Identification of the EXORDIUM gene family and functional analysis of VaEXO2a during cold stress response in Vitis amurensis

EXORDIUM (EXO) has been identified as an indicator of Brassinosteroid (BR)-responsive gene. However, the EXO gene family has not yet been identified and characterized in grapes. In this study, we identified 12 EXO gene family members in the grape genome, which were unevenly distributed on 5 chromosomes and included 5 collinear gene pairs. Except for the VvEXO6 gene, which contains two exons and one intron, the remaining VvEXO genes contain only one exon. All members of the VvEXO family contain the Phi_1 structural domain. Multi-species evolutionary analysis revealed that EXO proteins were clustered into five clades. Except for VvEXO6, VvEXO9, and VvEXO11, all other members contain a signal peptide. VvEXO9 and VvEXO11 also contain transmembrane structures. Subcellular localization prediction indicated that VvEXO is primarily located on the cell membrane. The analysis of low-temperature transcriptome data revealed that the VvEXO2a gene in grapes responds promptly to low-temperature stress. VaEXO2a, the homologous gene of VvEXO2a in Vitis amurensis, was induced by low temperature and BR. Functional validation was performed via VaEXO2a overexpression in transgenic 'Muscat Hamburg' grape roots, which resulted in enhanced tolerance to low-temperature stress. Moreover, based on the 591 differentially expressed genes identified, the downstream EXO target genes were mainly enriched in molecular functions such as antioxidant activity, oxidoreductase activity, and peroxidase activity. These results provide a reference for further exploration of the grape EXO gene family's functions and its involvement in stress tolerance. Bioinformatics identified 12 VvEXOs, with VaEXO2a positively regulating grape cold resistance.