Dynamic changes in DNA methylation occur in TE regions and affect cell proliferation during leaf-to-callus transition in Arabidopsis

Plant somatic cells can be reprogrammed into pluripotent cell mass, called callus, through a two-step in vitro tissue culture method. Incubation on callus-inducing medium triggers active cell proliferation to form a pluripotent callus. Notably, DNA methylation is implicated during callus formation, but a detailed molecular process regulated by DNA methylation remains to be fully elucidated. Here, we compared genome-wide DNA methylation profiles between leaf and callus tissues in Arabidopsis using whole-genome bisulphite-sequencing. Global distribution of DNA methylation showed that CHG methylation was increased, whereas CHH methylation was reduced especially around transposable element (TE) regions during the leaf-to-callus transition. We further analysed differentially expressed genes around differentially methylated TEs (DMTEs) during the leaf-to-callus transition and found that genes involved in cell cycle regulation were enriched and also constituted a coexpression gene network along with pluripotency regulators. In addition, a conserved DNA sequence analysis for upstream cis-elements led us to find a putative transcription factor associated with cell fate transition. CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) was newly identified as a regulator of plant regeneration, and consistently, the cca1lhy mutant displayed altered phenotypes in callus proliferation. Overall, these results suggest that DNA methylation coordinates cell cycle regulation during callus formation, and CCA1 may act as a key upstream coordinator at least in part in the processes.植物体细胞可通过两步体外组织培养方法重编程为多能细胞团，即愈伤组织（callus）。在愈伤组织诱导培养基上培养可触发活跃的细胞增殖，进而形成具有多能性的愈伤组织。值得注意的是，DNA甲基化（DNA methylation）参与愈伤组织形成过程，但由其调控的具体分子机制仍有待全面阐明。本研究利用全基因组亚硫酸氢盐测序（whole-genome bisulphite-sequencing）技术，对比了拟南芥（Arabidopsis）叶片与愈伤组织的全基因组DNA甲基化谱。DNA甲基化的整体分布分析显示，在叶片向愈伤组织转变过程中，CHG甲基化水平显著升高，而CHH甲基化水平则明显降低，尤其在转座因子（transposable element，TE）区域附近。本研究进一步分析了叶片向愈伤组织转变过程中，差异甲基化转座因子（differentially methylated TEs，DMTEs）附近的差异表达基因（differentially expressed genes），发现参与细胞周期调控（cell cycle regulation）的基因显著富集，且该类基因与多能性调控因子共同构成了共表达基因网络。此外，通过对上游顺式作用元件（upstream cis-elements）的保守DNA序列分析，本研究鉴定出一个与细胞命运转变（cell fate transition）相关的推定转录因子（putative transcription factor）。昼夜节律时钟关联蛋白1（CIRCADIAN CLOCK-ASSOCIATED 1，CCA1）被新鉴定为植物再生的调控因子；相应地，cca1lhy突变体在愈伤组织增殖过程中表现出异常表型。综上，本研究结果表明，DNA甲基化可协调愈伤组织形成过程中的细胞周期调控，且CCA1至少在部分过程中可作为关键的上游调控因子发挥作用。