Recent studies have shown that low doses of ethanol stimulate the maturation of some fruits. The present work showed that

spraying Cabernet Sauvignon grapes, with 5% ethanol at veraison enhances the anthocyanin accumulation. Veraison is the time

when the berries turn from green to purple. HPLC analysis showed a marked increase in the total concentrations of the derivatives

of delphinidin, cyanidin, petunidin, peonidin and malvidin from the fourth day after the ethanol treatment until harvest. This was

not linked to a difference in berry weight in comparison to controls. Two distinct expression patterns were found for anthocyanin

biosynthesis genes in the treated and untreated berries. For one group, consisting of chalcone synthase, flavanone-3-hydroxylase,

dihydroxyflavonol-4-reductase and leucoanthocyanidin dioxygenase, the expression was inhibited or unchanged by the ethanol

treatment, whereas for UDP glucose-flavonoid 3-O-glucosyltransferase (UFGT) there was a marked increase in expression from 1

to 20 days after ethanol treatment. These results suggest that the UFGT gene is a key factor in the observed anthocyanin

accumulation following ethanol treatment

Chervin, Christian

El-Kereamy, Ashraf

Ford, Christopher

Mondies, Hélène

Monje, Marie-Carmen

Moutounet, Michel

Nepveu, Françoise

Roustan, Jean-Paul

Souquet, Jean-Marc

Van Heeswijck, R.

English

Recent studies have shown that low doses of ethanol stimulate the maturation of some fruits. The present work showed that spraying Cabernet Sauvignon grapes, with 5% ethanol at veraison enhances the anthocyanin accumulation. Veraison is the time when the berries turn from green to purple. HPLC analysis showed a marked increase in the total concentrations of the derivatives of delphinidin, cyanidin, petunidin, peonidin and malvidin from the fourth day after the ethanol treatment until harvest. This was not linked to a difference in berry weight in comparison to controls. Two distinct expression patterns were found for anthocyanin biosynthesis genes in the treated and untreated berries. For one group, consisting of chalcone synthase, flavanone-3-hydroxylase, dihydroxyflavonol-4-reductase and leucoanthocyanidin dioxygenase, the expression was inhibited or unchanged by the ethanol treatment, whereas for UDP glucose-flavonoid 3-O-glucosyltransferase (UFGT) there was a marked increase in expression from 1 to 20 days after ethanol treatment. These results suggest that the UFGT gene is a key factor in the observed anthocyanin accumulation following ethanol treatment

Mondies, Hélène

Ford, Christopher M

van Heeswijck, Robyn

Open Archive Toulouse Archive Ouverte

    OATAO is an open access repository that collects the work of Toulouse researchers and makes it freely available over the web where possible Any correspondence concerning this service should be sent  to the repository administrator: tech-oatao@listes-diff.inp-toulouse.fr This is an author’s version published in: http://oatao.univ-toulouse.fr/23185  To cite this version:  El-Kereamy, Ashraf  and Chervin, Christian  and Souquet, Jean-Marc and Moutounet, Michel and Monje, Marie-Carmen  and Nepveu, Françoise and Mondies, H�l�ne  and Ford, Christopher M and van Heeswijck, Robyn and Roustan, Jean-Paul  Ethanol triggers grape gene expression leading to anthocyanin accumulation during berry ripening. (2002) Plant Science, 163 (3). 449-454. ISSN 0168-9452  Official URL: https://doi.org/10.1016/S0168-9452(02)00142-5  Ethanol triggers grape gene expression leading to anthocyaninaccumulation during berry ripeningAshraf El Kereamy a,1, Christian Chervin a,1,*, Jean-Marc Souquet b,Michel Moutounet b, Marie-Carmen Monje c, Franc¸oise Nepveu c, Helene Mondies a,Christopher M. Ford d, Robyn van Heeswijck d, Jean-Paul Roustan aa Ecole Nationale Supe´rieure Agronomique de Toulouse, BP 107, 31326 Castanet, Franceb UMR Sciences pour l’Œnologie, Laboratoire Biopolyme`res et Aroˆmes, INRA, place Viala, 34060 Montpellier Cedex, Francec Faculte´ des Sciences Pharmaceutiques, 35 chemin des Maraıˆchers, 31062 Toulouse Cedex 4, Franced Department of Horticulture, Viticulture and Oenology, The University of Adelaide, PMB 1, Glen Osmond, SA 5064, AustraliaAbstractRecent studies have shown that low doses of ethanol stimulate the maturation of some fruits. The present work showed thatspraying Cabernet Sauvignon grapes, with 5% ethanol at veraison enhances the anthocyanin accumulation. Veraison is the timewhen the berries turn from green to purple. HPLC analysis showed a marked increase in the total concentrations of the derivativesof delphinidin, cyanidin, petunidin, peonidin and malvidin from the fourth day after the ethanol treatment until harvest. This wasnot linked to a difference in berry weight in comparison to controls. Two distinct expression patterns were found for anthocyaninbiosynthesis genes in the treated and untreated berries. For one group, consisting of chalcone synthase, flavanone-3-hydroxylase,dihydroxyflavonol-4-reductase and leucoanthocyanidin dioxygenase, the expression was inhibited or unchanged by the ethanoltreatment, whereas for UDP glucose-flavonoid 3-O -glucosyltransferase (UFGT) there was a marked increase in expression from 1to 20 days after ethanol treatment. These results suggest that the UFGT gene is a key factor in the observed anthocyaninaccumulation following ethanol treatment. # 2002 Elsevier Science Ireland Ltd. All rights reserved.Keywords: Vitis vinifera ; Maturation; Ethanol; Anthocyanins; Glucosyltransferase1. IntroductionThe application of exogenous ethanol has beenreported to inhibit or promote tomato fruit ripening ina dose-dependent manner, correlated to ethylene evolu-tion [1]. Farag et al. [2] showed that ethanol incombination with an ethylene precursor enhancedanthocyanin content of cranberries. In a recent study[3], we observed that spraying an aqueous solution ofethanol onto grapes at veraison enhanced internalethylene production. We also noticed that berry skinextracts resulting from the ethanol treatment showedincreased absorbance at 520 nm. This is the wavelengthat which anthocyanins, the main colored pigments ofgrapes [4], have the maximum absorption. In this paper,we report HPLC analyses of berry skin extracts,performed to determine which anthocyanin speciesaccumulated after the ethanol treatment.In grapes a number of structural genes involved inanthocyanin biosynthesis have been cloned (PAL, CHS,CHI, DFR, F3H, LDOX, and UFGT) [5]. PAL standsfor phenylalanine ammonia-lyase, CHS for chalconesynthase, CHI for chalcone isomerase, F3H for flava-none-3-hydroxylase, DFR for dihydroxyflavonol-4-re-ductase, LDOX for leucoanthocyanidin dioxygenaseand UFGT for UDP glucose-flavonoid 3-O -glucosyl-Abbreviations: CHS, chalcone synthase; DFR, dihydroxyflavonol-4-reductase; F3H, flavanone-3-hydroxylase; LDOX,leucoanthocyanidin dioxygenase; UFGT, UDP glucose-flavonoid 3-O -glucosyltransferase.* Corresponding author. Collaborator via a fellowship under theOECD Co-operative Research Programme: Biological ResourceManagement for Sustainable Agriculture Systems.E-mail address: chervin@ensat.fr (C. Chervin).1 Joint first authors.transferase. Their transcription rates change accordingto the berry development state, and a marked increasewas observed at veraison for some of them, dependingon the cultivars [6,7]. It has been shown that some ofthese genes are regulated by treatments with auxinswhich inhibit ethylene action [8]. In addition, theexposure of fruits other than grapes to ethylene, resultedin an increased transcription of CHS and F3H [9,10].Thus, in addition to the HPLC analysis, we checkedthe effect of ethanol on the transcription of CHS, DFR,F3H, LDOX, and UFGT during berry developmentafter veraison.2. Material and methods2.1. Grapes and treatments at veraisonTreatments were carried out in 1999, on 10 individualvines of Cabernet Sauvignon (Vitis vinifera L.), graftedon 110 Richter (Vitis Berlandieri/Vitis rupestris), in avineyard close to Toulouse (France). The treatmentstook place 8/9 weeks after anthesis when 50% of berrieschanged color (first fortnight of August). Clusters weresprayed (back pump) with ethanol at 5% in water.Spraying was performed at a rate equivalent to 200 l/ha.Control vines were sprayed with water. Five clusters pertreatment were sampled, each on a separate vine, atvarious times after spraying (0, 1, 4, 20 and 56 days ‘atharvest’). Batches of twenty randomly chosen berrieswere used to calculate the berry weight. Berries werethen kept frozen at /80 8C.2.2. RNA and protein extractions, Northern and WesternblotsTotal RNA was extracted from the samples accordingto Boss et al. [7]. Aliquots of 15 mg of RNA weredenatured and run on a 1.2% agarose gel (containing 3%formaldehyde and 10% MOPS). Northern blot analysiswas performed using the available probes of CHS,DFR, F3H, LDOX, and UFGT genes cloned bySparvoli et al. [5]. The method of Boss et al. [7] wasused with the following modifications. The RNA wastransferred to a Gene Screen Plus†hybridizationtransfer membrane (NENTM Life Science Products,Boston, MA) with a vacuum blotter (mod. TDNA,Appligene, Illkirsch, France) for 3.5 h at 45/50 mbars.Then prehybridization was run for 4 h at 42 8C in 5xSSPE, 50% (w/v) deionized formamide, 1% SDS, 10%dextran-NaSO4 MW 500 000 and 10 mg/ml denaturedsalmon sperm. The membranes were hybridized for 15 hat 42 8C with the addition of denatured 32P-labelledcDNAs probes prepared by random primer labeling(Ready-To-Go DNA Labeling Kit, Amersham Pharma-cia Biotech, Uppsala, Sweden). The membrane was thenwashed twice for 10 min at 42 8C and then once for 15min at 42 8C and exposed to the autoradiography film(Hyper film, Amersham Pharmacia Biotech). The mem-branes were also hybridized with a 18S probe. Thehybridization signal intensity was assessed with theSigmaScan software (SPSS Inc, Chicago). To enablethe comparison, the hybridization signals were adjustedaccording to the mean 18S signal of each membrane,then the adjusted values were expressed as percentagesof the means of the day 0 signal (100%). Blots wereperformed in duplicates with different RNA samples,extracted from berries of different vines.Proteins were extracted as described previously [11]and Western blots and immunodetection were per-formed according to Ford et al. [12]. Equal proteinamounts (10 mg) were loaded onto each lane (data notshown).2.3. Grape skin extracts and HPLC analysesMethanolic extraction was performed according toBoss et al. [7]. Briefly, berries were individually thawedin warm water and skins were frozen immediately afterpeeling, then ground in mortar and pestle with liquidnitrogen; we used skins of 20 berries per replicaterandomly chosen among each batch of 5 clusters.Separation of the anthocyanins was performed by directHPLC analysis on a reversed stationary phase asdescribed by Cheynier et al. [13]. Anthocyanins wereanalysed at 520 nm with a diode array detector andidentified according to the retention times and UVspectral data. Quantifications were based on peak areasat 520 nm using a response factor calculated withmalvidin-3-glucoside. This compound was purchasedfrom Extrasynthese (Genay, France).Fig. 1. Effect of a 5% ethanol (EtOH) treatment at veraison (day 0) onberry weight up to harvest (day 56); n/3, error bars represent S.E.3. Results and discussion3.1. Berry weight and color of skin extractsThe berry weight did not changed between thecontrols and the treated berries over the ripening period(Fig. 1). These results confirmed our observations [3],which showed that ethanol induced an increase in theabsorbance at 520 nm of berry skin extracts without achange of berry weight at harvest.3.2. HPLC analysisThe HPLC analysis of berry skin extracts showed thatethanol sprayed at veraison stimulated the accumulationof the five main anthocyanins present in berry skins,namely delphinidin-3-glucoside, cyanidin-3-glucoside,petunidin-3-glucoside, peonidin-3-glucoside, and malvi-din-3-glucoside, from 4 days after treatment untilharvest (Fig. 2). Malvidin-3-glucoside derivatives werethe main pigments at harvest and their levels were nearlyFig. 2. Accumulation of the main anthocyanins in grape berry skins after spraying at veraison with 5% ethanol (EtOH). Each point is the sum ofmonoglucosides, acetylglucosides and p-coumarylglucosides; n/3, error bars represent S.E.three fold higher in ethanol treated berries than incontrols. There was no change in the relative distribu-tion between monoglucosides, acetylglucosides and p -coumarylglucosides (data not shown), which were pre-sent in percentages similar to those previously described[4].These results suggest that the increased anthocyaninlevels, previously observed in cranberries when treatedFig. 3. (A) Relative transcription of the five of anthocyanin genes, CHS, F3H, DFR, LDOX and UFGT, after an ethanol (5%) treatment at veraison(day 0) up to harvest (day 56), in comparison to controls. The inserted images show the most representative blots; n/2, error bars represent S.E. (B)Immunoblot of total soluble protein extracts with rabbit antibodies raised against rUFGT as described in Ford et al. [12].with a combination of an ethylene precursor andethanol [2], were not only due to ethanol increasingthe ethylene precursor diffusion through cell membranesas suggested by their authors, but may be partlydue to an effect of ethanol itself on anthocyaninbiosynthesis. In the next part of our work, we studiedthe transcription rate of some of the genes involved inthe anthocyanin synthesis, using available cDNAprobes.3.3. Gene transcription and expressionTreatment of Cabernet Sauvignon grapes with 5%ethanol at veraison resulted in some altered regulationof the transcription of anthocyanin biosynthesis genescompared to the controls. Ethanol treatment had nostimulating effect on the transcription of genes in theearly part of the anthocyanin biosynthesis pathway,including CHS, DFR, F3H and LDOX (Fig. 3).However, both Northern and Western blot analysesindicated that the UFGT gene was more highly ex-pressed than in controls from the first day after ethanoltreatment until 20 days later (Fig. 3). This ethanol effect,observed for the first time in grapes, could be indirectthrough an increase of C2H4 production [3], which isknown to induce UFGT activity in fruit [14]. Moreover,this effect could be partly due to the conversion of someethanol into acetaldehyde, which is more efficient thanethanol in inducing ethylene production in fruit,[1,15,16] or could involve a stress response in the treatedplants.These results provide new evidence that UFGT playsan important role in increasing the accumulation ofanthocyanins in red cultivars, as well as its role indetermining the difference between red and white grapes[6,17].In apples, it was reported that chalcone synthase doesnot play a regulatory role in anthocyanin synthesis,whereas like UFGT expression in grapevine, the UF-GaIT activity was correlated with anthocyanin synthesisduring fruit maturation [18]. This may be due to itseffect on anthocyanidin glycosylation, which usuallyconfers stability to anthocyanins [19].The pattern of gene expression in grape berry could beexplained in relation to anthocyanin regulatory genes(i.e. those that control the expression of the structuralgenes of the pathway). Two types of regulatory genesmay be present, one that controls the expression ofPAL, CHS, CHI, F3H, DFR and LDOX and anotherthat induces UFGT gene expression [7]. Sequenceanalysis showed that there was no difference either inthe coding region or in the promoter of the UFGT genebetween white and the red grape cultivars, suggestingdifferences in the UFGT regulatory factors [17]. Thesefactors which control the expression of the anthocyaninstructural genes, including UFGT, have been identifiedin other species, but have not been yet isolated in grapes.Thus, a possible explication of our results is that ethanoltreatment can affect the expression of UFGT regulatoryfactors.It is clear that UFGT genes, whose expression iscorrelated with anthocyanin synthesis, are regulated byregulatory genes that can respond to exogenous treat-ment such as auxin in grapes [8] and ethylene in apples[18]. However, the only paper describing the inductionof glycosyltransferase activity by ethanol comes from astudy on rats [20].In conclusion, the effect of ethanol sprayed atveraison on anthocyanin accumulation in grape berriesseems to be due to its stimulatory effect on UFGT genetranscription rather than on the other anthocyaningenes studied (CHS, DFR, F3H and LDOX). Thisincrease did not change the relative distribution amongthe different anthocyanin derivatives. However, wecannot exclude that other genes or cellular functionsare involved in this response, and we have not checkedthe enzyme activities corresponding to the genes forwhich northern analysis were conducted.These results offer new perspectives to improve berrycolor of grape varieties showing anthocyanin deficien-cies or of grapes cultivated in climate areas where fullmaturity is hard to reach. Ethanol, a simple organiccompound, could match the sustainable viticultureneeds. However, it is essential that we determine theoptimal ethanol dose according to the sprayer type.More work has been initiated to study the best dose andthe best moment for application of ethanol on grapeberries in order to achieve greater anthocyanin accumu-lation.AcknowledgementsWe are grateful to Dr Paul Boss for useful commentsabout the text and Northern analysis, to Dr CharlesRomieu for fruitful discussion, to Dr Chiara Tonelli forthe generous gift of the cDNA probes, to Dr Jose´Raynal for technical help and Drs Jean-Claude Pechand Mondher Bouzayen for the use of their laboratoryfacilities.References[1] J.C. Beaulieu, M.E. Saltveit, Inhibition or promotion of tomatofruit ripening by acetaldehyde and ethanol is concentrationdependent and varies with initial fruit maturity, J. Amer. Soc.Hort. Sci. 122 (1997) 392/398.[2] K.M. Farag, J.P. Palta, E.J. Stang, Ethanol enhances theeffectiveness of ethephon on anthocyanin production in cranberryfruits in the field, HortScience 27 (1992) 411/412.[3] C Chervin, A. Elkereamy, J.P. Roustan, J.D. Faragher, A.Latche, J.C. Pech, M. Bouzayen, An ethanol spray at veraisonenhances colour in red wines, Aust. J. Grape Wine Res. 7 (2001)144/145.[4] G. Mazza, Anthocyanins in grapes and grape products, Crit. Rev.Food Nutr. 35 (1995) 341/371.[5] F. Sparvoli, C. Martin, A. Scienza, G. Gavazzi, C. Tonelli,Cloning and molecular analysis of structural genes involved inflavonoid and stilbene biosynthesis in grape (Vitis Vinifera L.),Plant Mol. Biol. 24 (1994) 743/755.[6] P.K. Boss, C. Davies, S.P. Robinson, Expression of anthocyaninbiosynthesis pathway genes in red and white grapes, Plant Mol.Biol. 32 (1996) 565/569.[7] P.K. Boss, C. Davies, S.P. Robinson, Analysis of the expressionof anthocyanin pathway genes in developing Vitis vinifera L. cv.Shiraz grape berries and the implication for pathway regulation,Plant Physiol. 111 (1996) 1059/1066.[8] C. Davies, P.K. Boss, S.P. Robinson, Treatment of grape berries,a nonclimacteric fruit with a synthetic auxin, retards ripening andalters the expression of developmentally regulated genes, PlantPhysiol. 115 (1997) 1155/1161.[9] R. Ardi, I. Kobiler, B. Jacoby, N.T. Keen, D. Prusky, Involve-ment of epicatechin biosynthesis in the activation of the mechan-ism of resistance of avocado fruits to Colletotrichumgloeosporioides , Physiol. Mol. Plant Pathol. 53 (1998) 269/285.[10] A.K. Kanellis, G. Diallinas, Developmental and ripening regula-tion of gene expression of phenylpropanoid biosynthetic enzymesin melon fruit, Proceedings of ‘Postharvest physiology andpathology’, International Symposium, Agadir, Morocco, 16/21January 1994, Institut Agronomique et Veterinaire Hassan II,(1995) 353/360.[11] C.M. Ford, P.B. Høj, Multiple glucosyltransferase activities in thegrapevine Vitis vinifera L., Aust. J. Grape Wine Res. 4 (1998) 48/58.[12] C.M. Ford, P.K. Boss, P.B. Høj, Cloning and characterisation ofVitis vinifera UDP-glucose: flavonoid 3-O -glucosyltransferase, ahomologue of the enzyme encoded by the maize Bronze-1 locusthat may primarily serve to glucosylate anthocyanidins in vivo, J.Biol. Chem. 273 (1998) 9224/9233.[13] V. Cheynier, I. Hidalgo Arellano, J.M. Souquet, M. Moutounet,Estimation of the oxidative changes in phenolic compounds ofCarignane during winemaking, Am. J. Enol. Vitic. 48 (1997) 225/228.[14] G. Hrazdina, G.F. Parsons, L.R. Mattick, Physiological andbiochemical events during development and maturation of grapeberries, Am. J. Enol. Vitic. 35 (1984) 220/227.[15] F. Mencarelli, P. Savarese, M.E. Satveit, Ripening of kiwifruitexposed to ethanol and acetaldehyde vapors, Hort. Sci. 26 (1991)566/569.[16] E. Pesis, R. Marinansky, Carbon dioxide and ethylene productionby harvested grape berries in response to acetaldehyde andethanol, J. Amer. Soc. Hort. Sci. 117 (1992) 110/113.[17] S. Kobayashi, M. Ishimaru, C.K. Ding, H. Yakushji, N. Goto,Comparison of UDP-glucose: flavonoid 3-O -glucosyltransferase(UFGT) gene sequence between white grapes (Vitis vinifera ) andtheir sports with red skin, Plant Sci. 160 (2001) 543/550.[18] Z. Ju, L. Chenglian, Y. Yongbing, Activities of chalcone synthaseand UFGA1: flavonoid-3-O -glycosyltransferase in relation toanthocyanin synthesis in apple, Sci. Hort. 63 (1995) 175/185.[19] C.B. Do, F. Cormier, Y. Nicolas, Isolation and characterizationof UDP-glucose cyanidin 3-O -glucosyltransferase from grape cellsuspension cultures (Vitis vinifera L.), Plant Sci. 112 (1995) 43/51.[20] C.S. Lieber, E. Rubin, Ethanol increase glycosyl transferaseactivity in the hepatic golgi apparatus, Nature, New Biol. 243(125) (1973) 123/125.

Ethanol triggers grape gene expression leading to anthocyanin accumulation during berry ripening

Crossref

International audienceRecent studies have shown that low doses of ethanol stimulate the maturation of some fruits. The present work showed that spraying Cabernet Sauvignon grapes, with 5% ethanol at veraison enhances the anthocyanin accumulation. Veraison is the time when the berries turn from green to purple. HPLC analysis showed a marked increase in the total concentrations of the derivatives of delphinidin, cyanidin, petunidin, peonidin and malvidin from the fourth day after the ethanol treatment until harvest. This was not linked to a difference in berry weight in comparison to controls. Two distinct expression patterns were found for anthocyanin biosynthesis genes in the treated and untreated berries. For one group, consisting of chalcone synthase, flavanone-3-hydroxylase, dihydroxyflavonol-4-reductase and leucoanthocyanidin dioxygenase, the expression was inhibited or unchanged by the ethanol treatment, whereas for UDP glucose-flavonoid 3-O-glucosyltransferase (UFGT) there was a marked increase in expression from 1 to 20 days after ethanol treatment. These results suggest that the UFGT gene is a key factor in the observed anthocyanin accumulation following ethanol treatment

HAL: Hyper Article en Ligne

Ethanol triggers grape gene expression leading to anthocyaninaccumulation during berry ripeningAshraf El Kereamy a,1, Christian Chervin a,1,*, Jean-Marc Souquet b,Michel Moutounet b, Marie-Carmen Monje c, Franc¸oise Nepveu c, Helene Mondies a,Christopher M. Ford d, Robyn van Heeswijck d, Jean-Paul Roustan aa Ecole Nationale Supe´rieure Agronomique de Toulouse, BP 107, 31326 Castanet, Franceb UMR Sciences pour l’Œnologie, Laboratoire Biopolyme`res et Aroˆmes, INRA, place Viala, 34060 Montpellier Cedex, Francec Faculte´ des Sciences Pharmaceutiques, 35 chemin des Maraıˆchers, 31062 Toulouse Cedex 4, Franced Department of Horticulture, Viticulture and Oenology, The University of Adelaide, PMB 1, Glen Osmond, SA 5064, AustraliaAbstractRecent studies have shown that low doses of ethanol stimulate the maturation of some fruits. The present work showed thatspraying Cabernet Sauvignon grapes, with 5% ethanol at veraison enhances the anthocyanin accumulation. Veraison is the timewhen the berries turn from green to purple. HPLC analysis showed a marked increase in the total concentrations of the derivativesof delphinidin, cyanidin, petunidin, peonidin and malvidin from the fourth day after the ethanol treatment until harvest. This wasnot linked to a difference in berry weight in comparison to controls. Two distinct expression patterns were found for anthocyaninbiosynthesis genes in the treated and untreated berries. For one group, consisting of chalcone synthase, flavanone-3-hydroxylase,dihydroxyflavonol-4-reductase and leucoanthocyanidin dioxygenase, the expression was inhibited or unchanged by the ethanoltreatment, whereas for UDP glucose-flavonoid 3-O -glucosyltransferase (UFGT) there was a marked increase in expression from 1to 20 days after ethanol treatment. These results suggest that the UFGT gene is a key factor in the observed anthocyaninaccumulation following ethanol treatment.Keywords: Vitis vinifera ; Maturation; Ethanol; Anthocyanins; Glucosyltransferase1. IntroductionThe application of exogenous ethanol has beenreported to inhibit or promote tomato fruit ripening ina dose-dependent manner, correlated to ethylene evolu-tion [1]. Farag et al. [2] showed that ethanol incombination with an ethylene precursor enhancedanthocyanin content of cranberries. In a recent study[3], we observed that spraying an aqueous solution ofethanol onto grapes at veraison enhanced internalethylene production. We also noticed that berry skinextracts resulting from the ethanol treatment showedincreased absorbance at 520 nm. This is the wavelengthat which anthocyanins, the main colored pigments ofgrapes [4], have the maximum absorption. In this paper,we report HPLC analyses of berry skin extracts,performed to determine which anthocyanin speciesaccumulated after the ethanol treatment.In grapes a number of structural genes involved inanthocyanin biosynthesis have been cloned (PAL, CHS,CHI, DFR, F3H, LDOX, and UFGT) [5]. PAL standsfor phenylalanine ammonia-lyase, CHS for chalconesynthase, CHI for chalcone isomerase, F3H for flava-none-3-hydroxylase, DFR for dihydroxyflavonol-4-re-ductase, LDOX for leucoanthocyanidin dioxygenaseand UFGT for UDP glucose-flavonoid 3-O -glucosyl-Abbreviations: CHS, chalcone synthase; DFR, dihydroxyflavonol-4-reductase; F3H, flavanone-3-hydroxylase; LDOX,leucoanthocyanidin dioxygenase; UFGT, UDP glucose-flavonoid 3-O -glucosyltransferase.* Corresponding author. Collaborator via a fellowship under theOECD Co-operative Research Programme: Biological ResourceManagement for Sustainable Agriculture Systems.E-mail address: chervin@ensat.fr (C. Chervin).1 Joint first authors.transferase. Their transcription rates change accordingto the berry development state, and a marked increasewas observed at veraison for some of them, dependingon the cultivars [6,7]. It has been shown that some ofthese genes are regulated by treatments with auxinswhich inhibit ethylene action [8]. In addition, theexposure of fruits other than grapes to ethylene, resultedin an increased transcription of CHS and F3H [9,10].Thus, in addition to the HPLC analysis, we checkedthe effect of ethanol on the transcription of CHS, DFR,F3H, LDOX, and UFGT during berry developmentafter veraison.2. Material and methods2.1. Grapes and treatments at veraisonTreatments were carried out in 1999, on 10 individualvines of Cabernet Sauvignon (Vitis vinifera L.), graftedon 110 Richter (Vitis Berlandieri/Vitis rupestris), in avineyard close to Toulouse (France). The treatmentstook place 8/9 weeks after anthesis when 50% of berrieschanged color (first fortnight of August). Clusters weresprayed (back pump) with ethanol at 5% in water.Spraying was performed at a rate equivalent to 200 l/ha.Control vines were sprayed with water. Five clusters pertreatment were sampled, each on a separate vine, atvarious times after spraying (0, 1, 4, 20 and 56 days ‘atharvest’). Batches of twenty randomly chosen berrieswere used to calculate the berry weight. Berries werethen kept frozen at /80 8C.2.2. RNA and protein extractions, Northern and WesternblotsTotal RNA was extracted from the samples accordingto Boss et al. [7]. Aliquots of 15 mg of RNA weredenatured and run on a 1.2% agarose gel (containing 3%formaldehyde and 10% MOPS). Northern blot analysiswas performed using the available probes of CHS,DFR, F3H, LDOX, and UFGT genes cloned bySparvoli et al. [5]. The method of Boss et al. [7] wasused with the following modifications. The RNA wastransferred to a Gene Screen Plus†hybridizationtransfer membrane (NENTM Life Science Products,Boston, MA) with a vacuum blotter (mod. TDNA,Appligene, Illkirsch, France) for 3.5 h at 45/50 mbars.Then prehybridization was run for 4 h at 42 8C in 5xSSPE, 50% (w/v) deionized formamide, 1% SDS, 10%dextran-NaSO4 MW 500 000 and 10 mg/ml denaturedsalmon sperm. The membranes were hybridized for 15 hat 42 8C with the addition of denatured 32P-labelledcDNAs probes prepared by random primer labeling(Ready-To-Go DNA Labeling Kit, Amersham Pharma-cia Biotech, Uppsala, Sweden). The membrane was thenwashed twice for 10 min at 42 8C and then once for 15min at 42 8C and exposed to the autoradiography film(Hyper film, Amersham Pharmacia Biotech). The mem-branes were also hybridized with a 18S probe. Thehybridization signal intensity was assessed with theSigmaScan software (SPSS Inc, Chicago). To enablethe comparison, the hybridization signals were adjustedaccording to the mean 18S signal of each membrane,then the adjusted values were expressed as percentagesof the means of the day 0 signal (100%). Blots wereperformed in duplicates with different RNA samples,extracted from berries of different vines.Proteins were extracted as described previously [11]and Western blots and immunodetection were per-formed according to Ford et al. [12]. Equal proteinamounts (10 mg) were loaded onto each lane (data notshown).2.3. Grape skin extracts and HPLC analysesMethanolic extraction was performed according toBoss et al. [7]. Briefly, berries were individually thawedin warm water and skins were frozen immediately afterpeeling, then ground in mortar and pestle with liquidnitrogen; we used skins of 20 berries per replicaterandomly chosen among each batch of 5 clusters.Separation of the anthocyanins was performed by directHPLC analysis on a reversed stationary phase asdescribed by Cheynier et al. [13]. Anthocyanins wereanalysed at 520 nm with a diode array detector andidentified according to the retention times and UVspectral data. Quantifications were based on peak areasat 520 nm using a response factor calculated withmalvidin-3-glucoside. This compound was purchasedfrom Extrasynthese (Genay, France).Fig. 1. Effect of a 5% ethanol (EtOH) treatment at veraison (day 0) onberry weight up to harvest (day 56); n/3, error bars represent S.E.3. Results and discussion3.1. Berry weight and color of skin extractsThe berry weight did not changed between thecontrols and the treated berries over the ripening period(Fig. 1). These results confirmed our observations [3],which showed that ethanol induced an increase in theabsorbance at 520 nm of berry skin extracts without achange of berry weight at harvest.3.2. HPLC analysisThe HPLC analysis of berry skin extracts showed thatethanol sprayed at veraison stimulated the accumulationof the five main anthocyanins present in berry skins,namely delphinidin-3-glucoside, cyanidin-3-glucoside,petunidin-3-glucoside, peonidin-3-glucoside, and malvi-din-3-glucoside, from 4 days after treatment untilharvest (Fig. 2). Malvidin-3-glucoside derivatives werethe main pigments at harvest and their levels were nearlyFig. 2. Accumulation of the main anthocyanins in grape berry skins after spraying at veraison with 5% ethanol (EtOH). Each point is the sum ofmonoglucosides, acetylglucosides and p-coumarylglucosides; n/3, error bars represent S.E.three fold higher in ethanol treated berries than incontrols. There was no change in the relative distribu-tion between monoglucosides, acetylglucosides and p -coumarylglucosides (data not shown), which were pre-sent in percentages similar to those previously described[4].These results suggest that the increased anthocyaninlevels, previously observed in cranberries when treatedFig. 3. (A) Relative transcription of the five of anthocyanin genes, CHS, F3H, DFR, LDOX and UFGT, after an ethanol (5%) treatment at veraison(day 0) up to harvest (day 56), in comparison to controls. The inserted images show the most representative blots; n/2, error bars represent S.E. (B)Immunoblot of total soluble protein extracts with rabbit antibodies raised against rUFGT as described in Ford et al. [12].with a combination of an ethylene precursor andethanol [2], were not only due to ethanol increasingthe ethylene precursor diffusion through cell membranesas suggested by their authors, but may be partlydue to an effect of ethanol itself on anthocyaninbiosynthesis. In the next part of our work, we studiedthe transcription rate of some of the genes involved inthe anthocyanin synthesis, using available cDNAprobes.3.3. Gene transcription and expressionTreatment of Cabernet Sauvignon grapes with 5%ethanol at veraison resulted in some altered regulationof the transcription of anthocyanin biosynthesis genescompared to the controls. Ethanol treatment had nostimulating effect on the transcription of genes in theearly part of the anthocyanin biosynthesis pathway,including CHS, DFR, F3H and LDOX (Fig. 3).However, both Northern and Western blot analysesindicated that the UFGT gene was more highly ex-pressed than in controls from the first day after ethanoltreatment until 20 days later (Fig. 3). This ethanol effect,observed for the first time in grapes, could be indirectthrough an increase of C2H4 production [3], which isknown to induce UFGT activity in fruit [14]. Moreover,this effect could be partly due to the conversion of someethanol into acetaldehyde, which is more efficient thanethanol in inducing ethylene production in fruit,[1,15,16] or could involve a stress response in the treatedplants.These results provide new evidence that UFGT playsan important role in increasing the accumulation ofanthocyanins in red cultivars, as well as its role indetermining the difference between red and white grapes[6,17].In apples, it was reported that chalcone synthase doesnot play a regulatory role in anthocyanin synthesis,whereas like UFGT expression in grapevine, the UF-GaIT activity was correlated with anthocyanin synthesisduring fruit maturation [18]. This may be due to itseffect on anthocyanidin glycosylation, which usuallyconfers stability to anthocyanins [19].The pattern of gene expression in grape berry could beexplained in relation to anthocyanin regulatory genes(i.e. those that control the expression of the structuralgenes of the pathway). Two types of regulatory genesmay be present, one that controls the expression ofPAL, CHS, CHI, F3H, DFR and LDOX and anotherthat induces UFGT gene expression [7]. Sequenceanalysis showed that there was no difference either inthe coding region or in the promoter of the UFGT genebetween white and the red grape cultivars, suggestingdifferences in the UFGT regulatory factors [17]. Thesefactors which control the expression of the anthocyaninstructural genes, including UFGT, have been identifiedin other species, but have not been yet isolated in grapes.Thus, a possible explication of our results is that ethanoltreatment can affect the expression of UFGT regulatoryfactors.It is clear that UFGT genes, whose expression iscorrelated with anthocyanin synthesis, are regulated byregulatory genes that can respond to exogenous treat-ment such as auxin in grapes [8] and ethylene in apples[18]. However, the only paper describing the inductionof glycosyltransferase activity by ethanol comes from astudy on rats [20].In conclusion, the effect of ethanol sprayed atveraison on anthocyanin accumulation in grape berriesseems to be due to its stimulatory effect on UFGT genetranscription rather than on the other anthocyaningenes studied (CHS, DFR, F3H and LDOX). Thisincrease did not change the relative distribution amongthe different anthocyanin derivatives. However, wecannot exclude that other genes or cellular functionsare involved in this response, and we have not checkedthe enzyme activities corresponding to the genes forwhich northern analysis were conducted.These results offer new perspectives to improve berrycolor of grape varieties showing anthocyanin deficien-cies or of grapes cultivated in climate areas where fullmaturity is hard to reach. Ethanol, a simple organiccompound, could match the sustainable viticultureneeds. However, it is essential that we determine theoptimal ethanol dose according to the sprayer type.More work has been initiated to study the best dose andthe best moment for application of ethanol on grapeberries in order to achieve greater anthocyanin accumu-lation.AcknowledgementsWe are grateful to Dr Paul Boss for useful commentsabout the text and Northern analysis, to Dr CharlesRomieu for fruitful discussion, to Dr Chiara Tonelli forthe generous gift of the cDNA probes, to Dr Jose´Raynal for technical help and Drs Jean-Claude Pechand Mondher Bouzayen for the use of their laboratoryfacilities.References[1] J.C. Beaulieu, M.E. Saltveit, Inhibition or promotion of tomatofruit ripening by acetaldehyde and ethanol is concentrationdependent and varies with initial fruit maturity, J. Amer. Soc.Hort. Sci. 122 (1997) 392/398.[2] K.M. Farag, J.P. Palta, E.J. Stang, Ethanol enhances theeffectiveness of ethephon on anthocyanin production in cranberryfruits in the field, HortScience 27 (1992) 411/412.[3] C Chervin, A. Elkereamy, J.P. Roustan, J.D. Faragher, A.Latche, J.C. Pech, M. Bouzayen, An ethanol spray at veraisonenhances colour in red wines, Aust. J. Grape Wine Res. 7 (2001)144/145.[4] G. Mazza, Anthocyanins in grapes and grape products, Crit. Rev.Food Nutr. 35 (1995) 341/371.[5] F. Sparvoli, C. Martin, A. Scienza, G. Gavazzi, C. Tonelli,Cloning and molecular analysis of structural genes involved inflavonoid and stilbene biosynthesis in grape (Vitis Vinifera L.),Plant Mol. Biol. 24 (1994) 743/755.[6] P.K. Boss, C. Davies, S.P. Robinson, Expression of anthocyaninbiosynthesis pathway genes in red and white grapes, Plant Mol.Biol. 32 (1996) 565/569.[7] P.K. Boss, C. Davies, S.P. Robinson, Analysis of the expressionof anthocyanin pathway genes in developing Vitis vinifera L. cv.Shiraz grape berries and the implication for pathway regulation,Plant Physiol. 111 (1996) 1059/1066.[8] C. Davies, P.K. Boss, S.P. Robinson, Treatment of grape berries,a nonclimacteric fruit with a synthetic auxin, retards ripening andalters the expression of developmentally regulated genes, PlantPhysiol. 115 (1997) 1155/1161.[9] R. Ardi, I. Kobiler, B. Jacoby, N.T. Keen, D. Prusky, Involve-ment of epicatechin biosynthesis in the activation of the mechan-ism of resistance of avocado fruits to Colletotrichumgloeosporioides , Physiol. Mol. Plant Pathol. 53 (1998) 269/285.[10] A.K. Kanellis, G. Diallinas, Developmental and ripening regula-tion of gene expression of phenylpropanoid biosynthetic enzymesin melon fruit, Proceedings of ‘Postharvest physiology andpathology’, International Symposium, Agadir, Morocco, 16/21January 1994, Institut Agronomique et Veterinaire Hassan II,(1995) 353/360.[11] C.M. Ford, P.B. Høj, Multiple glucosyltransferase activities in thegrapevine Vitis vinifera L., Aust. J. Grape Wine Res. 4 (1998) 48/58.[12] C.M. Ford, P.K. Boss, P.B. Høj, Cloning and characterisation ofVitis vinifera UDP-glucose: flavonoid 3-O -glucosyltransferase, ahomologue of the enzyme encoded by the maize Bronze-1 locusthat may primarily serve to glucosylate anthocyanidins in vivo, J.Biol. Chem. 273 (1998) 9224/9233.[13] V. Cheynier, I. Hidalgo Arellano, J.M. Souquet, M. Moutounet,Estimation of the oxidative changes in phenolic compounds ofCarignane during winemaking, Am. J. Enol. Vitic. 48 (1997) 225/228.[14] G. Hrazdina, G.F. Parsons, L.R. Mattick, Physiological andbiochemical events during development and maturation of grapeberries, Am. J. Enol. Vitic. 35 (1984) 220/227.[15] F. Mencarelli, P. Savarese, M.E. Satveit, Ripening of kiwifruitexposed to ethanol and acetaldehyde vapors, Hort. Sci. 26 (1991)566/569.[16] E. Pesis, R. Marinansky, Carbon dioxide and ethylene productionby harvested grape berries in response to acetaldehyde andethanol, J. Amer. Soc. Hort. Sci. 117 (1992) 110/113.[17] S. Kobayashi, M. Ishimaru, C.K. Ding, H. Yakushji, N. Goto,Comparison of UDP-glucose: flavonoid 3-O -glucosyltransferase(UFGT) gene sequence between white grapes (Vitis vinifera ) andtheir sports with red skin, Plant Sci. 160 (2001) 543/550.[18] Z. Ju, L. Chenglian, Y. Yongbing, Activities of chalcone synthaseand UFGA1: flavonoid-3-O -glycosyltransferase in relation toanthocyanin synthesis in apple, Sci. Hort. 63 (1995) 175/185.[19] C.B. Do, F. Cormier, Y. Nicolas, Isolation and characterizationof UDP-glucose cyanidin 3-O -glucosyltransferase from grape cellsuspension cultures (Vitis vinifera L.), Plant Sci. 112 (1995) 43/51.[20] C.S. Lieber, E. Rubin, Ethanol increase glycosyl transferaseactivity in the hepatic golgi apparatus, Nature, New Biol. 243(125) (1973) 123/125.

Activities of chalcone synthase and UFGA1: ﬂavonoid-3-O-glycosyltransferase in relation to anthocyanin synthesis in apple,

An ethanol spray at veraisonenhances colour in red wines,

Analysis of the expression of anthocyanin pathway genes in developing Vitis vinifera L. cv. Shiraz grape berries and the implication for pathway regulation,

Anthocyanins in grapes and grape products,

Carbon dioxide and ethylene production by harvested grape berries in response to acetaldehyde and ethanol,

Cloning and characterisation of Vitis vinifera UDP-glucose: ﬂavonoid 3-O-glucosyltransferase, a homologue of the enzyme encoded by the maize Bronze-1 locus that may primarily serve to glucosylate anthocyanidins in vivo,

Cloning and molecular analysis of structural genes involved in ﬂavonoid and stilbene biosynthesis in grape (Vitis Vinifera

Comparison of UDP-glucose: ﬂavonoid 3-O-glucosyltransferase (UFGT) gene sequence between white grapes (Vitis vinifera) and their sports with red skin,

Developmental and ripening regulation of gene expression of phenylpropanoid biosynthetic enzymes in melon fruit,

Estimation of the oxidative changes in phenolic compounds of Carignane during winemaking,

Ethanol enhances the effectiveness of ethephon on anthocyanin production in cranberry fruits in the ﬁeld,

Ethanol increase glycosyl transferase activity in the hepatic golgi apparatus,

Expression of anthocyanin biosynthesis pathway genes in red and white grapes,

Inhibition or promotion of tomato fruit ripening by acetaldehyde and ethanol is concentration dependent and varies with initial fruit maturity,

Involvement of epicatechin biosynthesis in the activation of the mechanism of resistance of avocado fruits to Colletotrichum gloeosporioides,

Isolation and characterization of UDP-glucose cyanidin 3-O-glucosyltransferase from grape cell suspension cultures (Vitis vinifera L.), Plant Sci.

Multiple glucosyltransferase activities in the grapevine Vitis vinifera

Physiological and biochemical events during development and maturation of grape berries,

Ripening of kiwifruit exposed to ethanol and acetaldehyde vapors,

Treatment of grape berries, a nonclimacteric fruit with a synthetic auxin, retards ripening and alters the expression of developmentally regulated genes, Plant Physiol. 115

Ethanol triggers grape gene expression leading to anthocyanin

accumulation during berry ripening

Recent studies have shown that low doses of ethanol stimulate the maturation of some fruits. The present work showed that spraying Cabernet Sauvignon grapes, with 5% ethanol at veraison enhances the anthocyanin accumulation. Veraison is the time when the berries turn from green to purple. HPLC analysis showed a marked increase in the total concentrations of the derivatives of delphinidin, cyanidin, petunidin, peonidin and malvidin from the fourth day after the ethanol treatment until harvest. This was not linked to a difference in berry weight in comparison to controls. Two distinct expression patterns were found for anthocyanin biosynthesis genes in the treated and untreated berries. For one group, consisting of chalcone synthase, flavanone-3-hydroxylase, dihydroxyflavonol-4-reductase and leucoanthocyanidin dioxygenase, the expression was inhibited or unchanged by the ethanol treatment, whereas for UDP glucose-flavonoid 3-O-glucosyltransferase (UFGT) there was a marked increase in expression from 1 to 20 days after ethanol treatment. These results suggest that the UFGT gene is a key factor in the observed anthocyanin accumulation following ethanol treatment.Ashraf El Kereamy, Christian Chervin, Jean-Marc Souquet, Michel Moutounet, Marie-Carmen Monje, Françoise Nepveu, Helene Mondies, Christopher M. Ford, Robyn van Heeswijck and Jean-Paul Roustanhttp://www.elsevier.com/wps/find/journaldescription.cws_home/506030/description#descriptio

Ethanol triggers grape gene expression leading to anthocyanin accumulation during berry ripening

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Ethanol triggers grape gene expression leading to anthocyanin accumulation during berry ripening

Abstract

Similar works

Full text

Available Versions

Open Archive Toulouse Archive Ouverte

Crossref

HAL: Hyper Article en Ligne

Open Archive Toulouse Archive Ouverte

Adelaide Research &amp; Scholarship

Portail HAL Um (Université de Montpellier)

Archive Ouverte en Sciences de l'Information et de la Communication

Hal-Diderot

HAL Université de Toulouse, et Toulouse INP

Adelaide Research & Scholarship