Seo Hyeon Ung, Jang Cheol Seong
Plant Genomics Laboratory, Graduate School, Interdisciplinary Program in Smart Agriculture, Kangwon National University, Chuncheon, 24341, Republic of Korea.
Plant Mol Biol. 2025 May 6;115(3):63. doi: 10.1007/s11103-025-01581-x.
Salinity is a major problem due to the continuous increase in the salinization of agricultural lands, particularly, paddy fields. Using a forward genetics approach, salt-insensitive TILLING line 3, sitl3, was selected from a core population induced by gamma-ray irradiation. Under salt stress, sitl3 had greater fresh weight and chlorophyll content, and lower HO and Na contents than the wild-type. In the gene (LOC_Os07g46180) with two PWWP domains (named Oyza sativa PWWP4, OsPWWP4) of sitl3, a premature stop was caused by an SNP, and was named OsPWWP4p.Gly462* (a stop gain occurred from the 462th amino acid residue). The OsPWWP4 and substrate proteins (OsEULS2, OsEULS3, and OsEULD2) were identified using yeast two-hybrid, bimolecular fluorescence complementation, in vitro pull-down, and in vitro methyltransferase assays. Subcellular localization of OsPWWP4 and OsPWWP4p.Gly462*GFP-tagged proteins revealed they were both localized in the nucleus, while OsEULS2, OsEULS3, and OsEULD2 GFP-tagged proteins were found in the nucleus and cytosol of rice protoplasts. The expression levels of OsEULS2, OsEULS3, OsEULD2 under salt stress were higher in sitl3 than in wild-type plants. In contrast, OsPWWP4 expression was higher in the latter. Genes involved in the salt overly sensitive (SOS) pathway showed higher expression in the aerial tissues of silt3 than in the wild-type. CRISPR/Cas9-mediated OsPWWP4 knock-out transgenic plants showed salt tolerance phenotypes with low Na contents and low Na/K ratios. The data suggest that sitl3 is a valuable genetic resource for understanding protein post-translational regulation-related salinity tolerance mechanisms such as methyltransferase activities, and for improving salt tolerance in rice through breeding.
由于农田，特别是稻田的盐渍化不断加剧，盐度已成为一个主要问题。采用正向遗传学方法，从经伽马射线辐照诱导的核心群体中筛选出耐盐的定向诱导基因组局部突变（TILLING）品系3，即sitl3。在盐胁迫下，sitl3的鲜重和叶绿素含量更高，而过氧化氢（HO）和钠含量低于野生型。在sitl3中具有两个PWWP结构域的基因（LOC_Os07g46180）（命名为水稻PWWP4，OsPWWP4）中，一个单核苷酸多态性（SNP）导致了提前终止，命名为OsPWWP4p.Gly462*（从第462个氨基酸残基处发生了终止获得）。利用酵母双杂交、双分子荧光互补、体外下拉和体外甲基转移酶分析鉴定了OsPWWP4和底物蛋白（OsEULS2、OsEULS3和OsEULD2）。OsPWWP4和OsPWWP4p.Gly462*绿色荧光蛋白（GFP）标记蛋白的亚细胞定位显示它们都定位于细胞核，而OsEULS2、OsEULS3和OsEULD2 GFP标记蛋白在水稻原生质体的细胞核和细胞质中被发现。在盐胁迫下，sitl3中OsEULS2、OsEULS3、OsEULD2的表达水平高于野生型植株。相反，OsPWWP4在后者中的表达更高。参与盐过敏（SOS）途径的基因在sitl3地上组织中的表达高于野生型。CRISPR/Cas9介导的OsPWWP4敲除转基因植株表现出耐盐表型，钠含量低，钠/钾比低。数据表明，sitl3是理解蛋白质翻译后调控相关的耐盐机制（如甲基转移酶活性）以及通过育种提高水稻耐盐性的宝贵遗传资源。
