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Background: Zinc finger proteins (ZFPs) are important regulators in abiotic and biotic stress tolerance in plants. However, the role of the ZFPs in wheat responding to pathogen infection is poorly understood. Results: In this study, we found TaZFP8-5B was down-regulated by Puccinia striiformis f. sp. tritici (Pst) infection. TaZFP8-5B possesses a single C2H2-type zinc finger domain with a plant-specific QALGGH motif, and an EAR motif (LxLxL) at the C-terminus. The EAR motif represses the trans-activation ability of TaZFP8-5B. Knocking down the expression of TaZFP8 by virus-induced gene silencing increased wheat resistance to Pst, whereas TaZFP8-5B-overexpressing reduced wheat resistance to stripe rust and rice resistance to Magnaporthe oryzae, suggesting that TaZFP8-5B plays a negative role in the modulation of plant immunity. Using bimolecular fluorescence complementation, split-luciferase, and yeast two-hybrid assays, we showed that TaZFP8-5B interacted with a wheat calmodulin-like protein TaCML21. Knock-down of TaCML21 reduced wheat resistance to Pst. Conclusions: This study characterized the function of TaZFP8-5B and its interacting protein TaCML21. Our findings provide a new perspective on a regulatory module made up of TaCML21-TaZFP8-5B in plant immunity. [ABSTRACT FROM AUTHOR]

Calmodulins (CaMs) and calmodulin-like proteins (CMLs) belong to families of calcium-sensors that act as calcium ion (Ca2+) 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 CaMs and CMLs that are involved in tolerance to multiple abiotic stresses. [ABSTRACT FROM AUTHOR]

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]

Summary: It is well known that calcium, ethylene and abscisic acid (ABA) can regulate fruit ripening, however, their interaction in the regulation of fruit ripening has not yet been fully clarified. The present study found that the expression of the papaya calcium sensor CpCML15 was strongly linked to fruit ripening. CpCML15 could bind Ca2+ and served as a true calcium sensor. CpCML15 interacted with CpPP2C46 and CpPP2C65, the candidate components of the ABA signalling pathways. CpPP2C46/65 expression was also related to fruit ripening and regulated by ethylene. CpCML15 was located in the nucleus and CpPP2C46/65 were located in both the nucleus and membrane. The interaction between CpCML15 and CpPP2C46/65 was calcium dependent and further repressed the activity of CpPP2C46/65 in vitro. The transient overexpression of CpCML15 and CpPP2C46/65 in papaya promoted fruit ripening and gene expression related to ripening. The reduced expression of CpCML15 and CpPP2C46/65 by virus‐induced gene silencing delayed fruit colouring and softening and repressed the expression of genes related to ethylene signalling and softening. Moreover, ectopic overexpression of CpCML15 in tomato fruit also promoted fruit softening and ripening by increasing ethylene production and enhancing gene expression related to ripening. Additionally, CpPP2C46 interacted with CpABI5, and CpPP2C65 interacted with CpERF003‐like, two transcriptional factors in ABA and ethylene signalling pathways that are closely related to fruit ripening. Taken together, our results showed that CpCML15 and CpPP2Cs positively regulated fruit ripening, and their interaction integrated the cross‐talk of calcium, ABA and ethylene signals in fruit ripening through the CpCML15‐CpPP2Cs‐CpABI5/CpERF003‐like pathway. [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]

Summary: Transient and rapid increase in cytosolic Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ 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. [ABSTRACT FROM AUTHOR]

SUMMARY: Wheat yellow mosaic virus (WYMV) causes severe wheat viral disease in Asia. However, the viral suppressor of RNA silencing (VSR) encoded by WYMV has not been identified. Here, the P1 protein encoded by WYMV RNA2 was shown to suppress RNA silencing in Nicotiana benthamiana. Mutagenesis assays revealed that the alanine substitution mutant G175A of P1 abolished VSR activity and mutant Y10A VSR activity remained only in younger leaves. P1, but not G175A, interacted with gene silencing‐related protein, N. benthamiana calmodulin‐like protein (NbCaM), and calmodulin‐binding transcription activator 3 (NbCAMTA3), and Y10A interacted with NbCAMTA3 only. Competitive Bimolecular fluorescence complementation and co‐immunoprecipitation assays showed that the ability of P1 disturbing the interaction between NbCaM and NbCAMTA3 was stronger than Y10A, Y10A was stronger than G175A. In vitro transcript inoculation of infectious WYMV clones further demonstrated that VSR‐defective mutants G175A and Y10A reduced WYMV infection in wheat (Triticum aestivum L.), G175A had a more significant effect on virus accumulation in upper leaves of wheat than Y10A. Moreover, RNA silencing, temperature, and autophagy have significant effects on the accumulation of P1 in N. benthamiana. Taken together, WYMV P1 acts as VSR by interfering with calmodulin‐associated antiviral RNAi defense to facilitate virus infection in wheat, which has provided clear insights into the function of P1 in the process of WYMV infection. Significance Statement: This study reveals that the wheat yellow mosaic virus (WYMV) P1 acts as VSR by interfering with calmodulin‐associated antiviral RNAi defense and facilitates virus infection in wheat. Results of our research provide clear insights and solutions for further understanding the role of P1 in process of WYMV infection. [ABSTRACT FROM AUTHOR]

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]

Calmodulin-like proteins (CMLs) are one of the Ca2+ 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. [ABSTRACT FROM AUTHOR]

Pear (Pyrus bretschneideri), one of the most widely planted fruit trees in the world, is infected by pear ring rot disease, which is triggered by Botryosphaeria dothidea. Previous research has shown that exogenous calcium enhanced pear resistance to B. dothidea. To explore the molecular mechanism of calcium in pear pathogen resistance, we searched the differentially expressed genes (DEGs) between calcium and H2O treatment with B. dothidea inoculation in pear by using RNA-seq data. On the basis of the standard of a proportion of calcium/H2O fold change >2, and the false discovery rate (FDR) <0.05, 2,812 and 572 genes with significant differential expression were identified between the H2O and calcium treatments under B. dothidea inoculation at 2 days postinoculation (dpi) (D2) and 8 dpi (D8), respectively, indicating that significantly more genes in D2 responded to calcium treatment. Results of the gene annotation showed that DEGs were focused on plant-pathogen interactions, hormone signal transduction, and phenylpropanoid biosynthesis in D2. Moreover, transient silencing of PbrCML30 (pear calmodulin-like proteins 30), which had significantly higher expression in response to calcium than H2O treatments, conferred compromised resistance to B. dothidea. Exogenous calcium treatment slightly alleviated the symptoms of TRV2-PbrCML30 leaves compared with TRV2 leaves under inoculation, supporting its key role in pear resistance to B. dothidea. Overall, the information obtained in this study provides a possible mechanism of calcium in regulating pear resistance to B. dothidea. [ABSTRACT FROM AUTHOR]

Calcium signaling plays a crucial role as a second messenger in plant immune responses. However, the regulatory mechanisms that govern host responses to infections caused by Sclerotinia sclerotiorum and Phytophthora capsici remain unclear. This study investigated the expression pattern of Nicotiana benthamiana calmodulin-like protein 30 (NbCML30) following infections. Our results reveal a significant downregulation of NbCML30 expression after S. sclerotiorum infection, with no changes observed in NbCML30 expression following P. capsici infection. Importantly, transient silencing of NbCML30 using tobacco rattle virus-mediated methods promoted infections caused by both S. sclerotiorum and P. capsici. In contrast, transgenic plants overexpressing NbCML30 exhibited a suppressive effect on the infections caused by S. sclerotiorum and P. capsici. Subsequent transcriptome sequencing uncovered that NbCML30 silencing markedly upregulated the expression of host endogenous genes, particularly activating pathways associated with protein processing in endoplasmic reticulum (ER) associated degradation (ERAD) pathway. These findings highlight the affirmative regulatory role of NbCML30 in conferring resistance against necrotrophic and hemibiotrophic pathogens, shedding light on its potential inhibitory mechanism. In conclusion, our study provides a theoretical basis for crop resistance genetic breeding strategies by elucidating the regulatory function of NbCML30 in plant defense against these pathogens. • Silencing NbCML30 enhances susceptibility to S. sclerotiorum and P. capsici infections. • Silencing NbCML30 activates pathways associated with endoplasmic reticulum-associated degradation (ERAD). • NbCML30 overexpression inhibits S. sclerotiorum and P. capsici infection. [ABSTRACT FROM AUTHOR]

The effect of calcium chloride (CaCl 2) treatment on γ-aminobutyric acid (GABA) accumulation in fresh-cut cantaloupe and the involved mechanisms were investigated. The result showed that 1% (w/v) CaCl 2 treatment increased GABA content and activities of glutamate decarboxylase (GAD) and succinate semialdehyde dehydrogenase (SSADH), while decreased glutamate (Glu) content and GABA transaminase (GABA-T) activities in fresh-cut cantaloupe. CmCML11 and CmCAMTA5 expressions of CaCl 2 -treated fruit increased by 187.4% and 165.6% than control fruit in the initial 6 h. Besides, expressions of GABA shunt genes, including CmGAD1 , CmGAD2 , CmGABA-T and CmSSADH were also up-regulated by CaCl 2 treatment during early storage. Moreover, acting as a transcriptional activator, CmCAMTA5 could bind to the CG-box in promoters of CmGAD1 , CmGABA-T and CmSSADH and activate their transcription. Furthermore, the interaction between CmCML11 and CmCAMTA5 could enhance the transcriptional activation on GABA shunt genes which were regulated by CmCAMTA5. Collectively, our findings revealed that CaCl 2 treatment promoted GABA accumulation in fresh-cut cantaloupe via the combined effect of CmCML11 and CmCAMTA5 in the regulation of expressions of CmGAD1 , CmGABA-T , and CmSSADH in GABA shunt. [Display omitted] • CaCl 2 treatment could activate GABA shunt and enhance GABA content of fresh-cut cantaloupe. • CaCl 2 treatment up-regulated the expressions of CmCML11 and CmCAMTA5. • CmCML11 protein physically interacted with CmCAMTA5. • GABA shunt was positively regulated by Ca2+-CML11-CAMTA5 signal transduction pathway. [ABSTRACT FROM AUTHOR]

[Objectives] Calmodulin-like protein (CML), a class of Ca2+ binding proteins containing the EF-hand domain, are closely related to the division and morphogenesis of plant cells. Based on the cloned CML25-like gene which expressed specifically in the cucumber fruit, we studied its mechanism of regulating parthenocarpy. [Methods] RT-qPCR method was used to study the temporal and spatial characteristics of CML25-like expression. Plant expression vector pLP100-35S-CML25-like was constructed, the cucumber inbred line CCMC was transformed by Agrobacterium-mediated transformation method using cotyledon as explants, and the expression characteristics of the transgenic plants were studied. The phenotypes of transgenic plants were observed, and the expression changes of hormone-related genes in fruit-set period of transgenic plants were detected using RT-qPCR method. [Results] In transgenic plants, the expression level of CML25-like was the highest in young leaves and female flowers of cucumber, and the lowest in male flowers. Moreover, CML25-like was up-regulated expression during the fruit development, and down-regulated expression during the fruit abortion. Statistical results of traits showed that the parthenocarpy rate of transgenic plants of over-expressing CML25-like could reach to 98.25%, which increased 86% compared with control CCMC. And there were no significant differences in length and transverse diameter between the parthenocarpic fruits and control pollinated fruits. The results of RT-qPCR showed that 16 of the 22 hormone-related genes were significant up-regulated expression during the fruit setting of transgenic plants. [Conclusions] The active expression of CML25-like was closely related to fruit development, and CML25-like could induce parthenocarpy of cucumber and promote fruit enlargement. We inferred that CML25-like induced parthenocarpy by promoting the up-regulated expression of hormone-related genes in the ovaries of weakly parthenocarpic cucumber. [ABSTRACT FROM AUTHOR]

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]

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. [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]

Primary infection of a plant with a pathogen that causes high accumulation of salicylic acid in the plant typically via a hypersensitive response confers enhanced resistance against secondary infection with a broad spectrum of pathogens, including viruses. This phenomenon is called systemic acquired resistance (SAR), which is a plant priming for adaption to repeated biotic stress. However, the molecular mechanisms of SAR-mediated enhanced inhibition, especially of virus infection, remain unclear. Here, we show that SAR against cucumber mosaic virus (CMV) in tobacco plants (Nicotiana tabacum) involves a calmodulin-like protein, rgs-CaM. We previously reported the antiviral function of rgs-CaM, which binds to and directs degradation of viral RNA silencing suppressors (RSSs), including CMV 2b, via autophagy. We found that rgs-CaM-mediated immunity is ineffective against CMV infection in normally growing tobacco plants but is activated as a result of SAR induction via salicylic acid signaling. We then analyzed the effect of overexpression of rgs- CaM on salicylic acid signaling. Overexpressed and ectopically expressed rgs-CaM induced defense reactions, including cell death, generation of reactive oxygen species, and salicylic acid signaling. Further analysis using a combination of the salicylic acid analogue benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) and the Ca2+ ionophore A23187 revealed that rgs-CaM functions as an immune receptor that induces salicylic acid signaling by simultaneously perceiving both viral RSS and Ca2+ influx as infection cues, implying its autoactivation. Thus, secondary infection of SAR-induced tobacco plants with CMV seems to be effectively inhibited through 2b recognition and degradation by rgs-CaM, leading to reinforcement of antiviral RNA silencing and other salicylic acid-mediated antiviral responses. IMPORTANCE Even without an acquired immune system like that in vertebrates, plants show enhanced whole-plant resistance against secondary infection with pathogens; this so-called systemic acquired resistance (SAR) has been known for more than half a century and continues to be extensively studied. SAR-induced plants strongly and rapidly express a number of antibiotics and pathogenesis-related proteins targeted against secondary infection, which can account for enhanced resistance against bacterial and fungal pathogens but are not thought to control viral infection. This study showed that enhanced resistance against cucumber mosaic virus is caused by a tobacco calmodulin-like protein, rgs-CaM, which detects and counteracts the major viral virulence factor (RNA silencing suppressor) after SAR induction. rgs-CaM-mediated SAR illustrates the growth versus defense trade-off in plants, as it targets the major virulence factor only under specific biotic stress conditions, thus avoiding the cost of constitutive activation while reducing the damage from virus infection. [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]

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]

Both calcium ion (Ca 2+ ) and salicylic acid (SA) influence various stress responses in plants. In acidic soils, aluminum (Al) toxicity adversely affects crop yield. In this study, we determined the influences of Ca 2+ and SA on root elongation, Al accumulation, and citrate secretion in soybean plant. We also investigated the activity of antioxidative enzymes in Al-exposed soybean roots. Root elongation was severally inhibited when the roots were exposed to 30 μM Al. The Al-induced inhibition of root elongation was ameliorated by Ca 2+ and SA but aggravated by Ca 2+ channel inhibitor (VP), CaM antagonists (TFP), Ca 2+ chelator (EGTA), and SA biosynthesis inhibitor (PAC). Furthermore, 1.0 mM CaCl 2 and 10 μM SA reduced the accumulation of Al in roots, but their inhibitors stimulated the accumulation of Al in roots. Citrate secretion from these roots increased with the addition of either 1.0 mM CaCl 2 or 10 μM SA but did not increase significantly when treated with higher Ca 2+ concentration. Enzymatic analysis showed that Ca 2+ and SA stimulated the activities of superoxidase (SOD), peroxidase (POD), and ascorbate peroxidase (APX) in Al-treated roots. In addition, SA restored the inhibition of Ca 2+ inhibitors on root elongation and Al content. Thus, both Ca 2+ and SA contribute to Al tolerance in soybean. Furthermore, Ca 2+ supplements rapidly increased Al-induced accumulation of free-SA or conjugated SA (SAG), while Ca 2+ inhibitors delayed the accumulation of SA for more than 8 h. Within 4 h of treatment, SA increased cytosolic Ca 2+ concentration in Al-treated roots, and upregulated the expression of four genes that possibly encode calmodulin-like (CML) proteins. These findings indicate that SA is involved in Ca 2+ -mediated signal transduction pathways in Al tolerance. [ABSTRACT FROM AUTHOR]

The concentration alteration of cytosolic-free calcium ([ Ca2+]cyt) is a well-known secondary messenger in plants and plays important roles during pollen grain germination and tube elongation. Here we demonstrate that CML25, a member of calmodulin-like proteins, has Ca2+-binding activity and plays a role in pollen grain germination, tube elongation and seed setting. CML 25 transcript was abundant in mature pollen grains and pollen tubes, and its product CML25 protein was primarily directed to the cytoplasm. Two independent CML 25 loss-of-function T-DNA insertion mutants suffered a major reduction in both the rate of pollen germination and the elongation of the pollen tube. Also, pollen grains of cml25 mutants were less sensitive to the external K+ and Ca2+ concentration than wild-type pollen. The disruption of CML 25 increased the [ Ca2+]cyt in both the pollen grain and the pollen tube, which in turn impaired the Ca2+-dependent inhibition of whole-cell inward K+ currents in protoplasts prepared from these materials (pollen grain and pollen tube). Complementation of cml25-1 mutant resulted in the recovery of wild-type phenotype. Our findings indicate that CML25 is an important transducer in the Ca2+-mediated regulation of K+ influx during pollen germination and tube elongation. [ABSTRACT FROM AUTHOR]

Cryptosporidium parvum is an obligate protozoan parasite invading epithelial cells of small intestine of human and animals, and causing diarrheal disease. In apicomplexan parasites, calcium signaling can regulate many essential biological processes such as invasion and migration. As the main intracellular receptor for calcium ions, calmodulins control the activities of hundreds of enzymes and proteins. Calmodulin-like protein (CML) is an important member of the calmodulin family and may play a key role in C. parvum , however, the actual situation is still not clear. The present study aimed to identify the parasite interaction partner proteins of C. parvum calmodulin-like protein (Cp CML). By constructing the cpcml bait plasmid, 5 potential Cp CML - interacting proteins in C. parvum oocyst were screened by yeast-two-hybrid system (Y2H). Bimolecular fluorescence complementation (BiFC) and Co-immunoprecipitation (Co-IP) were performed as subsequent validations. Fibrillarin RNA methylase (FBL) was identified via this screening method as Cp CML interacting protein in C. parvum. The identification of this interaction made it possible to get a further understanding of the function of Cp CML and its contribution to the pathogenicity of C. parvum. [Display omitted] • Screening of the potentially interacting proteins of Cp CML in C. parvum. • Fibrillarin RNA methylase (FBL) was identified as one of the interacting proteins of Cp CML in C. parvum. • Fibrillarin RNA methylase (FBL) may mediate 2′-O-methylation of rRNA and assistant in the maturation of rRNA in C. parvum. [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]

Abstract: Insect herbivory on plants is a complex incident consisting of at least two different aspects, namely mechanical damage and chemical challenge, as feeding insects introduce oral secretions (OS) into the wounded tissue of the attacked plant. Mechanical wounding alone is sufficient to induce a set of defense-related reactions in host plants, but some early events such as membrane potential (Vm) changes and cytosolic Ca2+-elevations can be triggered only by herbivores suggesting that OS-derived molecules are involved in those processes. Following an assay-guided purification based on planar lipid bilayer membrane technique in combination with proteomic analysis, a porin-like protein (PLP) of most likely bacterial origin was determined from collected OS of Spodoptera littoralis larvae. PLP exhibited channel-forming activity. Further, early defense-related events in plant–insect interaction were evaluated by using a purified fraction and α-hemolysin (α-HL) as a commercial pore-forming compound. Both up-regulated the calmodulin-like CML42 in Arabidopsis thaliana, which only responds to oral secretion and not to wounding. An elevation of in vivo [Ca2+]cyt was not observed. Because membrane channel formation is a widespread phenomenon in plant–insect interactions, this PLP might represent an example for microbial compounds from the insect gut which are initially involved in plant–insect interactions. [Copyright &y& Elsevier]

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]

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: Calmodulin-like protein (CaLP) was believed to be involved in the shell formation of pearl oyster. However, no further study of this protein was ever performed. In this study, the in vitro crystallization experiment showed that CaLP can modify the morphology of calcite. In addition, aragonite crystals can be induced in the mixture of CaLP and a nacre protein (at 16 kDa), which was detected and purified from the EDTA-soluble matrix of nacre. These results agreed with that of immunohistological staining in which CaLP was detected not only in the organic layer sandwiched between nacre (aragonite) and the prismatic layer (calcite), but also around the prisms of the prismatic layer. Take together, we concluded that (1) CaLP, as a component of the organic layer, can induce the nucleation of aragonite through binding with the 16-kDa protein, and (2) CaLP may regulate the growth of calcite in the prismatic layer. [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]

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 H2O2. 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. [ABSTRACT FROM AUTHOR]

Calcium signals are crucial for the activation and coordination of signaling cascades leading to the establishment of plant defense mechanisms. Here, we studied the contribution of CML8, an Arabidopsis calmodulin-like protein in response to Ralstonia solanacearum and to pathogens with different lifestyles, such as Xanthomonas campestris pv. campestris and Phytophtora capsici. We used pathogenic infection assays, gene expression, RNA-seq approaches, and comparative analysis of public data on CML8 knockdown and overexpressing Arabidopsis lines to demonstrate that CML8 contributes to defense mechanisms against pathogenic bacteria and oomycetes. CML8 gene expression is finely regulated at the root level and manipulated during infection with Ralstonia, and CML8 overexpression confers better plant tolerance. To understand the processes controlled by CML8, genes differentially expressed at the root level in the first hours of infection have been identified. Overexpression of CML8 also confers better tolerance against Xanthomonas and Phytophtora, and most of the genes differentially expressed in response to Ralstonia are differentially expressed in these different pathosystems. Collectively, CML8 acts as a positive regulator against Ralstonia solanaceraum and against other vascular or root pathogens, suggesting that CML8 is a multifunctional protein that regulates common downstream processes involved in the defense response of plants to several pathogens. [ABSTRACT FROM AUTHOR]

• Six new proteins including OsERD2 have been identified as targets of OsCML16 in rice. • OsERD2 interacts with OsCaM1 via the 1–16 motif. • Calcium mediates the ABA-induced transcription profiles of target genes in rice. • Six target genes are involved in cold, drought, and salt stress responses in rice. Calmodulin-like proteins (CMLs) have been shown to play key regulatory roles in calcium signaling in plants. However, few bona-fide CMLs binding proteins have been characterized in rice, a monocot model plant. Here, through large-scale screening of a yeast-two hybrid (Y2H) cDNA library with OsCML16 as a bait, six new putative interacting partners of OsCML16 were discovered and confirmed by both pairwise Y2H and bimolecular fluorescence complementation (BiFC) assays. Interestingly, the in vitro peptide-binding assays manifested that OsERD2 could bind both OsCaM1 and OsCML16 whereas other five target proteins could specifically bind OsCML16 but not OsCaM1. Furthermore, Ca2+ and TFP, a calmodulin (CaM) antagonist, were involved in the ABA-induced transcription of OsCML16 and its target genes, and they were also obviously induced by cold, drought, and salt stresses. Taken together, our new findings have provided the basis for the novel signaling pathways of OsCML16 in the abiotic stress response in rice. [ABSTRACT FROM AUTHOR]