Comprehensive analysis of epigenetic modifications in alfalfa under cadmium stress

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.