Abstract
l-Galactono-1,4-lactone (GalL) dehydrogenase (GLDH) is an enzyme that catalyzes the last step of l-ascorbate (AsA) biosynthesis in plants. To re-evaluate the importance of the enzyme and the possibility of manipulating the AsA content in plants, a cDNA encoding GLDH from sweet potato was introduced into tobacco plants by Agrobacterium-mediated transformation under the control of a CaMV 35S promoter. Protein blot analysis revealed the elevation of GLDH protein contents in three GLDH-transformed lines. Furthermore, these transgenic lines showed 6- to 10-fold higher GLDH activities in the roots than the non-transformed plants, SR1. Despite the elevated GLDH activity, the AsA content in the leaves did not change in all lines; i.e., the AsA content in GLDH-transformed lines was 3–7 μmol g−1 FW, comparable to that in the non-transformed plants. Incubation of leaf discs in a GalL solution led to a rapid 2- to 3-fold increase in the AsA content in both GLDH-transformed and non-transformed plants in the same manner. These results suggest that the supply of GalL is a crucial factor for determining the AsA pool size and that the upstream genes in the AsA biosynthetic pathway are responsible for enhancing the AsA content in plants.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- AsA:
-
l-Ascorbate
- CaMV:
-
Cauliflower mosaic virus
- DHA:
-
Dehydroascorbate
- DTT:
-
Dithiothreitol
- Gal:
-
l-Galactose
- GDH:
-
Gal 1-dehydrogenase
- GalL:
-
l-Galactono-1,4-lactone
- GGHPGT:
-
GDP-l-galactose:hexose-1-phosphate guanylyltransferase
- GLDH:
-
GalL dehydrogenase
- GLO:
-
l-Gulono-1,4-lactone oxidase
- GME:
-
GDP-d-mannose-3′,5′-epimerase
- GMPH:
-
GDP-d-mannose pyrophosphorylase
- GPP:
-
Gal-1-phosphate phosphatase
- GulL:
-
l-Gulono-1,4-lactone
- MIO:
-
myo-Inositol oxygenase
References
Agius F, Gonzalez-Lamothe R, Caballero JL, Munoz-Blanco J, Botella MA, Valpuesta V (2003) Engineering increased vitamin C levels in plants by overexpression of a d-galacturonic acid reductase. Nat Biotechnol 21:177–181. doi:10.1038/nbt777
Alhagdow M, Mounet F, Gilbert L, Nunes-Nesi A, Garcia V, Just D, Petit J, Beauvoit B, Fernie AR, Rothan C, Baldet P (2007) Silencing of the mitochondrial ascorbate synthesizing enzyme l-galactono-1,4-lactone dehydrogenase affects plant and fruit development in tomato. Plant Physiol 145:1408–1422. doi:10.1104/pp.107.106500
Arrigoni O, de Tullio MC (2002) Ascorbic acid: much more than just an antioxidant. Biochim Biophys Acta 1569:1–9
Bartoli CG, Guiamet JJ, Kiddle G, Pastori G, Di Cagno R, Theodoulou FL, Foyer CH (2005) The relationship between l-galactono-1,4-lactone dehydrogenase (GalLDH) and ascorbate content in leaves under optimal and stress conditions. Plant Cell Environ 28:1073–1081. doi:10.1111/j.1365-3040.2005.01338.x
Bartoli CG, Yu J, Gomez F, Fernandez L, McIntosh L, Foyer CH (2006) Inter-relationships between light and respiration in the control of ascorbic acid synthesis and accumulation in Arabidopsis thaliana leaves. J Exp Bot 57:1621–1631. doi:10.1093/jxb/erl005
Bauw GJC, Davey MW, Ostergaard J, Van Montagu MCE (2002) Production of ascorbic acid in plants. US Patent 6,469,149
Conklin PL, Pallanca JE, Last RL, Smirnoff N (1997) l-Ascorbic acid metabolism in the ascorbate-deficient Arabidopsis mutant vtc1. Plant Physiol 115:1277–1285. doi:10.1104/pp.115.3.1277
Conklin PL, Saracco SA, Norris SR, Last RL (2000) Identification of ascorbic acid-deficient Arabidopsis thaliana mutants. Genetics 154:847–856
Davey MW, Gilot C, Persiau G, Østergaard J, Han Y, Bauw GC, Van Montagu MC (1999) Ascorbate biosynthesis in Arabidopsis cell suspension culture. Plant Physiol 121:535–543. doi:10.1104/pp.121.2.535
Davey MW, Franck C, Keulemans J (2004) Distribution, developmental and stress responses of antioxidant metabolism in Malus. Plant Cell Environ 27:1309–1320. doi:10.1111/j.1365-3040.2004.01238.x
Foyer CH, Rowell J, Walker D (1983) Measurement of the ascorbate content of spinach leaf protoplasts and chloroplasts during illumination. Planta 157:239–244. doi:10.1007/BF00405188
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 J 30:541–553. doi:10.1046/j.1365-313X.2002.01315.x
Imai T, Karita S, Shiratori G, Hattori M, Nunome T, Ôba K, Hirai M (1998) l-Galactono-γ-lactone dehydrogenase from sweet potato: purification and cDNA sequence analysis. Plant Cell Physiol 39:1350–1358
Isherwood FA, Mapson LW (1961) Biosynthesis of l-ascorbic acid in animals and plants. Ann N Y Acad Sci 92:6–20. doi:10.1111/j.1749-6632.1961.tb46103.x
Ishikawa T, Dowdle J, Smirnoff N (2006) Progress in manipulating ascorbic acid biosynthesis and accumulation in plants. Physiol Plant 126:343–355. doi:10.1111/j.1399-3054.2006.00640.x
Jackson GA, Wood RB, Prosser MV (1961) Conversion of l-galactono-γ-lactone into l-ascorbic acid by plants. Nature 191:282–283. doi:10.1038/191282a0
Jain AK, Nessler CL (2000) Metabolic engineering of an alternative pathway for ascorbic acid biosynthesis in plants. Mol Breed 6:73–78. doi:10.1023/A:1009680818138
Laing WA, Wright MA, Cooney J, Bulley SM (2007) The missing step of the l-galactose pathway of ascorbate biosynthesis in plants, an l-galactose guanyltransferase, increases leaf ascorbate content. Proc Natl Acad Sci USA 104:9534–9539. doi:10.1073/pnas.0701625104
Linster CL, Van Schaftingen E (2007) Vitamin C. Biosynthesis, recycling and degradation in mammals. FEBS J 274:1–22. doi:10.1111/j.1742-4658.2006.05607.x
Loewus FA, Jang R, Seegmiller CG (1956) The conversion of C14-labeled sugars to l-ascorbic acid in ripening strawberries. J Biol Chem 222:649–664
Lorence A, Chevone BI, Mendes P, Nessler CL (2004) myo-Inositol oxygenase offers a possible entry point into plant ascorbate biosynthesis. Plant Physiol 134:1200–1205. doi:10.1104/pp.103.033936
Mano J, Hideg E, Asada K (2004) Ascorbate in thylakoid lumen functions as an alternative electron donor to photosystem II and photosystem I. Arch Biochem Biophys 429:71–80. doi:10.1016/j.abb.2004.05.022
Mapson LW, Breslow E (1958) Biological synthesis of ascorbic acid: l-galactono-γ-lactone dehydrogenase. Biochem J 68:395–406
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15:473–497. doi:10.1111/j.1399-3054.1962.tb08052.x
Nishikimi M, Koshizaka T, Ozawa T, Yagi K (1988) Occurrence in humans and guinea pigs of the gene related to their missing enzyme l-gulono-γ-lactone oxidase. Arch Biochem Biophys 267:842–846. doi:10.1016/0003-9861(88)90093-8
Nishikimi M, Fukuyama R, Minoshima S, Shimizu N, Yagi K (1994) Cloning and chromosomal mapping of the human nonfunctional gene for l-gulono-γ-lactone oxidase, the enzyme for l-ascorbic acid biosynthesis missing in man. J Biol Chem 269:13685–13688
Nocter G, Foyer CH (1998) Ascorbate and glutathione: keeping active oxygen under control. Annu Rev Plant Physiol Plant Mol Biol 49:249–279. doi:10.1146/annurev.arplant.49.1.249
Ôba K, Ishikawa S, Nishikawa M, Mizuno H, Yamamoto T (1995) Purification and properties of l-galactono-γ-lactone dehydrogenase, a key enzyme for ascorbic acid biosynthesis, from sweet potato roots. J Biochem 117:120–124
Pallanca JE, Smirnoff N (1999) Ascorbic acid metabolism in pea seedlings. A comparison of d-glucosone, l-sorbosone, and l-galactono-1,4-lactone as ascorbate precursors. Plant Physiol 120:453–462. doi:10.1104/pp.120.2.453
Pallanca JE, Smirnoff N (2000) The control of ascorbic acid synthesis and turnover in pea seedlings. J Exp Bot 51:669–674. doi:10.1093/jexbot/51.345.669
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 15:939–951. doi:10.1105/tpc.010538
Pavet V, Olmos E, Kiddle G, Mowla S, Kumar S, Antoniw J, Alvarez ME, Foyer CH (2005) Ascorbic acid deficiency activates cell death and disease resistance responses in Arabidopsis. Plant Physiol 139:1291–1303. doi:10.1104/pp.105.067686
Radzio JA, Lorence A, Chevone BI, Nessler C (2003) l-Gulono-1,4-lactone oxidase expression rescues vitamin C-deficient Arabidopsis (vtc) mutants. Plant Mol Biol 53:837–844. doi:10.1023/B:PLAN.0000023671.99451.1d
Smirnoff N, Wheeler GL (2000) Ascorbic acid in plants: biosynthesis and function. Crit Rev Plant Sci 19:267–290. doi:10.1016/S0735-2689(00)80005-2
Smirnoff N, Conklin PL, Loewus FA (2001) Biosynthesis of ascorbic acid in plants: a renaissance. Annu Rev Plant Physiol Plant Mol Biol 52:437–467. doi:10.1146/annurev.arplant.52.1.437
Tamaoki M, Mukai F, Asai N, Nakajima N, Kubo A, Aono M, Saji H (2003) Light-controlled expression of a gene encoding l-galactono-γ-lactone dehydrogenase which affects ascorbate pool size in Arabidopsis thaliana. Plant Sci 164:1111–1117. doi:10.1016/S0168-9452(03)00122-5
Tokunaga T, Miyahara 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 220:854–863. doi:10.1007/s00425-004-1406-3
Wakimoto BT (1998) Beyond the nucleosome: epigenetic aspects of position-effect variegation in Drosophila. Cell 93:321–324. doi:10.1016/S0092-8674(00)81159-9
Wallrath IL (1998) Unfolding the mysteries of heterochromatin. Curr Opin Genet Dev 8:147–153. doi:10.1016/S0959-437X(98)80135-4
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 278:47483–47490. doi:10.1074/jbc.M309135200
Wolucka BA, Van Montagu M (2007) The VTC2 cycle and the de novo biosynthesis pathways for vitamin C in plants: an opinion. Phytochemistry 68:2602–2613. doi:10.1016/j.phytochem.2007.08.034
Acknowledgments
The authors wish to thank Prof. H. Mori, Nagoya University, for providing primer information for CaMV35S promoter and NOS terminator and Prof. M. Matsuoka, Nagoya University, for providing the anti-GLDH antibody. We also would like to thank Ms. H. Maeda for her technical assistance and Drs. S. Imanishi, M. Nagata, and T. Nunome of the National Institute of Vegetables, Ornamental Plants, and Tea for their critical discussion. This work was financially supported by the Biotechnology Plant Breeding Project of the Ministry of Agriculture, Forestry, and Fisheries, Japan.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Imai, T., Niwa, M., Ban, Y. et al. 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, 105–112 (2009). https://doi.org/10.1007/s11240-008-9466-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11240-008-9466-x