Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-2063, USA.
J Biol Chem. 2012 Feb 17;287(8):5434-45. doi: 10.1074/jbc.M111.312298. Epub 2011 Dec 13.
The plant phenylpropanoid pathway produces an array of metabolites that impact human health and the utility of feed and fiber crops. We previously characterized several Arabidopsis thaliana mutants with dominant mutations in REDUCED EPIDERMAL FLUORESCENCE 4 (REF4) that cause dwarfing and decreased accumulation of phenylpropanoids. In contrast, ref4 null plants are of normal stature and have no apparent defect in phenylpropanoid biosynthesis. Here we show that disruption of both REF4 and its paralog, REF4-RELATED 1 (RFR1), results in enhanced expression of multiple phenylpropanoid biosynthetic genes, as well as increased accumulation of numerous downstream products. We also show that the dominant ref4-3 mutant protein interferes with the ability of the PAP1/MYB75 transcription factor to induce the expression of PAL1 and drive anthocyanin accumulation. Consistent with our experimental results, both REF4 and RFR1 have been shown to physically associate with the conserved transcriptional coregulatory complex, Mediator, which transduces information from cis-acting DNA elements to RNA polymerase II at the core promoter. Taken together, our data provide critical genetic support for a functional role of REF4 and RFR1 in the Mediator complex, and for Mediator in the maintenance of phenylpropanoid homeostasis. Finally, we show that wild-type RFR1 substantially mitigates the phenotype of the dominant ref4-3 mutant, suggesting that REF4 and RFR1 may compete with one another for common binding partners or for occupancy in Mediator. Determining the functions of diverse Mediator subunits is essential to understand eukaryotic gene regulation, and to facilitate rational manipulation of plant metabolic pathways to better suit human needs.
植物苯丙烷途径产生一系列代谢物，这些代谢物会影响人类健康和饲料及纤维作物的利用。我们之前已经鉴定出几种拟南芥突变体，它们在 REDUCED EPIDERMAL FLUORESCENCE 4（REF4）中具有显性突变，导致矮小和苯丙烷积累减少。相比之下，ref4 缺失植株的株高正常，苯丙烷生物合成没有明显缺陷。在这里，我们表明 REF4 及其同源物 REF4-RELATED 1（RFR1）的破坏会导致多个苯丙烷生物合成基因的表达增强，以及许多下游产物的积累增加。我们还表明，显性 ref4-3 突变蛋白干扰 PAP1/MYB75 转录因子诱导 PAL1 表达和驱动花青素积累的能力。与我们的实验结果一致，REF4 和 RFR1 都已被证明与保守的转录共调节复合物 Mediator 发生物理关联，该复合物将来自顺式作用 DNA 元件的信息传递到核心启动子处的 RNA 聚合酶 II。综上所述，我们的数据为 REF4 和 RFR1 在 Mediator 复合物中的功能作用以及 Mediator 在维持苯丙烷稳态中的作用提供了重要的遗传支持。最后，我们表明野生型 RFR1 可显著减轻显性 ref4-3 突变体的表型，这表明 REF4 和 RFR1 可能相互竞争共同的结合伙伴或 Mediator 中的占位。确定不同 Mediator 亚基的功能对于理解真核生物基因调控以及促进对植物代谢途径的合理操作以更好地满足人类需求至关重要。
