Abstract
Cytoplasmic male sterile (CMS) systems have great potential in hybrid seed production in many plants. However, the incompatibility between the cytoplasmic and nuclear genes and the availability of fewer CMS lines limit the system's application in cotton heterosis. Therefore, the present study sequenced the mitochondrial (mt) genomes of a novel cotton (Gossypium hirsutum L.) CMS line J4A and its cytoplasmic nuclear homologous maintainer line J4B to investigate the mechanism underlying CMS and improve its application. A novel CMS gene, orf116b, was identified in the CMS line. Ectopic expression of orf116b in Escherichia coli suppressed growth, while its overexpression in Arabidopsis, rice, tobacco, and cotton led to complete or partial male sterility. Further analysis of anthers revealed mt disruption in J4A with higher levels of hydrogen peroxide (H2O2) and malondialdehyde (MDA), but lower levels of ATP and ribosomal protein (RP) than in J4B. Finally, a membrane-based yeast two-hybrid (MYTH) assay and bimolecular fluorescence complementation (BiFC) assays demonstrated that orf116b probably interacts with an anther-specific protein, LAT52. These observations collectively proved that orf116b is associated with early and stable pollen abortion in cotton, providing a foundation for further research on cotton fertility restoration and heterosis breeding.