The <Emphasis Type="Italic">Arabidopsis</Emphasis> calmodulin-like proteins AtCML30 and AtCML3 are targeted to mitochondria and peroxisomes,respectively

Calmodulin (CaM) is a ubiquitous sensor/transducer of calcium signals in eukaryotic organisms. While CaM mediated calcium regulation of cytosolic processes is well established, there is growing evidence for the inclusion of organelles such as chloroplasts, mitochondria and peroxisomes into the calcium/calmodulin regulation network. A number of CaM-binding proteins have been identified in these organelles and processes such as protein import into chloroplasts and mitochondria have been shown to be governed by CaM regulation. What have been missing to date are the mediators of this regulation since no CaM or calmodulin-like protein (CML) has been identified in any of these organelles. Here we show that two Arabidopsis CMLs, AtCML3 and AtCML30, are localized in peroxisomes and mitochondria, respectively. AtCML3 is targeted via an unusual C-terminal PTS1-like tripeptide while AtCML30 utilizes an N-terminal, non-cleavable transit peptide. Both proteins possess the typical structure of CaMs, with two pairs of EF-hand motifs separated by a short linker domain. They furthermore display common characteristics, such as calcium-dependent alteration of gel mobility and calcium-dependent exposure of a hydrophobic surface. This indicates that they can function in a similar manner as canonical CaMs. The presence of close homologues to AtCML3 and AtCML30 in other plants further indicates that organellar targeting of these CMLs is not a specific feature of Arabidopsis. The identification of peroxisomal and mitochondrial CMLs is an important step in the understanding how these organelles are integrated into the cellular calcium/calmodulin signaling pathways.     

CML9, an Arabidopsis calmodulin-like protein, contributes to plant innate immunity through a flagellin-dependent signalling pathway

Many stimuli such as hormones and elicitors induce changes in intracellular calcium levels to integrate information and activate appropriate responses. The Ca²⁺ signals are perceived by various Ca²⁺ sensors, and calmodulin (CaM) is one of the best characterized in eukaryotes. Calmodulin‐like (CML) proteins extend the Ca²⁺ 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 Ca²⁺ 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.     

Calmodulin-like Proteins from <Emphasis Type="Italic">Arabidopsis</Emphasis> and Tomato are Involved in Host Defense Against <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">tomato</Emphasis>

Complex signal transduction pathways underlie the myriad plant responses to attack by pathogens. Ca²⁺ is a universal second messenger in eukaryotes that modulates various signal transduction pathways through stimulus-specific changes in its intracellular concentration. Ca²⁺-binding proteins such as calmodulin (CaM) detect Ca²⁺ signals and regulate downstream targets as part of a coordinated cellular response to a given stimulus. Here we report the characterization of a tomato gene (APR134) encoding a CaM-related protein that is induced in disease-resistant leaves in response to attack by Pseudomonas syringae pv. tomato. We show that suppression of APR134 gene expression in tomato (Solanum lycopersicum), using virus-induced gene silencing (VIGS), compromises the plant’s immune response. We isolated APR134-like genes from Arabidopsis, termed CML42 and CML43, to investigate whether they serve a functionally similar role. Gene expression analysis revealed that CML43 is rapidly induced in disease-resistant Arabidopsis leaves following inoculation with Pseudomonas syringae pv. tomato. Overexpression of CML43 in Arabidopsis accelerated the hypersensitive response. Recombinant APR134, CML42, and CML43 proteins all bind Ca²⁺ in vitro. Collectively, our data support a role for CML43, and APR134 as important mediators of Ca²⁺-dependent signals during the plant immune response to bacterial pathogens. This work was supported by a research grant (WAS) and postgraduate scholarships (DC, SLD) from the Natural Science and Engineering Research Council of Canada, the National Science Foundation (IBN-0109633; GBM), and the Swedish Research Council (SKE).     

拟南芥钙调素定点突变基因分离及其在钙不依赖钙调素结合蛋白检测中的应用

动植物系统研究表明,钙调素不仅在结合钙离子时调节多种靶酶或靶蛋白的活性,而且没有钙离子结合时,还可以通过结合钙不依赖的钙调素结合蛋白,发挥多种生物学作用．然而,目前却没有体内分析钙调素与钙不依赖钙调素结合蛋白相互作用的方法．首先,采用定点突变的方式,得到了拟南芥钙调素亚型2的多个突变基因mCaM2,随后,大肠杆菌重组表达突变蛋白的电泳迁移率及⁴⁵Ca²⁺覆盖分析表明,得到了编码失去钙结合能力的钙调素的突变基因mCaM2₁₂₃₄, mCaM2₁₂₃₄突变钙调素中所有4个钙结合EF-hand结构域中的关键氨基酸谷氨酸均突变为谷氨酰胺．在酵母双杂交体系中,作为诱饵蛋白的突变钙调素mCaM2₁₂₃₄与我们前期体外方法报道的钙不依赖性钙调素结合蛋白AtIQD26存在相互作用．这将为钙不依赖性钙调素结合蛋白提供有用的体内研究工具,有利于我们全面认识钙-钙调素-钙调素结合蛋白信号途径．     

A novel rice calmodulin-like gene, <Emphasis Type="Italic">OsMSR2,</Emphasis> enhances drought and salt tolerance and increases ABA sensitivity in Arabidopsis

Many abiotic stimuli, such as drought and salt stresses, elicit changes in intracellular calcium levels that serve to convey information and activate adaptive responses. Ca²⁺ signals are perceived by different Ca²⁺ sensors, and calmodulin (CaM) is one of the best-characterized Ca²⁺ sensors in eukaryotes. Calmodulin-like (CML) proteins also exist in plants, but their functions at the physiological and molecular levels are largely unknown. In this report, we present data on OsMSR2 (Oryza sativa L. Multi-Stress-Responsive gene 2), a novel calmodulin-like protein gene isolated from rice Pei’ai 64S (Oryza sativa L.). Expression of OsMSR2 was strongly up-regulated by a wide spectrum of stresses, including cold, drought, and heat in different tissues at different developmental stages of rice, as revealed by both microarray and quantitative real-time RT-PCR analyses. Analysis of the recombinant OsMSR2 protein demonstrated its potential ability to bind Ca²⁺ in vitro. Expression of OsMSR2 conferred enhanced tolerance to high salt and drought in Arabidopsis (Arabidopsis thaliana) accompanied by altered expression of stress/ABA-responsive genes. Transgenic plants also exhibited hypersensitivity to ABA during the seed germination and post-germination stages. The results suggest that expression of OsMSR2 modulated salt and drought tolerance in Arabidopsis through ABA-mediated pathways.     

Genome-wide identification and analyses of the rice calmodulin and related potential calcium sensor proteins

Background  

A wide range of stimuli evoke rapid and transient increases in [Ca²⁺]_cyt in plant cells which are transmitted by protein sensors that contain EF-hand motifs. Here, a group of Oryza sativa L. genes encoding calmodulin (CaM) and CaM-like (CML) proteins that do not possess functional domains other than the Ca²⁺-binding EF-hand motifs was analyzed.     

Calmodulin isoforms in Arabidopsis encoded by multiple divergent mRNAs

Three new, unique cDNA sequences encoding isoforms of calmodulin (CaM) were isolated from an Arabidopsis cDNA library cloned in gt10. These sequences (ACaM-4, -5, and -6) represent members of the Arabidopsis CaM gene family distinct from the three DNA sequences previously reported. ACaM-4 and -6 encode full-length copies of CaM mRNAs of ca. 0.75 kb. The ACaM-5 sequence encodes a partial length copy of CaM mRNA that is lacking sequences encoding the amino-terminal 10 amino acids of mature CaM and the initiator methionine. The derived amino acid sequence of ACaM-5 is identical to the sequences encoded by two of the previously characterized ACaM cDNAs, and is identical to TCH-1 mRNA, whose accumulation was increased by touch stimulation. The polypeptides encoded by ACaM-4 and -6 differ from that encoded by ACaM-5 by six and two amino acid substititions, respectively. Most of the deduced amino acid sequence substitutions in the Arabidopsis CaM isoforms occurred in the fourth Ca²⁺-binding domain. Polymerase chain reaction amplification assays of ACaM-4, -5 and -6 mRNA sequences indicated that each accumulated in Arabidopsis leaf RNA fractions, but only ACaM-4 and -5 mRNAs were detected in silique total RNA. The six different CaM cDNA sequences each hybridize with unique Eco RI restriction fragments in genomic Southern blots of Arabidopsis DNA, indicating that these sequences were derived from distinct structural genes. Our results suggest that CaM isoforms in Arabidopsis may have evolved to optimize the interaction of this Ca²⁺-receptor protein with specific subsets of response elements.     

Characterization of a pathogen-induced calmodulin-binding protein: mapping of four Ca2+-dependent calmodulin-binding domains

Ca²⁺ and calmodulin (CaM), a key Ca²⁺ sensor in all eukaryotes, have been implicated in defense responses in plants. To elucidate the role of Ca²⁺ and CaM in defense signaling, we used ³⁵S-labeled CaM to screen expression libraries prepared from tissues that were either treated with an elicitor derived from Phytophthora megasperma or infected with Pseudomonas syringae pv. tabaci. Nineteen cDNAs that encode the same protein, pathogen-induced CaM-binding protein (PICBP), were isolated. The PICBP fusion proteins bound ³⁵S-CaM, horseradish peroxidase-labeled CaM and CaM-Sepharose in the presence of Ca²⁺ whereas EGTA, a Ca²⁺ chelator, abolished binding, confirming that PICBP binds CaM in a Ca²⁺-dependent manner. Using a series of bacterially expressed truncated versions of PICBP, four CaM-binding domains, with a potential CaM-binding consensus sequence of WSNLKKVILLKRFVKSL, were identified. The deduced PICBP protein sequence is rich in leucine residues and contains three classes of repeats. The PICBP gene is differentially expressed in tissues with the highest expression in stem. The expression of PICBP in Arabidopsis was induced in response to avirulent Pseudomonas syringae pv. tomato carrying avrRpm1. Furthermore, PICBP is constitutively expressed in the Arabidopsis accelerated cell death2-2 mutant. The expression of PICBP in bean leaves was also induced after inoculation with avirulent and non-pathogenic bacterial strains. In addition, the hrp1 mutant of Pseudomonas syringae pv. tabaci and inducers of plant defense such as salicylic acid, hydrogen peroxide and a fungal elicitor induced PICBP expression in bean. Our data suggest a role for PICBP in Ca²⁺-mediated defense signaling and cell-death. Furthermore, PICBP is the first identified CBP in eukaryotes with four Ca²⁺-dependent CaM-binding domains.     

Arabidopsis Calmodulin-binding Protein IQ67-Domain 1 Localizes to Microtubules and Interacts with Kinesin Light Chain-related Protein-1

Calcium (Ca²⁺) is a key second messenger in eukaryotes and regulates diverse cellular processes, most notably via calmodulin (CaM). In Arabidopsis thaliana, IQD1 (IQ67 domain 1) is the founding member of the IQD family of putative CaM targets. The 33 predicted IQD proteins share a conserved domain of 67 amino acids that is characterized by a unique arrangement of multiple CaM recruitment motifs, including so-called IQ motifs. Whereas IQD1 has been implicated in the regulation of defense metabolism, the biochemical functions of IQD proteins remain to be elucidated. In this study we show that IQD1 binds to multiple Arabidopsis CaM and CaM-like (CML) proteins in vitro and in yeast two-hybrid interaction assays. CaM overlay assays revealed moderate affinity of IQD1 to CaM2 (K_d ∼ 0.6 μm). Deletion mapping of IQD1 demonstrated the importance of the IQ67 domain for CaM2 binding in vitro, which is corroborated by interaction of the shortest IQD member, IQD20, with Arabidopsis CaM/CMLs in yeast. A genetic screen of a cDNA library identified Arabidopsis kinesin light chain-related protein-1 (KLCR1) as an IQD1 interactor. The subcellular localization of GFP-tagged IQD1 proteins to microtubules and the cell nucleus in transiently and stably transformed plant tissues (tobacco leaves and Arabidopsis seedlings) suggests direct interaction of IQD1 and KLCR1 in planta that is supported by GFP∼IQD1-dependent recruitment of RFP∼KLCR1 and RFP∼CaM2 to microtubules. Collectively, the prospect arises that IQD1 and related proteins provide Ca²⁺/CaM-regulated scaffolds for facilitating cellular transport of specific cargo along microtubular tracks via kinesin motor proteins.     

Structural Analysis of a Calmodulin Variant from Rice: THE C-TERMINAL EXTENSION OF OsCaM61 REGULATES ITS CALCIUM BINDING AND ENZYME ACTIVATION PROPERTIES*

OsCaM61 is one of five calmodulins known to be present in Oryza sativa that relays the increase of cytosolic [Ca²⁺] to downstream targets. OsCaM61 bears a unique C-terminal extension with a prenylation site. Using nuclear magnetic resonance (NMR) spectroscopy we studied the behavior of the calmodulin (CaM) domain and the C-terminal extension of OsCaM61 in the absence and presence of Ca²⁺. NMR dynamics data for OsCaM61 indicate that the two lobes of the CaM domain act together unlike the independent behavior of the lobes seen in mammalian CaM and soybean CaM4. Also, data demonstrate that the positively charged nuclear localization signal region in the tail in apo-OsCaM61 is helical, whereas it becomes flexible in the Ca²⁺-saturated protein. The extra helix in apo-OsCaM61 provides additional interactions in the C-lobe and increases the structural stability of the closed apo conformation. This leads to a decrease in the Ca²⁺ binding affinity of EF-hands III and IV in OsCaM61. In Ca²⁺-OsCaM61, the basic nuclear localization signal cluster adopts an extended conformation, exposing the C-terminal extension for prenylation or enabling OsCaM61 to be transferred to the nucleus. Moreover, Ser¹⁷² and Ala¹⁷³, residues in the tail, interact with different regions of the protein. These interactions affect the ability of OsCaM61 to activate different target proteins. Altogether, our data show that the tail is not simply a linker between the prenyl group and the protein but that it also provides a new regulatory mechanism that some plants have developed to fine-tune Ca²⁺ signaling events.     

Kinase activity and calmodulin binding are essential for growth signaling by the phytosulfokine receptor PSKR1

The cell growth‐promoting peptide phytosulfokine (PSK) is perceived by leucine‐rich repeat (LRR) receptor kinases. To elucidate PSK receptor function we analyzed PSKR1 kinase activity and binding to Ca²⁺ sensors and evaluated the contribution of these activities to growth control in planta. Ectopically expressed PSKR1 was capable of auto‐ and transphosphorylation. Replacement of a conserved lysine within the ATP‐binding region by a glutamate resulted in the inhibition of auto‐ and transphosphorylation kinase activities. Expression of the kinase‐inactive PSKR1(K762E) receptor in the pskr null background did not restore root or shoot growth. Instead, the mutant phenotype was enhanced suggesting that the inactive receptor protein exerts growth‐inhibitory activity. Bioinformatic analysis predicted a putative calmodulin (CaM)‐binding site within PSKR1 kinase subdomain VIa. Bimolecular fluorescence complementation analysis demonstrated that PSKR1 binds to all isoforms of CaM, more weakly to the CaM‐like protein CML8 but apparently not to CML9. Mutation of a conserved tryptophan (W831S) within the predicted CaM‐binding site strongly reduced CaM binding. Expression of PSKR1(W831S) in the pskr null background resulted in growth inhibition that was similar to that of the kinase‐inactive receptor. We conclude that PSK signaling requires Ca²⁺/CaM binding and kinase activity of PSKR1 in planta. We further propose that the inactivated kinase interferes with other growth‐promoting signaling pathway(s).     

Functional interaction of calmodulin with a plant cyclic nucleotide gated cation channel

A family of plant ligand gated nonselective cation channels (cngcs) can be activated by direct, and reversible binding of cyclic nucleotide. These proteins have a cytoplasm-localized cyclic nucleotide binding domain (CNBD) at the carboxy-terminus of the polypeptide. A portion of the cngc CNBD also acts as a calmodulin (CaM) binding domain (CaMBD). The objective of this work is to further characterize interaction of cyclic nucleotide and CaM in gating plant cngc currents. The three-dimensional structure of an Arabidopsis thaliana cngc (Atcngc2) CNBD was modeled, indicating cAMP binding to the Atcngc2 CNBD in a pocket formed by a β barrel structure appressing a shortened (relative to animal cngc CNBDs) αC helix. The Atcngc2 CaMBD was expressed as a fusion peptide linking blue and green fluorescent proteins, and used to quantify CaM (A. thaliana CaM isoform 4) binding. CaM bound the fusion protein in a Ca²⁺–dependent manner with a K_d of 7.6 nM and a Ca²⁺ binding K_d of 200 nM. Functional characterization (voltage clamp analysis) of Atcngc2 was undertaken by expression in human embryonic kidney cells. CaM reversed cAMP activation of Atcngc2 currents. This functional interaction was dependent on free cytosolic Ca²⁺. Increasing cytosolic Ca²⁺ was found to inhibit cAMP activation of the channel in the absence of added CaM. We conclude that the physical interaction of Ca²⁺/CaM with plant cngcs blocks cyclic nucleotide activation of these channels. Thus, the cytosolic secondary messengers CaM, cAMP, and Ca²⁺ can act in an integrated fashion to gate currents through these plant ion channels.     

The Arabidopsis calmodulin‐like protein,CML39, functions during early seedling establishment

During Ca²⁺ signal transduction, Ca²⁺‐binding proteins known as Ca²⁺ sensors function to decode stimulus‐specific Ca²⁺ signals into downstream responses. Plants possess extended families of unique Ca²⁺ sensors termed calmodulin‐like proteins (CMLs) whose cellular roles are not well understood. CML39 encodes a predicted Ca²⁺ sensor whose expression is strongly increased in response to diverse external stimuli. In the present study, we explored the biochemical properties of recombinant CML39, and used a reverse genetics approach to investigate its physiological role. Our data indicate that Ca²⁺ binding by CML39 induces a conformational change in the protein that results in an increase in exposed‐surface hydrophobicity, a property that is consistent with its predicted function as a Ca²⁺ sensor. Loss‐of‐function cml39 mutants resemble wild‐type plants under normal growth conditions but exhibit persistent arrest at the seedling stage if grown in the absence of sucrose or other metabolizable carbon sources. Under short‐day conditions, cml39 mutants display increased sucrose‐induced hypocotyl elongation. When grown in the dark, cml39 mutants show impaired hypocotyl elongation in the absence of sucrose. Promoter–reporter data indicate that CML39 expression is prominent in the apical hook in dark‐grown seedlings. Collectively, our data suggest that CML39 functions in Arabidopsis as a Ca²⁺ sensor that plays an important role in the transduction of light signals that promote seedling establishment.     

Pull-down of Calmodulin-binding Proteins

Calcium (Ca²⁺) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca²⁺ signaling is mediated through the calcium-binding protein known as calmodulin (CaM)^1,2. CaM is involved at multiple levels in almost all cellular processes, including apoptosis, metabolism, smooth muscle contraction, synaptic plasticity, nerve growth, inflammation and the immune response. A number of proteins help regulate these pathways through their interaction with CaM. Many of these interactions depend on the conformation of CaM, which is distinctly different when bound to Ca²⁺ (Ca²⁺-CaM) as opposed to its Ca²⁺-free state (ApoCaM)³.While most target proteins bind Ca²⁺-CaM, certain proteins only bind to ApoCaM. Some bind CaM through their IQ-domain, including neuromodulin⁴, neurogranin (Ng)⁵, and certain myosins⁶. These proteins have been shown to play important roles in presynaptic function⁷, postsynaptic function⁸, and muscle contraction⁹, respectively. Their ability to bind and release CaM in the absence or presence of Ca²⁺ is pivotal in their function. In contrast, many proteins only bind Ca²⁺-CaM and require this binding for their activation. Examples include myosin light chain kinase¹⁰, Ca²⁺/CaM-dependent kinases (CaMKs)¹¹ and phosphatases (e.g. calcineurin)¹², and spectrin kinase¹³, which have a variety of direct and downstream effects¹⁴.The effects of these proteins on cellular function are often dependent on their ability to bind to CaM in a Ca²⁺-dependent manner. For example, we tested the relevance of Ng-CaM binding in synaptic function and how different mutations affect this binding. We generated a GFP-tagged Ng construct with specific mutations in the IQ-domain that would change the ability of Ng to bind CaM in a Ca²⁺-dependent manner. The study of these different mutations gave us great insight into important processes involved in synaptic function^8,15. However, in such studies, it is essential to demonstrate that the mutated proteins have the expected altered binding to CaM.Here, we present a method for testing the ability of proteins to bind to CaM in the presence or absence of Ca²⁺, using CaMKII and Ng as examples. This method is a form of affinity chromatography referred to as a CaM pull-down assay. It uses CaM-Sepharose beads to test proteins that bind to CaM and the influence of Ca²⁺ on this binding. It is considerably more time efficient and requires less protein relative to column chromatography and other assays. Altogether, this provides a valuable tool to explore Ca²⁺/CaM signaling and proteins that interact with CaM.     

Reduced expressions of calmodulin genes and protein and reduced ability of calmodulin to activate plasma membrane Ca2+‐ATPase in the brain of protein undernourished rats: modulatory roles of selenium and zinc supplementation

The roles of protein undernutrition as well as selenium (Se) and zinc (Zn) supplementation on the ability of calmodulin (CaM) to activate erythrocyte ghost membrane (EGM) Ca²⁺‐ATPase and the calmodulin genes and protein expressions in rat's cortex and cerebellum were investigated. Rats on adequate protein diet and protein‐undernourished (PU) rats were fed with diet containing 16% and 5% casein, respectively, for a period of 10 weeks. The rats were then supplemented with Se and Zn at a concentration of 0.15 and 227 mg l⁻¹, respectively, in drinking water for 3 weeks. The results obtained from the study showed significant reductions in synaptosomal plasma membrane Ca²⁺‐ATPase (PMCA) activity, Ca²⁺/CaM activated EGM Ca²⁺ATPase activity and calmodulin genes and protein expressions in PU rats. Se or Zn supplementation improved the ability of Ca²⁺/CaM to activate EGM Ca²⁺‐ATPase and protein expressions. Se or Zn supplementation improved gene expression in the cerebellum but not in the cortex. Also, the activity of PMCA was significantly improved by Zn. In conclusion, it is postulated that Se and Zn might be beneficial antioxidants in protecting against neuronal dysfunction resulting from reduced level of calmodulin such as present in protein undernutrition. Copyright © 2016 John Wiley & Sons, Ltd.     

The emerging function of IQD proteins as scaffolds in cellular signaling and trafficking

Calcium (Ca²⁺) signaling modules are essential for adjusting plant growth and performance to environmental constraints. Differential interactions between sensors of Ca²⁺ dynamics and their molecular targets are at the center of the transduction process. Calmodulin (CaM) and CaM-like (CML) proteins are principal Ca²⁺-sensors in plants that govern the activities of numerous downstream proteins with regulatory properties. The families of IQ67-Domain (IQD) proteins are a large class of plant-specific CaM/CML-targets (e.g., 33 members in A. thaliana) which share a unique domain of multiple varied CaM retention motifs in tandem orientation. Genetic studies in Arabidopsis and tomato revealed first roles for IQD proteins related to basal defense response and plant development. Molecular, biochemical and histochemical analysis of Arabidopsis IQD1 demonstrated association with microtubules as well as targeting to the cell nucleus and nucleolus. In vivo binding to CaM and kinesin light chain-related protein-1 (KLCR1) suggests a Ca²⁺-regulated scaffolding function of IQD1 in kinesin motor-dependent transport of multiprotein complexes. Furthermore, because IQD1 interacts in vitro with single-stranded nucleic acids, the prospect arises that IQD1 and other IQD family members facilitate cellular RNA localization as one mechanism to control and fine-tune gene expression and protein sorting.