CitACOS5 is crucial for male fertility in citrus by influencing pollen exine formation.

Abstract

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 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 (MMC) stage, leading to the generation of abnormal tetrads and nonviable pollen. The transcriptomic analysis of anthers at the MMC stage showed a significant downregulation of the sporopollenin biosynthesis pathway genes in QS. Among them, CitACOS5 was pronouncedly downregulated in QS. The CRISPR/Cas9 mutants in Mini-citrus, 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.