Nitrate reductase BoNIA2b responds to nitric oxide to positively regulate the accumulation of Glucoraphanin and Sulforaphane in broccoli hairy roots.

Glucoraphanin (GRA) and sulforaphane (SF) are potent anticancer compounds. Understanding their accumulation mechanisms is crucial for increasing their levels in broccoli. This study aimed to investigate the role of nitric oxide (NO) in GRA and SF accumulation and identify key genes involved in this process. Low NO concentrations were found to promote GRA and SF accumulation. Transcriptome sequencing and Weighted Gene Co-expression Network Analysis (WGCNA) identified BoNIA2b, a nitrate reductase (NR) gene, as a key regulator of NO-mediated GRA and SF accumulation. Silencing BoNIA2b reduced endogenous NO levels and NR activity in hairy roots, while exogenous treatment with sodium nitroprusside (SNP) restored NO levels without affecting NR activity. Overexpression of BoNIA2b increased NO content and NR activity. Silencing BoNIA2b decreased GRA content, but SF levels remained unaffected. SNP treatment enhanced both GRA and SF accumulation, with GRA being more dependent on BoNIA2b. In BoNIA2b-overexpressing roots, both GRA and SF levels were significantly higher than in controls. Moreover, the interaction between BoNIA2b and BoMYB28 protein was confirmed through Y2H and luciferase complementation assays. These findings underscore BoNIA2b's role in NO-mediated regulation of GRA and SF accumulation in broccoli hairy roots, offering insights to enhancing the production of these anticancer compounds.