NAD(P)H oxidases were detected in suspension cultured cells of ginseng (Panax ginseng C.A. Meyer). The activities of these enzymes were induced by an elicitor (Cle) extracted from cell walls of Colletotrichum lagerarium. In addition, Cle induced an oxidative burst and enhanced the synthesis of saponin, activity of phenylalanine ammonialyase (PAL), accumulation of chalcone synthase (CHS) and the transcription of a hydroxyproline_rich glycoprotein gene (hrgp). Pre_treatments with DPI and quinacrine (two inhibitors of mammalian neutrophil plasma membrane NADPH oxidase) for 30 min prior to Cle addition blocked the NAD(P)H oxidase activity induced by Cle. These inhibitors also inhibited the release of H2O2, the synthesis of saponin, PAL activity and CHS accumulation. Our data revealed homology between plasma membrane NAD(P)H oxidases of mammalian neutrophil cells and ginseng suspension cells. They also indicated that Cle_activated NAD(P)H oxidases catalysed the release of H2O2 and that H2O2 was functioning as a second messenger stimulating PAL activity, saponin synthesis and hrgp transcription. Elevations of Ca2+ and protein phosphorylation/dephosphorylation were required for this defense process. We propose that NAD(P)H oxidases mediate the processes of Cle_induced defense responses in ginseng suspensions, and postulate the existence of a signalling cascade including extracellular Cle stimulation, activation of plasma membrane NAD(P)H oxidases, release of H2O2, and the intracellular responses of metabolism and gene transcription in ginseng suspension cells.
质膜NAD（P）H 氧化酶参予金瓜炭疽（Colletotrichum lagerarium）细胞壁激发子诱导人参细胞产生激发反应
胡向阳1 Steven J. Neill2 方建颖1 蔡伟明1* 汤章城1
（1. 中国科学院上海生命科学研究院植物生理生态研究所，上海 200032；
2. Centra for Research in Plant Science, University of the West of England, Bristol, Coldharbour Lane, Bristol BS16 1QY, United Kindom）
摘要： 在人参（Panax ginseng C. A. Meyer）悬浮细胞质膜上测出了NAD（P）H 氧化酶活性。这类NAD（P）H 氧化酶活性可以被金瓜炭疽细胞壁激发子（Cle）诱导。Cle 处理还能诱导人参悬浮细胞的氧迸发、促进人参悬浮细胞的皂苷合成、提高苯丙氨酸解氨酶（PAL）的活力、以及诱导查尔式酮酶（CHS）的累积和细胞壁上抗性相关蛋白基因脯氨酸富裕蛋白基因hrgp（Hydroxyprolin-rich glycoproteins）的表达。当用哺乳动物白细胞质膜NADPH氧化酶的特异性抑制剂二亚苯基碘（Diphenylene iodonium, DPI）与奎吖因（quinacrine）预处理人参悬浮细胞30 min 后，Cle诱导的H2O2 释放与Cle 激活的质膜NAD(P)H 氧化酶活性被抑制，同时Cle诱导的PAL活性及CHS 的积累下降，皂苷合成与hrgp的表达被抑制。由此推测：人参细胞质膜NAD(P)H 氧化酶与哺乳动物白细胞质膜NADPH氧化酶有很大的相似性。在Cle激发人参悬浮细胞产生氧迸发的过程中，NAD(P)H 氧化酶活性被诱导从而导致H2O2 的产生，H2O2 作为第二信使，激活苯丙氨酸途径，诱发人参皂苷的合成及hrgp防御基因的表达。这一过程中还涉及到Ca2+ 内流，胞内Ca2+浓度的升高，蛋白磷酸化与去磷酸化。人参细胞质膜NAD(P)H氧化酶在人参细胞对Cle的反应过程中起一种介导作用。因此可能存在由Cle刺激, NAD(P)H 氧化酶被诱导，H2O2释放，到人参细胞产生激发反应这样一个由外及内的级联反应。
关键词： NAD(P)H 氧化酶；人参；金瓜炭疽；激发子；信号转导
通讯作者E-mail: wmcai@iris.sipp.ac.cn
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