Calcium (Ca) is an ubiquitous key second messenger in plants, where it modulates many developmental and adaptive processes in response to various stimuli. Several proteins containing Ca binding domain have been identified in plants, including calmodulin (CaM) and calmodulin-like (CML) proteins, which play critical roles in translating Ca signals into proper cellular responses. In this work, a genome-wide analysis conducted in Vitis vinifera identified three CaM- and 62 CML-encoding genes. We assigned gene family nomenclature, analyzed gene structure, chromosomal location and gene duplication, as well as protein motif organization. The phylogenetic clustering revealed a total of eight subgroups, including one unique clade of VviCaMs distinct from VviCMLs. VviCaMs were found to contain four EF-hand motifs whereas VviCML proteins have one to five. Most of grapevine CML genes were intronless, while VviCaMs were intron rich. All the genes were well spread among the 19 grapevine chromosomes and displayed a high level of duplication. The expression profiling of VviCaM/VviCML genes revealed a broad expression pattern across all grape organs and tissues at various developmental stages, and a significant modulation in biotic stress-related responses. Our results highlight the complexity of CaM/CML protein family also in grapevine, supporting the versatile role of its different members in modulating cellular responses to various stimuli, in particular to biotic stresses. This work lays the foundation for further functional and structural studies on specific grapevine CaMs/CMLs in order to better understand the role of Ca-binding proteins in grapevine and to explore their potential for further biotechnological applications.Copyright © 2018 Elsevier Masson SAS. All rights reserved.

The use of (35)S-labeled calmodulin (CaM) to screen a corn root cDNA expression library has led to the isolation of a CaM-binding protein, encoded by a cDNA with sequence similarity to small auxin up RNAs (SAURs), a class of early auxin-responsive genes. The cDNA designated as ZmSAUR1 (Zea mays SAURs) was expressed in Escherichia coli, and the recombinant protein was purified by CaM affinity chromatography. The CaM binding assay revealed that the recombinant protein binds to CaM in a calcium-dependent manner. Deletion analysis revealed that the CaM binding site was located at the NH(2)-terminal domain. A synthetic peptide of amino acids 20-45, corresponding to the potential CaM binding region, was used for calcium-dependent mobility shift assays. The synthetic peptide formed a stable complex with CaM only in the presence of calcium. The CaM affinity assay indicated that ZmSAUR1 binds to CaM with high affinity (K(d) approximately 15 nM) in a calcium-dependent manner. Comparison of the NH(2)-terminal portions of all of the characterized SAURs revealed that they all contain a stretch of the basic alpha-amphiphilic helix similar to the CaM binding region of ZmSAUR1. CaM binds to the two synthetic peptides from the NH(2)-terminal regions of Arabidopsis SAUR-AC1 and soybean 10A5, suggesting that this is a general phenomenon for all SAURs. Northern analysis was carried out using the total RNA isolated from auxin-treated corn coleoptile segments. ZmSAUR1 gene expression began within 10 min, increased rapidly between 10 and 60 min, and peaked around 60 min after 10 microM alpha-naphthaleneacetic acid treatment. These results indicate that ZmSAUR1 is an early auxin-responsive gene. The CaM antagonist N-(6-aminohexyl)5-chloro-1-naphthalenesulfonamide hydrochloride inhibited the auxin-induced cell elongation but not the auxin-induced expression of ZmSAUR1. This suggests that calcium/CaM do not regulate ZmSAUR1 at the transcriptional level. CaM binding to ZmSAUR1 in a calcium-dependent manner suggests that calcium/CaM regulate ZmSAUR1 at the post-translational level. Our data provide the first direct evidence for the involvement of calcium/CaM-mediated signaling in auxin-mediated signal transduction.

To characterize the role of nitric oxide (NO) in the tolerance of Arabidopsis (Arabidopsis thaliana) to heat shock (HS), we investigated the effects of heat on three types of Arabidopsis seedlings: wild type, noa1(rif1) (for nitric oxide associated1/resistant to inhibition by fosmidomycin1) and nia1nia2 (for nitrate reductase [NR]-defective double mutant), which both exhibit reduced endogenous NO levels, and a rescued line of noa1(rif1). After HS treatment, the survival ratios of the mutant seedlings were lower than those of wild type; however, they were partially restored in the rescued line. Treatment of the seedlings with sodium nitroprusside or S-nitroso-N-acetylpenicillamine revealed that internal NO affects heat sensitivity in a concentration-dependent manner. Calmodulin 3 (CaM3) is a key component of HS signaling in Arabidopsis. Real-time reverse transcription-polymerase chain reaction analysis after HS treatment revealed that the AtCaM3 mRNA level was regulated by the internal NO level. Sodium nitroprusside enhanced the survival of the wild-type and noa1(rif1) seedlings; however, no obvious effects were observed for cam3 single or cam3noa1(rif1) double mutant seedlings, suggesting that AtCaM3 is involved in NO signal transduction as a downstream factor. This point was verified by phenotypic analysis and thermotolerance testing using seedlings of three AtCaM3-overexpressing transgenic lines in an noa1(rif1) background. Electrophoretic mobility-shift and western-blot analyses demonstrated that after HS treatment, NO stimulated the DNA-binding activity of HS transcription factors and the accumulation of heat shock protein 18.2 (HSP18.2) through AtCaM3. These data indicate that NO functions in signaling and acts upstream of AtCaM3 in thermotolerance, which is dependent on increased HS transcription factor DNA-binding activity and HSP accumulation.

Calmodulin(CaM)-regulated protein phosphorylation forms an important component of Ca(2+) signaling in animals but is less understood in plants. We have identified a CaM-binding receptor-like kinase from soybean nodules, GmCaMK1, a homolog of Arabidopsis CRLK1. We delineated the CaM-binding domain (CaMBD) of GmCaMK1 to a 24-residue region near the C-terminus, which overlaps with the kinase domain. We have demonstrated that GmCaMK1 binds CaM with high affinity in a Ca(2+)-dependent manner. We showed that GmCaMK1 is expressed broadly across tissues and is enriched in roots and developing nodules. Finally, we examined the CaMBDs of the five-member GmCaMK family in soybean, and orthologs present across taxa.Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Glucosinolates are a class of secondary metabolites with important roles in plant defense and human nutrition. To uncover regulatory mechanisms of glucosinolate production, we screened Arabidopsis thaliana T-DNA activation-tagged lines and identified a high-glucosinolate mutant caused by overexpression of IQD1 (At3g09710). A series of gain- and loss-of-function IQD1 alleles in different accessions correlates with increased and decreased glucosinolate levels, respectively. IQD1 encodes a novel protein that contains putative nuclear localization signals and several motifs known to mediate calmodulin binding, which are arranged in a plant-specific segment of 67 amino acids, called the IQ67 domain. We demonstrate that an IQD1-GFP fusion protein is targeted to the cell nucleus and that recombinant IQD1 binds to calmodulin in a Ca(2+)-dependent fashion. Analysis of steady-state messenger RNA levels of glucosinolate pathway genes indicates that IQD1 affects expression of multiple genes with roles in glucosinolate metabolism. Histochemical analysis of tissue-specific IQD1::GUS expression reveals IQD1 promoter activity mainly in vascular tissues of all organs, consistent with the expression patterns of several glucosinolate-related genes. Interestingly, overexpression of IQD1 reduces insect herbivory, which we demonstrated in dual-choice assays with the generalist phloem-feeding green peach aphid (Myzus persicae), and in weight-gain assays with the cabbage looper (Trichoplusia ni), a generalist-chewing lepidopteran. As IQD1 is induced by mechanical stimuli, we propose IQD1 to be novel nuclear factor that integrates intracellular Ca(2+) signals to fine-tune glucosinolate accumulation in response to biotic challenge.