Calmodulin-Related Proteins Step Out from the Shadow of Their Namesake

Abstract

Emerging roles for these proteins in plant development and stress response highlight their importance in plant signaling, and their functional diversity underscores the significance of Ca²⁺ as a second messenger in plants.

Like all eukaryotes, plants must be able to detect changes in their environment and respond accordingly. In broad terms, cells possess signal transduction pathways, mediating responses to internal and external cues that consist of three distinct nodes: stimulus perception, signal transduction, and subsequent physiological response. The transduction node exploits rapid and transient changes in the cellular concentrations of second messengers to directly or indirectly affect the activities of downstream effector proteins (metabolic enzymes, transcription factors, etc.) that ultimately coordinate cellular responses. Changes in metabolic activity, gene expression, and protein turnover collectively lead to adaptive physiological events that are appropriate for the initial stimulus. This model (perception, transduction, and response) is simplistic yet widely applicable. Among the common themes that have emerged in signal transduction research is the importance of calcium (Ca²⁺) as a second messenger in plant development and in response to a variety of environmental stresses (McAinsh and Pittman, 2009; DeFalco et al., 2010; Dodd et al., 2010). Given the diversity of stimuli known to evoke Ca²⁺ transients in cells, plants make an excellent model system for studying the mechanisms of Ca²⁺ signal transduction.

Ca²⁺ IN PLANT SIGNAL TRANSDUCTION

Given that elevated Ca²⁺ levels are potentially toxic to ATP metabolism, and that Ca²⁺ may precipitate negatively charged biological molecules (Spalding and Harper, 2011), cells maintain tight control over cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt). Cells actively pump Ca²⁺ into the extracellular space and into intracellular stores (e.g. vacuole and endoplasmic reticulum), leading to the establishment of a steep electrochemical gradient of Ca²⁺ between these stores and the cytosol (McAinsh and Pittman, 2009; Spalding and Harper, 2011). At resting levels, Ca²⁺ is present in the cytosol at concentrations ranging from 100 to 200 nm, whereas concentrations in Ca²⁺ stores are orders of magnitude higher, in some cases reaching millimolar levels (Gilroy et al., 1993). Thus, cells are poised for rapid influxes of Ca²⁺ down its electrochemical gradient into the cytosol. While extrusion of Ca²⁺ is a constant process, when local influx transiently exceeds efflux, Ca²⁺ signaling events can occur (Spalding and Harper, 2011).

In the paradigm of Ca²⁺ signaling, the detection of a given stimulus evokes rapid and transient movement of Ca²⁺ into the cytosol. In plants, this has been observed for a number of developmental cues (e.g. gravitropic responses; Toyota et al., 2008), as well as abiotic stresses (e.g. salt and drought stress; Kiegle et al., 2000), and in response to biotic signals (e.g. pathogen attack and microbial symbioses; Ali et al., 2007; Kosuta et al., 2008). To generate Ca²⁺ fluxes, stimulus perception is linked to the coordinated activities of Ca²⁺ channels and pumps. Given the multitude of stimuli evoking cytosolic Ca²⁺ transients in plants, it is important to consider how specific cellular responses might be derived from a common second messenger.

Two main hypotheses describe how specificity of Ca²⁺-guided response may be achieved in plant cells: the Ca²⁺ signature hypothesis and the Ca²⁺ switch hypothesis. Both hypotheses can claim supporting evidence (Scrase-Field and Knight, 2003; McAinsh and Pittman, 2009), and they are not mutually exclusive. In the Ca²⁺ signature hypothesis, stimuli perceived by the cell evoke Ca²⁺ transients in the cytosol of particular amplitude, frequency, and spatial distribution that are thought to elicit a stimulus-specific downstream response. Calcium oscillations (i.e. Ca²⁺ “signatures”) having specific spatial and temporal properties have been documented in root hairs in response to Nod factors or signals derived from arbuscular mycorrhizal fungi (Miwa et al., 2006; Oldroyd and Downie, 2006; Kosuta et al., 2008) and in guard cells exposed to abscisic acid (ABA), oxidative stress, and external Ca²⁺ (Allen et al., 2000, 2001). In the binary switch model, specificity in Ca²⁺ signaling is derived from other components in the signaling pathway (i.e. Ca²⁺ sensors, see below; Scrase-Field and Knight, 2003). This hypothesis is supported by the observation that different stimuli (e.g. salt and drought stress) induce highly similar cytosolic Ca²⁺ influxes but elicit different downstream responses (Knight et al., 1997). It is noteworthy that cells may also coordinate Ca²⁺-mediated responses by priming/depriming of Ca²⁺ sensors (Israelsson et al., 2006). This stimulus-specific modulation (for example, posttranslational modification) of Ca²⁺ sensor function could permit or restrict different downstream signaling pathways depending on the prevailing conditions. Regardless of whether Ca²⁺ signals operate as signatures, switches, or variations thereof, a requirement of any model is that a mechanism exists to transduce Ca²⁺ signals to downstream effectors. Diverse families of Ca²⁺-binding proteins, collectively known as Ca²⁺ sensors, fulfill the role of signal propagation and occupy a key position in signal transduction pathways.

Ca²⁺-BINDING PROTEINS AND Ca²⁺ SENSORS

While a few different Ca²⁺-binding domains have been identified, Ca²⁺ sensors are most commonly characterized by the presence of one or more EF-hand Ca²⁺-binding motifs. The EF-hand is highly conserved among Ca²⁺-binding proteins and EF-hand-containing proteins are encoded by all eukaryotic genomes (Gifford et al., 2007). The Arabidopsis (Arabidopsis thaliana) genome is predicted to contain about 250 EF-hand proteins, more than any other organism examined to date (Day et al., 2002). EF-hand motifs are not exclusive to Ca²⁺ sensors and are also found in proteins, termed Ca²⁺ buffers, involved in regulating intracellular Ca²⁺ concentrations rather than signal transduction directly (Schwaller, 2010).

The EF-hand is composed of a 29-amino acid helix-loop-helix structure, with the central 12 residues forming a turn-loop structure that is responsible for coordination of the Ca²⁺ ion (Pidcock and Moore, 2001; Gifford et al., 2007). Within the coordination loop, ligating residues are found at positions 1, 3, 5, 7, 9, and 12 (Yamniuk et al., 2008). The loop is enriched in negatively charged residues, and a Gly at position six allows the loop to wrap around the Ca²⁺ ion, a feature critical for high affinity binding (Gifford et al., 2007). Structural properties of the EF-hand allow for rapid on/off Ca²⁺ binding, permitting quick responses to changes in [Ca²⁺]_cyt, a property likely to be vital for interpreting Ca²⁺ signals.

In plants, the three largest families of EF-hand Ca²⁺ sensors that have been characterized are the calmodulin (CaM) and CaM-like (CML) proteins, calcineurin-B-like (CBL) proteins, and the Ca²⁺-dependent protein kinases (CDPKs, called CPKs in Arabidopsis). Among these groups, the CaMs/CMLs and CBLs are considered to be sensor relays that function via regulation of downstream targets. Conversely, the CDPKs, as catalytic proteins, are sensor responders in which Ca²⁺ binding often regulates their kinase activity. Notably, three of these families, the CMLs, CBLs, and CDPKs, are unique to plants and some protists (CDPKs only), highlighting the importance of Ca²⁺ signaling to plant growth and development.

CaM is the most thoroughly characterized Ca²⁺ sensor in plants, and its physiological roles have been recently reviewed (Bouche et al., 2005; DeFalco et al., 2010). CaM is a small (149 amino acids), acidic protein consisting of two globular domains, each containing a pair of Ca²⁺-binding EF-hand motifs. A short central linker region connects the globular domains of CaM and affords it considerable flexibility in solution. Plant genomes contain multiple loci encoding conserved CaM isoforms. For example, seven distinct genes in Arabidopsis encode four different protein isoforms (CaM2/CaM3/CaM5, CaM1/CaM 4, CaM6, and CaM7) that share a minimum 97% identity at the primary sequence level (McCormack and Braam, 2003). In addition to the conserved CaMs, plant genomes contain expanded families of CML genes. A 50-member CML family, subdivided into nine phylogenetic groups, has been annotated in Arabidopsis, and rice (Oryza sativa) has been found to possess 32 CMLs (McCormack and Braam, 2003; McCormack et al., 2005; Boonburapong and Buaboocha, 2007); such large CML families are thus likely found across plant taxa.

In Arabidopsis, CMLs vary in length from 83 to 330 amino acids and share between 16% and 75% amino acid identity with CaM2 at the primary sequence level. Like CaM, they are characterized as possessing exclusively Ca²⁺-binding EF-hands and no other known functional motifs (McCormack and Braam, 2003; McCormack et al., 2005). Thus, CMLs are predicted to serve as Ca²⁺ sensors in planta. Many eukaryotes possess Ca²⁺ sensors related to CaM, such as S100 proteins, myosin light chains, and other small EF-hand proteins, but the CMLs are a family unique to plants. In other words, the closest orthologs of CMLs in other eukaryotes tend to be conserved CaMs or proteins very similar to CaMs. Unlike conserved CaM, which has four EF-hands, CMLs contain variable numbers of EF-hands, ranging from one (AtCML1) to six (AtCML12). The importance of this variation in terms of differential Ca²⁺ binding and response among CMLs to distinct Ca²⁺ signals has been hypothesized but not well explored. One interesting possibility is that some CMLs, like animal S100 proteins, may dimerize to facilitate pairing of EF-hands and thus cooperative binding of Ca²⁺. Dimerization of a Ca²⁺-bound N-terminal fragment of CML34 has been observed in vitro (Song et al., 2004). Some CMLs have N-terminal extensions of varying length, the functional significance of which remains unclear but may be involved in subcellular targeting of the protein. In Arabidopsis, CML30 is localized to the mitochondria, and a noncleavable transit peptide was identified at its N terminus (Chigri et al., 2012).

Another distinction between CMLs and CaMs can be found in the central helical linker region. In CaM, this feature affords structural flexibility important for the conformational changes that accompany interaction with target proteins. It is thus interesting to note that a number of CMLs when compared to CaM possess longer linker regions between the two globular domains: this may alter CML flexibility and perhaps interaction with downstream targets. Ultimately, how the physical characteristics of CMLs compare to CaM and influence their cellular roles remains largely speculative, as little empirical work has been done to determine their Ca²⁺ binding or other structural properties.

STRUCTURAL PROPERTIES OF CaM AND CMLs

Detailed biophysical analyses of CaM have revealed both high affinity binding of Ca²⁺ as well as Ca²⁺-induced conformational changes within the protein. Studies indicate that CaM binds Ca²⁺ with affinities in the high nanomolar to low micromolar range (Gilli et al., 1998), suggesting that the EF-hands of CaM are not occupied by Ca²⁺ in a resting cells. These affinities are, however, within the range of [Ca²⁺]_cyt measured during Ca²⁺ transients (McAinsh and Pittman, 2009), indicating that CaM could respond dynamically to changes in [Ca²⁺]_cyt. Binding of Ca²⁺ by CaM leads to changes in the secondary and tertiary structure of the protein (Ikura et al., 1990). In the absence of Ca²⁺, EF-hands adopt a “closed” conformation and Ca²⁺ binding by the motif results in “opening” of the hand as indicated by an increase in the interhelical angle of the EF-hand. These changes in the shape of the EF-hand upon Ca²⁺ binding lead to an overall Ca²⁺-responsive change in the structure of CaM (and presumably other EF-hand Ca²⁺ sensors). CaM is a strongly α-helical protein (approximately 40% for apoCaM), and Ca²⁺ binding results in an overall increase in α-helical content (approximately 50% for Ca²⁺/CaM; Martin and Bayley, 1986). These structural changes are accompanied by a dramatic increase in the surface hydrophobicity of CaM that is important for target interactions (Yamniuk and Vogel, 2005).

Where CMLs have been characterized biochemically, they display properties similar, though not identical, to that of conserved CaM. The Ca²⁺-binding properties of Arabidopsis CML42 (Dobney et al., 2009) have been examined in detail. Analysis of Ca²⁺ affinities by isothermal titration calorimetry identified three Ca²⁺-binding sites in CML42, one at the N terminus and two at the C terminus. The second EF-hand in CML42 is degenerate, having substitutions for several of the Ca²⁺-ligating residues within the coordination loop (Dobney et al., 2009). Of the three identified Ca²⁺-binding sites in CML42, two show dissociation constants similar to that of CaM, while one (EF-hand 3) has an affinity of approximately 30 nm in the presence of Mg²⁺, suggesting that this EF-hand is permanently occupied by Ca²⁺ in resting cells. This EF-hand may thus play a structural role, while the remaining two Ca²⁺-binding sites may serve sensory functions (Dobney et al., 2009). Interestingly, Ca²⁺-responsive conformational changes in CML42 were distinct from those of CaM, as CML42 showed no Ca²⁺-dependent changes in secondary structure but did exhibit changes in tertiary structure as demonstrated by NMR spectroscopy. Finally, CML42, like CaM, undergoes a considerable Ca²⁺-responsive increase in surface hydrophobicity (Dobney et al., 2009).

The structure of the Ca²⁺-loaded N-terminal EF-hand pair of CML34 from Arabidopsis has been resolved by NMR spectroscopy (Song et al., 2004). The first EF-hand in the N-terminal region of CML34 showed similar structure to the equivalent motif in CaM in the presence of Ca²⁺, having an interhelical angle of approximately 102° (compared with approximately 104° for CaM). Interestingly, the second EF-hand of CML34 had an interhelical angle of approximately 147° in the presence of Ca²⁺ in contrast to an angle of 102° for CaM, suggesting differences in Ca²⁺-responsive conformational changes between CaM and CMLs (Song et al., 2004). Analysis of CML42 by NMR revealed similar conformation of two EF-hands in both the Ca²⁺-bound and the Ca²⁺-free state (Dobney et al., 2009). Overall, structural analysis of CMLs compared with CaM suggests that sequence divergence among CMLs leads to differential responses to Ca²⁺ binding in vitro, and it is tempting to speculate that these differences may be important for in vivo CML-specific functions, such as response to specific Ca²⁺ signals or target interaction.

INTERACTION OF CaMs AND CMLs WITH THEIR TARGETS

CaM has been described as a universal Ca²⁺ sensor because of the myriad downstream effectors that it has been found to interact with. In plants, CaM physically associates with numerous targets, including protein kinases (Oh et al., 2012) and phosphatases (Liu et al., 2007), metabolic enzymes (Baum et al., 1996), transcription factors (Du et al., 2009), heat shock proteins (Virdi et al., 2009), transporters (Luoni et al., 2006) and channels (Hua et al., 2003), as well as a variety of proteins of unknown function (Bouche et al., 2005). Detailed characterization of CaM-target interaction has revealed that rather than being conserved at the level of primary structure, CaM-binding domains (CaMBDs) display conserved secondary structure and typically consist of short (12–30 amino acids) contiguous stretches of amino acids that have a propensity to form amphipathic α-helices (Yamniuk and Vogel, 2005). These structures are able to interact with hydrophobic clefts in CaM that become exposed upon Ca²⁺ binding. Additionally, CaM-target complexes are stabilized by electrostatic interactions between CaM and the target CaMBD. Several features of the CaM molecule likely contribute to its ability to interact with diverse targets (Gifford et al., 2007; Rainaldi et al., 2007). First, the linker region that connects the terminal globular domains in CaM is flexible, allowing CaM to wrap around helical binding domains. Second, the hydrophobic clefts through which CaM interacts with its targets are enriched in Met residues, the flexible side chains of which impart the ability for multiple conformations within the hydrophobic cleft. Finally, CaM can also interact with some proteins in the Ca²⁺-free state, reflecting its versatility in signaling (Rainaldi et al., 2007). Variation in the structural features of the binding pocket likely defines target specificity of different CaM (and presumably CML) isoforms. This is the case for two soybean (Glycine max) CaM isoforms, sCaM1 and sCaM4, which share 90% and 78% amino acid identity with conserved CaM, respectively (Ishida et al., 2008). Binding of Ca²⁺ by the C-terminal globular domain of sCaM1 led to exposure of a larger hydrophobic cleft compared with sCaM4, and this correlated with differences in target interaction and activation by these two CaM isoforms.

Recently, protein microarrays were used to broadly identify CaM- and CML-interacting proteins (Popescu et al., 2007). In this study, 1,133 Arabidopsis proteins (recombinantly expressed in tobacco [Nicotiana tabacum] and purified) were screened for interaction with CaM1, CaM6, CaM7, CML8, CML9, CML10, and CML12. In total, 173 proteins were reported to bind at least one CaM or CML (Popescu et al., 2007). About 25% of putative targets interacted with a single CaM or CML isoform, and a similar proportion interacted with all CaMs and CMLs tested, indicating that there may be broad overlap among some CaM and CML effectors. (Popescu et al., 2007). Overall, this study suggests that upwards of 15% of the proteome may be regulated by CaMs or CMLs. However, this study should be interpreted cautiously, as the majority of CaM/CML targets identified remain to be validated by further experimentation. Nevertheless, protein-array-based screening may provide a useful platform for future isolation of CaM/CML-interacting proteins.

Although a few downstream targets for CMLs have been confirmed by genetic or in vivo studies (Table I), detailed analysis of CML-binding domains is sparse. One study, which identified a pseudo response regulator (PRR2) as a downstream target of CML9, found that a nearly full-length target was required for interaction to occur in yeast (Saccharomyces cerevisiae; Perochon et al., 2010). Furthermore, interaction of CML9 with PRR2 was not strongly Ca²⁺ dependent. In another study, Ca²⁺-dependent interaction of CML18 with the C terminus of AtNHX1 (a Na⁺/H⁺ exchanger) was mapped using a yeast two-hybrid method. Helical wheel projection of this region revealed an amphipathic α-helix structure (Yamaguchi et al., 2005), like those typically involved in CaM-target interactions. Interestingly, binding was specific for CML18, as a conserved isoform, CaM81, from petunia (Petunia hybrida) did not interact with AtNHX1 (Yamaguchi et al., 2005). By comparison, a CaMBD was recently identified in BRASSINOSTEROID INSENSITIVE1 (BRI1; a leucine-rich repeat receptor-like kinase), and interaction of CaM suppressed protein kinase activity of BRI1 (Oh et al., 2012). Intriguingly, CML8 was also able to suppress kinase activity but did not interact with a peptide designed against the BRI1 CaMBD (Oh et al., 2012). This result suggests that CMLs may regulate their targets in a manner reminiscent of CaM but via distinct interaction domains.

Table I. List of known CML-interacting proteins and evidence put forth for the interaction.

CML	Species	Target	Gene Ontology Molecular Function	Evidence	Reference
CML8	Arabidopsis	BRI1	Protein Ser/Thr kinase activity	Effect on auto-/transphosphorylation	Oh et al., 2012
CML9	Arabidopsis	PRR2	DNA binding	Yeast two hybrid, fluorescence resonance energy transfer	Perochon et al., 2010
CML12	Arabidopsis	PINOID	Protein Ser/Thr kinase activity	Yeast two hybrid, copurification	Benjamins et al., 2003
CML18	Arabidopsis	AtNHX1	Na+:H+ antiporter activity	Yeast two hybrid, coimmunoprecipitation	Yamaguchi et al., 2005
CML19	Arabidopsis	AtRAD4	Damaged DNA binding	Copurification	Liang et al., 2006
CML20	Arabidopsis	TONNEAU1	Protein binding	Yeast two hybrid, copurification, bimolecular fluorescence complementation	Azimzadeh et al., 2008
CML24	Arabidopsis	ATG4b	Peptidase activity	Yeast two hybrid, copurification, coimmunoprecipitation	Tsai et al., 2012
CML42	Arabidopsis	KIC	Ca2+ ion binding	Yeast two hybrid, copurification	Dobney et al., 2009
rgsCaM	Tobacco	HCProa	RNA binding	Yeast two hybrid, surface plasmon resonance	Anandalakshmi et al., 2000; Nakahara et al., 2012

^aHCPro is a viral protein; no endogenous targets have been identified for rgsCaM.

In another study, interaction of a tobacco CML, rgsCaM (for regulator of gene silencing CaM), with a viral protein, helper component protease (HCPro) was characterized in detail, and a 24-amino acid peptide corresponding to an RNA-binding domain (RNABD) was demonstrated to associate with rgsCaM (Nakahara et al., 2012). Unlike classical CaM-target binding, which relies largely on hydrophobic interactions, the rgsCaM-RNABD interaction was mediated via ionic charges on the RNABD and on rgsCaM. Taken together, the limited data available on CML-target interactions suggests that at least some CMLs may interact with targets in a manner distinct from that of conserved CaM; however, further analysis is required on the structural characteristics of CML-target protein complexes before any generalizations can be made.

CMLs FUNCTION IN PLANT DEVELOPMENT

Various approaches aimed at understanding CML function, including global expression profiling, genetic analysis, protein biochemistry, and identification of downstream targets, are beginning to reveal diverse roles among members of this large protein family in plant development as well as in response to biotic and abiotic stress (Fig. 1).

Figure 1.

Working model of CML function in plant cells. This model displays known and hypothetical roles (red dashed boxes) for CMLs in development and stress response. CMLs are distributed among various cellular compartments and in at least one case, relocate in response to stimuli (CML19, dashed arrow). The model draws primarily from studies using Arabidopsis, but a role for rgsCaM from tobacco is also presented. Physiological responses mediated by CMLs are displayed as yellow boxes. For many CMLs, their downstream targets (blue boxes) are unknown or it is unclear whether putative targets are involved in CML-mediated pathways (denoted by a question mark). The suggested physiological functions of the CMLs depicted are described in the text. Black and blunted arrows indicate positive or negative regulation, respectively.

CMLs have been implicated in regulation of cell shape and cell division. In Arabidopsis, loss of function of CML42 leads to an increase in the number of trichome branches compared with wild-type plants (Dobney et al., 2009). Furthermore, CML42 was found to interact with kinesin-like CaM-binding protein-interacting Ca²⁺-binding protein (KIC), a Ca²⁺-binding protein that modulates the activity of a kinesin-like CaM-binding motor protein (KCBP) to regulate trichome morphology (Reddy et al., 2004). KIC negatively regulates KCBP, and KIC overexpressing transgenics (35S::KIC) have reduced trichome branching. Given the opposing phenotypes in CML42 knockouts and 35S::KIC plants, CML42 may positively regulate KIC function (Dobney et al., 2009), but further work is required to clarify the physiological significance of this interaction. Two different CMLs, CML7 and CML25, are involved in root hair elongation (Won et al., 2009; Lin et al., 2011). Transgenic plants lacking CML7 or CML25 function had longer root hairs compared with the wild type. Interestingly, increased root hair elongation in CML25 knockouts was only observed under phosphate-deficient conditions, providing a potential link between Ca²⁺ signaling and phosphate starvation responses. CML20 (also known as CENTRIN1) interacts with TONNEAU1, a protein with homology to human centrosomal proteins that function in organization of cortical microtubule arrays (Azimzadeh et al., 2008). Mammalian centrins are important for microtubule organization; however, the functional significance of the CML20-TONNEAU1 interaction is unknown.

Two closely related CMLs in Arabidopsis, CML23 and CML24, play a role in the transition to flowering. CML23 and CML24 display similarly broad expression patterns and exhibit at least partial overlap in function. Analysis of Arabidopsis plants carrying missense mutations in CML23, CML24, or both genes displayed altered flowering responses to photoperiod (Tsai et al., 2007). Interestingly, some alleles of CML24 caused early-flowering responses, while others caused a late-flowering phenotype. Double mutants (CML23/CML24) were always late flowering. Furthermore, mutants showed altered levels of transcripts for CONSTANS, FLOWERING LOCUS C, and SUPRESSOR OF CONSTANS1 that correlated with either early- or late-flowering phenotypes (Tsai et al., 2007). Given opposing flowering phenotypes in CML24 and CML23/CML24 mutants, it will be interesting to see if these Ca²⁺ sensors interact with the same downstream targets and whether interactions with either CML23 or CML24 lead to differential regulation of the same signaling pathway.

In addition to a role in flowering, CML24 appears to be involved in the regulation of autophagy via interaction with ATG4b, a protein that functions in the formation of autophagosomes (Tsai et al., 2013). Analysis of ATG4b Cys protease activity in extracts from plants harboring missense mutations in CML24 indicated that CML24 could control ATG4b activity. CML24 mutants display altered phenotypes related to misregulation of autophagy, including larger autophagic bodies and sensitivity to prolonged darkness (Tsai et al., 2013). While the precise mechanism for CML24 regulation of ATG4b is unclear, on the basis of loss-of-function mutants, it was proposed that CML24 positively regulates ATG4b and thus autophagosome formation.

A number of CMLs, including CML24 and CML39 are induced by mechanical stimuli (Lee et al., 2005). More recently, jasmonic acid (JA) was implicated in mechanosensing (Chehab et al., 2012), as touch inducibility of CML39 (a strongly JA-responsive gene, see below) was at least partially lost in mutants impaired in JA biosynthesis. Furthermore, mutant analysis revealed a role for CML24 in mechanosensing in roots (Wang et al., 2011), whereas a role for CML39 in mechanosensing remains unclear.

CMLs FUNCTION IN ABIOTIC STRESS RESPONSE

A variety of abiotic stress responses are mediated by Ca²⁺ signaling, and thus it is no surprise that CMLs are involved in the associated signal transduction pathways. Changes in expression of many CMLs have been observed in response to abiotic stress stimuli. Promoter activities of CML37 and CML38 are responsive to a number of different treatments including salinity, drought, oxidative stress, and mechanical wounding (Vanderbeld and Snedden, 2007). A CML from rice, Oryza sativa Multi-Stress Responsive gene2 (OsMSR2), is induced by multiple abiotic stress stimuli, and overexpression of OsMSR2 in Arabidopsis enhanced tolerance to drought and salinity and increased sensitivity to exogenous ABA (Xu et al., 2011). OsMSR2 is an ortholog of Arabidopsis CML37, CML38, and CML39, lending support to the hypothesis that these CMLs function in stress responses in Arabidopsis. Promoter and transcript analysis of CML24 revealed increased expression in response to high or low temperature, oxidative stress, as well exposure to ABA (Delk et al., 2005). Similarly, CML9 transcript levels change in response to NaCl, cold, and dehydration. Salt-responsive expression of CML9 is dependent on both ABA synthesis and signaling (Magnan et al., 2008), suggesting that this CML participates in ABA-dependent stress response pathways. In general, gene expression analyses indicate that many CMLs are stress responsive. In the future, it will be important to identify the transcription factors controlling CML expression and to assess the extent to which transcriptional activity relates to changes in CML protein levels. Regardless, expression profiling has proven useful in guiding questions aimed at understanding the roles of stress-induced CMLs.

Genetic approaches have also helped elucidate CML function during stress response. Repression of CML24 expression by RNA silencing resulted in decreased sensitivity to ABA as well as enhanced resistance to various metal stresses, including elevated cobalt, molybdenum, zinc, and magnesium (Delk et al., 2005). Transgenic knockouts of CML9 displayed reduced germination rates in response to both NaCl and KCl but not mannitol and exhibited greater inhibition of germination by ABA (Magnan et al., 2008). Furthermore, reduced germination rates in response to salinity could be rescued by inhibition of ABA synthesis. Intriguingly, adult plants lacking CML9 function were more tolerant of both salt and drought stress relative to the wild type (Magnan et al., 2008). It seems clear that CML9 plays a role in regulating ABA-mediated stress responses; however, further work is required to shed light on the mechanisms leading to these apparently contradictory phenotypes in seeds and adult plants.

CML18 has also been implicated in salt stress signaling on the basis of its interaction with a vacuolar Na⁺/H⁺ antiporter, AtNHX1 (Yamaguchi et al., 2005). It is noteworthy that CML18 localizes to the vacuolar lumen, where it interacts with the C terminus of AtNHX1. AtNHX1 can transport both Na⁺ and K⁺, and association with CML18 reduces the Na⁺:K⁺ translocation ratio. The interaction between CML18 and AtNHX1 is pH dependent, with the pair dissociating at high pH (Yamaguchi et al., 2005). This is interesting given that vacuolar pH is known to increase under salt stress (Katsuhara et al., 1989). Thus, dissociation of CML18 from AtNHX1 is predicted to occur in response to salinity, thereby promoting Na⁺/H⁺ relative to K⁺/H⁺ antiport activity, leading to sequestration of Na⁺ in the vacuole.

Two different CMLs are involved in protection of Arabidopsis plants from UV damage. CML42 knockout transgenics have reduced flavonol levels and display increased sensitivity to UV stress compared with the wild type (Vadassery et al., 2012a). CML19 (also known as CENTRIN2) interacts with Arabidopsis RADIATION REPAIR4 (AtRAD4), a protein that functions in homologous DNA repair (Liang et al., 2006). CML19 protein levels are up-regulated by UV stress, and localization of CML19 to the nucleus is UV dependent. Suppression of CML19 expression by RNA silencing resulted in a UV-sensitive phenotype, and overexpressing transgenics displayed enhanced DNA repair (Molinier et al., 2004). It is currently unclear how CML19 regulates AtRAD4 activity; however, nuclear localization of CML19 and interaction with AtRAD4 is likely an important mechanism for limiting UV damage.

CMLs FUNCTION IN PATHOGEN AND HERBIVORE DEFENSE

Biotic stress is another area where several CMLs appear to play roles in Ca²⁺-mediated responses. In Arabidopsis, expression of CML9 transcript is elevated following challenge by avirulent pathogen, flagellin, or salicylic acid (SA). The expression of CML9 in response to avirulent pathogens is dependent upon FLAGELLIN SENSITITVE2 (the flagellin receptor) and production of SA (Leba et al., 2012). Analysis of CML9 knockout or overexpression transgenics revealed increased or decreased susceptibility, respectively, to multiple bacterial pathogens and altered dynamics of PATHOGENESIS RELATED GENE1 expression following pathogen challenge (Leba et al., 2012). These findings suggest that CML9 functions in Ca²⁺-based immune responses to bacterial infection. In addition to its developmental roles, CML24 also functions in innate immunity signaling in Arabidopsis. Infection by avirulent pathogen induces Ca²⁺ transients upstream of nitric oxide generation and the hypersensitive response (Ma et al., 2008). Both of these responses are suppressed by treatment of plants with CaM antagonists and are abolished in plants lacking CML24 function (Ma et al., 2008). These data define a role for CML24 in pathogen defense and indicate a link between Ca²⁺ and reactive oxygen signaling in planta. Another member of the CML family implicated in defense response is CML43. CML43 transcript accumulates within 6 h following infection with avirulent pathogen, and CML43 overexpression caused an accelerated hypersensitive response compared with wild-type plants (Chaisson et al., 2005). Moreover, suppression of the tomato (Solanum lycopersicum) ortholog of CML43, AvrPto-Pto-Prf responsive134, by virus-induced gene silencing increased susceptibility of tomato plants to infection by an avirulent strain of Pseudomonas syringae (Chaisson et al., 2005). These data suggest that CML43 and its tomato ortholog positively regulate pathogen defense and, importantly, demonstrate that CML function is conserved across taxa.

In addition to bacterial pathogen responses, CMLs also function in host defense against viral attack. Expression of a CML from tobacco, rgsCaM, is induced in response to inoculation of leaves with tobacco etch virus. Expression of rgsCaM was also elevated in transgenic tobacco expressing a viral protein, HCPro (Anandalakshmi et al., 2000). rgsCaM interacts with HCPro in yeast two-hybrid assays, and it was suggested that rgsCaM serves as an endogenous suppressor of gene silencing. HCPro is an RNA-silencing suppressor (RSS) that functions to repress posttranscriptional gene silencing as a viral mechanism to thwart plant defense against infection. The suppression activity of HCPro (and other RSS proteins) is achieved via an RNABD that sequesters antiviral double-stranded RNAs generated by the plant. Recently, the interaction between HCPro and rgsCaM was characterized, and on the basis of homology with an RSS from the human immunodeficiency virus, it was revealed that rgsCaM associates directly with the RNABD of certain viral RSS proteins, including HCPro (Nakahara et al., 2012). Interaction of rgsCaM with HCPro attenuated the double-stranded RNA-binding function of HCPro and resulted in targeting of the rgsCaM-HCPro complex to autolysosomes and subsequent degradation (Nakahara et al., 2012). This important result indicates that rgsCaM promotes defense by directly suppressing HCPro activity. In Arabidopsis, the expression of CML38, a putative ortholog of rgsCaM, is strongly up-regulated in transgenic plants ectopically expressing HCPro from the turnip mosaic virus (Endres et al., 2010). Although CML38 shares 44% amino acid identity with tobacco rgsCaM, interaction with HCPro, or any viral RSS protein, has not yet been reported. rgsCaM also shares strong sequence identity with Arabidopsis CML37 and CML39, and thus it will be interesting to see if either of these proteins are involved in defense against viral pathogens.

CMLs have also been linked to defense responses against herbivores based on transcript profiling and genetic analyses. Oral secretions from Spodoptera littoralis larvae were shown to induce the expression of a number of different CMLs (CML9, CML11, CML12, CML16, CML17, CML23, and CML42; Vadassery et al., 2012b). CML42 expression is stimulated by either application of S. littoralis oral secretions or direct larval feeding, with transcript levels reaching a peak 30 min and 1 h following treatment, respectively (Vadassery et al., 2012a). On the basis of this rapid induction, larval feeding studies were carried out on plants lacking CML42, and it was found that larval development was impaired on CML42 loss-of-function mutants. Furthermore, glucosinolate levels were higher in CML42 knockout plants (Vadassery et al., 2012a). As noted above, CML42 also functions in trichome development (Dobney et al., 2009), though it is currently unclear whether altered trichome morphology in CML42 mutants contributes to herbivore resistance. It is worth noting that CML12, CML37, and CML38 transcript levels rise rapidly in response to mechanical wounding (Vanderbeld and Snedden, 2007; Walley et al., 2007), suggesting that they may also be involved in herbivory responses, although this has not yet been tested.

CMLs ARE INVOLVED IN HORMONE SIGNALING

Expression analysis has indicated that many CMLs are induced by treatment of plants with a variety of hormones that regulate biotic stress responses, such as JA and SA. CML39 expression is responsive to methyl jasmonate in Arabidopsis seedlings (Vanderbeld and Snedden, 2007). Microarray data revealed that CML39 expression is primarily restricted to pollen but, following challenge by bacterial and fungal pathogens, is also expressed in rosette leaves, indicating that this putative Ca²⁺ sensor may play a role in both JA-mediated development and pathogen defense. In roots, both transcript and protein expression of CML43 is stimulated by SA treatment (K.W. Bender, S. Dobney, A. Ogunrinde, D. Chiasson, R.T. Mullen, H.J. Teresinsi, P.J. Singh, K. Munro, S.P. Smith, and W.A. Snedden, unpublished data), supporting a role for this CML in biotic defense responses. It seems likely a number of CMLs function in hormone-mediated biotic defense; however, specific roles for CMLs and other Ca²⁺ sensors downstream of pathogen challenge remain to be elucidated.

CMLs also participate in hormone signaling during development. CML12 (also known as TOUCH3) interacts with PINOID, a Ser/Thr protein kinase involved in regulation of PIN-FORMED1 (an auxin efflux carrier) localization (Benjamins et al., 2003). Phosphorylation of PIN proteins by PINOID drives them to the apical plasma membrane resulting in auxin efflux from the apical side of the cell (Friml et al., 2004). Ca²⁺-dependent binding of CML12 to PINOID negatively regulates kinase activity leading to reduced apical efflux of auxin (Benjamins et al., 2003). Potential regulation of the polarity of auxin efflux by CML12 is interesting given that CML12 transcription is induced by mechanical stimuli (Wright et al., 2002; Lee et al., 2005).

Recently, CML8 and conserved CaM were observed to interact with the brassinosteroid receptor BRI1. Coexpression of CML8, CaM6, or CaM7 with the cytosolic domain of BRI1 in Escherichia coli inhibited autophosphorylation of the kinase (Oh et al., 2012). Furthermore, transphosphorylation of E. coli proteins by BRI1 was repressed when coexpressed with CML8, CaM6, or CaM7. While the physiological consequences of these interactions remain to be determined, CML8 or the CaMs may serve to attenuate BRI1-mediated signal transduction in vivo (Oh et al., 2012).

The picture that is emerging for CMLs is that, like CaM, they function in a wide assortment of cellular process in plants. The participation of CMLs in various aspects of development and stress response (e.g. CML9, CML24, and CML42) is well supported by experimental evidence; yet, it remains largely unknown whether this occurs through regulation of distinct targets by these CMLs under different conditions and/or during distinct developmental stages. These gaps in our understanding of CML function highlight the need to identify CML targets: as putative regulatory proteins, it is imperative to determine which downstream effectors are under CML control. As a closing note, it worthwhile to reiterate that while often discussed in isolation, development and stress response are, of course, intimately related phenomena. The physiological responses elicited by adverse environmental conditions represent adaptive processes aimed at facilitating completion of the life cycle. Given the universality of Ca²⁺ signaling in these important processes, the remarkable diversity of Ca²⁺ sensors in plants is likely essential for successful coordination of development and reproduction in a dynamic and often stressful environment.

Glossary

[Ca²⁺]_cyt

cytosolic Ca²⁺ concentration

ABA

abscisic acid

CaMBD

calmodulin-binding domain

RNABD

RNA-binding domain

JA

jasmonic acid

SA

salicylic acid

RSS

RNA-silencing suppressor