AUTHOR=He Beixuan , Zhang Yanjie , Wang Lunuan , Guo Dandan , Jia Xinlei , Wu Jianhui , Qi Shuyi , Wu Hong , Gao Yue , Guo Meili 

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

JOURNAL=Frontiers in Plant Science

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2022.833811

DOI=10.3389/fpls.2022.833811

ISSN=1664-462X

ABSTRACT=<p>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, <italic>CtACO3</italic>, 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 <italic>CtACO3</italic> transcripts, <italic>CtACO3-1</italic> and <italic>CtACO3-2</italic>, and the latter was a splice variant of <italic>CtACO3</italic> that lacked 5’ coding sequences. The functions and underlying probable mechanisms of the two transcripts have been explored. The quantitative PCR data showed that <italic>CtACO3-1</italic> and <italic>CtACO3-2</italic> were predominantly expressed in the floret and increased with floret development. Subcellular localization results indicated that <italic>Ct</italic>ACO3-1 was localized in the cytoplasm, whereas <italic>Ct</italic>ACO3-2 was localized in the cytoplasm and nucleus. Furthermore, the overexpression of <italic>Ct</italic>ACO3-1 or <italic>Ct</italic>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 <italic>CtCHS1</italic>, <italic>CtF3H1</italic>, <italic>CtFLS1</italic>, and <italic>CtDFR1</italic> was considerably induced in the overexpression of <italic>CtACO3-1</italic> or <italic>CtACO3-2</italic> lines. An interesting phenomenon for <italic>Ct</italic>ACO3-2 protein suppressing the transcription of <italic>CtACO3-1</italic> might be related to the nucleus location of <italic>Ct</italic>ACO3-2. Yeast two-hybrid (Y2H), glutathione <italic>S</italic>-transferase (GST) pull-down, and BiFC experiments revealed that <italic>Ct</italic>ACO3-2 interacted with <italic>Ct</italic>CSN5a. In addition, the interactions between <italic>Ct</italic>CSN5a and <italic>Ct</italic>COI1, <italic>Ct</italic>COI1 and <italic>Ct</italic>JAZ1, <italic>Ct</italic>JAZ1 and <italic>Ct</italic>bHLH3 were observed by Y2H and GST pull-down methods, respectively. The above results suggested that the <italic>Ct</italic>ACO3-2 promoting flavonoid accumulation might be attributed to the transcriptional activation of flavonoid biosynthesis genes by <italic>Ct</italic>bHLH3, whereas the <italic>Ct</italic>bHLH3 might be regulated through <italic>Ct</italic>CSN5-<italic>Ct</italic>COI1-<italic>Ct</italic>JAZ1 signal molecules. Our study provided a novel insight of <italic>Ct</italic>ACO3 affected the flavonoid biosynthesis in safflower.</p>