Nitric oxide as signalling molecule in plants: nitrate reductase or alternative sources for NO?

基于大量关于NO在脊椎动物中的作用的研究和与之相关的研究，在过去的十年中，NO在植物中的潜在作用和产生得到了越来越多的关注(Lesem 2000)。这项建议的目的是找出植物通过什么反应，产生多少，何时何地产生NO，以及NO合成是如何调控的，重点是植物与病原菌的相互作用。我们打算把重点放在两个潜在的NO来源上。另一种是一氧化氮合酶，利用NAD(P)H、氧气和各种辅助因子，将L精氨酸转化为瓜氨酸，从而产生NO。其他产生NO的酶，如黄嘌呤氧化酶，也将被考虑(Nakagawa)。我们希望通过利用各种烟草、拟南芥和菠菜突变体和转化体来研究叶片中NO的产生对环境因素的响应以及对激发子和病原体的响应。我们将应用化学发光法(Kaiser)和荧光NO指示剂(DAF 2和DAF 2 DA，Nakagawa)进行气相NO测量，并使用传统的分析方法来确定酶活性和基因表达。Nakagawa小组还将分离NR的三个功能结构域，以找出哪个结构域参与NO的产生。

Based on and related to the tremendous amount of research on the role of NO in vertebrates, the potential role and production of NO in plants has gained increasing attention during the last decade (Leshem 2000). The aim of this proposal is to find out by what reactions, how much, when and where NO is produced by plants and how NO synthesis is regulated, with emphasis on plant-pathogen interactions. We intend to focus on two potential NO sources. One is nitrate reductase (NR), which produces NO by reduction of nitrite with NAD(P)H, the other one is nitric oxide synthase (NOS) which is supposed to produce NO by converting L-arginine into citrulline, using NAD(P)H, oxygen and various co-factors. Other NO-producing enzymes, like xanthine oxidases, will also be considered (Nakagawa). We wish to study NO production in leaves in response to environmental factors and in response to elicitors and pathogens by using various tobacco-, Arabidopsis- and spinach mutants and transformants over- or underexpressing nitrate- or nitrate-reductase. We will apply gas phase NO measurements by chemiluminescence detection (Kaiser) as well as fluorescent NO indicators (DAF 2 and DAF 2 DA, Nakagawa), and use conventional assays for determination of enzyme activities and gene expression. The Nakagawa group will also isolate the three functional domains of NR in order to find out which domain is involved in NO production.