Bestwick C S, Brown I R, Bennett M H, Mansfield J W
Department of Biological Sciences, Wye College, University of London, Kent, United Kingdom.
Plant Cell. 1997 Feb;9(2):209-21. doi: 10.1105/tpc.9.2.209.
The active oxygen species hydrogen peroxide (H2O2) was detected cytochemically by its reaction with cerium chloride to produce electron-dense deposits of cerium perhydroxides. In uninoculated lettuce leaves, H2O2 was typically present within the secondary thickened walls of xylem vessels. Inoculation with wild-type cells of Pseudomonas syringae pv phaseolicola caused a rapid hypersensitive reaction (HR) during which highly localized accumulation of H2O2 was found in plant cell walls adjacent to attached bacteria. Quantitative analysis indicated a prolonged burst of H2O2 occurring between 5 to 8 hr after inoculation in cells undergoing the HR during this example of non-host resistance. Cell wall alterations and papilla deposition, which occurred in response to both the wild-type strain and a nonpathogenic hrpD mutant, were not associated with intense staining for H2O2, unless the responding cell was undergoing the HR. Catalase treatment to decompose H2O2 almost entirely eliminated staining, but 3-amino-1,2,4-triazole (catalase inhibitor) did not affect the pattern of distribution of H2O2 detected. H2O2 production was reduced more by the inhibition of plant peroxidases (with potassium cyanide and sodium azide) than by inhibition of neutrophil-like NADPH oxidase (with diphenylene iodonium chloride). Results suggest that CeCl3 reacts with excess H2O2 that is not rapidly metabolized during cross-linking reactions occurring in cell walls; such an excess of H2O2 in the early stages of the plant-bacterium interaction was only produced during the HR. The highly localized accumulation of H2O2 is consistent with its direct role as an antimicrobial agent and as the cause of localized membrane damage at sites of bacterial attachment.
通过过氧化氢（H₂O₂）与氯化铈反应生成电子致密的过氢氧化铈沉积物，对活性氧物质过氧化氢进行了细胞化学检测。在未接种的生菜叶片中，H₂O₂通常存在于木质部导管的次生加厚壁内。用菜豆丁香假单胞菌野生型细胞接种会引发快速的超敏反应（HR），在此期间，在附着细菌相邻的植物细胞壁中发现了H₂O₂的高度局部积累。定量分析表明，在这个非寄主抗性例子中，接种后5至8小时，正在经历HR的细胞中H₂O₂会出现长时间的爆发。细胞壁改变和乳头状沉积物，这是对野生型菌株和无致病性的hrpD突变体的反应，与H₂O₂的强烈染色无关，除非响应细胞正在经历HR。用过氧化氢酶处理以分解H₂O₂几乎完全消除了染色，但3-氨基-1,2,4-三唑（过氧化氢酶抑制剂）并不影响检测到的H₂O₂的分布模式。与抑制嗜中性粒细胞样NADPH氧化酶（用二苯基碘鎓氯化物）相比，抑制植物过氧化物酶（用氰化钾和叠氮化钠）对H₂O₂产生的抑制作用更大。结果表明，CeCl₃与过量的H₂O₂反应，这种过量的H₂O₂在细胞壁发生的交联反应中不会迅速代谢；在植物与细菌相互作用的早期阶段，这种过量的H₂O₂仅在HR期间产生。H₂O₂的高度局部积累与其作为抗菌剂的直接作用以及作为细菌附着部位局部膜损伤原因的作用一致。
