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

我们已经确定了一个ON/OFF信号机制，是需要在拟南芥耐缺氧（缺氧）。Rop是一种单体RHO样G蛋白，其活化是乙醇脱氢酶（ADH）诱导和乙醇发酵所需的，但不足以耐受缺氧。缺氧耐受需要启动Rop信号传导以及随后通过GT3活化蛋白RopGAP 4终止Rop信号传导。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ADH诱导所需的H2 O2产生;此外，RopGAP 4对Rop信号的负调节减少了H2 O2诱导的损伤，降低了细胞活力并增加了缺氧耐受性。我们建议，增加胞浆游离钙，第二信使在缺氧反应，是必要的完成激活的Rop信号。 我们预测Rop信号和胞浆游离钙水平之间的相互作用决定了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响应于缺氧而启动Rop信号传导;（2）阐明胞质游离钙流在Rop活性和RopGAP 4基因表达的调节中的作用;（3）确定Rop介导的NADPH氧化酶的活化是否涉及Rop的重新定位并且需要胞质钙的增加;（4）测试H2 O2信号是否足以诱导ADH和RopGAP 4表达;和（5）探索参与缺氧耐受的Rop下游的信号传导机制。 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）使用总的和大的多聚核糖体mRNA分析由缺氧诱导的Rop信号传导调节的基因表达的变化;（2）筛选恢复ropgap 4-KO幼苗的缺氧耐受性的显性抑制突变;和（3）继续筛选基因陷阱系中改变ADH表达的突变。Rop在低氧胁迫反应中的正向和负向调节作用的阐明将使农作物的耐淹性工程化成为可能。本项目将阐明使植物细胞感知细胞氧耗尽并通过刺激发酵反应的机制。该项目将研究如何严格控制应激反应等同于生存。了解控制这种反应的信号过程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.