Mitochondrial gene orf224 triggers retrograde signaling and reprogramming of anther cell fate in Brassica napus Polima CMS.

Cytoplasmic male sterility (CMS) is widely used in rapeseed hybrid breeding. The Polima (pol) CMS system is associated with the mitochondrial chimeric gene orf224, yet how it disrupts early anther differentiation and leads to sterility remains unclear. We combined cytological analyses, ultrastructural observations, transcriptomics, proteomics, and metabolomics to examine young anther development in pol CMS, maintainer, and restorer lines, with particular focus on mitochondrial function, metabolism, and hormone homeostasis. In pol CMS, L2-derived anther cells failed to differentiate into the middle layer, tapetum, and microspore mother cells, with development arrested at stage S5. The ORF224 protein preferentially accumulated in young anthers and exhibited cytotoxicity, causing mitochondrial dysfunction, reduced ATP production, and activation of mitochondrial retrograde signaling. These changes induced extensive metabolic reprogramming, including ectopic activation of photosynthesis-like reaction, starch accumulation, elevated ROS, and a pronounced increase in abscisic acid (ABA). Our results demonstrate that pol CMS sterility results from ORF224-mediated mitochondrial stress that reprograms anther cell fate from reproductive to somatic development, with ABA reinforcing developmental arrest, while programmed cell death occurs as a late downstream consequence.