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Plants accumulate several metabolites in response to drought stress, including branched-chain amino acids (BCAAs). However, the roles of BCAAs in plant drought responses and the underlying molecular mechanisms for BCAA accumulation remain elusive. Here, we demonstrate that rice (Oryza sativa) DROUGHT-INDUCED BRANCHED-CHAIN AMINO ACID AMINOTRANSFERASE (OsDIAT) mediates the accumulation of BCAAs in rice in response to drought stress. An in vitro enzyme activity assay indicated that OsDIAT is a branched-chain amino acid aminotransferase, and subcellular localization analysis revealed that OsDIAT localizes to the cytoplasm. The expression of OsDIAT was induced in plants upon exposure to abiotic stress. OsDIAT-overexpressing (OsDIATOX) plants were more tolerant to drought stress, whereas osdiat plants were more susceptible to drought stress compared with nontransgenic (NT) plants. Amino acid analysis revealed that BCAA levels were higher in OsDIATOX but lower in osdiat compared with in NT plants. Finally, the exogenous application of BCAAs improved plant tolerance to osmotic stress compared with that in control plants. Collectively, these findings suggest that OsDIAT mediates drought tolerance by promoting the accumulation of BCAAs., Competing Interests: Conflict of interest statement. No conflict of interest is declared., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)