Skip to main content
SpringerLink
Log in

Antisense-mediated depletion of GMPase gene expression in tobacco decreases plant tolerance to temperature stresses and alters plant development

  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

In our previous work [1] we investigated the role of tomato GDP-mannose pyrophosphorylase (EC 2.7.7.22) in plants by overexpressing its gene in tobacco leaves and showed its function in AsA metabolism and detoxification of reactive oxygen species under temperature stresses. In this study, we use the antisense technique to block the endogenous GMPase gene expression in tobacco in order to further investigate its function. Northern and western blot analysis confirmed that the expression of endogenous tobacco GMPase mRNA and protein was inhibited by this antisense expression. Consequently, the activity of GMPase and the content of AsA in the leaves of antisense transgenic plants were markedly decreased. This was also the case for the activities of both chloroplastic SOD (superoxide dismutase EC 1.15.1.1), APX (ascorbate peroxidase EC 1.11.1.7) and the content of AsA in leaves of the transgenic plants. On the contrary, the contents of H2O2 and O2 −• were increased. Meanwhile, the net photosynthetic rate (Pn) and the maximal photochemical efficiency of PSII (Fv/Fm) also declined in the leaves of antisense plants. Under high or low temperature stresses, the seed germination rate of the antisense transgenic plants was significantly decreased in comparison with that of the wild-type tobacco. Interestingly, the antisense plants had smaller leaves and an earlier onset of flowering. In conclusion, the depletion of GMPase decreased the content of AsA, resulting in the plants susceptible to the oxidative damage caused by temperature stresses and subjected to developmental alternations.

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

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

APX:

Ascorbate peroxidase

AsA:

l-ascorbic acid

Fv/Fm :

The maximal photochemical efficiency of PSII

GMPase :

GDP-d-mannose pyrophosphorylase gene

GMPase:

GDP-d-mannose pyrophosphorylase

Pn :

Net photosynthetic rate

PPFD:

Photosynthetic photon flux density

ROS:

Reactive oxygen species

SOD:

Superoxide dismutase

References

  1. 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 

  2. Howarth CJ (2005) Genetic improvements of tolerance to high temperature. The Howarth Press, New York

    Google Scholar 

  3. Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55:373–399

    Article  PubMed  CAS  Google Scholar 

  4. Pitzschke A, Forzani C, Hirt H (2006) Reactive oxygen specie signaling in plants. Antioxidant Redox Signaling 8:1757–1764

    Article  CAS  Google Scholar 

  5. Ledford HK, Chin BL, Niyogi KK (2007) Acclimation to singlet oxygen stress in Chlamydomonas reinhardtii. Eukaryot Cell 6:919–930

    Article  PubMed  CAS  Google Scholar 

  6. Asada K, Takahashi M (1987) Production and scavenging of active oxygen radicals in photosynthesis, vol 9. In: Kyle DJ, Osmond CB, Arntzen CJ (eds) Photoinhibition. Elsevier, Amsterdam, pp 227–288

  7. Foyer CH, Theodoulou FL, Delrot S (2001) The function of inter- and intracellular glutathione transport systems in plants. Trends Plant Sci 4:486–492

    Article  Google Scholar 

  8. Conklin PL, Williams EH, Last RL (1996) Environmental stress sensitivity of an ascorbic acid-deficient Arabidopsis mutant. PNAS 93:9970–9974

    Article  PubMed  CAS  Google Scholar 

  9. Conklin PL, Pallanca JE, Last RL, Smirnoff N (1997) Lascorbic acid metabolism in the ascorbate deficient mutant vtc1. Plant Physiol 115:1277–1285

    Article  PubMed  CAS  Google Scholar 

  10. 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 

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

    PubMed  CAS  Google Scholar 

  12. Sanmartin M, Drogoudi PD, Lyons T, Pateraki I, Barnes J, Kanellis AK (2003) Over-expression of ascorbate oxidase in the apoplast of transgenic tobacco results in altered ascorbate and glutathione redox states and increased sensitivity to ozone. Planta 216:918–928

    PubMed  CAS  Google Scholar 

  13. Neubauer C, Yamamoto HY (1994) Membrane barriers and Mehler-peroxidase limit the ascorbate available for violaxanthin de-epoxidase activity in intact chloroplasts. Photosynth Res 39:137–147

    Article  CAS  Google Scholar 

  14. Blokhina O, Virolainen E, Fagerstedt K (2003) Antioxidant, oxidative damage and oxygen deprivation stress: a review. Ann Bot 91:179–194

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. Kato N, Esaka M (1999) Changes in ascorbate oxidase gene expression and ascorbate levels in cell division and cell elongation in tobacco cells. Physiol Plant 105:321–329

    Article  CAS  Google Scholar 

  18. Kato N, Esaka M (2000) Expansion of transgenic tobacco protoplasts expressing pumpkin ascorbate oxidase is more rapid than that of wild-type protoplasts. Planta 210:1018–1022

    Article  PubMed  CAS  Google Scholar 

  19. Garg OP, Kapoor V (1972) Retardation of leaf senescence by ascorbate. J Exp Bot 23:699–704

    Article  CAS  Google Scholar 

  20. Wheeler GL, Jones MA, Smirnoff N (1998) The biosynthetic pathway of vitamin C in higher plants. Nature 303:365–369

    Google Scholar 

  21. Keller R, Springer F, Renz R, 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 

  22. Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring gene into plants. Science 227:1229–1231

    Article  CAS  Google Scholar 

  23. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  24. Kampfenkel K, Van Montagu M, Inzé D (1995) Extraction and determination of ascorbate and dehydroascorbate from plant tissue. Anal Biochem 225:165–167

    Article  PubMed  CAS  Google Scholar 

  25. Robinson SP, Downton WJS, Millhouse JA (1983) Photosynthesis and ion content of leaves and isolated chloroplasts of salt-stressed spinach. Plant Physiol 73:238–242

    Article  PubMed  CAS  Google Scholar 

  26. Jiménez A, Hernández JA, Del Rio LA, Sevilla F (1997) Evidence for the presence of the ascorbate-glutathione cycle in mitochondria and peroxisomes of pea leaves. Plant Physiol 114:275–284

    PubMed  Google Scholar 

  27. Giannopolitis CN, Ries SK (1977) Superoxide dismutases: I. Occurrence in higher plants. Plant Physiol 59:309–314

    Article  PubMed  CAS  Google Scholar 

  28. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  29. Sairam PK, Srivastava GC (2002) Changes in antioxidant activity in sub-cellular fractions of tolerant and susceptible wheat genotypes in response to long term salt stress. Plant Sci 162:897–904

    Article  CAS  Google Scholar 

  30. Wang AG, Luo GH (1990) Quantitative relation between the reaction of hydroxylamine and superoxide anion radicals in plants. Plant Physiol Commun 6:55–57

    CAS  Google Scholar 

  31. Van KO, Snel JFH (1990) The use of chlorophyll fluorescence nomenclature in plant stress physiology. Photosynth Res 25:147–150

    Article  Google Scholar 

  32. Gatzek S, Wheeler GL, Smirnoff N (2002) Antisense suppression of L-galactose dehydro-genase in Arabidopsis thaliana provides evidence for its role in ascorbate synthesis and reveals light modulated l-galactose synthesis. Plant J 30(4):541–553

    Article  PubMed  CAS  Google Scholar 

  33. Asada K (1999) The water–water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annu Rev Plant Physiol Plant Mol Biol 50:601–639

    Article  PubMed  CAS  Google Scholar 

  34. Li XG, Meng QW, Jiang GQ, Zou Q (2003) The susceptibility of cucumber and sweet pepper to chilling under low irradiance is related to energy dissipation and water–water cycle. Photosynthetica 41:259–265

    Article  CAS  Google Scholar 

  35. Lukowitz W, Nickle TC, Meinke DW, Last RL, Conklin PL, Sommerville 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 

  36. Nishikawa K, Onodera A, Tominaga N (2006) Phytochelatins do not correlate with the level of Cd accumulation in Chlamydomonas spp. Chemosphere 63:1553–1559

    Article  PubMed  CAS  Google Scholar 

  37. Yang XH, Wen XG, Gong HM, Lu QT, Yang ZP, Tang YL, Liang Z, Lu CM (2007) Genetic engineering of the biosynthesis of glycinebetaine enhances thermotolerance of photosystem II in tobacco plants. Planta 225:719–733

    Article  PubMed  CAS  Google Scholar 

  38. Tyburski J, Tretyn A (2010) Ascorbate and glutathione in organogenesis, regeneration and differentiation in plant in vitro cultures. In Anjum NA et al. (eds.) In: Ascorbate-glutathione pathway and stress tolerance in plants. Springer, Dordrecht, pp 55–92

Download references

Acknowledgments

We thank Professor Qingwei Meng for the technical assistance during antibody preparation and Northern blotting. This work was supported by grants from the National Natural Science Foundation of China (31101554), the Natural Science Foundation of Zhejiang province (Y3110340 and LQ12C15002) and Zhejiang Higher School Innovative Research Team (T200916).

Author information

Authors and Affiliations

  1. Department of Horticulture, School of Agriculture and Food Science, Zhejiang A & F University, Lin’an, 311300, Zhejiang, People’s Republic China

    Hua-Sen Wang, Zhu-Jun Zhu, Zhen Feng & Chao Yu

  2. Tai’an Municipal Bureau of Agriculture, Tai’an, 271000, People’s Republic China

    Shi-Gang Zhang

Authors
  1. Hua-Sen Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhu-Jun Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhen Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shi-Gang Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Chao Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Hua-Sen Wang or Chao Yu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, HS., Zhu, ZJ., Feng, Z. et al. Antisense-mediated depletion of GMPase gene expression in tobacco decreases plant tolerance to temperature stresses and alters plant development. Mol Biol Rep 39, 10413–10420 (2012). https://doi.org/10.1007/s11033-012-1920-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-012-1920-5

Keywords

Search

Navigation