OsDIAT enhances cold tolerance in indica rice by modulating branched-chain amino acid metabolism

Low-temperature (LT) stress severely limits rice growth and productivity, particularly in the chilling-sensitive indica subspecies. Here, we investigated the physiological, transcriptomic, and metabolomic responses of two similar genetic backgrounds indica rice varieties but contrasting cold tolerance: Diantun502 and Diantun506. Under cold stress, Diantun502 exhibited higher survival rates, greater chlorophyll retention, and elevated accumulation of osmoprotectants, branched-chain amino acids (BCAAs), and stress-related phytohormones compared to Diantun506. Integrative multi-omics analysis revealed that amino acid metabolism, BCAA-related pathways, was prominently enriched among differentially expressed genes and metabolites. We identified OsDIAT (Os05g0244700), encoding a branched-chain amino acid aminotransferase (BCAT), as a key candidate gene. Sequence variation in Diantun506 introduced a premature stop codon and generated a truncated protein with negligible catalytic activity. In an F₂ population derived from Diantun502 × Diantun506, cold-tolerance phenotypes were closely associated with OsDIAT genotypes, supporting the contribution of the Diantun506 allele to reduced LT tolerance. Overexpression of OsDIAT enhanced LT tolerance, BCAT activity, BCAA accumulation, and stress-related hormone levels, whereas knockout produced the opposite effects. Exogenous isoleucine partially restored the survival of knockout plants and promoted JA-Ile accumulation, the JA-Ile/JA ratio, and OsJAR1 expression. Collectively, these findings demonstrate that OsDIAT enhances LT tolerance by maintaining BCAA homeostasis and supporting associated JA-Ile responses, and provide a useful genetic target and molecular marker for breeding cold-tolerant indica rice.