See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/254279891

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

CITATIONS READS

96 200

2 authors:

Kyle W. Bender Wayne A Snedden

University of Zurich Queen's University
31 PUBLICATIONS 945 CITATIONS 58 PUBLICATIONS 5,924 CITATIONS

SEE PROFILE SEE PROFILE

All content following this page was uploaded by Kyle W. Bender on 10 February 2015.

The user has requested enhancement of the downloaded file.

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
changes in their environment and respond accord-
ingly. In broad terms, cells possess signal transduction
pathways, mediating responses to internal and exter-
nal cues that consist of three distinct nodes: stimulus
perception, signal transduction, and subsequent physi-
ological 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 co-
ordinate cellular responses. Changes in metabolic ac-
tivity, gene expression, and protein turnover collectively
lead to adaptive physiological events that are appropri-
ate for the initial stimulus. This model (perception,
transduction, and response) is simplistic yet widely ap-
plicable. Among the common themes that have emerged
in signal transduction research is the importance of cal-
cium (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+ sig-
nal 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 cyto-
solic 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 concen-
trations ranging from 100 to 200 nM, whereas concen-
trations 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.
* Address correspondence to wayne.snedden@queensu.ca.
www.plantphysiol.org/cgi/doi/10.1104/pp.113.221069
486 Plant PhysiologyÒ, October 2013, Vol. 163, pp. 486–495, www.plantphysiol.org Ó 2013 American Society of Plant Biologists. All Rights Reserved.
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 ob-
served for a number of developmental cues (e.g. grav-
itropic 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 cyto-
solic 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 hy-
pothesis. Both hypotheses can claim supporting evi-
dence (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 re-
sponse. Calcium oscillations (i.e. Ca2+ “signatures”) hav-
ing 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 modifica-
tion) 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 propa-
gation 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 char-
acterized 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, in-
volved 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 resi-
dues, 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+ sig-
naling to plant growth and development.
CaM is the most thoroughly characterized Ca2+ sen-
sor 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
Plant Physiol. Vol. 163, 2013
Update on Calmodulin-Related Proteins
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 fami-
lies 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 exclu-
sively Ca2+-binding EF-hands and no other known func-
tional motifs (McCormack and Braam, 2003; McCormack
et al., 2005). Thus, CMLs are predicted to serve as Ca2+
sensors in planta. Many eukaryotes possess Ca2+ sen-
sors 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 inter-
esting 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 func-
tional 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 interac-
tion with target proteins. It is thus interesting to note
that a number of CMLs when compared to CaM pos-
sess 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 spec-
ulative, as little empirical work has been done to de-
termine their Ca2+ binding or other structural properties.
STRUCTURAL PROPERTIES OF CaM AND CMLs
Detailed biophysical analyses of CaM have re-
vealed both high affinity binding of Ca2+ as well as
487
Bender and Snedden
Ca2+-induced conformational changes within the pro-
tein. 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 a-helical
protein (approximately 40% for apoCaM), and Ca2+
binding results in an overall increase in a-helical con-
tent (approximately 50% for Ca2+/CaM; Martin and
Bayley, 1986). These structural changes are accompa-
nied by a dramatic increase in the surface hydropho-
bicity 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 isother-
mal 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 degen-
erate, 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+, sug-
gesting 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 struc-
ture 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 pres-
ence of Ca2+, having an interhelical angle of approxi-
mately 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,
488
suggesting differences in Ca2+-responsive conforma-
tional 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 sug-
gests 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), tran-
scription 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 am-
phipathic a-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 do-
mains 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, re-
flecting 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 dif-
ferences in target interaction and activation by these
two CaM isoforms.
Plant Physiol. Vol. 163, 2013

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 taba-
cum] 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 effec-
tors. (Popescu et al., 2007). Overall, this study suggests
that upwards of 15% of the proteome may be regu-
lated 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 ex-
perimentation. Nevertheless, protein-array-based screen-
ing 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 regu-
lator (PRR2) as a downstream target of CML9, found
that a nearly full-length target was required for inter-
action 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
a-helix structure (Yamaguchi et al., 2005), like those
typically involved in CaM-target interactions. Interest-
ingly, binding was specific for CML18, as a conserved
isoform, CaM81, from petunia (Petunia hybrida) did not
Table I. List of known CML-interacting proteins and evidence put forth for the interaction
Gene Ontology
CML Species Target
Molecular Function
CML8 Arabidopsis BRI1 Protein Ser/Thr
kinase activity
CML9 Arabidopsis PRR2 DNA binding
CML12 Arabidopsis PINOID Protein Ser/Thr
kinase activity
CML18 Arabidopsis AtNHX1 Na+:H+ antiporter
activity
CML19 Arabidopsis AtRAD4 Damaged DNA
binding
CML20 Arabidopsis TONNEAU1 Protein binding
CML24 Arabidopsis ATG4b Peptidase activity
CML42 Arabidopsis KIC Ca2+ ion binding
rgsCaM Tobacco HCProa RNA binding
a
HCPro is a viral protein; no endogenous targets have been identified for rgsCaM.
Plant Physiol. Vol. 163, 2013
Update on Calmodulin-Related Proteins
interact with AtNHX1 (Yamaguchi et al., 2005). By com-
parison, a CaMBD was recently identified in BRASSI-
NOSTEROID INSENSITIVE1 (BRI1; a leucine-rich repeat
receptor-like kinase), and interaction of CaM suppressed
protein kinase activity of BRI1 (Oh et al., 2012). Intrigu-
ingly, 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.
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 character-
istics of CML-target protein complexes before any gen-
eralizations 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).
Evidence Reference
Effect on auto-/transphosphorylation Oh et al., 2012
Yeast two hybrid, fluorescence resonance Perochon et al., 2010
energy transfer
Yeast two hybrid, copurification Benjamins et al., 2003
Yeast two hybrid, coimmunoprecipitation Yamaguchi et al., 2005
Copurification Liang et al., 2006
Yeast two hybrid, copurification, bimolecular Azimzadeh et al., 2008
fluorescence complementation
Yeast two hybrid, copurification, Tsai et al., 2012
coimmunoprecipitation
Yeast two hybrid, copurification Dobney et al., 2009
Yeast two hybrid, surface plasmon resonance Anandalakshmi et al., 2000;
Nakahara et al., 2012
489
Bender and Snedden

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 re-
duced trichome branching. Given the opposing phe-
notypes 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 dif-
ferent CMLs, CML7 and CML25, are involved in root
hair elongation (Won et al., 2009; Lin et al., 2011). Trans-
genic 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 mi-
crotubule organization; however, the functional signifi-
cance 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 mu-
tations in CML23, CML24, or both genes displayed al-
tered 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 al-
ways late flowering. Furthermore, mutants showed al-
tered levels of transcripts for CONSTANS, FLOWERING
LOCUS C, and SUPRESSOR OF CONSTANS1 that cor-
related with either early- or late-flowering phenotypes
(Tsai et al., 2007). Given opposing flowering pheno-
types 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 reg-
ulation of the same signaling pathway.
In addition to a role in flowering, CML24 appears to
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


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 reg-
ulation 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 in-
ducibility of CML39 (a strongly JA-responsive gene,
see below) was at least partially lost in mutants im-
paired in JA biosynthesis. Furthermore, mutant anal-
ysis 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 path-
ways. 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 over-
expression of OsMSR2 in Arabidopsis enhanced toler-
ance to drought and salinity and increased sensitivity to
exogenous ABA (Xu et al., 2011). OsMSR2 is an ortho-
log of Arabidopsis CML37, CML38, and CML39, lend-
ing support to the hypothesis that these CMLs function
in stress responses in Arabidopsis. Promoter and tran-
script 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 sig-
naling (Magnan et al., 2008), suggesting that this CML
participates in ABA-dependent stress response path-
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
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.
Plant Physiol. Vol. 163, 2013
Update on Calmodulin-Related Proteins
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, molyb-
denum, zinc, and magnesium (Delk et al., 2005). Trans-
genic 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 germina-
tion 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 mecha-
nisms leading to these apparently contradictory phe-
notypes in seeds and adult plants.
CML18 has also been implicated in salt stress sig-
naling 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+ trans-
location 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 re-
sponse 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 homol-
ogous 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 mech-
anism 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
491
Bender and Snedden
following challenge by avirulent pathogen, flagellin, or
salicylic acid (SA). The expression of CML9 in response
to avirulent pathogens is dependent upon FLAGEL-
LIN SENSITITVE2 (the flagellin receptor) and pro-
duction of SA (Leba et al., 2012). Analysis of CML9
knockout or overexpression transgenics revealed in-
creased or decreased susceptibility, respectively, to
multiple bacterial pathogens and altered dynamics of
PATHOGENESIS RELATED GENE1 expression fol-
lowing 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 in-
nate immunity signaling in Arabidopsis. Infection by
avirulent pathogen induces Ca2+ transients upstream
of nitric oxide generation and the hypersensitive re-
sponse (Ma et al., 2008). Both of these responses are
suppressed by treatment of plants with CaM antago-
nists 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 be-
tween Ca2+ and reactive oxygen signaling in planta.
Another member of the CML family implicated in
defense response is CML43. CML43 transcript accu-
mulates within 6 h following infection with avirulent
pathogen, and CML43 overexpression caused an accel-
erated 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 func-
tion is conserved across taxa.
In addition to bacterial pathogen responses, CMLs
also function in host defense against viral attack. Ex-
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
transgenic tobacco expressing a viral protein, HCPro
(Anandalakshmi et al., 2000). rgsCaM interacts with
HCPro in yeast two-hybrid assays, and it was sug-
gested that rgsCaM serves as an endogenous sup-
pressor of gene silencing. HCPro is an RNA-silencing
suppressor (RSS) that functions to repress posttrans-
criptional 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). In-
teraction of rgsCaM with HCPro attenuated the double-
stranded RNA-binding function of HCPro and resulted
in targeting of the rgsCaM-HCPro complex to
492
autolysosomes and subsequent degradation (Nakahara
et al., 2012). This important result indicates that rgsCaM
promotes defense by directly suppressing HCPro ac-
tivity. In Arabidopsis, the expression of CML38, a pu-
tative 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 pro-
tein, 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 lit-
toralis 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 ap-
plication 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 devel-
opment was impaired on CML42 loss-of-function mu-
tants. Furthermore, glucosinolate levels were higher in
CML42 knockout plants (Vadassery et al., 2012a). As
noted above, CML42 also functions in trichome devel-
opment (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, fol-
lowing challenge by bacterial and fungal pathogens,
is also expressed in rosette leaves, indicating that
this putative Ca 2+ 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;
Plant Physiol. Vol. 163, 2013

however, specific roles for CMLs and other Ca2+ sen-
sors downstream of pathogen challenge remain to be
elucidated.
CMLs also participate in hormone signaling during
development. CML12 (also known as TOUCH3) inter-
acts 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 ki-
nase activity leading to reduced apical efflux of auxin
(Benjamins et al., 2003). Potential regulation of the po-
larity 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 cy-
tosolic 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 ex-
perimental evidence; yet, it remains largely unknown
whether this occurs through regulation of distinct tar-
gets 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 pro-
teins, 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 re-
sponses 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 re-
markable diversity of Ca2+ sensors in plants is likely
essential for successful coordination of development
and reproduction in a dynamic and often stressful
environment.
Received May 7, 2013; accepted July 31, 2013; published August 2, 2013.
LITERATURE CITED
Ali R, Ma W, Lemtiri-Chlieh F, Tsaltas D, Leng Q, von Bodman S,
Berkowitz GA (2007) Death don’t have no mercy and neither does
calcium: Arabidopsis CYCLIC NUCLEOTIDE GATED CHANNEL2 and
innate immunity. Plant Cell 19: 1081–1095
Plant Physiol. Vol. 163, 2013
Update on Calmodulin-Related Proteins
Allen GJ, Chu SP, Harrington CL, Schumacher K, Hoffmann T, Tang YY,
Grill E, Schroeder JI (2001) A defined range of guard cell calcium
oscillation parameters encodes stomatal movements. Nature 411:
1053–1057
Allen GJ, Chu SP, Schumacher K, Shimazaki CT, Vafeados D, Kemper A,
Hawke SD, Tallman G, Tsien RY, Harper JF, et al (2000) Alteration of
stimulus-specific guard cell calcium oscillations and stomatal closing in
Arabidopsis det3 mutant. Science 289: 2338–2342
Anandalakshmi R, Marathe R, Ge X, Herr JM Jr, Mau C, Mallory A, Pruss
G, Bowman L, Vance VB (2000) A calmodulin-related protein that
suppresses posttranscriptional gene silencing in plants. Science 290:
142–144
Azimzadeh J, Nacry P, Christodoulidou A, Drevensek S, Camilleri C, Amiour
N, Parcy F, Pastuglia M, Bouchez D (2008) Arabidopsis TONNEAU1 pro-
teins are essential for preprophase band formation and interact with centrin.
Plant Cell 20: 2146–2159
Baum G, Lev-Yadun S, Fridmann Y, Arazi T, Katsnelson H, Zik M,
Fromm H (1996) Calmodulin binding to glutamate decarboxylase is
required for regulation of glutamate and GABA metabolism and normal
development in plants. EMBO J 15: 2988–2996
Benjamins R, Ampudia CS, Hooykaas PJJ, Offringa R (2003) PINOID-
mediated signaling involves calcium-binding proteins. Plant Physiol
132: 1623–1630
Boonburapong B, Buaboocha T (2007) Genome-wide identification and
analyses of the rice calmodulin and related potential calcium sensor
proteins. BMC Plant Biol 7: 4
Bouché N, Yellin A, Snedden WA, Fromm H (2005) Plant-specific
calmodulin-binding proteins. Annu Rev Plant Biol 56: 435–466
Chehab EW, Yao C, Henderson Z, Kim S, Braam J (2012) Arabidopsis touch-
induced morphogenesis is jasmonate mediated and protects against pests.
Curr Biol 22: 701–706
Chiasson D, Ekengren SK, Martin GB, Dobney SL, Snedden WA (2005)
Calmodulin-like proteins from Arabidopsis and tomato are involved in
host defense against Pseudomonas syringae pv. tomato. Plant Mol Biol 58:
887–897
Chigri F, Flosdorff S, Pilz S, Kölle E, Dolze E, Gietl C, Vothknecht UC
(2012) The Arabidopsis calmodulin-like proteins AtCML30 and AtCML3
are targeted to mitochondria and peroxisomes, respectively. Plant Mol
Biol 78: 211–222
Day I, Reddy V, Ali GS, Reddy ASN (2002) Analysis of EF-hand-
containing proteins in Arabidopsis. Genome Biol 3: research0056.0051–
research0056.0024
DeFalco TA, Bender KW, Snedden WA (2010) Breaking the code: Ca2+
sensors in plant signalling. Biochem J 425: 27–40
Delk NA, Johnson KA, Chowdhury NI, Braam J (2005) CML24, regulated
in expression by diverse stimuli, encodes a potential Ca2+ sensor that
functions in responses to abscisic acid, daylength, and ion stress. Plant
Physiol 139: 240–253
Dobney S, Chiasson D, Lam P, Smith SP, Snedden WA (2009) The
calmodulin-related calcium sensor CML42 plays a role in trichome
branching. J Biol Chem 284: 31647–31657
Dodd AN, Kudla J, Sanders D (2010) The language of calcium signaling.
Annu Rev Plant Biol 61: 593–620
Du L, Ali GS, Simons KA, Hou J, Yang T, Reddy ASN, Poovaiah BW
(2009) Ca 2+/calmodulin regulates salicylic-acid-mediated plant immu-
nity. Nature 457: 1154–1158
Endres MW, Gregory BD, Gao Z, Foreman AW, Mlotshwa S, Ge X, Pruss
GJ, Ecker JR, Bowman LH, Vance V (2010) Two plant viral suppressors
of silencing require the ethylene-inducible host transcription factor
RAV2 to block RNA silencing. PLoS Pathog 6: e1000729
Friml J, Yang X, Michniewicz M, Weijers D, Quint A, Tietz O, Benjamins
R, Ouwerkerk PBF, Ljung K, Sandberg G, et al (2004) A PINOID-
dependent binary switch in apical-basal PIN polar targeting directs
auxin efflux. Science 306: 862–865
Gifford JL, Walsh MP, Vogel HJ (2007) Structures and metal-ion-binding
properties of the Ca2+-binding helix-loop-helix EF-hand motifs. Biochem
J 405: 199–221
Gilli R, Lafitte D, Lopez C, Kilhoffer MC, Makarov A, Briand C, Haiech J
(1998) Thermodynamic analysis of calcium and magnesium binding to
calmodulin. Biochemistry 37: 5450–5456
Gilroy S, Bethke PC, Jones RL (1993) Calcium homeostasis in plants. J Cell
Sci 106: 453–461
493
Bender and Snedden
Hua BG, Mercier RW, Zielinski RE, Berkowitz GA (2003) Functional
interaction of calmodulin with a plant cyclic nucleotide gated cation
channel. Plant Physiol Biochem 41: 945–954
Ikura M, Kay LE, Bax A (1990) A novel approach for sequential assignment
of proton, carbon-13, and nitrogen-15 spectra of larger proteins: hetero-
nuclear triple-resonance three-dimensional NMR spectroscopy. Applica-
tion to calmodulin. Biochemistry 29: 4659–4667
Ishida H, Huang H, Yamniuk AP, Takaya Y, Vogel HJ (2008) The solution
structures of two soybean calmodulin isoforms provide a structural
basis for their selective target activation properties. J Biol Chem 283:
14619–14628
Israelsson M, Siegel RS, Young J, Hashimoto M, Iba K, Schroeder JI
(2006) Guard cell ABA and CO2 signaling network updates and Ca2+
sensor priming hypothesis. Curr Opin Plant Biol 9: 654–663
Katsuhara M, Kuchitsu K, Takeshige K, Tazawa M (1989) Salt stress-
induced cytoplasmic acidification and vacuolar alkalization in Ni-
tellopsis obtusa cells: in vivo 31P-nuclear magnetic resonance study. Plant
Physiol 90: 1102–1107
Kiegle E, Moore CA, Haseloff J, Tester MA, Knight MR (2000) Cell-type-
specific calcium responses to drought, salt and cold in the Arabidopsis
root. Plant J 23: 267–278
Knight H, Trewavas AJ, Knight MR (1997) Calcium signalling in Arabi-
dopsis thaliana responding to drought and salinity. Plant J 12: 1067–1078
Kosuta S, Hazledine S, Sun J, Miwa H, Morris RJ, Downie JA, Oldroyd
GED (2008) Differential and chaotic calcium signatures in the sym-
biosis signaling pathway of legumes. Proc Natl Acad Sci USA 105:
9823–9828
Leba L-J, Cheval C, Ortiz-Martín I, Ranty B, Beuzón CR, Galaud J-P,
Aldon D (2012) CML9, an Arabidopsis calmodulin-like protein, con-
tributes to plant innate immunity through a flagellin-dependent sig-
nalling pathway. Plant J 71: 976–989
Lee D, Polisensky DH, Braam J (2005) Genome-wide identification of
touch- and darkness-regulated Arabidopsis genes: a focus on calmodulin-
like and XTH genes. New Phytol 165: 429–444
Liang L, Flury S, Kalck V, Hohn B, Molinier J (2006) CENTRIN2 interacts
with the Arabidopsis homolog of the human XPC protein (AtRAD4) and
contributes to efficient synthesis-dependent repair of bulky DNA le-
sions. Plant Mol Biol 61: 345–356
Lin W-D, Liao Y-Y, Yang TJW, Pan C-Y, Buckhout TJ, Schmidt W (2011)
Coexpression-based clustering of Arabidopsis root genes predicts functional
modules in early phosphate deficiency signaling. Plant Physiol 155:
1383–1402
Liu HT, Li GL, Chang HUI, Sun DY, Zhou RG, Li B (2007) Calmodulin-
binding protein phosphatase PP7 is involved in thermotolerance in
Arabidopsis. Plant Cell Environ 30: 156–164
Luoni L, Bonza MC, De Michelis MI (2006) Calmodulin/Ca2+-ATPase
interaction at the Arabidopsis thaliana plasma membrane is dependent on
calmodulin isoform showing isoform-specific Ca2+ dependencies. Physiol
Plant 126: 175–186
Ma W, Smigel A, Tsai Y-C, Braam J, Berkowitz GA (2008) Innate immu-
nity signaling: cytosolic Ca2+ elevation is linked to downstream nitric
oxide generation through the action of calmodulin or a calmodulin-like
protein. Plant Physiol 148: 818–828
Magnan F, Ranty B, Charpenteau M, Sotta B, Galaud J-P, Aldon D (2008)
Mutations in AtCML9, a calmodulin-like protein from Arabidopsis thali-
ana, alter plant responses to abiotic stress and abscisic acid. Plant J 56:
575–589
Martin SR, Bayley PM (1986) The effects of Ca2+ and Cd2+ on the secondary
and tertiary structure of bovine testis calmodulin. A circular-dichroism
study. Biochem J 238: 485–490
McAinsh MR, Pittman JK (2009) Shaping the calcium signature. New
Phytol 181: 275–294
McCormack E, Braam J (2003) Calmodulins and related potential calcium
sensors of Arabidopsis. New Phytol 159: 585–598
McCormack E, Tsai Y-C, Braam J (2005) Handling calcium signaling:
Arabidopsis CaMs and CMLs. Trends Plant Sci 10: 383–389
Miwa H, Sun J, Oldroyd GED, Downie JA (2006) Analysis of calcium
spiking using a cameleon calcium sensor reveals that nodulation gene
expression is regulated by calcium spike number and the developmental
status of the cell. Plant J 48: 883–894
Molinier J, Ramos C, Fritsch O, Hohn B (2004) CENTRIN2 modulates
homologous recombination and nucleotide excision repair in Arabi-
dopsis. Plant Cell 16: 1633–1643
494
Nakahara KS, Masuta C, Yamada S, Shimura H, Kashihara Y, Wada TS,
Meguro A, Goto K, Tadamura K, Sueda K, et al (2012) Tobacco
calmodulin-like protein provides secondary defense by binding to and
directing degradation of virus RNA silencing suppressors. Proc Natl
Acad Sci USA 109: 10113–10118
Oh M-H, Kim HS, Wu X, Clouse SD, Zielinski RE, Huber SC (2012)
Calcium/calmodulin inhibition of the Arabidopsis BRASSINOSTEROID-
INSENSITIVE 1 receptor kinase provides a possible link between calcium
and brassinosteroid signalling. Biochem J 443: 515–523
Oldroyd GED, Downie JA (2006) Nuclear calcium changes at the core of
symbiosis signalling. Curr Opin Plant Biol 9: 351–357
Perochon A, Dieterle S, Pouzet C, Aldon D, Galaud J-P, Ranty B (2010)
Interaction of a plant pseudo-response regulator with a calmodulin-like
protein. Biochem Biophys Res Commun 398: 747–751
Pidcock E, Moore GR (2001) Structural characteristics of protein binding
sites for calcium and lanthanide ions. J Biol Inorg Chem 6: 479–489
Popescu SC, Popescu GV, Bachan S, Zhang Z, Seay M, Gerstein M,
Snyder M, Dinesh-Kumar SP (2007) Differential binding of calmodulin-
related proteins to their targets revealed through high-density
Arabidopsis protein microarrays. Proc Natl Acad Sci USA 104: 4730–
4735
Rainaldi M, Yamniuk AP, Murase T, Vogel HJ (2007) Calcium-dependent
and -independent binding of soybean calmodulin isoforms to the cal-
modulin binding domain of tobacco MAPK phosphatase-1. J Biol Chem
282: 6031–6042
Reddy VS, Day IS, Thomas T, Reddy ASN (2004) KIC, a novel Ca2+
binding protein with one EF-hand motif, interacts with a microtubule
motor protein and regulates trichome morphogenesis. Plant Cell 16:
185–200
Schwaller B (2010) Cytosolic Ca2+ buffers. Cold Spring Harb Perspect Biol
2: a004051
Scrase-Field SAMG, Knight MR (2003) Calcium: just a chemical switch?
Curr Opin Plant Biol 6: 500–506
Song J, Zhao Q, Thao S, Frederick RO, Markley JL (2004) Solution
structure of a calmodulin-like calcium-binding domain from Arabidopsis
thaliana. J Biomol NMR 30: 451–456
Spalding EP, Harper JF (2011) The ins and outs of cellular Ca2+ transport.
Curr Opin Plant Biol 14: 715–720
Toyota M, Furuichi T, Tatsumi H, Sokabe M (2008) Cytoplasmic calcium
increases in response to changes in the gravity vector in hypocotyls and
petioles of Arabidopsis seedlings. Plant Physiol 146: 505–514
Tsai Y-C, Delk NA, Chowdhury NI, Braam J (2007) Arabidopsis potential
calcium sensors regulate nitric oxide levels and the transition to flow-
ering. Plant Signal Behav 2: 446–454
Tsai Y-C, Koo Y, Delk NA, Gehl B, Braam J (2012) Calmodulin-related
CML24 interacts with ATG4b and affects autophagy progression in
Arabidopsis. Plant J 73: 325–335
Vadassery J, Reichelt M, Hause B, Gershenzon J, Boland W, Mithöfer A
(2012a) CML42-mediated calcium signaling coordinates responses to
Spodoptera herbivory and abiotic stresses in Arabidopsis. Plant Physiol
159: 1159–1175
Vadassery J, Scholz SS, Mithöfer A (2012b) Multiple calmodulin-like
proteins in Arabidopsis are induced by insect-derived (Spodoptera lit-
toralis) oral secretion. Plant Signal Behav 7: 1277–1280
Vanderbeld B, Snedden WA (2007) Developmental and stimulus-induced
expression patterns of Arabidopsis calmodulin-like genes CML37, CML38
and CML39. Plant Mol Biol 64: 683–697
Virdi AS, Thakur A, Dutt S, Kumar S, Singh P (2009) A sorghum 85kDa
heat stress-modulated protein shows calmodulin-binding properties
and cross-reactivity to anti-Neurospora crassa Hsp 80 antibodies. FEBS
Lett 583: 767–770
Walley JW, Coughlan S, Hudson ME, Covington MF, Kaspi R, Banu G,
Harmer SL, Dehesh K (2007) Mechanical stress induces biotic and
abiotic stress responses via a novel cis-element. PLoS Genet 3: 1800–
1812
Wang Y, Wang B, Gilroy S, Chehab EW, Braam J (2011) CML24 is involved
in root mechanoresponses and cortical microtubule orientation in Arabi-
dopsis. J Plant Growth Regul 30: 467–479
Won S-K, Lee Y-J, Lee H-Y, Heo Y-K, Cho M, Cho H-T (2009) cis-Element-
and transcriptome-based screening of root hair-specific genes and their
functional characterization in Arabidopsis. Plant Physiol 150: 1459–
1473
Plant Physiol. Vol. 163, 2013

Wright AJ, Knight H, Knight MR (2002) Mechanically stimulated TCH3
gene expression in Arabidopsis involves protein phosphorylation and
EIN6 downstream of calcium. Plant Physiol 128: 1402–1409
Xu G-Y, Rocha PS, Wang M-L, Xu M-L, Cui Y-C, Li L-Y, Zhu Y-X, Xia X
(2011) A novel rice calmodulin-like gene, OsMSR2, enhances drought
and salt tolerance and increases ABA sensitivity in Arabidopsis. Planta
234: 47–59
Yamaguchi T, Aharon GS, Sottosanto JB, Blumwald E (2005) Vacuolar
Na+/H+ antiporter cation selectivity is regulated by calmodulin from
Plant Physiol. Vol. 163, 2013
View publication stats
Update on Calmodulin-Related Proteins
within the vacuole in a Ca2+- and pH-dependent manner. Proc Natl
Acad Sci USA 102: 16107–16112
Yamniuk AP, Gifford JL, Linse S, Vogel HJ (2008) Effects of metal-
binding loop mutations on ligand binding to calcium- and integrin-
binding protein 1. Evolution of the EF-hand? Biochemistry 47:
1696–1707
Yamniuk AP, Vogel HJ (2005) Structural investigation into the differential
target enzyme regulation displayed by plant calmodulin isoforms. Bio-
chemistry 44: 3101–3111
495