Nemie-Feyissa Dugassa, Heidari Behzad, Blaise Mickael, Lillo Cathrine
University of Stavanger, Centre for Organelle Research, Faculty of Science and Technology, N-4036 Stavanger, Norway.
CARB Centre, Dept. of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10c, DK-8000 Aarhus C, Denmark.
Phytochemistry. 2015 Mar;111:21-6. doi: 10.1016/j.phytochem.2015.01.004. Epub 2015 Feb 4.
The two Arabidopsis basic-helix-loop-helix transcription factors GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) are positive regulators of anthocyanin biosynthesis, and form protein complexes (MBW complexes) with various R2R3 MYB transcription factors and a WD40 repeat protein TRANSPARENT TESTA GLABROUS1 (TTG1). In earlier studies, GL3, in contrast to EGL3, was shown to be essential for accumulation of anthocyanins in response to nitrogen depletion. This could not be fully explained by the strong induction of GL3 in response to nitrogen depletion because the EGL3 transcripts were constitutively at a relatively high level and transcripts levels of the two genes were similar under nitrogen depletion. Here the GL3 and EGL3 proteins were characterized with respect to their affinities for PRODUCTION OF ANTHOCYANIN PIGMENT2 (PAP2), a R2R3-MYB which is induced by nitrogen depletion and is part of MBW complexes promoting anthocyanin synthesis. GL3 and EGL3 were also tested for their binding to MYBL2, a negative regulator of anthocyanin synthesis and MBW complexes. Using heterologously expressed proteins and Microscale Thermophoresis, GL3 showed binding constants (Kd) of 3.5±1.7 and 22.7±3.7 μM, whereas EGL3 showed binding constants of 7.5±2.3 and 8.9±1.4 μM for PAP2 and MYBL2, respectively. This implies that MYBL2 will not inhibit a MBW complex containing GL3 as easily as for a complex containing EGL3. In transgenic plants where EGL3 reaches high concentrations compared with MYBL2 the equilibrium is shifted and MYBL2 is not likely to be an efficient competitor, hence anthocyanin formation could be restored by either EGL3 or GL3 genes when overexpressed by help of the 35S promoter. The present work underpins that GL3 is essential for anthocyanin accumulation under nitrogen depletion not only due to transcriptional activation, but also because of binding properties to proteins promoting or inhibiting the activity of the MBW complex.
拟南芥的两个碱性螺旋-环-螺旋转录因子GLABRA3（GL3）和GLABRA3增强子（EGL3）是花青素生物合成的正调控因子，它们与多种R2R3 MYB转录因子以及WD40重复蛋白TRANSPARENT TESTA GLABROUS1（TTG1）形成蛋白复合物（MBW复合物）。在早期研究中，与EGL3不同，GL3被证明对于响应氮素缺乏时花青素的积累至关重要。这无法完全通过氮素缺乏时GL3的强烈诱导来解释，因为EGL3转录本一直处于相对较高水平，并且在氮素缺乏条件下这两个基因的转录本水平相似。在此，对GL3和EGL3蛋白针对花青素色素2（PAP2）的亲和力进行了表征，PAP2是一种R2R3-MYB，受氮素缺乏诱导且是促进花青素合成的MBW复合物的一部分。还测试了GL3和EGL3与MYBL2的结合情况，MYBL2是花青素合成和MBW复合物的负调控因子。使用异源表达蛋白和微量热泳动技术，GL3对PAP2和MYBL2的结合常数（Kd）分别为3.5±1.7和22.7±3.7 μM，而EGL3对PAP2和MYBL2的结合常数分别为7.5±2.3和8.9±1.4 μM。这意味着MYBL2抑制含有GL3的MBW复合物的难度要高于抑制含有EGL3的复合物。在转基因植物中，与MYBL2相比EGL3达到高浓度时，平衡会发生偏移，MYBL2不太可能成为有效的竞争者，因此当通过35S启动子过表达时，EGL3或GL3基因都可以恢复花青素的形成。目前的工作表明，GL3对于氮素缺乏条件下花青素的积累至关重要，这不仅是由于转录激活，还因为其与促进或抑制MBW复合物活性的蛋白的结合特性。
