AR242-PP56-17 ARI 28 March 2005 18:28

Plant-SpecificCalmodulin-Binding ProteinsNicolas Bouche,1 Ayelet Yellin,2

Wayne A. Snedden,3 and Hillel Fromm2

1Institut National de la Recherche Agronomique, Institut Jean-Pierre Bourgin,Laboratoire de Biologie Cellulaire, 78026 Versailles, France;email: bouche@versailles.inra.fr2Department of Plant Sciences, Tel Aviv University, Tel Aviv, 69978 Israel;email: yellinay@post.tau.ac.il, hillelf@post.tau.ac.il3Department of Biology, Queen’s University, Kingston, Ontario, K7L 3N6, Canada;email: sneddenw@biology.queensu.ca

Annu. Rev. Plant Biol.2005. 56:435–66

doi: 10.1146/annurev.arplant.56.032604.144224

Copyright c© 2005 byAnnual Reviews. All rightsreserved

First published online as aReview in Advance onJanuary 18, 2005

1543-5008/05/0602-0435$20.00

Key Words

calcium, signal transduction, environmental stress, Arabidopsis

Abstract

Calmodulin (CaM) is the most prominent Ca2+ transducer in eukaryoticcells, regulating the activity of numerous proteins with diverse cellularfunctions. Many features of CaM and its downstream targets are sim-ilar in plants and other eukaryotes. However, plants possess a uniqueset of CaM-related proteins, and several unique CaM target proteins.This review discusses recent progress in identifying plant-specific CaM-binding proteins and their roles in response to biotic and abiotic stressesand development. The review also addresses aspects emerging from re-cent structural studies of CaM interactions with target proteins relevantto plants.

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Contents

INTRODUCTION: THELANGUAGE OF CALCIUMSIGNALING . . . . . . . . . . . . . . . . . . . . . 436Ca2+ as a Signal Carrier in Living

Organisms . . . . . . . . . . . . . . . . . . . . . .436Ca2+-Modulated Proteins in Plants . 437

CALMODULIN-TARGETINTERACTIONS . . . . . . . . . . . . . . . . 439Structural Analysis and Functional

Implications . . . . . . . . . . . . . . . . . . . . 439The Repertoire of CaM Target

Proteins in Plants . . . . . . . . . . . . . . . 441CALMODULIN AND PLANT

RESPONSE TO ABIOTICSTRESS . . . . . . . . . . . . . . . . . . . . . . . . . . 443Response to Osmotic Stress and

Salinity . . . . . . . . . . . . . . . . . . . . . . . . . 444Response to Cold and Heat

Stresses . . . . . . . . . . . . . . . . . . . . . . . . .445CaMBPs and Oxidative Stress . . . . . . 446Activation of Glutamate

Decarboxylase byEnvironmental Stresses andProduction of GABA . . . . . . . . . . . . 447

Tolerance to XenobioticCompounds . . . . . . . . . . . . . . . . . . . . 448

CALMODULIN AND PLANTRESPONSE TO BIOTICSTRESS . . . . . . . . . . . . . . . . . . . . . . . . . . 449CaMs and CMLs in Pathogen

Response . . . . . . . . . . . . . . . . . . . . . . . 449CaM Targets and Pathogen

Response . . . . . . . . . . . . . . . . . . . . . . . 451CALMODULIN AND PLANT

DEVELOPMENT . . . . . . . . . . . . . . . . 453CaMBPs Responding to Hormonal

Treatment . . . . . . . . . . . . . . . . . . . . . . 453CaM and CaMBPs Involved

in the Development ofPolarized Cells . . . . . . . . . . . . . . . . . .454

CONCLUDING REMARKS ANDFUTURE DIRECTIONS . . . . . . . . . 456

INTRODUCTION: THELANGUAGE OF CALCIUMSIGNALING

Ca2+ as a Signal Carrier in LivingOrganisms

All organisms continually monitor their envi-ronment and respond to changes with adap-tive mechanisms that are initiated at the molec-ular and biochemical levels and require thecoordination of cellular events to ensure thata response is appropriate for a given stimu-lus. Therefore, complex intra- and intercellularcommunication networks have evolved to con-vey information about a perceived stimulus tothe cellular machinery responsible for mediat-ing the responses. Organisms use various smallorganic and inorganic molecules, termed sec-ond messengers (e.g., Ca2+, cyclic nucleotides,phospholipids, sugars, amino acids), to encodeinformation and deliver it to downstream effec-tors, which decode signals and initiate cellularresponses including changes in enzyme activity,gene expression, transport across membranes,and cytoskeletal rearrangement. Ca2+ is one ofthe most prominent second messengers in eu-karyotes, and its roles as a signal carrier are thesubject of intensive investigations in both an-imals and plants. The reader is also referredto more specific reviews on different aspects ofCa2+ signaling in plants (73, 76, 145, 174).

In recent years, there has been a major ef-fort directed toward elucidating the informa-tion carried in the Ca2+ signals evoked by ex-ogenous stimuli. Spatial and temporal propa-gation of the Ca2+ signals, the amplitude ofthe signal, which is typically proportional to thestrength of the stimulus, and the frequency ofoscillations are all elements of information car-ried by the Ca2+ signal that must be decodedby the cellular machinery. Spatial distributionof the Ca2+ signal in the cell is controlled bya complex network of Ca2+-permeable chan-nels, Ca2+-antiporters, and Ca2+-pumps, whichoperate at the plasma membrane or in mem-branes of intracellular Ca2+ stores including theER, mitochondria, chloroplast, and the vacuole,

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and are regulated by different second messen-gers (reviewed in 76). Of particular interest isthe relationship between cytosolic and nuclearCa2+. Ca2+ signals in the cytosol and in the nu-cleus have distinct functions in both animals(13, 18, 71) and plants (169). Recent investi-gations (13, 132) suggest that although nuclearCa2+ signals in many cases reflect the patternsof cytosolic Ca2+, the nucleus can also generatestimulus-induced signals independently. Thereis evidence that the nucleus contains intrinsicCa2+ signaling machinery that can release Ca2+

locally in discrete nuclear regions. Echevarrıaet al. (54) identified a nucleoplasmic reticu-lum in epithelial cells, composing a branch-ing intranuclear network continuous with thenuclear envelope and the endoplasmic retic-ulum. These structures function as a nuclearCa2+-storing network that can give rise to local-ized Ca2+ gradients and thus Ca2+-dependentevents can be regulated differentially in the nu-cleus, just as they are in the cytosol. It is likelythat plants possess similar intranuclear Ca2+

stores.One important question concerning the role

of Ca2+ as a second messenger is how cells con-trol stimulus-response specificity. There is ev-idence that the frequency of Ca2+ oscillationscarries information that is decoded by cellulartargets in both plants and animals. In plantsthis was elegantly demonstrated by studyingthe Arabidopsis det3 mutant, which is unable toclose its stomata in response to exogenous Ca2+.However, stomatal closure could be restored indet3 by subjecting it to artificial Ca2+ oscilla-tions (4). Changes in the frequency or dura-tion of the Ca2+ oscillations influenced stom-atal aperture. These studies indicate a specificmechanism to translate Ca2+ signals into a cel-lular response (3, 5). The nature of these de-coders is intriguing. One can speculate that de-coding Ca2+ signals requires proteins that canrespond to Ca2+ oscillations by fine conforma-tional changes. De Koninck & Schulman (44)found that the CaM-dependent protein Kinase-II is sensitive to the frequency of Ca2+ oscil-lations in vitro in a way that is reflected inthe autonomous kinase activity, suggesting that

ER: endoplasmicreticulum

CaM: calmodulin

Calmodulin: CaM isa ubiquitous Ca2+sensor protein (16 to18 kD) with nocatalytic activity thatcan, upon bindingCa2+, activate targetproteins involved invarious cellularprocesses. The CaMprototype is comprisedof two globulardomains connectedwith a long flexiblehelix. Each globulardomain contains a pairof intimately linked EFhands. One EF handmotif is composed of aspecializedhelix-loop-helixstructure that bindsone molecule of Ca2+.

CaMKII is a decoder of the frequency of Ca2+

oscillations. Other studies showed that oscilla-tions increase both the efficacy and informa-tion content of Ca2+ signals that lead to geneexpression and cell differentiation (50). Certaintranscription factors are activated only by high-frequency Ca2+ oscillations, whereas othersmay be activated by infrequent oscillations (50)or both high-frequency and low-frequency os-cillations. A recent review addressed the role ofplant protein kinases in decoding Ca2+ signals(73). Given the importance of Ca2+-modulatedproteins in decoding Ca2+ signals, it is impor-tant to consider the repertoire of plant Ca2+-modulated proteins.

Ca2+-Modulated Proteins in Plants

Most proteins that function as transducers ofCa2+ signals contain a common structural mo-tif, the “EF hand” (125), which is a helix-loop-helix structure that binds a single Ca2+ ion.These motifs typically, but not exclusively, oc-cur in closely linked pairs, interacting throughantiparallel β-sheets (125). This arrangementis the basis for cooperativity in Ca2+ binding.The superfamily of EF-hand proteins is dividedinto several classes based on differences in num-ber and organization of EF-hand pairs, aminoacid sequences within or outside the motifs,affinity to Ca2+, and/or selectivity and affinityto target proteins (39, 125). Nakayama et al.(124) discussed the evolution of EF-hand pro-teins and divided them into 66 subfamilies. Dayet al. (42) reported a comprehensive bioinfor-matic search for EF-hand-containing proteinsin Arabidopsis. Other reviews discuss specificfamilies of EF-hand proteins in plants includingthe CaM superfamily (119) and protein kinases(72, 73). Note that there are other protein mo-tifs that bind Ca2+. One is the 70-amino acidannexin fold, which is present in members ofthe membrane-associated annexin subfamily ofCa2+ sensors. Another Ca2+-binding motif isthe C2 domain of about 130–145 amino acids,present in membrane-associated proteins, ofwhich over 140 are found in the Arabidopsisgenome. Reddy & Reddy (145) discussed the

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Calmodulin-likeprotein: CaM-likeproteins (CMLs) arecomposed mostly ofEF-handCa2+-binding motifs,with no otheridentifiable functionaldomains, and are atleast 16% identical toCaM, as defined byMacCormack &Braam (119). Plantspossess many moreCaMs and CMLs thanany other knownorganism. Forexample, Arabidopsispossesses six CAMloci, encoding threeCaM isoforms, and∼50 CML genes.

occurrence of these Ca2+-binding motifs inplant proteins in more detail.

In plants, several classes of EF-hand-containing Ca2+-modulated proteins wereidentified. The first of these is the family ofCaMs, which have different isoforms and CaM-related proteins. According to the definitionsof MacCormack & Braam (119), for CaM iso-forms and CaM-like proteins (CMLs), Ara-bidopsis possesses 6 CaM loci, defined as CAM,which encode 3 isoforms, and ∼50 additionalgenes, defined as CAM-like (CML), which aregenes encoding proteins composed mostly ofEF-hand Ca2+-binding motifs, with no otheridentifiable functional domains, and at least16% are identical to CaM. However, plantsalso possess species-specific CaM-related pro-teins that are not found in Arabidopsis. For ex-ample, petunia CaM PhCaM53 is highly sim-ilar to the rice CaM OSCaM61 because it hasa stretch of basic amino acids as an extensionat the C terminus. This extension undergoesprenylation in response to specific changes incarbon metabolism. PhCaM53 is targeted tothe nucleus in the dark, or to the plasma mem-brane in the light. However, if a carbon sourceis provided exogenously in the dark, PhCaM53will target to the plasma membrane (115, 147).There is no homolog of PhCaM53 in Arabidop-sis, although there are other CMLs in Arabidop-sis with different types of C-terminal extensionsthat might function similarly to PhCaM53. An-other example of a species-specific CaM-relatedprotein is the rgs-CaM, which was identifiedas a regulator of gene silencing in tobacco (6).The protein has 3 EF-hands and a 50-aminoacid long N-terminal extension. The Arabidop-sis genome does not contain a gene encodinga protein with a similar N-terminal extension.In cultivated hexaploid wheat, which has thelargest known family of CaM genes in a sin-gle plant (188), one CaM isoform, designatedTaCaM-III, lacks the first EF-hand and insteadcontains a hydrophobic domain with a tryp-tophan residue, which is typically absent fromCaM (188). One of the fascinating aspects ofCa2+ signaling in plants is the occurrence ofthis large family of CaM-related proteins with

species-specific isoforms, which is a sharp con-trast to the situation in animals with only oneCaM isoform encoded by three genes. It is likelythat N- or C-terminal extensions of CaMs andCMLs underlie specific physiological roles. Inaddition to the functional roles of CaM exten-sions, many plant CaM isoforms differ fromothers by a few specific amino acids substitu-tions. The functional physiological relevanceof these amino acid substitutions in providingstimulus-response specificity is beginning to beelucidated in vivo (75). In vitro studies supportthe idea that CaM isoforms differ in their abilityto activate target enzymes (34, 35, 95, 106–108).

Another family of EF-hand-containingCa2+-modulated proteins in plants is the cal-cineurin B-like proteins (CBLs). A member ofthis family is SOS3, a regulator of salt tolerance.It activates the SOS2 protein kinase (69), whichis a regulator of SOS1 and AtNHX1, the plasmamembrane and tonoplast Na+/H+ antiporter,respectively. In Arabidopsis CBLs are encodedby 10 genes (115) that activate protein kinasesrelated to the Suc-Non-Fermenting (SNF) pro-tein kinase from yeast. In plants these proteinkinases are referred to as CBL-Interacting Pro-tein Kinases (CIPKs), of which Arabidopsis pos-sesses 25 genes (115). A third family of EF-hand-containing Ca2+-sensor proteins in plantsare the SUB and SUL proteins involved in pho-tomorphogenic responses (68). The SUB1 pro-tein possesses a DNA-binding domain, whichsuggests that it may combine the function ofCa2+ sensing and transcriptional regulation,similar to the mammalian DREAM protein(27).

Other prominent Ca2+-modulated proteinsin plants are the Ca2+-regulated protein ki-nases, which possess a catalytic kinase do-main and a regulatory domain with EF-handor visinin-like Ca2+-binding motifs (72, 73).The Ca2+-dependent protein kinases (CDPKs;reviewed in 32), the Ca2+/CaM-regulated ki-nases, and the chimeric Ca2+ and Ca2+/CaMregulated kinases (CCaMKs), are among thisfamily of Ca2+-regulated protein kinases. TheCDPK family alone consists of 34 genes in Ara-bidopsis (72, 73).

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A bioinformatic approach (42) revealed nu-merous new potential plant Ca2+-binding pro-teins containing EF-hand domains, some ofwhich lack functional domains other than theCa2+-binding sites (e.g., recoverin-like pro-teins), whereas some include diverse functionalgroups. Day et al. (42) estimated that, over-all, Arabidopsis contains ∼250 Ca2+-responsiveEF-hand-containing proteins, and these likelymodulate the activity of an even larger reper-toire of downstream targets. Among theidentified Ca2+-responsive proteins, some arepredicted to be targeted to the chloroplast andmitochondria, others to the nucleus or cytosol.The estimated predicted number of EF-hand-containing proteins in Arabidopsis is the largestamong all organisms with complete genomessequenced (42). This may reflect the need ofplants, as sessile organisms, to modulate theirentire metabolism, growth, and development inresponse to changes in their environment.

CALMODULIN-TARGETINTERACTIONS

Structural Analysis and FunctionalImplications

CaM is one of the best characterized Ca2+-responsive proteins. It has no catalytic activ-ity of its own but, upon binding Ca2+, acti-vates numerous target proteins involved in avariety of cellular processes. CaM is an acidicEF-hand protein present in all eukaryotes. TheCaM prototype is composed of 148 amino acidsarranged in two globular domains connectedwith a long flexible helix (Figure 1a). Eachglobular domain contains a pair of intimatelylinked EF hands. The majority of known tar-get sites for CaM are composed of a stretch of12–30 contiguous amino acids with positivelycharged amphiphilic characteristics, variabilityin primary sequence, and a propensity to forman α-helix upon binding to CaM. Early X-raydiffraction and NMR studies of CaM provideda model for the structural basis of CaM-targetinteractions (39). Binding Ca2+ to CaM (Kd

in the range of 10−7 to 10−6 M) (Figure 1b)

NMR: nuclearmagnetic resonance

exposes two hydrophobic surfaces surroundedby negative charges, one in each globular do-main. Ca2+/CaM may then bind to its tar-gets mainly by hydrophobic interactions withlong hydrophobic side chains in the target sites.Electrostatic interactions contribute to the sta-bility of the CaM-target complex (CaM tar-gets are depicted in red in Figure 1). Thefirst 3D structures of CaM target peptide com-plexes to be resolved suggested that the twoglobular domains of CaM wrap around the tar-get, forming an almost globular structure (e.g.,CaMKII; Figure 1c). However, as more 3Dstructures of CaM/target complexes have beenresolved, different types of unexpected inter-actions have been revealed (77, 171, 181). Interms of CaM:target stochiometry, recent datarevealed ratios of 1:1 (Figure 1c,d; CaMKIIand a Ca2+-pump, respectively), 2:2 (Figure 1e;a potassium channel), and 1:2 [Figure 1f; petu-nia glutamate decarboxylase (GAD)]. Second,although some targets interact with both theC- and N-terminal lobes of CaM (Figure 1c,d,e,f ), others interact with only one lobe(Figure 1d). Also note that Ca2+-independentinteractions of CaM, or CaM complexes withjust two bound Ca2+ ions (e.g., Figure 1e), havebeen resolved (reviewed in 77). The latter isconsistent with genetic studies demonstratingthat a yeast mutant expressing a CaM unableto bind any Ca2+ can still rescue an otherwiselethal mutant that completely lacks CaM (58).Recent structural findings also support earliergenetic studies of yeast CaM mutants bearingone or more substitutions of phenylalanine toalanine (129). This report showed that muta-tions affecting specific cellular functions such asCaM localization and nuclear division were alllocated in the N-terminal lobe of CaM, whereasmutations implicated in actin organization andbud emergence were located in the C-terminalhalf (129).

The recent data from 3D structures revealedseveral types of CaM-binding motifs (77) andprovided the ability to cautiously predict, basedon amino acid sequence, protein interactionswith CaM (http://calcium.uhnres.utoronto.ca). However, there are clearly still unidentified

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Figure 1Ribbon presentationof the 3D structure ofCaM and CaM incomplex with Ca2+and target proteins.CaM is blue, CaMtargets are red, andCa2+ ions are green.The N-terminal lobeof CaM is orientedtoward the top, andthe C-terminal lobe istoward the bottom ofthe figures. Structuraldata are taken from theProtein Data Bank,accession codes:(a) apo-CaM (1CFD),(b) Ca2+/CaM(3CLN),(c) Ca2+/CaM/CaMKIIa (1CM1),(d) Ca2+/CaM/Ca2+pump (1CFF),(e) Ca2+/CaM/K+channel (1G4Y),(f) Ca2+/CaM/petuniaGAD (1NWD).Structures are derivedfrom X-ray diffractionanalysis of crystalstructures (a–c, e) orfrom NuclearMagnetic Resonance(NMR) analysis (d, f ).Figures were preparedwith the MOLMOLprogram(http://129.132.45.141/wuthrich/software/molmol/) (96).

CaM-binding motifs, and some proteins knownto bind CaM do not possess typical motifs. Re-cently, the first 3D structure of a CaM-bindingdomain (CaMBD) of a plant protein [glutamate

decarboxylase (GAD)] associated with CaMwas resolved (189). The 3D structure of thepetunia CaM-binding peptide associated withCaM (Figure 1f ) shows the ability of CaM to

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interact with two GAD CaMBDs, suggesting arole for CaM in GAD dimerization and possi-bly in the formation of a high molecular weightoligomeric complex. These large GAD com-plexes (∼500 kDa) are found in plants (16)and are stabilized by Ca2+ and destabilizedin the presence of Ca2+-chelators (16). Coim-munoprecipitation experiments show that CaMis associated with the native GAD complexin vivo (16). It remains unknown whetherCaM’s role in dimerization of GAD subunitsand in GAD activation are separable functions.Possibly, activation of GAD requires the forma-tion of a large oligomeric complex that dependson Ca2+/CaM.

Recently, the crystal structure of a kinesin-like CaM-binding protein (KCBP) from potatowas resolved (172). Together with biochemi-cal data, the crystal structure of KCBP suggeststhat Ca2+/CaM inhibits the binding of KCBPto microtubules by blocking their binding siteson KCBP.

The structures presented in Figure 1, to-gether with biochemical data from other stud-ies, reveal a number of protein activation/inhibition mechanisms by CaM, as depicted inFigure 2. Relieving autoinhibition has beendemonstrated with several CaM-target inter-actions, including plant and animal Ca2+-ATPases, CaM-dependent kinases, and plantGAD (156). Active-site remodeling is a newtype of mechanism revealed by analyzing theendema factor protein from Bacillus anthracis,the causative agent of anthrax, in which in-teraction with CaM bound to two Ca2+ ionsreorganizes the protein domains to constructthe binding site (reviewed in 77). A simi-lar mechanism may occur with other targetswith different numbers of bound Ca2+ ions.Dimerization at a 2:2 ratio of CaM:target wasrevealed by studying the 3D structure of CaMbound to the small conductance Ca2+-activatedpotassium (SK) channel. In this case, Ca2+ ionsare bound only to the N-terminal EF-handswhile the C-terminal EF motifs mediate tether-ing to the channel. Target activation may occurthrough dimerization and stabilization of a mul-timeric complex at a 1:2 ratio, as found for the

CaMBD:calmodulin-bindingdomain

Calmodulin-bindingdomain: Target sitesfor CaM are defined asCaMBDs and aretypically composed ofa stretch of 12–30contiguous aminoacids with positivelycharged amphiphiliccharacteristics.Although theirprimary sequencevaries, they usuallyform an α-helix uponbinding to CaM.Recent structuralstudies revealed newand unexpected typesof CaM-proteininteractions andCaMBDs.

CNGC:cyclic-nucleotide gatedchannel

plant GAD CaMBD with CaM (189). There isalso evidence for target inactivation by occupy-ing a ligand-binding site. This possible mech-anism emerges from studies of plant CNGCs.Inactivation of CNGCs by Ca2+/CaM occursin plants and in other organisms. However,in plant CNGCs the binding sites for CaMand Cyclic Nucleotide Monophosphates (cN-MPs) overlap (9), unlike in the mammalianproteins where their respective binding sitesare well separated. In the plant CNGCs (ofwhich 20 genes exist in the Arabidopsis genome)(118, 162), one of three conserved α-helicesthat constitute a conserved cNMP-binding do-main retained the ability to bind CaM (9). Func-tional analysis of a plant CNGC in a heterol-ogous system showed that Ca2+/CaM inhibitscNMP-mediated channel activation (80). Thisfunctional model might also occur with KCBP,where Ca2+/CaM occupies a domain that wouldotherwise bind microtubules (172).

Finally, note that because each plant pos-sesses a large number of CaM and CML pro-teins (e.g., more than 50 in Arabidopsis), it is notalways clear which of these proteins interactsin vivo with the identified CaMBPs. Interac-tion of different CMLs with the same CaMBPsmay occur in vivo under different physiologi-cal situations. These interactions will likely re-sult in different conformational changes of theCaMBPs, different sensitivities to Ca2+, anddifferent downstream cellular effects. A majorchallenge in the coming years will be to resolvethese numerous types of interactions and thecorresponding physiological responses.

The Repertoire of CaM TargetProteins in Plants

Molecular approaches for screening cDNA ex-pression libraries using labeled recombinantCaM as a probe (56, 110, 113) proved a pow-erful tool in identifying the cellular targets ofCaM in plants as well as in other organisms.Screening cDNA libraries derived from variousplant species, organs, and cell types, and fromplants exposed to a variety of stimuli, revealedmany clones encoding CaM-binding proteins

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(CaMBP). Using bioinformatics tools has re-cently enabled the identification of several newgenes encoding potential CaMBPs in the Ara-bidopsis genome (142). The reader is also re-ferred to recent reviews on the roles of CaMas a Ca2+ signal transducer in plants (142, 155,187, 195).

Table 1 (Follow the Supplemental Mate-rial link from the Annual Reviews home pageat http://www.annualreviews.org) is an up-dated version of the list of CaMBPs describedby Snedden & Fromm (155) summarizing therepertoire of plant proteins for which some ev-idence suggests regulation by (or associationwith) CaM. The table reflects the functional di-versity of plant CaMBPs and reveals an involve-ment in regulation of metabolism, cytoskeleton,ion transport, protein folding, transcription,protein phosphorylation and dephosphoryla-tion, phospholipid metabolism, and unknownfunctions. Considering that some of the pro-teins listed are encoded by relatively large genefamilies (e.g., 20 CNGC genes, 12 ACA genesin Arabidopsis), and given the potential for al-ternative splicing, the total number of differentCaMBPs in a plant could easily reach severalhundred.

Of the proteins listed in Table 1 more than20 are plant-specific CaMBPs with no obvioushomologs in other organisms (proteins markedas ∗). This group includes, for example, thechimeric Ca2+-resposive CaM-binding proteinkinases (CCaMKs), a PP7 ser/thr phosphatase,the pollen-specific protein NPG1, and others.

About 20 proteins have homologs in other or-ganisms but are likely regulated by CaM onlyin plants (proteins marked as ∗∗). This groupincludes, for example, glutamate decarboxylase(although it may be CaM regulated in yeast),apyrase, and NAD kinase. The remainder areproteins whose homologs are regulated by CaMin other organisms as well as in plants (pro-teins marked as ∗∗∗). Examples of this group in-clude CaM-regulated plasma membrane Ca2+-ATPases, protein kinases, and kinesin. In thisreview, we define plant-specific CaMBPs asthose lacking a highly homologous protein inother organisms (∗) or proteins whose probablehomologs in other organisms do not bind CaM(∗∗). The occurrence of many plant-specificCaMBPs likely reflects the extensive use of theCaM messenger system in plants, particularly inrelation to responses to biotic and abiotic stress,and in development, as described later.

CALMODULIN AND PLANTRESPONSE TO ABIOTIC STRESS

Signaling pathways involved in the responseto environmental stresses form interconnectednetworks in which Ca2+ plays a major role (re-viewed in 94). Many abiotic stimuli can inducea transient cytosolic Ca2+ increase (reviewed in76, 127, 174). Gene expression of Ca2+ sen-sors such as the CaMs and CMLs is often in-duced in response to various abiotic stresses (re-viewed in 155), as has been observed for otherCa2+ sensors, such as CDPKs (116), calcineurin

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 2Schematic presentation of the mechanisms of CaM activation or inhibition of target proteins. (a) Relievingautoinhibition: Ca2+/CaM binding to the target displaces the autoinhibitory domain (AID) from the activesite, thus allowing enzyme activation. (b) Active-site remodeling: Ca2+/CaM binding confers aconformational change that stabilizes the active site and allows enzyme activation. (c) Target dimerization:Two CaM molecules interact with two K+ channel domains. Upon Ca2+ binding the two channel domainsdimerize, thus opening the channel for ion transport. (d) One Ca2+/CaM binds two CaM-binding domains(CaMBDs) of petunia glutamate decarboxylase (GAD). One CaMBD interacts with the N-terminal lobe ofCaM, the other with the C-terminal lobe. (e) Ca2+/CaM binds to a site that coincides with the cyclicnucleotide monophosphate (cNMP)-binding site of plant cyclic-nucleotide gated channels (CNGCs). Thus,Ca2+/CaM may displace bound cNMP and/or prevent binding of cNMP, causing the closure of the channel.Green, target proteins; pink, N-terminal lobe of CaM; orange, C-terminal lobe of CaM; blue, Ca2+ ions;blue arrows point to the direction of ion movement through channels; purple, cNMP (cAMP or cGMP).

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B-like proteins (33), or annexins (104). For in-stance, three proteins encoded by genes in-duced by touch stimulation (TCH genes; i.e.,touch-induced) encode CaM or CML pro-teins (26). Out of the conserved CaMs and the∼50 CML genes present in Arabidopsis, sev-eral seem well represented by ESTs in librariesgenerated from plants subjected to stress orhormone treatment (119). Collectively, thesefindings support the idea that CaM and CMLproteins are involved in response to environ-mental stimuli likely via activation of specificCaMBPs. Evidence to support this hypothesisis discussed below.

Response to Osmotic Stressand Salinity

At the cellular level, one of the primary re-sponses to environmental stresses such as anhyperosmotic stress (i.e., water deficit or highsalinity) or a hypo-osmotic stress (i.e., an in-crease in environmental water potential) is atransient elevation of Ca2+ concentrations (re-viewed in 174). Changes in Ca2+ levels canbe monitored using transgenic plants express-ing Ca2+ reporters such as aequorin. In theseplants, repeated exposures to NaCl treatmentsprovoke prolonged alterations of both cytoplas-mic and apoplastic Ca2+ concentrations (57).This elevation of Ca2+ concentration is also ob-served in the cytoplasm when plants are exposedto hypo-osmotic stress (57). A stress-inducedCa2+ signal is likely detected by Ca2+ sen-sors such as annexins that are Ca2+-dependentmembrane-binding proteins (104) or proteinsof the salt overly sensitive (SOS) signaling path-way in which the Ca2+-binding protein SOS3can activate the SOS2 protein kinase (reviewedin 64, 193, 194). CaM also contributes to thesensing of Ca2+ signals caused by osmotic or saltstresses. The transcription of CaM genes is in-duced when cultured cells from tomato (49) oryoung mung bean Vigna radiate (20) are exposedto salinity. Transgenic tobacco plants expressinga heterologous bovine CaM germinate faster onmedium containing high levels of NaCl com-pared to the control (130). Thus, although CaM

is probably a regulatory component during re-sponses to osmotic and salinity stress, only a fewCaMBPs involved in these pathways have beenidentified.

Some CaMBPs are induced by salt or os-motic stress treatment, based on gene expres-sion data. In tobacco, a Ca2+/CaM-binding ki-nase (NtCBK2) (82) is induced in high-saltconditions and GA treatment whereas otherhormones (i.e., auxin, ABA), or exposure tocold or heat did not affect the expression ofthe NtCBK2 (81). In situ hybridizations re-vealed that NtCBK2 is expressed in tobacco an-ther, pistil, and embryo (81). In Physcomitrellapatens, screening a cDNA library with radiola-beled CaM revealed a novel class of CaMBP(MCamb) sharing homologies with the mam-malian ATP-sensitive potassium channels ofthe Kir family (161). The CaMBDs of bothMCamb1 and MCamb2 were mapped and ex-pression analysis revealed that MCamb1 is in-duced by mannitol and NaCl (161). There areother examples of CaMBPs whose transcriptionis induced by salt or osmotic stresses (Table 1),but the specificity of this activation is unclearand not exclusive to salinity or osmotic stress.For example, some members of the CAMTA(CaM-binding transcription activator) familyare induced by salt but also by ethylene, methyljasmonate, abscisic acid, H2O2, salicylic acid,UV, heat, and cold (185). As such, these proteinsare probably involved in multiple pathways as-sociated with environmental stress response.

The role of two CaMBPs has been specif-ically addressed in relation to salt or osmoticstresses. In Arabidopsis, ACA4 encodes a vac-uolar Ca2+-ATPase containing a CaMBD inthe N terminal part of the protein (59), unlikethe animal counterpart where the CaMBD islocated in the C terminal. The role of ACA4was addressed in a yeast strain deficient for thetransport of Ca2+ (i.e., lacking both endogenousCa2+-ATPases and the calcineurin regulatorysubunit B). Only a truncated form of ACA4,lacking the N-terminal CaMBD, could restorethe growth of the yeast mutant on mediumcontaining low Ca2+. ACA4 is an autoinhib-ited ATPase and, like the CaMBD, the AID is

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located in the N terminus. CaM binding likelyrelieves autoinhibition. Thus, the truncatedform of ACA4 can restore the growth ofyeast cells because the corresponding protein isconstitutively active. Quantitative RT-PCR ex-periments revealed that the ACA4 mRNAaccumulates in a dose-dependent manner inArabidopsis seedlings treated with increasingamounts of NaCl, raising the possibility thatACA4 could play a role in salt stress tolerance.Previous studies had shown that mRNA fromCa2+-ATPases accumulates upon NaCl treat-ment in tomato (175) and soybean (37). To fur-ther address this question, the growth of theyeast mutant expressing the full-length or thetruncated form of ACA4 was tested under dif-ferent stress conditions. Both conferred protec-tion against osmotic stress such as high levels ofNaCl, KCl, and mannitol. The most adverse ef-fect occurred in the deregulated form of ACA4(i.e., lacking the N terminus). Thus, it is likelythat during conditions of osmotic stress, ACA4is stimulated by Ca2+/CaM, leading to therapid replenishment of Ca2+ stores such as thevacuole.

Recently, a new CaMBP (AtCaMBP25) in-volved in osmotic stress regulation was iso-lated from Arabidopsis (133). AtCaMBP25 is anuclear-targeted protein that shows differentialaffinity to Arabidopsis CaM and CMLs. North-ern blot analysis revealed that AtCaMBP25expression is induced by mannitol treatment,high salinity, cold, and seedling dehydra-tion. The germination of transgenic seedlingsoverexpressing AtCaMBP25 was inhibited onmedium containing mannitol or salt, whereasantisense lines showed increased tolerance tothese stresses. Similarly, root elongation ofseedlings transferred from standard conditionsto medium containing mannitol or NaCl wasretarded for plants expressing the sense trans-gene and improved for antisense lines. How-ever, the response to drought and freezing wasnot altered in these transgenic plants, imply-ing that AtCaMBP25 plays a specific role in os-motic stress regulation. Overall, these data sug-gest that AtCAMBP25 is a negative regulator ofosmotic stress responses.

AID: autoinhibitorydomain

Response to Cold and Heat Stresses

Ca2+ is also involved in plant responses tocold treatment. Tobacco seedlings expressingaequorin reveal that cold and wind can initiatespecific Ca2+ signals that are spatially distinct(169). Genes encoding several CaMs and CMLsare induced by cold treatment and likely partici-pate in translating these signals (26, 169). In ad-dition, Ca2+ transporters have been implicatedin cold response. Cold acclimation enhances theactivity of a plasma membrane Ca2+-ATPasein winter rye leaves (136), and in Arabidopsisa Ca2+/H+ transporter (CAX1) controlled theexpression of cold-induced genes (28). Com-pared to wild-type plants, cax1 mutants showedenhanced freezing tolerance after cold acclima-tion. This directly correlated with the induc-tion of CBF/DREB1 genes that encode a fam-ily of transcription activators that mediate coldacclimation in Arabidopsis. CaM may functionas a negative regulator of cold-induced geneexpression given that cold-treated Arabidopsisplants overexpressing CaM3 show decreasedlevels of COR (cold on regulated) transcripts(164). As noted above, some CaMBPs respondto multiple environmental stimuli, includingcold treatment. Examples include proteins ho-mologous to mammalian K+ channels (161) andthe CAMTA transcription factors (185).

Cells exposed to elevated temperatures re-spond by activating specific signals that medi-ate the heat shock response and mobilize heatshock proteins (HSPs). Ca2+ and CaM are im-portant components in this process. In wheat,both the levels of CaM mRNA and CaM pro-tein increased after heat shock (112). In ad-dition, the expression of two HSP genes andthe accumulation of the corresponding pro-teins were upregulated by the addition of Ca2+

and downregulated by a chelator of Ca2+, Ca2+

channel blockers, or CaM antagonists (112).Therefore, changes in Ca2+ signals can mimicthe effect of heat shock and induce the expres-sion of HSP genes. Several CaMBPs functionduring the heat shock response. Two relatedhigh molecular weight FK506-binding proteins(FKBPs) (reviewed in 74) from wheat (FKBP73

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and FKBP77) function in protein folding as-sociated with their peptidyl prolyl cis-trans-isomerase activity. These FKBPs possess a pre-dicted CaMBD (Table 1) and their homologsin Arabidopsis bind CaM (142). FKBP77 expres-sion in wheat is induced by heat shock (97),and both wheat FKBPs form complexes withHSP90 (140). Recently, another FKBP fromArabidopsis (TWD1; i.e., AtFKBP42) boundboth HSP90 and CaM (86), a property that isshared with human FKBPs (152). FKBP73 canalso function in vitro as a molecular chaperone,a property also observed for some mammalianFKBPs (98). Transgenic wheat overexpressingFKBP77 present morphological abnormalitiesassociated with higher levels of HSP90 mRNA(99). In addition, cytosolic HSP70 from maizeinteracts with CaM, a phenomenon that in-hibits its intrinsic ATPase activity (159). CaMalso binds to a chloroplast chaperonin (182).Other CaMBPs of unknown function, whoseexpression is induced or suppressed by heatshock, have also been reported, namely TCB48(114) or TCB60 (41, 113) in tobacco and itsArabidopsis homologs (142). Taken together, thedata support a role for CaMBPs in modulatingplant adaptation to temperature stress. How-ever, more genetic and biochemical studies areneeded to assign specific roles to the variousCaMBPs and elucidate which pathways theyparticipate in.

CaMBPs and Oxidative Stress

Plants exposed to environmental stresses oftengenerate reactive oxygen intermediates (ROIs)whose levels must be tightly controlled to avoidcellular damage (reviewed in 7). There seemsto be a relationship between the levels of ROIsand Ca2+ in the cell. In tobacco cell cultures ex-pressing aequorin, a treatment with H2O2 trig-gers a biphasic Ca2+ elevation (103). Similarly,in Arabidopsis seedlings, Rentel & Knight (146)observed a biphasic Ca2+ signature composedof two independent peaks: an early one specificto cotyledons and a second one found in roots.More importantly, the magnitude of the Ca2+

signal correlates with the induction level of

genes responding to oxidative stress and ROIs(146). It is thus likely that redox changes in thecell generate a Ca2+ signal that can modulatecellular responses to oxidative stress by activat-ing specific genes, including certain CaMBPs.In addition, oxidative damage induced by heatstress in Arabidopsis seedlings is exacerbated bypretreating plants with CaM inhibitors (102),suggesting an important role for CaM in oxida-tive stress recovery. Some targets of CaM thatmight participate in oxidative stress responseare described below.

In plants, at least one class of ROI-scaveng-ing proteins seems to be regulated by CaM.Catalases (i.e., hydroperoxidases) are protec-tive enzymes that degrade hydrogen peroxide(H2O2) to water and oxygen. By screening acDNA expression library for CaMBPs, Yang &Poovaiah (186) isolated an Arabidopsis catalaseisoform (AtCat3). The CaM-binding region ofAtCat3 was mapped and the corresponding syn-thetic peptide binds a recombinant CaM ina Ca2+-dependent manner (186). Additionally,a catalase from tobacco leaves could be puri-fied using a CaM-sepharose column (186). Theactivity of the enzyme was stimulated abouttwofold by Ca2+/CaM, but not by Ca2+ or CaMalone. The regulation of catalase by CaM isprobably specific to plants because CaM hadno effect on catalases from sources such as As-pergillus niger, human erythrocytes, and bovineliver (186). In addition, CaM was detected withspecific antibodies in peroxisomes isolated frometiolated pumpkin cotyledons (186). Peroxi-somes are specialized organelles involved in thecatabolic oxidation of various biomolecules andthe resultant H2O2 is consumed by peroxyso-mal catalases. Another class of ROI-scavengingenzymes, the superoxide dismutases (SODs),was suggested to be CaMBPs. A decade ago,SOD from maize germs was shown to reversiblybind CaM immobilized on a column in a Ca2+-dependent manner (65). However, these find-ings were never confirmed in CaM-binding as-says with recombinant SODs or SODs purifiedfrom other sources.

H2O2 has a dual role as both a strong oxi-dant that can damage cellular components and

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as a second messenger. Therefore, H2O2 lev-els must be tightly controlled. H2O2 as a sig-nal mediates responses to biotic and abioticstresses and can activate various pathways in-volved in adaptation to environmental stress(126). In plants, the transcription of genes en-coding CaM-binding transcription factors isstimulated by H2O2. AtBTs (Arabidopsis thalianaBTB and TAZ domain proteins) represent anew family of proteins sharing homologies withtranscription factors (51). All five AtBT pro-teins bind CaM and the transcription of someisoforms is modified within an hour in leavesexposed to H2O2 or salicylic acid. AtBT1 is lo-calized to the nucleus and interacts with mem-bers of the fsh/Ring3 class of transcriptionregulators (51). Database searches reveal nohomologies between AtBT and gene sequencesof nonplant organisms. Similarly, some mem-bers of the CAMTA family of transcription fac-tors are also induced rapidly by H2O2 (185).Plant CAMTAs resemble a group of putativetranscription activators identified in the humangenome (101). CAMTAs are specific to multi-cellular organisms and possess a conserved do-main organization with a novel DNA-bindingregion termed CG-1, a TIG-like DNA-bindingdomain, and ankyrin repeats. Both human andArabidopsis members of this family can activatetranscription in yeast (24). Human CAMTA1functions as a suppressor of neuroblastoma (87)and is transcriptionally regulated in a cell-cycle-dependent manner (123). However, CaM bind-ing to human CAMTAs has yet to be demon-strated. The plant CAMTAs bind CaM in aCa2+-dependent manner (24, 183) and binda DNA motif containing a CGCG element(185). No function has been attributed to BTsor CAMTAs; therefore, the link between thesetranscription activators and the perception ofROI contents is presently unclear. AtBTs andCAMTA transcription factors are induced notonly by H2O2 but by many other treatments,including salicylic acid for AtBT (51) or UV-B, salt, ethylene, and wounding for AtCAMTAs(183). It is thus likely that these proteins are notdirectly involved in sensing ROIs, as are sometranscription factors described in yeast (46) and

GABA:γ -aminobutyric acid

bacteria (91), but rather are acting downstreamof the ROI signal. Finding their gene targetsshould help to reveal their function in ROI andother signaling pathways.

Finally, CaM may participate indirectly inregulating ROI content through the CaM-regulated GABA-shunt metabolic pathway (seebelow). In Arabidopsis, mutants disrupted in thelast enzyme (SSADH) of the pathway are moresensitive to environmental stress because theyare unable to scavenge H2O2 (21). The reac-tion catalyzed by SSADH can provide both suc-cinate and NADH to the respiratory chain. Itwas, therefore, hypothesized that the degrada-tion of GABA could limit the accumulation ofROIs under oxidative stress conditions that in-hibit certain enzymes of the TCA cycle.

Activation of GlutamateDecarboxylase by EnvironmentalStresses and Production of GABA

In plants, one of the best characterizedCa2+/CaM regulated pathways involved in abi-otic stress response is the GABA shunt (re-viewed in 23). The first of the three enzymesof the pathway is GAD, which catalyzes theconversion of glutamate to GABA. Cloning thepetunia GAD gene and characterizing the en-coded enzyme as a Ca2+/CaMBP (8, 15) re-vealed that GAD activity is upregulated byCa2+/CaM in many plant species, including Vi-cia faba (111), soybean (153), tobacco (16, 190,191), petunia (156), rice (12), asparagus (36),and Arabidopsis (167, 196). These observationsprovide a model to explain the rapid stimula-tion of GAD activity in response to biotic andabiotic stresses, which elicit changes in cytoso-lic Ca2+ concentrations (reviewed in 151, 154).Thus, the production of GABA via the stimula-tion of GAD by CaM is directly associated withcytosolic Ca2+ fluxes.

The interaction between GAD and CaMseems to be plant specific because none ofthe mammalian GADs identified possess aCa2+/CaMBD. Recently, a rice GAD was de-scribed (OsGAD2) that lacks a CaMBD (1).The physiological relevance of GAD activation

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by CaM was first addressed in tobacco by us-ing transgenic plants ectopically expressing ei-ther GAD or a truncated GAD that could notbind CaM (16). Disruption of the CaMBDof GAD resulted in constitutive GAD activ-ity (i.e., Ca2+-independent), abnormal steady-state levels of glutamate (low) and GABA (high)and aberrant plant development (16). Conse-quently, CaM tightly controls the activity ofGAD in vivo and this regulation is necessary fornormal plant development. Another approachto clarify the role of CaM-regulated GABA pro-duction in plants is by using Arabidopsis T-DNAinsertion mutants for the GAD genes. For ex-ample, disrupting the root-specific GAD1 generevealed that it plays a major role in GABA syn-thesis under normal growth conditions and inresponse to heat stress (22). However, the rolesof GAD and GABA in response to environmen-tal stresses remain to be clarified.

Tolerance to Xenobiotic Compounds

CaMBPs expressed ectopically in transgenicplants can change the sensitivity of the plantto toxic compounds. For example, CNGCsmay serve as entry pathways for certain heavymetals. CNGCs contain six putative trans-membrane domains, a pore region, a cyclic-nucleotide-binding domain, and a CaMBD (re-viewed in 162). These proteins are similar tothe mammalian cyclic nucleotide gated nonse-lective cation channels, which are activated bycyclic nucleotides and involved in Ca2+ signaltransduction (reviewed in 88). Tobacco trans-genic plants overexpressing a member of theCNGC family (i.e., NtCBP4) exhibited hy-persensitivity to Pb2+. These transgenic lineswere indistinguishable from WT plants un-der normal growth conditions but accumulatedPb2+ (11). Furthermore, transgenic lines ex-pressing a truncated form of NtCBP4, lackingthe CaMBD and part of the cyclic nucleotide-binding domain, displayed improved toleranceto Pb2+ associated with a lower Pb2+ content inthe plant tissues as compared to the WT (160).A similar phenotype was observed in Arabidop-sis mutant plants with a T-DNA inserted in the

AtCNGC1 gene, an ortholog of NtCBP4 (160).Thus, NtCBP4 and AtCNGC1 are probablyCa2+-permeable channels providing a routefor Pb2+ entry across the plasma membrane.Nevertheless, transgenic plants overexpressingmodified versions of CNGC proteins providenew tools to manipulate plant tolerance toheavy metals (10).

Apyrases are plasma membrane-associatedenzymes with their hydrolytic activity directedto the extracellular matrix where they hy-drolyze most nucleoside tri- and diphosphates.Functions of ecto-apyrases are not well de-fined in plants but are related to the hydrol-ysis of extracellular ATP and ADP. Althoughapyrases from animals have not been describedas CaMBPs, the activity of an endogenous peaapyrase (psNTP9) is stimulated by Ca2+/CaM(31), and the corresponding recombinant pro-tein binds CaM in a Ca2+-dependent manner(79). In Arabidopsis, only one of the two relatedapyrase genes cloned (i.e., AtAPY1) encodesa CaMBP (157). Transgenic Arabidopsis plantsoverexpressing the pea apyrase are more resis-tant to toxic concentrations of cycloheximide(163), plant growth regulators such as cytokinin(163), and herbicides of different chemicalclasses (176). WT plants grown in the presenceof apyrase inhibitors become more sensitiveto the herbicides (176). Thus, apyrase is in-volved in a multidrug resistance mechanism.Because the pea apyrase used in these studies isCaM regulated, it raises the interesting possibil-ity that CaM may regulate extracellular apyraseactivity. In addition, AtMRP1, a vacuolar mul-tidrug resistance-associated (MRP/ABCC)-likeABC transporter, was recently shown to possessa functional CaMBD (60) that can also inter-act with the FK506-binding protein TWD1.CaM had no effect on the binding of TWD1to the transporter in vitro, thus the mode ofaction of CaM versus TWD1 on AtMRP1 re-mains to be elucidated. Interestingly, TWD1itself has a putative CaMBD (86) and interactswith two other multidrug resistance-like trans-porters (AtPGP1 and AtPGP19). Arabidopsistwd1 mutants present a pleiotropic phenotypecharacterized by reduction of cell elongation

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and disorientated growth of all plant organs(61). It is thus likely that this protein is involvedin regulating multidrug resistance-associatedtransporters although the biochemical role ofCaM in this process is unclear.

CALMODULIN AND PLANTRESPONSE TO BIOTIC STRESS

Ca2+ likely functions as a second messengerduring plant responses to biotic stimuli such aspathogen attacks. This section discusses recentfindings that support a role for Ca2+ sensorssuch as CaM, CLMs, and their downstreamtargets in regulating the signaling events thatfollow biotic stimulus perception. For relateddiscussions on CDPKs or reviews on plant host-pathogen interactions, the reader is referred toa number of recent articles (116, 128, 177).

When a plant cell detects a pathogen in-vader, a cascade of defensive events is triggered.Typical rapid responses to pathogen attack in-clude ion fluxes, production of ROIs, cell wallfortification, synthesis of defense compounds,and changes in gene expression (reviewed in 40,128). Ca2+ influx into the cytosol is among theearliest of these responses and likely serves as amessenger to regulate specific downstream de-fense pathways. Numerous studies have shownthat influx across the plasma membrane fromextracellular stores is a key source of Ca2+ inresponse to elicitors or pathogens (19, 62, 66,109, 178, 197), but the participation of inter-nal Ca2+ stores is also likely important (92,103, 105). Note, however, that only a few ofthese studies (45, 66, 178) have been conductedin planta using living pathogens and althoughmuch has been learned using microbial elici-tor preparations (or analogs) and cell cultures,we are a long way from understanding the dy-namics of Ca2+ signaling during host-pathogeninteraction. Nevertheless, one of the most in-triguing findings that has emerged from thesestudies is that the cytosolic Ca2+ signal, whichis evoked by pathogen attack, differs consider-ably from the spiking and oscillation patternsoften seen during response to abiotic stimulisuch as cold, drought, or salinity (reviewed in

HR: hypersensitiveresponse

76, 127, 174). In general, most studies sug-gest that the elevation in cytosolic Ca2+ thatrapidly follows pathogen (or elicitor) percep-tion is sustained for a prolonged period al-though it may be biphasic in character (19, 45,66, 178). Importantly, the sustained level of cy-tosolic Ca2+ was not observed during compati-ble host-pathogen interactions, suggesting thatearly recognition of the pathogen is a prerequi-site for this particular Ca2+ signature. How suchan apparently simple signature contributes, ifat all, to the regulation of the myriad down-stream events such as the oxidative burst, theHR, the synthesis of defense compounds, andthe induction of defense-related genes, remainslargely unknown. And although Ca2+ has beenimplicated in many of these processes, typi-cally through the use of pharmacological agentssuch as Ca2+ ionophores or chelators, the evi-dence for a regulatory role for Ca2+ is largelycorrelative. However, in addition to findingsfrom Ca2+ imaging research, there is a grow-ing body of data from gene expression analy-ses, biochemical studies, and reverse geneticsto strongly suggest that Ca2+ and Ca2+ sen-sors and their downstream targets are impor-tant components in plant defense signal trans-duction. The following discussion focuses onrecent work on CaMs, CMLs, and CaM tar-gets during pathogen response. For the CMLfamily, attention focuses on Arabidopsis CMLs[as annotated by McCormack & Braam (119)]and their putative homologs in other plants.Most examples are drawn from studies usingmicrobial pathogens or elicitors, but a few ex-amples involving herbivory response are alsopresented.

CaMs and CMLs in PathogenResponse

A number of studies have shown the involve-ment of CaMs and CMLs during pathogenresponse. Two soybean CaMs, SCaM4 andSCaM5, which possess about 78% identity toa conserved CaM such as Arabidopsis CaM2,are rapidly induced by fungal elicitor prepa-rations and this induction can be mimicked

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SAR: systemicacquired resistance

PTGS:post-transcriptionalgene silencing

using Ca2+ ionophores or prevented by Ca2+

chelators (75). Elegant functional analyses con-firmed the involvement of these proteins inpathogen response by showing that overex-pression in tobacco resulted in enhanced lev-els of SAR-associated transcripts, spontaneousformation of necrotic lesions, and increasedbroad-spectrum pathogen resistance (75). Sev-eral CaMs were upregulated in tobacco car-rying the N-resistance gene by infection withtobacco mosaic virus (TMV) (180). Amongthe TMV-induced CaMs was a divergent iso-form, NtCaM13, which is a putative ortholog ofthe pathogen-inducible soybean CaMs, SCaM4and SCaM5, described above. Although post-translational levels of several conserved CaMswere induced by wounding treatments, Nt-CaM13 was repressed, suggesting stimulus-specific responses. Another pathogen-inducibleCML is Hra32 from Phaseolus vulgaris (84).Hra32 is about 53% identical to an Arabidop-sis NaCl-inducible protein, AtCP1 (84), andabout 23% identical to the conserved Arabidop-sis CaM2. When plants were inoculated with anincompatible bacterial pathogen, Hra32 tran-script levels were temporally correlated with theonset of HR symptoms. Infection with a com-patible pathogen resulted in a different tem-poral expression pattern of Hra32 that wascorrelated with cell death associated with dis-ease, whereas general stimuli such as wound-ing evoked transient Hra32 expression (83).CaM transcript levels were induced in tomatoin response to wounding or the wound signalmolecule systemin and were constitutively highin transgenic plants overexpressing systemin(17). Expression-profiling studies on Arabidopsisidentified a CML (At3g51920, herein referredto as CML9) as one of the earliest genes in-duced during a bacterial pathogen challenge(45). CML9 was also induced by other stimulibut in a temporally distinct manner. These au-thors speculated that the most rapidly inducedgenes may be part of an encoding signature ofexpression for infection that helps to establishthe pathogenic context of the infection. Thecell somehow decodes this information and re-sponds accordingly.

Another pathogen-induced CML was iden-tified in a high-throughput expression studyusing tobacco (carrying the tomato Cf-9 re-sistance gene) that was challenged with fun-gal elicitor preparations. Among the genesrapidly induced by elicitors from an incom-patible Cladosporium fulvum race was a CMLdesignated ACRE-31 (53). The ACRE-31 pro-tein shows about 30% identity to Arabidopsis-conserved CaM2 and about 60% identity toboth Arabidopsis CML42 and CML43. A similartranscript-profiling study, using the tomato andPseudomonas syringae pv tomato host-pathogensystem, identified a probable ortholog ofACRE-31, APR134, as an early upregulatedgene during pathogen response (122). Func-tional analysis of APR134 in tomato and CML43in Arabidopsis confirmed a role for these genesduring the plant immune response. Suppressionof APR134 expression using virus-induced genesilencing (VIGS) results in loss of resistanceto incompatible P. syringae pv tomato, whereasoverexpression of CML43 in Arabidopsis acceler-ates the HR (W.A. Snedden, unpublished data).

Another interesting study that shed lighton the roles of CaMs during pathogen re-sponse describes a novel CML from tobaccothat functions as a negative regulator of PTGS(6). PTGS is part of the defense strategy plantsuse against viral pathogens. rgs-CaM (regulatorof gene silencing CaM) is about 26% identi-cal to conserved Arabidopsis CaM2 at the aminoacid level and about 50% identical to ArabidopsisCML37. Interestingly, the unusual N-terminalextensions of rgs-CaM and CML37 are quitedifferent from one another. Tobacco rgs-CaMwas isolated by a two-hybrid screen using thehelper component protease (HC-Pro) from aplant potyvirus as bait. In tobacco, expression ofHC-Pro from a transgene, or viral infection, in-duces the expression of rgs-CaM and suppressesPTGS, suggesting that rgs-CaM has a negativeregulatory role in PTGS and is subverted dur-ing viral attack (6).

Collectively, the studies described aboveimplicate CaM and related CMLs in plantresponses to pathogens. Examining accessi-ble databases from global expression studies

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suggests that many unstudied CMLs are in-duced during plant-pathogen interaction (W.A.Snedden, unpublished data), and these datashould help direct future research efforts. Theseemingly paradoxical concept of inducing theexpression of a sensor for a signaling molecule,Ca2+, whose presence is rapidly evoked andtransitory, has not gone unnoticed. It has beensuggested that increased levels of Ca2+ sensorsduring stress response prime cells for subse-quent or long-term stimuli or create a stoichio-metric level of the sensors to regulate down-stream targets (195). Trewavas (166) suggestedthat the changes occurring in the levels of Ca2+

sensors upon the perception of stress stim-uli is comparable to the process of learningin other organisms. A recent study in animalcells suggests that effectors compete for CaMin vivo and this competition is coordinated tosimultaneously regulate the activities of varioustargets (165). Thus, given that CaMs and,presumably, CMLs are essentially noncatalyticregulators, identifying and characterizing theirtargets has become an important challenge.Our understanding of the role of CaM targetsin pathogen response has made considerableprogress in recent years.

CaM Targets and Pathogen Response

NAD kinase (NADK), which catalyzes thephosphorylation of NAD to NADP, was amongthe first CaM targets implicated in signal-ing during pathogen response in plants (70).NADKs are likely found in all organisms andwere recently cloned from a number of prokary-otes and eukaryotes (see 168 and referencestherein). It is unclear whether other eukaryotespossess CaM-binding NADKs. Although theroles of NADKs remain obscure, their prod-uct, NADP, may indirectly contribute to theNADPH pool utilized by the important defenseenzyme NADPH oxidase (135). A role in de-fense signaling for NADK was demonstrated(70). Transgenic tobacco plants overexpressinga mutant CaM, unable to be methylated at Lys-115 and that hyperactivates NADK, showed en-hanced levels of NADPH and an accelerated

production of ROIs in response to various elici-tors or an incompatible bacterial pathogen (70).Recently, cDNAs were isolated from Arabidop-sis encoding two isoforms of NADK, one ofwhich is a CaM-binding isoform that is uniqueto plants (168). This work should help facilitatefuture transgenic studies aimed at assessing theroles of NADKs in pathogen response. PlantCaM-regulated catalases (186) and NADKsmay function in concert to modulate ROI lev-els during Ca2+-mediated responses to externalstimuli.

GAD is a well studied CaM-activated en-zyme whose product, GABA, is rapidly syn-thesized in plants in response to various abi-otic stimuli (discussed above). Because GABAis an inhibitory neurotransmitter in insects itmay also serve as a chemical deterrent againstherbivory. Crawling or feeding by herbivorousinsects stimulated rapid GABA production intobacco and soybean (25) and transgenic plantsthat hyperaccumulate GABA received less her-bivory than control transgenics or wild-typeplants (117).

Further support for a role of CaM targetsin pathogen response comes from the recentidentification of MLO as a CaMBP (89, 90).The Mlo gene encodes a seven-transmembranereceptor-like protein that is unique to plants.Although related to mammalian G-protein-coupled receptors, MLO functions indepen-dently of heterotrimeric G-proteins (90). Themlo mutation is particularly interesting becauseit confers broad spectrum resistance in bar-ley against powdery mildew disease. This sug-gests that MLO negatively regulates this de-fense pathway in wild-type plants. Expression ofthe rice homolog of Mlo, OsMlo, is significantlyinduced by a fungal pathogen or various defensesignaling compounds and an influx of Ca2+ isnecessary but not sufficient for this induction(89). Importantly, MLO mutations that impairits ability to bind CaM compromise its defensesuppression activity in vivo (90). Future stud-ies should focus on elucidating the biochemicalactivity of MLO and assessing whether othermembers of the MLO family are also CaMtargets.

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In Arabidopsis, the expression of variousCAMTA genes is differentially induced by exter-nal stimuli, including signaling molecules suchas ethylene, salicylic acid, and hydrogen perox-ide, known to play a role during pathogen re-sponse (139, 185). A functional role for CAM-TAs in pathogen response has not yet beenestablished, but putative target genes, whosepromoters contain CAMTA-binding cis-ele-ments, include Ca2+-binding proteins andother targets known to participate in stimulus-response pathways (185).

An important recent development in therole of CaM targets during pathogen responsewas the isolation of an Arabidopsis nitric oxidesynthase (AtNOS1), which is a novel plantCaMBP (67). Although AtNOS1 is a CaM tar-get that catalyzes the synthesis of NO usingl-Arg as a substrate, it does not share signifi-cant sequence identity with mammalian NOScounterparts and thus represents a new classof NOS enzymes. NO is an important signal-ing molecule in animals and recently receivedsimilar attention in plant systems. AtNOS1 isinvolved in hormonal signaling associated withgrowth and development (67), and a role duringpathogen response is emerging. Previous stud-ies demonstrated a role for NO in the HR andthe induction of defense genes during pathogenresponse (47, 48, 52), but the source of the NOremained obscure, especially given the absenceof an obvious NOS ortholog in the Arabidop-sis genome. Thus, isolating a plant NOS wasa major breakthrough. NO production inducesCa2+ influx from intracellular stores during re-sponse to a fungal elicitor, suggesting a positivefeedback of sorts for this CaM-dependent en-zyme (100). Key evidence that AtNOS1 is animportant component in plant defense was re-cently provided by a study that examined theeffects of bacterial lipopolysaccharides on NOproduction and plant immunity (191a). Thiswork revealed that the strong, rapid burst ofNO observed during exposure of Arabidopsisto bacterial lipopolysaccharides was generatedby AtNOS1 and was responsible for regulat-ing downstream genes involved in pathogenresponse. Furthermore, mutant plants lacking

AtNOS1 were more susceptible to pathogenicbacteria (191a). Overall, the data suggest thatAtNOS1 plays an important role in plant im-munity. However, there is still much to learnabout the role of NO in different host-pathogensystems. Note that in addition to AtNOS1-mediated NO synthesis, there are other enzy-matic and nonenzymatic mechanisms of gener-ating NO in plants (99a). Consequently, it willrequire a multidisciplinary approach to eluci-date the contributions of the various sources toNO-based defense signaling in plants.

Several other CaMBPs were recently identi-fied where a role in pathogen response was sug-gested. PICBP encodes a plant-specific CaMBPof unknown biochemical function that is in-duced by incompatible bacterial strains as wellas by a number of defense-related signalingmolecules (141). It is noteworthy that PICBPis unique in possessing four Ca2+-dependentCaMBDs. Because PICBP is encoded by a sin-gle gene in Arabidopsis, reverse genetic stud-ies should help reveal its cellular role in de-fense (141). In a similar study, Ali et al. (2)identified additional CaM targets whose tran-script expression patterns were responsive topathogen infection or related stimuli. Severalgenes encoding isoforms of bean PvCBP-60were strongly induced by incompatible bacte-ria (2). The CMP-60 family (also known asTCBP60s, see Table 1) is made up of plant-specific CaMBPs with no homology to anyknown protein. Although their biochemicalfunction remains unknown, they are consis-tently among the most abundant proteins iso-lated in screening methods aimed at identifyingnovel CaM targets.

A CaM-binding cyclic-nucleotide gatedchannel, DND1 (AtCNGC2), was also recentlyshown to participate in defense signaling (38).Plants carrying a mutation in this gene fail toproduce the HR when challenged with aviru-lent P. syringae but display gene-for-gene resis-tance and constitutive SAR (38). The under-lying mechanism of the dnd1 phenotype andwhether DND1 participates directly or indi-rectly in pathogen response remains unclearbut it seems to be positioned downstream of

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salicylic acid in the signal cascade. Complemen-tation experiments using wild-type AtCNGC2restored the normal phenotype of dnd1 mutantsand it would be interesting to extend these stud-ies using AtCNGC2-carrying mutations in theCaMBD (9) to assess the functional importanceof this domain. Gene expression studies provideadditional support for a role of specific CNGCsin defense response (2). A second memberof this family, AtCNGC4, also participates inpathogen-response signaling (14). Plants car-rying mutations in either HLM1 (which en-codes AtCNGC4) or DND1 are phenotypi-cally similar but there are some key differencesin their responses to pathogen infection andin the expression patterns of these genes inwild-type plants, suggesting that although bothfunction during pathogen response, they donot play redundant roles (14). Another CaM-regulated transporter, SCA1, which encodes aCa2+-ATPase, is induced in response to a fun-gal elicitor (37). Given the diversity of CaM-regulated Ca2+ pumps and channels in plants(see Table 1), it will be a challenge to deter-mine which members contribute to the changesin Ca2+ dynamics observed in response to bioticstress.

Another novel CaMBP recently implicatedin pathogen response is the MAPK phos-phatase, NtMKP1 (179). MAPKs were previ-ously shown to participate in defense signalingand it is likely that their activity is regulatedby specific phosphatases (177). Consistent withthis suggestion, transgenic plants overexpress-ing NtMKP1 showed reduced activity of sev-eral defense-related MAPKs in response towounding. Although NtMKP1 showed differ-ential binding to several CaMs, the role of CaMbinding to NtMKP1 remains under investiga-tion. It will be interesting to see if any of theother recently described CaM-binding phos-phatases and kinases (see Table 1) have tar-gets involved in pathogen response. Recently,DMI3, an important gene required for symbi-otic nodule development in legumes, was shownto encode a kinase of the CCaMK class (120).This study reflects an importance of CaM tar-gets in other aspects of plant-microbe commu-

nication beyond that of classic host-pathogeninteraction.

It is likely that rapid advances in high-throughput transcript expression analyses willyield even more clues about which CaM tar-gets participate in host-pathogen interaction. Inaddition, as proteomic methods evolve, it maysoon be possible to screen an entire plant pro-teome for CaM targets, as was done with yeast(192). It will be particularly important to iden-tify the targets of the unique CMLs to help un-derstand why plants have evolved such a sophis-ticated array of these Ca2+ sensors. In general,an emphasis on functional analysis using thecombined tools of genetics, biochemistry, andmolecular and cell biology is needed to unravelthe complexity of Ca2+-mediated pathogenresponse.

CALMODULIN AND PLANTDEVELOPMENT

Much of our knowledge regarding a role forCaM in plant development has come from ge-netic studies. In particular, research on mu-tants with aberrant pollen tube growth or tri-chome morphogenesis has demonstrated thatCaMBPs are key components in these devel-opmental processes. These and other findingslinking CaMBPs and plant development arediscussed below.

CaMBPs Responding to HormonalTreatment

CaM interacts with at least one group of pro-teins directly involved in response to hormonalchanges: the small auxin up RNA (SAUR) pro-teins encoded by short unstable transcripts thataccumulate rapidly and specifically after auxintreatment. By screening a cDNA library witha radiolabeled CaM, Yang & Poovaiah (184)isolated a cDNA encoding a SAUR protein(ZmSAUR1). CaM-binding SAURs are widelydistributed in the plant kingdom, although theirphysiological role is unclear. Northern analysisconfirmed that ZmSAUR1 is an early auxin re-sponse gene, induced within 10 minutes after

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auxin treatment. Subsequently, another mem-ber of the SAUR family (ZmSAUR2) was iso-lated and shown to bind CaM (93). In addition,other genes encoding CaMBPs, such as mem-bers of the CAMTA transcription factor family,are induced by ethylene (183).

CaM and CaMBPs Involved in theDevelopment of Polarized Cells

CaM and specific CaMBPs are involved in thedevelopment of at least two types of polarizedcells specific to plants: pollen tubes and tri-chomes. Ca2+ fluxes are required for the nor-mal growth and elongation of pollen tubes;furthermore, the Ca2+ gradient oscillations inpollen are very well documented (reviewed in78). Unlike Ca2+, CaM and CaM mRNA dis-tribute uniformly throughout the pollen cell, asdemonstrated with fluorescently labeled CaMor CaM RNA microinjected in living tubes andmonitored by confocal microscopy (121). How-ever, using a fluorescent analogue of CaM re-vealed that the activity of CaM is higher in theapical tip of the tube and oscillates, followingthe distribution and oscillation pattern of cy-tosolic Ca2+ (137).

There are many potential CaM targets thatmight be required for pollen tube growth, sev-eral of which have been identified by reversegenetic approaches in Arabidopsis. One memberof the family of autoinhibited Ca2+-ATPases(ACA9) is of particular importance for nor-mal pollen tube growth and fertilization (150).Three alleles of the aca9 mutation present thesame phenotype: a reduced set of seeds dueto the poor growth of aca9 pollen tubes and alow frequency of fertilized ovules. When pollentubes reach an ovule, more than 50% fail to de-velop an embryo. ACA9 is specifically expressedin the male gametophyte (150). One hypothesisto explain the phenotype of aca9 mutants is thatACA9 is required to maintain the oscillationsof Ca2+ observed in the tips of pollen tubes.Other CaMBPs specifically expressed in pollenplay an essential role in pollen germination.The maize pollen calmodulin-binding protein(MPCBP) and NPG1 (no pollen germination1)

in Arabidopsis are members of a plant-specificfamily of CaMBPs containing tetratricopeptiderepeats (TPR), a type of protein-protein inter-action domain (63, 149). Although the exactfunction of these proteins is unknown, the npg1mutant allele is not transmitted through themale gametophyte because npg1 pollen germi-nation is arrested (63). Another gene necessaryfor pollen tube growth encodes a transaminase(GABA-T) that degrades GABA. Although nota CaMBP, GABA-T is one component of theGABA-shunt pathway that, in plants, is CaMregulated (reviewed in 23). The pollen-pistil in-teraction2 ( pop2) mutant is deficient in a GABA-T, which ultimately leads to growth inhibitionand misguidance of pop2 pollen tubes in pop2pistils (131). In WT plants, the level of GABAincreases along the path through which pollentubes travel, whereas in pop2 plants this gradientof GABA is disturbed (131). Thus, the growthof pollen tubes depends on GABA productionand degradation, a process at least partially con-trolled by GAD, a CaMBP. The disruption ofapyrases (see above) also inhibits pollen germi-nation in Arabidopsis (158). T-DNA knockoutswere isolated for each of the two Arabidopsisapyrases but they lacked a visible morpho-logical change. However, pollen from double-knockout mutants failed to germinate. Thedouble knockout can be complemented by ei-ther one of the two apyrases: AtAPY1, which is aCaMBP, and AtAPY2, which is not. Therefore,although apyrases are clearly needed for the de-velopment of pollen tubes, the role of CaM inthe regulation of AtAPY1 activity remains un-clear. In addition, CaM interacted with the cy-tosolic kinase domain of the S locus receptor ki-nase (SRK) involved in recognizing self-pollenduring the self-incompatibility response (170).A CaMBD was identified in SRK, but the roleof CaM in self-pollen recognition remains tobe clarified.

CaM binds a kinesin protein that inter-acts with microtubules to play a major rolein trichome morphogenesis. In Arabidopsis,ZWICHEL was identified by genetic screensfor altered trichome morphology. zwi mutantsare affected in trichome stalk expansion and

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branching, but not in other aspects of plantdevelopment. The ZWICHEL protein is akinesin-like CaMBP (AtKCBP). Ca2+/CaM in-hibits the motor activity of AtKCBP and its in-teraction with microtubules (reviewed in 138).KCBP also plays a major role in cotton fiberelongation (85, 134). Kinesins from other or-ganisms such as yeast, Caenorhabditis, or humando not contain a CaMBD. However, sea urchindoes possess a KCBP with a CaMBD at the C

terminus (148). The mechanism by which CaMinhibits plant KCBPs was addressed in a re-cent study where the CaMBD of a plant KCBPwas fused to various kinesins from Drosophilathat do not normally bind CaM (144). CaMwas able to regulate, in a Ca2+-dependentmanner, both the binding to microtubules ofthese chimeric kinesins and their microtubule-stimulated ATPase activity. Finally, the crys-tal structure of a KCBP from potato was

Figure 3Schematic presentation of stimulus-response signaling mediated by Ca2+/CaM. Various biotic and abioticstimuli evoke a transient change in cytosolic and/or organelle Ca2+ levels, which are transduced by CaMs,CaM-like proteins (CMLs), and other Ca2+ sensors. CaMs and CMLs interact with downstream effectorsthat modulate numerous biochemical and cellular functions (see Table 1 in the Supplemental Material linkin the online version of this chapter or at http://www.annualreviews.org/) for details of the proteins involvedin each depicted biochemical cellular activity). Exogenous stimuli also elicit signaling pathways independentof Ca2+. These may interact with the Ca2+ signaling pathways at various points of the transduction cascades.Physiological response to external stimuli is comprised of the combined molecular and biochemical changesin both the intracellular and extracellular regions and includes changes in the Ca2+ sensing machinery itself.

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resolved, suggesting that Ca2+/CaM blocks themicrotubule-binding sites on KCBP (172).KCBP is probably only one partner of a mul-tiple protein complex because it also interactswith a protein kinase (43) and two proteins in-volved in trichome cell morphogenesis: AN-GUSTIFOLIA (55) and KIC (143). KIC pos-sesses a Ca2+-binding EF-hand motif and bothCaM and KIC inhibit KCBP activity in a Ca2+-dependent manner, supporting a key role forCa2+ in trichome morphogenesis.

CONCLUDING REMARKSAND FUTURE DIRECTIONS

The past decade has been fruitful in identi-fying the repertoire of plant CaMs, CMLs,and CaMBPs. However, only in very few caseshave their physiological roles been revealedand, thus, more functional studies are needed.Figure 3 depicts a schematic presentation ofthe main components of the Ca2+/CaM mes-senger system in plants. Future research in thisfield will be driven by the developing tools ofreverse and forward genetics, bioinformatics,various high-throughput techniques for geneexpression analyses, and emerging technolo-gies in proteomics to study protein expres-

sion and interactions. There should also beemphasis on real-time in vivo cellular studiesof spatial and temporal protein dynamics andprotein-protein interaction using microscopytechniques with fluorescently tagged CaMs,CMLs, and CaMBPs. Given that stimulus-response specificity is likely governed in partby fine-tuning the localization and expressionof proteins that constitute the CaM messengersystem, the challenge ahead is to pinpoint thespecificities within the system and the intercon-nections with other signaling pathways. Thedynamics and regulators of intracellular Ca2+

signals should also be further studied. Math-ematical modeling of Ca2+ dynamics and re-sponse circuitry will help integrate the variousaspects of signal transduction into a mecha-nistic model of how information is processedwithin a cell. The ultimate goal is to take thecombined knowledge from related areas and,through a systems-biology approach, emergewith an understanding of how a plant per-ceives any given stimulus and reprograms itsmetabolic and developmental profiles to copeor adapt accordingly. As researchers target thatgoal, agricultural and environmental biotech-nology will continue to benefit from advancesin basic signal transduction research.

ACKNOWLEDGMENTS

We thank Zohar Bloom and Refael Ackermann for assistance in drawing the structural CaM-targetmodels.

LITERATURE CITED

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129. Ohya Y, Botstein D. 1994. Diverse essential functions revealed by complementing yeastcalmodulin mutants. Science 263:963–66

130. Olsson P, Yilmaz JL, Sommarin M, Persson S, Bulow L. 2004. Expression of bovine calmod-ulin in tobacco plants confers faster germination on saline media. Plant Sci. 166:1595–604

131. Palanivelu R, Brass L, Edlund AF, Preuss D. 2003. Pollen tube growth and guidance isregulated by POP2, an Arabidopsis gene that controls GABA levels. Cell 114:47–59

132. Pauly N, Knight MR, Thuleau P, van der Luit AH, Moreau M, et al. 2000. Control of freecalcium in plant cell nuclei. Nature 405:754–55

The paper describesa CaMBP(designatedAtCAMBP25)unique to plantsisolated by screeningan expression cDNAlibrary withradiolabeled CaM.CaMBP25 has anunknown activity.The protein islocalized in thenucleus and plays arole in the responseto ionic and nonionicosmotic stress. It isalso interesting thatCaMBP25 bindsdifferentially only tosome CaM isoforms.

133. Perruc E, Charpenteau M, Ramirez BC, Jauneau A, Galaud JP, et al. 2004. A novelcalmodulin-binding protein functions as a negative regulator of osmotic stress tol-erance in Arabidopsis thaliana seedlings. Plant J. 38:410–20

134. Preuss ML, Delmer DP, Liu B. 2003. The cotton kinesin-like calmodulin-binding proteinassociates with cortical microtubules in cotton fibers. Plant Physiol. 132:154–60

135. Pugin A, Frachisse JM, Tavernier E, Bligny R, Gout E, et al. 1997. Early events induced bythe elicitor cryptogein in tobacco cells: involvement of a plasma membrane NADPH oxidaseand activation of glycolysis and the pentose phosphate pathway. Plant Cell 9:2077–91

136. Puhakainen T, Pihakaski-Maunsbach K, Widell S, Sommarin M. 1999. Cold acclimationenhances the activity of plasma membrane Ca2+-ATPase in winter rye leaves. Plant Physiol.Biochem. 37:231–39

137. Rato C, Monteiro D, Hepler PK, Malho R. 2004. Calmodulin activity and cAMP signallingmodulate growth and apical secretion in pollen tubes. Plant J. 38:887–97

138. Reddy AS, Day IS. 2000. The role of the cytoskeleton and a molecular motor in trichomemorphogenesis. Trends Plant Sci. 5:503–5

139. Reddy AS, Reddy VS, Golovkin M. 2000. A calmodulin binding protein from Arabidopsisis induced by ethylene and contains a DNA-binding motif. Biochem. Biophys. Res. Commun.279:762–69

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140. Reddy RK, Kurek I, Silverstein AM, Chinkers M, Breiman A, Krishna P. 1998. High-molecular-weight FK506-binding proteins are components of heat-shock protein 90 hete-rocomplexes in wheat germ lysate. Plant Physiol. 118:1395–401

141. Reddy VS, Ali GS, Reddy AS. 2003. Characterization of a pathogen-induced calmodulin-binding protein: mapping of four Ca2+-dependent calmodulin-binding domains. Plant Mol.Biol. 52:143–59

This articleaddresses acombination of anexperimentalapproach andbioinformatic studiesto identify genesencoding CaMBPs inthe Arabidopsisgenome.

142. Reddy VS, Ali GS, Reddy ASN. 2002. Genes encoding calmodulin-binding proteinsin the Arabidopsis genome. J. Biol. Chem. 277:9840–52

143. Reddy VS, Day IS, Thomas T, Reddy AS. 2004. KIC, a novel Ca2+ binding protein withone EF-hand motif, interacts with a microtubule motor protein and regulates trichomemorphogenesis. Plant Cell 16:185–200

This study addressesthe mechanism bywhich CaM inhibitsplant KCBPs.

144. Reddy VS, Reddy AS. 2002. The calmodulin-binding domain from a plant kinesinfunctions as a modular domain in conferring Ca2+-calmodulin regulation to animalplus- and minus-end kinesins. J. Biol. Chem. 277:48058–65

145. Reddy VS, Reddy ASN. 2004. Proteomics of calcium-signaling components in plants. Phy-tochemistry 65:1745–76

146. Rentel MR, Knight MR. 2004. Oxidative stress-induced calcium signaling in Arabidopsisthaliana. Plant Physiol. 135:1471–79

147. Rodriguez-Concepcion M, Yalovsky S, Zik M, Fromm H, Gruissem W. 1999. The preny-lation status of a novel plant calmodulin directs plasma membrane or nuclear localizationof the protein. EMBO J. 18:1996–2007

148. Rogers GC, Hart CL, Wedaman KP, Scholey JM. 1999. Identification of kinesin-C, acalmodulin-binding carboxy-terminal kinesin in animal (Strongylocentrotus purpuratus) cells.J. Mol. Biol. 294:1–8

149. Safadi F, Reddy VS, Reddy ASN. 2000. A pollen-specific novel calmodulin-binding proteinwith tetratricopeptide repeats. J. Biol. Chem. 275:35457–70

This paper describesthe phenotype of theaca9 ArabidopsisT-DNA mutants.

150. Schiott M, Romanowsky SM, Baekgaard L, Jakobsen MK, Palmgren MG, HarperJF. 2004. A plant plasma membrane Ca2+ pump is required for normal pollen tubegrowth and fertilization. Proc. Natl. Acad. Sci. USA 101:9502–7

151. Shelp BJ, Bown AW, McLean MD. 1999. Metabolism and functions of gamma-aminobutyric acid. Trends Plant Sci. 4:446–52

152. Silverstein AM, Galigniana MD, Kanelakis KC, Radanyi C, Renoir JM, Pratt WB. 1999.Different regions of the immunophilin FKBP52 determine its association with the gluco-corticoid receptor, hsp90, and cytoplasmic dynein. J. Biol. Chem. 274:36980–86

153. Snedden WA, Arazi T, Fromm H, Shelp BJ. 1995. Calcium/calmodulin activation of soy-bean glutamate decarboxylase. Plant Physiol. 108:543–49

154. Snedden WA, Fromm H. 1999. Regulation of the γ -aminobutyrate-synthesizing enzyme,glutamate decarboxylase, by calcium-calmodulin: a mechanism for rapid activation in re-sponse to stress. In Plant Responses to Environmental Stresses: From Phytohormones to GenomeReorganization, ed. HR Lerner, pp. 549–74 . New York: Marcel Dekker

155. Snedden WA, Fromm H. 2001. Calmodulin as a versatile calcium signal transducer inplants. New Phytol. 151:35–66

156. Snedden WA, Koutsia N, Baum G, Fromm H. 1996. Activation of a recombinant petu-nia glutamate decarboxylase by calcium/calmodulin or by a monoclonal antibody whichrecognizes the calmodulin binding domain. J. Biol. Chem. 271:4148–53

157. Steinebrunner I, Jeter C, Song C, Roux SJ. 2000. Molecular and biochemical comparisonof two different apyrases from Arabidopsis thaliana. Plant Physiol. Biochem. 38:913–22

158. Steinebrunner I, Wu J, Sun Y, Corbett A, Roux SJ. 2003. Disruption of apyrases inhibitspollen germination in Arabidopsis. Plant Physiol. 131:1638–47

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159. Sun XT, Li B, Zhou GM, Tang WQ, Bai J, et al. 2000. Binding of the maize cytosolicHsp70 to calmodulin, and identification of calmodulin-binding site in Hsp70. Plant Physiol.41:804–10

160. Sunkar R, Kaplan B, Bouche N, Arazi T, Dolev D, et al. 2000. Expression of a truncatedtobacco NtCBP4 channel in transgenic plants, and disruption of the homologous ArabidopsisCNGC1 gene confer Pb2+ tolerance. Plant J. 24:533–42

161. Takezawa D, Minami A. 2004. Calmodulin-binding proteins in bryophytes: identificationof abscisic acid-, cold-, and osmotic stress-induced genes encoding novel membrane-boundtransporter-like proteins. Biochem. Biophys. Res. Commun. 317:428–36

162. Talke IN, Blaudez D, Maathuis FJ, Sanders D. 2003. CNGCs: prime targets of plant cyclicnucleotide signalling? Trends Plant Sci. 8:286–93

163. Thomas C, Rajagopal A, Windsor B, Dudler R, Lloyd A, Roux SJ. 2000. A role for ec-tophosphatase in xenobiotic resistance. Plant Cell 12:519–33

164. Townley HE, Knight MR. 2002. Calmodulin as a potential negative regulator of ArabidopsisCOR gene expression. Plant Physiol. 128:1169–72

165. Tran QK, Black DJ, Persechini A. 2003. Intracellular coupling via limiting calmodulin. J.Biol. Chem. 278:24247–50

166. Trewavas A. 1999. How plants learn. Proc. Natl. Acad. Sci. USA 96:4216–18167. Turano FJ, Fang TK. 1998. Characterization of two glutamate decarboxylase cDNA clones

from Arabidopsis. Plant Physiol. 117:1411–21168. Turner WL, Waller JC, Vanderbeld B, Snedden WA. 2004. Cloning and characterization

of two NAD kinases from Arabidopsis: identification of a calmodulin binding isoform. PlantPhysiol. 135:1243–55

169. van der Luit AH, Olivari C, Haley A, Knight MR, Trewavas AJ. 1999. Distinct calciumsignaling pathways regulate calmodulin gene expression in tobacco. Plant Physiol. 121:705–14

170. Vanoosthuyse V, Tichtinsky G, Dumas C, Gaude T, Cock JM. 2003. Interaction of calmod-ulin, a sorting nexin and kinase-associated protein phosphatase with the Brassica oleracea Slocus receptor kinase. Plant Physiol. 133:919–29

171. Vetter SW, Leclerc E. 2003. Novel aspects of calmodulin target recognition and activation.Eur. J. Biochem. 270:404–14

The paper describesthe first crystalstructure of a plantCaMBP (thekinesin-like proteinfrom potato) andhelps to explain themechanism by whichCaM inhibits themotor activity ofplant kinesin–likeproteins.

172. Vinogradova MV, Reddy VS, Reddy ASN, Sablin EP, Fletterick RJ. 2004. Crystalstructure of kinesin regulated by Ca2+-calmodulin. J. Biol. Chem. 279:23504–9

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apyrase overexpression in Arabidopsis thaliana. Nat. Biotechnol. 21:428–33177. Xing T, Ouellet T, Miki BL. 2002. Towards genomic and proteomic studies of protein

phosphorylation in plant-pathogen interactions. Trends Plant Sci. 7:224–30178. Xu H, Heath MC. 1998. Role of calcium in signal transduction during the hypersensitive

response caused by basidiospore-derived infection of the cowpea rust fungus. Plant Cell10:585–98

179. Yamakawa H, Katou S, Seo S, Mitsuhara I, Kamada H, Ohashi Y. 2004. Plant MAPKphosphatase interacts with calmodulins. J. Biol. Chem. 279:928–36

180. Yamakawa H, Mitsuhara I, Ito N, Seo S, Kamada H, Ohashi Y. 2001. Transcriptionallyand post-transcriptionally regulated response of 13 calmodulin genes to tobacco mosaicvirus-induced cell death and wounding in tobacco plant. Eur. J. Biochem. 268:3916–29

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181. Yamniuk AP, Vogel HJ. 2004. Calmodulin’s flexibility allows for promiscuity in its interac-tions with target proteins and peptides. Mol. Biotechnol. 27:33–57

182. Yang T, Poovaiah BW. 2000. Arabidopsis chloroplast chaperonin 10 is a calmodulin-bindingprotein. Biochem. Biophys. Res. Commun. 275:601–7

183. Yang T, Poovaiah BW. 2000. An early ethylene up-regulated gene encoding a calmodulin-binding protein involved in plant senescence and death. J. Biol. Chem. 275:38467–73

184. Yang T, Poovaiah BW. 2000. Molecular and biochemical evidence for the involvement ofcalcium/calmodulin in auxin action. J. Biol. Chem. 275:3137–43

185. Yang T, Poovaiah BW. 2002. A calmodulin-binding/CGCG box DNA-binding proteinfamily involved in multiple signaling pathways in plants. J. Biol. Chem. 277:45049–58

186. Yang T, Poovaiah BW. 2002. Hydrogen peroxide homeostasis: activation of plant catalaseby calcium/calmodulin. Proc. Natl. Acad. Sci. USA 99:4097–102

187. Yang T, Poovaiah BW. 2003. Calcium/calmodulin-mediated signal network in plants.Trends Plant Sci. 8:505–12

188. Yang T, Segal G, Abbo S, Feldman M, Fromm H. 1996. Characterization of the calmodulingene family in wheat: structure, chromosomal location, and evolutionary aspects. Mol. Gen.Genet. 252:684–94

The paper describesthe first 3D structureof a plant CaMBDassociated with CaM.NMR analysisrevealed that thestructure shows anovel type ofCaM-targetinteraction, with astochiometry of 1:2,suggesting a role forCaM in glutamatedecarboxylasesubunitdimerization, andpossibly in theformation of a largeroligomeric complex.

189. Yap KL, Yuan T, Mal TK, Vogel HJ, Ikura M. 2003. Structural basis for simultane-ous binding of two carboxy-terminal peptides of plant glutamate decarboxylase tocalmodulin. J. Mol. Biol. 328:193–204

190. Yevtushenko DP, McLean MD, Peiris S, Van Cauwenberghe OR, Shelp BJ. 2003. Cal-cium/calmodulin activation of two divergent glutamate decarboxylases from tobacco. J.Exp. Bot. 54:2001–2

191. Yun SJ, Oh SH. 1998. Cloning and characterization of a tobacco cDNA encodingcalcium/calmodulin-dependent glutamate decarboxylase. Mol. Cells 8:125–29

191a. Zeidler D, Zahringer U, Gerber I, Dubery I, Hartung T, et al. 2004. Innate immunity inArabidopsis thaliana: Lipopolysaccharides activate nitric oxide synthase (NOS) and inducedefense genes. Proc. Natl. Acad. Sci. USA 101:15811–16

192. Zhu H, Bilgin M, Bangham R, Hall D, Casamayor A, et al. 2001. Global analysis of proteinactivities using proteome chips. Science 293:2101–5

193. Zhu JK. 2002. Salt and drought stress signal transduction in plants. Annu. Rev. Plant Biol.53:247–73

194. Zhu JK. 2003. Regulation of ion homeostasis under salt stress. Curr. Opin. Plant Biol. 6:441–45

195. Zielinski RE. 1998. Calmodulin and calmodulin-binding proteins in plants. Annu. Rev. PlantPhysiol. Plant Mol. Biol. 49:697–725

196. Zik M, Arazi T, Snedden WA, Fromm H. 1998. Two isoforms of glutamate decarboxylasein Arabidopsis are regulated by calcium/calmodulin and differ in organ distribution. PlantMol. Biol. 37:967–75

197. Zimmermann S, Nurnberger T, Frachisse JM, Wirtz W, Guern J, et al. 1997. Receptor-mediated activation of a plant Ca2+-permeable ion channel involved in pathogen defense.Proc. Natl. Acad. Sci. USA 94:2751–55

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Contents ARI 29 March 2005 21:29

Annual Review ofPlant Biology

Volume 56, 2005

Contents

Fifty Good YearsPeter Starlinger � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

PhytoremediationElizabeth Pilon-Smits � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �15

Calcium Oxalate in Plants: Formation and FunctionVincent R. Franceschi and Paul A. Nakata � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �41

Starch DegradationAlison M. Smith, Samuel C. Zeeman, and Steven M. Smith � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �73

CO2 Concentrating Mechanisms in Algae: Mechanisms,Environmental Modulation, and EvolutionMario Giordano, John Beardall, and John A. Raven � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �99

Solute Transporters of the Plastid Envelope MembraneAndreas P.M. Weber, Rainer Schwacke, and Ulf-Ingo Flugge � � � � � � � � � � � � � � � � � � � � � � � � � � � � 133

Abscisic Acid Biosynthesis and CatabolismEiji Nambara and Annie Marion-Poll � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 165

Redox Regulation: A Broadening HorizonBob B. Buchanan and Yves Balmer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 187

Endocytotic Cycling of PM ProteinsAngus S. Murphy, Anindita Bandyopadhyay, Susanne E. Holstein,

and Wendy A. Peer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 221

Molecular Physiology of Legume Seed DevelopmentHans Weber, Ljudmilla Borisjuk, and Ulrich Wobus � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 253

Cytokinesis in Higher PlantsGerd Jürgens � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 281

Evolution of Flavors and ScentsDavid R. Gang � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 301

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Contents ARI 29 March 2005 21:29

Biology of Chromatin DynamicsTzung-Fu Hsieh and Robert L. Fischer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 327

Shoot BranchingPaula McSteen and Ottoline Leyser � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 353

Protein Splicing Elements and Plants: From Transgene Containmentto Protein PurificationThomas C. Evans, Jr., Ming-Qun Xu, and Sriharsa Pradhan � � � � � � � � � � � � � � � � � � � � � � � � � � � 375

Molecular Genetic Analyses of Microsporogenesis andMicrogametogenesis in Flowering PlantsHong Ma � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 393

Plant-Specific Calmodulin-Binding ProteinsNicolas Bouche, Ayelet Yellin, Wayne A. Snedden, and Hillel Fromm � � � � � � � � � � � � � � � � � � � � 435

Self-Incompatibility in PlantsSeiji Takayama and Akira Isogai � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 467

Remembering Winter: Toward a Molecular Understanding ofVernalizationSibum Sung and Richard M. Amasino � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 491

New Insights to the Function of Phytopathogenic Baterial Type IIIEffectors in PlantsMary Beth Mudgett � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 509

INDEXES

Subject Index � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 533

Cumulative Index of Contributing Authors, Volumes 46–56 � � � � � � � � � � � � � � � � � � � � � � � � � � � 557

Cumulative Index of Chapter Titles, Volumes 46–56 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 562

ERRATA

An online log of corrections to Annual Review of Plant Biology chapters may be found athttp://plant.annualreviews.org/

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