Proanthocyanidins (PAs) are fundamental nutritional metabolites in different types of grape products consumed by human beings. Although the biosynthesis of PAs in berry of Vitis vinifera has gained intensive investigations, the understanding of PAs in other Vitis species is limited. In this study, we report PA formation and characterization of gene expression involved in PA biosynthesis in leaves of V. bellula, a wild edible grape species native to south and south-west China. Leaves are collected at five developmental stages defined by sizes ranging from 0.5 to 5 cm in length. Analyses of thin layer chromatography (TLC) and high performance liquid chromatography-photodiode array detector (HPLC-PAD) show the formation of (+)-catechin, (？)-epicatechin, (+)-gallocatechin and (？)-epigallocatechin during the entire development of leaves. Analyses of butanol-HCl boiling cleavage coupled with spectrometry measurement at 550 nm show a temporal trend of extractable PA levels, which is characterized by an increase from 0.5 cm to 1.5 cm long leaves followed by a decrease in late stages. TLC and HPLC-PAD analyses identify cyanidin, delphinidin and pelargonidin produced from the cleavage of PAs in the butanol-HCl boiling, showing that the foliage PAs of V. bellula include three different types of extension units. Four cDNAs, which encode VbANR, VbDFR, VbLAR1 and VbLAR2, respectively, are cloned from young leaves. The expression patterns of VbANR and VbLAR2 but not VbLAR1 and VbDFR follow a similar trend as the accumulation patterns of PAs. Two cDNAs encoding VbMYBPA1 and VbMYB5a, the homologs of which have been demonstrated to regulate the expression of both ANR and LAR in V. vinifera, are also cloned and their expression profiles are similar to those of VbANR and VbLAR2. In contrast, the expression profiles of MYBA1 and 2 homologs involved in anthocyanin biosynthesis are different from those of VbANR and VbLAR2. Our data show that both ANR and LAR branches are involved in PA biosynthesis in leaves of V. bellula.
References
[1]
Xie, D.-Y.; Dixon, R.A. Proanthocyanidin biosynthesis—Still more questions than answers? Phytochemistry 2005, 66, 2127–2144, doi:10.1016/j.phytochem.2005.01.008.
[2]
Dixon, R.A.; Xie, D.Y.; Sharma, S.B. Proanthocyanidins—A final frontier in flavonoid research? New Phytol. 2005, 165, 9–28, doi:10.1111/j.1469-8137.2004.01217.x.
Stringer, S.J.; Perkins Veazie, P.M.; Marshall, D.A. Natraceutical profile of selected muscadine (Witis rotundifolia michx.) cultivars and breeding lines. Hortscience 2005, 40, 1068.
[5]
Radvanyi, J., Jr.; Stojanovic, B.J.; Drapala, W.J.; Overcash, J.P.; Hegwood, C.P., Jr. Composition and quality of juices and wines of eight Vitis rotundifolia Michx. cultivars. Am. J. Enol. Vitic. 1980, 31, 316–322.
[6]
Weinges, K.; Piretti, M.V. Proanthocyanidins. 20. Isolation of C30H26O12-Procyanidin-B1 from grapes. Annalen Der Chemie-Justus Liebig 1971, 748, 218, doi:10.1002/jlac.19717480125.
[7]
Bagchi, D.; Garg, A.; Krohn, R.L.; Bagchi, M.; Bagchi, D.J.; Balmoori, J.; Stohs, S.J. Protective effects of grape seed proanthocyanidins and selected antioxidants against TPA-induced hepatic and brain lipid peroxidation and DNA fragmentation, and peritoneal macrophage activation in mice. Gen. Pharmacol. 1998, 30, 771–776, doi:10.1016/S0306-3623(97)00332-7.
[8]
Kennedy, J.A.; Hayasaka, Y.; Vidal, S.; Waters, E.J.; Jones, G.P. Composition of grape skin proanthocyanidins at different stages of berry development. J. Agric. Food Chem. 2001, 49, 5348–5355, doi:10.1021/jf010758h.
[9]
Natella, F.; Belelli, F.; Gentili, V.; Ursini, F.; Scaccini, C. Grape seed proanthocyanidins prevent plasma postprandial oxidative stress in humans. J. Agric. Food Chem. 2002, 50, 7720–7725, doi:10.1021/jf020346o.
[10]
Renaud, S.; de Lorgeril, M. Wine, alcohol, platelets, and the French paradox for coronary heart disease. The Lancet 1992, 339, 1523, doi:10.1016/0140-6736(92)91277-F.
[11]
Somers, T.C. Wine tannins-isolation of condensed flavonoid pigments by gel-filtration. Nature 1966, 209, 368–370, doi:10.1038/209368a0.
[12]
Yamakoshi, J.; Kataoka, S.; Koga, T.; Ariga, T. Proanthocyanidin-rich extract from grape seeds attenuates the development of aortic atherosclerosis in cholesterol-fed rabbits. Atherosclerosis 1999, 142, 139–149, doi:10.1016/S0021-9150(98)00230-5.
[13]
Sato, M.; Ray, P.S.; Maulik, G.; Maulik, N.; Engelman, R.M.; Bertelli, A.A. E.; Bertelli, A.; Das, D.K. Myocardial protection with red wine extract. J. Cardiovascul. Pharmacol. 2000, 35, 263–268, doi:10.1097/00005344-200002000-00013.
[14]
Sanchez-Moreno, C.; Cao, G.; Ou, B.; Prior, R.L. Anthocyanin and proanthocyanidin content in selected white and red Wines. Oxygen radical absorbance capacity comparison with nontraditional wines obtained from Highbush blueberry. J. Agric. Food Chem. 2003, 51, 4889–4896, doi:10.1021/jf030081t.
[15]
Jordao, A.M.; Goncalves, F.J.; Correia, A.C.; Cantao, J.; Rivero-Perez, M.D.; SanJose, M.L.G. Proanthocyanidin content, antioxidant capacity and scavenger activity of Portuguese sparkling wines (Bairrada Appellation of Origin). J. Sci. Food Agric. 2010, 90, 2144–2152.
Sato, M.; Maulik, G.; Ray, P.S.; Bagchi, D.; Das, D.K. Cardioprotective effects of grape seed proanthocyanidin against ischemic reperfusion injury. J. Mol. Cell. Cardiol 1999, 31, 1289–1297, doi:10.1006/jmcc.1999.0961.
[18]
Tanner, G.J.; Francki, K.T; Abrahams, S.; Watson, J.M.; Larkin, P.J.; Ashton, A.R. Ashton, .Proanthocyanidin biosynthesis in plants. Purification of legume leucoanthocyanidin reductase and molecular cloning of its cDNA. J. Biol Chem. 2003, 278, 31647–31656.
[19]
Peng, Q.Z.; Zhu, Y.; Liu, Z.; Du, C.; Li, K.G.; Xie, D.Y. An integrated approach to demonstrating the ANR pathway of proanthocyanidin biosynthesis in plants. Planta 2012, 236, 901–918, doi:10.1007/s00425-012-1670-6.
[20]
Xie, D-Y.; Sharma, S.B.; Paiva, N.L.; Ferreira, D.; Dixon, R.A. Role of anthocyanidin reductase, encoded by BANYULS in plant flavonoid biosynthesis. Science 2003, 299, 396–399, doi:10.1126/science.1078540.
[21]
Gagné, S.; Lacampagne, S.; Claisse, O.; Gény, L. Leucoanthocyanidin reductase and anthocyanidin reductase gene expression and activity in flowers, young berries and skins of Vitis vinifera L. cv. Cabernet-Sauvignon during development. Plant Physiol. Biochem. 2009, 47, 282–290, doi:10.1016/j.plaphy.2008.12.004.
[22]
Bogs, J.; Downey, M.O.; Harvey, J.S.; Ashton, A.R.; Tanner, G.J.; Robinson, S.P. Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves. Plant Physiol. 2005, 139, 652–663, doi:10.1104/pp.105.064238.
[23]
Gargouri, M.; Manigand, C.; Mauge, C.; Granier, T.; Langlois d'Estaintot, B.; Cala, O.; Pianet, I.; Bathany, K.; Chaudiere, J.; Gallois, B. Structure and epimerase activity of anthocyanidin reductase from Vitis vinifera. Acta Crystallogr. Sect. D—Biol. Crystallogr. 2009, 65, 989–1000, doi:10.1107/S0907444909025013.
[24]
Pfeiffer, J.; Kuhnel, C.; Brandt, J.; Duy, D.; Punyasiri, P.A.N.; Forkmann, G.; Fischer, T.C. Biosynthesis of flavan 3-ols by leucoanthocyanidin 4-reductases and anthocyanidin reductases in leaves of grape (Vitis vinifera L.), apple (Malus x domestica Borkh.) and other crops. Plant Physiol. Biochem. 2006, 44, 323–334, doi:10.1016/j.plaphy.2006.06.001.
[25]
Bogs, J.; Jaffe, F.W.; Takos, A.M.; Walker, A.R.; Robinson, S.P. The grapevine transcription factor VvMYBPA1 regulates proanthocyanidin synthesis during fruit development. Plant Physiol. 2007, 143, 1347–1361, doi:10.1104/pp.106.093203.
[26]
Xie, D.-Y.; Sharma, S.B.; Wright, E.; Wang, Z.-Y.; Dixon, R.A. Metabolic engineering of proanthocyanidins through co-expression of anthocyanidin reductase and the PAP1 MYB transcription factor. Plant J. 2006, 45, 895–907, doi:10.1111/j.1365-313X.2006.02655.x.
[27]
Lacampagne, S.; Gagne, S.; Geny, L. Involvement of abscisic acid in controlling the proanthocyanidin biosynthesis pathway in grape skin: New elements regarding the regulation of tannin composition and leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) activities and expression. J. Plant Growth Regul. 2010, 29, 81–90, doi:10.1007/s00344-009-9115-6.
[28]
Deluc, L.; Barrieu, F.; Marchive, C.; Lauvergeat, V.; Decendit, A.; Richard, T.; Carde, J.-P.; Merillon, J.-M.; Hamdi, S. Characterization of a grapevine R2R3-MYB transcription factor that regulates the phenylpropanoid pathway. Plant Physiol.200 , 499–511.
[29]
Kobayashi, S.; Yamamoto, N.G.; Hirochika, H. Association of VvmybA1 gene expression with anthocyanin production in grape (Vitis vinifera) skin - color mutants. J. Jpn. Soc. Hortic. Sci. 2005, 74, 196–203, doi:10.2503/jjshs.74.196.
[30]
Jeong, S.T.; Goto-Yamamoto, N.; Hashizume, K.; Kobayashi, S.; Esaka, M. Expression of VvmybA1 gene and anthocyanin accumulation in various grape organs. Am. J. Enol. Vitic. 2006, 57, 507–510.
[31]
Yakushiji, H.; Kobayashi, S.; Goto-Yamamoto, N.; Jeong, S.T.; Sueta, T.; Mitani, N.; Azuma, A. A skin color mutation of grapevine, from black-skinned Pinot Noir to white-skinned Pinot Blanc, is caused by deletion of the functional VvmybA1 allele. Biosci. Biotechnol. Biochem. 2006, 70, 1506–1508, doi:10.1271/bbb.50647.
This, P.; Lacombe, T.; Cadle-Davidson, M.; Owens, C.L. Wine grape (Vitis vinifera L.) color associates with allelic variation in the domestication gene VvmybA1. Theor. Appl. Genet. 2007, 114, 723–730, doi:10.1007/s00122-006-0472-2.
[34]
Güner, A.; Gyulai, G.; Tóth, Z.; Ba？l？1, G.A.; Szabó, Z.; Gyulai, F.; Bittsánszky, A.; Waters, L., Jr.; Heszky, L. Grape (Vitis vinifera) seeds from Antiquity and the Middle Ages Excavated in Hungary - LM and SEM analysis. Anadolu. Univ. J. Sci. Technol. 2009, 10, 205–213.
[35]
Gris, E.F.; Mattivi, F.; Ferreira, E.A.; Vrhovsek, U.; Pedrosa, R.C.; Bordignon-Luiz, M.T. Proanthocyanidin profile and antioxidant capacity of Brazilian Vitis vinifera red wines. Food Chem. 2011, 126, 213–220.
[36]
Spranger, I.; Sun, B.; Mateus, A. Chemical characterization and antioxidant activities of oligomeric and polymeric procyanidin fractions from grape seeds. Food Chem. 2008, 108, 519–532, doi:10.1016/j.foodchem.2007.11.004.
[37]
Vitseva, O.; Varghese, S.; Chakrabarti, S.; Folts, J.; Freedman, J. Grape seed and skin extracts inhibit platelet function and release of reactive oxygen intermediates. J. Cardiovascul. Pharmacol. 2005, 46, 445–451, doi:10.1097/01.fjc.0000176727.67066.1c.
[38]
Souquet, J.-M.; Cheynier, V.; Brossaud, F.; Moutounet, M. Polymeric proanthocyanidins from grape skins. Phytochemistry 1996, 43, 509–512.
[39]
Fuleki, T.; Ricardo-da-Silva, J.M. Catechin and procyanidin composition of seeds from grape cultivars grown in Ontario. J. Agric. Food Chem. 1997, 45, 156–1160.
[40]
Gabetta, B.; Fuzzati, N.; Griffini, A.; Lolla, E.; Pace, R.; Ruffilli, T.; Peterlongo, F. Characterization of proanthocyanidins from grape seeds. Fitoterapia 2000, 71, 162–175, doi:10.1016/S0367-326X(99)00161-6.
Castillo-Munoz, N.; Fernandez-Gonzalez, M.; Gomez-Alonso, S.; Garcia-Romero, E.; Hermosin-Gutierrez, I. Red-color related phenolic composition of Garnacha Tintorera (Vitis vinifera L.) grapes and red wines. J. Agric. Food Chem. 2009, 57, 7883–7891, doi:10.1021/jf9002736.
[44]
He, F.; He, J.J.; Pan, Q.H.; Duan, C.Q. Mass-spectrometry evidence confirming the presence of pelargonidin-3-O-glucoside in the berry skins of Cabernet Sauvignon and Pinot Noir (Vitis vinifera L.). Aust. J. Grape Wine Res. 2010, 16, 464–468, doi:10.1111/j.1755-0238.2010.00107.x.
[45]
He, J.J.; Liu, Y.X.; Pan, Q.H.; Cui, X.Y.; Duan, C.Q. Different anthocyanin profiles of the skin and the pulp of Yan73 (Muscat Hamburg x Alicante Bouschet) grape berries. Molecules 2010, 15, 1141–1153, doi:10.3390/molecules15031141.
[46]
Zhao, Q.; Duan, C.Q.; Wang, J. Anthocyanins profile of grape berries of Vitis amurensis, its hybrids and their wines. Int J. Mol. Sci. 2010, 11, 2212–2228, doi:10.3390/ijms11052212.
[47]
Punyasiri, P.A.N.; Abeysinghe, I.S.B.; Kumar, V.; Treutter, D.; Duy, D.; Gosch, C.; Martens, S.; Forkmann, G.; Fischer, T.C. Flavonoid biosynthesis in the tea plant Camellia sinensis: Properties of enzymes of the prominent epicatechin and catechin pathways. Arch. Biochem. Biophys. 2004, 431, 22–30, doi:10.1016/j.abb.2004.08.003.
[48]
Shen, G.A.; Pang, Y.Z.; Wu, W.S.; Liu, X.F.; Zhao, L.X.; Sun, X.F.; Tang, K.X. Isolation and characterization of a putative anthocyanidin reductase gene from Ginkgo biloba. J. Plant Physiol. 2006, 163, 224–227, doi:10.1016/j.jplph.2005.06.012.
[49]
Terrier, N.; Torregrosa, L.; Ageorges, A.; Vialet, S.; Verries, C.; Cheynier, V.; Romieu, C. Ectopic expression of VvMybPA2 promotes proanthocyanidin biosynthesis in grapevine and suggests additional targets in the pathway. Plant Physiol. 2009, 149, 1028–1041.
[50]
Deluc, L.; Bogs, J.; Walker, A.R.; Ferrier, T.; Decendit, A.; Merillon, J.-M.; Robinson, S.P.; Barrieu, F. The transcription factor VvMYB5b contributes to the regulation of anthocyanin and proanthocyanidin biosynthesis in developing grape berries. Plant Physiol. 2008, 147, 2041–2053, doi:10.1104/pp.108.118919.
[51]
Walker, A.R.; Lee, E.; Bogs, J.; McDavid, D.A.J.; Thomas, M.R.; Robinson, S.P. White grapes arose through the mutation of two similar and adjacent regulatory genes. Plant J. 2007, 49, 772–785, doi:10.1111/j.1365-313X.2006.02997.x.
[52]
Azuma, A.; Kobayashi, S.; Goto-Yamamoto, N.; Shiraishi, M.; Mitani, N.; Yakushiji, H.; Koshita, Y. Color recovery in berries of grape (Vitis vinifera L.) 'Benitaka', a bud sport of 'Italia', is caused by a novel allele at the VvmybA1 locus. Plant Sci. 2009, 176, 470–478, doi:10.1016/j.plantsci.2008.12.015.
[53]
Dereeper, A.; Guignon, V.; Blanc, G.; Audic, S.; Buffet, S.; Chevenet, F.; Dufayard, J.-F.; Guindon, S.; Lefort, V.; Lescot, M.; et al. Phylogeny.fr: Robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 2008, 36 (Suppl 2), W465–W469, doi:10.1093/nar/gkn180.
