Skip to main content
Log in

Elevating vitamin C content via overexpression of myo-inositol oxygenase and l-gulono-1,4-lactone oxidase in Arabidopsis leads to enhanced biomass and tolerance to abiotic stresses

  • Biotechnology/Genetic Transformation/Functional Genomics
  • Published:
In Vitro Cellular & Developmental Biology - Plant Aims and scope Submit manuscript

Abstract

l-ascorbic acid (vitamin C) is an abundant metabolite in plant cells and tissues. Ascorbate functions as an antioxidant, as an enzyme cofactor, and plays essential roles in multiple physiological processes including photosynthesis, photoprotection, control of cell cycle and cell elongation, and modulation of flowering time, gene regulation, and senescence. The importance of this key molecule in regulating whole plant morphology, cell structure, and plant development has been clearly established via characterization of low vitamin C mutants of Arabidopsis, potato, tobacco, tomato, and rice. However, the consequences of elevating ascorbate content in plant growth and development are poorly understood. Here, we demonstrate that Arabidopsis lines overexpressing a myo-inositol oxygenase or an l-gulono-1,4-lactone oxidase, containing elevated ascorbate, display enhanced growth and biomass accumulation of both aerial and root tissues. To our knowledge, this is the first study demonstrating such a marked positive effect in plant growth in lines engineered to contain elevated vitamin C content. In addition, we present evidence showing that these lines are tolerant to a wide range of abiotic stresses including salt, cold, and heat. Total ascorbate content of the transgenic lines remained higher than those of controls under the abiotic stresses tested. Interestingly, exposure to pyrene, a polycyclic aromatic hydrocarbon and known inducer of oxidative stress in plants, leads to stunted growth of the aerial tissue, reduction in the number of root hairs, and inhibition of leaf expansion in wild type plants, while these symptoms are less severe in the overexpressers. Our results indicate the potential of this metabolic engineering strategy to develop crops with enhanced biomass, abiotic stress tolerance, and phytoremediation capabilities.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.
Figure 7.

Similar content being viewed by others

References

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Alford SR, Rangarajan P, Williams P, Gillaspy GE (2012) myo-inositol oxygenase is required for responses to low energy conditions in Arabidopsis thaliana. Front Plant Sci 3:1–11

    Article  Google Scholar 

  • Alkio M, Tabuchi TM, Wang X, Colon-Carmona A (2005) Stress responses to polycyclic aromatic hydrocarbons in Arabidopsis include growth inhibition and hypersensitive response-like symptoms. J Exp Bot 56:2983–2994

    Article  PubMed  CAS  Google Scholar 

  • Athar HR, Khan A, Ashraf M (2008) Exogenously applied ascorbic acid alleviates salt-induced oxidative stress in wheat. Environ Exp Bot 63:224–231

    Article  CAS  Google Scholar 

  • Barth C, De Tullio M, Conklin PL (2006) The role of ascorbic acid in the control of flowering time and the onset of senescence. J Exp Bot 57:1657–1665

    Article  PubMed  CAS  Google Scholar 

  • Bartoli CG, Yu J, Gomez F, Fernandez I, McIntosh L, Foyer CH (2006) Inter-relationships between light and respiration in the control of ascorbic acid biosynthesis and accumulation in Arabidopsis thaliana. J Exp Bot 57:1621–1631

    Article  PubMed  CAS  Google Scholar 

  • Boyes DC, Zayed AM, Ascenzi R, McCaskill AJ, Hoffman NE, Davis KR, Görlach J (2001) Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput functional genomics in plants. Plant Cell 13:1499–1510

    PubMed  CAS  Google Scholar 

  • Bulley S, Wright M, Rommens C, Yan H, Rassam M et al. (2012) Enhancing ascorbate in fruits and tubers through over-expression of the L-galactose pathway gene GDP-L-galactose phosphorylase. Plant Biotechnol J 10:390–397

    Article  PubMed  CAS  Google Scholar 

  • Chen Z, Gallie DR (2005) Increasing tolerance to ozone by elevating foliar ascorbic acid confers greater protection against ozone than increasing avoidance. Plant Physiol 138:1673–1689

    Article  PubMed  CAS  Google Scholar 

  • Chen Z, Gallie DR (2006) Dehydroascorbate reductase affects leaf growth, development, and function. Plant Physiol 142:775–787

    Article  PubMed  CAS  Google Scholar 

  • Chen Z, Gallie DR (2008) Dehydroascorbate reductase affects non-photochemical quenching and photosynthetic performance. J Biol Chem 283: 21347–21361

    Article  PubMed  CAS  Google Scholar 

  • 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 27:959–970

    Article  CAS  Google Scholar 

  • Conklin PL, Williams EH, Last RL (1996) Environmental stress sensitivity of an ascorbic acid-deficient Arabidopsis mutant. Proc Natl Acad Sci USA 93:9970–9974

    Article  PubMed  CAS  Google Scholar 

  • Debolt S, Melino V, Ford CM (2007) Ascorbate as a biosynthetic precursor in plants. Ann Bot 99:3–8

    Article  PubMed  CAS  Google Scholar 

  • De Tullio MC (2012) Beyond the antioxidant: the double life of vitamin C. Subcell Biochem 56:49–65

    PubMed  Google Scholar 

  • De Tullio MC, Arrigoni O (2004) Hopes, disillusions, and more hopes from vitamin C. Cell Molec Life Scien 61:209–219

    Article  Google Scholar 

  • Dolatabadian A, Modarres Sanavy SAM, Sharifi M (2009) Alleviation of water deficit stress effects by foliar application of ascorbic acid on Zea mays L. J Agron 195:347–355

    CAS  Google Scholar 

  • Eltayeb AE, Kawano N, Badawi GH, Kaminaka H, Sanekata T et al. (2006) Enhanced tolerance to ozone and drought stresses in transgenic tobacco over-expressing dehydroascorbate reductase in cytosol. Physiol Plant 127:57–65

    Article  CAS  Google Scholar 

  • Eltayeb AE, Kawano N, Badawi GH, Kaminaka H, Sanekata T et al. (2007) Overexpression of monodehydroascorbate reductase in transgenic tobacco confers enhanced tolerance to ozone, salt, and polyethylene glycol stresses. Planta 225:1255–1264

    Article  PubMed  CAS  Google Scholar 

  • Eltayeb AE, Yamamoto S, Eltayeb ME, Yin L, Tsujimoto H, Tanaka K (2011) Transgenic potato overexpressing Arabidopsis cytosolic AtDHAR1 showed higher tolerance to herbicide, drought, and salt stresses. Breeding Sci 61:3–10

    Article  CAS  Google Scholar 

  • Endres S, Tenhaken R (2009) Myo-inositol oxygenase controls the level of myo-inositol in Arabidopsis, but does not increase ascorbic acid. Plant Physiol 149:1042–1049

    Article  PubMed  CAS  Google Scholar 

  • Endres S, Tenhaken R (2011) Downregulation of the myo-inositol oxygenase gene family has no effect on cell wall composition in Arabidopsis. Planta 234:157–169

    Article  PubMed  CAS  Google Scholar 

  • Foyer CH, Pellny TK, Locato V, De Gara L (2009) Analysis of redox relationships in the plant cell cycle: determinations of ascorbate, glutathione and poly (ADP ribose) polymerase (PARP) in plant cell cultures. Methods Mol Biol 476:193–209

    Google Scholar 

  • Frei M, Wissuwa M, Pariasca-Tanaka J, Chen CP, Südekum KH, Kohno Y (2012) Leaf ascorbic acid level—is it really important for ozone tolerance in rice? Plant Physiol Biochem 59:63–70

    Article  PubMed  CAS  Google Scholar 

  • Gilbert L, Alhagdow M, Nunes-Nesi A, Quemener B, Guillon F et al. (2009) 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

    Article  PubMed  CAS  Google Scholar 

  • Goggin FL, Avila CA, Lorence A (2010) Vitamin C content in plants is modified by insects and influence susceptibility to herbivory. BioEssays 32:777–790

    Article  PubMed  CAS  Google Scholar 

  • Guo Z, Tan H, Zhu Z, Lu S, Zhou B (2005) Effect of intermediates on ascorbic acid and oxalate biosynthesis of rice and in relation to its stress resistance. Plant Physiol Biochem 43:955–962

    Article  PubMed  CAS  Google Scholar 

  • Hamada AM, Al-Hakimi ABM (2009) Hydroponic treatment with ascorbic acid decreases the effect of salinity injury in two soybean cultivars. Phyton 49:43–62

    CAS  Google Scholar 

  • Horemans N, Potters G, De Wilde L, Caubergs RJ (2003) Division of tobacco bright yellow-2 cell cultures. Plant Physiol 133:361–367

    Article  PubMed  CAS  Google Scholar 

  • Huang C, He W, Guo J, Chang X, Su P, Zhang L (2005) Increased sensitivity to salt stress in an ascorbate-deficient Arabidopsis mutant. J Exp Bot 56:3041–3049

    Article  PubMed  CAS  Google Scholar 

  • Kanter U, Usadel B, Guerineau F, Li Y, Pauly M, Tenhaken R (2005) The inositol oxygenase gene family of Arabidopsis is involved in the biosynthesis of nucleotide sugar precursors for cell-wall matrix polysaccharides. Planta 221:243–254

    Article  PubMed  CAS  Google Scholar 

  • Keller R, Springer F, Renz A, Kossmann J (1999) Antisense inhibition of the GDP-mannose pyrophosphorylase reduces the ascorbate content in transgenic plants leading to developmental changes during senescence. Plant J 19:131–141

    Article  PubMed  CAS  Google Scholar 

  • Kwon SY, Choi SM, Ahn YO, Lee HS, Lee HB et al. (2003) Enhanced stress-tolerance of transgenic tobacco plants expressing a human dehydroascorbate reductase gene. J Plant Physiol 160:347–353

    Article  PubMed  CAS  Google Scholar 

  • Lee YP, Kim SH, Bang JW, Lee HS, Kwak SS, Kwon SY (2007) Enhanced tolerance to oxidative stress in transgenic tobacco plants expressing three antioxidant enzymes in chloroplasts. Plant Cell Rep 26:591–598

    Article  PubMed  CAS  Google Scholar 

  • Li F, Wu QY, Sun YL, Wang LY, Yang XH, Meng QW (2010) Overexpression of chloroplastic monodehydroascorbate reductase enhance tolerance to temperature and methyl viologen-mediated oxidative stress. Physiol Plant 139:421–434

    PubMed  CAS  Google Scholar 

  • Li Q, Li Y, Li C, Yu X (2012) Enhanced ascorbic acid accumulation through overexpression of dehydroascorbate reductase confers tolerance to methyl viologen and salt stresses in tomato. Czech J Genet Plant Breed 48:74–86

    Google Scholar 

  • Lichtenthaler, HK (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol 148:350–382

    CAS  Google Scholar 

  • Liu H, Weisman D, Ye Y, Cui B, Huang Y, Colon-Carmona A, Wang Z (2009) An oxidative stress response to polycyclic aromatic hydrocarbon exposure is rapid and complex in Arabidopsis thaliana. Plant Sci 176:375–382

    Article  CAS  Google Scholar 

  • Liu Y, Yu L, Wang R (2011) Level of ascorbic acid in transgenic rice for L-galactono-1,4-lactone dehydrogenase overexpressing or suppressed is associated with plant growth and seed set. Acta Physiol Plant 33:1353–1363

    Article  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Lorence A, Nessler CL (2007) Pathway engineering of the plant vitamin C metabolic network. In Verpoorte R, Alfermann A, Johnson TS (eds) Applications of Plant Metabolic Engineering. Springer, Dordrecht, pp 197–217

    Chapter  Google Scholar 

  • Maruta T, Tanouchi A, Tamoi M, Yabuta Y, Yoshimura K et al. (2010) Arabidopsis chloroplastic ascorbate peroxidase isoenzymes play a dual role in photoprotection and gene regulation under photooxidative stress. Plant Cell Physiol 51:190–200

    Article  PubMed  CAS  Google Scholar 

  • Mittler R, Blumwald E (2010) Genetic engineering for modern agriculture: challenges and perspectives. Annu Rev Plant Biol 61:443–462

    Article  PubMed  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Olmos E, Kiddle G, Pellny TK, Kumar S, Foyer CH (2006) Modulation of plant morphology, root architecture, and cell structure by low vitamin C in Arabidopsis thaliana. J Exp Bot 57:1645–1655

    Article  PubMed  CAS  Google Scholar 

  • Pavet V, Olmos E, Kiddle G, Kumar S, Antoniw J et al. (2005) Ascorbic acid deficiency activates cell death and disease resistance in Arabidopsis thaliana. Plant Physiol 139:1291–1303

    Article  PubMed  CAS  Google Scholar 

  • Potters G, De Gara L, Asard H, Horemans N (2002) Ascorbate and glutathione: guardians of the cell cycle, partners in crime? Plant Physiol Biochem 40:537–548

    Article  CAS  Google Scholar 

  • Queval G, Noctor G (2007) A plate reader method for the measurement of NAD, NADP, glutathione, and ascorbate in tissue extracts: Application to redox profiling during Arabidopsis rosette development. Anal Biochem 363:58–69

    Article  PubMed  CAS  Google Scholar 

  • Radzio JA, Lorence A, Chevone BI, Nessler CL (2003) L-Gulono-1,4-lactone oxidase expression rescues vitamin C-deficient Arabidopsis (vtc) mutants. Plant Molec Biol 53:837–844

    Article  CAS  Google Scholar 

  • Shalata A, Neumann PM (2001) Exogenous ascorbic acid (vitamin C) increases resistance to salt stress and reduced lipid peroxidation. J Exp Bot 52: 2207–2211

    PubMed  CAS  Google Scholar 

  • Smirnoff N (2011) Vitamin C: the metabolism and functions of ascorbic acid in plants. Adv Bot Res 59:107–177

    CAS  Google Scholar 

  • Tamaoki M, Mukai F, Asai N, Nakajimi N, Kubo A et al. (2003) Light-controlled expression of a gene encoding L-galactono-lactone dehydrogenase which affects ascorbate pool size in Arabidopsis thaliana. Plant Sci 16:1111–1117

    Article  Google Scholar 

  • Torabinejad J, Donahue JL, Gunesekera BN, Allen-Daniels MJ, Gillaspy GE (2009) VTC4 is a bifunctional enzyme that affects myo-inositol and ascorbate biosynthesis in plants. Plant Physiol 150:951–961

    Article  PubMed  CAS  Google Scholar 

  • Tóth SZ, Nagy V, Puthur JT, Kovács L, Garab G (2011) The physiological role of ascorbate as photosystem II electron donor: protection against photoinactivation in heat-stressed leaves. Plant Physiol 156:382–392

    Article  PubMed  Google Scholar 

  • Upadhyaya HCP, Akula N, Young KE, Chun SC, Kim DH, Park SW (2010) Enhanced ascorbic acid accumulation in transgenic potato confers tolerance to various abiotic stresses. Biotechnol Lett 32:321–330

    PubMed  Google Scholar 

  • Ushimaru T, Nakagawa T, Fujioka Y, Daicho K, Naito M et al. (2006) Transgenic Arabidopsis plants expressing the rice dehydroascorbate reductase gene are resistant to salt stress. J Plant Physiol 163:1179–1184

    Article  PubMed  CAS  Google Scholar 

  • Veljovic-Jovanovic SD, Pignocchi C, Noctor G, Foyer CH (2001) Low ascorbic acid in the vtc-1 mutant of Arabidopsis is associated with decreased growth and intracellular redistribution of the antioxidant system. Plant Physiol 127:426–435

    Article  PubMed  CAS  Google Scholar 

  • Wang Z, Xiao Y, Chen W, Tang K, Zhang L (2010) Increased vitamin C content accompanied by an enhanced recycling pathway confers oxidative stress tolerance in Arabidopsis. JIPB 52:400–409

    PubMed  CAS  Google Scholar 

  • Yin L, Wanf S, Eltayeb AE, Uddin MI, Yamamoto Y et al. (2010) Overexpression of dehydroascorbate reductase, but not monodehydroascorbate reductase, confers tolerance to aluminum stress in transgenic tobacco. Planta 231:609–621

    Article  PubMed  CAS  Google Scholar 

  • Zhang C, Liu J, Zhang Y, Cai X, Gong P et al. (2011) Overexpression of SIGMEs leads to ascorbate accumulation with enhanced oxidative stress, cold, and salt tolerance in tomato. Plant Cell Rep 30:389–398

    Article  PubMed  CAS  Google Scholar 

  • Zhang W, Lorence A, Gruszewski HA, Chevone BI, Nessler CL (2009) AMRI, an Arabidopsis gene that coordinately and negatively regulates the mannose/L-galactose ascorbic acid biosynthetic pathway. Plant Physiol 150:942–950

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors would like to thank Dr. Brenda Winkel (Virginia Tech, Blacksburg, VA) for her gift of wild type Arabidopsis seeds, G Wilson, J Yactayo-Chang, and J Martínez-Quintana, for their contribution to plant care, P Vasu for assistance with chlorophyll measurements, and SI Aboobucker for critical reading of the text. This study was supported at AL Laboratory with funds from the Arkansas Biosciences Institute, the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000, and a sub-award from the Arkansas INBRE program (National Center for Research Resources (5P20RR016460-11) and the National Institute of General Medical Sciences (8P20GM103429-11) from the National Institutes of Health). K Lisko thanks the Molecular Biosciences PhD program for a scholarship. Preliminary work at CN Laboratory was supported by the Interagency Metabolic Engineering Program (National Science Foundation—Metabolic Biochemistry and Integrative Plant Biology, fund IBN118612, and US Department of Agriculture, fund 2002-3S321-11600).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Argelia Lorence.

Additional information

Editor: J. Finer

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lisko, K.A., Torres, R., Harris, R.S. et al. Elevating vitamin C content via overexpression of myo-inositol oxygenase and l-gulono-1,4-lactone oxidase in Arabidopsis leads to enhanced biomass and tolerance to abiotic stresses. In Vitro Cell.Dev.Biol.-Plant 49, 643–655 (2013). https://doi.org/10.1007/s11627-013-9568-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11627-013-9568-y

Keywords

Navigation