CitACOS5, a fatty acyl-CoA synthetase, is crucial for male fertility in citrus by influencing pollen exine formation

Male sterility (MS) holds great potential and value for breeding seedless cultivars of fruit trees. However, functional characterization of MS-related genes has been difficult in perennial fruit crops, because of the extended juvenile period and difficulties in genetic transformation. In this study, we compared anther development of 'Qianyang Seedless' (QS), a natural seedless bud sport (somatic mutation) of Ponkan mandarin (Citrus reticulata Blanco) with a stable MS phenotype, with that of the seedy wild-type 'Egan No. 1' (EG). We observed significant abnormalities in QS at the microspore mother cell stage, leading to the generation of abnormal tetrads and non-viable pollen. A transcriptomic analysis of anthers at the microspore mother cell stage showed a significant down-regulation of the sporopollenin biosynthesis pathway genes in QS. Among them, CitACOS5 showed pronounced down-regulation in QS. CRISPR/Cas9 mutants of Mini-citrus (Fortunella hindsii), cr-acos5, exhibited complete MS phenotypes, characterized by the absence of pollen grains due to defects in pollen wall formation and tapetum degradation. Biochemical assays validated that CitMYB80 directly bound to the promoter of CitACOS5 and activated its expression. Our results suggest that CitACOS5 is crucial for male fertility in citrus by influencing pollen exine formation, which offers potential target genes for seedless citrus breeding.