Wang Ying, Xun Hongwei, Lv Jiameng, Ju Wanting, Jiang Yuhui, Wang Meng, Guo Ruihong, Zhang Mengru, Ding Xiaoyang, Liu Bao, Xu Chunming
Key Laboratory of Molecular Epigenetics of the Ministry of Education (MOE), Northeast Normal University, Changchun, China.
Jilin Academy of Agricultural Sciences (Northeast Agricultural Research Center of China), Changchun, China.
Plant Biotechnol J. 2025 May;23(5):1585-1600. doi: 10.1111/pbi.14606. Epub 2025 Jan 31.
Cytosine methylation (mCG) is an important heritable epigenetic modification, yet its functions remain to be fully defined in important crops. This study investigates mCG in soybean following the loss-of-function mutation of two GmMET1 genes. We generated knockout mutants of GmMET1s by CRISPR-Cas9 and conducted comprehensive methylome and transcriptome analyses. Our findings unravel the functional redundancy of the two GmMET1s, with GmMET1b being more critically involved in maintaining mCG levels, and complete knockout of both copies is lethal. We establish that genome-wide mCG levels scale with aggregated expression of GmMET1s. We identify a set of mCG-regulated genes whose expression levels were quantitatively modulated by upstream, body, or downstream mCG. Moreover, we find genes that were negatively regulated by upstream or body mCG are enriched in specific biological processes such as that of jasmonic acid metabolism. Notably, >80% of the differentially methylated genes (DMGs) in the mutants also exist as DMGs in natural soybean populations. Phenotypically, mutants that are heterozygous for GmMET1a and homozygous for GmMET1b knockouts (GmMET1aGmMET1b) exhibited early flowering, which was inherited by their selfed progeny (GmMET1aGmMET1b) with otherwise normal growth and development. Moreover, mutation of either GmMET1s, with slight reduction of mCG levels and similar phenotypes compared to the wild type under normal conditions, showed enhanced tolerance to cold and drought stresses. Together, our results underscore highly orchestrated regulatory effects of mCG on gene expression in soybean, which dictates growth, development and stress responses, implicating its utility in the improvement of soybean for better adaptability and higher yield.
胞嘧啶甲基化（mCG）是一种重要的可遗传表观遗传修饰，但其在重要作物中的功能仍有待充分明确。本研究在两个GmMET1基因功能丧失突变后对大豆中的mCG进行了研究。我们通过CRISPR-Cas9技术生成了GmMET1s的敲除突变体，并进行了全面的甲基化组和转录组分析。我们的研究结果揭示了两个GmMET1s的功能冗余，其中GmMET1b在维持mCG水平方面发挥更关键的作用，两个拷贝的完全敲除是致死的。我们确定全基因组mCG水平与GmMET1s的聚合表达呈比例关系。我们鉴定出一组mCG调控基因，其表达水平受到上游、基因体或下游mCG的定量调节。此外，我们发现受上游或基因体mCG负调控的基因在茉莉酸代谢等特定生物学过程中富集。值得注意的是，突变体中>80%的差异甲基化基因（DMG）在天然大豆群体中也作为DMG存在。在表型上，GmMET1a杂合且GmMET1b敲除纯合的突变体（GmMET1aGmMET1b）表现出早花，其自交后代（GmMET1aGmMET1b）继承了这一性状，且生长发育正常。此外，任一GmMET1s发生突变，与正常条件下的野生型相比，mCG水平略有降低且表型相似，但对寒冷和干旱胁迫的耐受性增强。总之，我们的结果强调了mCG对大豆基因表达具有高度协调的调控作用，这种调控作用决定了大豆的生长、发育和胁迫反应，暗示了其在改良大豆以提高适应性和产量方面的实用性。
