Genome-Wide Identification and Functional Characterization of CaGRAS43 in Enhancing Cold Stress Tolerance in Pepper

Cold stress severely limits plant growth and crop productivity, necessitating the identification of key regulatory genes that enhance tolerance. In this study, we performed a genome-wide identification and expression analysis of the GRAS transcription factor family in pepper under cold stress. In total, 64 GRAS genes (CaGRAS1-CaGRAS64) were identified in the pepper genome and distributed across 10 different chromosomes. Additional in silico analysis provided key insights on the identified CaGRAS genes, including their conserved domains, exon-intron organization, evolutionary relationships, gene ontology and synteny, and protein-protein interaction network. Further, their expression analysis under cold stress suggested that CaGRAS43 could be a candidate gene regulating cold stress response. Subsequent silencing of CaGRAS43 via virus-induced gene silencing (VIGS) increased plant sensitivity to low temperatures, resulting in elevated reactive oxygen species (ROS) accumulation, higher malondialdehyde (MDA) levels, increased relative electrolyte leakage (REL), and reduced antioxidant enzyme activity. In contrast, transient overexpression of CaGRAS43 in pepper and stable overexpression in Arabidopsis significantly improved cold tolerance, maintained chlorophyll content, reduced ROS and membrane damage, and enhanced the expression of cold-responsive and antioxidant defense genes. These results suggested that CaGRAS43 functions as a positive regulator of cold stress tolerance, orchestrating both physiological and molecular mechanisms. Moreover, the results of this study highlight CaGRAS43 as a promising candidate for engineering cold-resilient pepper and other crops.