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）引入大气以来，活性氧中间体（ROI）一直是生命中不受欢迎的伙伴。与氧气相反，这些部分还原或活化的O2衍生物具有高活性和毒性，可导致细胞氧化破坏。在人类中，ROI在衰老、癌症和各种退行性疾病等过程中发挥着关键作用。在植物和作物中，ROI是干旱、热冲击、寒冷和盐胁迫下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调节了植物的许多不同过程，包括应激反应、发育和生长。这种冲突在细胞中是如何解决的在很大程度上是未知的。尽管如此，很明显，细胞中的ROI水平需要严格控制。我们的目标是识别和表征控制植物中ROI积累的不同细胞机制。我们提出了一种独特的方法，利用突变体，缺乏关键的ROI解毒酶，诱导ROI在特定细胞区室的积累。我们假设具有抑制去除ROI能力的突变体将积累ROI并激活其ROI响应机制。因此，对这些突变体基因表达的研究将使我们能够识别和表征一些控制植物ROI积累的关键调控基因。此外，我们的方法将使我们能够简化ROI与许多不同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植物ROI代谢的不同基因。基于我们的初步结果，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。拟南芥具有抑制ROI清除能力的突变体将在受控条件下（使用时间-过程设计）诱导植物内部ROI胁迫。分子分析结合生理和生化测量将用于识别在ROI积累时细胞中激活的调控基因。表达锌指蛋白和其他roi调控基因的转基因植物将在琼脂板上筛选其对氧化胁迫的耐受性。我们筛选的特定基因的功能将在转基因植物和突变体中确定。将开发一种基因筛选来识别roi传感器。数据、方法和生物材料一经确认，将立即向公众开放。该项目将招收20名本科生，包括少数民族学生，并向他们介绍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，以分享知识并提高对ROI及其在植物和其他生物中的重要作用的认识。将开发一套高中教学工具包，其中包括一个视频演示和几个基本实验，展示ROI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。耐ROI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）可以被许多不同的研究小组和科学家使用，以确定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，增强我们对植物正常代谢和环境胁迫期间ROI功能的全面了解。

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.