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

我们已经确定了拟南芥耐缺氧(缺氧)所需的开/关信号机制。Rop是一种单体Rho样G蛋白，它的激活是酒精脱氢酶(ADH)诱导和乙醇发酵所必需的，但不足以耐缺氧。耐缺氧需要启动Rop信号，然后通过GTP酶激活蛋白RopGAP4终止Rop信号。我们的数据支持一个模型，在该模型中，Rop信号促进ADH诱导所需的NADPH氧化酶活性和H_2O_2的产生；此外，RopGAP4对Rop信号的负调控减少了H_2O_2诱导的损伤，延长了细胞存活，并提高了耐缺氧能力。我们认为，细胞内游离钙的增加是完成Rop信号的激活所必需的，它是对缺氧做出反应的第二信使。我们预测，Rop信号和胞内游离钙水平之间的相互作用控制着胁迫耐受所必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启动了Rop信号对缺氧的反应；(2)阐明细胞内游离钙通量在调节Rop活性和RopGAP4基因表达中的作用；(3)确定Rop介导的NADPH氧化酶的激活是否涉及Rop的重新定位和需要细胞内钙的增加；(4)测试H_2O_2信号是否足以诱导ADH和RopGAP4的表达；以及(5)探索Rop下游参与耐缺氧的信号机制。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)利用总的和大的多聚体mRNA分析受缺氧诱导的Rop信号调控的基因表达的变化；(2)筛选恢复ropgap4-KO幼苗耐缺氧的显性抑制突变；以及(3)继续筛选改变ADH表达的基因陷阱系。阐明Rop的正负调控在低氧胁迫反应中的作用，将有助于作物耐淹的工程研究。本项目将阐明植物细胞感受细胞氧耗竭并通过刺激发酵作出反应的机制。该项目将研究严格控制压力反应如何等同于生存。对控制这种反应的信号传递过程的了解将使科学家能够操纵洪水耐受性。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有更多的相关性。

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.