Identification of metabolic pathways associated with cytoplasmic male sterility in cotton (Gossypium hirsutum L.) using Data-Independent Acquisition-based proteomic profiling.

Cotton (Gossypium hirsutum L.) is a globally important cash crop. Cytoplasmic male sterility (CMS) is a key biological tool for hybrid seed production. However, a systematic, stage-specific proteomic profile of CMS anther development in cotton remains lacking. To address this, we employed, for the first time in cotton CMS research, a high-resolution quantitative proteomic approach using data-independent acquisition mass spectrometry (DIA-MS) to compare the CMS line C2P5A and its maintainer line C2P5B across three critical anther developmental stages: pollen mother cell (Pms), tetrad stage (Tds), and mononuclear stage (Ms). A total of 498 significantly differentially expressed proteins (DEPs) were identified, with 194 upregulated and 304 downregulated. Bioinformatic analysis revealed that these DEPs are significantly enriched in key metabolic pathways essential for anther development, including glycolysis/gluconeogenesis, pyruvate metabolism, the tricarboxylic acid (TCA) cycle, starch and sucrose metabolism, and fatty acid degradation. Notably, we identified 12 aldehyde dehydrogenases (ALDHs) and several cytochrome P450 proteins associated with reactive oxygen species (ROS) homeostasis and pollen wall formation. This study presents a systematic, stage-resolved proteomic atlas of CMS in cotton using DIA-MS, which reveals that widespread dysregulation of central energy and secondary metabolite pathways underpins the sterility phenotype. Our integrated multi-omics and functional validation approach provides novel molecular insights into CMS mechanisms and pinpoints potential targets for hybrid breeding.