of glyoxalase-I from a higher plant;upregulation by stress, Plant Mol. Biol. 29 (1995)1223–1233.

[139] R. Deswal, S.K. Sopory, Glyoxalase I from Brassicajuncea is a calmodulin stimulated protein, Biochim.Biophys. Acta 1450 (1999) 460–467.

[140] W.-L. Hsieh, W.S. Pierce, H. Sze, Calcium-pumpingATPases in vesicles from carrot cells, Plant Physiol. 97(1991) 1535–1544.

[141] R.J. Cyr, Calcium/calmodulin affects microtubule sta-bility in lysed protoplasts, J. Cell Sci. 100 (1991) 311–317.

[142] D.D. Fisher, S. Gilroy, R.J. Cyr, Evidence for oppos-ing effects of calmodulin on cortical microtubules,Plant Physiol. 112 (1996) 1079–1087.

[143] R.C. Moore, N.A. Durso, R.J. Cyr, Elongation factor-1alpha stabilizes microtubules in a calcium/calmodulin-dependent manner, Cell Motil. Cytoskel. 41 (1998)168–180.

[144] M. Gong, Z.-G. Li, Calmodulin-binding proteins fromZea mays germs, Phytochemistry 40 (1995) 1335–1339.

[145] V. Mermall, P.L. Post, M.S. Mooseker, Unconven-tional myosins in cell movement, membrane traffic, andsignal transduction, Science 279 (1998) 527–533.

[146] A.E. Knight, J. Kendrick-Jones, A myosin-like proteinfrom a higher plant, J. Mol. Biol. 231 (1993) 148–154.


[147] M. Kinkema, H. Wang, J. Schiefelbein, Molecularanalysis of the myosin gene family in Arabidopsisthaliana, Plant Mol. Biol. 26 (1994) 1139–1153.

[148] T. Shimmen, E. Yokota, Physiological and biochemicalaspects of cytoplasmic streaming, Int. Rev. Cytol. 155(1994) 97–140.

[149] T. Kohno, T. Shimmen, Accelerated sliding of pollentube organelles along Characeae actin bundles regu-lated by Ca2+, J. Cell. Biol. 106 (1988) 1539–1543.

[150] T. Kohno, T. Shimmen, Mechanism of Ca2+ inhibitionof cytoplasmic streaming in lily pollen tubes, J. Cell.Sci. 91 (1988) 501–509.

[151] E. Yokota, S. Muto, T. Shimmen, Inhibitory regulationof higher-plant myosin by Ca2+ ions, Plant Physiol.119 (1999) 231–240.

[152] E. Yokota, C. Yukawa, S. Muto, S. Sonobe, T. Shim-men, Biochemical and immunocytochemical characteri-zation of two types of myosins in cultured tobaccobright yellow-2 cells, Plant Physiol. 121 (1999) 525–534.

[153] S. Higashi-Fujime, R. Ishikawa, H. Iwasawa, O.Kagami, E. Kurimoto, K. Kohama, T. Hozumi, Thefastest actin-based motor protein from the green algae,Chara, and its distinct mode of interaction with actin,FEBS Lett. 375 (1995) 151–154.

[154] Y. Tominaga, R. Wayne, H.Y.L. Tung, M. Tazawa,Phosphorylation-dephosphorylation is involved inCa2+ -controlled cytoplasmic streaming of characeancells, Protoplasma 136 (1987) 161–169.

[155] D.W. McCurdy, A.C. Harmon, Phosphorylation of aputative myosin light chain in Chara by calcium-depen-dent protein kinase, Protoplasma 171 (1992) 85–88.

[156] C.H. Putnam-Evans, Calcium-dependent protein kinaseis localized with F-actin in plant cells, Cell Motil.Cytoskel. 12 (1989) 12–22.

[157] J. Kudla, Q. Xu, K. Harter, W. Gruissem, S. Luan,Genes for calcineurin B-like proteins in Arabidopsis aredifferentially regulated by stress signals, Proc. Natl.Acad. Sci. USA 96 (1999) 4718–4723.

[158] J. Shi, K.N. Kim, O. Ritz, V. Albrecht, R. Gupta, K.Harter, S. Luan, J. Kudla, Novel protein kinases asso-ciated with calcineurin B-like calcium sensors in Ara-bidopsis, Plant Cell. 11 (1999) 2393–2406.

[159] S.J. Wu, L. Ding, J.-K. Zhu, SOS1, a genetic locusessential for salt tolerance and potassium acquisition,Plant Cell. 8 (1996) 617–627.

[160] J. Liu, J.K. Zhu, An Arabidopsis mutant that requiresincreased calcium for potassium nutrition and salt tol-erance, Proc. Natl. Acad. Sci. USA 94 (1997) 14960–14964.

[161] J. Liu, M. Ishitani, U. Halfter, C.S. Kim, J.K. Zhu,The Arabidopsis thaliana SOS2 gene encodes a proteinkinase that is required for salt tolerance, Proc. Natl.Acad. Sci. USA 97 (2000) 3730–3734.

[162] U. Halfter, M. Ishitani, J.K. Zhu, The ArabidopsisSOS2 protein kinase physically interacts with and isactivated by the calcium-binding protein SOS3, Proc.Natl. Acad. Sci. USA 97 (2000) 3735–3740.

[163] J.M. Pardo, M.P. Reddy, S. Yang, A. Maggio, G.H.Huh, T. Matsumoto, M.A. Coca, M. Paino-D’Urzo, H.Koiwa, D.J. Yun, A.A. Watad, R.A. Bressan, P.M.

Hasegawa, Stress signaling through Ca2+/calmodulin-dependent protein phosphatase calcineurin mediatessalt adaptation in plants, Proc. Natl. Acad. Sci. USA95 (1998) 9681–9686.

[164] R.A. Bressan, P.M. Hasegawa, J.M. Pardo, Plants usecalcium to resolve salt stress, Trends Plant Sci. 3 (1998)411–412.

[165] M. Rodriguez-Concepcion, S. Yalovsky, M. Zik, H.Fromm, W. Gruissem, The prenylation status of anovel plant calmodulin directs plasma membrane ornuclear localization of the protein, EMBO J. 18 (1999)1996–2007.

[166] C. Xiao, H. Xin, A. Dong, C. Sun, K. Cao, A novelcalmodulin-like protein gene in rice which has an un-usual prolonged C-terminal sequence carrying a puta-tive prenylation site, DNA Res. 6 (1999) 179–181.

[167] M. Rodriguez-Concepcion, S. Yalovsky, W. Gruissem,Protein prenylation in plants: old friends and newtargets, Plant Mol. Biol. 39 (1999) 865–870.

[168] J. Braam, M.L. Sistrunk, D.H. Polisensky, W. Xu,M.M. Purugganan, D.M. Antosiewicz, P. Campbell,K.A. Johnson, Plant responses to environmental stress:regulation and functions of the Arabidopsis TCHgenes, Planta 203 (1997) S35–41.

[169] M.L. Sistrunk, D.M. Antosiewicz, M.M. Purugganan,J. Braam, Arabidopsis TCH3 encodes a novel calcium-binding protein and shows environmentally inducedand tissue specific regulation, Plant Cell. 6 (1994)1553–1565.

[170] G. Frandsen, F. Muller-Uri, M. Nielsen, J. Mundy, K.Skriver, Novel plant Ca(2+)-binding protein expressedin response to abscisic acid and osmotic stress, J. Biol.Chem. 271 (1996) 343–348.

[171] J. Leung, M. Bouvier-Durand, P.C. Morris, D. Guer-rier, F. Chefdor, J. Giraudat, Arabidopsis ABA re-sponse gene ABI1: features of a calcium-modulatedprotein phosphatase, Science 264 (1994) 1448–1452.

[172] K. Meyer, M.P. Leube, E. Grill, A protein phosphatase2C involved in ABA signal transduction in Arabidopsisthaliana, Science 264 (1994) 1452–1455.

[173] H.J. Jang, K.T. Pih, S.G. Kang, J.H. Lim, J.B. Jin,H.L. Piao, I. Hwang, Molecular cloning of a novelCa2+-binding protein that is induced by NaCl stress,Plant Mol. Biol. 37 (1998) 839–847.

[174] C. Bowler, R. Fluhr, The role of calcium and activatedoxygens as signals for controlling cross-tolerance,Trends Plant Sci. 5 (2000) 241–246.

[175] G. Arimura, R. Ozawa, T. Shimoda, T. Nishioka, W.Boland, J. Takabayashi, Herbivory-induced volatileselicit defence genes in lima bean leaves, Nature 406(2000) 512–515.

[176] D.R. Bergey, C.A. Ryan, Wound- and systemin-in-ducible calmodulin gene expression in tomato leaves,Plant Mol. Biol. 40 (1999) 815–823.

[177] D. Flego, M. Pirhonen, H. Saarilahti, T.K. Palva, E.T.Palva, Control of virulence gene expression by plantcalcium in the phytopathogen Erwinia caroto6ora, Mol.Microbiol. 25 (1997) 831–838.

[178] J.L. Jakobek, J.A. Smith-Becker, P.B. Lindgren, Abean cDNA expressed during a hypersensitive reactionencodes a putative calcium-binding protein, Mol. PlantMicrob. Interact. 12 (1999) 712–719.


[179] D.C. Boyes, J. Nam, J.L. Dangl, The Arabidopsisthaliana RPM1 disease resistance gene product is aperipheral plasma membrane protein that is degradedcoincident with the hypersensitive response, Proc. Natl.Acad. Sci. USA 95 (1998) 15849–15854.

[180] M. Grant, J. Mansfield, Early events in host-pathogeninteractions, Curr. Opin. Plant Biol. 2 (1999) 312–319.

[181] T. Romeis, P. Piedras, S. Zhang, D.F. Klessig, H. Hirt,J.D. Jones, Rapid A6r9- and Cf-9-dependent activationof MAP kinases in tobacco cell cultures and leaves:convergence of resistance gene, elicitor, wound, andsalicylate responses, Plant Cell. 11 (1999) 273–287.

[182] T. Romeis, P. Piedras, J.D. Jones, Resistance gene-de-pendent activation of a calcium-dependent protein ki-nase in the plant defense response, Plant Cell. 12 (2000)803–816.

[183] T. Keller, H.G. Damude, D. Werner, P. Doerner, R.A.Dixon, C. Lamb, A plant homolog of the neutrophilNADPH oxidase gp91phox subunit gene encodes aplasma membrane protein with Ca2+ binding motifs,Plant Cell. 10 (1998) 255–266.

[184] E. Schiebel, M. Bornens, In search of a function forcentrins, Trends Cell Biol. 5 (1995) 197–201.

[185] J.K. Zhu, P.M. Bressan, P.M. Hasegawa, An Atriplexnummularia cDNA sequence relatedness to the algalcaltractin gene, Plant Physiol. 99 (1992) 1743–1745.

[186] M.C. Cordeiro, R. Piqueras, D.E. de Oliveira, C.Castresana, Characterization of early induced genes inArabidopsis thaliana responding to bacterial inocula-tion: identification of centrin and of a novel proteinwith two regions related to kinase domains, FEBS Lett.434 (1998) 387–393.

[187] V. Stoppin-Mellet, J. Canaday, A.-M. Lambert, Char-acterization of microsome-associated tobacco BY-2centrins, Eur. J. Cell. Biol. 78 (1999) 842–848.

[188] L.M. Blackman, J.D. Harper, R.L. Overall, Localiza-tion of a centrin-like protein to higher plant plasmodes-mata, Eur. J. Cell. Biol. 78 (1999) 297–304.

[189] A. Dietrich, J.E. Mayers, K. Hahlbrock, Fungal elicitortriggers rapid, transient and specific protein phosphory-lation in parsley cell suspension cultures, J. Biol. Chem.265 (1990) 6360–6368.

[190] A.F. Monroy, F. Sarhan, R.S. Dhindsa, Cold-inducedchanges in freezing tolerance, protein phosphorylation,and gene expression, Plant Physiol. 102 (1993) 1227–1235.

[191] J.M. Christie, G.L. Jenkins, Distinct UV-B and UV-A/blue light signal transduction pathways induce chalconesynthase gene expression in Arabidopsis cells, PlantCell. 8 (1996) 1555–1567.

[192] S. Grabski, E. Arnoys, B. Bush, M. Schindler, Regula-tion of actin tension in plant cells by kinases andphosphatases, Plant Physiol. 116 (1998) 279–290.

[193] K. Takahashi, M. Isobe, S. Muto, Mastoparan inducesan increase in cytosolic calcium ion concentration andsubsequent activation of protein kinases in tobaccosuspension culture cells, Biochim. Biophys. Acta 1401(1998) 339–346.

[194] A.C. Harmon, M. Gribskov, J.F. Harper, CDPKs- akinase for every Ca2+ signal?, Trends Plant Sci. 5(2000) 154–159.

[195] A.C. Harmon, C. Putnam-Evans, M.J. Cormier, Acalcium-dependent but calmodulin-independent proteinkinase from soybean, Plant Physiol. 83 (1987) 830–837.

[196] J.F. Harper, M.R. Sussman, G.E. Schaller, C. Putnam-Evans, H. Charbonneau, A.C. Harmon, A calcium-de-pendent protein kinase with a regulatory domainsimilar to calmodulin, Science 252 (1991) 951–954.

[197] T. Urao, T. Katagiri, T. Mizoguchi, K. Yamaguchi-Shinozaki, N. Hayashida, K. Shinozaki, Two genesthat encode Ca(2+ )-dependent protein kinases are in-duced by drought and high-salt stresses in Arabidopsisthaliana, Mol. Gen. Genet. 244 (1994) 331–340.

[198] J.R. Botella, J.M. Arteca, M. Somodevilla, R.N.Arteca, Calcium-dependent protein kinase gene expres-sion in response to physical and chemical stimuli inmungbean (Vigna radiata), Plant Mol. Biol. 30 (1996)1129–1137.

[199] J.F. Harper, B.M. Binder, M.R. Sussman, Calcium andlipid regulation of an Arabidopsis protein kinase ex-pressed in Escherichia coli, Biochemistry 32 (1993)3282–3290.

[200] B.M. Binder, J.F. Harper, M.R. Sussman, Characteri-zation of an Arabidopsis calmodulin-like domainprotein kinase purified from Escherichia coli using anaffinity sandwich technique, Biochemistry 33 (1994)2033–2041.

[201] P.K. Farmer, J.H. Choi, Calcium and phospholipidactivation of a recombinant calcium-dependent proteinkinase (DcCPK1) from carrot (Daucus carota L.),Biochim. Biophys. Acta 1434 (1999) 6–17.

[202] J. Sheen, Ca2+-dependent protein kinases and stresssignal transduction in plants, Science 274 (1996) 1900–1902.

[203] B. Watillon, R. Kettmann, P. Boxus, A. Burny, Acalcium/calmodulin-binding serine/threonine proteinkinase homologous to the mammalian type II calcium/calmodulin-dependent protein kinase is expressed inplant cells, Plant Physiol. 101 (1993) 1381–1384.

[204] D. Takezawa, S. Ramachandran, V. Paranjape, B.W.Poovaiah, Dual regulation of a chimeric plant Serine/threonine kinase by calcium and clacium/calmodulin, J.Biol. Chem. 271 (1996) 8126–8132.

[205] W. Wang, B.W. Poovaiah, Interaction of plantchimeric calcium/calmodulin-dependent protein kinasewith a homolog of eukaryotic elongation factor-1alpha,J. Biol. Chem. 274 (1999) 12001–12008.

[206] P.V. Satyanarayanan, C.R. Cremo, B.W. Poovaiah,Plant chimeric Ca2+/calmodulin-dependent protein ki-nase: Role of the neural visinin-like domain in regulat-ing autophosphorylation and calmodulin affinity, J BiolChem. 275 (2000) 30417–30422.

[207] G.B. Clark, S.J. Roux, Annexins of plant cells, PlantPhysiol. 109 (1995) 1133–1139.

[208] E.K. Lim, M.R. Roberts, D.J. Bowles, Biochemicalcharacterization of tomato annexin p35. Independenceof calcium binding and phosphatase activities, J. Biol.Chem. 273 (1998) 34920–34925.

[209] F. Baluska, J. Samaj, R. Napier, D. Volkmann, Maizecalreticulin localizes preferentially to plasmodesmata inroot apex, Plant J. 19 (1999) 481–488.


[210] M. Michalak, P. Mariani, M. Opas, Calreticulin, amultifunctional Ca2+ binding chaperone of the endo-plasmic reticulum, Biochem. Cell Biol. 76 (1998) 779–785.

[211] T. Furuyama, V.A. Dzelzkalns, A novel calcium-bindingprotein is expressed in Brassica pistils and anthers latein flower development, Plant Mol. Biol. 39 (1999)729–737.

[212] F. Safadi, D.L. Mykles, A.S.N. Reddy, Partial purifica-tion and characterization of a Ca2+-dependentproteinase from Arabidopsis Roots, Arch. Biochem.Biophys. 348 (1997) 143–151.

[213] P.K. Hepler, R.O. Wayne, Calcium and plant develop-ment, Ann. Rev. Plant Physiol. 36 (1985) 397–439.

[214] B.W. Poovaiah, A.S.N. Reddy, Calcium messenger sys-tem in plants, CRC Crit. Rev. Plant Sci. 6 (1987) 47–103.

[215] A. Leonardi, D.S. Heimovaara, M. Wang, Differentialinvolvement of abscisic acid in dehydration and osmoticstress in rice cell suspension, Physiol. Plant 93 (1995)31–37.

[216] J. Braam, Regulated expression of the calmodulin-re-lated TCH genes in cultured Arabidopsis cells: Inductionby calcium and heat shock, Proc. Natl. Acad. Sci. USA89 (1992) 3213–3216.

[217] V. Raz, R. Fluhr, Calcium requirement for ethylene-de-pendent responses, Plant Cell. 4 (1992) 1123–1130.

[218] A.F. Monroy, F. Sarhan, R.S. Dhindsa, Cold-inducedchanges in freezing tolerance, protein phosphorylation,and gene expression, Plant Physiol. 102 (1993) 1227–1235.

[219] E. Lam, M. Benedyk, N.-H. Chua, Characterization ofphytochrome-regulated gene expression in a photoau-totrophic cell suspension: possible role for calmodulin,Mol. Cell. Biol. 9 (1989) 4819–4823.

[220] G. Neuhaus, C. Bowler, R. Kern, N.-H. Chua, Calcium/calmodulin-dependent and-independent phytochromesignal transduction pathways, Cell 73 (1993) 937–952.

[221] G. Neuhaus, C. Bowler, K. Hiratsuka, H. Yamagata,N.H. Chua, Phytochrome-regulated repression of geneexpression requires calcium and cGMP, EMBO J. 16(1997) 2554–2564.

[222] J. Leon, E. Rojo, E. Titarenko, J.J. Sanchez-Serrano,Jasmonic acid-dependent and -independent wound sig-nal transduction pathways are differentially regulated byCa2+/calmodulin in Arabidopsis thaliana, Mol. Gen.Genet. 258 (1998) 412–419.

[223] M.R. Knight, N.D. Read, A.K. Campbell, A.J. Tre-wawas, Imaging calcium dynamics in living plants usingsemi-synthetic recombinant aequorins, J. Cell. Biol. 121(1993) 83–90.

[224] E. Kiegle, C.A. Moore, J. Haseloff, M.A. Tester, M.R.Knight, Cell-type-specific calcium responses to drought,salt and cold in the Arabidopsis root, Plant J. 23 (2000)267–278.

[225] M.R. McAnish, A.M. Hetherington, Encoding specific-ity in Ca2+ signalling systems, Trends Plant Sci. 3 (1998)32–36.

[226] P.K. Hepler, Tip growth in pollen tubes: Calcium leadsthe way, Trends Plant Sci. 2 (1997) 79–80.

[227] A. Trewavas, How plants learn, Proc. Natl. Acad. Sci.USA 96 (1999) 4216–4218.

[228] I. Staxen, C. Pical, L.T. Montgomery, J.E. Gray, A.M.Hetherington, M.R. McAinsh, Abscisic acid inducesoscillations in guard-cell cytosolic free calcium thatinvolve phosphoinositide-specific phospholipase C, Proc.Natl. Acad. Sci. USA 96 (1999) 1779–1784.

[229] A. Haley, A.J. Russell, N. Wood, A.C. Allan, M. Knight,A.K. Campbel, A.J. Trewavas, Effects of mechanicalsignalling on plant cell cytosolic calcium, Proc. Natl.Acad. Sci. USA 92 (1995) 4124–4128.

[230] R.Y. Tsien, A. Miyawaki, Seeing the machinery of livecells, Science 280 (1998) 1954–1955.

[231] P.J. Krysan, J.C. Young, F. Tax, M.R. Sussman, Iden-tification of transferred DNA insertions within Ara-bidopsis genes involved in signal transduction and iontransport, Proc. Natl. Acad. Sci. USA 93 (1996) 8145–8150.

[232] M. Schena, R.A. Heller, T.P. Theriault, K. Konrad, E.Lachenmeier, R.W. Davis, Microarrays: biotechnology’sdiscovery platform for functional genomics, TrendsBiotechnol. 16 (1998) 301–306.

[233] Y. Saijo, S. Hata, J. Kyozuka, K. Shimamoto, K. Izui,Over-expression of a single Ca2+-dependent proteinkinase confers both cold and salt/drought tolerance onrice plants, Plant J. 23 (2000) 319–327.

[234] M.J. Chrispeels, L. Holuigue, R. Latorre, S. Luan, A.Orellana, H. Pena-Cortes, N.V. Raikhel, P.C. Ronald,A. Trewavas, Signal transduction networks and thebiology of plant cells, Biol. Res. 32 (1999) 35–60.

[235] D. Takezawa, A rapid induction by elicitors of themRNA encoding CCD-1, a 14kDa Ca2+-bindingprotein in wheat cultured cells, Plant Mol. Biol. 42 (2000)807–817.

[236] V. Ling, R.E. Zielinski, Isolation of an ArabidopsiscDNA sequence encoding a 22 kDa calcium-bindingprotein (CaBP-22) related to calmodulin, Plant Mol.Biol. 22 (1993) 207–214.

[237] M.L. Khan, C.B. Gogonea, Z.K. Siddiqui, M.Y. Ali, R.Kikuno, K. Nishikawa, S.S. Siddiqui, Molecular cloningand expression of the Caenorhabditis elegans klp-3, anortholog of C terminus motor kinesins Kar3 and ncd, JMol. Biol. 270 (1997) 627–639.

[238] F. Ferreira, E. Engel, P. Briza, K. Richter, C. Ebner, M.Breitenbach, Characterization of recombinant Bet v 4, abirch pollen allergen with two EF-hand calcium-bindingdomains, Int. Arch. Allergy Immunol. 118 (1999) 304–305.

[239] T. Furumoto, N. Ogawa, S. Hata, K. Izui, Plant calcium-dependent protein kinase-related kinases (CRKs) do notrequire calcium for their activities, FEBS Lett. 396 (1996)147–151.

[240] A.S.N. Reddy, I.S. Day, The role of cytoskeleton and amolecular motor in trichrome morphogenesis, TrendsPlant Sci. (2000) (in press).

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