Integrated physiological, proteomic, and phosphoproteomic analyses of 24-epibrassinolide-mediated yield protection...

Complete submergence during panicle differentiation can severely reduce rice yield, yet the physiological and molecular responses associated with yield protection by 24-epibrassinolide (EBR) remain unclear. A two-year pot experiment evaluated submergence-sensitive Jiyou 3 and submergence-tolerant Nongxiangzhan under a non-submerged control (CK), complete submergence without EBR (SUB), and complete submergence following foliar pretreatment with 0.06, 0.12, or 0.24 mg L^-1 EBR. Submergence lasted four days. Yield and physiological traits were assessed in both cultivars, whereas tandem mass tag-based proteomic and phosphoproteomic analyses were confined to Nongxiangzhan under CK, SUB, and 0.12 mg L^-1 EBR (E12). Across cultivars and years, the greatest yield recovery among EBR treatments was observed under E12. Relative to SUB, E12 increased grain yield per plant by 33.79%-42.03%, primarily through higher grains per panicle and seed-setting rate. E12 increased superoxide dismutase and ascorbate peroxidase activities while reducing hydrogen peroxide and malondialdehyde accumulation. This improved redox status coincided with partial recovery of net photosynthetic rate and Rubisco activity. Higher phosphofructokinase, pyruvate decarboxylase, and alcohol dehydrogenase activities indicated greater glycolytic and ethanolic-fermentation capacity. Nitrogen assimilation was partially maintained, and submergence-induced leaf hormone changes were moderated. Proteomic and phosphoproteomic responses converged on photosynthesis, carbon metabolism, redox regulation, and signaling. Integrated analysis identified 14 proteins with concordant abundance and phosphorylation increases, including components of glycolysis, photosynthetic electron transfer, and antioxidant defense, whereas 74.95% of E12-responsive phosphorylation sites occurred without significant abundance changes. EBR-mediated yield preservation was associated primarily with improved redox homeostasis, maintenance of photosynthetic carbon assimilation, and adjustment of carbon metabolism under oxygen limitation, while nitrogen assimilation and hormone regulation may have supported reproductive development. These findings support further multi-environment field evaluation of EBR pretreatment as a candidate management option to reduce yield loss from short-duration complete submergence during rice panicle differentiation.