Metabolic and Gene Expression Profile Reveal Genetic Basis of Reproductive Stage Drought Tolerance in a Drought‐Defying Rice Mutant

ABSTRACT Mutation breeding provides a powerful route to generate novel genetic variation and to uncover genes underlying stress tolerance. Identifying mechanisms that sustain rice productivity under water‐limited conditions is a central challenge in plant biology. We developed a gamma‐ray–mutagenised population of Oryza sativa L. cv. IR64 and systematically screened the M 2 , M 3 and M 4 generations for drought tolerance at the seedling, pre‐flowering and post‐flowering stages. Through this multi‐stage, multi‐generational, and multi‐environment phenotypic selection, we identified a drought‐defying (DD) mutant line that displayed pronounced drought tolerance with an approximately threefold yield advantage under drought over the wild type (WT). Physiological analyses revealed that, under drought stress, the DD mutant consistently showed enhanced physiological performance, accumulated higher levels of osmolytes, and maintained a more robust antioxidant defence system than the WT. Metabolomic and targeted gene expression analyses showed enhanced accumulation of drought‐responsive metabolites (with 46% of the significant metabolites in the DD mutant displaying a response similar to N22) associated with coordinated upregulation of stress‐responsive pathways, including DREB ( OsDREB2A, OsDREB2B ), glyoxalases ( OsGLYI‐11.2, OsGLYII.2, OsGLYIII ) and trehalose‐6‐phosphate synthase ( OsTPS ), in the DD mutant relative to the WT. Notably, MutMap + analysis, supported by improved IR64 genome assembly, pinpointed the aldo‐keto reductase OsAKR candidate gene on chromosome 4 as a major contributor to the drought‐tolerant phenotype. Elevated expression of OsAKR in the DD mutant was associated with enhanced detoxification of reactive carbonyl species, including methylglyoxal (MG) and malondialdehyde (MDA), under drought stress. Together, these findings provide mechanistic insight into redox and carbonyl homeostasis in drought tolerance and establish this mutant as a valuable genetic resource for dissecting and improving stress resilience in rice.