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Calmodulins (CaMs) and calmodulin-like proteins (CMLs) belong to families of calcium-sensors that act as calcium ion (Ca 2+ ) signal-decoding proteins and regulate downstream target proteins. As a tropical halophyte, Canavalia rosea shows great resistance to multiple abiotic stresses, including high salinity/alkalinity, extreme drought, heat, and intense sunlight. However, investigations of calcium ion signal transduction involved in the stress responses of C. rosea are limited. The CaM and CML gene families have been identified and characterized in many other plant species. Nevertheless, there is limited available information about these genes in C. rosea . In this study, a bioinformatic analysis, including the gene structures, conserved protein domains, phylogenetic relationships, chromosome distribution, and gene synteny, was comprehensively performed to identify and characterize CrCaMs and CrCMLs . A spatio-temporal expression assay in different organs and environmental conditions was then conducted using the RNA sequencing technique. Additionally, several CrCaM and CrCML members were then cloned and functionally characterized using the yeast heterogeneous expression system, and some of them were found to change the tolerance of yeast to heat, salt, alkalinity, and high osmotic stresses. The results of this study provide a foundation for understanding the possible roles of the CrCaM and CrCML genes, especially for halophyte C. rosea 's natural ecological adaptability for its native habitats. This study also provides a theoretical basis for further study of the physiological and biochemical functions of plant CaM s and CML s that are involved in tolerance to multiple abiotic stresses.

Transient and rapid increase in cytosolic Ca 2+ plays a crucial role in plant-pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). Cyclic nucleotide-gated channels (CNGCs) have been implicated in mediating this Ca 2+ influx; however, their regulatory mechanisms remain poorly understood. Here, we have found that AVRblb2 requires the calmodulin (CaM) and calmodulin-like (CML) proteins as co-factors to interact with the NbCNGCs, resulting in the formation of AVRblb2-CaM/CML-NbCNGCs complex. Furthermore, CaM and CML are dissociated from NbCNGC18 during PTI response to increase Ca 2+ influx; however, Avrblb2 inhibits calcium channel activation by disrupting the release of CaM and CML from NbCNGC18. Following recognition of PAMP, NbCNGC18 forms active heteromeric channels with other NbCNGCs, which may give selectivity of CNGC complex against diverse signals for fine-tuning of cytosolic Ca 2+ level to mediate appropriate responses. Silencing of multiple NbCNGCs compromised the function of AVRblb2 on the pathogenicity of Phytophthora infestans, confirming that AVRblb2 contributes to pathogen virulence by targeting CNGCs. Our findings provide new insights into the regulation of CNGCs in PTI and the role of pathogen effectors in manipulating host cell physiology to promote infection., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

Calcium signalling, including pulse, amplitude, and duration, is essential for plant development and response to various stimuli. However, the calcium signalling should be decoded and translated by calcium sensors. In plants, three classes of calcium-binding proteins have been identified as calcium sensors, including calcium-dependent protein kinase (CDPK), calcineurin B-like protein (CBL), and calmodulin (CaM). Calmodulin-like proteins (CMLs), which have several EF-hands, also serve as specific calcium sensors and can sense, bind, and interpret the calcium signal during the plant's growth and defense decision-making processes. In recent decades, the function of CMLs in plant development and response to various stimuli has been systematically reviewed, shedding light on the molecular mechanism of plant CML-mediated networks in calcium signal transduction. Here, by providing an overview of CML expression and biological function in plants, we demonstrate that growth-defense trade-offs occur during calcium sensing, an aspect that has not been well studied in recent years., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Calmodulin ( CaM ) and calmodulin-like ( CML ) genes are widely involved in plant growth and development and mediating plant stress tolerance. However, the whole genome scale studies about CaM and CML gene families have not been done in wheat, and the possible functions of most wheat CaM/CML gene members are still unknown. In this study, a total of 18 TaCaM and 230 TaCML gene members were identified in wheat genome. Among these genes, 28 TaCaM/CML gene members have 74 duplicated copies, while 21 genes have 48 transcript variants, resulting in 321 putative TaCaM/CML transcripts totally. Phylogenetic tree analysis showed that they can be classified into 7 subfamilies. Similar gene structures and protein domains can be found in members of the same gene cluster. The TaCaM/CML genes were spread among all 21 chromosomes with unbalanced distributions, while most of the gene clusters contained 3 homoeologous genes located in the same homoeologous chromosome group. Synteny analysis showed that most of TaCaM/CMLs gene members can be found with 1-4 paralogous genes in T. turgidum and Ae. Tauschii . High numbers of cis -acting elements related to plant hormones and stress responses can be observed in the promoters of TaCaM/CMLs . The spatiotemporal expression patterns showed that most of the TaCaM/TaCML genes can be detected in at least one tissue. The expression levels of TaCML17, 21, 30, 50, 59 and 75 in the root or shoot can be up-regulated by abiotic stresses, suggesting that TaCML17, 21, 30, 50, 59 and 75 may be related with responses to abiotic stresses in wheat. The spatiotemporal expression patterns of TaCaM/CML genes indicated they may be involved widely in wheat growth and development. Our results provide important clues for exploring functions of TaCaMs/CMLs in growth and development as well as responses to abiotic stresses in wheat in the future.

Calmodulin-like proteins (CMLs) are one of the Ca 2+ sensors in plants, but the functions of most CMLs remain unknown. The regulation of cold tolerance and flowering time by MtCML42 in Medicago truncatula and the underlying mechanisms were investigated using MtCML42-overexpressing plants and cml42 Medicago mutants with a Tnt1 retrotransposon insertion. Compared with the wild type (WT), MtCML42-overexpressing lines had increased cold tolerance, whereas cml42 mutants showed decreased cold tolerance. The impaired cold tolerance in cml42 could b complemented by MtCML42 expression. The transcript levels of MtCBF1, MtCBF4, MtCOR413, MtCAS15, MtLTI6A, MtGolS1 and MtGolS2 and the concentrations of raffinose and sucrose were increased in response to cold treatment, whereas higher levels were observed in MtCML42-overexpressing lines and lower levels were observed in cml42 mutants. In addition, early flowering with upregulated MtFTa1 and downregulated MtABI5 transcripts was observed in MtCML42-overexpressing lines, whereas delayed flowering with downregulated MtFTa1 and upregulated MtABI5 was observed in cml42. MtABI5 expression could complement the flowering phenotype in the Arabidopsis mutant abi5. Our results suggest that MtCML42 positively regulates MtCBF1 and MtCBF4 expression, which in turn upregulates the expression of some COR genes, MtGolS1 and MtGolS2, which leads to raffinose accumulation and increased cold tolerance. MtCML42 regulates flowering time through sequentially downregulating MtABI5 and upregulating MtFTa1 expression., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Calmodulin-like (CML) proteins are primary calcium sensors and function in plant growth and response to stress stimuli. However, so far, the function of plant CML proteins, including tomato, is still unclear. Previously, it was found that a tomato ( Solanum lycopersicum ) CML, here named SlCML39 , was significantly induced by high temperature (HT) at transcription level, but its biological function is scarce. In this study, the characteristics of SlCML39 and its role in HT tolerance were studied. SlCML39 encodes a protein of 201 amino acids containing four EF hand motifs. Many cis-acting elements related to plant stress and hormone response appear in the promoter regions of SlCML39 . SlCML39 is mainly expressed in the root, stem, and leaf and can be regulated by HT, cold, drought, and salt stresses as well as ABA and H 2 O 2 . Furthermore, heterologous overexpression of SlCML39 reduces HT tolerance in Arabidopsis thaliana at the germination and seedling growth stages. To better understand the molecular mechanism of SlCML39 , the downstream gene network regulated by SlCML39 under HT was analyzed by RNA-Seq. Interestingly, we found that many genes involved in stress responses as well as ABA signal pathway are down-regulated in the transgenic seedlings under HT stress, such as KIN1 , RD29B , RD26 , and MAP3K18 . Collectively, these data indicate that SlCML39 acts as an important negative regulator in response to HT stress, which might be mediated by the ABA signal pathway.

Optimal temperature and light are both necessary conditions for coral survival. Light enhances calcification, and thermal stress disrupts Ca 2+ homeostasis. As calcium is involved in many important metabolic activities, in this study, we cloned the calmodulin-like protein (CaLP) gene of one of the scleractinian corals, Galaxea astreata. We also detected the relative mRNA expression levels of gaCaLP using the calcium channel blocker verapamil and CaCl 2 treatment under conditions of light and dark, and compared expression levels under controlled temperature conditions. Full-length gaCaLP cDNA comprised 1290 nucleotides and contained 498 bp open reading frame that encoded a protein with 165 amino acids. With CaCl 2 , expression levels of gaCaLP only increased in the presence of light, suggesting that light may be a restrictive factor in CaLP expression when sufficient calcium is available in the environment. In addition, after verapami treatment, we noted that a down regulation of gaCaLP, suggesting that the expression of CaLP is closely related to extracellular Ca 2+ influx. Under temperature stress at both high (30 °C) and low (20 °C) temperatures, expression levels of gaCaLP showed an initial increase, followed by a decreasing trend as treatment progressed. Expression levels reached their maximum value at 24 h. This result showed that CaLP participated in a temperature stress response, and Ca 2+ homeostasis was disrupted during stress. The findings of the present study will help determine the function and regulatory mechanisms of gaCaLP.

In their natural environment, plants have to continuously face constraints such as biotic and abiotic stresses. To achieve their life cycle, plants have to perceive and interpret the nature, but also the strength of environmental stimuli to activate appropriate physiological responses. Nowadays, it is well established that signaling pathways are crucial steps in the implementation of rapid and efficient plant responses such as genetic reprogramming. It is also reported that rapid raises in calcium (Ca 2+ ) levels within plant cells participate in these early signaling steps and are essential to coordinate adaptive responses. However, to be informative, calcium increases need to be decoded and relayed by calcium-binding proteins also referred as calcium sensors to carry-out the appropriate responses. In a recent study, we showed that CML8, an Arabidopsis calcium sensor belonging to the calmodulin-like (CML) protein family, promotes plant immunity against the phytopathogenic bacteria Pseudomonas syringae pv tomato (strain DC3000). Interestingly, other CML proteins such as CML9 were also reported to contribute to plant immunity using the same pathosystem. In this addendum, we propose to discuss about the specific contribution of these 2 CMLs in stress responses.

In addition to the well-known Ca(2+) sensor calmodulin, plants possess many calmodulin-like proteins (CMLs) that are predicted to have specific roles in the cell. Herein, we described the biochemical and biophysical characterization of recombinant Arabidopsis thaliana CML14. We applied isothermal titration calorimetry to analyze the energetics of Ca(2+) and Mg(2+) binding to CML14, and nuclear magnetic resonance spectroscopy, together with intrinsic and ANS-based fluorescence, to evaluate the structural effects of metal binding and metal-induced conformational changes. Furthermore, differential scanning calorimetry and limited proteolysis were used to characterize protein thermal and local stability. Our data demonstrate that CML14 binds one Ca(2+) ion with micromolar affinity (Kd ∼ 12 µM) and the presence of 10 mM Mg(2+) decreases the Ca(2+) affinity by ∼5-fold. Although binding of Ca(2+) to CML14 increases protein stability, it does not result in a more hydrophobic protein surface and does not induce the large conformational rearrangement typical of Ca(2+) sensors, but causes only localized structural changes in the unique functional EF-hand. Our data, together with a molecular modelling prediction, provide interesting insights into the biochemical properties of Arabidopsis CML14 and may be useful to direct additional studies aimed at understanding its physiological role., (© 2016 The Protein Society.)

Plants and animals can recognize the invasion of pathogens through their perception of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Plant PRRs identified have been exclusively receptor-like kinases/proteins (RLK/Ps), and no RLK/P that can detect viruses has been identified to date. RNA silencing (RNA interference, RNAi) is regarded as an antiviral basal immunity because the majority of plant viruses has RNA as their genomes and encode RNA silencing suppressor (RSS) proteins to counterattack antiviral RNAi. Many RSSs were reported to bind to double-stranded RNAs (dsRNAs), which are regarded as viral PAMPs. We have recently identified a tobacco calmodulin (CaM)-like protein, rgs-CaM, as a PRR that binds to diverse viral RSSs through its affinity for the dsRNA-binding domains. Because rgs-CaM seems to target RSSs for autophagic degradation with self-sacrifice, the expression level of rgs-CaM is important for antiviral activity. Here, we found that the rgs-CaM expression was induced immediately (within 1 h) after wounding at a wound site on tobacco leaves. Since the invasion of plant viruses is usually associated with wounding, and several hours are required for viruses to replicate to a detectable level in invaded cells, the wound-induced expression of rgs-CaM seems to be linked to its antiviral function, which should be ready before the virus establishes infection. CaMs and CaM-like proteins usually transduce calcium signals through their binding to endogenous targets. Therefore, rgs-CaM is a unique CaM-like protein in terms of binding to exogenous targets and functioning as an antiviral PRR.

Plant calmodulins (CaMs) and calmodulin-like proteins (CMLs) mediate Ca2+ signaling in response to abiotic stresses. Manipulation of this signaling in crops could increase stress tolerance. We review methods for detecting Ca2+ signals, regulatory roles of CaMs and CMLs, binding targets, and Ca2+ networks under abiotic stress in organelles. [ABSTRACT FROM AUTHOR]

The root‐knot nematode Meloidogyne graminicola secretes effectors into rice tissues to modulate host immunity. Here, we characterised MgCRT1, a calreticulin protein of M. graminicola, and identified its target in the plant. In situ hybridisation showed MgCRT1 mRNA accumulating in the subventral oesophageal gland in J2 nematodes. Immunolocalization indicated MgCRT1 localises in the giant cells during parasitism. Host‐induced gene silencing of MgCRT1 reduced the infection ability of M. graminicola, while over‐expressing MgCRT1 enhanced rice susceptibility to M. graminicola. A yeast two‐hybrid approach identified the calmodulin‐like protein OsCML31 as an interactor of MgCRT1. OsCML31 interacts with the high mobility group protein OsHMGB1 which is a conserved DNA binding protein. Knockout of OsCML31 or overexpression of OsHMGB1 in rice results in enhanced susceptibility to M. graminicola. In contrast, overexpression of OsCML31 or knockout of OsHMGB1 in rice decreases susceptibility to M. graminicola. The GST‐pulldown and luciferase complementation imaging assay showed that MgCRT1 decreases the interaction of OsCML31 and OsHMGB1 in a competitive manner. In conclusion, when M. graminicola infects rice and secretes MgCRT1 into rice, MgCRT1 interacts with OsCML31 and decreases the association of OsCML31 with OsHMGB1, resulting in the release of OsHMGB1 to enhance rice susceptibility. Summary statement: A plant‐pathogenic nematode effector MgCRT1 interacts with OsCML31 and decreases the association of OsCML31 with OsHMGB1 in a competitive manner, resulting in the release of OsHMGB1 to enhance rice susceptibility. [ABSTRACT FROM AUTHOR]

Calmodulins (CaMs) and Calmodulin-like proteins (CMLs) are vital in plant growth, development, and stress responses. However, CaMs and CMLs have not been fully identified and characterized in brown algae, which has been evolving independently of the well-studied green plant lineage. In this study, whole-genome searches revealed one SjCaM and eight SjCMLs in Saccharina japonica, and one EsCaM and eleven EsCMLs in Ectocarpus sp. SjCaM and EsCaM encoded identical protein products and shared 88.59–89.93% amino acid identities with Arabidopsis thaliana AtCaMs, thereby indicating that brown algae CaMs retained a similar Ca2+ sensors function as in plants. The phylogenetic and gene structure analysis results showed that there was significant divergence in the gene sequences among brown algae CMLs. Furthermore, evolutionary analysis indicated that the function of brown alga CMLs was relatively conserved, which may be related to the fact that brown algae do not need to face complex environments like terrestrial plants. Regulatory elements prediction and the expression analysis revealed the probable functioning of SjCaM/CML genes in gametophyte development and the stress response in S. japonica. In addition, the SjCaM/SjCMLs interacting proteins and chemicals were preliminarily predicted, suggesting that SjCaM/SjCMLs might play putative roles in Ca2+/CaM-mediated growth and development processes and stimulus responses. Therefore, these results will facilitate our understanding of the evolution of brown algae CaMs/CMLs and the functional identification of SjCaM/SjCMLs. [ABSTRACT FROM AUTHOR]

Calmodulin (CaM) and calmodulin-like (CML) genes are widely involved in plant growth and development and mediating plant stress tolerance. However, the whole genome scale studies about CaM and CML gene families have not been done in wheat, and the possible functions of most wheat CaM/CML gene members are still unknown. In this study, a total of 18 TaCaM and 230 TaCML gene members were identified in wheat genome. Among these genes, 28 TaCaM/CML gene members have 74 duplicated copies, while 21 genes have 48 transcript variants, resulting in 321 putative TaCaM/CML transcripts totally. Phylogenetic tree analysis showed that they can be classified into 7 subfamilies. Similar gene structures and protein domains can be found in members of the same gene cluster. The TaCaM/CML genes were spread among all 21 chromosomes with unbalanced distributions, while most of the gene clusters contained 3 homoeologous genes located in the same homoeologous chromosome group. Synteny analysis showed that most of TaCaM/CMLs gene members can be found with 1–4 paralogous genes in T. turgidum and Ae. Tauschii. High numbers of cis-acting elements related to plant hormones and stress responses can be observed in the promoters of TaCaM/CMLs. The spatiotemporal expression patterns showed that most of the TaCaM/TaCML genes can be detected in at least one tissue. The expression levels of TaCML17, 21, 30, 50, 59 and 75 in the root or shoot can be up-regulated by abiotic stresses, suggesting that TaCML17, 21, 30, 50, 59 and 75 may be related with responses to abiotic stresses in wheat. The spatiotemporal expression patterns of TaCaM/CML genes indicated they may be involved widely in wheat growth and development. Our results provide important clues for exploring functions of TaCaMs/CMLs in growth and development as well as responses to abiotic stresses in wheat in the future.

Until now, no completely effective parasite‐specific drugs or vaccines have been approved for the treatment of cryptosporidiosis. Through the separation and identification of the sporozoite membrane protein of Cryptosporidium parvum (C. parvum), 20 related proteins were obtained. Among them, a calmodulin‐like protein (CML) has a similar functional domain‐exchange factor hand (EF‐hand) motif as calmodulin proteins (CaMs), so it may play a similarly important role in the invasion process. A 663 bp full gene encoding the C. parvum calmodulin‐like protein (CpCML) was inserted in pET28a vector and expressed in Escherichia coli. An immunofluorescence assay showed that CpCML was mainly located on the surface of the sporozoites. Three‐week‐old female BALB/c mice were used for modelling the immunoreactions and immunoprotection of recombinant CpCML (rCpCML) against artificial Cryptosporidium tyzzeri infections. The results indicated a significantly increased in anti‐CpCML antibody response, which was induced by the immunized recombinant protein. Compared to rP23 (recombinant P23), GST6P‐1 (expressed by pGEX‐6P‐1 transfected E. coli), GST4T‐1 (expressed by pGEX‐4T‐1 transfected E. coli), glutathione (GSH), adjuvant and blank control groups, rCpCML‐immunized mice produced specific spleen cell proliferation in addition to different production levels of IL‐2, IFN‐γ, TNF‐α, IL‐4 and IL‐5. Additionally, immunization with rCpCML led to 34.08% reduction of oocyst shedding in C. tyzzeri infected mice faeces which was similar to rP23. These results suggest that CpCML may be developed as a potential vaccine candidate antigen against cryptosporidiosis. [ABSTRACT FROM AUTHOR]

Calmodulins (CAMs) and calmodulin-like proteins (CMLs) can participate in the regulation of various physiological processes via sensing and decoding Ca2+ signals. To reveal the characteristics of the CAM/CML family in Ginkgo biloba, a comprehensive analysis was performed at the genome-wide level. A total of 26 CAMs/CMLs, consisting of 5 GbCAMs and 21 GbCMLs, was identified on 11 out of 12 chromosomes in G. biloba. They displayed a certain degree of multiplicity in their sequences, albeit with conserved EF hands. Collinearity analysis suggested that tandem rather than segmental or whole-genome duplications were likely to play roles in the evolution of the Ginkgo CAM/CML family. Furthermore, GbCAMs/GbCMLs were grouped into higher, lower, and moderate expression in magnitude. The cis-acting regulatory elements involved in phytohormone-responsiveness within GbCAM/GbCML promotors may explain their varied expression profiles. The ectopic expression of a GbCML gene (Gb_30819) in transgenic Arabidopsis led to phenotypes with significantly shortened root length and seedling height, and decreased yields of both pods and seeds. Moreover, an electrophoresis mobility shift assay demonstrated the Ca2+-binding activity of Gb_30819 in vitro. Altogether, these results contribute to insights into the characteristics of the evolution and expression of GbCAMs/GbCMLs, as well as evidence for Ca2+-CAM/CML pathways functioning within the ancient gymnosperm G. biloba. [ABSTRACT FROM AUTHOR]

Calmodulins (CAMs) and calmodulin-like proteins (CMLs) can participate in the regulation of various physiological processes via sensing and decoding Ca2+ signals. To reveal the characteristics of the CAM/CML family in Ginkgo biloba, a comprehensive analysis was performed at the genome-wide level. A total of 26 CAMs/CMLs, consisting of 5 GbCAMs and 21 GbCMLs, was identified on 11 out of 12 chromosomes in G. biloba. They displayed a certain degree of multiplicity in their sequences, albeit with conserved EF hands. Collinearity analysis suggested that tandem rather than segmental or whole-genome duplications were likely to play roles in the evolution of the Ginkgo CAM/CML family. Furthermore, GbCAMs/GbCMLs were grouped into higher, lower, and moderate expression in magnitude. The cis-acting regulatory elements involved in phytohormone-responsiveness within GbCAM/GbCML promotors may explain their varied expression profiles. The ectopic expression of a GbCML gene (Gb_30819) in transgenic Arabidopsis led to phenotypes with significantly shortened root length and seedling height, and decreased yields of both pods and seeds. Moreover, an electrophoresis mobility shift assay demonstrated the Ca2+-binding activity of Gb_30819 in vitro. Altogether, these results contribute to insights into the characteristics of the evolution and expression of GbCAMs/GbCMLs, as well as evidence for Ca2+-CAM/CML pathways functioning within the ancient gymnosperm G. biloba.

The calmodulin (CaM) and calmodulin-like (CML) proteins are major calcium sensors that play a critical role in environmental stimulus response in plants. Nevertheless, the CaM/CML proteins from the specific plants with extreme tolerance to abiotic stresses remained so far uncharacterized. In this study, 66 candidate proteins (three NsCaMs and sixty-three NsCMLs) were identified from the halophyte Nitraria sibirica , which can withstand an extreme salinity. Bioinformatic analysis of upstream cis -acting elements predicted the potential involvement of NsCaM/CMLs in abiotic stress responses and various hormone responses. Additionally, the Nitraria sibirica transcriptome revealed that 17 and 7 NsCML s were significantly upregulated under 100 mM or 400 mM NaCl treatment. Transcription of most salt-responsive genes was similarly upregulated under cold stress, yet downregulated under drought treatment. Moreover, predictive subcellular localization analysis suggested that the stress-responsive NsCML proteins mainly localize at the cellular membrane and within the nucleus. Furthermore, transgenic overexpression of two NsCML s (NISI03G1136 and NISI01G1645) was found to mitigate H 2 O 2 accumulation caused by salt stress. These results provide insights into the potential function of Nitraria sibirica CaM/CML proteins, which could aid the investigation of molecular mechanisms of extreme tolerance to abiotic stresses in halophytes. [ABSTRACT FROM AUTHOR]

Abstract In plants, many target proteins of calmodulins (CaMs) have been identified in cellular metabolism and responses. However, calmodulin-like proteins (CMLs) and their target proteins have not been discovered in stress responses in rice. In this study, a novel CC-NBS-LRR protein was obtained in screening a cold stress rice seedlings yeast cDNA library with OsCML16 as bait. Furthermore, yeast two-hybrid and BiFC assays demonstrated that the full length, CC region in the N-terminus and LRR in the C-terminus of Pi304 protein could interact with OsCML16. More interestingly, OsCML16 bound to the 1–10 motif rather than 1–14 motif in the Ca2+ or Mg2+ dependent manner in vitro. In addition, transcript levels of OsCML16 and OsPi304 were induced more markedly in Nipponbare than in 9311 under cold stress. Taken together, these data indicates that they are involved in the cold stress signaling and response in rice. Highlights • OsCML16 binds to both Ca2+ and Mg2+ in vitro. • OsCML16 interacts with a novel CC-NBS-LRR protein OsPi304. • OsCML16 binds to the 1-10 motif in a Ca2+/Mg2+-dependent manner in vitro. • OsCML16 binds to LRR domain in a Ca2+/Mg2+-independent manner in vitro. • OsPi304 and OsCML16 genes are induced by cold stress. [ABSTRACT FROM AUTHOR]

Calmodulin-like protein 19 (CML19) is an Arabidopsis centrin that modulates nucleotide excision repair (NER) by binding to RAD4 protein, the Arabidopsis homolog of human Xeroderma pigmentosum complementation group C protein. Although the necessity of CML19 as a part of the RAD4 plant recognition complex for functional NER is known at a cellular level, little is known at a molecular level. Herein, we used a combination of biophysical and biochemical approaches to investigate the structural and ion and target-peptide binding properties of CML19. We found that CML19 possesses four Ca 2+ -specific binding sites, two of high affinity in the N-terminal domain and two of low affinity in the C-terminal domain. Binding of Ca 2+ to CML19 increases its alpha-helix content, stabilizes the tertiary structure, and triggers a conformational change, resulting in the exposure of a hydrophobic patch instrumental for target protein recognition. Using bioinformatics tools we identified a CML19-binding site at the C-terminus of RAD4, and through in vitro binding experiments we analyzed the interaction between a 17-mer peptide representing this site and CML19. We found that the peptide shows a high affinity for CML19 in the presence of Ca 2+ (stoichiometry 1:1) and the interaction primarily involves the C-terminal half of CML19. [ABSTRACT FROM AUTHOR]

The AP2/ ERF family is a plant-specific transcription factor family whose members have been associated with various developmental processes and stress tolerance. Here, we functionally characterized the drought-inducible Os ERF48, a group Ib member of the rice ERF family with four conserved motifs, CMI-1, -2, -3 and -4. A transactivation assay in yeast revealed that the C-terminal CMI-1 motif was essential for Os ERF48 transcriptional activity. When Os ERF48 was overexpressed in an either a root-specific ( ROX O s ERF 48) or whole-body ( OX O s ERF 48) manner, transgenic plants showed a longer and denser root phenotype compared to the nontransgenic ( NT) controls. When plants were grown on a 40% polyethylene glycol-infused medium under in vitro drought conditions, ROX O s ERF 48 plants showed a more vigorous root growth than OX O s ERF 48 and NT plants. In addition, the ROX O s ERF 48 plants exhibited higher grain yield than OX O s ERF 48 and NT plants under field-drought conditions. We constructed a putative Os ERF48 regulatory network by cross-referencing ROX O s ERF 48 root-specific RNA-seq data with a co-expression network database, from which we inferred the involvement of 20 drought-related genes in Os ERF48-mediated responses. These included genes annotated as being involved in stress signalling, carbohydrate metabolism, cell-wall proteins and drought responses. They included, Os CML16, a key gene in calcium signalling during abiotic stress, which was shown to be a direct target of Os ERF48 by chromatin immunoprecipitation- qPCR analysis and a transient protoplast expression assay. Our results demonstrated that Os ERF48 regulates Os CML16, a calmodulin-like protein gene that enhances root growth and drought tolerance. [ABSTRACT FROM AUTHOR]

L. japonicus, a model plant of legumes plants, is widely used in symbiotic nitrogen fixation. A large number of studies on it have been published based on the genetic, biochemical, structural studies. These results are secondhand reports that CaM is a key regulator during Rhizobial infection. In plants, there are multiple CaM genes encoding several CaM isoforms with only minor amino acid differences. Moreover, the regulation mechanism of this family of proteins during rhizobia infection is still unclear. In the current study, a family of genes encoding CaMs and CMLs that possess only the Ca2+-binding EF-hand motifs were analyzed. Using ML and BI tree based on amino acid sequence similarity, seven loci defined as CaMs and 19 CMLs, with at least 23% identity to CaM, were identified. The phylogenetics, gene structures, EF hand motif organization, and expression characteristics were evaluated. Seven CaM genes, encoding only 4 isoforms, were found in L. japonicus. According to qRT-PCR, four LjCaM isoforms are involved in different rhizobia infection stages. LjCaM1 might be involved in the early rhizobia infection epidermal cells stage. Furthermore, additional structural differences and expression behaviors indicated that LjCMLs may have different potential functions from LjCaMs. [ABSTRACT FROM AUTHOR]

• A novel CgCaLP-2 gene was identified in Crassostrea gigas and it underwent alternative splicing. • Two protein isoforms were detected and the 23 kD form mainly responded to long-term CO2 exposure. • CgCaLP-2 redistributed to nuclear membrane after long-term CO2 exposure. • The expression of a transcription factor CgSIX4 was associated with CgCaLP-2 under CO2 exposure and in vivo knock down further verified their relevance. Marine organisms need to adapt to improve organismal fitness under ocean acidification (OA). Recent studies have shown that marine calcifiers can achieve acclimation by stimulating calcium binding/signaling pathways. Here, a CaM-like gene (CgCaLP-2) from oyster Crassostrea gigas which typically responded to long-term CO 2 exposure (two months) rather than short-term exposure (one week) was characterized. The cloned cDNA was 678 bp and was shorter than the retrieved sequence from NCBI (1125 bp). The two sequences, designated as CgCaLP-2-v1 and CgCaLP-2-v2 , were demonstrated to be different splice variants by the genome sequence analysis. Western blotting analysis revealed two bands of 23 kD and 43 kD in mantle and hemocytes, corresponding to predicted molecular weight of CgCaLP-2-v1 and CgCaLP-2-v2, respectively. The isoform CgCaLP-2-v1 (the 23 kD band) was highly stimulated in response to long-term CO 2 exposure (42-day and 56-day treatment) in hemocytes and mantle tissue. The fluorescence signal of CgCaLP-2 in mantle and hemocytes became more intensive after long-term CO 2 exposure. Besides, in hemocytes, CgCaLP-2 presented a higher localization on the nuclear membrane after long-term CO 2 exposure (56 d). The target gene network of CgCaLP-2 was predicted, and a transcription factor (TF) gene annotated as Homeobox protein SIX4 (CgSIX4) showed a similar expressive trend to CgCaLP-2 during CO 2 exposure. Suppression of CgCaLP-2 via RNA interference significantly reduced the mRNA expression of CgSIX4. The results suggested that CgCaLP-2 might mediate the Ca2+-CaLP-TF signal transduction pathway under long-term CO 2 exposure. This study serves as an example to reveal that alternative splicing is an important mechanism for generation multiple protein isoforms and thus shape the plastic responses under CO 2 exposure, providing new insight into the potential acclimation ability of marine calcifiers to future OA. [ABSTRACT FROM AUTHOR]

Transient changes in intracellular Ca2+ concentration have been well recognized to act as cell signals coupling various environmental stimuli to appropriate physiological responses with accuracy and specificity in plants. Calmodulin (CaM) and calmodulin-like proteins (CMLs) are major Ca2+ sensors, playing critical roles in interpreting encrypted Ca2+ signals. Ca2+-loaded CaM/CMLs interact and regulate a broad spectrum of target proteins such as channels/pumps/antiporters for various ions, transcription factors, protein kinases, protein phosphatases, metabolic enzymes and proteins with unknown biochemical functions. Many of the target proteins of CaM/CMLs directly or indirectly regulate plant responses to environmental stresses. Basic information about stimulus-induced Ca2+ signal and overview of Ca2+ signal perception and transduction are briefly discussed in the beginning of this review. How CaM/CMLs are involved in regulating plant responses to abiotic stresses are emphasized in this review. Exciting progress has been made in the past several years, such as the elucidation of Ca2+/CaM-mediated regulation of AtSR1/CAMTA3 and plant responses to chilling and freezing stresses, Ca2+/CaM-mediated regulation of CAT3, MAPK8 and MKP1 in homeostasis control of ROS signals, discovery of CaM7 as a DNA-binding transcription factor regulating plant response to light signals. However, many key questions in Ca2+/CaM-mediated signaling warrant further investigation. Ca2+/CaM-mediated regulation of most of the known target proteins is presumed based on their interaction. The downstream targets of CMLs are mostly unknown, and how specificity of Ca2+ signaling could be realized through the actions of CaM/CMLs and their target proteins is largely unknown. Future breakthroughs in Ca2+/CaM-mediated signaling will not only improve our understanding of how plants respond to environmental stresses, but also provide the knowledge base to improve stress-tolerance of crops.

The tobacco calmodulin-like protein rgs-CaM is involved in host defense against virus and is reported to possess an associated RNA silencing suppressor activity. Rgs-CaM is also believed to act as an antiviral factor by interacting and targeting viral silencing suppressors for autophagic degradation. Despite these functional data, calcium interplay in the modulation of rgs-CaM is still poorly understood. Here we show that rgs-CaM displays a prevalent alpha-helical conformation and possesses three functional Ca 2+ -binding sites. Using computational modeling and molecular dynamics simulation, we demonstrate that Ca 2+ binding to rgs-CaM triggers expansion of its tertiary structure with reorientation of alpha-helices within the EF-hands. This conformational change leads to the exposure of a large negatively charged region that may be implicated in the electrostatic interactions between rgs-CaM and viral suppressors. Moreover, the k d values obtained for Ca 2+ binding to the three functional sites are not within the affinity range of a typical Ca 2+ sensor. [ABSTRACT FROM AUTHOR]

Transient changes in intracellular Ca2+ concentration have been well recognized to act as cell signals coupling various environmental stimuli to appropriate physiological responses with accuracy and specificity in plants. Calmodulin (CaM) and calmodulin-like proteins (CMLs) are major Ca2+ sensors, playing critical roles in interpreting encrypted Ca2+ signals. Ca2+-loaded CaM/CMLs interact and regulate a broad spectrum of target proteins such as channels/pumps/antiporters for various ions, transcription factors, protein kinases, protein phosphatases, metabolic enzymes, and proteins with unknown biochemical functions. Many of the target proteins of CaM/CMLs directly or indirectly regulate plant responses to environmental stresses. Basic information about stimulus-induced Ca2+ signal and overview of Ca2+ signal perception and transduction are briefly discussed in the beginning of this review. How CaM/CMLs are involved in regulating plant responses to abiotic stresses are emphasized in this review. Exciting progress has been made in the past several years, such as the elucidation of Ca2+/CaM-mediated regulation of AtSR1/CAMTA3 and plant responses to chilling and freezing stresses, Ca2+/CaM-mediated regulation of CAT3, MAPK8 and MKP1 in homeostasis control of reactive oxygen species signals, discovery of CaM7 as a DNA-binding transcription factor regulating plant response to light signals. However, many key questions in Ca2+/CaM-mediated signaling warrant further investigation. Ca2+/CaM-mediated regulation of most of the known target proteins is presumed based on their interaction. The downstream targets of CMLs are mostly unknown, and how specificity of Ca2+ signaling could be realized through the actions of CaM/CMLs and their target proteins is largely unknown. Future breakthroughs in Ca2+/CaM-mediated signaling will not only improve our understanding of how plants respond to environmental stresses, but also provide the knowledge base to improve stress-tolerance of crops. [ABSTRACT FROM AUTHOR]

Many signalling pathways in plants are regulated by the second messenger calcium (Ca2+ ). In the standard model, Ca2+ -sensor proteins, such as CaM (calmodulin), detect Ca2+ signals and subsequently regulate downstream targets to advance the signal transduction cascade. In addition to CaM, plants possess many CMLs (CaM-like proteins) that are predicted to function as Ca2+ sensors, but which remain largely uncharacterized. In the present study, we examined the biochemical properties, subcellular localization and tissue-specific distribution of Arabidopsis CML43. Our data indicate that CML43 displays characteristics typical of Ca2+ sensors, including high-affinity Ca2+ binding, conformational changes upon Ca2+ binding that expose hydrophobic regions and stabilization of structure in the presence of Mg2+ or Ca2+ . In vivo localization analysis demonstrates that CML43 resides in cytosolic and nuclear compartments. Transgenic plants expressing a CML43:GUS (β-glucoronidase) promoter reporter gene revealed that CML43 promoter activity is restricted almost exclusively to root tips under normal growth conditions. GUS reporter activity in these transgenic plants was strongly increased when exposed to the defence compound SA (salicylic acid). Furthermore, immunoblot analysis revealed that the CML43 protein accumulates following treatment with SA. Collectively, our findings suggest that CML43 functions as a Ca2+ sensor in root tips during both normal growth and plant immune response. [ABSTRACT FROM AUTHOR]

Plants are often subjected to various environmental stresses that lead to deleterious effects on growth, production, sustainability, etc. The information of the incoming stress is read by the plants through the mechanism of signal transduction. The plant Ca2+ serves as secondary messenger during adaptations to stressful conditions and developmental processes. A plethora of Ca2+ sensors and decoders functions to bring about these changes. The cellular concentrations of Ca2+, their subcellular localization, and the specific interaction affinities of Ca2+ decoder proteins all work together to make this process a complex but synchronized signaling network. In this review, we focus on the versatility of these sensors and decoders in the model plant Arabidopsis as well as plants of economical importance. Here, we have also thrown light on the possible mechanism of action of these important components. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

Calmodulin (CaM) is a highly conserved and ubiquitous Ca2+-binding protein regulating intracellular Ca2+ concentration by acting as a sensor of this divalent cation in eukaryotic cells. Being such a very important signal sensor, CaM is susceptible to undergo many posttranslational modifications. One of these important modifications is its phosphorylation. Our previous investigations showed that CaM and calmodulin-like protein (CaLP) cloned from Pinctada fucata have many different characteristics in spite of their high similarity to each other. We have narrowed down that the C-terminal domains of CaM and CaLP are responsible for their discrepant subcellular localizations and shuttling of CaLP when it is co-transfected with p21Cip1, which is commonly considered as an important cell cycle regulating protein. In this study, we first predicted the potential phosphorylation site responsible for the shuttling and confirmed by fluorescence confocal microscopy. Together with fluorescence activated cell sorter analysis, we further investigated the releasing ability of wild type and point mutated CaLP from arrested cell cycle caused by p21Cip1 overexpression. By performing pull-down analysis and phosphorylation status of CaLP in cytoplasm fraction of transfected COS-7 cells with CaLP alone and phosphorylation status of CaLP in nuclear fraction of co-transfected COS-7 cells with CaLP and p21Cip, we propose that the CaLP staying in the cytoplasm is in the state of phosphorylation, but when p21Cip1 is overexpressed in mammalian cells, some signal triggers CaLP dephosphorylation and translocation into the nucleus. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Pearl oyster Pinctada fucata calmodulin-like protein (PfCaLP), containing an extra tail (D150-K161) at the C-terminal, is a novel protein involved in the regulation of oyster calcium metabolism. The purpose of this study is to gain insight into the conformational characteristics of the N/C-domain of PfCaLP, especially the detailed contribution of the extra tail to the Ca2+/Mg2+-induced conformational changes, the stability of the intact PfCaLP molecule and its C-domain, as well as to the interdomain communications in PfCaLP. Our results demonstrate that a strong interaction exists between the hydrophilic tail and the C-domain of PfCaLP. The extra tail, through affecting the C-domain conformational changes, further influences the migration rate, conformational changes, N/C-domain interactions and exposure of the hydrophobic patches of the intact PfCaLP molecule. Furthermore, the tail could actively regulate the stability of PfCaLP and its C-domain. Our studies are helpful to explain our previous finding that the tail plays important roles in PfCaLP-target interaction in the oyster calcium metabolism. [Copyright &y& Elsevier]

Abstract: Calmodulin (CaM) plays a very important role in many physiological processes and is highly conserved in different species. In a previous study, we successfully cloned CaM and a novel calmodulin-like protein (CaLP) with an extra C-terminal sequence from the pearl oyster Pinctada fucata and then expressed in Escherichia coli. In this research, we used fluorescence confocal microscopy to analyze the protein–protein interaction between CaM/CaLP and p21Cip1, which is cloned from mammalian cells, to show the different characteristics of these two proteins in vivo. The fluorescence confocal microscopy showed that the C-terminal globular domain together with the extra tail of CaLP is very important in CaLP''s sequestration in cytoplasm. The most interesting phenomenon is that transfection of p21Cip1 can stimulate translocation of CaLP from the cytoplasm to the nucleus, but this is not the case for CaM. Fluorescence confocal microscopy and co-immunoprecipitation on different mutants of CaLP with p21Cip1 indicated that the C-terminal globular domain of CaLP is responsible for the trafficking of CaLP from cytoplasm to nucleus. [Copyright &y& Elsevier]

Abstract: Calmodulin-like protein (CLP) is a specific light chain of unconventional myosin-10 (Myo10) and enhances Myo10-dependent filopodial extension. Here we show that phenylalanine-795 in the third IQ domain (IQ3) of Myo10 is critical for CLP binding. Remarkably, mutation of F795 to alanine had little effect on calmodulin binding to IQ3. Fluorescence microscopy and time-lapse video microscopy showed that HeLa cells expressing CLP and transiently transfected with GFP-Myo10-F795A exhibited significantly shorter filopodia and decreased intrafilopodial motility compared to wildtype GFP-Myo10-transfected cells. Thus, F795 represents a unique anchor for CLP and is essential for CLP-mediated Myo10 function in filopodial extension and motility. Structured summary: MINT-6595901: GST-IQ3 Myo10 (uniprotkb:Q9HD67) binds (MI:0407) to CLP (uniprotkb:P27482) by pull down (MI:0096) MINT-6596000: CLP (uniprotkb:P27482) and GST-IQ3 Myo10 (uniprotkb:Q9HD67) bind (MI:0407) by classical fluorescence spectroscopy (MI:0017) MINT-6596013: CaM (uniprotkb:P62158) and GST-IQ3 Myo10 (uniprotkb:Q9HD67) bind (MI:0407) by classical fluorescence spectroscopy (MI:0017) MINT-6595938:GST-IQ3 Myo10 (uniprotkb:Q9HD67) binds (MI:0407) to CaM (uniprotkb:P62158) by pull down (MI:0096) [Copyright &y& Elsevier]

Abstract: Myosin-10 (Myo10) is involved in processes ranging from filopodial formation and extension to spindle orientation during cell division. Myo10 contains three IQ motifs that bind calmodulin and calmodulin-like protein (CLP) as light chains. We recently found that CLP expression up-regulates Myo10, leading to increased Myo10-dependent cell motility and filopodial extension [R.D. Bennett, et al., J. Biol. Chem. 282 (2007) 3205–3212]. CLP-dependent Myo10 up-regulation occurs without increase in Myo10 mRNA. We followed Myo10 degradation in vivo and in vitro and found that it was unaffected by CLP. Myo10 decayed rapidly with a half-life of ∼2.5h. Using an in vitro transcription/translation system we determined that CLP increased Myo10 translation, resulting in a higher relative accumulation of Myo10 in the presence than in the absence of CLP. Our data suggest that CLP functions to increase translation of Myo10 possibly by acting as a chaperone for the emerging Myo10 heavy chain protein. [Copyright &y& Elsevier]

Abstract: Calmodulin (CaM) and calmodulin-like protein (CaLP) are two proteins involved in biomineralization. Their localizations in Pinctada fucata mantle epithelia were studied by Western blot (WB) analysis of the nuclear/cytosol fraction of primary cultured P. fucata mantle cells and immunogold electron microscopy. The results showed a completely different distribution of these two proteins at the subcellular level. CaM was distributed throughout both the nucleus and cytoplasm of the mantle epithelium but CaLP was distributed only in the cytoplasm. The functions of these two proteins in biomineralization were investigated by shell regeneration. During this process, the expressions of CaM and CaLP were greatly enhanced in different organelles of the mantle epithelium. Overexpression of these two proteins and a mutant of calmodulin-like protein (M-CaLP) that lacks an extra C-terminal tail in MC3T3-E1 promoted the mRNA expression of osteopontin, a biomineralization marker for osteoblasts. All of the results indicated that CaM and CaLP have completely different distributions in the mantle epithelium and affect the biomineralization process at different levels. The extra C-terminal tail of CaLP is important for its functions in biomineralization in P. fucata. [Copyright &y& Elsevier]

Abstract: Oyster (Pinctada fucata) calmodulin-like protein (CaLP), containing a C-terminally extra hydrophilic tail (150D–161K), is a novel protein involved in the regulation of oyster calcium metabolism. To investigate the importance of the extra fragment to the Ca2+/Mg2+-dependent conformational changes in the intact CaLP molecule and the interactions between CaLP and its target proteins, a truncated CaLP mutant (M-CaLP) devoid of the extended C-terminus was constructed and overexpressed in Escherichia coli. The conformational characteristics of M-CaLP were studied by CD and fluorescence spectroscopy and compared with those of the oyster CaM and CaLP. The far-UV CD results reveal that the extra tail has a strong effect on the Ca2+-induced, but a relatively weak effect on the Mg2+-induced conformational changes in CaLP. However, upon Ca2+ or Mg2+ binding, only slight changes for intrinsic phenylalanine and tyrosine fluorescence spectra between M-CaLP and CaLP are observed. Our results also indicate that the extra tail can significantly decrease the exposure of the hydrophobic patches in CaLP. Additionally, affinity chromatography demonstrates that the target binding of CaLP is greatly influenced by its additional tail. All our results implicate that the extra tail may play some important roles in the interactions between CaLP and its targets in vivo. [Copyright &y& Elsevier]

Calcium metabolism in oysters is a very complicated and highly controlled physiological and biochemical process. However, the regulation of calcium metabolism in oyster is poorly understood. Our previous study showed that calmodulin (CaM) seemed to play a regulatory role in the process of oyster calcium metabolism. In this study, a full-length cDNA encoding a novel calmodulin-like protein (CaLP) with a long C-terminal sequence was identified from pearl oyster Pinctada fucata, expressed in Escherichia coli and characterized in vitro. The oyster CaLP mRNA was expressed in all tissues tested, with the highest levels in the mantle that is a key organ involved in calcium secretion. In situ hybridization analysis reveals that CaLP mRNA is expressed strongly in the outer and inner epithelial cells of the inner fold, the outer epithelial cells of the middle fold, and the dorsal region of the mantle. The oyster CaLP protein, with four putative Ca2+-binding domains, is highly heat-stable and has a potentially high affinity for calcium. CaLP also displays typical Ca2+-dependent electrophoretic shift, Ca2+-binding activity and significant Ca2+-induced conformational changes. Ca2+-dependent affinity chromatography analysis demonstrated that oyster CaLP was able to interact with some different target proteins from those of oyster CaM in the mantle and the gill. In summary, our results have demonstrated that the oyster CaLP is a novel member of the CaM superfamily, and suggest that the oyster CaLP protein might play a different role from CaM in the regulation of oyster calcium metabolism. [ABSTRACT FROM AUTHOR]