Mutation of a RING E3 ligase, OsDIRH2, enhances drought tolerance in rice with low stomata density

Drought is a major environmental stress factor that negatively affects rice growth and yield. From a forward genetic perspective, we selected a drought-insensitive TILLING line (ditl4) from a gamma-ray-induced core mutant population (M₁₀). Under drought conditions, ditl4 exhibited greater fresh weight, survival rate, chlorophyll, proline, and soluble sugar contents, and lower H₂O₂ and MDA levels than wild-type (WT). In addition, the activities of antioxidant enzymes, such as superoxide dismutase, catalase, and peroxidase, were higher in ditl4 than in the WT. In the relative water loss assay, dilt4 showed significantly decreased leaf curling and water loss compared to WT. Also, the ratio of "closed" stomata aperture was increased in ditl4 under drought stress, suggesting reduced transpiration to prevent water loss. The ditl4 mutant showed decreased stomatal conductance, transpiration, and CO₂ assimilation and increased water use efficiency due to the low density of stomata. Whole-genome resequencing analysis of dilt4 identified a single nucleotide polymorphism (SNP) in OsDIRH2 (LOC_Os11g39640), annotated as a RING-H2 type E3 ligase, resulting in a premature stop codon. CRISPR/Cas9-mediated knock-out mutants (OsDIRH2a and OsDIRH2b) enhanced drought tolerance by lowering stomatal density compared to empty vector control plants. These findings suggested that ditl4 with low stomatal density would be useful as a genetic resource for a drought-tolerant breeding program to improve water-use efficiency.