Chervin, C.

Roustan, J. P.

Tira-Umphon, A.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 46 (4), 210–211 (2007)Correspondence to: Dr C. CHERVIN, UMR 990, Génomique et Biotechnologie des Fruits, INP, ENSAT, INRA, Univer-sité de Toulouse, F-31320 Castanet Tolosan, France. Fax: +33-5-6219-3573. E-mail: chervin@ensat.fr Research NoteThe stimulation by ethylene of the UDP glucose-flavonoid 3-O-glucosyltransferase (UFGT) in grape tissues is independent from the MybA transcription factorsA. TIRA-UMPHON, J. P. ROUSTAN and C. CHERVINUMR 990, Génomique et Biotechnologie des Fruits, INP, EN-SAT, INRA, Université de Toulouse, Castanet Tolosan, FranceK e y   w o r d s :  C2H4, glycosyltransferase, grapevine, anthocyanin.Introduction:  Grapes are regarded as non-climacteric fruit (CHERVIN et al. 2004 and references herein), in which ethylene evolution is very low and for which the ripening process seems to occur independently of ethylene produc-tion. However, a recent study has shown that grape berry tissues have a fully functional pathway for ethylene syn-thesis, that this pathway is activated just before veraison when red berries start to accumulate anthocyanins, and that ethylene perception is critical for some berry changes asso-ciated with ripening, including anthocyanin accumulation (CHERVIN et al. 2004). This last conclusion was supported by the fact that anthocyanin accumulation in the berry skins was inhibited when a specific inhibitor of ethylene recep-tors, 1-methylcyclopropene was applied to berries just be-fore veraison, at the same time as ethylene production was activated in the berry.The control of anthocyanin accumulation during the ripening phase in red grape berries is thought to be great-ly dependent on UFGT activity (KOBAYASHI et al. 2001, YAKUSHIJI et al. 2006). This enzyme plays a key role in sta-bilising the aglycone moiety of the anthocyanins (PIFFAUT et al. 1994) and may be essential for their transport to the vacuole. We have shown previously that exogenous ethyl-ene could induce grape ufgt expression (EL-KEREAMY et al. 2003), confirming observations about the commercial use of ethylene precursor to boost grape skin colour.The anthocyanin biosynthesis in model plants for the anthocyanin pathway such as petunia and maize is control-led by Myc and Myb transcription factors, and indeed, one transcription factor of this family, MybA1, was shown to be critical for the regulation of ufgt expression (YAKUSHIJI et al. 2006 and refs therein) and anthocyanin accumulation in grape tissues. WALKER et al. (2007) showed that two iso-forms of this transcription factor, MybA1 and MybA2 are involved in anthocyanin accumulation in grapevine. The question remains whether the ethylene signal is acting on grape ufgt expression through the activation of mybA expression or via ethylene responsive transcription factors. Materials and Methods: 'Cabernet Sauvignon' grape-vines used for the ethylene experiments are grafted on '110 Richter' rootstocks and grown in Toulouse, South-West of France, in a non-irrigated vineyard. Full bloom occurred around mid-June. The ethylene was applied for 24 h at dif-ferent times after full bloom, at an initial concentration of 40 µl·l-1, three biological replicates were then collected and stored at -80 °C. All other sampling processes and the RNA extractions were performed as described in EL-KEREAMY et al. (2003). Suspensions of purple grape cells of 'Gamay' were grown as described previously (TRIANTAPHYLIDES et al. 1993). Cells were vacuum-filtered onto Whatman n°1 filter paper and set upon 5 ml fresh MS-based cell culture medium in small Petri dishes. Cells were then sprayed with one ml of a sterile solution of 7 mM 2-chloroethylphos-phonic acid (2-CEPA), a precursor of ethylene, the controls were sprayed with sterile water. Incubation time was 6 h before freezing samples for RNA extraction, performed as described above.The qRT-PCR analyses were performed according to EL-SHARKAWY et al. (2005), with the following modifica-tions. Oligonucletide sequences of mybA primers were VvmybA1c(F), 5’- GCAAGCCTCAGGACAGAA -3’ and VvmybA1c(R), 5’- AAGCCCACATCAAATGGAAAA-3’. They would amplify both VvmybA1 (AB097923) and VvmybA2 (AB097924) isoforms. Oligonucletide sequenc-es for the ufgt primers were  Vvufgt(F) 5’-GGCTTTT-GTCACACATTGCG-3’ and Vvufgt(R) 5’-AAAAAG-GGCCTGCAAATCAA-3’. The myb et ufgt mRNA levels were expressed as transcript accumulation indexes relative to a control gene ef1α, as previously shown (TERRIER et al. 2005). The LSD value between treatments was calculated at the 5 % level using a one way ANOVA (SigmaStat, Sys-tat Software Inc., San Jose, CA). The ethylene cis-elements were estimated by homol-ogy search using PLACE database, http://www.dna.affrc.go.jp/PLACE/signalscan.html or PlantCARE database, http://bioinformatics.psb.ugent.be/webtools/plantcare/html/Results and Discussion: The application of gaseous ethylene significantly stimulated the accumulation of ufgt mRNA in berries when applied 8 and 9 weeks after full bloom (Fig. 1 a). These observations made in 'Cabernet Sauvignon' berry tissues were confirmed in another culti-var, under different experimental conditions, the 'Gamay' cell suspension sprayed with the 2-CEPA, an ethylene pre-cursor (Fig. 1c). These results confirmed previous observa-tions, either the enhancement of anthocyanin accumulation after spraying 2-CEPA onto the grape clusters (EL-KEREAMY et al. 2003), or the inhibition of anthocyanin accumulation following a treatment of the clusters with the ethylene in-hibitor, 1-methylcyclopropene (CHERVIN et al. 2004). However no significant difference was seen for the ac-cumulation mybA mRNAs in the same experimental condi-tions (Fig. 1 b and c). This absence of responsiveness to an ethylene signal was further confirmed by the analysis  211 A. TIRA-UMPHON et al.of the VvmybA1 promoter, GenBank AB242302 (the se-quence of the VvmybA2 promoter was not available when we wrote this article), which showed three ethylene cis-elements (ethylene response elements, ERE) in the -2 kb upstream region (Fig. 2 a). The closest GCCGCC box, one of the most potent EREs (FUJIMOTO et al. 2000), locates around -2100 bp. The closest ATTTnAAA  box, the other common ERE, locates at -850 bp from mybA1 start codon (Fig. 2). In comparison there are seven EREs in the ufgt promoter, GenBank AY955269, (Fig. 2). The GCCGCC sequence was found at -160 bp from the ufgt start codon, a good place to be active (Dr Ohme-Takagi, pers. comm. and analyses of ethylene responsive promoters, data not shown). Two other ERE sequences, ATTTnAAA are rea-sonably close to the gene start codon, located between -450 and -350 bp. In another ethylene responsive promoter, they were shown to be active when placed at -500 bp (ITZHAKI et al. 1994). Here we used the sequence of the Shiraz ufgt promoter, which has 97 to 99 % homology to the grapevine ufgt promoter sequences isolated from the 'Kyoho', 'Italia', 'Ruby Oku', 'Muscat of Alexandria' and 'Flame Muscat' cultivars (KOBAYASHI et al. 2001), and 95 % to 'Caber-net Sauvignon' (GenBank AY919624). The alignment of various ufgt promoter sequences has already been shown (KOBAYASHI et al. 2001). So we conclude that the stimulation of ufgt expression by ethylene is independent from mybA expression. But it does not preclude the importance of MybA in controlling the ufgt expression. Indeed, Dr. GOTO-YAMAMOTO found that the MybA protein binds to a region of the ufgt pro-moter around -428 to -303 bp from the start codon (pers. comm.) and recent works confirm the role of MybA in stimulating anthocyanin accumulation (YAKUSHIJI et al. 2006, WALKER et al. 2007). To our knowledge this is the Fig. 2: Locations of ethylene responsive elements (cis-acting) on the promoter of mybA1 (AB242302) and on the promoter of ufgt (AY955269) of Vitis vinifera. The nucleotide numbering is rela-tive to the start codon (0). Cis-elements were estimated by homol-ogy search using PLACE and PlantCARE databases. Fig. 1: Effects of exogenous ethylene to a) the ufgt mRNA levels, to b) the mybA1 mRNA levels in 'Cabernet Sauvignon' berries ac-cording to time after full bloom, and to c) mRNAs of both genes in 'Gamay' cell suspensions. The mRNA levels were determined by qRT- PCR analyses and are expressed as the transcript accu-mulation index relative to ef1-α. Each bar represents the mean of three biological replicates, and LSD bars were calculated at the 0.05 level for differences between the treatments (control or ethylene).first report addressing the responsiveness of myb expres-sion to an ethylene signal.The authors wish to acknowledge the INRA-INP/ENSAT, and the Midi-Pyrénées regional council for a partial funding of this collaborative work, the Domaine de Candie (Régie Agricole de la Ville de Toulouse) for the grapes and the team of CRGS platform of Genopole Toulouse where the qRT-PCR analyses were performed. CHERVIN, C.; EL-KEREAMY, A.; ROUSTAN, J. P.; LATCHE, A.; LAMON, J.; BOUZAYEN, M.; 2004:  Ethylene seems required for the ripening of grape, a non-climacteric fruit. Plant Sci. 167, 1301-1305.EL-KEREAMY, A.; CHERVIN, C.; ROUSTAN, J. P.; CHEYNIER, V.; SOUQUET, J. M.; MOUTOUNET, M.; RAYNAL, J.; FORD, C. M.; LATCHE, A.; PECH, J. C.; BOUZAYEN, M.; 2003: Exogenous ethylene stimulates the long-term expression of genes related to anthocyanin biosynthesis in grape berries. Physiol. Plant. 119, 175-182.EL-SHARKAWY, I.; MANRÍQUEZ, D.; FLORES, B. F.; REGAD, F.; BOUZAYEN, M.; LATCHÉ, A.; PECH, J. P.; 2005: Functional characterization of a melon alcohol acyl-transferase gene family involved in the biosynthesis of ester volatiles. Identification of the crucial role of a threonine resi-due for enzyme activity. Plant Mol. Biol. 59, 345-362.FUJIMOTO, S. Y.; OHTA, M.; USUI, A.; SHINSHI, H.; OHME-TAKAGI, M.; 2000: Arabidopsis ethylene-responsive element binding factors act as tran-scriptional activators or repressors of GCC box-mediated gene ex-pression. Plant Cell 12, 393-404.KOBAYASHI, S.; ISHIMARU, M.; DING, C. K.; YAKUSHIJI, H.; GOTO, N.; 2001: Comparison of UDP-glucose:flavonoid 3-O-glucosyltransferase (ufgt) gene sequences between white grapes (Vitis vinifera) and their sports with red skin. Plant Sci. 160, 543-550.PIFFAUT, B.; KADER, F.; GIRARDIN, M.; METCHE, M.; 1994: Comparative degradation pathways of malvidin 3,5-diglucoside after enzymatic and thermal treatments. Food Chem. 50, 115-120.TERRIER , N.; GLISSANT, D.; GRIMPLET, J.; BARRIEU,  F.; ABBAL, P.; COU-TURE, C.; AGEORGES, A.; ATANASSOVA, R.; LÉON, C.; RENAUDIN, J. P.; DÉDALDÉCHAMP, F.; ROMIEU, C.; DELROT, S.; HAMDI, S.; 2005: Iso-gene specific oligo arrays reveal multifaceted changes in gene ex-pression during grape berry (Vitis vinifera, L.) development. Planta 222, 832-847.TRIANTAPHYLIDÈS, C.; NESPOULOUS, L.; CHERVIN, C.; 1993: Ammonium re-quirement for radiation-induced accumulation of polyamines in sus-pension cultured grape cells. Physiol. Plant. 87, 389-395.WALKER A. R.; LEE, E.; BOGS, J.; MCDAVID, D. A. J.; THOMAS, M. R.; ROB-INSON, S. P.; 2007. White grapes arose through the mutation of two similar and adjacent regulatory genes. Plant J. 49, 772-785.YAKUSHIJI, H.; KOBAYASHI, S.; GOTO-YAMAMOTO, N.; TAE JEONG, S.; SUETA, T.; MITANI, N.; AZUMA, A.; 2006: A skin color mutation of grape-vine, from black-skinned Pinot Noir to white-skinned Pinot Blanc, is caused by deletion of the functional VvmybA1 allele. Biosci. Bio-tech. Biochem. 70, 1506-1508.Received March 9, 2007MybATime after full bloom (weeks)6 7 8 9 100.000.020.041.001.502.002.50UfgtTranscript AccumulationIndex0.000.010.020.030.040.050.06ControlEthyleneLSD0.05LSD0.05abGene namesUfgt MybA0.00.20.40.60.81.0LSD0.05cTranscript AccumulationIndex-2100 -1400 -850 -160 -1200 -930 -770 --450 -375 00mybA1ufgt:  GCCGCC box: ATTTnAAA boxEthylene Responsive Elements (ERE)-1200 

The stimulation by ethylene of the UDP glucose-flavonoid 3-O-glucosyltransferase (UFGT) in grape tissues is independent from the MybA transcription factors

https://ojs.openagrar.de/index.php/VITIS/article/download/4259/4211

The stimulation by ethylene of the UDP glucose-flavonoid 3-O-glucosyltransferase (UFGT) in grape tissues is independent from the MybA transcription factors

Abstract

Similar works

Full text

Available Versions

JKI Open Journal Systems (Julius Kühn-Institut)