Both Two CtACO3 Transcripts Promoting the Accumulation of the Flavonoid Profiles in Overexpressed Transgenic Safflower

Abstract

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,CtACO3, 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 twoCtACO3transcripts,CtACO3-1andCtACO3-2, and the latter was a splice variant ofCtACO3that lacked 5’ coding sequences. The functions and underlying probable mechanisms of the two transcripts have been explored. The quantitative PCR data showed thatCtACO3-1andCtACO3-2were predominantly expressed in the floret and increased with floret development. Subcellular localization results indicated thatCtACO3-1 was localized in the cytoplasm, whereasCtACO3-2 was localized in the cytoplasm and nucleus. Furthermore, the overexpression ofCtACO3-1 orCtACO3-2 in transgenic safflower lines significantly increased the accumulation of quinochalcones and flavonols. The expression of the flavonoid pathway genes showed an upward trend, withCtCHS1,CtF3H1,CtFLS1, andCtDFR1was considerably induced in the overexpression ofCtACO3-1orCtACO3-2lines. An interesting phenomenon forCtACO3-2 protein suppressing the transcription ofCtACO3-1might be related to the nucleus location ofCtACO3-2. Yeast two-hybrid (Y2H), glutathioneS-transferase (GST) pull-down, and BiFC experiments revealed thatCtACO3-2 interacted withCtCSN5a. In addition, the interactions betweenCtCSN5a andCtCOI1,CtCOI1 andCtJAZ1,CtJAZ1 andCtbHLH3 were observed by Y2H and GST pull-down methods, respectively. The above results suggested that theCtACO3-2 promoting flavonoid accumulation might be attributed to the transcriptional activation of flavonoid biosynthesis genes byCtbHLH3, whereas theCtbHLH3 might be regulated throughCtCSN5-CtCOI1-CtJAZ1 signal molecules. Our study provided a novel insight ofCtACO3 affected the flavonoid biosynthesis in safflower.