Chilling-Mediated DNA Methylation Changes during Dormancy and Its Release Reveal the Importance of Epigenetic Regulation during Winter Dormancy in Apple (<i>Malus</i> x <i>domestica</i> Borkh.)

Winter dormancy is a well known mechanism adopted by temperate plants, to mitigate the chilling temperature of winters. However, acquisition of sufficient chilling during winter dormancy ensures the normal phenological traits in subsequent growing period. Thus, low temperature appears to play crucial roles in growth and development of temperate plants. Apple, being an important temperate fruit crop, also requires sufficient chilling to release winter dormancy and normal phenological traits, which are often associated with yield and quality of fruits. DNA cytosine methylation is one of the important epigenetic modifications which remarkably affect the gene expression during various developmental and adaptive processes. In present study, methylation sensitive amplified polymorphism was employed to assess the changes in cytosine methylation during dormancy, active growth and fruit set in apple, under differential chilling conditions. Under high chill conditions, total methylation was decreased from 27.2% in dormant bud to 21.0% in fruit set stage, while no significant reduction was found under low chill conditions. Moreover, the demethylation was found to be decreased, while methylation increased from dormant bud to fruit set stage under low chill as compared to high chill conditions. In addition, RNA-Seq analysis showed high expression of DNA methyltransferases and histone methyltransferases during dormancy and fruit set, and low expression of DNA glcosylases during active growth under low chill conditions, which was in accordance with changes in methylation patterns. The RNA-Seq data of 47 genes associated with MSAP fragments involved in cellular metabolism, stress response, antioxidant system and transcriptional regulation showed correlation between methylation and their expression. Similarly, bisulfite sequencing and qRT-PCR analysis of selected genes also showed correlation between gene body methylation and gene expression. Moreover, significant association between chilling and methylation changes was observed, which suggested that chilling acquisition during dormancy in apple is likely to affect the epigenetic regulation through DNA methylation.冬季休眠是温带植物为抵御冬季低温胁迫所采用的一种广为人知的适应性机制。然而，植物在冬季休眠期间需积累足够的低温累积量，才能在后续生育期展现正常的物候特性。由此可见，低温在温带植物的生长发育过程中发挥着至关重要的作用。苹果作为重要的温带果树作物，同样需要积累足够的低温以打破冬季休眠并保障正常物候特性，而这些特性往往与果实的产量和品质密切相关。DNA胞嘧啶甲基化是一类重要的表观遗传修饰方式，可在多种发育与适应过程中显著调控基因的表达。本研究采用甲基化敏感扩增多态性（methylation sensitive amplified polymorphism, MSAP）技术，分析不同低温累积条件下苹果在休眠期、营养生长期以及坐果期的胞嘧啶甲基化变化。在高低温累积条件下，总甲基化率从休眠芽期的27.2%降至坐果期的21.0%；而在低低温累积条件下，未检测到显著的甲基化水平下降。此外，相较于高低温累积条件，低低温累积条件下从休眠芽期至坐果期的去甲基化水平有所降低，而甲基化水平则呈上升趋势。此外，RNA测序（RNA-Seq）分析结果显示，在低低温累积条件下，DNA甲基转移酶与组蛋白甲基转移酶在休眠期和坐果期呈高表达，而DNA糖基化酶在营养生长期呈低表达，这与甲基化模式的变化趋势一致。针对与MSAP片段相关的47个参与细胞代谢、胁迫响应、抗氧化系统以及转录调控的基因的RNA-Seq数据显示，其甲基化水平与基因表达量之间存在显著相关性。同样，对筛选出的基因进行亚硫酸氢盐测序与实时定量聚合酶链反应（qRT-PCR）分析，结果也证实了基因体甲基化与基因表达之间的相关性。此外，本研究还发现低温累积与甲基化变化之间存在显著关联，这表明苹果休眠期的低温累积可能通过DNA甲基化途径调控表观遗传过程。