Both Two CtACO3 Transcripts Promoting the Accumulation of the Flavonoid Profiles in Overexpressed Transgenic Safflower.
Both Two CtACO3 Transcripts Promoting the Accumulation of the Flavonoid Profiles in Overexpressed Transgenic Safflower.
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DOI：
10.3389/fpls.2022.833811
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发表时间：
2022
影响因子：
5.6
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中科院分区：
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红花小花中独特的黄酮类化合物喹诺查尔酮，如羟基红花黄A（HSYA）和红花素，在治疗心脑血管疾病方面表现出良好的药理作用，但黄酮类生物合成的调节机制尚不清楚。本研究发现乙烯信号通路所需的关键酶基因CtACO3与红花不同小花发育时期的黄酮生物合成呈正相关，并具有两个CtACO3转录本，CtACO3-1和CtACO3-2，后者是CtACO3缺失5'编码序列的剪接变体。两个转录本的功能和潜在的可能机制已被探索。定量PCR数据显示，CtACO3-1和CtACO3-2主要在花中表达，并随着花的发育而增加。亚细胞定位结果表明CtACO3-1定位于细胞质，而CtACO3-2定位于细胞质和细胞核。此外，转基因红花品系中CtACO3-1或CtACO3-2的过度表达显着增加了喹啉查尔酮和黄酮醇的积累。类黄酮途径基因的表达呈上升趋势，其中CtCHS1、CtF3H1、CtFLS1和CtDFR1在CtACO3-1或CtACO3-2系的过表达中显着诱导。 CtACO3-2 蛋白抑制 CtACO3-1 转录的一个有趣现象可能与 CtACO3-2 的核位置有关。酵母双杂交 (Y2H)、谷胱甘肽 S-转移酶 (GST) 下拉和 BiFC 实验表明 CtACO3-2 与 CtCSN5a 相互作用。此外，分别通过Y2H和GST pull-down方法观察了CtCSN5a与CtCOI1、CtCOI1与CtJAZ1、CtJAZ1与CtbHLH3之间的相互作用。上述结果表明，CtACO3-2促进黄酮类物质积累可能是由于CtbHLH3对类黄酮生物合成基因的转录激活所致，而CtbHLH3可能是通过CtCSN5-CtCOI1-CtJAZ1信号分子进行调控的。我们的研究提供了 CtACO3 影响红花中类黄酮生物合成的新见解。
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 two CtACO3 transcripts, CtACO3-1 and CtACO3-2, and the latter was a splice variant of CtACO3 that lacked 5’ coding sequences. The functions and underlying probable mechanisms of the two transcripts have been explored. The quantitative PCR data showed that CtACO3-1 and CtACO3-2 were predominantly expressed in the floret and increased with floret development. Subcellular localization results indicated that CtACO3-1 was localized in the cytoplasm, whereas CtACO3-2 was localized in the cytoplasm and nucleus. Furthermore, the overexpression of CtACO3-1 or CtACO3-2 in transgenic safflower lines significantly increased the accumulation of quinochalcones and flavonols. The expression of the flavonoid pathway genes showed an upward trend, with CtCHS1, CtF3H1, CtFLS1, and CtDFR1 was considerably induced in the overexpression of CtACO3-1 or CtACO3-2 lines. An interesting phenomenon for CtACO3-2 protein suppressing the transcription of CtACO3-1 might be related to the nucleus location of CtACO3-2. Yeast two-hybrid (Y2H), glutathione S-transferase (GST) pull-down, and BiFC experiments revealed that CtACO3-2 interacted with CtCSN5a. In addition, the interactions between CtCSN5a and CtCOI1, CtCOI1 and CtJAZ1, CtJAZ1 and CtbHLH3 were observed by Y2H and GST pull-down methods, respectively. The above results suggested that the CtACO3-2 promoting flavonoid accumulation might be attributed to the transcriptional activation of flavonoid biosynthesis genes by CtbHLH3, whereas the CtbHLH3 might be regulated through CtCSN5-CtCOI1-CtJAZ1 signal molecules. Our study provided a novel insight of CtACO3 affected the flavonoid biosynthesis in safflower.