The rice transcription factor S40-14 regulates chloroplast and ROS-related gene networks and promotes dark-induced leaf senescence.

Leaf senescence triggers major metabolic changes that recycle resources and ensure plant survival and is sensitive to environmental conditions. In this study, we characterized OsS40-14 using CRISPR/Ca9 mutants and overexpression lines to address its function in rice (Oryza sativa) flag leaf senescence. The oss40-14 mutants displayed a stay-green leaf phenotype under dark treatment, with preserved chlorophylls and photosynthetic capacity and reduced reactive oxygen species (ROS) levels. In contrast, OsS40-14 overexpression lines exhibited accelerated leaf senescence. Transcriptome analysis of dark-treated flag leaf samples revealed that the 1585 differentially expressed genes in oss40-14 vs WT were enriched in macromolecular metabolism, photosynthesis, and stress responses. In addition, tsCUT&Tag-seq analysis using GFP-tagged OsS40-14 revealed 2311 genomic loci bound by OsS40-14, with approximately 40.95% of the binding signal enriched at transcription start sites. The consensus binding motif of OsS40-14 was identified as TACCCACAAGACAC, with a seed sequence of 'ACCCA'. Finally, integrated analysis of the transcriptome and tsCUT&Tag-seq datasets revealed that 153 OsS40-14-targeted candidates comprised 66 potentially repressed genes enriched in plastid organization and photosynthetic processes, and 41 putatively activated genes associated with stress signaling and senescence. Overall, our results suggest that OsS40-14 fine-tunes the photosynthetic machinery and ROS homeostasis during developmental senescence and under dark conditions, thereby accelerating leaf senescence, reducing photosynthetic assimilation and grain filling in rice.