The genome-wide identification and salt tolerance analysis of AOX2 subfamily genes in Cucurbita pepo

Secondary salinization in protected agriculture has emerged as a major constraint on the yield and quality of Cucurbita pepo. Therefore, identifying key salt tolerant genes and elucidating their functions are of great theoretical and practical significance for cultivating new salt tolerant varieties through molecular breeding methods to address salt stress issues in protected agriculture. The alternative oxidase (AOX) contributes not only to plant growth and development, but also to stress tolerance. In this study, two AOX2-type candidate genes were identified and characterized in Cucurbita pepo . Phylogenetic analysis confirmed their classification as AOX2 members, designated CpeAOX2a and CpeAOX2b . Promoter sequence analysis revealed multiple stress- and hormone-responsive cis-elements in both CpeAOX2a and CpeAOX2b , suggesting their potential roles in adversity responses. RT-qPCR analysis showed that both genes were significantly upregulated under salt stress, however, CpeAOX2b was more sensitive and strongly responsive to salt stress. Heterologous overexpression of CpeAOX2a in Arabidopsis did not significantly improve its salt tolerance, while overexpression of CpeAOX2b in Arabidopsis enhanced salt tolerance, which was reflected in improved growth, increased Fv/Fm ratios, higher proline and chlorophyll levels, reduced REL and MDA content, stronger antioxidant enzyme activities, and upregulation of stress-related genes compared to WT plants. Furthermore, virus-induced gene silencing of CpeAOX2b reduced salt tolerance in Cucurbita pepo . These findings demonstrate that CpeAOX2b is crucial for salt stress tolerance in Cucurbita pepo and provide novel genetic targets for breeding salt-tolerant varieties.