He Beixuan, Zhang Yanjie, Wang Lunuan, Guo Dandan, Jia Xinlei, Wu Jianhui, Qi Shuyi, Wu Hong, Gao Yue, Guo Meili
Department of Pharmacognosy, College of Pharmacy, Naval Medical University (Second Military Medical University), Shanghai, China.
Department of Cardiology, Changhai Hospital of Naval Medical University (Second Military Medical University), Shanghai, China.
Front Plant Sci. 2022 Apr 6;13:833811. doi: 10.3389/fpls.2022.833811. eCollection 2022.
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.
红花小花中独特的黄酮类化合物——喹诺查耳酮，如羟基红花黄色素A（HSYA）和红花苷，在治疗心脑血管疾病方面显示出优异的药理作用，然而，黄酮类生物合成的调控机制在很大程度上尚不清楚。在本研究中，发现乙烯信号通路所需的关键酶基因在红花不同小花发育时期与黄酮类生物合成呈正相关，且有两个转录本，ACO3-1和ACO3-2，后者是ACO3-1缺少5'编码序列的剪接变体。已对这两个转录本的功能及其潜在机制进行了探索。定量PCR数据表明，ACO3-1和ACO3-2主要在小花中表达，并随着小花发育而增加。亚细胞定位结果表明，ACO3-1定位于细胞质，而ACO3-2定位于细胞质和细胞核。此外，在转基因红花品系中过表达ACO3-1或ACO3-2显著增加了喹诺查耳酮和黄酮醇的积累。黄酮类途径基因的表达呈上升趋势，CHS、CHI、F3H和FLS在ACO3-1或ACO3-2过表达品系中显著上调。ACO3-2蛋白抑制CHS转录这一有趣现象可能与ACO3-2的细胞核定位有关。酵母双杂交（Y2H）、谷胱甘肽-S-转移酶（GST）下拉和双分子荧光互补（BiFC）实验表明，ACO3-2与CSN5a相互作用。此外，分别通过Y2H和GST下拉方法观察到CSN5a与COI1、COI1与JAZ1、JAZ1与bHLH3之间的相互作用。上述结果表明，ACO3-2促进黄酮类积累可能归因于bHLH3对黄酮类生物合成基因的转录激活，而bHLH3可能通过CSN5-COI1-JAZ1信号分子进行调控。我们的研究为ACO3影响红花中黄酮类生物合成提供了新的见解。
