CHG Hypomethylation of BoCYP83A1 May Contribute to Methyl Jasmonate-Induced Sulforaphane Accumulation in Broccoli

Sulforaphane (SFN) is widely recognized for its health-promoting benefits. While several studies have reported that methyl jasmonate (MeJA) enhances SFN accumulation, the role of epigenetic regulation remains unclear. Here, we performed an integrated analysis of transcriptome, whole-genome bisulfite sequencing, and small RNA-seq data from broccoli florets and leaves under MeJA treatment and control conditions. Our results revealed that MeJA-responsive differentially methylated regions (DMRs) predominantly occurring in the CHG context. Integrative analysis of methylation and siRNA data indicated that changes in MeJA-responsive CHH methylation were associated with 24-nucleotide siRNA clusters. Furthermore, inhibiting DNA methylation using 5-azacytidine treatment in broccoli seedlings increased SFN content, validating the role of DNA hypomethylation in this process. By combining transcriptional regulatory network analysis with DMR data, we identified the MeJA-responsive CHG-hypomethylated gene BoCYP83A1. The McrBC-PCR assay confirmed reduced methylation in the BoCYP83A1 promoter region. The function of BoCYP83A1 in MeJA-induced SFN biosynthesis was validated using RNA interference in broccoli. Further evidence demonstrated that the transcription factors BoMYB68 and BoIAA18 promote BoCYP83A1 expression by directly binding to its promoter. Our findings not only provide novel insights into the epigenetic regulation underlying MeJA-induced SFN accumulation in plants but also facilitate molecular breeding for the development of high-SFN crops.