The GhWRKY65–GhAOC3A Module Enhances the Chilling Tolerance in Cotton by Regulating Jasmonic Acid Accumulation

Allene oxide cyclase (AOC) is an integral enzyme in jasmonic acid (JA) biosynthesis and a key regulator of plant stress responses. However, the regulatory mechanisms and enzymatic activities of cotton AOC under stress responses remain poorly understood. In our research, using BLASTP and conserved domain validation, we identified 68 AOC gene members across 10 Gossypium species. Phylogenetic, gene-structure, and syntenic analyses showed high evolutionary conservation of AOC genes, accompanied by lineage-specific expansion. Promoter sequence analysis indicated that the AOCs contained abundant cis-elements associated with hormone signaling, abiotic-stress response, and transcription factor (TF) regulation. Expression profiling revealed tissue-specific and stress-responsive expression patterns among GhAOC genes, with GhAOC3A showing the most pronounced upregulation under chilling stress. GhAOC3A was identified as a chloroplast-localized protein based on the subcellular localization assay. GhAOC3A overexpression enhanced cotton chilling tolerance with elevated JA accumulation, stronger antioxidant defense, stable osmotic homeostasis, and retained chlorophyll content, and VIGS results provided supportive evidence for the function of GhAOC3A in chilling tolerance. Among the predicted TFs that could regulate the expression of GhAOC3A, GhWRKY65 was further identified and confirmed to bind to the W-box element and activate GhAOC3A transcription. Overexpression of GhWRKY65 significantly enhanced cotton chilling tolerance, as well as elevated GhAOC3A expression and increased JA accumulation. Conversely, silencing GhWRKY65 compromised cotton chilling tolerance. Collectively, these findings suggest that the GhWRKY65-GhAOC3A regulatory module positively modulates cotton chilling tolerance and JA levels. Our study provides insights into the transcriptional network associated with plant stress response and offers effective candidates for breeding chilling-tolerant cotton materials.