5-methyl cytosine is widespread in plant genomes in both CG and non-CG contexts. During replication, hemi-methylation on parental DNA strands guides symmetric CG methylation on nascent strands, but non-CG methylation requires modified histones and small RNA guides. Here, we used immortalized Arabidopsis cell suspensions to sort replicating nuclei and determine genome-wide cytosine methylation dynamics during the plant cell cycle. We find that symmetric mCG and mCHG are selectively retained in actively dividing cells in culture, whereas mCHH is depleted. mCG becomes transiently asymmetric during S phase but is rapidly restored in G2, whereas mCHG remains asymmetric throughout the cell cycle. Hundreds of loci gain ectopic CHG methylation, as well as 24-nt small interfering RNAs (siRNAs) and histone H3 lysine dimethylation (H3K9me2), without gaining CHH methylation. This suggests that spontaneous epialleles that arise in plant cell cultures are stably maintained by siRNA and H3K9me2 independent of the canonical RNA-directed DNA methylation (RdDM) pathway. In contrast, loci that fail to produce siRNA may be targeted for demethylation when the cell cycle arrests. Comparative analysis with methylomes of various tissues and cell types suggests that loss of small-RNA-directed non-CG methylation during DNA replication promotes germline reprogramming and epigenetic variation in plants propagated as clones.

5-甲基胞嘧啶在 CG 和非 CG 环境中广泛存在于植物基因组中。在复制过程中，亲本 DNA 链上的半甲基化引导新生链上的对称 CG 甲基化，但非 CG 甲基化需要修饰的组蛋白和小 RNA 引导。在这里，我们使用了永生的拟南芥细胞悬浮液对复制的细胞核进行分类并确定植物细胞周期期间的全基因组胞嘧啶甲基化动态。我们发现对称的 mCG 和 mCHG 选择性地保留在培养中的活跃分裂细胞中，而 mCHH 被耗尽。mCG 在 S 期变得短暂不对称，但在 G2 期迅速恢复，而 mCHG 在整个细胞周期中保持不对称。数百个位点获得异位 CHG 甲基化，以及 24-nt 小干扰 RNA (siRNA) 和组蛋白 H3 赖氨酸二甲基化 (H3K9me2)，而没有获得 CHH 甲基化。这表明植物细胞培养物中出现的自发表观等位基因由 siRNA 和 H3K9me2 稳定维持，独立于典型的 RNA 指导的 DNA 甲基化 (RdDM) 途径。相比之下，当细胞周期停滞时，不能产生 siRNA 的基因座可能会成为去甲基化的目标。与各种组织和细胞类型的甲基化组的比较分析表明，DNA 复制过程中小 RNA 指导的非 CG 甲基化的丧失促进了作为克隆繁殖的植物的种系重编程和表观遗传变异。