Dihydroflavonol 4-reductase (DFR, EC 1.1.1.219) is a rate-limited enzyme in the biosynthesis of anthocyanins and condensed tannins (proanthocyanidins) that catalyzes the reduction of dihydroflavonols to leucoanthocyanins. In this study, two full-length transcripts encoding for PtrDFR1 and PtrDFR2 were isolated from Populus trichocarpa. Sequence alignment of the two PtrDFRs with other known DFRs reveals the homology of these genes. The expression profile of PtrDFRs was investigated in various tissues of P. trichocarpa. To determine their functions, two PtrDFRs were overexpressed in tobacco (Nicotiana tabacum) via Agrobacterium-mediated transformation. The associated color change in the flowers was observed in all 35S:PtrDFR1 lines, but not in 35S:PtrDFR2 lines. Compared to the wild-type control, a significantly higher accumulation of anthocyanins was detected in transgenic plants harboring the PtrDFR1. Furthermore, overexpressing PtrDFR1 in Chinese white poplar (P. tomentosa Carr.) resulted in a higher accumulation of both anthocyanins and condensed tannins, whereas constitutively expressing PtrDFR2 only improved condensed tannin accumulation, indicating the potential regulation of condensed tannins by PtrDFR2 in the biosynthetic pathway in poplars.
References
[1]
Winkel-Shirley B (2001) It takes a garden. How work on diverse plant species has contributed to an understanding of flavonoid metabolism. Plant Physiol 127: 1399–1404.
[2]
Martens S, Knott J, Seitz CA, Janvari L, Yu SN, et al. (2003) Impact of biochemical pre-studies on specific metabolic engineering strategies of flavonoid biosynthesis in plant tissues. Biochem Eng J 14: 227–235.
[3]
Manach C, Scalbert A, Morand C, Remesy C, Jimenez L (2004) Polyphenols: food sources and bioavailability. Am J Clin Nutr 79: 727–747.
[4]
Dixon RA, Steele CL (1999) Flavonoids and isoflavonoids: An old mine for metabolic engineering. Trends in Plant Sci 4: 394–400.
[5]
Dixon RA, Xie DY, Sharma SB (2005) Proanthocyanidins – a final frontier in flavonoid research. New Phytol 165: 9–28.
[6]
Kristiansen KN, Rohde W (1991) Structure of the Hordeum vulgare gene encoding dihydroflavonol-4-reductase and molecular analysis of ant18 mutants blocked in flavonoid synthesis. Mol Gen Genet 230: 49–59.
[7]
Peters D, Constabel CP (2002) Molecular analysis of herbivore-induced condensed tannin synthesis: Cloning, expression of dihydroflavonol reductase from trembling aspen (Populus tremuloides). Plant J 32: 701–712.
[8]
Xie DY, Jackson LA, Cooper JD, Ferreira D, Paiva NL (2004) Molecular and biochemical analysis of two cDNA clones encoding dihydroflavonol-4-reductase from Medicago truncatula. Plant Physiology 134(3): 979–994.
[9]
Olsen O, Wang X, von Wettstein D (1993) Sodium azide mutagenesis: preferential generation of A.T→G.C transitions in the barley Ant18 gene. Proc Natl Acad Sci USA 90: 8043–8047.
[10]
Shirley BW, Kubasek WL, Storz G, Bruggemann E, Koornneef M, et al. (1995) Analysis of Arabidopsis mutants deficient in flavonoid biosynthesis. Plant J 8: 659–671.
[11]
Beld M, Martin C, Huits H, Stuitje AR, Gerats AG (1989) Flavonoid synthesis in Petunia hybrida: Partial characterization of dihydroflavonol-4-reductase genes. Plant Mol Biol 13: 491–502.
[12]
Sparvoli F, Martin C, Scienza A, Gavazzi G, Tonelli C (1994) Cloning and molecular analysis of structural genes involved in flavonoid and stillbene biosynthesis in grape (Vitis vinifera L.). Plant Mol Biol 24: 743–755.
[13]
Liew CF, Loh CS, Gho CJ, Lim SH (1998) The isolation, molecular characterization and expression of dihydroflavonol 4-reductase cDNA in the orchid, Bromheadia finlaysoniana. Plant Sci 135: 161–169.
[14]
Fischer TC, Halbwirth H, Meisel B, Stich K, Forkmann G (2003) Molecular cloning substrate specificity of the functionally expressed dihydroflavonol 4-reductase from Malus domestica and Pyrus communis cultivars and the consequences for flavonoid metabolism. Arch Biochem Biophys 412: 223–230.
[15]
Lo Piero AR, Puglisi I, Petrone G (2006) Gene characterization, analysis of expression and in vitro synthesis of dihydroflavonol 4-reductase from Citrus sinensis (L.) Osbeck. Phytochemist 67: 684–695.
[16]
Devic M, Guilleminot J, Debeaujon I, Bechtold N, Bensaude E, et al. (1999) The BANYULS gene encodes a DFR-like protein and is a marker of early seed coat development. Plant J 19: 387–398.
[17]
Inagaki Y, Johzuka-Hisatomi Y, Mori T, Takahashi S, Hayakawa Y, et al. (1999) Genomic organization of the genes encoding dihydroflavonol 4-reductase for flower pigmentation in the Japanese and common morning glories. Gene 226: 181–188.
[18]
Ostergaard L, Lauvergeat VV, Naested H, Mattsson O, Mundy J (2001) Two differentially regulated Arabidopsis genes define a new branch of the DFR superfamily. Plant Sci 160: 463–472.
[19]
Taylor G (2002) Populus: Arabidopsis for forestry. Do we need a model tree? Ann Bot 90: 681–689.
[20]
Jansson S, Douglas CJ (2007) Populus: a model system for plant biology. Annu Rev Plant Biol 58: 435–458.
[21]
Sterky F, Bhalerao RR, Unneberg P, Segerman B, Nilsson P, et al. (2004) A Populus EST resource for plant functional genomics. PNAS 101(38): 13951–6.
[22]
Tuskan GA, DiFazio S, Jansson S, Bohlmann J, Grigoriev I, et al. (2006) The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 313: 1596–1604.
[23]
Tsai CJ, Harding SA, Tschaplinski TJ, Lindroth RL, Yuan Y (2006) Genome-wide analysis of the structural genes regulating defense phenylpropanoid metabolism in Populus. New Phytol 172: 47–62.
[24]
Gou J, Strauss SH, Tsai CJ, Fang K, Chen Y, et al. (2010) Gibberellins regulate lateral root formation in Populus through interactions with auxin and other hormones. Plant Cell 22: 623–63.
Jia Z, Gou J, Sun Y, Yuan L, Tang Q, et al. (2011) Enhanced resistance to fungal pathogens in transgenic Populus tomentosa Carr. by overexpression of an nsLTP-like antimicrobial protein gene from motherwort (Leonurus japonicus). Tree Physiol 30: 1599–1605.
[27]
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15: 473–497.
[28]
Horsch RB, Fry J, Hoffmann N, Neidermeyer J, Rogers SG, et al. (1988) Leaf disc transformation. In: Gelvin SB, Schilperoort R, editors. Plant Molecular Biology Manual. 1 p. Kluwer Academic Publishers, Dordrecht, The Netherlands.
[29]
Jia ZC, Sun YM, Yuan L, Tian Q, Luo KM (2010) The chitinase gene (Bbchit1) from Beauveria bassiana enhances resistance to Cytospora chrysosperma in Populus tomentosa Carr. Biotechnol Lett 32: 1325–1332.
[30]
Lloyd G, McCown B (1980) Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture. Combined Proc Int Plant Propagators Soc 30: 421–427.
[31]
Luo KM, Zheng XL, Chen YQ, Xiao YH, Zhao DG, et al. (2006) The maize Knotted1 gene as a positive selectable marker gene is effective for Agrobacterium-mediated transformation in tobacco. Plant Cell Rep 25: 403–409.
[32]
Gou J, Ma C, Kadmiel M, Gai Y, Strauss S, et al. (2011) Tissue-specific expression of Populus C19 GA 2-oxidases differentially regulate above- and below-ground biomass growth through control of bioactive GA concentrations. New Phytol. 5 Aug 2011, DOI: 10.1111/j.1469-8137.2011.03837.x.
[33]
Pang YZ, Peel GJ, Wright E, Wang ZY, Dixon RA (2007) Early steps in proanthocyanidin biosynthesis in the model legume Medicago truncatula. Plant Physiol 145: 601–615.
[34]
Swain T, Hillis WEJ (1959) Phenolic constituents of Prunus domestica I. Quantitative analysis of phenolic constituents. J Sci Food Agric 10: 63.
[35]
Price ML, Van Scoyoc S, Butler LG (1978) A critical evaluation of the vanillin reaction as an assay for tannin in sorghum grain. J Agri Food Chem 26: 1214–1218.
[36]
Waterman PG, Mole S (1994) Analysis of Phenolic Plant Metabolites in Methods in Ecology. Oxford (UK): Blackwell. 85 p.
[37]
Johnson E, Yi H, Shin B, Oh BJ, Cheong H, et al. (1999) Cymbidium hybrida dihydroflavonol 4-reductase does not efficiently reduce dihydrokaempferol to produce pelargonidin-type anthocyanins. Plant J 19: 81–85.
[38]
Lacombe E, Hawkins S, Doorsselaere JV, Piquemal J, Goffner D, et al. (1997) Cinnamoyl CoA reductase, the first committed enzyme of the lignin branch biosynthetic pathway: cloning, expression and phylogenetic relationships. Plant J 11: 429–441.
[39]
Aida R, Yoshida K, Kondo T, Kishimoto S, Shibata M (2000) Copigmentation gives bluer flowers on transgenic torenia plants with the antisense dihydroflavonol-4-reductase gene. Plant Sci 160: 49–56.
[40]
Polashock JJ, Griesbach RJ, Sullivan RF, Vorsa N (2002) Cloning of a cDNA encoding the cranberry dihydroflavonol-4-reductase (DFR) and expression in transgenic tobacco. Plant Sci 163: 241–251.
[41]
Davies KM, Schwinn KE, Deroles SC, Manson DG, Lewis DH, et al. (2003) Enhancing anthocyanin production by altering competition for substrate between flavonol synthase and dihydroflavonol 4-reductase. Euphytica 131: 259–268.
[42]
Bevan MW (1984) Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res 12: 8711–8721.
[43]
Driebe EM, Whitham TG (2000) Cottonwood hybridization affects tannin and nitrogen content of leaf litter and alters decomposition. Oecologia 123: 99–107.
[44]
Whitha TG, Young WP, Martinsen GD, Gehring CA, Schweitzer JA, et al. (2003) Community and ecosystem genetics: A consequence of the extended phenotype. Ecology 84: 559–573.
[45]
Lindroth RL, Hwang SY (1996) Diversity, redundancy and multiplicity in chemical defense systems of aspen. In: Romeo JT, Saunders JA, Barbosa P, editors. “Recent Advances in Phytochemistry.”. 25 p. Plenum Press, New York.
[46]
Schultz JC, Baldwin IT (1982) Oak leaf quality declines in response to defoliation by gypsy moth larvae. Science 217: 149–151.
[47]
Donaldson JR, Lindroth RL (2007) Genetics, environment, and their interaction determine efficacy of chemical defense in trembling aspen. Ecology 88: 729–739.
[48]
Bavage AD, Davies IG, Robbins MP, Morris P (1997) Expression of an Antirrhinum dihydroflavonol reductase gene results in changes in condensed tannin structure and accumulation in root cultures of Lotus corniculatus (birds foot trefoil). Plant Mol Biol 35: 443–458.
[49]
Robbins MP, Bavage AD, Strudwicke C, Morris P (1998) Genetic manipulation of condensed tannins in higher plants. II. Analysis of birdsfoot trefoil plants harboring antisense dihydroflavonol reductase constructs. Plant Physiol 116: 1133–1144.
[50]
Shimada N, Sasaki R, Sato S, Kaneko T, Tabata S, et al. (2005) A comprehensive analysis of six dihydroflavonol 4-reductases encoded by a gene cluster of the Lotus japonicus genome. J Exp Bot 56: 2573–2585.