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Ectopic overexpression of peach GDP-d-mannose pyrophosphorylase and GDP-d-mannose-3′,5′-epimerase in transgenic tobacco

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Abstract

We created transgenic tobacco overexpressing peach GDP-d-mannose pyrophosphorylase (PpGMPH) and GDP-d-mannose-3′,5′-epimerase (PpGME), which are involved in the main l-ascorbate (Asc) biosynthetic pathway in plants. Despite an apparent increase in enzymatic activity in the PpGMPH-overexpressing primary transgenic (T0) lines, no significant increase in Asc pool size was observed in leaves. For PpGME-overexpressing T0 lines, Asc pool size was also unchanged, although transcriptional overexpression was confirmed. To reinforce the biosynthetic pathway from d-mannose-1-phosphate to GDP-l-galactose, we further created PpGMPH- and PpGME-double overexpressing crossed transgenic (dOx T1) lines. Transcriptional overexpression of PpGMPH and PpGME transgenes was confirmed, and the Asc content in the dOx T1 lines was slightly elevated in young leaves, albeit not significantly. Western blot analyses revealed protein amounts of GMPH and GME were similar among dOx T1, parental T0 and non-transgenic (SR1) lines in three tissue types tested: leaves, flower buds and immature fruits. Moreover, a similar amount of GMPH and GME proteins were detected in young leaves (YL) and old leaves (OL), although Asc content in YL is roughly double as that in OL. In vitro assay using recombinant PpGME suggested the equilibrium of GME reaction is unfavorable to forward Asc biosynthesis at this step. Finally, to investigate substrate availability in relation to Asc pool size, we added exogenous GDP-d-mannose (5 mM) or l-galactose (5 mM) to tobacco leaf discs; the former resulted in no increase in Asc content, whereas a significant increase was observed in the latter, not only in the dOx T1 lines, but also in the original line SR1. Similar treatments were also conducted under high light, which resulted in a further increase in the increment of Asc content in the l-galactose-fed discs irrespective of transgene expression level. Taken together, both l-galactose availability and light rather than upper biosynthetic gene expression levels of GMPH/GME are critical determinant of Asc content in tobacco leaves, indicating complex modulation of this compound.

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Abbreviations

Asc:

l-Ascorbate

CaMV:

Cauliflower mosaic virus

DHA:

Dehydroascorbate

dOx T1:

Double overexpressing crossed transgenic

DTT:

Dithiothreitol

FB:

Flower buds

GalUAR:

d-Galacturonate reductase

GDH:

l-Galactose dehydrogenase

GGP:

GDP-l-galactose phophorylase

GLDH:

l-Galactono-1,4-lactone dehydrogenase

GME:

GDP-d-mannose-3′,5′-epimerase

GMPH:

GDP-d-mannose pyrophosphorylase

GPP:

l-Galactose-1-phosphate phosphatase

IF:

Immature fruits

Km:

Kanamycin

MS:

Murashige and Skoog’s

OL:

Old leaves

ROS:

Reactive oxygen species

SDS:

Sodium dodecyl sulfate

TLC:

Thin layer chromatography

YL:

Young leaves

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 Biotech 21:177–181

    Article  CAS  Google Scholar 

  • 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

    Google Scholar 

  • Arrigoni O, De Tullio MC (2002) Ascorbic acid: much more than just an antioxidant. Biochim Biophys Acta 1569:1–9

    Article  PubMed  CAS  Google Scholar 

  • Badejo AA, Tanaka N, Esaka M (2008) Analysis of GDP-d-mannose 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

    Article  PubMed  CAS  Google Scholar 

  • Badejo AA, Fujikawa Y, Esaka M (2009) Gene expression of ascorbic acid biosynthesis related enzymes of the Smirnoff-Wheeler pathway in acerola (Malpighia glabra). J Plant Physiol 166:652–660

    Article  PubMed  CAS  Google Scholar 

  • Badejo AA, Wada K, Gao Y, Maruta T, Sawa Y, Shigeoka S, Ishikawa T (2012) Translocation and the alternative d-galacturonate pathway contribute to increasing the ascorbate level in ripening tomato fruits together with the d-mannose/l-galactose pathway. J Exp Bot 63:229–239

    Article  PubMed  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, Gómez F, Fernández 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

    Article  PubMed  CAS  Google Scholar 

  • Bulley SM, Rassam M, Hoser D, Otto W, Schünemann 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 guanyltransferase is a major control point of vitamin C biosynthesis. J Exp Bot 60:765–778

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Conklin PL, Norris SR, Wheeler GL, Williams EH, Smirnoff N, Last RL (1999) Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis. Proc Natl Acad Sci USA 96:4198–4203

    Article  PubMed  CAS  Google Scholar 

  • Conklin PL, Saracco SA, Norris SR, Last RL (2000) Identification of ascorbic acid-deficient Arabidopsis thaliana mutants. Genetics 154:847–856

    PubMed  CAS  Google Scholar 

  • Conklin PL, Gatzek S, Wheeler GL, Dowdle J, Raymond MJ, Rolinski S, Isupov M, Littlechild JA, Smirnoff N (2006) Arabidopsis thaliana VTC4 encodes l-galactose-1-P phosphatase, a plant ascorbic acid biosynthetic enzyme. J Biol Chem 281:15662–15670

    Article  PubMed  CAS  Google Scholar 

  • Cronje C, George GM, Fernie AR, Bekker J, Kossmann J, Bauer R (2012) Manipulation of l-ascorbic acid biosynthesis pathways in Solanum lycopersicum: elevated GDP-mannose pyrophosphorylase activity enhances l-ascorbate levels in red fruit. Planta 235:553–564

    Article  PubMed  CAS  Google Scholar 

  • Davey MW, Van Montagu M, Inzé D, Sanmartin M, Kanellis A, Smirnoff N, Benzie IJJ, Strain JJ, Favell D, Fletcher J (2000) Plant l-ascorbic acid: chemistry, function, metabolism, bioavailability and effects of processing. J Sci Food Agric 80:825–860

    Article  CAS  Google Scholar 

  • Dowdle J, Ishikawa T, Gatzek S, Rolinski S, Smirnoff N (2007) Two genes in Arabidopsis thaliana encoding GDP-l-galactose phosphorylase are required for ascorbate biosynthesis and seedling viability. Plant J 52:673–689

    Article  PubMed  CAS  Google Scholar 

  • Fiocchetti F, D’Amore R, De Palma M, Bertini L, Caruso C, Caporale C, Testa A, Cristinzio G, Saccardo F, Tucci M (2008) Constitutive over-expression of two wheat pathogenesis-related genes enhances resistance of tobacco plants to Phytophthora nicotianae. Plant Cell, Tissue Organ Cult 92:73–84

    Article  Google Scholar 

  • Foyer CH, Noctor G (2005) Redox homeostasis and antioxidant signaling: a metabolic interface between stress perception and physiological responses. Plant Cell 17:1866–1875

    Article  PubMed  CAS  Google Scholar 

  • Foyer CH, Noctor G (2009) Redox regulation in photosynthetic organisms: signaling, acclimation, and practical implications. Antiox Redox Signal 11:861–905

    Article  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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 AR, Lahaye M, Rothan C, Baldet P (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 

  • Hancock RD, Viola R (2005) Biosynthesis and catabolism of l-ascorbic acid in plants. Crit Rev Plant Sci 24:167–188

    Article  CAS  Google Scholar 

  • Horsch RB, Fry J, Hoffmann N, Neidermeyer J, Rogers SG, Fraley RT (1989) Leaf disc transformation. In: Gelvin SB, Schilperoort RA (eds) Plant molecular biology manual. Kluwer, Dordrecht, pp A5/1–A5/9

    Google Scholar 

  • Imai T, Ban Y, Terakami S, Yamamoto T, Moriguchi T (2009a) l-Ascorbate biosynthesis in peach: cloning of six l-galactose pathway-related genes and their expression during peach fruit development. Physiol Plant 136:139–149

    Article  PubMed  CAS  Google Scholar 

  • Imai T, Niwa M, Ban Y, Hirai M, Ôba K, Moriguchi T (2009b) Importance of the l-galactonolactone pool for enhancing the ascorbate content revealed by l-galactonolactone dehydrogenase-overexpressing tobacco plants. Plant Cell, Tissue Organ Cult 96:105–112

    Article  CAS  Google Scholar 

  • Ioannidi E, Kalamaki MS, Engineer C, Pateraki I, Alexandrou D, Mellidou I, Giovannonni J, Kanellis AK (2009) Expression profiling of ascorbic acid-related genes during tomato fruit development and ripening and in response to stress conditions. J Exp Bot 60:663–678

    Article  PubMed  CAS  Google Scholar 

  • Ishikawa T, Dowdle J, Smirnoff N (2006) Progress in manipulating ascorbic acid biosynthesis and accumulation in plants. Physiol Plant 126:343–355

    Article  CAS  Google Scholar 

  • Ishikawa T, Nishikawa H, Gao Y, Sawa Y, Shibata H, Yabuta Y, Maruta T, Shigeoka S (2008) The pathway via d-galacturonate/l-galactonate is significant for ascorbate biosynthesis in Euglena gracilis: identification and functional characterization of aldonolactonase. J Biol Chem 283:31133–31141

    Article  PubMed  CAS  Google Scholar 

  • Ko WH, Su CC, Chen CL, Chao CP (2009) Control of lethal browning of tissue culture plantlets of Cavendish banana cv. Formosana with ascorbic acid. Plant Cell, Tissue Organ Cult 96:137–141

    Article  Google Scholar 

  • Li M-J, Ma F-W, Zhang M, Pu F (2008) Distribution and metabolism of ascorbic acid in apple fruits (Malus domestica Borkh cv. Gala). Plant Sci 174:606–612

    Article  CAS  Google Scholar 

  • Linster CL, Clarke SG (2008) l-Ascorbate biosynthesis in higher plants: the role of VTC2. Trends Plant Sci 13:567–573

    Article  PubMed  CAS  Google Scholar 

  • Linster CL, Van Schaftingen E (2007) Vitamin C: biosynthesis, recycling and degradation in mammals. FEBS J 274:1–22

    Article  PubMed  CAS  Google Scholar 

  • Linster CL, Gomez TA, Christensen KC, Adler LN, Young BD, Brenner C, Clarke SG (2007) Arabidopsis VTC2 encodes a GDP-l-galactose phosphorylase, the last unknown enzyme in the Smirnoff–Wheeler pathway to ascorbic acid in plants. J Biol Chem 282:18879–18885

    Article  PubMed  CAS  Google Scholar 

  • Linster CL, Adler LN, Webb K, Christensen KC, Brenner C, Clarke SG (2008) A second GDP-l-galactose phosphorylase in Arabidopsis en route to vitamin C. Covalent intermediate and substrate requirements for the conserved reaction. J Biol Chem 283:18483–18492

    Article  PubMed  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 

  • Lukowitz W, Nickle TC, Meinke DW, Last RL, Conklin PL, Somerville CR (2001) Arabidopsis cyt1 mutants are deficient in a mannose-1-phosphate guanylyltransferase and point to a requirement of N-linked glycosylation for cellulose biosynthesis. Proc Natl Acad Sci USA 98:2262–2267

    Article  PubMed  CAS  Google Scholar 

  • Melino VJ, Soole KL, Ford CM (2009) Ascorbate metabolism and the developmental demand for tartaric and oxalic acids in ripening grape berries. BMC Plant Biol 9:145

    Article  PubMed  Google Scholar 

  • Müller-Moulé P (2008) An expression analysis of the ascorbate biosynthesis enzyme VTC2. Plant Mol Biol 68:31–41

    Article  PubMed  Google Scholar 

  • Müller-Moulé P, Golan T, Niyogi KK (2004) Ascorbate-deficient mutants of Arabidopsis grow in high light despite chronic photooxidative stress. Plant Physiol 134:1163–1172

    Article  PubMed  Google Scholar 

  • Nakagawa T, Kurose T, Hino T, Tanaka K, Kawamukai M, Niwa Y, Toyooka K, Matsuoka K, Jinbo T, Kimura T (2007) Development of series of Gateway binary vectors, pGWBs, for releasing efficient construction of fusion genes for plant transformation. J Biosci Bioeng 104:34–41

    Article  PubMed  CAS  Google Scholar 

  • Noctor G, Foyer CH (1998) Ascorbate and glutathione: keeping active oxygen under control. Ann Rev Plant Physiol Plant Mol Biol 49:249–279

    Article  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Qin C, Qian W, Wang W, Wu Y, Yu C, Jiang X, Wang D, Wu P (2008) GDP-mannose pyrophosphorylase is a genetic determinant of ammonium sensitivity in Arabidopsis thaliana. Proc Natl Acad Sci USA 105:18308–18313

    Article  PubMed  CAS  Google Scholar 

  • Smirnoff N (2000) Ascorbic acid: metabolism and functions of a multi-facetted molecule. Curr Opin Plant Biol 3:229–235

    PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Voxeur A, Gilbert L, Rihouey C, Driouich A, Rothan C, Baldet P, Lerouge P (2011) Silencing of the GDP-d-mannose 3,5-epimerase affects the structure and cross-linking of the pectic polysaccharide rhamnogalacturonan II and plant growth in tomato. J Biol Chem 286:8014–8020

    Article  PubMed  CAS  Google Scholar 

  • Wang HS, Yu C, Zhu ZJ, Yu XC (2011) Overexpression in tobacco of a tomato GMPase gene improves tolerance to both low and high temperature stress by enhancing antioxidation capacity. Plant Cell Rep 30:1029–1040

    Article  PubMed  CAS  Google Scholar 

  • Watanabe K, Suzuki K, Kitamura S (2006) Characterization of a GDP-d-mannose 3′,5′-epimerase from rice. Phytochemistry 67:338–346

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Wolucka BA, Davey MW, Boerjan W (2001) A high-performance liquid chromatography radio method for determination of l-ascorbic acid and guanosine 5′-diphosphate-l-galactose, key metabolites of the plant vitamin C pathway. Anal Biochem 294:161–168

    Article  PubMed  CAS  Google Scholar 

  • Yabuta Y, Mieda T, Rapolu M, Nakamura A, Motoki T, Maruta T, Yoshimura K, Ishikawa T, Shigeoka S (2007) Light regulation of ascorbate biosynthesis is dependent on the photosynthetic electron transport chain but independent of sugars in Arabidopsis. J Exp Bot 58:2661–2671

    Article  PubMed  CAS  Google Scholar 

  • Zhang C, Liu J, Zhang Y, Cai X, Gong P, Zhang J, Wang T, Li H, Ye Z (2010) Overexpression of SlGMEs leads to ascorbate accumulation with enhanced oxidative stress, cold, and salt tolerance in tomato. Plant Cell Rep 30:389–398

    Article  PubMed  Google Scholar 

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Author notes

  1. Yusuke Ban

    Present address: Grape and Persimmon Research Division, NIFTS, NARO, Akitsu, Higashi-hiroshima, 739-2494, Hiroshima, Japan

Authors and Affiliations

  1. National Institute of Fruit Tree Science (NIFTS), National Agriculture and Food Research Organization (NARO), Fujimoto, Tsukuba, 305-8605, Ibaraki, Japan

    Tsuyoshi Imai, Toshiya Yamamoto & Takaya Moriguchi

  2. Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennodai, Tsukuba, 305-8572, Ibaraki, Japan

    Tsuyoshi Imai, Yusuke Ban, Toshiya Yamamoto & Takaya Moriguchi

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  1. Tsuyoshi Imai
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  2. Yusuke Ban
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  4. Takaya Moriguchi
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Correspondence to Tsuyoshi Imai.

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Imai, T., Ban, Y., Yamamoto, T. et al. Ectopic overexpression of peach GDP-d-mannose pyrophosphorylase and GDP-d-mannose-3′,5′-epimerase in transgenic tobacco. Plant Cell Tiss Organ Cult 111, 1–13 (2012). https://doi.org/10.1007/s11240-012-0165-2

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  • DOI: https://doi.org/10.1007/s11240-012-0165-2

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