Molecular Cloning and Function Analysis of an Anthocyanidin Synthase Gene from Ginkgo biloba, and Its Expression in Abiotic Stress Responses

  • Xu, Feng (College of Horticulture and Gardening, Yangtze University) ;
  • Cheng, Hua (College of Horticulture and Gardening, Yangtze University) ;
  • Cai, Rong (College of Horticulture and Gardening, Yangtze University) ;
  • Li, Lin Ling (College of Life Science and Engineering, Huanggang Normal University) ;
  • Chang, Jie (College of Horticulture and Gardening, Yangtze University) ;
  • Zhu, Jun (College of Horticulture and Gardening, Yangtze University) ;
  • Zhang, Feng Xia (College of Horticulture and Gardening, Yangtze University) ;
  • Chen, Liu Ji (College of Horticulture and Gardening, Yangtze University) ;
  • Wang, Yan (College of Horticulture and Gardening, Yangtze University) ;
  • Cheng, Shu Han (College of Horticulture Science and Engineering, Shandong Agricultural University) ;
  • Cheng, Shui Yuan (College of Life Science and Engineering, Huanggang Normal University)
  • Received : 2008.04.28
  • Accepted : 2008.09.09
  • Published : 2008.12.31


Anthocyanidin synthase (ANS, leucoanthocyanidin oxygenase), a 2-oxoglutarate iron-dependent oxygenase, catalyzed the penultimate step in the biosynthesis of the anthocyanin class of flavonoids, from the colorless leucoanthocyanidins to the colored anthocyanidins. The full-length cDNA and genomic DNA sequences of ANS gene (designated as GbANS) were isolated from Ginkgo biloba for the first time. The full-length cDNA of GbANS contained a 1062-bp open reading frame (ORF) encoding a 354-amino-acid protein. The genomic DNA analysis showed that GbANS gene had three exons and two introns. The deduced GbANS protein showed high identities to other plant ANSs. The conserved amino acids (H-X-D) ligating ferrous iron and residues (R-X-S) participating in 2-oxoglutarate binding were found in GbANS at the similar positions like other ANSs. Southern blot analysis indicated that GbANS belonged to a multi-gene family. The expression analysis by real-time PCR showed that GbANS expressed in a tissue-specific manner in G. biloba. GbANS was also found to be up-regulated by all of the six tested abiotic stresses, UV-B, abscisic acid, sucrose, salicylic acid, cold and ethylene, consistent with the promoter region analysis of GbANS. The recombinant protein was successfully expressed in E. coli strain with pET-28a vector. The in vitro enzyme activity assay by HPLC indicated that recombinant GbANS protein could catalyze the formation the cyanidin from leucocyanidin and conversion of dihydroquercetin to quercetin, suggesting GbANS is a bifunctional enzyme within the anthocyanidin and flavonol biosynthetic pathway.


abiotic stresses;anthocyanidin synthase;enzyme activity;GbANS;gene expression;Ginkgo biloba;in vitro


Supported by : Natural Science Foundation of Hubei Province, Youth Talent Foundation of Hubei Province


  1. Almeida, J.R., D'Amico, E., Preuss, A, Carbone, F., de Vos, C.H., Deiml, B., Mourgues, F., Perrotta, G., Fischer, T.C., Bovy, AG., et al. (2007). Characterization of major enzymes and genes involved in flavonoid and proanthocyanidin biosynthesis during fruit development in strawberry (Fragaria $\times$ananassa). Arch. Biochem. Biophys. 465, 61-71
  2. Cheng, L., Xu, Y., Grotewold, E., Jin, Z., Wu, F., Fu, C., and Zhao, D. (2007). Characterization of anthocyanidin synthase (ANS) gene and anthocyanidin in rare medicinal plant-Saussurea me dusa. Plant Cell Tiss. Organ. Cult. 89, 63-73
  3. Christie, P.J., Alfenito, M.R., and Walbot, V. (1994). Impact of lowtemperature stress on general phenylpropanoid and anthocyanin pathways: Enhancement of transcript abundance and anthocyanin pigmentation in maize seedlings. Planta 194,541-549
  4. Cui, Y., Magill, R., Frederiksen, R., and Magill, C. (1996). Chalcone synthase and phenylalanine ammonia-lyase mRNA levels following exposure of sorghum seedlings to three fungal pathogens. Physiol. Mol. Plant Pahol. 48, 187-199
  5. Fofana, B., Mcnally, D.J., Labbe, C., Boulanger, R., Benhamou, N., Seguin, A, and Belanger, R.R. (2002). Milsana-induced resistance in powdery mildew-infected cucumber plants correlates with the induction of chalcone synthase and chalcone isomerase. Physiol. Mol. Plant Pahol. 61, 121-132
  6. Holton, TA, and Cornish, E.C. (1995). Genetic and biochemistry of anthocyanin biosynthesis. Plant Cell 7, 1071-1083
  7. Jain, M., Nijhawan, A, Tyagi, AK., and Khurana, J.P. (2006). Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem. Biophys. Res. Commun. 345, 646-651
  8. Kim, S., Bang, H., Yoo, K., and Pike, L.M. (2006). Identification of the fourth allele of the ANS (anthocyanidin synthase) gene and its effect on red color intensity in onions (Allium cepa). Euphytica 149,45-51
  9. Kush, A, Goyvaerts, E., Chye, M.L., and Chua, N.H. (1990). Laticifer-specific gene expression in Hevea brasiliensis (rubber tree). Proc. Natl. Acad. Sci. USA 87,1787-1790
  10. Lesnick, M.L., and Chandler, V.L. (1998). Activation of the maize anthocyanin gene a2 is mediated by an element conserved in many anthocyanin promoters. Plant Physiol. 117, 437-445
  11. Nakatsuka, T., Nishihara, M., Mishiba, K., and Yamamura, S. (2005). Two different mutations are involved in the formation of white-flowered gentian plants. Plant Sci. 169,949-958
  12. Parejo, I., Viladomat, F., Bastida, J., Schmeda-Hirschmann, G., Burillo, J., and Codina, C. (2004). Bioguided isolation and identification of the nonvolatile antioxidant compounds from fennel (Foeniculum vulgare MilL) waste. J. Agric. Food Chem. 52, 1890-1897
  13. Pelletier, MK, Murrell, J.R., and Shirley, BW. (1997). Charcterizaion of flavonol synthase and leucoanthocyanidin dioxygenase genes in Arabidopsis. Plant Physiol. 113,1437-1445
  14. Roach, PL, Clifton, I.J., Fulop, V., Harlos, K., Barton, G.J., Hajdu, J., Andersson, I., Schofield, C.J., and Baldwin, J.E. (1995). Crystal structure of isopenicillin N synthase is the first from a new structural family of enzymes. Nature 375, 700-704
  15. Rosati, C., Cadle, A, Duron, M., Ingouff, M., and Simoneau, P. (1999). Molecular characterization of the anthocyanidin synthase gene in Forsyhia x intermedia reveals organ-specific expression during flower development. Plant Sci. 149,73-79
  16. Sierpina, V.S., Wollschlaeger, B., and Blumentha, M. (2003). Ginkgo bilobe. Am. Family Physician 68, 923-927
  17. Skriver, K., and Mundy, J. (1990). Gene expression in response to abscisic acid and osmotic stress. Plant Cell 2, 503-512
  18. Winkel-Shirley, B. (2001). Flavonoids biosynthesis: a colorful rnodel for genetics, biochernistry, cell biology, and biotechnology. Plant Physiol. 126, 485-493
  19. Xu, F., Cheng, SY., Cheng, S.H., Wang, Y., and Du, HW. (2007). Time course of expression of chalcone synthase gene in Ginkgo biloba. J. Plant Physiol. Mol. BioI. 33,309-317
  20. Xu, F., Cai, R., Cheng, S., Du, H., Wang, Y., and Cheng, S. (2008a). Molecular cloning, characterization and expression of phenylalanine ammonia-lyase gene from Ginkgo biloba. Afr J. Biothenol. 7,721-729
  21. Akihisa, T., Tokuda, H., Ukiya, M., lizuka, M., Schneider, S., Ogasawara, K., Mukainaka, T., Iwatsuki, K., Suzuki, T., and Nishino, H. (2003). Chalcones, coumarins, and flavanones from the exudate of Angelica keiskei and their chemopreventive effects. Cancer Lett. 201, 133-137
  22. Kobayashi, S., Ishimaru, M., Hiraoka, K., and Honda, C. (2002). Myb-related genes of the Kyoho grape (Vitis labruscana) regulate anthocyanin biosynthesis. Planta 215,924-933
  23. Reddy, AM., Reddy, V.S., Scheffler, BE, Wienand, U., and Reddy, AR. (2007). Novel trangenic rice overexpression anthocyanidin synthase accumulates a mixture of flavonoids leading to an increased antioxidant potential. Metab. Eng. 9,95-111
  24. Cheng, S., Wang, Y., Li J., Gu, M., and Shu, H. (2002). Study on the synthesis and metabolism of the flavonoids in Ginkgo biloba leaf. Sci entia Silvae Sinicae 38, 60-63 (in Chinese with an English Abstract)
  25. Xu, F., Cai, R., Cheng, S., Chen, L., and Cheng, S. (2008b). Molecular cloning, characterization and expression of atpA and atpB genes from Ginkgo bilobe. Biologia 63, 526-534
  26. Boss, PK, Davies, C., and Robinson, S.P. (1996). Expression of anthocyanin biosynthesis pathway genes in red and white grapes. Plant Mol. BioI. 32, 565-569
  27. Grotewold, E. (2006). The genetics and biochemistry of floral pigments. Annu. Rev. Plant BioI. 57, 761-780
  28. Saitou, N., and Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. BioI. Evol. 4, 406-425
  29. Wilrnouth, R.C., Turnbull, J.J., Welford, RW.o., Clifton, I.J., Prescott, AG., and Schofield, C.J. (2002). Structure and rnechanisrn of anthocyanidin synthase frorn Arabidopsis thaliana. Structure 10, 93-103
  30. Prescott, AG., and Lloyd, M.D. (2000). The iron (II) and 2-oxoaciddependent dioxygenases and their role in metabolism. Nat. Prod. Rep. 17, 367-383
  31. Jansson, S., Meyer-Gauen, G., Cerff, R., and Martin, W. (1994). Nucleotide distribution in gymnosperm nuclear sequence suggests a model for GC-content change in land-plant nuclear genomes. J. Mol. Evol. 39, 34-46
  32. Pang, Y., Peel, G.J., Wright, E., Wang, Z., and Dixon, RA (2007). Early steps in proanthocyanidin biosynthesis in the model legume Medicago truncatula. Plant Physiol. 145,601-615
  33. Dixon, RA, Xie, D.Y., and Sharma, S.B. (2005). Proanthocyanidins--a final frontier in flavonoid research? New Phytol. 165, 9-28
  34. Hara, M., Oki, K., Hoshino, K., and Kuboi, T. (2003). Enhancement of anthocyanin biosynthesis by sugar in radish (Raphanus setivus) hypocotyl. Plant Sci. 164,259-265
  35. Zhang, Z., Barlow, J.N., Baldwin, J.E., and Schofiel, C.J. (1997). Metal-catalyzed oxidation and mutagenesis studies on the iron (II) binding site of 1-aminocyclopropane-1-carboxylate oxidase. Biochemistry 36, 15999-16007
  36. Liao, Z.H., Chen, M., Guo, L., Gong, Y., Tan, F., Sun, X., and Tang, K. (2004). Rapid isolation of high-quality total RNA from Taxus and Ginkgo. Prep. Biochem. Biotechnol. 34, 209-214
  37. Tapia, G., Verdugo, I., Yanez, M., Ahumada, I., Theoduloz, C., Cordero, C., Poblete F., Gonzalez, E., and Ruiz-Lara, S. (2005). Involvement of ethylene in stress-Induced expression of the TLC1.1 retrotransposon from Lycopersicon chilense Dun. Plant Physiol. 138,2075-2086
  38. Pang, Y., Shen, G., Wu, W., Liu, X., Lin, J., Tan, F., Sun, X., and Tang, K. (2005). Characterization and expression of chalcone synthase gene from Ginkgo biloba. Plant Sci. 168,1525-1531
  39. Redman, J., Whitcraft, J., Johnson, C., and Arias, J. (2002). Abiotic and biotic stress differentially stimulate as-1 element activity in Arabidopsis. Plant Cell Rep. 21,180-185
  40. Shen, G., Pang, Y., Wu, W., Liu, X., Zhao, L., Sun, X., and Tang, K. (2006b). Isolation and characterization of a putative anthocyanindin reductase gene from Ginkgo biloba. J. Plant Physiol. 163,224-227
  41. Turnbull, J.J., Nakajirna, J., Welford, RW., Yarnazaki, M., Saito, K., and Schofield, C.J. (2004). Mechanistic studies on three 2oxoglutarate-dependent oxygenases of flavonoid biosynthesis: anthocyanidin synthase, flavonol synthase, and flavanone 3beta-hydroxylase. J. BioI. Chern. 279,1206-1216
  42. Xie, D.Y., Sharma, S.B., Paiva, N.L., Ferreira, D., and Dixon, RA (2003). Role of anthocyanidin reductase, encoded by BANYULS in plant flavonoid biosynthesis. Science 299, 396-399
  43. Shen, G., Pang, Y., Wu, W., Deng, Z., Zhao, L., Cao, Y., Sun, X., and Tang, K. (2006a). Cloning and characterization of a flavanone 3-hydroxylase gene from Ginkgo biloba. Biosci. Rep. 26, 19-29
  44. Thompson, J.D., Higgins, D.G., and Gibson, 1.J. (1994). Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673-4680
  45. Lukacin, R., and Britsch, L. (1997). Identification of strictly conserved histidine and arginine residues as part of the active site in Petunia hybrida flavanone 3b-hydroxylase. Eur. J. Biochem. 249,748-757
  46. van der Meer, I.M., Stuitjie, AR., and Mol, N.J.M. (1993). Regulation of general phenylpropanoid and flavonoid gene expression. In Control of Plant Gene Expression, D.P.S. Verrna, ed. (Boca Raton, FL, USA: CRC Press), pp. 125-155
  47. Xie, DY., and Dixon, RA (2005). Proanthocyanidin biosynthesisstill more questions than answers? Phytochemistry 6, 62127-62144
  48. Mol, J., Grotewold, E., and Koes, R. (1998). How genes paint flowers and seeds. Trends Plant Sci. 3, 212-217
  49. Prescott, AG., Stamford, N.P., Wheeler, G., and Firmin, JL (2002). In vitro properties of a recombinant flavonol synthase from Arabidopsis thaliana. Phytochemistry 60, 589-593
  50. Ardi, R., Kobiler, I., Jacoby, B., Keen, NT., and Prusky, D. (1998). Involvement of epicatechin biosynthesis in the activation of the mechanism of resistance of avocado fruits to Colletotrichum gloeosporioides. Physiol. Mol. Plant Pathol. 53, 269-285
  51. Mori, K., Sugaya, S., and Gemma, H. (2005). Decreased anthocyanin bisynthesis in grape berries grown under elevated night tempera-ture condition. Sci. Hort. 105,319-330
  52. EI-Kereamya, A, Chervin, C., Roustan, J., Cheynier, V., Michel, Souquet, J., Moutounet, M., Raynal, J., Ford, C., Latche, A, Pech, J., et al. (2003). Exogenous ethylene stimulates the long-term expression of genes related to anthocyanin biosynthesis in grape berries. Physiol. Plant 119, 175-182
  53. Reyes, J.C., Muro-Pastor, M.I., and Florencio, F. (2004). The GATA family of transcription factors in Arabidopsis and rice. J. Plant Physiol. 134, 1718-1732
  54. Finkelstein, R.R., and Lynch, T.J. (2000). The Arabidopsis abscisic acid response gene ABI5 encodes a basic leucine zipper transcription factor. Plant Cell 12, 599-609
  55. Gaffney, T., Friedrich, L., Vernooij, B., Negrotto, D., Nye, D., Uknes, S., et al. (1993). Requirement of salicylic acid for the induction of systemic acquired resistance. Science 261,754-756
  56. Pool-Zobel, BL, Bub, A, Schroder, N., and Rechkemmer, G. (1999). Anthocyanins are potent antioxidants in model systems but do not reduce endogenous oxidative DNA damage in human colon cells. Eur. J. Nutr. 38, 227-234
  57. Turnbull, J.J., Sobey, w'J., Aplin, R.T., Hassan, A, Firnin, J.L., Sscofield, C.J., and Prescott, AG. (2000). Are anthocyanidins the irnrnediate products of anthocyanidin synthase? Chern. Cornrnun.2473-2474
  58. Arukwe, A (2006). Toxicological housekeeping genes: do they really keep the house? Environ. Sci. Technol. 40,7944-7949
  59. Prescott, AG., and John, P. (1996). Dioxygenases: molecular structure and role in plant metabolism. Annu. Rev. Plant Physiol. Plant Mol. BioI. 47, 245-271
  60. van Beek, TA (2002). Chernical analysis of Ginkgo biloba leaves and extracts. J. Chrornatogr. A 967, 21-55
  61. Abe, H., Urao, 1., Ito, 1., Seki, M., Shinozaki, K., and YamaguchiShinozaki, K. (2003). Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid sig naling. Plant Cell 15,63-78
  62. Buchel, AS., Brederode, F.T., Bol, J.F., and Linthorst, H.J.M. (1999). Mutation of GT-1 binding sites in the Pr-1A promoter influences the level of inducible gene expression in vivo. Plant Mol. BioI. 40, 387-396
  63. Cheng, S., Wang, Y., Li, J., Gu, M., and Shu, H. (2001). The relationships between flavonoids and other related compositions in Ginkgo biloba leaf during its anabolism. J. Huazhong Agric. Univ. 20,474-477
  64. Harborne, J.B., and Williams, CA (2001). Anthocyanins and other flavonoids. Nat. Prod. Rep. 18,310-333
  65. Hirner, AA, Veit, S., and Seitz, H.U. (2001). Regulation of anthocyanin biosynthesis in UV-A-irradiated cell cultures of carrot and in organs of intact carrot plants. Plant Sci. 161,315-332
  66. Kim, S., Binzel, M.L., Yoo, K.S., Park, S., and Pike, L.M. (2004). Pink (P), a new locus responsible for a pink trait in onions (Allium cepa) resulting from natural mutations of anthocyanidin synthase. Mol. Genet. Genomics 272, 18-27
  67. Gollop, R., Farhi, S., and Perl, A (2001). Regulation of the leucoanthocyanidin dioxygenase gene expression in vitis vtnitere: Plant Sci. 161,579-588
  68. Tsukaya, H., Ohshima, 1., Naito, S., Chino, M., and Komeda, Y. (1991). Sugar-dependent expression of the CHS-A gene for chalcone synthase from Petunia in transgenic Arabidopsis. Plant Physiol. 97, 1414-1421