Aydin Abdullah, Yerlikaya Bayram Ali, Yerlikaya Seher, Yilmaz Nisa Nur, Kavas Musa
Department of Agricultural Biotechnology, Faculty of Agriculture, Ondokuz Mayis University, Samsun, Turkey.
Plant Genome. 2025 Mar;18(1):e20542. doi: 10.1002/tpg2.20542.
Synergistic and antagonistic relationships between cytokinins and other plant growth regulators are important in response to changing environmental conditions. Our study aimed to determine the functions of SlHP2 and SlHP3, two members of cytokinin signaling in tomato, in drought stress response using CRISPR/Cas9-mediated mutagenesis. Ten distinct genome-edited lines were generated via Agrobacterium tumefaciens-mediated gene transfer and confirmed through Sanger sequencing. Stress experiments were conducted with two of these lines (slhp2,3-10 and slhp2,3-11), which harbored homozygous mutations in both genes. The responses of two lines carrying homozygous mutations in both genes under polyethylene glycol (PEG)-induced stress were examined using morphological, physiological, biochemical, and molecular methods. The genome-edited lines demonstrated enhanced water retention, reduced stomatal density, and less oxidative damage compared to the wild-type plants under PEG-induced stress. Moreover, the slhp2,3 double mutant plants exhibited improved root growth, showcasing their superior drought tolerance over wild-type plants by accessing deeper water sources and maintaining hydration in water-limited environments. To investigate the involvement of cytokinin signaling regulators and genes associated with stomatal formation and differentiation, the expression of genes (Speechless [SPCH], FAMA, MUTE, TMM, HB25, HB31, RR6, RR7, and Solyc02g080860) was assessed. The results revealed that all regulators were downregulated, with SPCH, TMM, RR7, and RR6 showing significant reductions under PEG-induced stress. These results emphasize the promise of utilizing CRISPR/Cas9 to target cytokinin signaling pathways, enhancing drought tolerance in tomatoes through improvements in water retention and root growth, along with a reduction in stomatal density and malondialdehyde content.
细胞分裂素与其他植物生长调节剂之间的协同和拮抗关系对于应对不断变化的环境条件至关重要。我们的研究旨在利用CRISPR/Cas9介导的诱变技术，确定番茄中细胞分裂素信号传导的两个成员SlHP2和SlHP3在干旱胁迫响应中的功能。通过根癌农杆菌介导的基因转移产生了10个不同的基因组编辑株系，并通过桑格测序进行了确认。对其中两个在两个基因中都携带纯合突变的株系（slhp2,3-10和slhp2,3-11）进行了胁迫实验。使用形态学、生理学、生物化学和分子方法研究了这两个在两个基因中都携带纯合突变的株系在聚乙二醇（PEG）诱导的胁迫下的反应。与野生型植物相比，在PEG诱导的胁迫下，基因组编辑株系表现出更强的保水能力、更低的气孔密度和更少的氧化损伤。此外，slhp2,3双突变体植物表现出更好的根系生长，通过获取更深层的水源并在水分有限的环境中保持水分，显示出其比野生型植物更强的耐旱性。为了研究细胞分裂素信号调节因子以及与气孔形成和分化相关的基因的参与情况，评估了基因（无言语[SPCH]、FAMA、MUTE、TMM、HB25、HB31、RR6、RR7和Solyc02g080860）的表达。结果显示，所有调节因子均下调，其中SPCH、TMM、RR7和RR6在PEG诱导的胁迫下显著降低。这些结果强调了利用CRISPR/Cas9靶向细胞分裂素信号通路的前景，通过改善保水能力和根系生长、降低气孔密度和丙二醛含量来提高番茄的耐旱性。
