Calcium and Calmodulin-Mediated Regulation of Gene Expression in ...

Molecular Plant

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Volume 2

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Number 1

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Pages 13–21

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January 2009

REVIEW ARTICLE

Calcium and Calmodulin-Mediated Regulation of Gene Expression in Plants Min Chul Kim1, Woo Sik Chung, Dae-Jin Yun and Moo Je Cho Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center and Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701, Korea

ABSTRACT Sessile plants have developed a very delicate system to sense diverse kinds of endogenous developmental cues and exogenous environmental stimuli by using a simple Ca21 ion. Calmodulin (CaM) is the predominant Ca21 sensor and plays a crucial role in decoding the Ca21 signatures into proper cellular responses in various cellular compartments in eukaryotes. A growing body of evidence points to the importance of Ca21 and CaM in the regulation of the transcriptional process during plant responses to endogenous and exogenous stimuli. Here, we review recent progress in the identification of transcriptional regulators modulated by Ca21 and CaM and in the assessment of their functional significance during plant signal transduction in response to biotic and abiotic stresses and developmental cues. Key words:

Abiotic/environmental stress; calcium signaling/transport; gene expression.

INTRODUCTION Plant cells have developed an elaborate system for perceiving their endogenous and exogenous environments and eventually producing proper physiological responses. Plants employ the divalent cation calcium (Ca2+) as a second messenger in relaying these endogenous (developmental) and exogenous (environmental) signals to appropriate cellular responses. Calcium alone specifically encodes a myriad of distinct signals by using spatial and temporal Ca2+ spikes as well as the frequency and amplitude of Ca2+ oscillations (Evans et al., 2001; Rudd and Franklin-Tong, 2001; Sanders et al., 2002; Ng and McAinsh, 2003; White and Broadley, 2003). These ‘Ca2+ signatures’ are decoded by several types of Ca2+ sensor proteins that contain a high-affinity Ca2+binding motif, the ‘EF-hand’ motif. Three major classes of Ca2+ sensors have been identified in plants. The first of these is CaM, which is a ubiquitous and Ca2+-binding protein that is highly conserved in eukaryotes (Snedden and Fromm, 2001; Yang and Poovaiah, 2003). The calcium-dependent protein kinase (CDPK) represents a second class of Ca2+ sensor; this protein contains a catalytic kinase domain and EF-hand motifs (Cheng et al., 2002). The third type of EF-hand-containing Ca2+modulated protein is the SOS3 family of proteins also referred to as a calcineurin B-like protein (CBL), which is a plant-specific Ca2+ sensor (Luan et al., 2002; Zhu, 2002). The binding of Ca2+ to Ca2+ sensors triggers a change in conformation and/or enzymatic activity, followed by the participation of these activated Ca2+/Ca2+ sensor complexes in the induction of appropriate physiological cellular responses via modulation of the functions of the binding/target proteins involved in a plethora of cellular processes, including ion transport, metabolism, post-translational protein modifications and gene expression.

Calmodulin is a small acidic protein that contains four EFhands and is one of the best characterized Ca2+ sensors. The binding of Ca2+ to CaM induces a conformational change of this protein, which results in exposure of hydrophobic surfaces surrounded by negative charges and contributes to hydrophobic and electrostatic interactions with its target proteins (Snedden and Fromm, 1998; Hoeflich and Ikura, 2002). Although CaM has no enzymatic activity of its own, the Ca2+/ CaM complex is able to regulate a variety of cellular processes by modulating the activities of numerous target proteins. To date, more than 50 different types of CaM-binding proteins have been identified and their physiological functions are implicated in diverse aspects of cellular processes, including regulation of metabolism, cytoskeleton function, phytohormone signaling, ion transport, protein folding, protein phosphorylation and dephosphorylation, phospholipid metabolism, and transcriptional regulation (Snedden and Fromm, 2001; Yang and Poovaiah, 2003; Bouche´ et al., 2005). In addition to the intracellular (cytosolic and nuclear) functions of CaM, recent evidence suggests the presence of extracellular CaM and CaM-binding proteins in plants, as well as the regulation of important physiological functions by extracellular CaM, including proliferation of suspension-cultured cells, pollen

1 To whom correspondence should be addressed. E-mail [email protected], fax +82-55-751 5420, tel. +82-55-751-5428.

ª The Author 2009. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS. doi: 10.1093/mp/ssn091, Advance Access publication 6 January 2009 Received 21 November 2008; accepted 21 November 2008

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Calcium Signaling and Transcription

germination and tube growth, and stomatal closure (Ma et al., 2000; Chen et al., 2004; Mao et al., 2005; Wang et al., 2008). The Ca2+/CaM complex mediates plant responses to developmental cues and environmental biotic and abiotic stimuli via two distinct pathways. First, the Ca2+/CaM complex directly binds to and modulates the activity of numerous target proteins that participate in the control of a variety of cellular functions. Alternatively, the Ca2+/CaM complex indirectly triggers cellular responses by regulating the expression of genes encoding downstream effectors. In this review, we will focus on recent advances in the investigation of the role of Ca2+ and CaM in the regulation of gene expression.

SENSING SIGNALS IN THE NUCLEUS Ca2+ Signaling in the Nucleus Even though the nucleus is separated from the cytosol by the nuclear envelope, it is still capable of communicating with the cytosol through nuclear pores. For this reason, it has been largely assumed that an elevation of the cytosolic Ca2+ levels in response to stimuli can influence nuclear calcium signaling via simple diffusion through the nuclear pore; however, recent empirical evidence suggests that the nuclei of plant cells also possess a specific Ca2+ signaling mechanism. The nucleus exhibits a distinct and independent Ca2+ signature when compared with the cytosol, in response to blue light, osmotic, mechanical, and thermal stimuli, as well as to the Ca2+ stimulating agent mastoparan (Baum et al., 1999; Pauly et al., 2000, 2001; Xiong et al., 2004). Moreover, the identification of Ca2+ channels in the nuclear envelope provides further evidence supporting the existence of a nuclear-specific Ca2+ signaling pathway in plants (Grygorczyk and Grygorczyk, 1998; White, 2000; Brie`re et al., 2006). Similar to what is observed in the cytosol, Ca2+ spikes in the nucleus contributes to the regulation of several nuclear-specific cellular processes, which include DNA replication, DNA degradation during programmed cell death, cell cycle regulation, and transcription (Snedden and Fromm, 2001; White and Broadley, 2003).

CaM Is a Primary Nuclear Ca2+ Signal Decoder The nuclear Ca2+ signature encoding various stimuli is decoded in part by EF-hand motif-containing Ca2+ sensors, which include CaM and CDPK (Snedden and Fromm, 2001; Harper et al., 2004). Calcium-dependent protein kinase proteins are members of the serine/threonine protein kinase family and are specifically expressed in plants and some protozoans (Harper et al., 2004). Sub-cellular localization analysis has implicated a role for these proteins in various intracellular compartments, including the nucleus. Two Arabidopsis CDPK isoforms, AtCPK3 and AtCPK4, have a nuclear and cytosolic distribution, which suggests a possible function in the nucleus (Dammann et al., 2003). The CDPK isolated from cultured groundnut (Arachis hypogea) cells contains a nuclear localization signal (NLS) sequence in its junction domain, which is sufficient for the translocation of the Arachis CDPK (AhCPK2) into

the nucleus in response to osmotic stress (Raichaudhuri et al., 2006). A similar mode of nuclear translocation in response to NaCl stress has been observed for McCDPK1, which is a CDPK isolated from the common ice plant Mesembryanthemum crystallinum (Patharkar and Cushman, 2000). The physical interaction of plant CDPKs with putative DNA-binding proteins and their co-localization in the nucleus suggest a possible role for CDPK in the regulation of gene expression (Patharkar and Cushman, 2000; Rodriguez Milla et al., 2006). In addition to the role of CDPK, the presence of CaM in the nucleus and the identification of several nuclear molecules as CaM-binding proteins provide evidence that supports a pivotal role for CaM in the decoding of Ca2+ signaling, both in the cytosol and in the nucleus. Early immunocytochemical observations conducted using CaM-specific antibodies in several plant species, including pea and barley, revealed the localization of CaM in the nucleus (Dauwalder et al., 1986; Schuurink et al., 1996). The isolation of a novel type of CaM from petunia, which contains a prenylation site at its C-terminus, provided more evidence for the role of CaM in the nucleus (Rodrı´guezConcepcioń et al., 1999). These results suggested that the prenylation of this CaM, termed CaM53, can be affected by the sugar status of the cell. In addition, sugar-deficient conditions lead to the translocation of non-prenylated CaM53 protein into the nucleus, where it may play a role in the Ca2+-mediated sugarsensing pathway in plant cells (Rodrı´guez-Concepcioń et al., 1999). Moreover, the importance of CaM as a nuclear Ca2+ decoder is reflected in the identification of numerous nuclear CaM-binding proteins. The pea nuclear apyrase binds to CaM in a Ca2+-dependent manner and is activated by Ca2+/CaM binding (Chen et al., 1987; Hsieh et al., 2000). Another Ca2+ -dependent plant-specific CaM-binding nuclear protein, the potato CaM-binding protein (PCBP), was identified by screening an expression library prepared from developing potato tubers, which implies an important role for Ca2+/CaM-mediated nuclear signaling in tuberization (Reddy et al., 2002a). Recently, a novel type of nuclear CaM-binding protein (AtCaMBP25) was reported in Arabidopsis. The analysis of gain- and loss-offunction mutants of this protein indicates that AtCaMBP25 functions as a negative regulator of osmotic stress responses (Perruc et al., 2004). Finally, the strongest evidence supporting a crucial role for CaM as a nuclear Ca2+ signaling decoder arises from the identification of a variety of transcription factors regulated directly or indirectly by CaM (Snedden and Fromm, 2001; Yang and Poovaiah, 2003; Bouche´ et al., 2005); thus, accumulating evidence strongly suggests a role for CaM in the mediation of cellular responses to developmental and environmental stimuli via regulation of gene expression.

RESPONSE TO NUCLEAR SIGNALS: TRANSCRIPTIONAL ASPECT Ca2+-Binding Transcription Factors The first identified Ca2+-binding transcription factor, designated AtNIG1 (Arabidopsis thaliana NaCl-inducible gene 1),

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was recently described in plants (Kim and Kim, 2006). AtNIG1 is a basic helix-loop-helix-type transcription factor that contains an EF-hand motif. In addition, the bacterially expressed recombinant AtNIG1 protein binds to the canonical E-box element (CANNTG), which is found in the promoter region of several salt stress-responsive genes, which include CBFs (Crepeat/DRE-Binding Factors) (Shinwari et al., 1998; Chinnusamy et al., 2003). The expression of the AtNIG1 gene is induced by NaCl treatment and AtNIG1 loss-of-function mutant plants are more sensitive to salt stress and abscisic acid (ABA), which suggests that AtNIG1 participates in salt stress signaling by regulating the expression of salt stress-responsive genes (Kim and Kim, 2006); however, the biological role of Ca2+ binding on the activity of AtNIG1 remains unclear. A Ca2+-binding transcription repressor protein named DREAM (Down-stream Regulatory Element Antagonist Modulator) was first identified in humans a decade ago (Carrioń et al., 1999). In low nuclear Ca2+ level conditions, DREAM binds to the DRE silence element of the prodynorphin gene and suppresses its expression. In contrast, the elevation of nuclear Ca2+ levels in response to pain stimulus or related neuronal depolarization leads to the binding of Ca2+ to four EF-hand motifs of DREAM and results in the dissociation of Ca2+-bound DREAM from the DRE element, which, in turn, triggers the transcription of target genes (Ikura et al., 2002; Osawa et al., 2005). The identification of Ca2+-binding transcription factors in plants and in animals implies the conservation of the Ca2+ signaling pathway among different kingdoms.

CaM Functions as a Transcriptional Regulator In addition to AtNIG1, another interesting type of Ca2+ -binding transcription factor was identified in plants, CAM7. One of the Arabidopsis CaM isoforms functions as a transcriptional regulator (Kushwaha et al., 2008). The CAM7 protein, also named ZBF3, was identified together with two other Zbox binding transcription factors, ZBF1/MYC2 and ZBF2/ GBF1, in a ligand-binding screen (Yadav et al., 2005; Mallappa et al., 2006). CAM7 binds to Z-/G-box elements located in the promoter of light-responsive genes, including CAB1 and RBCS1A, both in vitro and in vivo, and triggers their expression. Consistently, the expression of CAB1 and RBCS1A is upregulated by CAM7 overexpression in transgenic plants and is down-regulated in loss-of-function mutant plants. Moreover, ectopic expression of CAM7 causes hyperphotomorphogenic growth, irrespective of light quality. Taken together, these results suggest that CAM7 participates in the Ca2+-mediated light-signaling pathway by regulating photomorphogenic growth and light-responsive gene expression (Kushwaha et al., 2008). Although the biological role played by Ca2+ binding in the transcriptional function of CAM7 remains to be elucidated, this work is valuable in that it supports this potential role for CAM7. The mutational analysis of the EF-hand region of CAM7 revealed that the EF-hand Ca2+-binding motif also plays an important role in determining target specificity. The dual role of the EF-hand region in Ca2+- and DNA binding

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suggests that the binding of Ca2+ to the EF-hand motif may lead to a conformational change of this region, which, in turn, may affect the DNA-binding activity of CAM7, as was observed for the DREAM protein (Ikura et al., 2002; Osawa et al., 2005).

Ca2+/CaM-Mediated Transcriptional Regulation: Direct Regulation In plants, the TGA3 transcription factor, which is a member of the basic leucine zipper transcription factor family, was first identified by its physical interaction with Ca2+-loaded CaM (Szymanski et al., 1996). The TGA3 protein binds to the C-/G-box sequence elements found in the promoter of the Arabidopsis calmodulin isoform Cam-3. This binding of the Ca2+/CaM complex to TGA3 enhances its DNA-binding activity. However, the signaling pathway involving TGA3 has not been defined. The largest and the best characterized CaM-binding transcription factor family in plants includes proteins that contain a CG-1 DNA-binding domain. The existence of this novel type of DNA-binding protein was first identified during the isolation of a partial cDNA clone encoding a sequence-specific DNA-binding protein from parsley, which is named CG-1 (da Costa e Silva, 1994). Parsley CG-1 binds to a CGCG DNA sequence motif, the transcription of which is promoted by UV irradiation; however, it was not until other plant CG-1 orthologs were identified as CaM-binding proteins that it was established that this novel type of DNA-binding protein family binds to CaM. CG-1 homologs have been identified in various plant species, including Arabidopsis, rice, tobacco, and rapeseed (Reddy et al., 2000; Yang and Poovaiah, 2000; Bouche´ et al., 2002; Yang and Poovaiah, 2002; Choi et al., 2005), as well as in other organisms, which include humans and Drosophila (Finkler et al., 2007). All CG-1 family members contain a typical CG-1 DNA-binding domain in their N-terminus, a TIG nonspecific DNA-binding domain, several ankyrin (ANK) repeats (involved in protein–protein interactions), and CaM-binding motifs localized at the C-terminus (Finkler et al., 2007). The CG-1 proteins possess two different types of CaM-binding motifs; a Ca2+-dependent CaM-binding domain and a Ca2+-independent CaM-binding (or Ca2+-dependent CaMdissociation) domain, termed the IQ motif. In contrast to the Ca2+-dependent CaM-binding motif, which has been characterized in various CG-1 family members, the binding of CaM to the IQ motif in Ca2+-free conditions was only verified in an in-vitro CaM overlay assay using the rice CG-1 homolog, OsCBT. However, the role played by Ca2+-independent CaM binding in the function of OsCBT remains to be elucidated (Choi et al., 2005). Several independent empirical observations indicate that the CG-1 proteins function as transcriptional activators of downstream gene expression (Bouche´ et al., 2002; Mitsuda et al., 2003; Choi et al., 2005). Moreover, a physiological meaning for Ca2+/CaM binding in the function of CG-1 transcription activators was deduced from the analysis of OsCBT-mediated transcription regulation in the presence or absence of CaM using a protoplast system. Co-transfection with a CaM gene suppressed the transcriptional activation mediated by OsCBT,

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which seems to indicate that the Ca2+/CaM complex functions as a negative regulator of the activity of OsCBT (Choi et al., 2005). The analysis of the gene expression patterns of CG-1 family members in plants may provide valuable clues toward the understanding of the biological roles of these proteins. The expression of NtER1 (Nicotiana tobaccum ethyleneresponsive Gene1) and EICBP (Ethylene Induced Calmodulin Binding Protein), which are CG-1 family members isolated from tobacco and Arabidopsis, respectively, is up-regulated in response to phytohormone ethylene (Reddy et al., 2000; Yang and Poovaiah, 2000). Moreover, the transcripts of NtER1 are more abundant in senescing tissues, which indicates that NtER1 may participate in the ethylene signaling pathway involved in plant senescence and cell death (Yang and Poovaiah, 2000). In addition, the expression patterns of six Arabidopsis CG-1 family members (named AtSRs; Arabidopsis thaliana signal-responsive genes/AtCAMTAs; Arabidopsis thaliana CaM-binding Transcription Activators) were extensively examined by RT–PCR. These six genes show differential induction patterns in response to various abiotic stresses (e.g. heat, cold, UVB, salt, and wounding), hormones (e.g. ethylene and ABA), and defense signaling molecules (e.g. methyl jasmonate, H2O2, and salicylic acid) (Yang and Poovaiah, 2002). The first genetic evidence of a biological function for a CG-1 transcriptional activator family member was only reported recently. The camta3 mutants, which consist of loss-of-function alleles of one of the Arabidopsis CAMTA family members (CAMTA3), show enhanced spontaneous lesion formation, increased expression of defense-related genes, and resistance to pathogens, which suggest that CAMTA3 is also involved in biotic defense responses (Galon et al., 2008). Furthermore, the analysis of the gene expression profile in the camta3 mutant revealed that the WRKY transcription factor may be a direct downstream target of CAMTA3. The results of all analyses of the plant CG-1 family members suggest that these transcription activators may play important roles in Ca2+/CaM-mediated plant responses to both abiotic and biotic environmental stresses and developmental stimuli. In response to hyperosmotic stresses (e.g. salt and drought stresses), plants activate various signaling pathways that promote the sensing of the stimuli and trigger the adequate cellular responses, which include Ca2+ signaling, protein phosphorylation cascades, and ABA signaling (Zhu, 2002). The Ca2+ signatures also play an important role in sensing the saline environment. The salt-overly-sensitive (SOS) pathway is one of the best-characterized pathways that participate in the decoding of the salt stress-mediated Ca2+ signatures (Chinnusamy et al., 2004; Mahajan et al., 2008). In addition, a growing body of evidence indicates that CaM is a major sensory molecule involved in the decoding of Ca2+ signatures associated with salt stress (Bouche´ et al., 2005). A divergent type of soybean CaM isoform (GmCaM4) was recently reported to mediate saltinduced Ca2+ signaling by activating an R2R3-type MYB transcription factor, which is an upstream regulator of a set of salt- and dehydration-responsive genes (Yoo et al., 2005).

CaM binds to the R2R3 DNA-binding domain of the AtMYB2 protein in a Ca2+-dependent manner, which, in turn, enhances not only the DNA-binding activity of AtMYB2, but also the AtMYB2-mediated transcriptional activation. Moreover, overexpression of GmCaM4 in Arabidopsis leads to constitutive expression of salt- and dehydration-responsive genes, which include P5CS1, ADH1, and rd22, and confers salt tolerance in transgenic plants. More interestingly, the expression of GmCaM4 is induced not only by salt stress, but also by pathogens; constitutive expression of GmCaM4 is sufficient to trigger the expression of an array of defense-related genes and confers broad-spectrum resistance to bacterial, fungal and viral pathogens in transgenic tobacco plants (Heo et al., 1999). These results suggest that GmCaM4 plays a crucial role in both abiotic and biotic signaling pathways. However, the exact physiological role of this divergent type of CaM isoform in plant defense responses to pathogens warrants clarification. The WRKY protein family consists of a large number of zincfinger-type transcription factors. The 74 WRKY family members are found in the Arabidopsis genome and their biological functions are mainly implicated in plant defense responses, which include the activation of systemic acquired resistance ¨ lker and Somssich, 2004). One of the Arabidopsis WRKY fam(U ily members, AtWRKY7, was recently reported to interact with CaM in a Ca2+-dependent manner via the N-terminal C-region ([K/R]EPRVAV[Q/R]SEVD[I/V]L). Moreover, Ca2+-loaded CaM was also shown to bind to the WRKY group IId family members, which contain the C-region, using a CaM overlay filter assay (Park et al., 2005). Interestingly, the analysis of gainand loss-of-function mutants suggests a biological function for AtWRKY7 in plant defense responses to pathogen invasion (Kim et al., 2006). The AtWRKY7 loss-of-function mutants exhibit enhanced resistance to a virulent strain of bacteria. In addition, transgenic plants that constitutively overexpress AtWRKY7 exhibit enhanced susceptibility to bacterial pathogens, which is concomitant with a reduction in the expression of defense-related genes. This suggests a negative regulatory role for AtWRKY7 in plant defense responses to bacterial pathogens. Even though a direct biological link between the role of AtWRKY7 in plant defense responses and the regulation of AtWRKY7 function by CaM binding is currently lacking, the analysis of this correlation may provide important biological clues as to the role of the Ca2+/CaM complex in plant defense signaling pathways. In contrast to the well defined role of the Ca2+/CaM complex in transcriptional regulation during plant response to environmental stresses, its role in plant development is poorly understood. A NAC domain-containing transcription factor, termed CBNAC (CaM-binding NAC protein) was identified recently as a CaM-binding protein via the screening of an Arabidopsis cDNA expression library. The function of the Ca2+/CaM complex in CBNAC-mediated transcriptional regulation was characterized (Kim et al., 2007). The NAC protein family comprises a large group of plant-specific transcription factors. It has been suggested that NAC proteins play a role in the development of

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embryos, shoot apical meristems, and floral organs and also in auxin-mediated lateral root formation. NAC proteins have also been implicated in plant responses to biotic and abiotic stresses (Olsen et al., 2005). A Ca2+-dependent CaM-binding domain was mapped to the C-terminus of the CBNAC protein and their physical interaction was confirmed in yeast. The random binding site selection method was used to identify a CBNAC-binding sequence (CBNACBS) containing a core GCTT motif. Furthermore, a transient assay using a synthetic promoter containing a CBNACBS revealed that CBNAC functions as a transcriptional repressor of target gene expression and that CaM promotes the transcriptional repression mediated by the CBNAC protein (Kim et al., 2007). The identification of target genes of the CBNAC transcription factor will help elucidate the physiological functions of this protein as well as the binding of CaM to CBNAC.

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Ca2+/CaM-Mediated Transcriptional Regulation: Indirect Regulation In addition to the direct binding of Ca2+ or of the Ca2+/CaM complex to transcription factors and ensuing modulation of their functions, which include DNA-binding or transcription activities (summarized in Table 1), the Ca2+/CaM complex may also regulate gene expression indirectly by modulating posttranslational modification of transcription factors (Snedden and Fromm, 2001). It is well known that, in animals, this indirect transcriptional regulation that is mediated by CaM is mainly accomplished through CaM kinases and the CaMdependent protein phosphatase calcineurin (Ikura et al., 2002). Two recent reports suggest that, in plants, CaM also participates indirectly in the regulation of gene expression by acting through a CaM-binding protein kinase and a CaMbinding protein phosphatase (Liu et al., 2007, 2008). Liu

Table 1. Calcium- and Calmodulin-Binding Transcription Factors in Plants Protein

Properties

References

– Arabidopsis basic helix-loop-helix protein; binds to E-box element (CANNTG); single EF-hand motif

(Kim and Kim, 2006)

Ca2+-binding transcription factors

AtNIG1

– Induced expression in response to NaCl; atnig1-1 knockout mutant shows enhanced sensitivity to salt stress

CAM7 (ZBF3, Z-boxbinding transcription factor3)

– Arabidopsis calmodulin isoforms; binds to Z-/G-box light-responsive elements

(Kushwaha et al., 2008)

- Promotes the expression of light-responsive genes and photomorphogenic growth

CaM-binding transcription factors – Arabidopsis basic leucine zipper protein; binds to C/G-box element of Arabidopsis CaM isoforms (Cam-3) promoter TGA3

– CaM binding enhances the DNA-binding activity of TGA3

CG-1 family transcription activators (CAMTA/SR/ EICBP/NtER1/OsCBT)

– Contain a novel type of CG-1 DNA-binding domain; binds to DNA sequence containing core CGCG motif – Found in various organisms including plants, humans, and flies – Ca2+-dependent CaM binding and also Ca2+-independent CaM binding (OsCBT); the binding of Ca2+/CaM complex suppresses OsCBT-mediated transcriptional activation

(Szymanski et al., 1996)

(Reddy et al., 2000; Yang and Poovaiah, 2000; Bouche´ et al., 2002; Yang and Poovaiah, 2002; Choi et al., 2005; Finkler et al., 2007; Galon et al., 2008)

– Involved in various signaling pathways in response to abiotic, biotic, and hormonal stimuli and camta3, loss-of-function mutant of one of the Arabidopsis CAMTA family showed enhanced spontaneous lesion formation, PR gene expression, and resistance to pathogens AtMYB2

– Arabidopsis R2R3-type transcription factor; regulates expression of salt- and dehydration-responsive genes, which include P5CS1, ADH1, and rd22

(Yoo et al., 2005)

– Binding to the Ca2+/CaM complex enhances its DNA-binding and transcriptional activator activities AtWRKY7

– Arabidopsis zinc-finger-type transcription factor; binds to the W-box element [(C/T)TGAC(T/C)]

(U¨lker and Somssich, 2004; Park et al., 2005; Kim et al., 2006)

– The Ca2+/CaM complex binds to the N-terminal C-region of AtWRKY7 – Functions as a transcriptional repressor and enhances plant susceptibility to bacterial pathogen CBNAC

– Arabidopsis NAC transcription factor; binds to DNA sequences that contain a core GCTT motif – Functions as a transcriptional repressor and its activity is enhanced by Ca2+-dependent binding to CaM

(Kim et al., 2007)

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et al. (2007) showed that AtPP7, the first plant protein Ser/Thr phosphatase ever described, interacts with both CaM and the heat shock transcription factor (HSF), which regulates the expression of heat shock protein (HSP) genes. The expression of the Arabidopsis thaliana PP7 gene is very rapidly induced in response to heat shock. In addition, pp7 knockout mutants are more sensitive to heat stress than wild-type plants, whereas transgenic plants overexpressing AtPP7 exhibit increased thermotolerance with elevated levels of HSP genes in heat shock conditions (Liu et al., 2007). In addition to the action of the CaM-binding protein phosphatase, an involvement of a CaM-binding protein kinase in the regulation of transcription has also been proposed. The Arabidopsis CaM-binding protein kinase AtCBK3 phosphorylates the AtHSFA1a transcription factor in vitro, in the presence of Ca2+ and CaM conditions. Moreover, the binding of CaM to AtCBK3 induces its phosphorylation activity upon AtHSFA1a (Liu et al., 2008). This observation is consistent with the previous results, which indicate that phosphorylation of human HSF1

by Ca2+/CaM-dependent protein kinase II (CaMKII) induces its transcriptional activity (Holmberg et al., 2001). Taken together, those results suggest a biological role for the Ca2+/CaM complex in the heat-shock signaling pathway of plants. The completion of the sequencing of several genomes and the development of bioinformatic approaches have enabled a more efficient identification of CaM-binding proteins in plants. A large number of new Ca2+-dependent CaM-binding proteins were identified from Arabidopsis via extensive screening of expression libraries and searching for homologs to other known plant or animal CaM-binding proteins (Reddy et al., 2002b). One of these newly identified CaM-binding proteins is a pirin-like protein similar to human pirin. The human pirin protein interacts with the CCAAT box binding nuclear factor I (NFI/CTF1) and with the oncoprotein Bcl-3, and it has been suggested that pirin may participate in NF-jB-dependent transcriptional regulation (Wendler et al., 1997; Pang et al., 2004); however, the physiological role of the pirin-like protein in plants is still not clear.

Figure 1. Mechanisms of Transcriptional Regulation Mediated by Ca2+ and CaM in Plants. The Ca2+ and CaM elicited by developmental cues and environmental stresses mediate plant responses by regulating the transcriptional process in various ways. (A) In the simplest case, Ca2+ directly binds to the transcription factor (TF) and modulates its activity. (B) The Ca2+-loaded CaM binds to DNA and functions as a TF. (C) Most commonly, the Ca2+/CaM complex interacts with TFs and modulates their function. (D) The Ca2+/CaM complex indirectly regulates transcription by participating in the multi-component transcriptional machinery, which is composed of the Ca2+/CaM complex, TFs, and transcription factor-binding protein (TFBP). The latter functions as a bridge between the Ca2+/ CaM complex and TFs. (E) Finally, Ca2+/CaM regulates gene expression via the modulation of the phosphorylation status of TFs, effected by a CaM-binding protein kinase and a CaM-binding protein phosphatase. PM, plasma membrane; NE, nuclear envelop; CaM, calmodulin; TF, transcription factor; TFBP, transcription factor-binding protein; PK, protein kinase; PP, protein phosphatase; P, phosphate.

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A plant-specific family of nuclear proteins designated as AtBTs (Arabidopsis thaliana BTB and TAZ domain proteins) have been identified as Ca2+-dependent CaM-binding proteins (Du and Poovaiah, 2004). Yeast two-hybrid analysis revealed that some members of the AtBT family interact with members of the fsh/Ring3 class transcription regulators. The structural features of AtBT proteins and their interaction with other transcriptional regulators indicate that the AtBT family may participate in transcriptional modulation via an association with multi-component transcriptional machinery, and not through direct binding to DNA, as suggested in animals (Chan and La Thangue, 2001). Moreover, the expression patterns of the various members of the AtBT family suggest that AtBTs may play a role in transcriptional regulation in response to H2O2 and salicylic acid stresses (Du and Poovaiah, 2004). Recent evidence seems to suggest that Ca2+-loaded CaM also regulates gene expression by modulating the transcription elongation reaction. The Ca2+-loaded CaM interacts with DRL1 (DEFORMED ROOTS AND LEAVES1) protein, which is homologous to the yeast TOT4/KTI12 proteins. In turn, this molecule associates with the RNA polymerase II transcription elongation complex (Nelissen et al., 2003). Abrogation of DRL1 function causes defects in the development of organs and meristems in Arabidopsis.

CONCLUSIONS AND PERSPECTIVES Precisely controlled gene expression in challenging environmental conditions and during dynamic developmental processes is critical to the triggering of proper cellular responses. Plants use Ca2+ signature and CaM, which is a prominent Ca2+ signature decoder, in the delicate regulation of gene expression in response to endogenous or exogenous stimuli. As summarized in Figure 1, Ca2+ and CaM participate in transcriptional regulation by performing various regulatory actions on transcription factors. (A) The elevated Ca2+ in the nucleus (either diffused from the cytosol or released from nuclear Ca2+ reservoirs in response to stimuli) binds directly to transcription factors and modulates their activity. (B) Ca2+-loaded CaM binds directly to promoter sequences and regulates gene expression, which implies that CaM functions as a transcription factor. (C) Most commonly, the Ca2+/CaM complex interacts with transcription factors and modulates either their DNA-binding or transcriptional activities. (D) The Ca2+/CaM complex indirectly regulates transcription by associating with the multi-component transcriptional machinery. For example, the Ca2+/CaM complex binds to transcription factor-binding protein (TFBP) and, in turn, this trimeric complex interacts with transcription factors and modulates their function. The TFBP can function as a bridge between Ca2+/ CaM and transcription factors. (E) Finally, the Ca2+/CaM complex regulates gene expression by modulating the phosphorylation status of transcription factors. This indirect regulation is achieved by a CaM-binding protein kinase and a CaMbinding protein phosphatase. In the last decade, a great

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number of CaM-binding proteins, including transcriptional regulators, have been identified and their biological functions have been extensively investigated in plants. Moreover, studies of gene expression and characterization of loss- and gain-offunction mutants indicate that transcriptional regulation mediated by Ca2+ and CaM may play an important role in relaying a diverse range of endogenous and exogenous signals (e.g. hormones, pathogens, salt, light, heat, cold, and wounding) to proper plant responses. However, very little is known about the specific physiological roles played by transcriptional regulators that are modulated by Ca2+/CaM or about how CaM regulates the biological function of these regulator molecules. Molecular genetic analysis of knockout and gain-of-function mutants for these molecules, followed by transcriptome, proteome, and interactome analyses using mutants, will allow the understanding of the functional significance of CaM and its target transcriptional regulators in plant signaling pathways.

FUNDING This work was supported by grants from the Environmental Biotechnology National Core Research Center (R15-2003-012-010010) funded by the Ministry of Education, Science and Technology of Korea; the Basic Research Program (R01-2006-000-10035-0] of KOSEF; and the Biogreen21 Project (20070301034030) of the Rural Development Administration.

ACKNOWLEDGMENTS We thank Ray A. Bressan and Sung Cheol Koo for critical reading and insightful comments. No conflict of interest declared.

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