Plant Genomics Laboratory, Interdisciplinary Program in Smart Agriculture, Kangwon National University, Chuncheon, Republic of Korea.
Molecular Plant Physiology Laboratory, Department of Plant Life & Resource Sciences, Sangji University, Wonju, Republic of Korea.
Physiol Plant. 2024 Sep-Oct;176(5):e14565. doi: 10.1111/ppl.14565.
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 (M). Under drought conditions, ditl4 exhibited greater fresh weight, survival rate, chlorophyll, proline, and soluble sugar contents, and lower HO 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 CO 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.
干旱是一种主要的环境胁迫因素，会对水稻的生长和产量产生负面影响。从正向遗传学的角度来看，我们从伽马射线诱导的核心突变体群体（M）中选择了一个抗旱 TILLING 系（ditl4）。在干旱条件下，ditl4 的鲜重、存活率、叶绿素、脯氨酸和可溶性糖含量较高，HO 和 MDA 水平较低，而野生型（WT）则较低。此外，抗氧化酶如超氧化物歧化酶、过氧化氢酶和过氧化物酶的活性在 ditl4 中高于 WT。在相对水分损失测定中，与 WT 相比，dilt4 的叶片卷曲和水分损失明显减少。此外，在干旱胁迫下，dilt4 中的“关闭”气孔孔径比例增加，表明蒸腾减少以防止水分流失。由于气孔密度较低，ditl4 突变体表现出较低的气孔导度、蒸腾作用和 CO 同化作用，以及较高的水分利用效率。对 dilt4 的全基因组重测序分析发现，OsDIRH2（LOC_Os11g39640）中的一个单核苷酸多态性（SNP），注释为 RING-H2 型 E3 连接酶，导致提前出现终止密码子。CRISPR/Cas9 介导的敲除突变体（OsDIRH2a 和 OsDIRH2b）通过降低气孔密度提高了抗旱性，与空载体对照植物相比。这些发现表明，具有低气孔密度的 ditl4 可用作耐旱性育种计划的遗传资源，以提高水利用效率。
