Both Two Transcripts Promoting the Accumulation of the Flavonoid Profiles in Overexpressed Transgenic Safflower

The unique flavonoids, quinochalcones, such as hydroxysafflor yellow A (HSYA) and carthamin, in the floret of safflower showed an excellent pharmacological effect in treating cardiocerebral vascular disease, yet the regulating mechanisms governing the flavonoid biosynthesis are largely unknown. In this study, , the key enzyme genes required for the ethylene signaling pathway, were found positively related to the flavonoid biosynthesis at different floret development periods in safflower and has two transcripts, and , and the latter was a splice variant of that lacked 5' coding sequences. The functions and underlying probable mechanisms of the two transcripts have been explored. The quantitative PCR data showed that and were predominantly expressed in the floret and increased with floret development. Subcellular localization results indicated that ACO3-1 was localized in the cytoplasm, whereas ACO3-2 was localized in the cytoplasm and nucleus. Furthermore, the overexpression of ACO3-1 or ACO3-2 in transgenic safflower lines significantly increased the accumulation of quinochalcones and flavonols. The expression of the flavonoid pathway genes showed an upward trend, with , , , and was considerably induced in the overexpression of or lines. An interesting phenomenon for ACO3-2 protein suppressing the transcription of might be related to the nucleus location of ACO3-2. Yeast two-hybrid (Y2H), glutathione -transferase (GST) pull-down, and BiFC experiments revealed that ACO3-2 interacted with CSN5a. In addition, the interactions between CSN5a and COI1, COI1 and JAZ1, JAZ1 and bHLH3 were observed by Y2H and GST pull-down methods, respectively. The above results suggested that the ACO3-2 promoting flavonoid accumulation might be attributed to the transcriptional activation of flavonoid biosynthesis genes by bHLH3, whereas the bHLH3 might be regulated through CSN5-COI1-JAZ1 signal molecules. Our study provided a novel insight of ACO3 affected the flavonoid biosynthesis in safflower.