Using a Functional Genomics Approach to Study ROI Metabolism in Plants

自从大约20亿年前光合生物将氧气（O2）引入大气层以来，活性氧中间体（ROI）一直是生命不受欢迎的伴侣。与氧气相反，这些部分还原或活化的O2衍生物具有高度反应性和毒性，可导致细胞的氧化破坏。在人类中，ROI在衰老和癌症以及不同的退行性疾病等过程中发挥着关键作用。在植物和作物中，ROI是在诸如干旱、热激、寒冷和盐胁迫期间伤害和产量损失的主要原因。尽管它们具有毒性，但最近的研究表明ROI调节植物的许多不同过程，包括胁迫反应，发育和生长。这种冲突在细胞中如何解决在很大程度上是未知的。尽管如此，很明显，细胞中的ROI水平需要严格调节。我们的目标是确定和表征不同的细胞机制，控制投资回报率的积累在植物中。我们提出了一种独特的方法，使用突变体，缺乏关键的ROI解毒酶，以诱导特定的细胞隔室中的ROI的积累。我们假设，突变体抑制能力，以消除投资回报率将积累投资回报率和激活其投资回报率的反应机制。因此，在这些突变体中的基因表达的研究将使我们能够识别和表征一些关键的调控基因，控制投资回报率在植物中的积累。此外，我们的方法将使我们能够简化ROI和许多不同的细胞途径之间存在的复杂相互作用，并分离和表征直接参与调节ROI代谢的不同基因。根据我们的初步结果，我们假设锌指基因家族的成员在调节植物ROI代谢中发挥着核心作用。我们将确定它们如何控制ROI代谢，以及它们如何适应ROI反应基因的层次结构。 具有抑制R 0 I的能力的拟南芥突变体将用于在受控条件下诱导植物中的内部R 0 I胁迫（使用时程设计）。结合生理和生化测量的分子分析将用于鉴定ROI积累时细胞中激活的调控基因。将在琼脂平板上筛选表达锌指蛋白和其他ROI调节基因的转基因植物对氧化胁迫的耐受性。通过我们的筛选鉴定的特定基因的功能将在转基因植物和突变体中确定。将开发一种识别ROI传感器的基因筛选。数据、方法和生物材料一经确认，将立即向公众提供。多达20名本科生，包括少数民族，将被招募到该项目，并介绍了植物生物学的现代研究技术。将为本科生开发一门在线课程，以分享知识，提高对ROI及其在植物和其他生物中所起重要作用的认识。将开发一个高中教学工具包，其中包括一个视频演示和几个基本实验，证明投资回报率对植物保护的重要性。公众可以通过专门为该项目开发的网站访问该项目。许多不同的研究小组和科学家可以使用ROI耐受植物、ROI相关基因数据库和ROI响应流程图（在本研究过程中开发）来确定ROI在多大程度上和以何种方式参与他们正在研究的生物过程/途径。它们将作为一种宝贵的资源，并增强我们对植物在正常代谢过程中以及环境胁迫期间ROI功能的整体理解。

Ever since the introduction of oxygen (O2) into our atmosphere by photosynthetic organisms, about two billion years ago, reactive oxygen intermediates (ROI) have been the unwelcome companions of life. In contrast to oxygen, these partially reduced or activated derivatives of O2 are highly reactive and toxic and can lead to the oxidative destruction of cells. In humans ROI play a key role in processes such as aging and cancer and in different degenerative diseases. In plants and crops ROI are the main cause of injury and yield loss during stresses such as drought, heat shock, cold and salinity. In spite of their toxicity, recent studies suggested that ROI regulate many different processes in plants including stress-response, development and growth. How this conflict is resolved in cells is largely unknown. Nonetheless, it is clear that the level of ROI in cells needs to be tightly regulated. Our goal is to identify and characterize the different cellular mechanisms that control ROI accumulation in plants. We propose a unique approach of using mutants, deficient in key ROI-detoxifying enzymes, to induce the accumulation of ROI in specific cellular compartments. We hypothesize that mutants with suppressed capability to remove ROI will accumulate ROI and activate their ROI-response mechanisms. The study of gene expression in these mutants will therefore enable us to identify and characterize some of the key regulatory genes that control ROI accumulation in plants. Moreover, our approach will enable us to simplify the complex interactions that exist between ROI and many different cellular pathways, and to isolate and characterize different genes directly involved in regulating ROI metabolism in plants. Based on our preliminary results we hypothesize that members of the zinc-finger gene family play a central role in regulating ROI metabolism in plants. We will determine how they control ROI metabolism and how they fit into the hierarchy of the ROI-response genes. Arabidopsis mutants with suppressed capability to scavenge ROI will be used to induce an internal ROI stress in plants under controlled conditions (using a time-course design). Molecular analysis coupled with physiological and biochemical measurements will be used to identify regulatory genes activated in cells upon ROI accumulation. Transgenic plants expressing zinc-finger proteins and other ROI-regulatory genes will be screened for their tolerance to oxidative stress on agar plates. The function of specific genes, identified by our screen, will be determined in transgenic plants and mutants. A genetic screen to identify ROI-sensors will be developed. Data, methods, and biological material will be made available to the public as soon as they are confirmed. Up to 20 undergraduate students, including minorities, will be recruited into the project and introduced to modern research techniques in plant biology. An online course for undergraduate students will be developed to share knowledge and enhance awareness to ROI and the important role they play in plants and other organisms. A high school teaching kit that includes a video presentation and several basic experiments demonstrating the importance of ROI to plant protection will be developed. Public access to the project will be ensured through a website specifically developed for the project.The ROI-tolerant plants, the database of ROI-associated genes and the flow-chart of ROI response (developed during the course of this study), could be used by many different research groups and scientists to determine to what extent and in what manner ROI are involved in the biological process/pathway they are studying. They would serve as a valuable resource and enhance our overall understanding of ROI function in plants during normal metabolism, as well as during periods of environmental stress.