Epigenetics plays an important role in plant growth and development and in environmental adaptation. Alfalfa, an important forage crop, is rich in nutrients. However, little is known about the molecular regulatory mechanisms underlying the response of alfalfa to cadmium (Cd) stress. Here, we performed DNA methylation (5mC), RNA methylation (m⁶A) and transcriptomic sequencing analyses of alfalfa roots under Cd stress. Whole-genome methylation sequencing and transcriptomic sequencing revealed that Cd stress reduced DNA methylation levels. Moreover, a reduced 5mC methylation level was associated with decreased expression of several DNA methyltransferase genes. Compared with those under normal (CK) conditions, the m⁶A modification levels under Cd stress were greater and were positively correlated with gene expression in alfalfa roots. We also found a negative correlation between the 5mC level and the m⁶A level, especially in CG and CHG contexts. In yeast, the overexpression of MsNARMP5 (natural resistance-associated macrophage protein) and MsPCR2 (plant cadmium resistance 2), which are modified by 5mC or m⁶A, significantly increased Cd stress tolerance. These results provide candidate genes for future studies on the mechanism of Cd stress tolerance in alfalfa roots and valuable information for studying heavy metal stress in alfalfa breeding.

中文翻译：

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镉胁迫下紫花苜蓿表观遗传修饰的综合分析


表观遗传学在植物生长发育以及环境适应中起着重要作用。紫花苜蓿是一种重要的饲料作物，营养物质丰富。然而，人们对紫花苜蓿对镉 （Cd） 胁迫反应的分子调控机制知之甚少。在这里，我们对 Cd 胁迫下的紫花苜蓿根进行了 DNA 甲基化 （5mC） 、 RNA 甲基化 （m⁶A） 和转录组测序分析。全基因组甲基化测序和转录组测序显示 Cd 应激降低了 DNA 甲基化水平。此外，5mC 甲基化水平降低与几个 DNA 甲基转移酶基因的表达降低有关。与正常 （CK） 条件下相比，Cd 胁迫下 m⁶A 修饰水平更大，且与紫花苜蓿根系的基因表达呈正相关。我们还发现 5mC 水平和 m⁶A 水平之间存在负相关，尤其是在 CG 和 CHG 环境中。在酵母中，经 5 mC 或 m⁶A 修饰的 MsNARMP5 （天然抗性相关巨噬细胞蛋白） 和 MsPCR2 （植物抗镉 2） 的过表达显著提高了 Cd 胁迫耐受性。这些结果为未来研究紫花苜蓿根系耐镉胁迫机制提供了候选基因，为研究紫花苜蓿育种中的重金属胁迫提供了有价值的信息。