The chimeric gene orf610a reduces cotton pollen fertility by impairing the assembly of ATP synthase

SummaryCytoplasmic male sterility (CMS) serves as a pivotal tool for exploiting hybrid vigour and studying nuclear‐cytoplasmic interactions. Despite its long‐standing use in cotton breeding, the underlying mechanisms of the CMS‐D2 system remain elusive. Our study unravelled the role of the mitochondrial chimeric gene orf610a in reducing fertility in cotton through its interaction with ATP synthase subunit D (atpQ). Using yeast two‐hybrid, bimolecular luciferase complementation, and transgenic overexpression studies, we identified a unique interaction between orf610a and atpQ, which disturbs the assembly of ATP synthase. This interaction leads to a decrease in ATP levels, an increase in H2O2 production, and mitochondrial dysfunctions, which are associated with pollen abortion. Transcriptomic and biochemical analyses of three independent overexpression lines identified 1711 differentially expressed genes (DEGs), among which 10 were related to reactive oxygen species (ROS) and ATP production. Phenotypic analysis confirmed that orf610a expression causes abnormal anther development and reduced pollen viability, contributing to sterility. Notably, SEM and TEM analyses highlighted structural anomalies in the pollen of orf610a‐overexpressing lines, supporting the detrimental impacts of altered ATP synthase function. Our findings suggest that orf610a's interaction with ATP synthase components disrupts normal mitochondrial function and energy production, leading to male sterility in cotton. Understanding the molecular interactions involved in CMS can aid in developing strategies to manipulate sterility for crop improvement, offering insights into mitochondrial‐nuclear interactions that could impact future breeding programmes.