Comprehensive analysis of pepper (Capsicum annuum) RAV genes family and functional identification of CaRAV1 under chilling stress

Despite its known significance in plant abiotic stress responses, the role of the RAV gene family in the response of Capsicum annuum to chilling stress remains largely unexplored. In this study, we identified and characterized six members of the CaRAV gene subfamily in pepper plants through genome-wide analysis. Subsequently, the CaRAV subfamily was classified into four branches based on homology with Arabidopsis thaliana, each exhibiting relatively conserved domains within the branch. We discovered that light response elements accounted for the majority of CaRAVs, whereas low-temperature response elements were specific to the NGA gene subfamily. After pepper plants were subjected to chilling stress, qRT‒PCR analysis revealed that CaRAV1, CaRAV2 and CaNGA1 were significantly induced in response to chilling stress, indicating that CaRAVs play a role in the response to chilling stress. Using virus-induced gene silencing (VIGS) vectors, we targeted key members of the CaRAV gene family. Under normal growth conditions, the MDA content and SOD enzyme activity of the silenced plants were slightly greater than those of the control plants, and the REC activity was significantly greater than that of the control plants. The levels of MDA and electrolyte leakage were greater in the silenced plants after they were exposed to chilling stress, and the POD and CAT enzyme activities were significantly lower than those in the control, which was particularly evident under repeated chilling stress. In addition, the relative expression of CaPOD and CaCAT was greater in V2 plants upon repeated chilling stress, especially CaCAT was significantly greater in V2 plants than in the other two silenced plants, with 3.29 and 1.10 increases within 12 and 24 h. These findings suggest that CaRAV1 and CaNGA1 positively regulate the response to chilling stress. Silencing of key members of the CaRAV gene family results in increased susceptibility to chilling damage and reduced antioxidant enzyme activity in plants, particularly under repeated chilling stress. This study provides valuable information for understanding the classification and putative functions of RAV transcription factors in pepper plants.