Transcriptome sequencing data analysis.

Male sterility (MS) plays a crucial role in plant reproduction and hybrid breeding as it is associated with pollen viability and release. However, the regulatory mechanisms governing anther dehiscence in peppers remain poorly characterized. Thus, this study identified the pepper C2H2 family transcription factor CaZAT5 and characterized its function. The results indicated that CaZAT5 represses transcriptional activity and is predominantly expressed during pepper flower development. Silencing CaZAT5 in pepper led to early flowering, whereas its overexpression (OE) in tomato delayed flowering. Moreover, CaZAT5 negatively regulated vegetative growth by suppressing CaSOC1 expression, thereby affecting pollen morphology and viability. Histological analyses revealed that the anthers of CaZAT5-OE plants exhibited abnormal mitosis, resulting in both enlarged and shrunken pollen grains. Additionally, CaZAT5 overexpression inhibited anther dehiscence during pollen maturation, affecting pollen release. The consequent reduction in pollen viability and inhibited anther dehiscence decreased fruit set and yield in the plants. Transcriptome (RNA-seq) analysis revealed that CaZAT5 overexpression suppressed the expression of genes involved in cell wall loosening, degradation, and secondary wall thickening in the anthers. DAP-seq, Y1H, Dual-LUC, and EMSA identified potential CaZAT5-regulated genes involved in anther dehiscence, including cell wall degradation genes (CaPG and CaBG4) and the expansin gene CaExpA13. Collectively, these findings suggest that CaZAT5 modulates flowering time, pollen development, and anther dehiscence by regulating the expression of genes related to flowering and cell wall loosening and degradation. These findings contribute to a more comprehensive understanding of the potential role of CaZAT5 in regulating flowering time and male fertility.