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.

Fruit ripening is a complex, tightly regulated process that affects fruit flavor, nutritional value, and shelf life. Citrus is a typical non-climacteric fruit, as citrus fruits ripen independently of ethylene. However, the precise regulatory mechanism underlying this process remains to be elucidated. To dissect the epigenetic and transcriptional basis of citrus ripening, we performed integrative analyses of genome-wide histone modifications (H3K27ac, H3K4me3, and H3K27me3), chromatin accessibility, and transcriptome profiles throughout fruit ripening in Citrus reticulata. We constructed a transcriptional regulatory network encompassing 52 key transcription factors that orchestrate citrus fruit ripening. Further, we identified the key role of abscisic acid (ABA) in modulating sucrose and citric acid metabolism and uncovered the underlying transcriptional and epigenetic regulatory network during fruit ripening in C. reticulata. The findings demonstrate that CrHSFA6B and CrbZIP5b interact with the promoters of CrACO1 (Aconitase 1) and CrACO3 and CrSPSF1 (Sucrose Phosphate Synthase F1), regulating their expression to affect the sucrose and citric acid metabolism and fruit ripening. This study provides insights into the epigenomic dynamics of citrus fruit ripening and identifies a putative regulatory cascade centered on ABA, CrbZIP5b, and CrHSFA6B that modulates key processes in citrus ripening.

Citrus fruits are widely cultivated all over the world. Due to climatic conditions, citrus fruits are frequently exposed to periodic low temperatures, which poses a serious threat to their yield and quality. Cold not only restricts plant growth and deteriorates fruit quality but also leads to fruit abscission and tree mortality, posing severe constraints on large-scale citrus production. The TIFY family gene plays crucial roles in plant development and stress adaptation. However, the genome-wide identification and functional analysis of TIFY genes in cold stress adaptation of citrus plants remain largely unexplored. Here, we performed a systematic genome-wide analysis of the TIFY family in sweet orange (Citrus sinensis (L.) Osbeck) and identified 14 CsTIFY members. We conducted a comprehensive study on the protein characteristics, phylogenetic relationships, gene structure, chromosome distribution, promoter cis-acting elements, and subcellular localization of these genes. Phylogenetic analysis classified the CsTIFYs into ZML (ZML1–ZML4), JAZ (JAZ1–JAZ7), PPD (JAZ8, JAZ9), and TIFY (TIFY1) subfamilies, and they are distributed on seven chromosomes. Collinearity analysis revealed that segmental duplication is the primary driver for CsTIFY family expansion. Expression profiling under cold stress identified JAZ1, JAZ2, and JAZ3 as the most cold-inducible members. All three CsTIFY proteins are targeted to the nucleus, as confirmed by subcellular localization analysis. Overexpression of JAZ1, JAZ2, or JAZ3 in citrus calli significantly enhanced cold sensibility. Collectively, this study elucidates the gene function of CsTIFYs under cold stress and provides new insight for molecular breeding of cold-tolerant citrus varieties.