Bolwell G Paul, Bindschedler Laurence V, Blee Kristopher A, Butt Vernon S, Davies Dewi R, Gardner Sarah L, Gerrish Chris, Minibayeva Farida
Division of Biochemistry, School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX, UK.
J Exp Bot. 2002 May;53(372):1367-76.
The oxidative burst, the generation of reactive oxygen species (ROS) in response to microbial pathogen attack, is a ubiquitous early part of the resistance mechanisms of plant cells. It has also become apparent from the study of a number of plant-pathogen interactions and those modelled by elicitor treatment of cultured cells that there may be more than one mechanism operating. However, one mechanism may be dominant in any given species. NADPH oxidases have been implicated in a number of systems and have been cloned and characterized. However, the enzyme system which is the major source of ROS in French bean (Phaseolus vulgaris) cells treated with a cell wall elicitor from Colletotrichum lindemuthianum, appears to be dependent on an exocellular peroxidase. The second component, the extracellular alkalinization, occurs as a result of the Ca(2+) and proton influxes and the K(+) efflux common to most elicitation systems as one of the earliest responses. The third component, the actual reductant/substrate, has remained elusive. The low molecular weight compound composition of apoplastic fluid was compared before and after elicitation. The substrate only becomes available some min after elicitation and can be extracted, so that by comparing the profiles by LC-MS it has been possible to identify possible substrates. The mechanism has proved to be complex and may involve a number of low molecular weight components. Stimulation of H(2)O(2) production was observed with saturated fatty acids such as palmitate and stearate without concomitant oxylipin production. This biochemical evidence is supported by immunolocalization studies on papillae forming at bacterial infection sites that show the peroxidase isoform present at sites of H(2)O(2) production revealed by cerium chloride staining together with the cross-linked wall proteins and callose and callose synthase. The peroxidase has been cloned and expressed in Pichia pastoris and has been shown to catalyse the oxidation reaction with the same kinetics as the purified enzyme. Furthermore, Arabidopsis plants transformed heterologously using the French bean peroxidase in antisense orientation have proved to be highly susceptible to bacterial and fungal pathogens. Thus it is possible that Arabidopsis is another species with the potential to mount an apoplastic oxidative burst and these transformed plant lines may be useful to identify the peroxidase that is responsible.
氧化爆发，即植物细胞响应微生物病原体攻击而产生活性氧（ROS），是植物细胞抗性机制中普遍存在的早期环节。从对多种植物 - 病原体相互作用以及通过激发子处理培养细胞所建立的模型研究中也可以明显看出，可能存在多种作用机制。然而，在任何特定物种中，可能有一种机制占主导地位。NADPH氧化酶已在多个系统中被涉及，并已被克隆和表征。然而，在用来自菜豆炭疽菌的细胞壁激发子处理的菜豆（Phaseolus vulgaris）细胞中，作为ROS主要来源的酶系统似乎依赖于一种胞外过氧化物酶。第二个组成部分，细胞外碱化，是大多数激发系统中常见的Ca(2+)和质子内流以及K(+)外流的结果，是最早的反应之一。第三个组成部分，实际的还原剂/底物，仍然难以捉摸。比较了激发前后质外体流体的低分子量化合物组成。底物在激发后几分钟才变得可用并且可以被提取，因此通过液相色谱 - 质谱比较图谱，有可能识别可能的底物。事实证明该机制很复杂，可能涉及多种低分子量成分。观察到饱和脂肪酸如棕榈酸和硬脂酸可刺激H(2)O(2)的产生，且不伴随氧脂的产生。关于在细菌感染部位形成的乳头的免疫定位研究支持了这一生化证据，该研究表明，通过氯化铈染色显示的H(2)O(2)产生部位存在的过氧化物酶同工型与交联的壁蛋白、胼胝质和胼胝质合酶在一起。该过氧化物酶已在毕赤酵母中克隆并表达，并已证明其催化氧化反应的动力学与纯化酶相同。此外，用反义方向的菜豆过氧化物酶进行异源转化的拟南芥植株已被证明对细菌和真菌病原体高度敏感。因此，拟南芥有可能是另一种具有进行质外体氧化爆发潜力的物种，这些转化的植物品系可能有助于鉴定负责的过氧化物酶。
