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 (OsDIAT^OX) 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 OsDIAT^OX 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.

Abiotic stresses severely affect plant growth and productivity. To cope with abiotic stresses, plants have evolved tolerance mechanisms that are tightly regulated by reprogramming transcription factors (TFs). APETALA2/ethylene-responsive factor (AP2/ERF) transcription factors are known to play an important role in various abiotic stresses. However, our understanding of the molecular mechanisms remains incomplete. In this study, we identified the role of OsERF83, a member of the AP2/ERF transcription factor family, in response to drought stress. OsERF83 is a transcription factor localized to the nucleus and induced in response to various abiotic stresses, such as drought and abscisic acid (ABA). Overexpression of OsERF83 in transgenic plants (OsERF83^OX) significantly increased drought tolerance, with higher photochemical efficiency in rice. OsERF83^OX was also associated with growth retardation, with reduced grain yields under normal growth conditions. OsERF83 is predominantly expressed in the vascular tissue of all organs. Transcriptome analysis revealed that OsERF83 regulates drought response genes, which are related to the transporter (OsNPF8.10, OsNPF8.17, OsLH1), lignin biosynthesis (OsLAC17, OsLAC10, CAD8D), terpenoid synthesis (OsTPS33, OsTPS14, OsTPS3), cytochrome P450 family (Oscyp71Z4, CYP76M10), and abiotic stress-related genes (OsSAP, OsLEA14, PCC13-62). OsERF83 also up-regulates biotic stress-associated genes, including PATHOGENESIS-RELATED PROTEIN (PR), WALL-ASSOCIATED KINASE (WAK), CELLULOSE SYNTHASE-LIKE PROTEIN E1 (CslE1), and LYSM RECEPTOR-LIKE KINASE (RLK) genes. Our results provide new insight into the multiple roles of OsERF83 in the cross-talk between abiotic and biotic stress signaling pathways.

Plants have evolved sophisticated defense systems to enhance drought tolerance. These include the microRNA (miRNA) group of small noncoding RNAs that act as post‐transcriptional regulators; however, details of the mechanisms by which they confer drought tolerance are not well understood. Here, we show thatosa‐MIR171f, a member ofosa‐MIR171gene family, is mainly expressed in response to drought stress and regulates the transcript levels ofSCARECROW‐LIKE6‐I(SCL6‐I) andSCL6‐IIin rice (Oryza sativa). TheSCL6genes are known to be involved in shoot branching and flag leaf morphology.Osa‐MIR171f‐overexpressing (osa‐MIR171f‐OE) transgenic plants showed reduced drought symptoms compared with non‐transgenic (NT) control plants under both field drought and polyethylene glycol (PEG)‐mediated dehydration stress conditions. Transcriptome analysis ofosa‐MIR171f‐OE plants andosa‐mir171f‐knockout (K/O) lines generated by clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) revealed thatosa‐mature‐miR171a‐f (osa‐miR171) regulates the expression of flavonoid biosynthesis genes, consequently leading to drought tolerance. This upregulation in theosa‐MIR171f‐OE plants, which did not occur in NT control plants, was observed under both normal and drought conditions. Our findings indicate thatosa‐miR171 plays a role in drought tolerance by regulatingSCL6‐IandSCL6‐IItranscript levels.

Drought is one of the major environmental stresses adversely affecting crop productivity worldwide. Precise characterization of genes involved in drought response is necessary to develop new crop varieties with enhanced drought tolerance. Previously, we identified 66 drought-induced miRNAs in rice plants. For the further functional investigation of the miRNAs, we applied recombinant codon-optimized Cas9 (rCas9) for rice with single-guide RNAs specifically targeting mature miRNA sequences or sites required for the biogenesis of mature miRNA. A total of 458 T₀ transgenic plants were analyzed to determine the frequency and type of mutations induced by CRISPR/rCas9 on 13 independent target miRNAs. The average mutation frequency for 13 genes targeted by single guide RNAs (sgRNAs) in T₀ generation was 59.4%, including mono-allelic (8.54%), bi-allelic (11.1%), and hetero-allelic combination (39.7%) mutations. The mutation frequency showed a positive correlation with Tm temperature of sgRNAs. For base insertion, one base insertion (99%) was predominantly detected in transgenic plants. Similarly, one base deletion accounted for the highest percentage, but there was also a significant percentage of cases in which more than one base was deleted. The deletion of more than two bases in OsmiR171f and OsmiR818b significantly reduced the level of corresponding mature miRNAs. Further functional analysis using CRISPR/Cas9-mediated mutagenesis confirmed that OsmiR818b is involved in drought response in rice plants. Overall, this study suggests that the CRISPR/rCas9 system is a powerful tool for loss-of-function analysis of miRNA in rice.