Li Yueqing, Shan Xiaotong, Gao Ruifang, Yang Song, Wang Shucai, Gao Xiang, Wang Li
Key Laboratory of Molecular Epigenetics of MOE, Changchun, China.
Institute of Genetics and Cytology, Northeast Normal University, Changchun, China.
Sci Rep. 2016 Jul 28;6:30514. doi: 10.1038/srep30514.
The MBW complex, comprised by R2R3-MYB, basic helix-loop-helix (bHLH) and WD40, is a single regulatory protein complex that drives the evolution of multiple traits such as flavonoid biosynthesis and epidermal cell differentiation in plants. In this study, two IIIf Clade-bHLH regulator genes, FhGL3L and FhTT8L, were isolated and functionally characterized from Freesia hybrida. Different spatio-temporal transcription patterns were observed showing diverse correlation with anthocyanin and proanthocyanidin accumulation. When overexpressed in Arabidopsis, FhGL3L could enhance the anthocyanin accumulation through up-regulating endogenous regulators and late structural genes. Unexpectedly, trichome formation was inhibited associating with the down-regulation of AtGL2. Comparably, only the accumulation of anthocyanins and proanthocyanidins was strengthened in FhTT8L transgenic lines. Furthermore, transient expression assays demonstrated that FhGL3L interacted with AtPAP1, AtTT2 and AtGL1, while FhTT8L only showed interaction with AtPAP1 and AtTT2. In addition, similar activation of the AtDFR promoter was found between AtPAP1-FhGL3L/FhTT8L and AtPAP1- AtGL3/AtTT8 combinations. When FhGL3L was fused with a strong activation domain VP16, it could activate the AtGL2 promoter when co-transfected with AtGL1. Therefore, it can be concluded that the functionality of bHLH factors may have diverged, and a sophisticated interaction and hierarchical network might exist in the regulation of flavonoid biosynthesis and trichome formation.
由R2R3-MYB、基本螺旋-环-螺旋（bHLH）和WD40组成的MBW复合体是一种单一的调控蛋白复合体，它推动了植物中多个性状的进化，如类黄酮生物合成和表皮细胞分化。在本研究中，从杂交小苍兰中分离出两个III f进化枝bHLH调控基因FhGL3L和FhTT8L，并对其进行了功能表征。观察到不同的时空转录模式，显示出与花青素和原花青素积累的不同相关性。当在拟南芥中过表达时，FhGL3L可以通过上调内源调控因子和晚期结构基因来增强花青素的积累。出乎意料的是，毛状体形成受到抑制，这与AtGL2的下调有关。相比之下，在FhTT8L转基因株系中，仅花青素和原花青素的积累得到增强。此外，瞬时表达分析表明，FhGL3L与AtPAP1、AtTT2和AtGL1相互作用，而FhTT8L仅与AtPAP1和AtTT2相互作用。此外，在AtPAP1-FhGL3L/FhTT8L和AtPAP1-AtGL3/AtTT8组合之间发现了对AtDFR启动子的类似激活。当FhGL3L与强激活域VP16融合时，与AtGL1共转染时它可以激活AtGL2启动子。因此，可以得出结论，bHLH因子的功能可能已经分化，并且在类黄酮生物合成和毛状体形成的调控中可能存在复杂的相互作用和层次网络。
