Positive and Negative Regulation of Rop Signaling in the Response to Oxygen Deprivation

我们已经确定了拟南芥（Arabidopsis thaliana）耐受缺氧（缺氧）所需的一个ON/OFF信号机制。Rop是一种单体rho样g蛋白，它的激活是乙醇脱氢酶（ADH）诱导和乙醇发酵所必需的，但对缺氧耐受却不够。对缺氧的耐受需要启动Rop信号以及随后由GTPase激活蛋白RopGAP4终止Rop信号。我们的数据支持一个模型，其中rop信号促进NADPH氧化酶活性和诱导ADH所需的H2O2产生；此外，RopGAP4负向调控Rop信号，可降低h2o2诱导的损伤，延长细胞活力，提高缺氧耐受性。我们认为，胞质游离钙的增加是完成Rop信号激活所必需的，这是响应氧剥夺的第二个信使。我们预测Rop信号和胞质游离钙水平之间的相互作用决定了胁迫耐受所需的基因表达的变化。我们的目标是：(1)确定线粒体信号是否在缺氧时启动Rop信号；(2)阐明胞质游离钙通量在Rop活性和RopGAP4基因表达调控中的作用；(3)确定Rop介导的NADPH氧化酶激活是否涉及Rop的重新定位，是否需要增加胞质钙；(4)检测H2O2信号是否足以诱导ADH和RopGAP4表达；(5)探索Rop下游参与缺氧耐受的信号机制。我们将使用基因组和遗传分析来有效地研究这个信号网络。这些研究将包括：(1)利用总和大多体mRNA分析缺氧诱导的Rop信号调节的基因表达变化；(2)筛选恢复ropgap4-KO幼苗缺氧耐受性的显性抑制突变；(3)继续筛选改变ADH表达的基因诱捕系。阐明Rop在低氧胁迫响应中的正调控和负调控作用，为作物耐涝工程提供了理论依据。这个项目将阐明机制，允许植物细胞感知细胞氧气的消耗，并通过刺激发酵作出反应。该项目将研究如何严格控制应激反应等于生存。了解控制这种反应的信号传导过程将使科学家能够操纵洪水耐受能力。由于植物的应激反应与植物和动物的低氧应激反应之间的相似性，这些结果可能与植物和动物生物学具有额外的相关性。

We have identified an ON/OFF signaling mechanism that is required for tolerance of oxygen deprivation (anoxia) in Arabidopsis thaliana. Activation of Rop, a monomeric RHO-like G-protein, is required for alcohol dehydrogenase (ADH) induction and ethanolic fermentation but is insufficient for anoxia tolerance. Tolerance of anoxia requires initiation of Rop signaling as well as the subsequent termination of Rop signaling by the GTPase activating protein RopGAP4. Our data support a model in which Rop-signaling promotes NADPH oxidase activity and H2O2 production required for ADH induction; in addition the negative regulation of Rop signaling by RopGAP4 reduces H2O2-induced damage, prolongs cell viability and increases anoxia tolerance. We propose that an increase in cytosolic free calcium, a second messenger in response to oxygen deprivation, is necessary to complete the activation of Rop signaling. We predict that interplay between Rop signaling and cytosolic free calcium levels governs changes in gene expression necessary for stress tolerance. Our goals are to: (1) Determine if a mitochondrial signal initiates Rop signaling in response to oxygen deprivation; (2) Elucidate the role of cytosolic free calcium fluxes in the regulation of Rop activity and RopGAP4 gene expression; (3) Determine whether Rop-mediated activation of NADPH oxidase involves the relocation of Rop and requires an increase in cytosolic calcium; (4) Test whether an H2O2 signal is sufficient to induce ADH and RopGAP4 expression; and (5) Explore the signaling mechanisms downstream of Rop that are involved in anoxia tolerance. We will use genomic and genetic analyses to effectively study this signaling network. These studies will include: (1) Analysis of changes in gene expression regulated by anoxia-induced Rop signaling using total and large polysomal mRNA; (2) A screen for dominant suppressor mutations that restore anoxia tolerance to ropgap4-KO seedlings; and (3) Continued screening of gene-trap lines for mutations altering ADH expression. The elucidation of the role of positive and negative regulation of Rop in the low-oxygen stress response should enable the engineering of flooding tolerance in crop plants.This project will elucidate the mechanisms that allow plant cells to sense a depletion of cellular oxygen and respond by stimulating fermentation. The project will study how the strict control of the stress response is tantamount for survival. The understanding of the signaling process that controls this response will enable scientists to manipulate flooding tolerance. The results may have additional relevance to plant and animal biology, due to similarities between stress responses in plants and the low-oxygen stress response in plants and animals.