Insights into the mechanism of chlorophyll and carotenoid metabolism regulated by BoPIF4 and BobHLH66 in broccoli

Postharvest broccoli tends to fade from green to yellow, which is closely associated with pigment metabolism. Numerous genes involved in chlorophyll metabolism and carotenoid biosynthesis pathway in broccoli have been identified, but the underlying transcriptional regulation mechanism of these genes remains unclear. Here, two bHLH transcription factors, designated as BoPIF4 and BobHLH66, were characterized from broccoli, which were conspicuously induced during senescence. BoPIF4 and BobHLH66 were both localized exclusively in nucleus and had transactivation ability. Intriguingly, these two transcriptional regulators BoPIF4/BobHLH66 separately recognized the binding elements (G-box and GCACGTGC) in the promoters of BoCAO and BoHYD, and facilitated their transcription. Furthermore, the transient expression assay of BoPIF4/BobHLH66 in 'Naihan-Youxiu' broccoli and calli demonstrated that they were the positive regulators of BoCAO and BoHYD. The functions of BoPIF4 and BobHLH66 have been further verified in the genetic transformation system of Arabidopsis thaliana. Collectively, these findings suggest that BoPIF4/BobHLH66 may function as a vital component of the senescence-regulating module in broccoli through transcriptional activation of BoCAO and BoHYD.