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The Ca(2+) ion is recognized as a crucial second messenger in signaling pathways coupling the perception of environmental stimuli to plant adaptive responses. Indeed, one of the earliest events following the perception of environmental changes (temperature, salt stress, drought, pathogen, or herbivore attack) is intracellular variation of free calcium concentrations. These calcium variations differ in their spatio-temporal characteristics (subcellular location, amplitude, kinetics) with the nature and strength of the stimulus and, for this reason, they are considered as signatures encrypting information from the initial stimulus. This information is believed to drive a specific response by decoding via calcium-binding proteins. Based on recent examples, we illustrate how individual calcium sensors from the calcium-dependent protein kinase and calmodulin-like protein families can integrate inputs from various environmental changes. Focusing on members of these two families, shown to be involved in plant responses to both abiotic and biotic stimuli, we discuss their role as key hubs and we put forward hypotheses explaining how they can drive the signaling pathways toward the appropriate plant responses.

MAPK phosphatases (MKPs) are negative regulators of MAPKs in eukaryotes and play key roles in the regulation of different cellular processes. However in plants, little is known about the regulation of these Dual Specific Phosphatases (DSPs) by Ca(2+) and calmodulin (CaM). Here, we showed that the wheat MKP (TMKP1) harboring a calmodulin (CaM) binding domain, binds to CaM in a Ca(2+)-dependent manner. In addition, TMKP1 exhibited a phosphatase activity in vitro that is specifically enhanced by Mn(2+) and to a lesser extent by Mg(2+), but without any synergistic effect between the two bivalent cations. Most interestingly, CaM/Ca(2+) complex inhibits the catalytic activity of TMKP1 in a CaM-dose dependent manner. However, in the presence of Mn(2+) this activity is enhanced by CaM/Ca(2+) complex. These dual regulatory effects seem to be mediated via interaction of CaM/Ca(2+) to the CaM binding domain in the C-terminal part of TMKP1. Such effects were not reported so far, and raise a possible role for CaM and Mn(2+) in the regulation of plant MKPs during cellular response to external signals., (Copyright © 2014 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.)

Calcium is a universal messenger involved in the modulation of diverse developmental and adaptive processes in response to various physiological stimuli. Ca(2+) signals are represented by stimulus-specific Ca(2+) signatures that are sensed and translated into proper cellular responses by diverse Ca(2+) binding proteins and their downstream targets. Calmodulin (CaM) and calmodulin-like (CML) proteins are primary Ca(2+) sensors that control diverse cellular functions by regulating the activity of various target proteins. Recent advances in our understanding of Ca(2+)/CaM-mediated signalling in plants have emerged from investigations into plant defence responses against various pathogens. Here, we focus on significant progress made in the identification of CaM/CML-regulated components involved in the generation of Ca(2+) signals and Ca(2+)-dependent regulation of gene expression during plant immune responses. This article is part of a Special Issue entitled: 12th European Symposium on Calcium., (Copyright © 2013 Elsevier B.V. All rights reserved.)

An increase in cellular calcium ion (Ca(2+)) concentration is now acknowledged to be one of the earliest events occurring during the induction of plant defence responses to a wide variety of pathogens. Sphingoid long-chain bases (LCBs) have also been recently demonstrated to be important mediators of defence-related programmed cell death during pathogen attack. Here, we present recent data highlighting how Ca(2+) and LCBs may be interconnected to regulate cellular processes which lead either to plant susceptibility or to resistance mechanisms. This article is part of a Special Issue entitled: 12th European Symposium on Calcium., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Many stimuli such as hormones and elicitors induce changes in intracellular calcium levels to integrate information and activate appropriate responses. The Ca(2+) signals are perceived by various Ca(2+) sensors, and calmodulin (CaM) is one of the best characterized in eukaryotes. Calmodulin-like (CML) proteins extend the Ca(2+) toolkit in plants; they share sequence similarity with the ubiquitous and highly conserved CaM but their roles at physiological and molecular levels are largely unknown. Knowledge of the contribution of Ca(2+) decoding proteins to plant immunity is emerging, and we report here data on Arabidopsis thaliana CML9, whose expression is rapidly induced by phytopathogenic bacteria, flagellin and salicylic acid. Using a reverse genetic approach, we present evidence that CML9 is involved in plant defence by modulating responses to bacterial strains of Pseudomonas syringae. Compared to wild-type plants, the later responses normally observed upon flagellin application are altered in knockout mutants and over-expressing transgenic lines. Collectively, using PAMP treatment and P. syringae strains, we have established that CML9 participates in plant innate immunity., (© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.)

Plants have evolved complex signaling networks to respond to their fluctuating environment and adapt their growth and development. Calcium-dependent signaling pathways play key role in the onset of these adaptive responses. In plant cells, the intracellular calcium transients are triggered by numerous stimuli and it is supposed that the large repertory of calcium sensors present in higher plants could contribute to integrate these signals in physiological responses. Here, we present data on CML9, a calmodulin-like protein that appears to be involved in plant responses to both biotic and abiotic stress. Using a reverse genetic approach based on gain and loss of function mutants, we present here data indicating that this CML might also be involved in root growth control in response to the flagellin, a pathogen-associated molecular pattern (PAMP) also involved in plant immunity.

Calcium is a key second messenger in signaling pathways associated with developmental and adaptive processes in plants. Stimulus-specific calcium signals, considered as calcium signatures, are translated into appropriate cellular responses through the action of various calcium-binding proteins and downstream effectors. We review here recent progress made in calcium signaling in the nucleus of plant cell. Experimental evidences show that nuclei can generate calcium signals on their own and point out the importance of calcium in the regulation of gene transcription. Future directions are given concerning the need to elucidate the mechanisms involved in the regulation of nuclear calcium homeostasis, the conversion of calcium signals into transcriptional responses or other fundamental downstream nuclear functions. Overall, a better understanding of nuclear signaling will be useful to get an integrated picture of the signaling network of the plant cell.

Calmodulin (CaM) is a primary calcium sensor in all eukaryotes. It binds calcium and regulates the activity of a wide range of effector proteins in response to calcium signals. The list of CaM targets includes plant-specific proteins whose functions are progressively being elucidated. Plants also possess numerous calmodulin-like proteins (CMLs) that appear to have evolved unique functions. Functional studies of CaM and CMLs in plants highlight the importance of this protein family in the regulation of plant development and stress responses by converting calcium signals into transcriptional responses, protein phosphorylation or metabolic changes. This review summarizes some of the significant progress made by biochemical and genetic studies in identifying the properties and physiological functions of plant CaMs and CMLs. We discuss emerging paradigms in the field and highlight the areas that need further investigation., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

The RD20 gene encodes a member of the caleosin family, which is primarily known to function in the mobilization of seed storage lipids during germination. In contrast to other caleosins, RD20 expression is early-induced by water deficit conditions and we recently provided genetic evidence for its positive role in drought tolerance in Arabidopsis. RD20 is also responsive to pathogen infection and is constitutively expressed in diverse tissues and organs during development suggesting additional roles for this caleosin. This addendum describes further exploration of phenotypic alterations in T-DNA insertional rd20 mutant and knock-out complemented transgenic plants in the context of early development and susceptibility to a phytopathogenic bacteria. We show that the RD20 gene is involved in ABA-mediated inhibition of germination and does not play a significant role in plant defense against Pseudomonas syringae.

Plants overcome water deficit conditions by combining molecular, biochemical and morphological changes. At the molecular level, many stress-responsive genes have been isolated, but knowledge of their physiological functions remains fragmentary. Here, we report data for RD20, a stress-inducible Arabidopsis gene that belongs to the caleosin family. As for other caleosins, we showed that RD20 localized to oil bodies. Although caleosins are thought to play a role in the degradation of lipids during seed germination, induction of RD20 by dehydration, salt stress and ABA suggests that RD20 might be involved in processes other than germination. Using plants carrying the promoter RD20::uidA construct, we show that RD20 is expressed in leaves, guard cells and flowers, but not in root or in mature seeds. Water deficit triggers a transient increase in RD20 expression in leaves that appeared predominantly dependent on ABA signaling. To assess the biological significance of these data, a functional analysis using rd20 knock-out and overexpressing complemented lines cultivated either in standard or in water deficit conditions was performed. The rd20 knock-out plants present a higher transpiration rate that correlates with enhanced stomatal opening and a reduced tolerance to drought as compared with the wild type. These results support a role for RD20 in drought tolerance through stomatal control under water deficit conditions.

Calmodulin (CaM) plays a crucial role in the regulation of diverse cellular processes by modulating the activities of numerous target proteins. Plants possess an extended CaM family including numerous CaM-like proteins (CMLs), most of which appear to be unique to plants. We previously demonstrated a role for CML9 in abiotic stress tolerance and seed germination in Arabidopsis thaliana. We report here the isolation of PRR2, a pseudo-response regulator as a CML9 interacting protein by screening an expression library prepared from Arabidopsis seedlings with CML9 as bait in a yeast two-hybrid system. PRR2 is similar to the response regulators of the two-component system, but lacks the invariant residue required for phosphorylation by which response regulators switch their output response, suggesting the existence of alternative regulatory mechanisms. PRR2 was found to bind CML9 and closely related CMLs but not a canonical CaM. Mapping analyses indicate that an almost complete form of PRR2 is required for interaction with CML9, suggesting a recognition mode different from the classical CaM-target peptide complex. PRR2 contains several features that are typical of transcription factors, including a GARP DNA recognition domain, a Pro-rich region and a Golden C-terminal box. PRR2 and CML9 as fusion proteins with fluorescent tags co-localized in the nucleus of plant cells, and their interaction in the nuclear compartment was validated in planta by using a fluorophore-tagged protein interaction assay. These findings suggest that binding of PRR2 to CML9 may be an important mechanism to modulate the physiological role of this transcription factor in plants., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Many stimuli, such as hormones and abiotic stress factors, elicit changes in intracellular calcium levels that serve to convey information and activate appropriate responses. The Ca2+ signals are perceived by different Ca2+ receptors, and calmodulin (CaM) is one of the best-characterized Ca2+ sensors in eukaryotes. Calmodulin-like (CML) proteins also exist in plants; they share sequence similarity with the ubiquitous and highly conserved CaM, but their roles at the physiological and molecular levels are largely unknown. We present data on Arabidopsis thaliana CML9 (AtCML9) that exhibits 46% amino acid sequence identity with CaM. AtCML9 transcripts are found in all major organs, and a putative AtCML9 regulatory region confers reporter gene expression at various sites, including root apex, stomata, hydathodes and trichomes. AtCML9 expression is rapidly induced by abiotic stress and abscisic acid (ABA) in young seedlings, and by using cml9 knock-out mutants we present evidence that AtCML9 plays essential roles in modulating responses to salt stress and ABA. Seed germination and seedling growth for the mutant lines present a hypersensitive response to ABA that could be correlated with enhanced tolerance to salt stress and water deficit. Mutations of the AtCML9 gene also alter the expression of several stress-regulated genes, suggesting that AtCML9 is involved in salt stress tolerance through its effects on the ABA-mediated pathways.

The calmodulin (CaM) family is a major class of calcium sensor proteins which collectively play a crucial role in cellular signaling cascades through the regulation of numerous target proteins. Although CaM is one of the most conserved proteins in all eukaryotes, several features of CaM and its downstream effector proteins are unique to plants. The continuously growing repertoire of CaM-binding proteins includes several plant-specific proteins. Plants also possess a particular set of CaM isoforms and CaM-like proteins (CMLs) whose functions have just begun to be elucidated. This review summarizes recent insights that help to understand the role of this multigene family in plant development and adaptation to environmental stimuli.

A cDNA clone (LeCRK1), encoding a novel isoform of calcium-dependent protein kinase (CDPK), was isolated by screening a tomato (Lycopersicon esculentum) cDNA library. The protein derived from the full-length sequence indicated that it belongs to the family of CDPK-related kinases (CRKs) and the predicted amino acid sequence shows a modular organization of the protein consisting of different characteristic domains. The kinase domain of LeCRK1 shares a high degree of similarity with the catalytic domain of CDPKs. In contrast to canonical members of the family, LeCRK1 has a degenerate sequence in the C-terminal calmodulin-like domain. LeCRK1 protein was shown to be a functional kinase, but, consistent with the lack of calcium-binding activity, its autophosphorylation activity did not require calcium. LeCRK1 harbours an amphiphilic amino acid region revealed to be a functional calmodulin-binding site by in vitro assay. A putative myristoylation/palmitoylation sequence has been identified at the N-terminus. Expressing an LeCRK1::GFP fusion protein in the protoplast resulted in its targeting to the plasma membrane. Site-directed mutagenesis of critical amino acids of the myristoylation/palmitoylation consensus sites led to the accumulation of the mutated protein in the cytoplasm, suggesting that the native protein is anchored to the plasma membrane by acylated residues. Expression studies revealed significant accumulation of LeCRK1 transcripts during fruit ripening, although transcripts were also detected in stem, leaf, and flower. LeCRK1 mRNA level in leaves was slightly induced by ethylene and salicylic acid, and upon mechanical wounding and cold treatment. It is noteworthy that LeCRK1 mRNAs were undetectable in different tomato-ripening natural mutants such as NR, Rin, and Nor, suggesting a role in the ripening process.

A clone for a novel Arabidopsisthaliana calmodulin (CaM)-binding protein of 25 kDa (AtCaMBP25) has been isolated by using a radiolabelled CaM probe to screen a cDNA expression library derived from A. thaliana cell suspension cultures challenged with osmotic stress. The deduced amino acid sequence of AtCaMBP25 contains putative nuclear localization sequences and shares significant degree of similarity with hypothetical plant proteins only. Fusion of the AtCaMBP25 coding sequence to reporter genes targets the hybrid protein to the nucleus. Bacterially expressed AtCaMBP25 binds, in a calcium-dependent manner, to a canonical CaM but not to a less conserved isoform of the calcium sensor. AtCaMBP25 is encoded by a single-copy gene, whose expression is induced in Arabidopsis seedlings exposed to dehydration, low temperature or high salinity. Transgenic plants overexpressing AtCaMBP25 exhibits an increased sensitivity to both ionic (NaCl) and non-ionic (mannitol) osmotic stress during seed germination and seedling growth. By contrast, transgenic lines expressing antisense AtCaMBP25 are significantly more tolerant to mannitol and NaCl stresses than the wild type. Thus, the AtCaMBP25 gene functions as a negative effector of osmotic stress tolerance and likely participates in stress signal transduction pathways.

Screening a cDNA expression library with a radiolabelled calmodulin (CaM) probe led to the isolation of AtCaMRLK, a receptor-like kinase (RLK) of Arabidopsis thaliana. AtCaMRLK polypeptide sequence shows a modular organization consisting of the four distinctive domains characteristic of receptor kinases: an amino terminal signal sequence, a domain containing seven leucine-rich repeats, a single putative membrane-spanning segment and a protein kinase domain. Using truncated versions of the protein and a synthetic peptide, we demonstrated that a region of 23 amino acids, located near the kinase domain of AtCaMRLK, binds CaM in a calcium-dependent manner. Real-time binding experiments showed that AtCaMRLK interacted in vitro with AtCaM1, a canonical CaM, but not with AtCaM8, a divergent isoform of the Ca2+ sensor. The bacterially expressed kinase domain of the protein was able to autophosphorylate and to phosphorylate the myelin basic protein, using Mn2+ preferentially to Mg2+ as an ion activator. Site-directed mutagenesis of the conserved lysine residue (Lys423) to alanine, in the kinase subdomain II, resulted in a complete loss of kinase activity. CaM had no influence on the autophosphorylation activity of AtCaMRLK. AtCaMRLK was expressed in reproductive and vegetative tissues of A. thaliana, except in leaves. Disruption in the AtCaMRLK coding sequence by insertion of a DsG transposable element in an Arabidopsis mutant did not generate a discernible phenotype. The CaM-binding motif of AtCaMRLK was found to be conserved in several other members of the plant RLK family, suggesting a role for Ca2+/CaM in the regulation of RLK-mediated pathways.

Activation of the tobacco gene hsr203 is rapid, highly localized, specific for incompatible plant-pathogen interactions, and strongly correlated with programmed cell death occurring in response to diverse pathogens. Functional characterization of hsr203 gene product has shown that HSR203 is a serine hydrolase that displays esterase activity. We show here that transgenic tobacco plants deficient in HSR203 protein exhibit an accelerated hypersensitive response when inoculated with an avirulent strain of Ralstonia solanacearum. This response was accompanied by a maximal level of cell death and a drastic inhibition of in planta bacterial growth. Transgenic plants deficient in HSR203 were also found to show increased resistance in a dosage-dependent manner to Pseudomonas syringae pv. pisi, another avirulent bacterial pathogen, and to virulent and avirulent races of Phytophthora parasitica, a fungal pathogen of tobacco, but not to different virulent bacteria. Surprisingly, expression of another hsr gene, hsr515, and that of the defence genes PR1-a and PR5, was strongly reduced in the transgenic lines. Our results suggest that hsr203 antisense suppression in tobacco can have pleiotropic effects on HR cell death and defence mechanisms, and induces increased resistance to different pathogens.

We have recently reported the isolation of a tobacco gene, hsr 203J, whose transcripts accumulate during the hypersensitive reaction, a plant response associated with resistance to pathogens. We present and discuss here some structural and biochemical properties of the gene product. Nucleotide sequence analysis has shown that the hsr 203J gene contains an open reading frame coding for a polypeptide of 335 amino acids. The predicted amino acid sequence contains the GXSXG motif characteristic of serine hydrolases, and displays limited but significant similarity to lipases and esterases of prokaryotic origin. The hsr 203J gene was expressed in Escherichia coli, and the recombinant protein, purified to near homogeneity, was able to degrade p-nitrophenylbutyrate, a general substrate for carboxylesterases. The enzyme was unable to hydrolyze lipids, and was active on short-chain acyl esters only. The hydrolytic activity was abolished by diisopropyl fluorophosphate and a derivative of isocoumarin, as expected for a member of the serine hydrolase family. Sequence similarities between the tobacco esterase and expressed sequence tags in databases suggest the existence of members of this enzyme family in various plant species.

A bean cDNA clone that specifies a 70 kDa heat-shock protein (hsp70) has been isolated and sequenced. The nucleotide sequence analysis shows that the cDNA could encode a 72 kDa protein that is highly related to prokaryotic and mitochondrial members of the hsp70 family. The predicted protein was found to contain an amino-terminal extension typical of transit sequences. The in vitro transcription/translation product of the cDNA behaved as a 72 kDa polypeptide as predicted from the longest open reading frame. This polypeptide could be imported into isolated mitochondria and recovered as a 68 kDa product. The imported protein is identical in size to a mitochondrial protein that cross-reacts with hsp70-specific antibodies. The import data and Western blot analysis suggest that the cDNA clone encodes a mitochondrial member of the hsp70 family. Electrophoretic and immunoblot analysis reveal that the protein is loosely associated to the mitochondrial envelope and also exists as discrete soluble protein aggregates of about 270 and 420 kDa. Hsp70 of bean mitochondria can be in vitro phosphorylated on threonine residues in a calcium-dependent manner, and the modified protein was detected as an oligomer of about 160 kDa only. The data are discussed with respect to the chaperone function of hsp70 in mitochondria.

Crystals of the hexadecameric form of ribulose-bisphosphate carboxylase used to solve the structure of the enzyme are composed of protein substantially crosslinked by a disulfide bond between pairs of large subunits. Conditions leading to the selective formation of dimers of the large subunits are described. The stability and specificity of the intra-dimeric crosslink was used to confirm that only one cysteine residue, Cys247 of neighboring large subunits, is involved in the bridge. The ability to generate this disulfide selectively, or alternatively replace the cysteine by site-directed mutagenesis, has led us to conclude that there is no effect of these changes on any of the critical kinetic parameters of the enzyme. The benign effect of the oxidation indicates that the crystal structures of the ribulose-bisphosphate carboxylase, particularly of the active site, are a true representation of the native enzyme.

Ribulose-1,5-bisphosphate carboxylase/oxygelase (RuBPCase) was studied in melon leaves infected by Colletotrichum lagenarium, a fungal pathogen of melons. Electrophoretic analysis of melon leaf proteins indicated a strong effect of infection on RuBPCase, the subunits of which gradually disappeared during the different stages of infection. Enzyme activity also declined 4 d after inoculation and its content, measured by immunoelectrophoresis, decreased to a similar extent. Synthesis of the large and small subunits of RuBPCase was followed by in-vivo pulse-labeling experiments. A drastic decrease in the rate of RuBPCase-subunit synthesis occurred 3 d after inoculation and preceded the appearance of disease symptoms. There was an apparent coordination of the synthesis of the two subunits under these conditions.