RbohF-mediated ROS production is required for tapetal PCD and pollen maturation in rice

Reactive oxygen species (ROS) function as critical signaling molecules during plant reproductive development, particularly in regulating tapetal programmed cell death (PCD). However, the molecular mechanisms controlling ROS production in rice anthers remain incompletely understood. In this study, we characterized the role of RbohF, a rice respiratory burst oxidase homolog, using CRISPR/Cas9 knockout lines. Loss of RbohF resulted in severe defects in anther development, including impaired pollen maturation and reduced fertility. Histological analyses revealed progressive degeneration of microspores and abnormal tapetal persistence in the mutant. ROS staining demonstrated that superoxide and hydrogen peroxide accumulation was significantly reduced and delayed in rbohf anthers, particularly at the young microspore stage. Consistently, TUNEL assays showed delayed tapetal PCD in the mutant, indicating that RbohF-mediated ROS production is required for the timely initiation of tapetal degeneration. Gene expression analyses further suggested that RbohF functions downstream of major transcriptional regulators of pollen development. Notably, the male-sterile phenotype of rbohf mutants was exacerbated at cooler temperatures, highlighting a role for RbohF in buffering environmental stress. Together, these findings establish RbohF as a key regulator of ROS homeostasis, integrating developmental and environmental signals to ensure proper pollen development in rice.