Biologia Plantarum

, Volume 56, Issue 3, pp 451–457

Engineering ascorbic acid biosynthetic pathway in Arabidopsis leaves by single and double gene transformation

  • Y. Zhou
  • Q. C. Tao
  • Z. N. Wang
  • R. Fan
  • Y. Li
  • X. F. Sun
  • K. X. Tang


Six genes, which encode enzymes involved in ascorbic acid (AsA) biosynthesis, including guanosine diphosphate (GDP)-mannose pyrophosphorylase (GMP), GDP-mannose-3′,5′-epimerase (GME), GDP-galactose guanylyltransferase (GGT), L-galactose-1-phosphate phosphatase (GPP), L-galactose dehydrogenase (GDH) and L-galactono-1,4-lactone dehydrogenase (GLDH) were transformed into Arabidopsis thaliana, to evaluate the contribution of each gene to AsA accumulation. Additionally, two combinations, GGT-GPP and GGT-GLDH, were co-transformed into Arabidopsis with a reliable double-gene transformation system. AsA content of GGT transgenic lines was 2.9-fold higher as compared to the control, and co-transformation led up to 4.1-fold AsA enhancement. These results provided further evidence that GGT is the key enzyme in plant AsA biosynthesis.

Additional key words

GDP-L-galactose guanyltransferase transgenic plants vitamin C 



L-galactose dehydrogenase


guanosine diphosphate


GDP-galactose guanylyltransferase


L-galactono-1,4-lactone dehydrogenase




GDP-mannose pyrophosphorylase


L-galactose-1-phosphate phosphatase


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  1. Agius, F., Gonzalez-Lamothe, R., Caballero, J.L., Munoz-Blanco, J., Botella, M.A., Valpuesta, V.: Engineering increased vitamin C levels in plants by overexpression of a D-galacturonic acid reductase. — Natur. Biotechnol. 21: 177–181, 2003.CrossRefGoogle Scholar
  2. Alhagdow, M., Mounet, F., Gilbert, L., Nunes-Nesi, A., Garcia, V., Just, D., Petit, J., Beauvoit, B., Fernie, A.R., Rothan, C., Baldet, P.: Silencing of the mitochondrial ascorbate synthesizing enzyme L-galactono-1,4-lactone dehydrogenase affects plant and fruit development in tomato. — Plant Physiol. 145: 1408–1422, 2007.PubMedCrossRefGoogle Scholar
  3. Arrigoni, O., De Tullio, M.: The role of ascorbic acid in cell metabolism: between gene-directed functions and unpredictable chemical reactions. — J. Plant Physiol. 157: 481–488, 2000.CrossRefGoogle Scholar
  4. Ausubel, F., Brent, R., Kingston, R., Moore, D., Seidman, J., Smith, J., Struhl, K.: Short Protocols in Molecular Biology. 3rd Ed. — John Wiley&Son, New York 1995.Google Scholar
  5. Badejo, A., Tanaka, N., Esaka, M.: Analysis of GDP-Dmannose pyrophosphorylase gene promoter from acerola (Malpighia glabra) and increase in ascorbate content of transgenic tobacco expressing the acerola gene. — Plant Cell Physiol. 49: 126–132, 2008.PubMedCrossRefGoogle Scholar
  6. Barth, C., De Tullio, M., Conklin, P.: The role of ascorbic acid in the control of flowering time and the onset of senescence. — J. exp. Bot. 57: 1657–1665, 2006.PubMedCrossRefGoogle Scholar
  7. Bartoli, C., Guiamet, J., Kiddle, G., Pastori, G.., Di Cagno, R., Theodoulou, F., Foyer, C.: Ascorbate content of wheat leaves is not determined by maximal L-galactono-1,4-lactone dehydrogenase (GalLDH) activity under drought stress. — Plant Cell Environ. 28: 1073–1081, 2005.CrossRefGoogle Scholar
  8. Bulley, S., Rassam, M., Hoser, D., Otto, W., Schunemann, N., Wright, M., MacRae, E., Gleave, A., Laing, W.: Gene expression studies in kiwifruit and gene over-expression in Arabidopsis indicates that GDP-L-galactose guanyltransferase is a major control point of vitamin C biosynthesis. — J. exp. Bot. 60: 765–778, 2009.PubMedCrossRefGoogle Scholar
  9. Conklin, P., Gatzek, S., Wheeler, G., Dowdle, J., Raymond, M., Rolinski, S., Isupov, M., Littlechild, J., Smirnoff, N.: Arabidopsis thaliana VTC4 encodes L-galactose-1-phosphatase, a plant ascorbic acid biosynthetic enzyme. — J. biol. Chem. 281: 15662–15670, 2006.PubMedCrossRefGoogle Scholar
  10. Conklin, P., Norris, S., Wheeler, G., Williams, E., Smirnoff, N., Last, R.: Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis. — Proc. nat. Acad. Sci. USA 96: 4198–4203, 1999.PubMedCrossRefGoogle Scholar
  11. Davey, M., Van Montagu, M., Inze, D., Sanmartin, M., Kanellis, A., Smirnoff, N., Benzie, I., Strain, J., Favell, D., Fletcher, J.: Plant L-ascorbic acid: chemistry, function, metabolism, bioavailability and effects of processing. — J. Sci. Food Agr. 80: 825–860, 2000.CrossRefGoogle Scholar
  12. De Tullio, M., Liso, R., Arrigoni, O.: Ascorbic acid oxidase: an enzyme in search of a role. — Biol. Plant. 48: 161–166, 2004.CrossRefGoogle Scholar
  13. Dowdle, J., Ishikawa, T., Gatzek, S., Rolinski, S., Smirnoff, N.: Two genes in Arabidopsis thaliana encoding GDP-Lgalactose phosphorylase are required for ascorbate biosynthesis and seedling viability. — Plant J. 52: 673–689, 2007.PubMedCrossRefGoogle Scholar
  14. Gatzek, S., Wheeler, G., Smirnoff, N.: Antisense suppression of l-galactose dehydrogenase in Arabidopsis thaliana provides evidence for its role in ascorbate synthesis and reveals light modulated l-galactose synthesis. — Plant J. 30: 541–553, 2002.PubMedCrossRefGoogle Scholar
  15. Gilbert, L., Alhagdow, M., Nunes-Nesi, A., Quemener, B., Guillon, F., Bouchet, B., Faurobert, M., Gouble, B., Page, D., Garcia, V., Petit, J., Stevens, R., Causse, M., Fernie, A., Lahaye, M., Rothan, C., Baldet, P.: GDP-D-mannose 3,5-epimerase (GME) plays a key role at the intersection of ascorbate and non-cellulosic cell-wall biosynthesis in tomato. — Plant J. 60: 499–508, 2009.PubMedCrossRefGoogle Scholar
  16. Imai, T., Karita, S., Shiratori, G., Hattori, M., Nunome, T., Oba, K., Hirai, M.: L-galactono-gamma-lactone dehydrogenase from sweet potato: purification and cDNA sequence analysis. — Plant Cell Physiol. 39: 1350–1358, 1998.PubMedGoogle Scholar
  17. Ishikawa, T., Dowdle, J., Smirnoff, N.: Progress in manipulating ascorbic acid biosynthesis and accumulation in plants (vol 126, pg 343, 2006). — Physiol. Plant. 129: 831–831, 2007.CrossRefGoogle Scholar
  18. Jain, A., Nessler, C.: Metabolic engineering of an alternative pathway for ascorbic acid biosynthesis in plants. — Mol. Breed. 6: 73–78, 2000.CrossRefGoogle Scholar
  19. Laing, W., Bulley, S., Wright, M., Cooney, J., Jensen, D., Barraclough, D., MacRae, E.: A highly specific Lgalactose-1-phosphate phosphatase on the path to ascorbate biosynthesis. — Proc. nat. Acad. Sci. USA 101: 16976–16981, 2004.PubMedCrossRefGoogle Scholar
  20. Laing, W., Wright, M., Cooney, J., Bulley, S.: The missing step of the L-galactose pathway of ascorbate biosynthesis in plants, an L-galactose guanyltransferase, increases leaf ascorbate content. — Proc. nat. Acad. Sci. USA 104: 9534–9539, 2007.PubMedCrossRefGoogle Scholar
  21. Li, Y., Zhou, Y., Wang, Z., Sun, X., Tang, K.: Engineering tocopherol biosynthetic pathway in Arabidopsis leaves and its effect on antioxidant metabolism. — Plant Sci. 178: 312–320. 2010.CrossRefGoogle Scholar
  22. Linster, C., Clarke, S.: L-Ascorbate biosynthesis in higher plants: the role of VTC2. — Trends Plant Sci. 13: 567–573, 2008.PubMedCrossRefGoogle Scholar
  23. Linster, C., Gomez, T., Christensen, K., Adler, L., Young, B., Brenner, C., Clarke, S.: Arabidopsis VTC2 encodes a GDPL-galactose phosphorylase, the last unknown enzyme in the Smirnoff-Wheeler pathway to ascorbic acid in plants. — J. biol. Chem. 282: 18879–18885, 2007.PubMedCrossRefGoogle Scholar
  24. Loewus, F.: Biosynthesis and metabolism of ascorbic acid in plants and of analogs of ascorbic acid in fungi. — Phytochemistry 52: 193–210, 1999.CrossRefGoogle Scholar
  25. Loewus, F., Loewus, M.: Biosynthesis and Metabolism of Ascorbic-Acid in Plants. — Crc Critical Rev. in Plant Sci. 5:101–119, 1987CrossRefGoogle Scholar
  26. Lorence, A., Chevone, B., Mendes, P., Nessler, C.: myo-inositol oxygenase offers a possible entry point into plant ascorbate biosynthesis. — Plant Physiol. 134: 1200–1205, 2004.PubMedCrossRefGoogle Scholar
  27. Muller-Moule, P., Golan, T., Niyogi, K.: Ascorbate-deficient mutants of Arabidopsis grow in high light despite chronic photooxidative stress. — Plant Physiol. 134: 1163–1172, 2004.PubMedCrossRefGoogle Scholar
  28. MichaŁowicz, J., Urbanek, H., Bukowska, B., Duda, W.: The effect of 2,4-dichlorophenol and pentachlorophenol on antioxidant system in the leaves of Phalaris arudinacea. — Biol. Plant. 54: 597–600, 2010.CrossRefGoogle Scholar
  29. Pastori, G., Kiddle, G., Antoniw, J., Bernard, S., Veljovic-Jovanovic, S., Verrier, P.J., Noctor, G., Foyer, C.H.: Leaf vitamin C contents modulate plant defense transcripts and regulate genes that control development through hormone signaling. — Cell 15: 939–951, 2003.Google Scholar
  30. Reuhs, B., Glenn, J., Stephens, S., Kim, J., Christie, D., Glushka, J., Zablackis, E., Albersheim, P., Darvill, A., O’Neill, M.: L-Galactose replaces l-fucose in the pectic polysaccharide rhamnogalacturonan II synthesized by the Lfucose-deficient mur1 Arabidopsis mutant. — Planta 219: 147–157, 2004.PubMedCrossRefGoogle Scholar
  31. Smirnoff, N.: Ascorbate biosynthesis and function in photoprotection. — Phil. Trans. roy. Soc London B Biol. Sci. 355: 1455–1464, 2000.CrossRefGoogle Scholar
  32. Smirnoff, N., Wheeler, G.: Ascorbic acid in plants: biosynthesis and function. — Crit. Rev. Biochem. mol. Biol. 35: 291–314, 2000.PubMedCrossRefGoogle Scholar
  33. Tokunaga, T., Miyahara, K., Tabata, K., Esaka, M.: Generation and properties of ascorbic acid-overproducing transgenic tobacco cells expressing sense RNA for l-galactono-1,4-lactone dehydrogenase. — Planta 220: 854–863, 2005.PubMedCrossRefGoogle Scholar
  34. Wang, Z., Xiao, Y., Chen, W., Tang, K., Zhang, L.: Increased vitamin C content accompanied by an enhanced recycling pathway confers oxidative stress tolerance in Arabidopsis. — J. Integr. Plant Biol. 52: 400–409, 2010.PubMedCrossRefGoogle Scholar
  35. Wheeler, G., Jones, M., Smirnoff, N.: The biosynthetic pathway of vitamin C in higher plants. — Nature 393: 365–369, 1998.PubMedCrossRefGoogle Scholar
  36. Wolucka, B., Persiau, G., Van Doorsselaere, J., Davey, M., Demol, H., Van de Kerckhove, J., Van Montagu, M., Zabeau, M., Boerjan, W.: Partial purification and identification of GDP-mannose 3″,5″-epimerase of Arabidopsis thaliana, a key enzyme of the plant vitamin C pathway. — Proc. nat. Acad. Sci. USA 98: 14843–14848, 2001.PubMedCrossRefGoogle Scholar
  37. Wolucka, B., Van Montagu, M.: GDP-mannose 3′,5′-epimerase forms GDP-L-gulose, a putative intermediate for the de novo biosynthesis of vitamin C in plants. — J. biol. Chem. 278: 47483–47490, 2003.PubMedCrossRefGoogle Scholar
  38. Wolucka, B., Van Montagu, M.: The VTC2 cycle and the de novo biosynthesis pathways for vitamin C in plants: An opinion. — Phytochemistry 68: 2602–2613, 2007.PubMedCrossRefGoogle Scholar
  39. Zhang, X., Henriques, R., Lin, S., Niu, Q., Chua, N.: Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. — Nat. Protoc. 1: 641–646, 2006.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Y. Zhou
    • 1
  • Q. C. Tao
    • 1
  • Z. N. Wang
    • 1
  • R. Fan
    • 1
  • Y. Li
    • 1
  • X. F. Sun
    • 1
  • K. X. Tang
    • 2
  1. 1.State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan-SJTU-Nottingham Plant Biotechnology R&D CenterFudan UniversityShanghaiP.R. China
  2. 2.Plant Biotechnology Research Center, School of Agriculture and Biology, Fudan-SJTU-Nottingham Plant Biotechnology R&D CenterShanghai Jiao Tong UniversityShanghaiP.R. China

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