Michael Smith Laboratories, Department of Botany, and Wine Research Centre, University of British Columbia, Vancouver, British Columbia, Canada.
Planta. 2020 Feb 6;251(3):60. doi: 10.1007/s00425-020-03350-0.
The phosphorylation status of MYB75 at T-131 affects protein stability, flavonoid profiles, and patterns of gene expression. The Arabidopsis transcription factor Myeloblastosis protein 75 (MYB75, AT1G56650) is known to act as a positive transcriptional regulator of genes required for flavonoid and anthocyanin biosynthesis. MYB75 was also shown to negatively regulate lignin and other secondary cell wall biosynthetic genes (Bhargava et al. in Plant Physiol 154(3):1428-1438, 2010). While transcriptional regulation of MYB75 has been described in numerous publications, little is known about post-translational control of MYB75 protein function. In a recent publication, light-induced activation of a MAP kinase (MPK4, AT4G01370) in Arabidopsis was reported to lead to MYB75 phosphorylation at two canonical MPK target sites, threonines, T-126 and T-131. This double phosphorylation event positively influenced MYB75 protein stability (Li et al. in Plant Cell 28(11):2866-2883, 2016). We have examined this phenomenon through use of phosphomutant forms of MYB75 and found that MYB75 is phosphorylated primarily at T-131, and that the phosphorylation of MYB75 recombinant protein in vitro can be catalyzed by multiple MAP kinases, including MPK3 (AT3G45640), MPK6 (AT2G43790), MPK4 and MPK11 (AT1G01560). We also demonstrate that MYB75 can bind to a large number of Arabidopsis MPK's in vitro, suggesting it could be a target of multiple signalling pathways. The impact of MYB75 phosphorylation at T-131 on the function of this transcription factor, in terms of localization, stability, and protein-protein interactions with known binding partners was examined in transgenic lines expressing phosphomimic and phosphonull versions of MYB75, to capture the behaviour of permanently phosphorylated and unphosphorylated MYB75 protein, respectively. In addition, we describe how ectopic over-expression of different phosphovariant forms of MYB75 (MYB75, MYB75, and MYB75) affects flavonoid biochemical profiles and global changes of gene expression in the corresponding transgenic Arabidopsis plants.
在 T-131 处磷酸化的 MYB75 影响蛋白稳定性、类黄酮特征和基因表达模式。拟南芥的髓细胞瘤蛋白 75（MYB75，AT1G56650）作为黄酮类和花青素生物合成所需基因的正转录调控因子是已知的。MYB75 还被证明可以负调控木质素和其他次生细胞壁生物合成基因（Bhargava 等人，植物生理学 154（3）：1428-1438，2010）。虽然 MYB75 的转录调控已在许多出版物中进行了描述，但对 MYB75 蛋白功能的翻译后控制知之甚少。在最近的一篇论文中，报道了拟南芥中 MAP 激酶（MPK4，AT4G01370）的光诱导激活导致 MYB75 在两个典型的 MPK 靶位点，苏氨酸 T-126 和 T-131 处磷酸化。这种双重磷酸化事件正向影响 MYB75 蛋白稳定性（Li 等人，植物细胞 28（11）：2866-2883，2016）。我们通过使用 MYB75 的磷酸突变形式检查了这种现象，发现 MYB75 主要在 T-131 处被磷酸化，并且体外 MYB75 重组蛋白的磷酸化可以由多种 MAP 激酶催化，包括 MPK3（AT3G45640），MPK6（AT2G43790），MPK4 和 MPK11（AT1G01560）。我们还证明，MYB75 可以在体外与大量的拟南芥 MPK 结合，这表明它可能是多种信号通路的靶标。通过在表达 MYB75 的磷酸模拟和磷酸缺失突变体的转基因系中，研究了 MYB75 在 T-131 处磷酸化对该转录因子的功能的影响，包括定位、稳定性以及与已知结合伙伴的蛋白-蛋白相互作用，分别捕获了永久性磷酸化和非磷酸化 MYB75 蛋白的行为。此外，我们描述了异位过表达不同磷酸化形式的 MYB75（MYB75、MYB75 和 MYB75）如何影响相应的转基因拟南芥植物中的类黄酮生化特征和基因表达的全局变化。
