Original article Ethylene- and ozone-induced regulation of a grapevine resveratrol synthase gene: different responsive promoter regions Bernhard Grimmig a,1 , M. Nuria Gonzalez-Perez a,c , Gerhard Welzl b , Josep Penuelas c , Roland Schubert a,2 , Rüdiger Hain d , Bernd Heidenreich a , Christian Betz a,3 , Christian Langebartels a , Dieter Ernst a, *, Heinrich Sandermann Jr a a Institute of Biochemical Plant Pathology, GSF—National Research Center for Environment and Health, 85764 Neuherberg, Germany b CREAF, Facultat di Ciencies, Universitat Autonoma, 08193 Barcelona, Spain c Institute of Biomathematics and Biometry, GSF—National Research Center for Environment and Health, 85764 Neuherberg, Germany d Molecular Target Research and Biotechnology, Agricultural Centre Monheim, Bayer AG, 51368 Leverkusen, Germany Received 28 February 2002; accepted 7 May 2002 Abstract Stilbene synthases (STSs) are enzymes that play a critical role in the biosynthesis of stilbenes, phytoalexins that are induced by various biotic and abiotic stressors like pathogen attack, UV radiation or ozone exposure. To investigate the molecular basis for ozone- and ethylene-induced plant stress responses, we analysed the full size promoter of the grapevine resveratrol synthase gene (Vst1). Transgenic tobacco plants harbouring a Vst1 promoter::-glucuronidase (GUS) reporter construct showed an increased GUS activity upon ozone (0.3 µll –1 , 8 h) or ethylene (10 µll –1 , 8 h) treatment. Using the fluorimetric GUS assay, deletion analysis of the 5'-flanking sequence identified a minimal ethylene-responsive region between –280 and –40 bp upstream of the translation start codon. This region differs from the ozone-responsive Vst1 promoter region, suggesting that ethylene is not directly involved in signalling ozone-induced stilbene biosynthesis. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Ethylene; Nicotiana tabacum; Ozone; Pathogen; Promoter; Resveratrol; Stilbene 1. Introduction Plants respond to ozone by the induction of several defence reactions at the biochemical, as well as at the genetic or transcriptional, level [10,14,22,23]. However, the complex signal transduction chain resulting in ozone- induced gene activation has not yet been identified. Ozone decomposes in the cell wall and plasmalemma, and an oxidative burst resulting in the formation of reactive oxygen intermediates might also be involved in the signal transmis- sion [14,21,24]. Furthermore, it is assumed that ethylene [19,28], salicylic acid [12,20,27] and jasmonate [12,17,18] act as additional messengers for ozone-induced gene ex- pression [21]. If ozone and ethylene both induce the expression of identical genes, it could be predicted that similar regulatory components of the resveratrol synthase gene (Vst1) promoter are induced. We have previously confirmed ozone- and ethylene- induced expression of the grapevine Vst1 gene promoter in transgenic tobacco, indicating that important components of the environmentally regulated signal chain are conserved between Vitis vinifera L. and Nicotiana tabacum L., despite the fact that biosynthesis of resveratrol-type phytoalexins is normally missing in tobacco [5,25]. Moreover, 5'-deletion analysis of the Vst1 promoter identified an ozone-responsive region that differs from the basal pathogen-responsive Abbreviations: ANOVA, analysis of variance; GUS, -glucuronidase; MU, methylumbelliferone; RT-PCR, reverse-transcribed polymerase chain reaction; STS, stilbene synthase; Vst1, resveratrol synthase gene * Corresponding author. E-mail address: ernst@gsf.de (M. Nuria Gonzalez-Perez). 1 Present address: Bayer AG, GB Pflanzenschutz, Forschung/MW, Bayer AG, 51368 Leverkusen, Germany. 2 Present address: Section of Forest Genetics, Center of Life and Food Sciences Weihenstephan, Technical University of Munich, Am Hochanger 13, 85354 Freising, Germany. 3 Present address: MWG-BIOTECH, Anzinger Strasse 7, 85560 Ebers- berg, Germany. Plant Physiol. Biochem. 40 (2002) 865–870 www.elsevier.com/locate/plaphy © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 9 8 1 - 9 4 2 8 ( 0 2 ) 0 1 4 4 8 - 1