Anthocyanins in cornflower (Centaurea cyanus) is catalysed by a set of biosynthesis genes, however, the potential mechanism of transcriptional regulation remains unclear. In the present study, we traced the dynamic changes of petal colour development from white to violet and finally to blue on the same petal in cornflower. Pigment analysis showed that anthocyanin accumulation dramatically increased with petal colour development. Subsequently, nine libraries from above three colour regions were constructed for RNA-seq and 105,506 unigenes were obtained by de novo assembling. The differentially expressed genes among three colour regions were significantly enriched in the phenylpropanoid biosynthesis and flavonoid biosynthesis pathways, leading to the excavation and analysis of 46 biosynthesis genes involved in this process. Furthermore, four R2R3-CcMYBs clustered into subgroup 4 or subgroup 6 and one CcbHLH1 clustered into IIIf subgroup were screened out by phylogenetic analysis with Arabidopsis homologues. The promoters of flavanone 3-hydroxylase (CcF3H) and dihydroflavonol 4-reductase (CcDFR) were further isolated to investigate upstream regulation mechanism. CcMYB6-1 significantly upregulated the activity of above two promoters and stimulated anthocyanin accumulation by dual luciferase assay and transient expression in tobacco leaves, and its activity was obviously enhanced when co-infiltrated with CcbHLH1. Moreover, both yeast two-hybrid and bimolecular fluorescence complementation assays indicated the protein-protein interaction between these two activators. Based on these obtained results, it reveals that CcMYB6-1 and CcbHLH1 are two novel transcription factors synergistically involved in regulating anthocyanin biosynthesis. This study provides insights into the regulatory mechanism of anthocyanin accumulation in cornflower.

中文翻译：

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CcMYB6-1和CcbHLH1，两个新的转录因子，协同参与调节矢车菊的花色苷生物合成。

矢车菊中的花青素（Centaurea cyanus）是由一组生物合成基因催化的，但是，转录调控的潜在机制仍不清楚。在本研究中，我们追踪了矢车菊同一花瓣上花瓣颜色发展的动态变化，从白色到紫色，最后到蓝色。颜料分析表明，花色苷的积累随着花瓣颜色的发展而急剧增加。随后，从三个以上颜色区域构建了9个用于RNA-seq的文库，并通过从头组装获得了105,506个单基因。三个颜色区域之间差异表达的基因在苯丙烷类生物合成和类黄酮生物合成途径中显着富集，从而导致挖掘和分析了该过程中涉及的46个生物合成基因。此外，通过用拟南芥同源物进行系统发育分析，筛选出4个聚类为亚组4或亚组6的R2R3-CcMYB和一个聚类为IIIf亚组的CcbHLH1。黄烷酮3-羟化酶（CcF3H）和二氢黄酮醇4-还原酶（CcDFR）的启动子被进一步分离，以研究上游调节机制。CcMYB6-1通过双重荧光素酶检测和烟草瞬时表达在烟草叶片中显着上调了上述两个启动子的活性并刺激了花色苷的积累，与CcbHLH1共同浸润其活性明显增强。此外，酵母双杂交和双分子荧光互补测定都表明这两种活化剂之间的蛋白质相互作用。根据这些获得的结果，它揭示了CcMYB6-1和CcbHLH1是协同参与调节花色苷生物合成的两个新的转录因子。这项研究提供了对矢车菊中花色苷积累的调控机制的见解。