Yan Xiaohao, Zhou Yeling, Gan Shijie, Guo Zhiyu, Liang Jiansheng
Shenzhen Key Laboratory of Plant Genetic Engineering and Molecular Design, Institute of Plant and Food Science, Southern University of Science and Technology, Shenzhen, 518055, China.
Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, 518055, China.
Plant J. 2025 Aug;123(4):e70436. doi: 10.1111/tpj.70436.
DNA methylation (5-methylcytosine, 5mC) is a key epigenetic regulator of genome stability and stress adaptation in plants. However, the functional role of its oxidative derivative, 5-hydroxymethylcytosine (5hmC), remains poorly understood in plant systems, largely due to its low abundance and unresolved enzymatic origins. Here, we integrated ACE-seq (APOBEC-coupled epigenetic sequencing) with an optimized Tn5mC-seq (transposase-based library preparation in the context of whole-genome bisulfite sequencing, WGBS) approach to generate the first single-base resolution map of 5hmC in rice (Oryza sativa), unveiling its stress-responsive dynamics and regulatory interplay with 5mC during drought adaptation. Genome-wide profiling revealed a basal 5hmC level of ~0.03 (defined as the ratio of C/(C + T) at each site), with drought triggering a pronounced reduction in 5hmC abundance and locus number, followed by incomplete recovery post-rehydration. Unlike 5mC, which accumulates in heterochromatin, 5hmC preferentially localized to euchromatic regions, including promoters, exons, and intergenic elements, and exhibited enrichment at ABA-responsive transcription factors (e.g., OsATAF1, bZIP50). Strikingly, drought induced an antagonistic relationship between 5hmC and 5mC, with the latter increasing globally to reinforce transposon silencing. Multi-omics analyses demonstrated that 5hmC depletion in promoters correlated with transcriptional downregulation, while its accumulation in gene bodies (notably 5'-UTRs) suppressed stress-responsive genes. These findings highlight 5hmC's bifunctional regulatory capacity, contingent on genomic context, and its role in balancing transcriptional plasticity with genome stability during stress. Our work establishes 5hmC as a dynamic epigenetic mark in plant environmental adaptation and provides a foundation for leveraging DNA hydroxymethylation in crop resilience engineering.
DNA甲基化（5-甲基胞嘧啶，5mC）是植物基因组稳定性和应激适应的关键表观遗传调节因子。然而，其氧化衍生物5-羟甲基胞嘧啶（5hmC）在植物系统中的功能作用仍知之甚少，这主要是由于其丰度较低且酶起源尚未明确。在这里，我们将ACE-seq（载脂蛋白B编辑复合体耦合表观遗传测序）与优化的Tn5mC-seq（基于转座酶的全基因组亚硫酸氢盐测序文库制备，WGBS）方法相结合，生成了水稻（Oryza sativa）中首张5hmC的单碱基分辨率图谱，揭示了其在干旱适应过程中的应激反应动态以及与5mC的调控相互作用。全基因组分析显示，5hmC的基础水平约为0.03（定义为每个位点的C/(C + T)比值），干旱引发5hmC丰度和位点数量显著降低，复水后恢复不完全。与在异染色质中积累的5mC不同，5hmC优先定位于常染色质区域，包括启动子、外显子和基因间元件，并在脱落酸响应转录因子（如OsATAF1、bZIP50）处富集。引人注目的是，干旱诱导了5hmC和5mC之间的拮抗关系，后者在全局范围内增加以加强转座子沉默。多组学分析表明，启动子中5hmC的缺失与转录下调相关，而其在基因体（特别是5'-非翻译区）中的积累抑制了应激反应基因。这些发现突出了5hmC在基因组背景下的双功能调节能力，及其在应激期间平衡转录可塑性与基因组稳定性方面的作用。我们的工作确立了5hmC作为植物环境适应中的动态表观遗传标记，并为在作物抗逆性工程中利用DNA羟甲基化提供了基础。
