The natural Msc-4 allele confers genic male sterility via conserved and divergent gene regulatory mechanisms in pepper.

Utilization of genic male sterility (GMS) significantly reduces production costs and ensures the high varietal purity of hybrid seeds, thereby demonstrating considerable potential for hybrid seed production in pepper (Capsicum annuum). In this study, we report the characterization of a pepper GMS mutant, male sterility from China-4 (msc-4), which exhibits aborted pollen grains without detectable exine. Strikingly, the vegetative growth of these pepper plants was unaffected. Genome-based mapping and functional analysis of msc-4 revealed that a nonsense mutation in a cytochrome P450 family gene, CYP703A2/Msc-4, results in pollen development failure and subsequent GMS. Msc-4 transcripts specifically accumulated in the tapetum and microspores of developing anthers. Comparative transcriptome analysis suggested that fatty acid and sporopollenin metabolism are severely impaired in msc-4 mutants. Furthermore, we demonstrated that the AMS-MYB80-CYP703A2/Msc-4 regulatory cascade is generally conserved, albeit with some differences, in pepper. Importantly, mutations in the tomato and cucumber CYP703A2 orthologs conferred male sterility without growth penalty, strongly supporting that CYP703A2 genes are ideal targets for the generation of GMS germplasms. Overall, our findings shed light on the molecular basis of Msc-4 in pepper, demonstrating that mutagenesis of CYP703A2 genes can be readily adapted for a broad range of vegetable crops.