Transgenic Research

, Volume 17, Issue 2, pp 171–180 | Cite as

Enhancing salt tolerance in a crop plant by overexpression of glyoxalase II

  • Sneh L. Singla-Pareek
  • Sudesh Kumar Yadav
  • Ashwani Pareek
  • M. K. Reddy
  • S. K. Sopory
Original Paper


Earlier we have shown the role of glyoxalase overexpression in conferring salinity tolerance in transgenic tobacco. We now demonstrate the feasibility of same in a crop like rice through overproduction of glyoxalase II. The rice glyoxalase II was cloned in pCAMBIA1304 and transformed into rice (Oryza sativa cv PB1) via Agrobacterium. The transgenic plants showed higher constitutive activity of glyoxalase II that increased further upon salt stress, reflecting the upregulation of endogenous glyoxalase II. The transgenic rice showed higher tolerance to toxic concentrations of methylglyoxal (MG) and NaCl. Compared with non-transgenics, transgenic plants at the T1 generation exhibited sustained growth and more favorable ion balance under salt stress conditions.


Functional validation Glyoxalase II overexpression Methylglyoxal Rice transgenics Salinity tolerance 



We thank Professor Ray Wu, Cornell University, USA for valuable suggestions and critical reading of the manuscript. Thanks are also due to Drs. F White and BW Porter, Kansas State University, USA for the initial glyoxalase II clone and Drs. V. Rajamani and J. K. Tripathi, JNU, New Delhi for extending help in the work related to ionic content measurements. The financial support by the Department of Biotechnology (DBT, New Delhi) Rice Network Project, DBT Post-Doc fellowship to SKY and grants from the International Centre for Genetic Engineering and Biotechnology is duly acknowledged.


  1. Allen RE, Lo TW, Thornalley PJ (1993) A simplified method for the purification of human red blood cell glyoxalase. I. Characteristics, immunoblotting, and inhibitor studies. J Protein Chem 12:111–119PubMedCrossRefGoogle Scholar
  2. Arnon DI (1949) Copper enzymes in isolated chloroplasts: polyphenol oxidase in Beta vulgaris. Plant Physiol 24:1–15PubMedCrossRefGoogle Scholar
  3. Bito A, Haider M, Hadler I, Breitenbach M (1997) Identification and phenotypic analysis of two glyoxalase II encoding genes from Saccharomyces cerevisiae, GLO2 and GLO4, and intracellular localization of the corresponding proteins. J Biol Chem 272:21509–21519PubMedCrossRefGoogle Scholar
  4. 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 72:248–254PubMedCrossRefGoogle Scholar
  5. Cordell PA, Futers TS, Grants PJ, Pease RJ (2004) The Human hydroxyacylglutathione hydrolase (HAGH) gene encodes both cytosolic and mitochondrial forms of glyoxalase II. J Biol Chem 279:28653–28661PubMedCrossRefGoogle Scholar
  6. Deswal R, Sopory SK (1991) Purification and partial characterization of glyoxalase I from a higher plant, B. juncea. FEBS Lett 282:277–280PubMedCrossRefGoogle Scholar
  7. Deswal R, Chakravarty TN, Sopory SK (1993) The glyoxalase system in higher plants: regulation in growth and differentiation. Biochem Soc Trans 21:527–530PubMedGoogle Scholar
  8. Epstein E (1998) How calcium enhances plant salt tolerance. Science 280:1906–1907PubMedCrossRefGoogle Scholar
  9. Espartero J, Sanchez-Aguayo I, Pardo JM (1995) Molecular characterization of glyoxalase I from a higher plant: upregulation by stress. Plant Mol Biol 29:1223–1233PubMedCrossRefGoogle Scholar
  10. Fan L, Zheng S, Wang X (1997) Antisense suppression of phospholipase Dα retards abscisic acid- and ethylene-promoted senescence of postharvest Arabidopsis leaves. Plant Cell 9:2183–2196PubMedCrossRefGoogle Scholar
  11. Garg AK, Kim JK, Owens TG, Ranwala AP, Choi YD, Kochian LV, Wu RJ (2002) Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses. Proc Natl Acad Sci USA 99:15898–15903PubMedCrossRefGoogle Scholar
  12. Inoue Y, Kimura A (1996) Identification of the structural gene for glyoxalase I from Saccharomyces cerevisiae. J Biol Chem 271:25958–25965PubMedCrossRefGoogle Scholar
  13. Irsch T, Krauth-Siegel RL (2004) Glyoxalase II of African trypanosomes is trypanothione-dependent. J Biol Chem 279:22209–22217PubMedCrossRefGoogle Scholar
  14. Jain M, Choudhary D, Kale RK, Bhalla-Sarin N (2002) Salt- and glyphosate-induced increase in glyoxalase I activity in cell lines of groundnut (Arachis hypogaea). Physiol Plant 114:499–505PubMedCrossRefGoogle Scholar
  15. Johansen KS, Svendsen II, Rasmussen SK (2000) Purification and cloning of the two domain glyoxalase I from wheat bran. Plant Sci 155:11–20PubMedCrossRefGoogle Scholar
  16. Kalapos MP, Garzo T, Antonie F, Mandl J (1992) Accumulation of S-D-lactoylglutathione and transient decrease of glutathione level caused by methylglyoxal load in isolated hepatocytes. Biochim Biophys Acta 1135:159–164PubMedCrossRefGoogle Scholar
  17. Kim NS, Umezawa Y, Ohmura S, Kato S (1993) Human glyoxalase I: cDNA cloning, expression and sequence similarity to glyoxalase I from Pseudomonas putida. J Biol Chem 268:11217–11221PubMedGoogle Scholar
  18. Kocsy G, Galiba G, Brunold C (2001) Role of glutathione in adaptation and signaling during chilling and cold acclimation in plants. Physiol Plant 113:158–164PubMedCrossRefGoogle Scholar
  19. Kumar S, Singla-Pareek SL, Reddy MK, Sopory SK (2003) Glutathione: biosynthesis, homeostasis and its role in abiotic stresses. J Plant Biol 30:179–187Google Scholar
  20. Maiti MK, Krishnasamy S, Owen HA, Makaroff CA (1997) Molecular characterization of glyoxalase II from Arabidopsis thaliana. Plant Mol Biol 35:471–481PubMedCrossRefGoogle Scholar
  21. May MJ, Vernoux T, Leaver C, Van Montagu M, Inze D (1998) Glutathione homeostasis in plants: implications for environmental sensing and plant development. J Expt Bot 49:1102–1116CrossRefGoogle Scholar
  22. McLellan AC, Phillips SA, Thornalley PJ (1993) The assay of S-D-lactoylglutathione in biological systems. Anal Biochem 211:37–43PubMedCrossRefGoogle Scholar
  23. Noctor G, Arisi ACM, Jouanin L, Kunert KJ, Rennenberg H, Foyer CH (1998) Glutathione: biosynthesis, metabolism and relationship to stress tolerance explored in transformed plants. J Expt Bot 49:623–647CrossRefGoogle Scholar
  24. Norton SJ, Talesa V, Yuan WJ, Principato GB (1990) Glyoxalase I and glyoxalase II from Aloe vera: purification, characterization and comparison with animal glyoxalases. Biochem Int 22:411–418PubMedCrossRefGoogle Scholar
  25. Papoulis A, Al-Abed Y, Bucala R (1995) Identification of N2-(1-carboxylethyl) guanine (CEG) as a guanine advanced glycosylation endproduct. Biochemistry 34:648–655PubMedCrossRefGoogle Scholar
  26. Paulus C, Knollner B, Jacobson H (1993) Physiological and biochemical characterization of glyoxalase I, a general marker for cell proliferation, from a soybean cell suspension. Planta 189:561–566PubMedCrossRefGoogle Scholar
  27. Phillips SA, Thornalley PJ (1993) The formation of methylglyoxal from triose phosphates. Investigation using a specific assay for methylglyoxal. Eur J Biochem 212:101–105PubMedCrossRefGoogle Scholar
  28. Ramaswamy O, Guha-Mukherjee S, Sopory SK (1983) Presence of glyoxalase I in pea. Biochem Inter 7:307–318Google Scholar
  29. Ridderstrom M, Mannervik B (1996) Optimized heterologous expression of the human zinc enzyme glyoxalase I. Biochem J 314:463–467PubMedGoogle Scholar
  30. Ridderstrom M, Mannervik B (1997) Molecular cloning and characterization of the thiolesterase glyoxalase II from Arabidopsis thaliana. Biochem J 322:449–454PubMedGoogle Scholar
  31. Ridderstrom M, Saccucci F, Hellman U, Bergman T, Principato G, Mannervik B (1996) Molecular cloning, heterologous expression and characterization of human glyoxalase II. J Biol Chem 271:319–323PubMedCrossRefGoogle Scholar
  32. Rus A, Yokoi S, Sharkhuu A, Reddy M, Lee BH, Matsumoto TK, Koiwa H, Zhu JK, Bressan RA, Hasegawa PM (2001) AtHKT1 is a salt tolerance determinant that controls Na(+) entry into plant roots. Proc Natl Acad Sci USA 98:14150–14155PubMedCrossRefGoogle Scholar
  33. Saxena M, Bisht R, Roy SD, Sopory SK, Bhalla-Sarin N (2005) Cloning and characterization of a mitochondrial glyoxalase II from Brassica juncea that is upregulated by NaCl, Zn, and ABA. Biochem Biophys Res Commun 336:813–819PubMedCrossRefGoogle Scholar
  34. Singla-Pareek SL, Reddy MK, Sopory SK (2003) Genetic engineering of the glyoxalase pathway in tobacco leads to enhanced salinity tolerance. Proc Natl Acad Sci USA 100:14672–14677PubMedCrossRefGoogle Scholar
  35. Singla-Pareek SL, Yadav SK, Pareek A, Reddy MK, Sopory SK (2006) Transgenic tobacco overexpressing glyoxalase pathway enzymes grow and set viable seeds in zinc spiked soils. Plant Physiol 140:613–623PubMedCrossRefGoogle Scholar
  36. Skipsey M, Andrews CJ, Townson JK, Jepson I, Edwards R (2000) Cloning and characterization of glyoxalase I from Soybean. Arch Biochem Biophys 374:261–268PubMedCrossRefGoogle Scholar
  37. Talesa V, Rosi G, Contenti S, Mangiabene C, Lupattelli M, Norton SJ, Giovannini E, Principato GB (1990) Presence of glyoxalase II in mitochondria from spinach leaves: comparison with the enzyme from cytosol. Biochem Inter 22:1115–1120Google Scholar
  38. Thornalley PJ (1990) The glyoxalase system: new developments towards functional characterization of a metabolic pathway fundamental to biological life. Biochem J 269:1–11PubMedGoogle Scholar
  39. Thornalley PJ (1993) The glyoxalase system in health and disease. Mol Asp Med 14:287–371CrossRefGoogle Scholar
  40. Thornalley PJ (1996) Pharmacology of methylglyoxal: formation, modification of proteins and nucleic acids, and enzymatic detoxification-a role in pathogenesis and antiproliferative chemotherapy. Gen Pharmacol 27:565–573PubMedGoogle Scholar
  41. Veena, Reddy VS, Sopory SK (1999) Glyoxalase I from Brassica juncea: molecular cloning, regulation and its over-expression confer tolerance in transgenic tobacco under stress. Plant J 17:385–395PubMedCrossRefGoogle Scholar
  42. Yadav SK, Singla-Pareek SL, Ray M, Reddy MK, Sopory SK (2005a) Methylglyoxal levels in plants under salinity stress are dependent on glyoxalase I and glutathione. Biochem Biophys Res Commun 337:61–67CrossRefGoogle Scholar
  43. Yadav SK, Singla-Pareek SL, Reddy MK, Sopory SK (2005b) Methylglyoxal detoxification by glyoxalase system: A survival strategy during environmental stresses. Physiol Mol Biol Plants 11:1–11Google Scholar
  44. Yadav SK, Singla-Pareek SL, Reddy MK, Sopory SK (2005c) Transgenic tobacco plants overexpressing glyoxalase enzymes resist an increase in methylglyoxal and maintain higher reduced glutathione levels under salinity stress. FEBS Lett 579:6265–6271CrossRefGoogle Scholar
  45. Yadav SK, Singla-Pareek SL, Kumar M, Pareek A, Saxena M, Sarin NB, Sopory SK (2007) Characterization and functional validation of glyoxalase II from rice. Protein Expr Purif 51:126–132PubMedCrossRefGoogle Scholar
  46. Zivy M, Thiellement H, de Vienne D, Hofmann JP (1983) Study on nuclear and cytoplasmic genome expression in wheat by two-dimensional gel electrophoresis. Theor Appl Genet 66:1–7CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Sneh L. Singla-Pareek
    • 1
  • Sudesh Kumar Yadav
    • 1
    • 3
  • Ashwani Pareek
    • 2
  • M. K. Reddy
    • 1
  • S. K. Sopory
    • 1
  1. 1.Plant Molecular Biology GroupInternational Centre for Genetic Engineering and BiotechnologyNew DelhiIndia
  2. 2.Stress Physiology and Molecular Biology Laboratory, School of Life SciencesJawaharlal Nehru UniversityNew DelhiIndia
  3. 3.Biotechnology DivisionInstitute of Himalayan Bioresource TechnologyPalampurIndia

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