DNA Methylation Inhibitor 5-Azacytidine Promotes Leaf Senescence in Pak Choi (Brassica rapa subsp. chinensis) by Regulating Senescence-Related Genes

Leaf senescence is strictly regulated by multiple internal factors and external environmental signals, with the epigenetic modification being an important element among them. However, the epigenetic mechanism of leaf senescence is largely unknown in horticultural crops, especially the leaf vegetable pak choi, which easily senesces, and becomes yellow post-harvest. In this study, we found that the expression of DNA methyltransferases (BcMET1, BcSUVH4, BcDRM2, BcRDR2, and BcCMT3) of pak choi decreased during storage. The preliminary results showed that its senescence process was accompanied by DNA methylation changes. Moreover, treatment with 500 μM 5-Azacytidine (5-Aza) (DNA methylation inhibitor) can promote the senescence of pak choi leaves by (1) increasing the degradation of chlorophyll (Chl) and its derivatives, (2) increasing the activities of Mg-dechelatase (MDCase), pheophytinase (PPH) and pheophorbide a oxygenase (PAO), and (3) inducing the expression of senescence-related genes (BcSAG12, BcNYC1, BcSGR1, BcSGR2, BcPPH1, BcPPH2, BcPAO, and BcRCCR), thereby accelerating the senescence of the pak choi leaves. Further studies showed that DNA demethylation occurred in the promoter regions of BcSGR2 and BcSAG12 during storage, with the bisulfite sequencing detection showing that their degrees of methylation decreased. Therefore, our findings help us understand how epigenetic modifications affect the storage tolerance of leafy vegetables, which is highly significant for cultivating anti-senescent vegetable varieties.