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

摘要 包括人类在内的许多活着的有机体都进化出了感知和响应矿物质水平的机制 营养素或氨基酸。在本应用中，我们提出使用拟南芥植物硫(S)营养传感 和信号作为一种实验系统来剖析转运受体介导的营养感知 机械装置。我们先前的研究已经确定硫酸盐转运蛋白SULTR1；2是一种受体。这 在真核生物中发现了一种双功能转运蛋白受体，称为转运体 并有望通过对其进行传感和运输来提高养分的利用效率 功能。然而，植物如何利用受体SULTR1；2在基因控制中转导S信号 表达仍然是一个中心未回答的问题，并成为本项目的重点。我们的 最近的研究揭示了一种保守的转录控制捷径模型，它将Rho家族的小 GTP酶对RNA聚合酶II(POL II)C-末端结构域(CTD)丝氨酸的磷酸化 人类。POL II CTD含有不同数量的保守七肽重复序列(Y1S2P3T4S5P6S7)。 每种氨基酸都可以进行不同的翻译后修饰。因此，翻译后的 修改模式很复杂，统称为CTD代码。在CTD代码中，Ser2和Ser5 基因的磷酸化对于启动转录和完成转录是非常重要的。 周而复始。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，帮助招募POL II 核心推动者。相反，在快捷模型中，Rho GTPase信号直接针对POL II CTD Ser2和Ser5被磷酸化，从而可以迅速带来大规模的基因表达变化。我们 现在有初步证据表明ROP2 GTP酶的活性，它是植物特有的成员之一 Rho GTP酶的ROP亚家族以及CTD Ser2和Ser5的磷酸化水平受S状态的影响。 此外，在SULTR1；2突变体中，ROP GTP酶活性更高。因此，我们假设ROP2及其 功能冗余的ROP4在SULTR1；2介导的S感知和信号转导中起负调控作用。这个 拟议的工作旨在填补从SULTR1；2到POL II转录的主要空白。具体地说，目标1是测试 ROP2和ROP4GTP酶在SULTR1；2介导的S传感和信号转导中的负调控作用 使用遗传和生化方法。目的2是检验ROP2介导的POL II捷径的假设 S的回答需要模型。在目标3中，我们建议测试由ROP2控制SLIM1转录因子 活性和ROP2介导的POL II CTD编码调制协同作用，以实现最高生产力 S缺乏诱导S应答基因的转录。拟议中的研究不仅将导致小说 对植物如何利用营养转运体SULTR1；2和ROP信号有效切换的机械学见解 控制基因表达，也为其他生物体的转录调控提供了一个范例。

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.