Functional characterization of melatonin-responsive GhMYC5 in cotton drought tolerance

Drought stress imposes severe constraints on cotton yield and fiber quality. Prior research has shown that exogenous melatonin (MT) application effectively enhances drought tolerance in cotton. Through transcriptome sequencing and weighted gene co-expression network analysis (WGCNA), we identified GhMYC5, a basic helix-loop-helix (bHLH) transcription factor, as a melatonin-responsive hub gene governing drought response. To clarify the functional roles and molecular mechanisms of GhMYC5, virus-induced gene silencing (VIGS) was employed to suppress GhMYC5 expression in cotton, alongside the generation of heterologous overexpression lines in Arabidopsis thaliana. Phenotypic and physiological assessments demonstrated that silencing GhMYC5 substantially compromised drought tolerance. Under drought conditions, GhMYC5-silenced plants showed significantly decreased peroxidase (POD) and catalase (CAT) activities, leaf relative water content (RWC), and proline (PRO) accumulation, while malondialdehyde (MDA) levels were markedly increased. In contrast, transgenic Arabidopsis overexpressing GhMYC5 exhibited improved drought resistance. Transcriptomic profiling identified 9,525 differentially expressed genes (DEGs) in GhMYC5-silenced plants under drought stress. Gene Ontology analysis indicated that these DEGs were primarily linked to protein kinase-related biological processes. Importantly, key genes and kinases associated with Ca²⁺ signaling, abscisic acid (ABA) and jasmonic acid (JA) crosstalk, and the mitogen-activated protein kinase (MAPK) cascade pathway exhibited substantial alterations. Overall, these findings suggest that GhMYC5 serves as a positive regulator in cotton drought stress response, with its regulatory mechanism closely associated with ABA, JA, and MAPK signaling pathways. This study offers novel genetic resources and a theoretical foundation for elucidating molecular mechanisms underlying cotton drought tolerance, thereby facilitating the development of drought-resistant cotton cultivars.