Abstract：The COR413 (cold-regulated) gene, which encodes a plant-specific cold responsive protein, plays a critical role in cold acclimation. To determine the functional characterization of COR413 genes in response to cold stress, integrated grapevine (Vitis vinifera) genomic approaches of data mining and homologous cloning were used to obtain a total of 4 COR413 gene family members from cold-tolerant Vitis amurensis, designated as VaCOR413-PM2A、-PM1X1、-IM1 and -PM2B, respectively. Sequence analysis revealed that the 4 COR413 family members from V. amurensis showed significant nucleotide (97.2%~100%) and amino acid (99.5%~100%) similarity with those from V. vinifera, and the nucleotide differences were several single nucleotide substitutions. All COR413 proteins from V. amurensis contained the conserved domain WCOR413, several transmembrane motifs and Serine/Threonine/Tyrosine phosphorylation sties, where a predicted N-terminal signal peptide was only found in VaCOR413-PM1X1 and a glycosylphosphatidylinositol (GPI) anchor was only observed in VaCOR413-PM2B. The 4 members of COR413 from 2 grapevine species can be grouped into 2 distinct clusters, where COR413-PM2A, -PM1X1 and -PM2B were grouped together and -IM1 fall into another cluster. Moreover, tissue-specific expression profile of COR413 gene family members and their responses to cold stress were analyzed in both V. amurensis and V. vinifera cv. Cabernet Sauvignon. The results showed that VaCOR413-PM2A, -PM1X1, -IM1 and -PM2B displayed different tissue-specific features that were also not similar to those from V. vinifera. Compared to the corresponding gene expression profiles in V. vinifera, VaCOR413-PM2A, -PM1X1, -IM1 and -PM2B were detected in all tissues of V. amurensis with different expression intensities, among which VaCOR413-PM2A and -PM1X1 showed a relatively high expression in shoots, tendrils, flowers and young leaves, whereas no signals of VvCOR413-PM1X1 and -IM1 were detected in V. vinifera. Analysis of the gene expression profiles during the time-course of cold stress demonstrated the existence of 2 groups with different expression profiles in V. amurensis and 3 groups in V. vinifera. In one group of V. amurensis containing VaCOR413-PM2A, -PM2B and -PM1X1, a similar expression pattern was observed in with a decreased tendency following the time-course of cold stress, while VaCOR413-IM1 displayed a distinct expression profiles from those in group Ⅰ, and a high level of VaCOR413-IM1 expression was detected 24 h after cold stress with 9.57-fold than that of unstressed control. In V. vinifera, the expression profile of VvCOR413-PM2A was similar to VvCOR413-PM2B and their gene induction reached a maximum at 3 h in response to cold stress. VvCOR413-PM1X1 was rapid and transient in response to cold treatment, reached a maximum at 1 h, and then decreased. VvCOR413-IM1 displayed a high level of expression at 3 h of cold stress with 3.65-fold than that of control, quickly decreased at 6 h and maintained a relatively low level to the cold stress. Data from cold stress treatment suggested that VaCOR413-PM2A, -PM1X1 and -PM2B which were grouped together, displayed down-regulated expression in response to cold stress and the degree of expression level of these genes were generally lower than those from V. vinifera. Interestingly, VaCOR413-IM1 categorized into another group, showed a significant induction feature 24 h after cold stress. The results provide reference basis for exploring the possible function and mechanism of the members of the family in response to cold stress, which lays the theoretical for cold-(frozen-) tolerant molecular improvement in grapevine.
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