Acta Physiologiae Plantarum

, Volume 35, Issue 5, pp 1617–1624 | Cite as

Overexpression of Rosa roxburghii l-galactono-1,4-lactone dehydrogenase in tobacco plant enhances ascorbate accumulation and abiotic stress tolerance

  • Wei Liu
  • Hua-Ming An
  • Man Yang
Original Paper


l-Galactono-1, 4-lactone dehydrogenase (GalLDH; EC is the last key enzyme in the putative l-ascorbic acid (AsA) biosynthetic pathway of higher plants. To evaluate the effect of the gene on manipulating AsA accumulation, a cDNA encoding GalLDH (RrGalLDH, Acc. No. AY643403), isolated from Rosa roxburghii fruit known to be rich in AsA, was introduced into tobacco plants by Agrobacterium-mediated transformation under CaMV 35S constitutive promoter in the present study. Southern blotting revealed the stable integration of the transgene with single copy in four independent transgenic lines, among which, L3 and L4 showed the significantly enhanced RrGalLDH transcript levels, GalLDH activities and AsA accumulations as compared to untransformed (WT) plants. So, we developed the AsA-overproducing tobacco plant by overexpressing GalLDH. As exposed to salt stress (100 mM NaCl), these AsA-overproducing transgenic lines were found to grow better with increased shoot length and fresh weight than WT. Furthermore, L3, which demonstrated the highest AsA accumulation (2.1-fold higher than WT) and expression level of RrGalLDH, showed a higher resistance to oxidative stress caused by paraquat when compared to WT. These results further justify that the overexpression of GalLDH gene confers an elevated AsA accumulation and tolerance against environmental stresses.


l-Ascorbic acid l-Galactono-1 4-Lactone dehydrogenase Overexpression Rosa roxburghii Tratt. Abiotic stress Nicotiana tabacum L. 



The authors would like to thank Prof. Xiaopeng Wen for help in revising our English composition and Guizhou Key Laboratory of Agricultural Bioengineering for providing the tobacco material. This work was supported by the National Natural Science Foundation of China (31060257), the Excellent Youth Scientific and Technological Talent Cultivation Program (200704), and the special fund project for the outstanding talents in science and education of Guizhou Province, P. R. China (201012).

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Agius F, Gonzlezolez LR, Caballer JL (2003) Engineering increased vitamin C levels in plants by over- expression of a d-galacturonic acid reductase. Nat Biotechnol 21:177–181PubMedCrossRefGoogle Scholar
  2. Alhagdow M, Mounet F, Gilbert L, Nunes-Nesi A, Garcia V, Just D, Petit J, Beauvoit B, Fernie AR, Rothan C (2007) Silencing of the mitochondrial ascorbate synthesizing enzyme l-galactono-1,4-lactone dehydrogenase affects plant and fruit development in tomato. Plant Physiol 145:1408–1422PubMedCrossRefGoogle Scholar
  3. An HM, Chen LG, Fan WG (2004) cDNA fragment cloning of l-galactono-1, 4-lactone dehydrogenase and its expression in different organs of R. roxburghii Tratt. Agri Sci in China 11:807–811Google Scholar
  4. An HM, Fan WG, Chen LG, Asghar S, Liu QL (2007) Molecular characterisation and expression of l-galactono-1,4-lactone dehydrogenase and l-ascorbic acid accumulation during fruit development in Rosa roxburghii. J Hortic Sci Biotech 82:627–635Google Scholar
  5. Arrigoni O, de Tullio MC (2002) Ascorbic acid: much more than just an antioxidant. Biochem Biophys Acta 1569:1–9PubMedCrossRefGoogle Scholar
  6. Badawi GH, Kawano N, Yamauchi Y, Shimada E, Sasaki R, Kubo A, Tanaka K (2004) Over-expression of ascorbate peroxidase in tobacco chloroplasts enhances the tolerance to salt stress and water deficit. Physiol Plant 121:131–238CrossRefGoogle Scholar
  7. Bartoli CG, Pastori GM, Foyer CH (2000) Ascorbate biosynthesis in mitochondria is linked to the electron transport chain between complexes III and IV. Plant Physiol 123:335–343PubMedCrossRefGoogle Scholar
  8. Bauw GJC, Davey MW, Ostergaard J, Van Montagu MCE (2002) Production of ascorbic acid in plants. US Patent 6,469,149Google Scholar
  9. Bulley S, Rassam M, Hose D, Otto W, Schunemann N, Wright M, Macrae E, Gleave A, Laing W (2009) Gene expression studies in kiwifruit and gene over-expression in Arabidopsis indicates that GDP-L-galactose guanyl transferase is a major control point of vitamin C biosynthesis. J Exp Bot 60:765–778PubMedCrossRefGoogle Scholar
  10. Bulley S, Wright M, Rommens C, Yan Hua, HuaYan M, Wang KL, Andre C, Brewster D, Karunairetnam S, Allan AC, Laing WA (2011) Enhancing ascorbate in fruits and tubers through over-expression of the L-galactose pathway gene GDP-L-galactose phosphorylase. Plant Biotechnol J 10:390–397PubMedCrossRefGoogle Scholar
  11. Chang SJ, Jeff P, John C (1993) A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep 11:113–116CrossRefGoogle Scholar
  12. Chen Z, Young TE, Ling J, Chang SC, Gallie DR (2003) Increasing vitamin C content of plants through enhanced ascorbate recycling. Proc Natl Acad Sci USA 100:3525–3530PubMedCrossRefGoogle Scholar
  13. Citterio S, Sgorbati S, Scippa S, Sparvoli E (1994) Ascorbic acid effect on the onset of cell proliferation in pea root. Physiol Plant 92:601–607CrossRefGoogle Scholar
  14. Conklin PL (2001) Recent advance in the role and biosynthesis of ascorbic acid in plants. Plant Cell Environ 24:383–394CrossRefGoogle Scholar
  15. Conklin PL, Barth C (2004) Ascorbic acid, a familiar small molecule intertwined in the response of plants to ozone, pathogens, and the onset of senescence. Plant Cell Environ 27:959–970CrossRefGoogle Scholar
  16. Davey MW, Montagu VM, Inzh D, Sanmartin M, Kanellis A (2000) Plant l-ascorbic acid: chemistry, function, metabolism, bioavailability and effects of processing. Sci Food Agric 80:825–860CrossRefGoogle Scholar
  17. Eltayeb A, Kawano N, Badawi G, Kaminaka H, Sanekata T, Shibahara T, Inanaga S, Tanaka K (2007) Overexpression of monodehydroascorbate reductase in transgenic tobacco confers enhanced tolerance to ozone, salt and polyethylene glycol stresses. Planta 225:1255–1264PubMedCrossRefGoogle Scholar
  18. Fan WG, Xia GL, Luo YC (1997) Utilization of Rosa roxburghii resources and its development strategy in Guizhou province. Southwest China. J Agric Sci 10:109–115Google Scholar
  19. Gatzek S, Wheeler GL, Smirnoff N (2002) 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 Physiol 30:541–553Google Scholar
  20. He ZF, Niu AZ, Xiang XH, Wang SM (1984) A study on the nutrition and variation in the vitamin C content in the fruits of Rosa roxburghii Tratt. Acta Hortic Sinica 11:271–273Google Scholar
  21. Hemavathi, Upadhyaya CP, Ko EY, Nookaraju A, Kim HS, Heung J, Oh MO, Reddy AC, Chun SC, Kim DH, Park SW (2009) Over-expression of strawberry d-galacturonic acid reductase in potato leads to accumulation of vitamin C with enhanced abiotic stress tolerance. Plant Sci 177:659–667CrossRefGoogle Scholar
  22. Hemavathi, Upadhyaya CP, Akula N, Young KE, Chun SC, Kim DH, Park SW (2010) Enhanced ascorbic acid accumulation in transgenic potato confers tolerance to various abiotic stress. Biotechnol Lett 32:321–330PubMedCrossRefGoogle Scholar
  23. Imai T, Niwa M, Ban Y (2009) Importance of the l-galactonolactone pool for enhancing the ascorbate content revealed by l-galactonolactone dehydrogenase-overexpressing tobacco plants. Plant Cell Tiss Organ Cult 96:105–112CrossRefGoogle Scholar
  24. Jain AK, Nessler CL (2000) Metabolic engineering of an alternative pathway for ascorbic acid biosynthesis in plants. Mol Breed 6:73–78CrossRefGoogle Scholar
  25. Jones H, Gallois P, Marinho P (1995) Leaf disk transformation using Agrobacterium tumefaciens expression of heterologous genes in tobacco. In: Walker JM (ed) Plant gene transfer and expression protocols, vol 49. Methods in molecular biology, pp 39–48CrossRefGoogle Scholar
  26. Kwon SY, Ahn YO, Lee HS, Kwak SS (2001) Biochemical characterization of transgenic tobacco plants expressing a human dehydroascorbate reductase gene. J Biochem Mol Biol 34:316–321Google Scholar
  27. Li M, Liang D, Pu F, Ma F, Hou C, Lu T (2009) Ascorbate levels and the activity of key enzymes in ascorbate biosynthesis and recycling in the leaves of 22 Chinese persimmon cultivars. Sci Hortic 120:250–256CrossRefGoogle Scholar
  28. Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol 148:350–382CrossRefGoogle Scholar
  29. Loewus FA (1999) Biosynthesis and metabolism of ascorbic acid in plants and of analogs of ascorbic acid in fungi. Phytochemistry 52:193–210CrossRefGoogle Scholar
  30. Lorence A, Chevone BI, Mendes P, Nessler CL (2004) Myo-inositol oxygenase offers a possible entry point into plant ascorbate biosynthesis. Plant Physiol 134:1200–1205PubMedCrossRefGoogle Scholar
  31. Loscos J, Matamoros MA, Becana M (2008) Ascorbate and homoglutathione metabolism in common bean nodules under stress conditions and during natural senescence. Plant Physiol 146:1282–1292PubMedCrossRefGoogle Scholar
  32. Meyer P (1995) Understanding and controlling transgene expression. Trends Biotechnol 13:332–337CrossRefGoogle Scholar
  33. Millar AH, Mittova V, Kiddle G, Heazlewood JL, Bartoli CG, Theodoulou FL, Foyer CH (2003) Control of ascorbate synthesis by respiration and its implications for stress responses. Plant Physiol 133:443–447PubMedCrossRefGoogle Scholar
  34. Noctor G, Foyer CH (1998) Ascorbate and glutathione: keeping active oxygen under control. Ann Rev Plant Physiol Mol Biol 49:249–279CrossRefGoogle Scholar
  35. Ôba K, Ishikawa S, Nishikawa M, Mizuno H, Yamamoto T (1995) Purification and properties of l-galactono-1,4-lactone dehydrogenase, a key enzyme for ascorbic acid biosynthesis, from sweet potato root. J Biol Chem 117:120–124Google Scholar
  36. Padayatty SJ, Katz A, Wang Y, Eck P, Kwon O, Lee JH, Chen S, Corpe C, Dutta A, Dutta SK, Levine M (2003) Vitamin C as an antioxidant: a valuation of its role in disease prevention. J Am Coll Nutri 22:18–35Google Scholar
  37. Pastori GM, Kiddle G, Antoniw J, Bernard S, Veljovic-Jovanovic S, Verrier PJ, Noctor G, Foyer CH (2003) Leaf vitamin C contents modulate plant defense transcripts and regulate genes that control development through hormone signaling. Plant Cell 15:939–951PubMedCrossRefGoogle Scholar
  38. Pateraki I, Sanmartin M, Kalamak MS, Gerasopoulos D (2004) Moleclar characterization and expression studies during melon fruit development and ripening of l-galactono-1, 4-lactone dehydrogenase. J Exp Bot 55:1623–1633PubMedCrossRefGoogle Scholar
  39. Porebski SL, Bailey G, Baum BR (1997) Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Mol Biol Rep 15:8–15CrossRefGoogle Scholar
  40. Radzio JA, Lorence A, Chevone BI, Nessler CL (2003) Gulono-1,4-lactone oxidase expression rescues vitamin C-deficient Arabidopsis (vtc) mutants. Plant Mol Biol 53:837–844PubMedCrossRefGoogle Scholar
  41. Shi SG, Ma FM, Li YH, Feng FJ, Shang ZZ (2011) Overexpression of l-galactono-1, 4-lactone dehydrogenase (GLDH) in Lanzhou lily (Lilium davidii var. unicolor) via particle bombardment- mediated transformation. In Vitro Cell Dev Biol Plant 1:1–6Google Scholar
  42. Spiker S, Thompson WF (1996) Nuclear matric attachment regions and transgene expression in plants. Plant Physiol 110:15–21PubMedGoogle Scholar
  43. Tabata K, Ôba K, Suzuki K, Esaka M (2001) Generation and properties of ascorbic acid-deficient transgenic tobacco cells expressing antisense RNA for l-galactono-1,4-lactone dehydrogenase. Plant J 27:139–148PubMedCrossRefGoogle Scholar
  44. Takahama U, Oniki T (1992) Regulations of peroxidase-dependent oxidation of phenolics in the apoplast of spinach leaves by ascorbate. Plant Cell Physiol 33:379–387Google Scholar
  45. Tokunaga T, Miyaharal K, Tabata K, Esaka M (2005) Generation and properties of ascorbic acid-overproducing transgenic tobacco cells expressing sense RNA for l-galactono-1,4-lactone dehydrogenase. Planta 220:854–863PubMedCrossRefGoogle Scholar
  46. Ushimaru T, Nakagawa T, Fujioka Y, Daicho K, Naito M, Yamuchi Y, Nonaka H, Amako K, Yamawaki K, Murata N (2006) Transgenic Arabidopsis plants expressing the rice dehydroascorbate reductase gene are resistant to salt stress. J Plant Physiol 163:1179–1184PubMedCrossRefGoogle Scholar
  47. Wen XP, Pang XM, Deng XX (2004) Characterization of genetic relationships of Rosa roxburghii Tratt and its relatives using morphological traits, RAPD and AFLP markers. J Hortic Sci Biotechnol 79:189–196Google Scholar
  48. Wheeler GL, Jones MA, Smirnoff N (1998) The biosynthetic pathway of vitamin C in higher plants. Nature 393:65–369Google Scholar
  49. Wolucka BA, Van Montagu M (2003) 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 278:47483–47490PubMedCrossRefGoogle Scholar
  50. Wolucka BA, Van Montagu M (2007) The VTC2 cycle and the de novo biosynthesis pathways for vitamin C in plants: an opinion. Phytochemistry 68:2602–2613PubMedCrossRefGoogle Scholar
  51. Zhang C, Liu J, Zhang Y, Cai X, Gong P, Zhang J, Wang T, Li H, Ye Z (2011) Overexpression of SlGMEs leads to ascorbate accumulation with enhanced oxidative stress, cold and salt tolerance in tomato. Plant Cell Rep 30:389–398PubMedCrossRefGoogle Scholar

Copyright information

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2013

Authors and Affiliations

  1. 1.Guizhou Engineering Research Center for Fruit Crops, Agricultural CollegeGuizhou UniversityGuiyangPeople’s Republic of China

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