Exogenous ethylene stimulates the long-term expression of genes related to anthocyanin biosynthesis in grape berries Ashraf El-Kereamy a , Christian Chervin a, *, Jean-Paul Roustan a , Veronique Cheynier b , Jean-Marc Souquet b , Michel Moutounet b , Jose´ Raynal a , Christopher Ford c , Alain Latche´ a , Jean-Claude Pech a and Mondher Bouzayen a a UMR 990, Ge´nomique et Biotechnologie des Fruits, INRA-INP/ENSA Toulouse, BP 107, 31326 Castanet, France b UMR Sciences pour l’Œnologie, Laboratoire Biopolyme`res et Aro ˆmes, INRA, place Viala, 34060 Montpellier Cdx 1, France c School of Agriculture and Wine, The University of Adelaide, PMB 1, Glen Osmond SA 5064, Australia *Corresponding author, e-mail: chervin@flora.ensat.fr Received 11 December 2002; revised 21 February 2003 The treatment of grape berries (Vitis vinifera L. cv. Cabernet Sauvignon) with the ethylene-releasing compound, 2-chloro- ethylphosphonic acid (2-CEPA), at veraison is a method known to enhance grape skin colour. We observed that it produced a 6-fold increase, up to 30 pmol g 1 FW, of the cluster internal ethylene compared to untreated controls within the 24 h following treatment. This ethylene upsurge was associated with increased levels of chalcone synthase (CHS) and flavanone 3-hydroxylase (F3H) transcripts, which persisted over the following 20 days. Transcript levels of leucoanthocyanidin dioxygenase (LDOX) and UDP glucose-flavonoid 3-O-glucosyl transferase (UFGT) were similarly enhanced by 2-CEPA, although to a lesser extent. The effect on UFGT was confirmed at the protein level by an immunoblot analysis. The transcript accumulation of dihydro- flavonol 4-reductase (DFR) was unaffected by 2-CEPA treat- ment. Examination of the levels of CHS, F3H and UFGT mRNAs in berries during bunch exposure to ethylene, revealed elevated levels of each transcript within the first 6 h of treat- ment when compared to nonethylene-treated controls. HPLC analyses of berry skin extracts showed that levels of each of the anthocyanins analysed (delphinidin, cyanidin, petunidin, peonidin and malvidin) increased over the 10 days following the ethylene burst, and decreased thereafter. However, antho- cyanin levels at harvest were still higher in ethylene treated grapes than in controls. This data is the first evidence that ethylene triggers gene expression related to anthocyanin synthesis in grapes, and in addition, our results also confirm the existence of other regulatory modes in the anthocyanin biosynthetic pathway. Introduction Grapes, the world largest fruit crop (Mazza 1995), have been classified as non-climacteric due to the lack of an obvious increase in ethylene production and a concomi- tant increase in respiration at the onset of ripening (also called veraison, Coombe and Hale 1973). However, small increases in respiration rate and internal ethylene con- centration have been observed by other authors, taking into account the variations in gases dissolved in grape tissues at veraison (Alleweldt and Koch 1977). At this time, intensive anthocyanin synthesis is triggered in the subepidermal layer in the berries of red cultivars (Hrazdina et al. 1984). The biosynthesis of anthocyanins proceeds by a series of ordered chemical reactions cata- lysed by enzymes produced during berry development and after veraison (Boss et al. 1996). cDNAs derived from seven of the genes encoding these enzymes were cloned from grape seedlings by Sparvoli et al. (1994): phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), dihydroflavonol 4-reductase (DFR), flavanone 3-hydroxylase (F3H), leucoanthocyanidin dioxygenase (LDOX) and UDP glucose-flavonoid 3-O-glucosyl transferase (UFGT). The signal transduction pathways of the expression of these genes in grapes are yet to be identified. In a recent paper, PHYSIOLOGIA PLANTARUM 119: 175–182. 2003 Copyright # PhysiologiaPlantarum2003 PrintedinDenmark–allrightsreserved ISSN0031-9317 Abbreviations – 2-CEPA, 2-chloroethylphosphonic acid; CHI, chalcone isomerase; CHS, chalcone synthase; DFR, dihydroflavonol 4-reductase; F3H, flavanone 3-hydroxylase; LDOX, leucoanthocyanidin dioxygenase; OD, optical density; PAL, phenylalanine ammoni- alyase; UFGT, UDP glucose-flavonoid 3-O-glucosyltransferase. Physiol. Plant. 119, 2003 175