ABSTRACT

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.

摘要

通过两步体外组织培养方法，植物体细胞可以重编程为多能细胞团，称为愈伤组织。在愈伤组织诱导培养基上孵育会触发活跃的细胞增殖，形成多能愈伤组织。值得注意的是，愈伤组织形成过程中涉及 DNA 甲基化，但 DNA 甲基化调节的详细分子过程仍有待充分阐明。在这里，我们使用全基因组亚硫酸氢盐测序比较了拟南芥叶片和愈伤组织之间的全基因组 DNA 甲基化谱。DNA 甲基化的整体分布表明，在叶向愈伤组织转变过程中，CHG 甲基化增加，而 CHH 甲基化减少，尤其是在转座元件 (TE) 区域周围。我们进一步分析了叶向愈伤组织转变过程中差异甲基化TE（DMTE）周围的差异表达基因，发现参与细胞周期调节的基因被富集，并且与多能性调节因子一起构成了共表达基因网络。此外，对上游顺式元件的保守DNA序列分析使我们发现了与细胞命运转变相关的推定转录因子。昼夜节律时钟相关 1 (CCA1) 最近被鉴定为植物再生的调节因子，并且一致地，cca1lhy突变体在愈伤组织增殖中表现出改变的表型。总体而言，这些结果表明 DNA 甲基化在愈伤组织形成过程中协调细胞周期调节，并且 CCA1 可能至少在部分过程中充当关键的上游协调员。