Salicylic Acid Regulates Arabidopsis Microbial Pattern Receptor Kinase Levels and Signaling

In Arabidopsis thaliana, responses to pathogen-associated molecular patterns (PAMPs) are mediated by cell surface pattern recognition receptors (PRRs) and include the accumulation of reactive oxygen species, callose deposition in the cell wall, and the generation of the signal molecule salicylic acid (SA). SA acts in a positive feedback loop with ACCELERATED CELL DEATH6 (ACD6), a membrane protein that contributes to immunity. This work shows that PRRs associate with and are part of the ACD6/SA feedback loop. ACD6 positively regulates the abundance of several PRRs and affects the responsiveness of plants to two PAMPs. SA accumulation also causes increased levels of PRRs and potentiates the responsiveness of plants to PAMPs. Finally, SA induces PRR- and ACD6-dependent signaling to induce callose deposition independent of the presence of PAMPs. This PAMP-independent effect of SA causes a transient reduction of PRRs and ACD6-dependent reduced responsiveness to PAMPs. Thus, SA has a dynamic effect on the regulation and function of PRRs. Within a few hours, SA signaling promotes defenses and downregulates PRRs, whereas later (within 24 to 48 h) SA signaling upregulates PRRs, and plants are rendered more responsive to PAMPs. These results implicate multiple modes of signaling for PRRs in response to PAMPs and SA.     

Calcium/Calmodulin-Dependent Protein Kinase Is Negatively and Positively Regulated by Calcium,Providing a Mechanism for Decoding Calcium Responses during Symbiosis Signaling

The establishment of symbiotic associations in plants requires calcium oscillations that must be decoded to invoke downstream developmental programs. In animal systems, comparable calcium oscillations are decoded by calmodulin (CaM)–dependent protein kinases, but symbiotic signaling involves a calcium/CaM–dependent protein kinase (CCaMK) that is unique to plants. CCaMK differs from the animal CaM kinases by its dual ability to bind free calcium, via calcium binding EF-hand domains on the protein, or to bind calcium complexed with CaM, via a CaM binding domain. In this study, we dissect this dual regulation of CCaMK by calcium. We find that calcium binding to the EF-hand domains promotes autophosphorylation, which negatively regulates CCaMK by stabilizing the inactive state of the protein. By contrast, calcium-dependent CaM binding overrides the effects of autophosphorylation and activates the protein. The differential calcium binding affinities of the EF-hand domains compared with those of CaM suggest that CCaMK is maintained in the inactive state at basal calcium concentrations and is activated via CaM binding during calcium oscillations. This work provides a model for decoding calcium oscillations that uses differential calcium binding affinities to create a robust molecular switch that is responsive to calcium concentrations associated with both the basal state and with oscillations.     

Nitrated Cyclic GMP Modulates Guard Cell Signaling in Arabidopsis

Nitric oxide (NO) is a ubiquitous signaling molecule involved in diverse physiological processes, including plant senescence and stomatal closure. The NO and cyclic GMP (cGMP) cascade is the main NO signaling pathway in animals, but whether this pathway operates in plant cells, and the mechanisms of its action, remain unclear. Here, we assessed the possibility that the nitrated cGMP derivative 8-nitro-cGMP functions in guard cell signaling. Mass spectrometry and immunocytochemical analyses showed that abscisic acid and NO induced the synthesis of 8-nitro-cGMP in guard cells in the presence of reactive oxygen species. 8-Nitro-cGMP triggered stomatal closure, but 8-bromoguanosine 3′,5′-cyclic monophosphate (8-bromo-cGMP), a membrane-permeating analog of cGMP, did not. However, in the dark, 8-bromo-cGMP induced stomatal opening but 8-nitro-cGMP did not. Thus, cGMP and its nitrated derivative play different roles in the signaling pathways that lead to stomatal opening and closure. Moreover, inhibitor and genetic studies showed that calcium, cyclic adenosine-5′-diphosphate-ribose, and SLOW ANION CHANNEL1 act downstream of 8-nitro-cGMP. This study therefore demonstrates that 8-nitro-cGMP acts as a guard cell signaling molecule and that a NO/8-nitro-cGMP signaling cascade operates in guard cells.     

Constitutively Active Mitogen-Activated Protein Kinase Versions Reveal Functions of Arabidopsis MPK4 in Pathogen Defense Signaling

Plant mitogen-activated protein kinases (MAPKs) are involved in important processes, including stress signaling and development. In a functional yeast screen, we identified mutations that render Arabidopsis thaliana MAPKs constitutively active (CA). Importantly, CA-MAPKs maintain their specificity toward known activators and substrates. As a proof-of-concept, Arabidopsis MAPK4 (MPK4) function in plant immunity was investigated. In agreement with the phenotype of mpk4 mutants, CA-MPK4 plants were compromised in pathogen-induced salicylic acid accumulation and disease resistance. MPK4 activity was found to negatively regulate pathogen-associated molecular pattern-induced reactive oxygen species production but had no impact on callose deposition, indicating that CA-MPK4 allows discriminating between processes regulated by MPK4 activity from processes indirectly affected by mpk4 mutation. Finally, MPK4 activity was also found to compromise effector-triggered immunity conditioned by the Toll Interleukin-1 Receptor–nucleotide binding (NB)–Leu-rich repeat (LRR) receptors RPS4 and RPP4 but not by the coiled coil–NB-LRR receptors RPM1 and RPS2. Overall, these data reveal important insights on how MPK4 regulates plant defenses and establishes that CA-MAPKs offer a powerful tool to analyze the function of plant MAPK pathways.     

Focus Issue on Calcium Signaling: Calcium-Dependent Protein Kinase CPK6 Positively Functions in Induction by Yeast Elicitor of Stomatal Closure and Inhibition by Yeast Elicitor of Light-Induced Stomatal Opening in Arabidopsis

Yeast elicitor (YEL) induces stomatal closure that is mediated by a Ca²⁺-dependent signaling pathway. A Ca²⁺-dependent protein kinase, CPK6, positively regulates activation of ion channels in abscisic acid and methyl jasmonate signaling, leading to stomatal closure in Arabidopsis (Arabidopsis thaliana). YEL also inhibits light-induced stomatal opening. However, it remains unknown whether CPK6 is involved in induction by YEL of stomatal closure or in inhibition by YEL of light-induced stomatal opening. In this study, we investigated the roles of CPK6 in induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening in Arabidopsis. Disruption of CPK6 gene impaired induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening. Activation by YEL of nonselective Ca²⁺-permeable cation channels was impaired in cpk6-2 guard cells, and transient elevations elicited by YEL in cytosolic-free Ca²⁺ concentration were suppressed in cpk6-2 and cpk6-1 guard cells. YEL activated slow anion channels in wild-type guard cells but not in cpk6-2 or cpk6-1 and inhibited inward-rectifying K⁺ channels in wild-type guard cells but not in cpk6-2 or cpk6-1. The cpk6-2 and cpk6-1 mutations inhibited YEL-induced hydrogen peroxide accumulation in guard cells and apoplast of rosette leaves but did not affect YEL-induced hydrogen peroxide production in the apoplast of rosette leaves. These results suggest that CPK6 positively functions in induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening in Arabidopsis and is a convergent point of signaling pathways for stomatal closure in response to abiotic and biotic stress.Stomata, formed by pairs of guard cells, play a critical role in regulation of plant CO₂ uptake and water loss, thus critically influencing plant growth and water stress responsiveness. Guard cells respond to a variety of abiotic and biotic stimuli, such as light, drought, and pathogen attack (Israelsson et al., 2006; Shimazaki et al., 2007; Melotto et al., 2008).Elicitors derived from microbial surface mimic pathogen attack and induce stomatal closure in various plant species such as Solanum lycopersicum (Lee et al., 1999), Commelina communis (Lee et al., 1999), Hordeum vulgare (Koers et al., 2011), and Arabidopsis (Arabidopsis thaliana; Melotto et al., 2006; Khokon et al., 2010). Yeast elicitor (YEL) induces stomatal closure in Arabidopsis (Klüsener et al., 2002; Khokon et al., 2010; Salam et al., 2013). Our recent studies showed that YEL inhibits light-induced stomatal opening and that protein phosphorylation is involved in induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening (Salam et al., 2013).Cytosolic Ca²⁺ has long been recognized as a conserved second messenger in stomatal movement (Shimazaki et al., 2007; Roelfsema and Hedrich 2010; Hubbard et al., 2012). Elevation of cytosolic free Ca²⁺ concentration ([Ca2+]_cyt) is triggered by influx of Ca²⁺ from apoplast and release of Ca²⁺ from intracellular stores in guard cell signaling (Leckie et al., 1998; Hamilton et al., 2000; Pei et al., 2000; Garcia-Mata et al., 2003; Lemtiri-Chlieh et al., 2003). The influx of Ca²⁺ is carried by nonselective Ca²⁺-permeable cation (I_Ca) channels that are activated by plasma membrane hyperpolarization and H₂O₂ (Pei et al., 2000; Murata et al., 2001; Kwak et al., 2003). Elevation of [Ca2+]_cyt activates slow anion (S-type) channels and down-regulates inward-rectifying potassium (K_in) channels in guard cells (Schroeder and Hagiwara, 1989; Grabov and Blatt, 1999). The activation of S-type channels is a hallmark of stomatal closure, and the suppression of K_in channels is favorable to stomatal closure but not to stomatal opening (Pei et al., 1997; Kwak et al., 2001; Xue et al., 2011; Uraji et al., 2012).YEL induces stomatal closure with extracellular H₂O₂ production, intracellular H₂O₂ accumulation, activation of I_Ca channels, and transient [Ca2+]_cyt elevations (Klüsener et al., 2002; Khokon et al., 2010). However, it remains to be clarified whether YEL activates S-type channels and inhibits K_in channels in guard cells.Calcium-dependent protein kinases (CDPKs) are regulators in Ca²⁺-dependent guard cell signaling (Mori et al., 2006; Zhu et al., 2007; Geiger et al., 2010, 2011; Zou et al., 2010; Munemasa et al., 2011; Brandt et al., 2012; Scherzer et al., 2012). In guard cells, CDPKs regulate activation of S-type and I_Ca channels and inhibition of K_in channels (Mori et al., 2006; Zou et al., 2010; Munemasa et al., 2011). A CDPK, CPK6, positively regulates activation of S-type channels and I_Ca channels without affecting H₂O₂ production in abscisic acid (ABA)- and methyl jasmonate (MeJA)-induced stomatal closure (Mori et al., 2006; Munemasa et al., 2011). CPK6 phosphorylates and activates SLOW ANION CHANNEL-ASSOCIATED1 expressed in Xenopus spp. oocyte (Brandt et al., 2012; Scherzer et al., 2012). These findings underline the role of CPK6 in regulation of ion channel activation and stomatal movement, leading us to test whether CPK6 regulates the induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening.In this study, we investigated activation of S-type channels and inhibition of K_in channels by YEL and roles of CPK6 in induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening. For this purpose, we examined the effects of mutation of CPK6 on induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening, activation of I_Ca channels, transient [Ca2+]_cyt elevations, activation of S-type channels, inhibition of K_in channels, H₂O₂ production in leaves, and H₂O₂ accumulation in leaves and guard cells.     

The Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-Related Protein Kinases Are Required for Elicitor-Induced Oxidative Burst and Immunity

Plant immunity is activated through complex and cross-talking transduction pathways that include a mitogen-activated protein kinase phosphorylation cascade. Here, we have investigated the role in immunity of the Arabidopsis (Arabidopsis thaliana) gene subfamily that encodes the mitogen-activated protein triple kinases indicated as ARABIDOPSIS NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-RELATED PROTEIN KINASE1 (ANP1), ANP2, and ANP3. For this study, we used representative danger signals (elicitors) belonging to the classes of the damage- and pathogen-associated molecular patterns, i.e. oligogalacturonides, linear fragments derived from the plant cell wall homogalacturonan, and the peptide elf18 derived from the bacterial elongation factor thermo-unstable. Analyses of single and double as well as conditional triple mutants show that ANPs are required for elicitor-triggered defense responses and protection against the necrotrophic fungus Botrytis cinerea. Notably, ANPs are also required for both the elicitor-induced oxidative burst and the transduction of the hydrogen peroxide signal but not for the inhibition of auxin-induced gene expression, indicating that this response can be uncoupled from the activation of defense responses. Our findings point to ANPs as key transduction elements that coordinate damage- and pathogen-associated molecular pattern-triggered immunity and orchestrate reactive oxygen species accumulation and signaling.Plants are continually exposed to microbial pathogens and, like animals, activate the innate immune system to respond properly and in a timely manner (Boller and He, 2009). Plants also rely on the structure of the cell wall that acts as a physical barrier against the microbial invasion (De Lorenzo et al., 2001). In their attempts to penetrate plant tissues, pathogens need to efficiently degrade the cell wall (Lionetti et al., 2010). Once the plant cell wall is breached, pathogens encounter the host plasma membrane, where pattern recognition receptors (PRRs) sense the presence of nonself molecules (pathogen-associated molecular patterns [PAMPs]) and activate the so-called PAMP-triggered immunity (PTI; Dodds and Rathjen, 2010). Endogenous molecules, released during infection or mechanical wounding and usually referred to as damage-associated molecular patterns (DAMPs), are also recognized by PRRs as danger signals and contribute to the activation of the plant immune response (Schwessinger and Ronald, 2012). Representative PAMPs are the peptides elf18 and flg22, derived from the bacterial elongation factor thermo-unstable (EF-Tu) and flagellin, respectively (Gómez-Gómez and Boller, 2000; Zipfel et al., 2006). Among the best characterized DAMPs are oligogalacturonides (OGs), linear molecules of 10 to about 16 α-1,4-d-galactopyranosyluronic acid residues released upon fragmentation of homogalacturonan, which is an important component of the plant cell wall (Ferrari et al., 2013). In Arabidopsis (Arabidopsis thaliana), elf18 and flg22 are recognized by the transmembrane leucine-rich repeat receptor kinases EF-TU RECEPTOR (EFR) and FLAGELLIN-SENSING2, respectively (Gómez-Gómez and Boller, 2000; Zipfel et al., 2006). OGs, instead, are recognized by the WALL-ASSOCIATED KINASE1 (WAK1), a receptor kinase containing epidermal growth factor-like repeats (Brutus et al., 2010).Activation of PRRs leads to a prompt induction of ion fluxes, an oxidative burst, and defense gene expression and to late responses such as callose deposition, seedling growth inhibition, and protection against further pathogen attack. An overlap but also some distinctive features exist between responses induced by PAMPs and DAMPs. For example, flg22 and OGs generate an extracellular oxidative burst mediated by RESPIRATORY BURST OXIDASE HOMOLOG D (RbohD) and induce protection against the necrotrophic fungus Botrytis cinerea independently of the ethylene, jasmonic acid, and salicylic acid pathways and of the RbohD-mediated production of reactive oxygen species (ROS; Zhang et al., 2007; Galletti et al., 2008). The inhibition of auxin responses is another feature shared by PAMPs and DAMPs (Savatin et al., 2011); in the case of OGs, the inhibition of auxin responses has been described as a true antagonism (Branca et al., 1988; Bellincampi et al., 1993; Savatin et al., 2011). On the other hand, microarray analyses indicate that late responses to the two classes of elicitors are considerably different (Denoux et al., 2008).In plants, as in animals, immunity is activated through complex and cross-talking transduction pathways that include a mitogen-activated protein (MAP) kinase (MAPK) phosphorylation cascade (Rodriguez et al., 2010). A MAPK cascade consists of a core module of three kinases that perform sequential phosphorylation reactions: a MAP kinase kinase kinase (MAP3K) activates, by phosphorylation, a MAP kinase kinase (MAP2K), which activates a MAPK. Sixty MAP3Ks, 10 MAP2Ks, and 20 MAPKs are encoded by the Arabidopsis genome (Ichimura et al., 2002), leading to a complexity that hampers the characterization of this transduction system. Three immune-related MAPK modules have been identified. A module comprising the MAP3K MEKK1, the MAP2Ks MKK1 and MKK2, and the MAPK MPK4 (MEKK1/MKK1-MKK2/MPK4) negatively controls defense responses (Kong et al., 2012; Rasmussen et al., 2012) by negatively regulating the expression of the MAP3K MEKK2, which triggers a salicylate (SA)-dependent autoimmunity response when the cascade is compromised (Berriri et al., 2012; Su et al., 2013). Two other modules, MEKK1-MAPKKKα/MKK4-MKK5/MPK3-MPK6 (Ren et al., 2008) and MKK9/MPK3-MPK6 (Xu et al., 2008), mediate the activation of defense responses, including the synthesis of ethylene and camalexin, i.e. a phytoalexin with antimicrobial activity. The only MAPK elements shown so far to participate in the response to DAMPs are the Arabidopsis MPK3 and MPK6. Both are phosphorylated within minutes upon elicitation with OGs and flg22, but only MPK6 is required for full elicitor-induced up-regulation of defense genes and protection against B. cinerea (Galletti et al., 2011).The subfamily of MAP3Ks indicated as ARABIDOPSIS NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1 (NPK1)-RELATED PROTEIN KINASEs (ANPs) includes three members, ANP1, ANP2, and ANP3, that were initially identified for their homology with the tobacco (Nicotiana tabacum) NPK1 (Jouannic et al., 1999; Sasabe and Machida, 2012). NPK1 regulates cytokinesis (Nakashima et al., 1998) as well as the effector-triggered immunity and development in Nicotiana benthamiana (Jin et al., 2002). ANPs have also been reported to be involved in the inhibition of auxin-induced gene expression. Constitutively active forms of ANPs, obtained by deletion of the C-terminal regulatory domain (ΔANPs) and expressed in protoplasts, negatively regulate the activity of the auxin-inducible soybean (Glycine max) GRETCHEN HAGEN3 promoter and activate the hydrogen peroxide signaling pathway (Kovtun et al., 2000). Therefore, ANPs have been proposed as a molecular link between oxidative stress and auxin signal transduction. However, we have previously shown that double knockout (KO) anp mutants exhibit a normal auxin/OG antagonism (Savatin et al., 2011), thus providing no support to this conclusion. Our work left unanswered whether this was due to the presence of a functional third ANP member or to a lack of involvement of ANPs in elicitor/auxin antagonism and, in general, in the response to OGs, because a main role of ANP1 in response to flg22 had been previously ruled out (Asai et al., 2002).The anp2 anp3 double mutant displays developmental defects related to cytokinesis as well as up-regulation of stress-related genes, while an anp triple KO mutant was never obtained, probably because ANPs are essential for plant development (Krysan et al., 2002). The phenotype of the anp2 anp3 mutant is similar to that of mpk4 mutant, suggesting that ANPs and MPK4 may be part of the same transduction pathway and act as negative regulators of defense responses (Beck et al., 2011).We show here that ANP genes are not involved in elicitor-auxin antagonism but are required for DAMP and PAMP signal transduction. Single and double mutants as well as conditional triple mutants, which were generated in this work, are defective in defense responses to both OGs and elf18. Notably, ANPs are required for both elicitor-induced generation of ROS and response to ROS. Our study points to ANPs as key regulators of plant immunity.     

The Arabidopsis Ethylene/Jasmonic Acid-NRT Signaling Module Coordinates Nitrate Reallocation and the Trade-Off between Growth and Environmental Adaptation

Stresses decouple nitrate assimilation and photosynthesis through stress-initiated nitrate allocation to roots (SINAR), which is mediated by the nitrate transporters NRT1.8 and NRT1.5 and functions to promote stress tolerance. However, how SINAR communicates with the environment remains unknown. Here, we present biochemical and genetic evidence demonstrating that in Arabidopsis thaliana, ethylene (ET) and jasmonic acid (JA) affect the crosstalk between SINAR and the environment. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that ethylene response factors (ERFs), including OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59, bind to the GCC boxes in the NRT1.8 promoter region, while ETHYLENE INSENSITIVE3 (EIN3) binds to the EIN3 binding site motifs in the NRT1.5 promoter. Genetic assays showed that cadmium and sodium stresses initiated ET/JA signaling, which converged at EIN3/EIN3-Like1 (EIL1) to modulate ERF expression and hence to upregulate NRT1.8. By contrast, ET and JA signaling mediated the downregulation of NRT1.5 via EIN3/EIL1 and other, unknown component(s). SINAR enhanced stress tolerance and decreased plant growth under nonstressed conditions through the ET/JA-NRT1.5/NRT1.8 signaling module. Interestingly, when nitrate reductase was impaired, SINAR failed to affect either stress tolerance or plant growth. These data suggest that SINAR responds to environmental conditions through the ET/JA-NRT signaling module, which further modulates stress tolerance and plant growth in a nitrate reductase-dependent manner.     

Focus Issue on Calcium Signaling: Recent Advances in Calcium/Calmodulin-Mediated Signaling with an Emphasis on Plant-Microbe Interactions

Calcium/calmodulin-mediated signaling contributes in diverse roles in plant growth, development, and response to environmental stimuli.During calcium (Ca2+) signaling, decoding the stimulus-response coupling involves a set of Ca2+ sensor proteins or Ca2+-binding proteins (DeFalco et al., 2010a; Kudla et al., 2010). These proteins usually possess one or more classical helix-loop-helix elongation factor (EF) hand motifs. Three major types of Ca2+-sensor proteins in plants are calmodulin (CaM)/CaM-like proteins, calcium-dependent protein kinases (CDPKs), and calcineurin B-like proteins. As compared with animals, plant genomes encode more diversified Ca2+ sensors; with the exception of canonic CaM, all other types of Ca2+ sensors (CaM-like proteins, CDPKs, and calcineurin B-like proteins) are plant specific. The large population and unique structural composition of Ca2+-binding proteins and the diversity of the target proteins regulated by the Ca2+ sensors reflect the complexity of Ca2+ signaling, which helps plants adapt to the changing environment. This update will be limited primarily to discussions on CaM and CaM-binding proteins and the recent advances in Ca2+/CaM-mediated signaling.CaM is a conserved Ca2+-binding protein found in all eukaryotes. The discovery of CaM can be traced back to the 1970s. An activator of cyclic nucleotide phosphodiesterase was shown to be involved in the regulation of cAMP concentration, which was stimulated by Ca2+ (Kakiuchi and Yamazaki, 1970; Cheung, 1971). The activator was found to bind Ca2+ and was eventually named “calmodulin,” an abbreviation of Ca2+-modulated protein. Since its discovery over 40 years ago, CaM has been regarded as a model Ca2+-binding protein and has been subjected to intensive studies in biochemistry, cell biology, and molecular biology because of its importance in almost all aspects of cellular regulation (Poovaiah and Reddy, 1987, 1993; Bouche et al., 2005; DeFalco et al., 2010a; Du et al., 2011; Reddy et al., 2011b). Disruption or depletion of the single copy of the CaM gene in yeast (Saccharomyces cerevisiae) results in a recessive lethal mutation (Davis et al., 1986), suggesting that CaM has a critical role in eukaryotic cells.The structure of CaM has been well studied, and the prototype of CaM found in all eukaryotes has 149 amino acids with two globular domains, each containing two EF hands connected by a long flexible helix (Meador et al., 1993; Zhang et al., 1995; Yun et al., 2004; Ishida et al., 2009). As more and more genomes are sequenced, it is becoming clear that CaM belongs to a small gene family in plants. In the model plant Arabidopsis (Arabidopsis thaliana), seven CaM genes encode for four highly conserved isoforms (CaM1/4, CaM2/3/5, CaM6, and CaM7) that differ in only one to five amino acid residues. Loss-of-function mutations of individual CaMs indicate that the different CaMs may have overlapping yet different functions. For example, a loss of function in Arabidopsis AtCaM2 affects pollen germination (Landoni et al., 2010). Phenotypic analysis showed that in normal growth conditions, atcam2-2 plants were indistinguishable from the wild type, while genetic analysis showed a reduced transmission of the atcam2-2 allele through the male gametophyte, and in vitro pollen germination revealed a reduced level of germination in comparison with the wild type. However, the atcam3 knockout mutant showed a clear reduction in thermotolerance after heat treatment at 45°C for 50 min (Zhang et al., 2009). Overexpression of AtCaM3 in either the atcam3 knockout or wild-type background significantly rescued or increased the thermotolerance, respectively. Further analysis of individual CaM mutants under different stress conditions should reveal more on the functional significance of individual CaM genes.