Genome-wide identification of GhAOX gene family and its expression and regulation in cotton CMS line Jin A
Introduction Alternative oxidase (AOX) is a core functional protein that mitigates excessive mitochondrial reactive oxygen species (ROS) accumulation and modulates plant stress adaptation, and its biological activity is tightly controlled by sophisticated regulatory networks.The cotton cytoplasmic male sterile (CMS) line Jin A represents an optimal model for dissecting the functional characteristics of GhAOX genes, as it exhibits pronounced ROS overaccumulation and aberrant GhAOX transcriptional profiles in anthers during the critical developmental stage of pollen mother cell degeneration, relative to its maintainer Jin B. Methods We conducted a genome-wide identification of the GhAOX gene family in upland cotton (Gossypium hirsutum) and systematically characterized the molecular features of GhAOX members in Jin A at the transcriptional, translational, post-translational modification, and gene structural levels. Results A total of eight GhAOX genes were identified, among which GhAOX2b1/2 and GhAOX2c1/2 displayed anther-preferential expression patterns and were significantly upregulated in response to multiple abiotic stresses, including osmotic stress (PEG), salinity (NaCl), cold (4℃) and heat (37℃). Functional validation via overexpression and virus-induced gene silencing (VIGS) assays verified the ROS-scavenging capacity of GhAOX. Notably, in Jin A, despite elevated GhAOX protein abundance, its enzymatic activity was substantially compromised, which was attributed to perturbed transcriptional regulation, isoform-specific differential activation by tricarboxylic acid (TCA) cycle intermediates, and reduced abundance of mitochondrial effector molecules. Discussion This dysregulation was likely implicated in the aberrant ROS accumulation underlying the pollen abortion phenotype in Jin A. Collectively, this study establishes a comprehensive molecular framework for elucidating the multi-level regulatory mechanisms of AOX in response to intracellular microenvironmental perturbations and uncovers its pivotal role in regulating reproductive fitness in upland cotton.