Role of ROP GTPases in sulfate transceptor SULTR1;2-regulated sulfur nutrient sensing

摘要 包括人类在内的许多生物体已经进化出了感知和响应矿物质水平的机制 营养素或氨基酸。在这个应用中，我们建议使用拟南芥植物硫（S）营养感测 和信号传导作为一个实验系统来剖析运输受体介导的营养传感 机制等我们先前的研究已经鉴定了硫酸盐转运蛋白SULTR1; 2作为受体。这 在真核生物中，越来越多的人发现了一种具有双重功能的转运受体，称为转运体 生物，并预计将通过执行其传感和运输来提高养分利用效率 功能协调发展的然而，植物如何利用SULTR1; 2受体来调控S信号在基因调控中的作用， 表达仍然是一个尚未回答的核心问题，并成为本项目的重点。我们 最近的研究揭示了一个保守的转录调控的快捷模式， RNA聚合酶II（Pol II）的GTP酶C-末端结构域（CTD）跨植物、真菌和真菌的Ser磷酸化 人类Pol II CTD含有不同数量的保守七肽重复序列（Y1S2P3T4S5P6S7）， 其中每个氨基酸可以进行不同的翻译后修饰。因此，翻译后 修改模式是复杂的，统称为CTD代码。在CTD代码中，Ser2和Ser5 磷酸化沿着基因对于启动转录和完成转录非常重要 周期在经典的转录控制模型中，当被Rho或Ras GTP酶激活时，MAP 激酶级联激活与基因特异性顺式元件结合的转录因子，并帮助募集Pol II， 核心启动子相比之下，在捷径模型中，Rho GT3信号直接靶向Pol II CTD， Ser2和Ser5磷酸化，从而可以迅速引起大规模的基因表达变化。我们 现在有初步的证据表明，ROP 2 GT3的活性，这是植物特有的成员， Rho GTP酶的ROP亚家族以及CTD Ser2和Ser5磷酸化水平受S状态影响。 此外，ROP GT3活性在SULTR 1; 2突变体中更高。因此，我们假设ROP 2及其 功能冗余的ROP 4在SULTR1; 2介导的S传感和信号传导中起负调节剂的作用。的 建议的工作旨在填补从SULTR1; 2到Pol II转录的主要空白。具体来说，目标1是测试 ROP2和ROP4 GTP酶在SULTR1; 2介导的S感受和信号传导中起负调节剂的作用 使用遗传学和生物化学方法。目的2是检验ROP2介导的Pol II捷径 S响应需要模型。在目标3中，我们建议测试ROP 2控制的SLIM 1转录因子 活性和ROP2介导的Pol II CTD编码调节协同作用， S-缺陷诱导的S-反应基因的转录。这项研究不仅会带来新的 植物如何利用营养素转运体SULTR1; 2和ROP信号转导开关，以有效地 控制基因表达，而且还为其他生物体中的转录调节提供范例。

ABSTRACT Many living organisms including humans have evolved mechanisms to sense and respond to levels of mineral nutrients or amino acids. In this application, we propose to use Arabidopsis plant sulfur (S) nutrient sensing and signaling as an experimental system to dissect transporting receptor-mediated nutrient sensing mechanisms. Our prior research has led to identification of sulfate transporter SULTR1;2 as a receptor. This type of dual-function transporting receptor, termed transceptor, has been increasingly found in eukaryotic organisms and is expected to increase nutrient use efficiency by performing both its sensing and transporting functions. However, how plants use the transceptor SULTR1;2 to transduce the S signal in the control of gene expression has remained a central unanswered question and becomes the focus of the present project. Our recent studies have revealed a conserved shortcut model of transcriptional control that links Rho family small GTPases to RNA polymerase II (Pol II) C-terminal domain (CTD) Ser phosphorylation across plants, fungi and humans. The Pol II CTD contains various number of conserved heptad peptide repeats (Y1S2P3T4S5P6S7) in which each amino acid can be subjected to different posttranslational modifications. Thus, the posttranslational modification pattern is complex and collectively called the CTD code. Among the CTD code, Ser2 and Ser5 phosphorylation along the gene is very important for initiating transcription and completing the transcription cycle. In the classical model of transcriptional control, upon activation by Rho or Ras GTPases, the MAP kinase cascade activates transcription factors that bind to gene-specific cis-elements and helps recruit Pol II to the core promoter. In contrast, in the shortcut model, Rho GTPase signaling directly targets the Pol II CTD Ser2 and Ser5 phosphorylation and thus can rapidly bring about large-scale gene expression changes. We now have preliminary evidence indicating that activity of ROP2 GTPase, which is a member of plant-unique ROP subfamily of Rho GTPases, and levels of CTD Ser2 and Ser5 phosphorylation are impacted by S status. In addition, ROP GTPase activity is higher in SULTR1;2 mutants. Therefore, we hypothesize that ROP2 and its functionally redundant ROP4 act as negative regulators in SULTR1;2-mediated S sensing and signaling. The proposed work aims to fill the major gaps from SULTR1;2 to Pol II transcription. Specifically, Aim 1 is to test that ROP2 and ROP4 GTPases act as negative regulators in SULTR1;2-mediated S sensing and signaling using genetic and biochemical approaches. Aim 2 is to test the hypothesis that ROP2-mediated Pol II shortcut model is required for S response. In Aim 3, we propose to test that ROP2-controlled SLIM1 transcription factor activity and ROP2-mediated Pol II CTD code modulation act cooperatively to achieve the most productive transcription of S-deficiency induced S-response genes. The proposed study will not only lead to novel mechanistic insights into how plants use nutrient transceptor SULTR1;2 and ROP signaling switch to efficiently control gene expression but also provide a paradigm for transcriptional regulation in other organisms.