Temperature-sensitive genic male sterility (TGMS) is the foundation of two-line hybrid rice breeding, with fertility transition primarily regulated by temperature during the thermosensitive stage of microsporogenesis. Although temperature is the primary regulator of fertility transition, photoperiod modulates sterility expression by influencing the duration of exposure during the thermosensitive phase. This study investigated the interactive effects of temperature and photoperiod on a tms5-derived TGMS red rice line (EC720903 and derivatives) through integrated morphological, biochemical, and targeted metabolomic analyses. Elevated temperature induced complete male sterility, which wasaccompanied with increased reactive oxygen species (ROS) accumulation, lipid peroxidation, membrane damage, hormonal imbalance, and metabolic reprogramming of amino acids and phenolic compounds. Photoperiod extension alone induced moderate pollen sterility, while its combination with elevated temperature further intensified oxidative and metabolic perturbations, resulting in the greatest reduction in pollen viability. The combined temperature × photoperiod treatment was characterized by enhanced accumulation of H₂O₂, and malondialdehyde, together with pronounced hormonal dysregulation and coordinated changes in amino acid and phenylpropanoid metabolism, indicating disruption of tapetal function and pollen development. Collectively, the findings demonstrate that temperature and photoperiod interact to regulate fertility expression through coordinated physiological and metabolic responses, with temperature acting as the primary determinant and photoperiod functioning as a complementary regulator of sterility. The observed interaction further suggests that photoperiod management may help maintain sterility expression when environmental temperatures fluctuate near the critical sterility threshold, thereby improving the stability of TGMS-based hybrid seed production under variable climatic conditions.
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