Univ, de Bordeaux, Institut des Sciences de la Vigne et du Vin, UMR 1287 Ecophysiologie et Génomique Fonctionnelle de la Vigne, 210 Chemin de Leysotte, 33882 Villenave d'Ornon, France.
BMC Plant Biol. 2011 Aug 23;11:117. doi: 10.1186/1471-2229-11-117.
Flavonoid pathway is spatially and temporally controlled during plant development and the transcriptional regulation of the structural genes is mostly orchestrated by a ternary protein complex that involves three classes of transcription factors (R2-R3-MYB, bHLH and WDR). In grapevine (Vitis vinifera L.), several MYB transcription factors have been identified but the interactions with their putative bHLH partners to regulate specific branches of the flavonoid pathway are still poorly understood.
In this work, we describe the effects of a single amino acid substitution (R69L) located in the R2 domain of VvMYB5b and predicted to affect the formation of a salt bridge within the protein. The activity of the mutated protein (name VvMYB5b(L), the native protein being referred as VvMYB5b(R)) was assessed in different in vivo systems: yeast, grape cell suspensions, and tobacco. In the first two systems, VvMYB5b(L) exhibited a modified trans-activation capability. Moreover, using yeast two-hybrid assay, we demonstrated that modification of VvMYB5b transcriptional properties impaired its ability to correctly interact with VvMYC1, a grape bHLH protein. These results were further substantiated by overexpression of VvMYB5b(R) and VvMYB5b(L) genes in tobacco. Flowers from 35S::VvMYB5b(L) transgenic plants showed a distinct phenotype in comparison with 35S::VvMYB5b(R) and the control plants. Finally, significant differences in transcript abundance of flavonoid metabolism genes were observed along with variations in pigments accumulation.
Taken together, our findings indicate that VvMYB5b(L) is still able to bind DNA but the structural consequences linked to the mutation affect the capacity of the protein to activate the transcription of some flavonoid genes by modifying the interaction with its co-partner(s). In addition, this study underlines the importance of an internal salt bridge for protein conformation and thus for the establishment of protein-protein interactions between MYB and bHLH transcription factors. Mechanisms underlying these interactions are discussed and a model is proposed to explain the transcriptional activity of VvMYB5(L) observed in the tobacco model.
类黄酮途径在植物发育过程中受到时空调控，结构基因的转录调控主要由涉及三类转录因子（R2-R3-MYB、bHLH 和 WDR）的三元蛋白复合物进行协调。在葡萄（Vitis vinifera L.）中，已经鉴定出几种 MYB 转录因子，但它们与假定的 bHLH 伙伴的相互作用以调节类黄酮途径的特定分支仍然知之甚少。
在这项工作中，我们描述了位于 VvMYB5b 的 R2 结构域中单个氨基酸取代（R69L）的影响，该取代预计会影响蛋白质内盐桥的形成。突变蛋白（命名为 VvMYB5b(L)，天然蛋白称为 VvMYB5b(R)）的活性在不同的体内系统中进行了评估：酵母、葡萄细胞悬浮液和烟草。在前两个系统中，VvMYB5b(L)表现出修饰的反式激活能力。此外，使用酵母双杂交测定，我们证明 VvMYB5b 转录特性的修饰会损害其与葡萄 bHLH 蛋白 VvMYC1 正确相互作用的能力。通过在烟草中过表达 VvMYB5b(R)和 VvMYB5b(L)基因，进一步证实了这些结果。与 VvMYB5b(R)和对照植物相比，35S::VvMYB5b(L)转基因植物的花朵表现出明显不同的表型。最后，观察到类黄酮代谢基因的转录丰度存在显著差异，同时伴随着色素积累的变化。
总之，我们的研究结果表明，VvMYB5b(L)仍然能够结合 DNA，但与突变相关的结构后果会影响该蛋白激活一些类黄酮基因转录的能力，方法是修饰与它的共同伙伴的相互作用。此外，这项研究强调了内部盐桥对蛋白质构象的重要性，从而对 MYB 和 bHLH 转录因子之间的蛋白质-蛋白质相互作用的建立具有重要意义。讨论了这些相互作用的机制，并提出了一个模型来解释在烟草模型中观察到的 VvMYB5(L)的转录活性。
