Arabidopsis thaliana defense - related protein ELI 3 is an aromatic alcohol : NADP 1 oxidoreductase ( benzyl alcohol dehydrogenase y disease resistance y fungal elicitor )

We expressed a cDNA encoding the Arabidopsis thaliana defense-related protein ELI3-2 in Escherichia coli to determine its biochemical function. Based on a protein database search, this protein was recently predicted to be a mannitol dehydrogenase [Williamson, J. D., Stoop, J. M. H., Massel, M. O., Conkling, M. A. & Pharr, D. M. (1995) Proc. Natl. Acad. Sci. USA 92, 7148–7152]. Studies on the substrate specificity now revealed that ELI3-2 is an aromatic alcohol: NADP1 oxidoreductase (benzyl alcohol dehydrogenase). The enzyme showed a strong preference for various aromatic aldehydes as opposed to the corresponding alcohols. Highest substrate affinities were observed for 2-methoxybenzaldehyde, 3-methoxybenzaldehyde, salicylaldehyde, and benzaldehyde, in this order, whereas mannitol dehydrogenase activity could not be detected. These and previous results support the notion that ELI3-2 has an important role in resistance-related aromatic acid-derived metabolism. Plant defense toward potential pathogens encompasses a wide array of mechanisms, some leading to the rapid reinforcement of preexisting structural barriers, others to the de novo synthesis of a large diversity of defense-related compounds via transcriptional activation of the corresponding genes. In recent years, numerous plant genes potentially involved in the pathogen defense response have been isolated. However, many of them were detected by various differential screening approaches solely on the basis of enhanced expression levels without knowledge of the biochemical functions of the encoded proteins (1–3). In several cases, functional identification was subsequently achieved, for example, by expression in Escherichia coli or yeast, by the use of specific antibodies or by genetic complementation studies. Often, however, inference of function was merely based on deduced amino acid sequence similarity to known proteins, a valuable but not unequivocal means of identification. The eli3 gene was originally identified as part of the defense response in parsley [Petroselinum crispum (Pc)] (4). Expression of this gene was shown to be rapidly and transiently stimulated in cultured parsley cells upon treatment with a cell wall preparation (elicitor) from the phytopathogenic fungus Phytophthora sojae, and histochemical studies revealed local and rapid accumulation of ELI3 mRNA around fungal infection sites in parsley leaves (5). The presence of this gene in other plant species, such as potato (Solanum tuberosum), alfalfa (Medicago sativa), and Arabidopsis thaliana (At) was also demonstrated (6). Subsequently, two sequence-related counterparts of eli3 in parsley were isolated from A. thaliana (Ateli3-1 and Ateli3-2), and their expression was shown to be induced by fungal elicitor in cultured A. thaliana cells (6). Additional evidence for an important role of the eli3 gene product in plant disease resistance came from genetic studies demonstrating that eli3 expression was dependent on the presence of the RPMI resistance gene in A. thaliana (7). The RPM1 locus confers resistance to Pseudomonas syringae strains carrying the corresponding avirulence (avr) gene avrRpm1 (8). The ELI3 cDNAs from parsley and A. thaliana share 67% nucleotide and 70% deduced amino acid sequence identity (7). At the time of their isolation, no related sequences were found in the various data bases. Meanwhile, several plant cinnamylalcohol dehydrogenases (CAD) with similarity to the deduced ELI3 proteins have been reported (9). Based on this similarity, it was possible that the eli3 gene encodes a CAD. Recently, however, Williamson et al. (10) suggested that ELI3 is a mannitol dehydrogenase (MTD). Again, this was based on sequence similarity to an MTD from celery [Apium graveolens (Ag), AgMTD]. This proposal prompted speculation as to the functional relevance of eli3 gene expression (10), particularly in an accompanying commentary (11), which, in view of our present data, is no longer tenable. We now provide evidence that ELI3-2 from A. thaliana is a novel type of aromatic (preferably benzyl) alcohol dehydrogenase with substrate specificity distinct from both CAD and MTD. MATERIALS AND METHODS Materials. Actinobacillus mannitol dehydrogenase was purchased from Sigma; NAD1, NADH, NADP1, and NADPH were from Boehringer Mannheim. The following aldehydes and alcohols were obtained from Aldrich: cinnamaldehyde, cinnamyl alcohol, sinapaldehyde, coniferaldehyde, coniferyl alcohol, 3,5-dimethoxy-4-hydroxybenzaldehyde, 2-methoxybenzyl alcohol, 3-methoxybenzyl alcohol, salicylaldehyde b-Oglucoside, and 4-coumaryl alcohol. Acetaldehyde, D-mannitol, benzaldehyde, benzyl alcohol, salicylaldehyde, 2-hydroxybenzyl alcohol, 2-methoxybenzaldehyde, 3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 4-hydroxybenzaldehyde, 3,4dimethoxybenzaldehyde, and vanillin were from Merck, and capronaldehyde was from Fluka. 4-Coumaraldehyde was a kind gift from J. Grima-Pettenati (Toulouse, France) and W. Heller (Neuherberg, Germany). All substrates were dissolved in methoxyethanol. Expression in E. coli.One of two established, closely related A. thaliana ELI3 cDNAs, AtELI3-2, containing the entire coding region (7), was cloned into the expression vector pQE50 (Qiagen, Düsseldorf, Germany), and the resulting construct, pQE50-ELI3-2, was introduced into the E. coli host strain SURE. The transformed bacterial cells were grown at 378C in Luria–Bertani medium containing 1 mM ZnCl2 and 50 mgyml The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: Pc, Petroselinum crispum; At, Arabidopsis thaliana; Ag, Apium graveolens; CAD, cinnamyl-alcohol dehydrogenase; BAD, benzyl alcohol dehydrogenase; Eu, Eucalyptus gunnii; MTD, mannitol dehydrogenase; IPTG, isopropyl b-D-thiogalactoside. *To whom reprint requests should be addressed at:Max Planck Institut für Züchtungsforschung, Department of Biochemistry, Carl von Linné Weg 10, D-50829 Köln, Germany. e-mail: [email protected]. †Present address: Bayer AG, Landwirtschaftszentrum, Geb. 6240, D-51368 Leverkusen, Germany.