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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]

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]

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]

• 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]

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]

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) 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]

• 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]