We have analyzed the spatial and temporal expression of defense responses induced in attached tobacco leaves treated with a Phytophthora megasperma glycoprotein that was previously shown to be an efficient elicitor of the hypersensitive reaction. The infiltrated tissue (zone 1), the surrounding tissue (zone 2), which is 5 mm in width, and tissue at a distance >2 cm from zone 2 (zone 3) were analyzed separately. Cell death occurred only in zone 1 and was completed by 14 h. Defense gene expression was induced in zones 1 and 2 with striking differential patterns, but not in zone 3. There was a rapid (1 to 4 h) induction of genes of the phenylpropanoid, salicylic acid, and ses-quiterpenoid pathways in zones 1 and 2. However, it was strong and transient in the former and of lesser extent but sustained in the latter. High amounts of scopoletin, a phenylpropanoid metabolite, were found synthesized in zone 2. Pathogenesis-related (PR) transcripts and the corresponding PR proteins accumulated in high amounts in zone 2 rather late after the treatment (24 h). Salicylic acid was synthesized in both zone 1 and zone 2. Using a radioactively labeled elicitor, we demonstrated that the elicitor remained strictly localized to zone 1. The results provide strong evidence that an endogenous cell-to-cell signaling mechanism was triggered and occurred between the elici-tor-treated cells (zone 1) and the surrounding cells (zone 2), where it induces strong defense responses without cell death.

Salicylic acid (SA) is a key endogenous component of local and systemic disease resistance in plants. In this study, we investigated the role of benzoic acid (BA) as precursor of SA biosynthesis in tobacco (Nicotiana tabacum cv Samsun NN) plants undergoing a hypersensitive response following infection with tobacco mosaic virus or in tobacco cell suspensions elicited with β-megaspermin, an elicitor from Phytophthora megasperma. We found a small pool of conjugated BA in healthy leaves and untreated cell suspensions of tobacco, whereas free BA levels were barely detectable. Infection of plants with tobacco mosaic virus or elicitation of cells led to a rapid de novo synthesis and accumulation of conjugated BA, whereas free BA was weakly induced. In presence of diphenylene iodonium, an inhibitor of superoxide anion formation, SA accumulation was abolished in elicited cells and much higher BA levels were concomitantly induced, mainly as a conjugated form. Furthermore, piperonylic acid, an inhibitor of cinnamate-4-hydroxylase was used as a powerful tool to redirect the metabolic flow from the main phenylpropanoid pathway into the SA biosynthetic branch. Under these conditions, in vivo labeling and radioisotope dilution experiments with [14C]trans-cinnamic acid as precursor clearly indicated that the free form of BA produced in elicited tobacco cells is not the major precursor of SA biosynthesis. The main conjugated form of BA accumulating after elicitation of tobacco cells was identified for the first time as benzoyl-glucose. Our data point to the likely role of conjugated forms of BA in SA biosynthesis.

No abstract

Recherche Scientifique, 28 ~ frans-Cinnamate 4-hydroxylase (C4H) is a plant-specific cytochrome (P450) that is encoded by the gene CYP73A and catalyzes the second step of the multibranched phenylpropanoid pathway. lncreases in C4H activity in response to physical and chemical stresses have been well documented, but the mechanism of these increases has never been studied i n detail. This paper reports on the regulatory mechanism controlling C4H activity in Jerusalem artichoke (Helianfhus tuberosus) tubers in response to wounding and chemical treatments. We compared induction of C4H and other P450-catalyzed activities. C4H was moderately induced by chemicals relative t o other P450s. lncreases in enzyme activity, C4H protein, and transcripts were quantified and compared in tuber tissue 48 h after wounding and chemical treatments. Our data suggest that induction of the enzyme activity results primarily from gene activation. lime-course experiments were performed after wounding and aminopyrine treatment. Compared with wounded tissues, aminopyrine triggered an additional and delayed peak of transcript accumulation. l h e timing of the induced changes in activity, protein, and transcripts confirms that C4H induction results primarily from an increase in CYP73Al mRNA, in both wounded and aminopyrine-treated tissues. However, posttranscriptional mechanisms might also contribute to the regulation of C4H activity.P450s are ubiquitous enzymes that catalyze the activation of molecular oxygen and the insertion of one of its atoms into physiological and artificial substrates (Porter and Coon, 1991). In plants P450s perform many oxygenation reactions in secondary metabolism, in sterols and fatty acid derivative synthesis, and in the detoxification of xenobiotics (Bolwell et al., 1994;Durst and OKeefe, 1995). More than 16 P450-catalyzed reactions have been reported in the pathway leading to the biosynthesis of phenylpropanoids (Werck-Reichhart, 1995). The influx of metabolites into the pathway is controlled by a sequence of three catalytic steps leading from Phe to activated 4-coumaroyl COA. The second reaction of the sequence is catalyzed by a P450 called C4H. Complete cDNA sequences were recently reported for the enzymes from Jerusalem artichoke (Helianthus tuberosus; Teutsch et al., 1993), mung bean (Vigna radiata; Mizutani et al., 1993), and alfalfa (Medicago sativa;

The ability of a plant cytochrome P450 to bind and metabolise plant endogenous molecules and xenobiotics was investigated. The work was performed on the yeast-expressed CYP73A1, a cinnamate 4-hydroxylase isolated from Helianthus tuberosus. CYP73 controls the general phenylpropanoid pathway and is likely to be one of the most abundant sources of P450 in the biosphere. The enzyme shows a high selectivity toward plant secondary metabolites. Nevertheless, it oxygenates several small and planar xenobiotics with low efficiency, including an herbicide (chlorotoluron). One xenobiotic molecule, 2-naphthoic acid, is hydroxylated with an efficiency comparable to that of the physiological substrate. This reaction was used to devise a fluorimetric test for the rapid measurement of enzyme activity. A series of herbicidal molecules (hydroxybenzonitriles) are shown to bind the active site without being metabolised. These molecules behave as strong competitive inhibitors of CYP73 with a K(i) in the same micromolar range as the K(m) for the physiological substrate. It is proposed that their inhibition of the phenylpropanoid pathway reinforces their other phytotoxic effects at the level of the chloroplasts. All our results indicate a strong reciprocal interaction between plant P450s and xenobiotics.