The Arabidopsis gp91-phox Gene Family and NADPH Oxidase Function

植物不能移动来保护自己免受环境中的入侵。它们也不像脊椎动物那样拥有复杂的、循环的、基于细胞的免疫防御系统。然而，植物已经进化出一种识别病原体的免疫系统，该系统启动一系列细胞反应，共同阻止入侵者或对胁迫情况做出反应。活性氧(ROI)的产生是植物受病原菌侵染后的早期反应之一。产生的ROI可能直接参与了对入侵病原体的杀灭，但也可以作为诱导进一步防御的信号。质膜结合的NADPH氧化酶可能是植物ROI的来源。这种氧化酶，也被称为呼吸爆发氧化酶(RBO)，最初在哺乳动物巨噬细胞中被描述，是一种介导微生物死亡的多酶复合体。这种蛋白质是NADPH氧化酶的酶促亚单位，它使用分子氧来生成超氧化物。拟南芥有一个10个成员的Atrboh(拟南芥呼吸爆发氧化酶同源)基因家族，与被称为gp91Phox的动物蛋白同源。然而，NADPH氧化酶的精确亚基结构及其激活的调控似乎与哺乳动物巨噬细胞不同。越来越明显的是，ROI不是一个刽子手，而是一个信号，介导防御的激活以及对环境和发育过程的其他反应。在哺乳动物中，gp91Phox的多种亚型在不同的细胞类型中发挥作用，并发挥不同的功能。类似地，植物中不同的NADPH氧化酶也可能在不同的组织和对不同环境刺激的反应中调节质外体ROI的产生。例如，我们的团队已经证明，AtrbohD和AtrbohF负责在防御反应期间产生ROI，并且通过这些Atrboh家族成员产生的ROI在气孔关闭期间作为ABA信号的第二信使是必需的。此外，AtrbohC介导的氧化爆发通过激活允许钙进入和离开细胞的通道来调节植物根毛形成期间的细胞扩张。有趣的是，一氧化氮(NO)，另一种在哺乳动物巨噬细胞中介导细胞死亡的反应分子，可能与ROI一起调节植物的反应。研究表明，与病原体的相互作用会触发NO的释放，而ROI和NO似乎会协同作用来控制过敏反应。本项目的目标是利用功能基因组学工具，破译植物NADPH氧化酶基因家族的功能。需要检验的假设是，Atrboh的不同成员，植物NADPH氧化酶的组成部分，在防御反应和发育过程中控制ROI的产生，PCD和其他对环境的反应。ROI和NO(PCD的另一个重要调节因子)和其他反应之间的联系也将被调查。Atrboh基因的鉴定也为研究激活植物NADPH氧化酶的机制奠定了基础。这一点特别重要，因为哺乳动物NADPH氧化酶的重要调控元件似乎不存在于植物氧化酶中。将进行研究以确定ROI信号的更多成分，特别是那些参与调节Atrboh功能的信号转导途径的早期步骤，以及可能的ROI依赖信号的靶点或介体。这些研究将有助于进一步了解ROI在抗病反应中的作用，以及在ROI和NO介导的一系列发育反应中的作用。

Plants cannot move to defend themselves from aggressions in their environment. Also they do not posses a complex, circulating, cell-based immunological system of defense like vertebrates. However, plants have evolved an immune system for pathogen recognition that initiates a set of cellular responses that collectively stop the intruder or can react and respond to stress situations. Production of Reactive Oxygen Species (ROI) is one of the earlier responses observed after pathogen infection in plants. ROI produced may directly participate in the killing of the invading pathogens, but could also act as a signal that induces further defenses. A plasma membrane bound NADPH oxidase is the likely source of ROI in plants. This oxidase, also known as the respiratory burst oxidase (RBO), was initially described in mammalian macrophages and is a multienzymatic complex that mediate the killing of microbes. This protein is the enzymatic subunit of the NADPH oxidase that uses molecular oxygen to make superoxide. Arabidopsis has a 10 member Atrboh (Arabidopsis thaliana respiratory burst oxidase homologs), gene family, homologous to the animal protein called gp91phox. However, the precise subunit structure of the NADPH oxidase and the regulation of its activation appear to be different than in mammalian macrophages.It is becoming evident that ROI is not an executioner but a signal that mediate the activation of the defenses as well as other responses to the environment and developmental processes. In mammals, multiple isoforms of gp91phox act in different cell types, and perform different functions. Similarly, different NADPH oxidases in plants may also mediates production of apoplastic ROI in different tissues and in responses to different environmental stimuli. For example, our group has shown that AtrbohD and AtrbohF are responsible for the production of ROI during the defense response and that ROI generated through these members of the Atrboh family are required as second messengers in ABA signaling during stomata closure. Also, the oxidative burst mediated by AtrbohC regulates plant cell expansion during root hair formation through the activation of channels that allow calcium to move in and out of the cell.. Interestingly, Nitric Oxide (NO), another reactive molecule that mediates cell death in mammalian macrophages, may regulate plant responses in conjunction to ROI. Studies show that interaction with pathogens trigger NO release, and ROI and NO seem to, then, work synergistically to control the hypersensitive response.The goal of this project is to decipher, using functional genomics tools, the functions of the plant NADPH oxidase gene family. The hypothesis to be tested is that different members of Atrboh, components of the plant NADPH oxidase, control production of ROI during defense response and in developmental processes, PCD and other responses to the environment. The connections between ROI and NO, another important regulator of PCD and other responses, will be also investigated. The identification of the Atrboh genes also enables to study the mechanism that activates the plant NADPH oxidase. This is particularly relevant since important regulatory elements of the mammalian NADPH oxidase appear to be absent in the plant oxidase. Studies will be conducted to identify more components of the ROI signaling, especially those involved in earlier steps of the signal transduction pathway that regulate Atrboh function, as well as putative targets or mediators of ROI-dependent signaling. These studies will allow to further understand the function of ROI during disease resistance responses, as well as in a range of developmental responses mediated by ROI and NO.