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

Article in Plant Physiology · August 2013

DOI: 10.1104/pp.113.221069 · Source: PubMed

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Update on Importance of Calmodulin-Related Proteins

Calmodulin-Related Proteins Step Out from the Shadow

of Their Namesake

Kyle W. Bender and Wayne A. Snedden*

Department of Plant Biology, University of Illinois, Urbana, Illinois 61801 (K.W.B.); and Department of
Biology, Queen’s University, Kingston, Ontario, Canada K7L 3N6 (W.A.S.)

Like all eukaryotes, plants must be able to detect While extrusion of Ca2+ is a constant process, when
changes in their environment and respond accord- local influx transiently exceeds efflux, Ca2+ signaling
ingly. In broad terms, cells possess signal transduction events can occur (Spalding and Harper, 2011).
pathways, mediating responses to internal and exter- In the paradigm of Ca2+ signaling, the detection of a
nal cues that consist of three distinct nodes: stimulus given stimulus evokes rapid and transient movement
perception, signal transduction, and subsequent physi- of Ca2+ into the cytosol. In plants, this has been ob-
ological response. The transduction node exploits rapid served for a number of developmental cues (e.g. grav-
and transient changes in the cellular concentrations of itropic responses; Toyota et al., 2008), as well as abiotic
second messengers to directly or indirectly affect the stresses (e.g. salt and drought stress; Kiegle et al., 2000),
activities of downstream effector proteins (metabolic and in response to biotic signals (e.g. pathogen attack
enzymes, transcription factors, etc.) that ultimately co- and microbial symbioses; Ali et al., 2007; Kosuta et al.,
ordinate cellular responses. Changes in metabolic ac- 2008). To generate Ca2+ fluxes, stimulus perception is
tivity, gene expression, and protein turnover collectively linked to the coordinated activities of Ca2+ channels and
lead to adaptive physiological events that are appropri- pumps. Given the multitude of stimuli evoking cyto-
ate for the initial stimulus. This model (perception, solic Ca2+ transients in plants, it is important to consider
transduction, and response) is simplistic yet widely ap- how specific cellular responses might be derived from a
plicable. Among the common themes that have emerged common second messenger.
in signal transduction research is the importance of cal- Two main hypotheses describe how specificity of
cium (Ca2+) as a second messenger in plant development Ca2+-guided response may be achieved in plant cells:
and in response to a variety of environmental stresses the Ca2+ signature hypothesis and the Ca2+ switch hy-
(McAinsh and Pittman, 2009; DeFalco et al., 2010; Dodd pothesis. Both hypotheses can claim supporting evi-
et al., 2010). Given the diversity of stimuli known to dence (Scrase-Field and Knight, 2003; McAinsh and
evoke Ca2+ transients in cells, plants make an excellent Pittman, 2009), and they are not mutually exclusive. In
model system for studying the mechanisms of Ca2+ sig- the Ca2+ signature hypothesis, stimuli perceived by the
nal transduction. cell evoke Ca2+ transients in the cytosol of particular
amplitude, frequency, and spatial distribution that are
thought to elicit a stimulus-specific downstream re-
Ca2+ IN PLANT SIGNAL TRANSDUCTION sponse. Calcium oscillations (i.e. Ca2+ “signatures”) hav-
ing specific spatial and temporal properties have been
Given that elevated Ca2+ levels are potentially toxic documented in root hairs in response to Nod factors or
to ATP metabolism, and that Ca2+ may precipitate signals derived from arbuscular mycorrhizal fungi
negatively charged biological molecules (Spalding and (Miwa et al., 2006; Oldroyd and Downie, 2006; Kosuta
Harper, 2011), cells maintain tight control over cyto- et al., 2008) and in guard cells exposed to abscisic acid
solic Ca2+ concentration ([Ca2+]cyt). Cells actively pump (ABA), oxidative stress, and external Ca2+ (Allen et al.,
Ca2+ into the extracellular space and into intracellular 2000, 2001). In the binary switch model, specificity in
stores (e.g. vacuole and endoplasmic reticulum), leading Ca2+ signaling is derived from other components in the
to the establishment of a steep electrochemical gradient signaling pathway (i.e. Ca2+ sensors, see below; Scrase-
of Ca2+ between these stores and the cytosol (McAinsh Field and Knight, 2003). This hypothesis is supported
and Pittman, 2009; Spalding and Harper, 2011). At by the observation that different stimuli (e.g. salt and
resting levels, Ca2+ is present in the cytosol at concen- drought stress) induce highly similar cytosolic Ca2+
trations ranging from 100 to 200 nM, whereas concen- influxes but elicit different downstream responses (Knight
trations in Ca2+ stores are orders of magnitude higher, et al., 1997). It is noteworthy that cells may also coordinate
in some cases reaching millimolar levels (Gilroy et al., Ca2+-mediated responses by priming/depriming of Ca2+
1993). Thus, cells are poised for rapid influxes of Ca2+ sensors (Israelsson et al., 2006). This stimulus-specific
down its electrochemical gradient into the cytosol. modulation (for example, posttranslational modifica-
tion) of Ca2+ sensor function could permit or restrict
* Address correspondence to [email protected]. different downstream signaling pathways depending
www.plantphysiol.org/cgi/doi/10.1104/pp.113.221069 on the prevailing conditions. Regardless of whether
486 Plant PhysiologyÒ, October 2013, Vol. 163, pp. 486–495, www.plantphysiol.org Ó 2013 American Society of Plant Biologists. All Rights Reserved.
 Update on Calmodulin-Related Proteins

Ca2+ signals operate as signatures, switches, or variations encode four different protein isoforms (CaM2/CaM3/
thereof, a requirement of any model is that a mechanism CaM5, CaM1/CaM 4, CaM6, and CaM7) that share a
exists to transduce Ca2+ signals to downstream effectors. minimum 97% identity at the primary sequence level
Diverse families of Ca2+-binding proteins, collectively (McCormack and Braam, 2003). In addition to the
known as Ca2+ sensors, fulfill the role of signal propa- conserved CaMs, plant genomes contain expanded fami-
gation and occupy a key position in signal transduction lies of CML genes. A 50-member CML family, subdivided
pathways. 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,
Ca2+-BINDING PROTEINS AND Ca2+ SENSORS
2007); such large CML families are thus likely found
While a few different Ca2+-binding domains have across plant taxa.
been identified, Ca2+ sensors are most commonly char- In Arabidopsis, CMLs vary in length from 83 to 330
acterized by the presence of one or more EF-hand Ca2+- amino acids and share between 16% and 75% amino
binding motifs. The EF-hand is highly conserved among acid identity with CaM2 at the primary sequence level.
Ca2+-binding proteins and EF-hand-containing proteins Like CaM, they are characterized as possessing exclu-
are encoded by all eukaryotic genomes (Gifford et al., sively Ca2+-binding EF-hands and no other known func-
2007). The Arabidopsis (Arabidopsis thaliana) genome is tional motifs (McCormack and Braam, 2003; McCormack
predicted to contain about 250 EF-hand proteins, more et al., 2005). Thus, CMLs are predicted to serve as Ca2+
than any other organism examined to date (Day et al., sensors in planta. Many eukaryotes possess Ca2+ sen-
2002). EF-hand motifs are not exclusive to Ca2+ sensors sors related to CaM, such as S100 proteins, myosin
and are also found in proteins, termed Ca2+ buffers, in- light chains, and other small EF-hand proteins, but the
volved in regulating intracellular Ca2+ concentrations CMLs are a family unique to plants. In other words,
rather than signal transduction directly (Schwaller, 2010). the closest orthologs of CMLs in other eukaryotes tend
The EF-hand is composed of a 29-amino acid helix- to be conserved CaMs or proteins very similar to CaMs.
loop-helix structure, with the central 12 residues forming Unlike conserved CaM, which has four EF-hands,
a turn-loop structure that is responsible for coordination CMLs contain variable numbers of EF-hands, ranging
of the Ca2+ ion (Pidcock and Moore, 2001; Gifford et al., from one (AtCML1) to six (AtCML12). The importance
2007). Within the coordination loop, ligating residues are of this variation in terms of differential Ca2+ binding
found at positions 1, 3, 5, 7, 9, and 12 (Yamniuk et al., and response among CMLs to distinct Ca2+ signals has
2008). The loop is enriched in negatively charged resi- been hypothesized but not well explored. One inter-
dues, and a Gly at position six allows the loop to wrap esting possibility is that some CMLs, like animal S100
around the Ca2+ ion, a feature critical for high affinity proteins, may dimerize to facilitate pairing of EF-hands
binding (Gifford et al., 2007). Structural properties of the and thus cooperative binding of Ca2+. Dimerization of a
EF-hand allow for rapid on/off Ca2+ binding, permitting Ca2+-bound N-terminal fragment of CML34 has been
quick responses to changes in [Ca2+]cyt, a property likely observed in vitro (Song et al., 2004). Some CMLs have
to be vital for interpreting Ca2+ signals. N-terminal extensions of varying length, the func-
In plants, the three largest families of EF-hand Ca2+ tional significance of which remains unclear but may
sensors that have been characterized are the calmodulin be involved in subcellular targeting of the protein. In
(CaM) and CaM-like (CML) proteins, calcineurin-B-like Arabidopsis, CML30 is localized to the mitochondria,
(CBL) proteins, and the Ca2+-dependent protein kinases and a noncleavable transit peptide was identified at
(CDPKs, called CPKs in Arabidopsis). Among these groups, its N terminus (Chigri et al., 2012).
the CaMs/CMLs and CBLs are considered to be sensor Another distinction between CMLs and CaMs can
relays that function via regulation of downstream targets. be found in the central helical linker region. In CaM,
Conversely, the CDPKs, as catalytic proteins, are sensor this feature affords structural flexibility important for
responders in which Ca2+ binding often regulates their the conformational changes that accompany interac-
kinase activity. Notably, three of these families, the CMLs, tion with target proteins. It is thus interesting to note
CBLs, and CDPKs, are unique to plants and some protists that a number of CMLs when compared to CaM pos-
(CDPKs only), highlighting the importance of Ca2+ sig- sess longer linker regions between the two globular
naling to plant growth and development. domains: this may alter CML flexibility and perhaps
CaM is the most thoroughly characterized Ca2+ sen- interaction with downstream targets. Ultimately, how
sor in plants, and its physiological roles have been the physical characteristics of CMLs compare to CaM
recently reviewed (Bouche et al., 2005; DeFalco et al., and influence their cellular roles remains largely spec-
2010). CaM is a small (149 amino acids), acidic protein ulative, as little empirical work has been done to de-
consisting of two globular domains, each containing a termine their Ca2+ binding or other structural properties.
pair of Ca2+-binding EF-hand motifs. A short central
linker region connects the globular domains of CaM
STRUCTURAL PROPERTIES OF CaM AND CMLs
and affords it considerable flexibility in solution. Plant
genomes contain multiple loci encoding conserved CaM Detailed biophysical analyses of CaM have re-
isoforms. For example, seven distinct genes in Arabidopsis vealed both high affinity binding of Ca2+ as well as
Plant Physiol. Vol. 163, 2013 487
Bender and Snedden

Ca2+-induced conformational changes within the pro- suggesting differences in Ca2+-responsive conforma-
tein. Studies indicate that CaM binds Ca2+ with affinities tional changes between CaM and CMLs (Song et al.,
in the high nanomolar to low micromolar range (Gilli 2004). Analysis of CML42 by NMR revealed similar
et al., 1998), suggesting that the EF-hands of CaM are conformation of two EF-hands in both the Ca2+-bound
not occupied by Ca2+ in a resting cells. These affinities and the Ca2+-free state (Dobney et al., 2009). Overall,
are, however, within the range of [Ca2+]cyt measured structural analysis of CMLs compared with CaM sug-
during Ca2+ transients (McAinsh and Pittman, 2009), gests that sequence divergence among CMLs leads to
indicating that CaM could respond dynamically to differential responses to Ca2+ binding in vitro, and it is
changes in [Ca2+]cyt. Binding of Ca2+ by CaM leads to tempting to speculate that these differences may be
changes in the secondary and tertiary structure of the important for in vivo CML-specific functions, such as
protein (Ikura et al., 1990). In the absence of Ca2+, response to specific Ca2+ signals or target interaction.
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
INTERACTION OF CaMs AND CMLs WITH
the EF-hand. These changes in the shape of the EF-hand THEIR TARGETS
upon Ca2+ binding lead to an overall Ca2+-responsive
change in the structure of CaM (and presumably other CaM has been described as a universal Ca2+ sensor
EF-hand Ca2+ sensors). CaM is a strongly a-helical because of the myriad downstream effectors that it has
protein (approximately 40% for apoCaM), and Ca2+ been found to interact with. In plants, CaM physically
binding results in an overall increase in a-helical con- associates with numerous targets, including protein
tent (approximately 50% for Ca2+/CaM; Martin and kinases (Oh et al., 2012) and phosphatases (Liu et al.,
Bayley, 1986). These structural changes are accompa- 2007), metabolic enzymes (Baum et al., 1996), tran-
nied by a dramatic increase in the surface hydropho- scription factors (Du et al., 2009), heat shock proteins
bicity of CaM that is important for target interactions (Virdi et al., 2009), transporters (Luoni et al., 2006) and
(Yamniuk and Vogel, 2005). channels (Hua et al., 2003), as well as a variety of
Where CMLs have been characterized biochemically, proteins of unknown function (Bouche et al., 2005).
they display properties similar, though not identical, to Detailed characterization of CaM-target interaction has
that of conserved CaM. The Ca2+-binding properties of revealed that rather than being conserved at the level
Arabidopsis CML42 (Dobney et al., 2009) have been of primary structure, CaM-binding domains (CaMBDs)
examined in detail. Analysis of Ca2+ affinities by isother- display conserved secondary structure and typically
mal titration calorimetry identified three Ca2+-binding consist of short (12–30 amino acids) contiguous stretches
sites in CML42, one at the N terminus and two at the of amino acids that have a propensity to form am-
C terminus. The second EF-hand in CML42 is degen- phipathic a-helices (Yamniuk and Vogel, 2005). These
erate, having substitutions for several of the Ca2+- structures are able to interact with hydrophobic clefts
ligating residues within the coordination loop (Dobney in CaM that become exposed upon Ca2+ binding.
et al., 2009). Of the three identified Ca2+-binding sites Additionally, CaM-target complexes are stabilized by
in CML42, two show dissociation constants similar to electrostatic interactions between CaM and the target
that of CaM, while one (EF-hand 3) has an affinity of CaMBD. Several features of the CaM molecule likely
approximately 30 nM in the presence of Mg2+, sug- contribute to its ability to interact with diverse targets
gesting that this EF-hand is permanently occupied by (Gifford et al., 2007; Rainaldi et al., 2007). First, the
Ca2+ in resting cells. This EF-hand may thus play a linker region that connects the terminal globular do-
structural role, while the remaining two Ca2+-binding mains in CaM is flexible, allowing CaM to wrap around
sites may serve sensory functions (Dobney et al., 2009). helical binding domains. Second, the hydrophobic
Interestingly, Ca2+-responsive conformational changes clefts through which CaM interacts with its targets are
in CML42 were distinct from those of CaM, as CML42 enriched in Met residues, the flexible side chains of
showed no Ca2+-dependent changes in secondary struc- which impart the ability for multiple conformations
ture but did exhibit changes in tertiary structure as within the hydrophobic cleft. Finally, CaM can also
demonstrated by NMR spectroscopy. Finally, CML42, interact with some proteins in the Ca2+-free state, re-
like CaM, undergoes a considerable Ca2+-responsive flecting its versatility in signaling (Rainaldi et al., 2007).
increase in surface hydrophobicity (Dobney et al., 2009). Variation in the structural features of the binding
The structure of the Ca2+-loaded N-terminal EF-hand pocket likely defines target specificity of different CaM
pair of CML34 from Arabidopsis has been resolved by (and presumably CML) isoforms. This is the case for
NMR spectroscopy (Song et al., 2004). The first EF-hand two soybean (Glycine max) CaM isoforms, sCaM1 and
in the N-terminal region of CML34 showed similar sCaM4, which share 90% and 78% amino acid identity
structure to the equivalent motif in CaM in the pres- with conserved CaM, respectively (Ishida et al., 2008).
ence of Ca2+, having an interhelical angle of approxi- Binding of Ca2+ by the C-terminal globular domain of
mately 102° (compared with approximately 104° for sCaM1 led to exposure of a larger hydrophobic cleft
CaM). Interestingly, the second EF-hand of CML34 compared with sCaM4, and this correlated with dif-
had an interhelical angle of approximately 147° in the ferences in target interaction and activation by these
presence of Ca2+ in contrast to an angle of 102° for CaM, two CaM isoforms.
488 Plant Physiol. Vol. 163, 2013
 Update on Calmodulin-Related Proteins

Recently, protein microarrays were used to broadly interact with AtNHX1 (Yamaguchi et al., 2005). By com-
identify CaM- and CML-interacting proteins (Popescu parison, a CaMBD was recently identified in BRASSI-
et al., 2007). In this study, 1,133 Arabidopsis proteins NOSTEROID INSENSITIVE1 (BRI1; a leucine-rich repeat
(recombinantly expressed in tobacco [Nicotiana taba- receptor-like kinase), and interaction of CaM suppressed
cum] and purified) were screened for interaction with protein kinase activity of BRI1 (Oh et al., 2012). Intrigu-
CaM1, CaM6, CaM7, CML8, CML9, CML10, and ingly, CML8 was also able to suppress kinase activity but
CML12. In total, 173 proteins were reported to bind at did not interact with a peptide designed against the BRI1
least one CaM or CML (Popescu et al., 2007). About CaMBD (Oh et al., 2012). This result suggests that CMLs
25% of putative targets interacted with a single CaM or may regulate their targets in a manner reminiscent of
CML isoform, and a similar proportion interacted with CaM but via distinct interaction domains.
all CaMs and CMLs tested, indicating that there may In another study, interaction of a tobacco CML,
be broad overlap among some CaM and CML effec- rgsCaM (for regulator of gene silencing CaM), with a
tors. (Popescu et al., 2007). Overall, this study suggests viral protein, helper component protease (HCPro) was
that upwards of 15% of the proteome may be regu- characterized in detail, and a 24-amino acid peptide
lated by CaMs or CMLs. However, this study should corresponding to an RNA-binding domain (RNABD)
be interpreted cautiously, as the majority of CaM/CML was demonstrated to associate with rgsCaM (Nakahara
targets identified remain to be validated by further ex- et al., 2012). Unlike classical CaM-target binding, which
perimentation. Nevertheless, protein-array-based screen- relies largely on hydrophobic interactions, the rgsCaM-
ing may provide a useful platform for future isolation RNABD interaction was mediated via ionic charges on
of CaM/CML-interacting proteins. the RNABD and on rgsCaM. Taken together, the limited
Although a few downstream targets for CMLs have data available on CML-target interactions suggests
been confirmed by genetic or in vivo studies (Table I), that at least some CMLs may interact with targets in a
detailed analysis of CML-binding domains is sparse. manner distinct from that of conserved CaM; however,
One study, which identified a pseudo response regu- further analysis is required on the structural character-
lator (PRR2) as a downstream target of CML9, found istics of CML-target protein complexes before any gen-
that a nearly full-length target was required for inter- eralizations can be made.
action to occur in yeast (Saccharomyces cerevisiae; Perochon
et al., 2010). Furthermore, interaction of CML9 with
PRR2 was not strongly Ca2+ dependent. In another
CMLs FUNCTION IN PLANT DEVELOPMENT
study, Ca2+-dependent interaction of CML18 with the
C terminus of AtNHX1 (a Na+/H+ exchanger) was Various approaches aimed at understanding CML
mapped using a yeast two-hybrid method. Helical function, including global expression profiling, genetic
wheel projection of this region revealed an amphipathic analysis, protein biochemistry, and identification of
a-helix structure (Yamaguchi et al., 2005), like those downstream targets, are beginning to reveal diverse
typically involved in CaM-target interactions. Interest- roles among members of this large protein family in
ingly, binding was specific for CML18, as a conserved plant development as well as in response to biotic and
isoform, CaM81, from petunia (Petunia hybrida) did not abiotic stress (Fig. 1).

Table I. List of known CML-interacting proteins and evidence put forth for the interaction
Gene Ontology
CML Species Target Evidence Reference
Molecular Function
CML8 Arabidopsis BRI1 Protein Ser/Thr Effect on auto-/transphosphorylation Oh et al., 2012
kinase activity
CML9 Arabidopsis PRR2 DNA binding Yeast two hybrid, fluorescence resonance Perochon et al., 2010
energy transfer
CML12 Arabidopsis PINOID Protein Ser/Thr Yeast two hybrid, copurification Benjamins et al., 2003
kinase activity
CML18 Arabidopsis AtNHX1 Na+:H+ antiporter Yeast two hybrid, coimmunoprecipitation Yamaguchi et al., 2005
activity
CML19 Arabidopsis AtRAD4 Damaged DNA Copurification Liang et al., 2006
binding
CML20 Arabidopsis TONNEAU1 Protein binding Yeast two hybrid, copurification, bimolecular Azimzadeh et al., 2008
fluorescence complementation
CML24 Arabidopsis ATG4b Peptidase activity Yeast two hybrid, copurification, Tsai et al., 2012
coimmunoprecipitation
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
a
HCPro is a viral protein; no endogenous targets have been identified for rgsCaM.

Plant Physiol. Vol. 163, 2013 489

Bender and Snedden

CMLs have been implicated in regulation of cell in organization of cortical microtubule arrays (Azimzadeh
shape and cell division. In Arabidopsis, loss of function et al., 2008). Mammalian centrins are important for mi-
of CML42 leads to an increase in the number of trichome crotubule organization; however, the functional signifi-
branches compared with wild-type plants (Dobney et al., cance of the CML20-TONNEAU1 interaction is unknown.
2009). Furthermore, CML42 was found to interact with Two closely related CMLs in Arabidopsis, CML23
kinesin-like CaM-binding protein-interacting Ca2+- and CML24, play a role in the transition to flowering.
binding protein (KIC), a Ca2+-binding protein that CML23 and CML24 display similarly broad expression
modulates the activity of a kinesin-like CaM-binding patterns and exhibit at least partial overlap in function.
motor protein (KCBP) to regulate trichome morphology Analysis of Arabidopsis plants carrying missense mu-
(Reddy et al., 2004). KIC negatively regulates KCBP, and tations in CML23, CML24, or both genes displayed al-
KIC overexpressing transgenics (35S::KIC) have re- tered flowering responses to photoperiod (Tsai et al.,
duced trichome branching. Given the opposing phe- 2007). Interestingly, some alleles of CML24 caused early-
notypes in CML42 knockouts and 35S::KIC plants, flowering responses, while others caused a late-flowering
CML42 may positively regulate KIC function (Dobney phenotype. Double mutants (CML23/CML24) were al-
et al., 2009), but further work is required to clarify the ways late flowering. Furthermore, mutants showed al-
physiological significance of this interaction. Two dif- tered levels of transcripts for CONSTANS, FLOWERING
ferent CMLs, CML7 and CML25, are involved in root LOCUS C, and SUPRESSOR OF CONSTANS1 that cor-
hair elongation (Won et al., 2009; Lin et al., 2011). Trans- related with either early- or late-flowering phenotypes
genic plants lacking CML7 or CML25 function had longer (Tsai et al., 2007). Given opposing flowering pheno-
root hairs compared with the wild type. Interestingly, types in CML24 and CML23/CML24 mutants, it will be
increased root hair elongation in CML25 knockouts was interesting to see if these Ca2+ sensors interact with the
only observed under phosphate-deficient conditions, same downstream targets and whether interactions
providing a potential link between Ca2+ signaling and with either CML23 or CML24 lead to differential reg-
phosphate starvation responses. CML20 (also known as ulation of the same signaling pathway.
CENTRIN1) interacts with TONNEAU1, a protein with In addition to a role in flowering, CML24 appears to
homology to human centrosomal proteins that function be involved in the regulation of autophagy via interaction

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.

490 Plant Physiol. Vol. 163, 2013

 Update on Calmodulin-Related Proteins

with ATG4b, a protein that functions in the formation Genetic approaches have also helped elucidate CML
of autophagosomes (Tsai et al., 2013). Analysis of function during stress response. Repression of CML24
ATG4b Cys protease activity in extracts from plants expression by RNA silencing resulted in decreased
harboring missense mutations in CML24 indicated that sensitivity to ABA as well as enhanced resistance to
CML24 could control ATG4b activity. CML24 mutants various metal stresses, including elevated cobalt, molyb-
display altered phenotypes related to misregulation denum, zinc, and magnesium (Delk et al., 2005). Trans-
of autophagy, including larger autophagic bodies genic knockouts of CML9 displayed reduced germination
and sensitivity to prolonged darkness (Tsai et al., rates in response to both NaCl and KCl but not mannitol
2013). While the precise mechanism for CML24 reg- and exhibited greater inhibition of germination by ABA
ulation of ATG4b is unclear, on the basis of loss- (Magnan et al., 2008). Furthermore, reduced germina-
of-function mutants, it was proposed that CML24 tion rates in response to salinity could be rescued by
positively regulates ATG4b and thus autophagosome inhibition of ABA synthesis. Intriguingly, adult plants
formation. lacking CML9 function were more tolerant of both salt
A number of CMLs, including CML24 and CML39 and drought stress relative to the wild type (Magnan
are induced by mechanical stimuli (Lee et al., 2005). et al., 2008). It seems clear that CML9 plays a role in
More recently, jasmonic acid (JA) was implicated in regulating ABA-mediated stress responses; however,
mechanosensing (Chehab et al., 2012), as touch in- further work is required to shed light on the mecha-
ducibility of CML39 (a strongly JA-responsive gene, nisms leading to these apparently contradictory phe-
see below) was at least partially lost in mutants im- notypes in seeds and adult plants.
paired in JA biosynthesis. Furthermore, mutant anal- CML18 has also been implicated in salt stress sig-
ysis revealed a role for CML24 in mechanosensing in naling on the basis of its interaction with a vacuolar
roots (Wang et al., 2011), whereas a role for CML39 in Na+/H+ antiporter, AtNHX1 (Yamaguchi et al., 2005).
mechanosensing remains unclear. 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+ trans-
CMLs FUNCTION IN ABIOTIC STRESS RESPONSE
location ratio. The interaction between CML18 and
A variety of abiotic stress responses are mediated by AtNHX1 is pH dependent, with the pair dissociating at
Ca2+ signaling, and thus it is no surprise that CMLs are high pH (Yamaguchi et al., 2005). This is interesting
involved in the associated signal transduction path- given that vacuolar pH is known to increase under salt
ways. Changes in expression of many CMLs have been stress (Katsuhara et al., 1989). Thus, dissociation of
observed in response to abiotic stress stimuli. Promoter CML18 from AtNHX1 is predicted to occur in re-
activities of CML37 and CML38 are responsive to a sponse to salinity, thereby promoting Na+/H+ relative
number of different treatments including salinity, to K+/H+ antiport activity, leading to sequestration of
drought, oxidative stress, and mechanical wounding Na+ in the vacuole.
(Vanderbeld and Snedden, 2007). A CML from rice, Two different CMLs are involved in protection of
Oryza sativa Multi-Stress Responsive gene2 (OsMSR2), is Arabidopsis plants from UV damage. CML42 knockout
induced by multiple abiotic stress stimuli, and over- transgenics have reduced flavonol levels and display
expression of OsMSR2 in Arabidopsis enhanced toler- increased sensitivity to UV stress compared with the
ance to drought and salinity and increased sensitivity to wild type (Vadassery et al., 2012a). CML19 (also known
exogenous ABA (Xu et al., 2011). OsMSR2 is an ortho- as CENTRIN2) interacts with Arabidopsis RADIATION
log of Arabidopsis CML37, CML38, and CML39, lend- REPAIR4 (AtRAD4), a protein that functions in homol-
ing support to the hypothesis that these CMLs function ogous DNA repair (Liang et al., 2006). CML19 protein
in stress responses in Arabidopsis. Promoter and tran- levels are up-regulated by UV stress, and localization of
script analysis of CML24 revealed increased expression CML19 to the nucleus is UV dependent. Suppression
in response to high or low temperature, oxidative stress, of CML19 expression by RNA silencing resulted in a
as well exposure to ABA (Delk et al., 2005). Similarly, UV-sensitive phenotype, and overexpressing transgenics
CML9 transcript levels change in response to NaCl, displayed enhanced DNA repair (Molinier et al., 2004).
cold, and dehydration. Salt-responsive expression of It is currently unclear how CML19 regulates AtRAD4
CML9 is dependent on both ABA synthesis and sig- activity; however, nuclear localization of CML19 and
naling (Magnan et al., 2008), suggesting that this CML interaction with AtRAD4 is likely an important mech-
participates in ABA-dependent stress response path- anism for limiting UV damage.
ways. In general, gene expression analyses indicate
that many CMLs are stress responsive. In the future, it
will be important to identify the transcription factors
CMLs FUNCTION IN PATHOGEN AND
controlling CML expression and to assess the extent to
HERBIVORE DEFENSE
which transcriptional activity relates to changes in CML
protein levels. Regardless, expression profiling has proven Biotic stress is another area where several CMLs
useful in guiding questions aimed at understanding appear to play roles in Ca2+-mediated responses. In
the roles of stress-induced CMLs. Arabidopsis, expression of CML9 transcript is elevated
Plant Physiol. Vol. 163, 2013 491
Bender and Snedden

following challenge by avirulent pathogen, flagellin, or autolysosomes and subsequent degradation (Nakahara
salicylic acid (SA). The expression of CML9 in response et al., 2012). This important result indicates that rgsCaM
to avirulent pathogens is dependent upon FLAGEL- promotes defense by directly suppressing HCPro ac-
LIN SENSITITVE2 (the flagellin receptor) and pro- tivity. In Arabidopsis, the expression of CML38, a pu-
duction of SA (Leba et al., 2012). Analysis of CML9 tative ortholog of rgsCaM, is strongly up-regulated in
knockout or overexpression transgenics revealed in- transgenic plants ectopically expressing HCPro from
creased or decreased susceptibility, respectively, to the turnip mosaic virus (Endres et al., 2010). Although
multiple bacterial pathogens and altered dynamics of CML38 shares 44% amino acid identity with tobacco
PATHOGENESIS RELATED GENE1 expression fol- rgsCaM, interaction with HCPro, or any viral RSS pro-
lowing pathogen challenge (Leba et al., 2012). These tein, has not yet been reported. rgsCaM also shares strong
findings suggest that CML9 functions in Ca2+-based sequence identity with Arabidopsis CML37 and CML39,
immune responses to bacterial infection. In addition to and thus it will be interesting to see if either of these
its developmental roles, CML24 also functions in in- proteins are involved in defense against viral pathogens.
nate immunity signaling in Arabidopsis. Infection by CMLs have also been linked to defense responses
avirulent pathogen induces Ca2+ transients upstream against herbivores based on transcript profiling and
of nitric oxide generation and the hypersensitive re- genetic analyses. Oral secretions from Spodoptera lit-
sponse (Ma et al., 2008). Both of these responses are toralis larvae were shown to induce the expression of a
suppressed by treatment of plants with CaM antago- number of different CMLs (CML9, CML11, CML12,
nists and are abolished in plants lacking CML24 CML16, CML17, CML23, and CML42; Vadassery et al.,
function (Ma et al., 2008). These data define a role for 2012b). CML42 expression is stimulated by either ap-
CML24 in pathogen defense and indicate a link be- plication of S. littoralis oral secretions or direct larval
tween Ca2+ and reactive oxygen signaling in planta. feeding, with transcript levels reaching a peak 30 min
Another member of the CML family implicated in and 1 h following treatment, respectively (Vadassery
defense response is CML43. CML43 transcript accu- et al., 2012a). On the basis of this rapid induction,
mulates within 6 h following infection with avirulent larval feeding studies were carried out on plants
pathogen, and CML43 overexpression caused an accel- lacking CML42, and it was found that larval devel-
erated hypersensitive response compared with wild-type opment was impaired on CML42 loss-of-function mu-
plants (Chaisson et al., 2005). Moreover, suppression of tants. Furthermore, glucosinolate levels were higher in
the tomato (Solanum lycopersicum) ortholog of CML43, CML42 knockout plants (Vadassery et al., 2012a). As
AvrPto-Pto-Prf responsive134, by virus-induced gene noted above, CML42 also functions in trichome devel-
silencing increased susceptibility of tomato plants to opment (Dobney et al., 2009), though it is currently
infection by an avirulent strain of Pseudomonas syringae unclear whether altered trichome morphology in CML42
(Chaisson et al., 2005). These data suggest that CML43 mutants contributes to herbivore resistance. It is worth
and its tomato ortholog positively regulate pathogen noting that CML12, CML37, and CML38 transcript
defense and, importantly, demonstrate that CML func- levels rise rapidly in response to mechanical wounding
tion is conserved across taxa. (Vanderbeld and Snedden, 2007; Walley et al., 2007),
In addition to bacterial pathogen responses, CMLs suggesting that they may also be involved in herbivory
also function in host defense against viral attack. Ex- responses, although this has not yet been tested.
pression 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
CMLs ARE INVOLVED IN HORMONE SIGNALING
transgenic tobacco expressing a viral protein, HCPro
(Anandalakshmi et al., 2000). rgsCaM interacts with Expression analysis has indicated that many CMLs
HCPro in yeast two-hybrid assays, and it was sug- are induced by treatment of plants with a variety of
gested that rgsCaM serves as an endogenous sup- hormones that regulate biotic stress responses, such as
pressor of gene silencing. HCPro is an RNA-silencing JA and SA. CML39 expression is responsive to methyl
suppressor (RSS) that functions to repress posttrans- jasmonate in Arabidopsis seedlings (Vanderbeld and
criptional gene silencing as a viral mechanism to thwart Snedden, 2007). Microarray data revealed that CML39
plant defense against infection. The suppression activity expression is primarily restricted to pollen but, fol-
of HCPro (and other RSS proteins) is achieved via an lowing challenge by bacterial and fungal pathogens,
RNABD that sequesters antiviral double-stranded is also expressed in rosette leaves, indicating that
RNAs generated by the plant. Recently, the interaction this putative Ca 2+ sensor may play a role in both
between HCPro and rgsCaM was characterized, and on JA-mediated development and pathogen defense. In
the basis of homology with an RSS from the human roots, both transcript and protein expression of CML43
immunodeficiency virus, it was revealed that rgsCaM is stimulated by SA treatment (K.W. Bender, S. Dobney,
associates directly with the RNABD of certain viral RSS A. Ogunrinde, D. Chiasson, R.T. Mullen, H.J. Teresinsi,
proteins, including HCPro (Nakahara et al., 2012). In- P.J. Singh, K. Munro, S.P. Smith, and W.A. Snedden,
teraction of rgsCaM with HCPro attenuated the double- unpublished data), supporting a role for this CML in
stranded RNA-binding function of HCPro and resulted biotic defense responses. It seems likely a number of
in targeting of the rgsCaM-HCPro complex to CMLs function in hormone-mediated biotic defense;
492 Plant Physiol. Vol. 163, 2013
 Update on Calmodulin-Related Proteins

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