Plant Molecular Biology Reporter

, Volume 30, Issue 4, pp 894–903 | Cite as

Molecular Cloning and Characterization of γ-Glutamyl Cysteine Synthetase (VrγECS) from Roots of Vigna radiata (L.) Wilczek Under Progressive Drought Stress and Recovery

  • Debashree Sengupta
  • Golla Ramesh
  • Shalini Mudalkar
  • Koppolu Raja Rajesh Kumar
  • Pulugurtha Bharadwaja Kirti
  • Attipalli R. Reddy
Original Paper


Glutathione is an essential redox buffer and an antioxidant in majority of higher plants, imparting tolerance against abiotic stress. The rate-limiting enzyme, gamma-glutamyl cysteine synthetase (γECS), plays an important role in regulation of glutathione biosynthesis under adverse environmental conditions including drought. To understand the role of γECS in an economically important food legume, Vigna radiata (L.) Wilczek, under progressive drought stress, we cloned and derived the full-length cDNA sequence and denoted it as VrγECS. Real-time PCR analysis of VrγECS in the roots of V. radiata, during progressive drought stress and recovery, indicated a stable expression pattern of the gene. However, the VrγECS enzyme activity altered differentially during varying water-deficit conditions and recovery period, reflecting the existence of some post-transcriptional or post-translational regulatory system for the enzyme. Linear regression analysis between H2O2 and lipid peroxidation as well as H2O2 and VrγECS enzyme activity during drought stress and recovery demonstrates the delicate inter-relationships and putative regulatory mechanisms operating in the root system under adverse conditions. The present study could contribute towards understanding the complex regulation of γECS in glutathione biosynthesis in an important food legume under drought stress.


Drought Gamma-glutamyl cysteine synthetase Glutathione Mungbean Regulatory mechanisms 



Days after onset of stress treatment


Days after re-watering




Photosynthetic photon flux density


Rapid amplification of cDNA ends


Reactive oxygen species


Gamma-glutamyl cysteine synthetase



We thank Prof. N. Nadarajan, Tamil Nadu Agricultural University (TNAU), Coimbatore, India, for providing Vigna radiata seeds. We also thank DST-FIST facility of our department and CREBB facility of School of Life Sciences. DS and SM acknowledge the fellowship from Council of Scientific and Industrial Research (CSIR) and University Grant Commission (UGC), New Delhi, India, respectively. KRRK was supported by CSIR and Dr. D.S. Kothari Postdoctoral fellowship from UGC.

Supplementary material

11105_2011_398_Fig5_ESM.jpg (213 kb)
Supplementary Fig. S1

The complete cDNA sequence of pTZ57RT-encoded Vigna radiata γECS (JPEG 212 kb)

11105_2011_398_MOESM1_ESM.tif (149 kb)
High resolution image (TIFF 148 kb)
11105_2011_398_Fig6_ESM.jpg (321 kb)
Supplementary Fig. S2

Multiple alignment of VrγECS precursor protein with γECS of P. vulgaris, P. sativum, A. thaliana, B. napus, B. juncea and R. communis. The alignment clearly demonstrates the conservation of the cleavage site (IVAA) of the chloroplast target peptide among different plants (JPEG 321 kb)

11105_2011_398_MOESM2_ESM.tif (613 kb)
High resolution image (TIFF 613 kb)


  1. Aravind P, Prasad MNV (2005) Modulation of cadmium-induced oxidative stress in Ceratophyllum demersum by zinc involves ascorbate-glutathione cycle and glutathione metabolism. Plant Physiol Biochem 43:107–116PubMedCrossRefGoogle Scholar
  2. Ball L, Accotto GP, Bechtold U, Creissen G, Funck D, Jimenez A, Kular B, Leyland N, Mejia-Carranza J, Reynolds H, Karpinski S, Mullineaux PM (2004) Evidence for a direct link between glutathione biosynthesis and stress defense gene expression in Arabidopsis. Plant Cell 16:2448–2462PubMedCrossRefGoogle Scholar
  3. 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
  4. Castillo FJ (1996) Antioxidative protection in the in the inducible CAM plant Sedum album L. following the imposition of severe water stress and recovery. Oecologia 107:469–477CrossRefGoogle Scholar
  5. Chen J, Goldsborough PB (1994) Increased activity of γ-g]utamylcysteine synthetase in tomato cells selected for cadmium tolerance. Plant Physiol 106:233–239PubMedGoogle Scholar
  6. Chen KM, Gong HJ, Chen GC, Wang SM, Zhang CL (2004) Gradual drought under field conditions influences the glutathione metabolism, redox balance and energy supply in spring wheat. J Plant Growth Regul 23:20–28CrossRefGoogle Scholar
  7. Cobbett CS (2000) Phytochelatins and their roles in heavy metal detoxification. Plant Physiol 123:825–832PubMedCrossRefGoogle Scholar
  8. Combet C, Jambon M, Deleage G, Geourjon C (2002) Geno3D: automatic comparative molecular modelling of protein. Bioinformatics 18:213–214PubMedCrossRefGoogle Scholar
  9. Corpet F (1988) Multiple sequence alignment with hierarchical clustering. Nucl Acids Res 16:10881–10890PubMedCrossRefGoogle Scholar
  10. Creissen G, Firmin J, Fryer M, Kular B, Leyland N, Reynolds H, Pastori G, Wellburn F, Baker N, Wellburn A, Mullineaux P (1999) Elevated glutathione biosynthetic capacity in the chloroplasts of transgenic tobacco plants paradoxically causes increased oxidative stress. Plant Cell 11:1277–1292PubMedGoogle Scholar
  11. Cummins I, Dixon DP, Freitag-Pohl S, Skipsey M, Edwards R (2011) Multiple roles for plant glutathione transferases in xenobiotic detoxification. Drug Metab Rev 43:266–280PubMedCrossRefGoogle Scholar
  12. Davies WJ, Zhang J (1991) Roots signals and the regulation of growth and development of plant in dry soil. Annu Rev Plant Physiol Plant Mol Biol 42:55–76CrossRefGoogle Scholar
  13. Dhindsa RS (1991) Drought stress enzymes of glutathione metabolism, oxidation injury and protein synthesis in Tortula ruralis. Plant Physiol 83:816–819CrossRefGoogle Scholar
  14. Diao G, Wang Y, Wang C, Yang C (2011) Cloning and functional characterization of a novel glutathione-S-transferase gene from Limonium bicolor. Plant Mol Biol Rep 29:77–87CrossRefGoogle Scholar
  15. Emanuelsson O, Nielsen H, von Heijne G (1999) ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci 8:978–984PubMedCrossRefGoogle Scholar
  16. Ennahli S, Earl HJ (2005) Physiological limitations to photosynthetic carbon assimilation in cotton under water stress. Crop Sci 45:2374–2382CrossRefGoogle Scholar
  17. Foyer CH, Noctor G (2005) Redox homeostasis and antioxidant signaling: a metabolic interface between stress perception and physiological responses. Plant Cell 17:1866–1875PubMedCrossRefGoogle Scholar
  18. Foyer CH, Souriau N, Perret S, Lelandais M, Kunert KJ, Pruvost C, Jouanin L (1995) Over-expression of glutathione reductase but not glutathione synthetase leads to increases in antioxidant capacity and improved photosynthesis in poplar (Populus tremula × P. alba) trees. Plant Physiol 109:1047–1057PubMedCrossRefGoogle Scholar
  19. Fu J, Huang B (2001) Involvement of antioxidants and lipid peroxidation in the adaptation of two cool-season grasses to localized drought stress. Environ Exp Bot 45:105–114PubMedCrossRefGoogle Scholar
  20. George S, Parida A (2010) Characterization of an oxidative stress inducible non specific lipid transfer protein coding cDNA and its promoter from drought tolerant plant Prosopis juliflora. Plant Mol Biol Rep 28:32–40CrossRefGoogle Scholar
  21. Gromes R, Hothorn M, Lenherr ED, Rybin V, Scheffzek K, Rausch T (2008) The redox switch of γ-glutamylcysteine ligase via a reversible monomer-dimer transition is a mechanism unique to plants. Plant J 54:1063–1075PubMedCrossRefGoogle Scholar
  22. Gόmez LD, Vanacker H, Buchner P, Noctor G, Foyer CH (2004) Intercellular distribution of glutathione synthesis in Maize leaves and its response to short-term chilling. Plant Physiol 134:1662–1671CrossRefGoogle Scholar
  23. Hell R, Bergmann L (1990) y-Glutamylcysteine synthetase in higher plants: catalytic properties and subcellular localization. Planta 180:603–612CrossRefGoogle Scholar
  24. Hicks LM, Cahoon RE, Bonner ER, Rivard RS, Sheffield J, Jez JM (2007) Thiol-based regulation of redox-active glutamate cysteine ligase from Arabidopsis thaliana. Plant Cell 19:2653–2661PubMedCrossRefGoogle Scholar
  25. Hothorn M, Wachter A, Gromes R, Stuwe T, Rausch T, Scheffzek K (2006) Structural basis for the redox control of plant glutamate cysteine ligase. J Biol Chem 281:27557–27565PubMedCrossRefGoogle Scholar
  26. Innocenti G, Pucciariello C, Gleuher ML, Hopkins J, de Stefano M, Delledonne M, Puppo A, Baudouin E, Frendo P (2007) Glutathione synthesis is regulated by nitric oxide in Medicago truncatula roots. Planta 225:1597–1602PubMedCrossRefGoogle Scholar
  27. Jez JM, Cahoon RE, Chen S (2004) Arabidopsis thaliana glutamate-cysteine ligase functional properties, kinetic mechanisms and regulation of activity. J Biol Chem 279:33463–33470PubMedCrossRefGoogle Scholar
  28. Jiang T, Fountain J, Davis G, Kemerait R, Scully B, Lee RD, Guo B (2011) Root morphology and gene expression analysis in response to drought stress in maize (Zea mays). Plant Mol Biol Rep. doi: 10.1007/s11105-011-0347-9
  29. Klapheck S (1988) Homoglutathione: isolation, quantification and occurrence in legumes. Physiol Plant 74:727–732CrossRefGoogle Scholar
  30. Klapheck S, Chrost B, Starke J, Zimmermann H (1992) γ-Glutamyl-cysteinylserine—a new homologue of glutathione in plants of the family Poaceae. Bot Acta 105:174–179Google Scholar
  31. Kocsy G, Galiba G, Brunold C (2001) Role of glutathione in adaptation and signalling during chilling and cold acclimation in plants. Physiol Plant 113:158–164PubMedCrossRefGoogle Scholar
  32. Kopriva S (2006) Regulation of sulfate assimilation in Arabidopsis and beyond. Ann Bot 97:479–495PubMedCrossRefGoogle Scholar
  33. Lawn RJ, Ahn CS (1985) Mung bean (Vigna radiata (L.) Eilczek/Vigna mungo (L.) Hepper). In: Summerfield RJ, Roberts EH (eds) Grain legume crops. William Collins Sons & Co Ltd, London, pp 584–623Google Scholar
  34. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2 −ΔΔCt method. Methods 25:402–408Google Scholar
  35. Loscos J, Matamoros MA, Becana M (2008) Ascorbate and homoglutathione metabolism in common bean nodules under stress conditions and during natural senescence. Plant Physiol 146:1282–1292PubMedCrossRefGoogle Scholar
  36. Maurel C, Simonneau T, Sutka M (2010) The significance of roots as hydraulic rheostats. J Exp Bot 61:3191–3198PubMedCrossRefGoogle Scholar
  37. May MJ, Leaver CJ (1993) Oxidative stimulation of glutathione synthesis in Arabidopsis thaliana suspension cultures. Plant Physiol 103:621–627PubMedGoogle Scholar
  38. May MJ, Leaver CJ (1994) Arabidopsis thaliana γ-glutamyl cysteine synthetase is structurally unrelated to mammalian, yeast and Escherichia coli homologs. Proc Natl Acad Sci USA 91:10059–10063PubMedCrossRefGoogle Scholar
  39. May MJ, Vernoux T, Sanchez-Fernandez R, Van Montagu M, Inze D (1998) Evidence for posttranscriptional activation of gammaglutamylcysteine synthetase during plant stress responses. Proc Natl Acad Sci USA 95:12049–12054PubMedCrossRefGoogle Scholar
  40. Mehta A, Magalhães BS, Souza DSL, Vasconcelos EAR, Silva LP, Grossi-deSa MF, Franco OL, da Costa PHA, Rocha TL (2008) Rooteomics: the challenge of discovering plant defense-related proteins in roots. Curr Protein Pept Sci 9:108–116PubMedCrossRefGoogle Scholar
  41. Mittler R, Vanderauwera S, Gollery M, Van Breusegem F (2004) Reactive oxygen gene network of plants. Trends Plant Sci 9:490–498PubMedCrossRefGoogle Scholar
  42. Mittova V, Theodoulou FL, Kiddle G, Gomez L, Volokita M, Tal M, Foyer CH, Guy M (2003) Coordinate induction of glutathione biosynthesis and glutathione-metabolizing enzymes is correlated with salt tolerance in tomato. FEBS Lett 554:417–421PubMedCrossRefGoogle Scholar
  43. 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 Exp Bot 49:623–647Google Scholar
  44. Noctor G, Gomez L, Vanacker H, Foyer CH (2002) Interactions between biosynthesis, compartmentation and transport in the control of glutathione homeostasis and signalling. J Exp Bot 53:1283–1304PubMedCrossRefGoogle Scholar
  45. Ogawa K (2005) Glutathione-associated regulation of plant growth and stress responses. Antioxid Redox Signal 7:973–981PubMedCrossRefGoogle Scholar
  46. Peng H, Feng Y, Zhang H, Wei X, Liang S (2011) Molecular cloning and characterization of glycine- and proline- rich proteins (GPRPs) in Soybean. Plant Mol Biol Rep. doi: 10.1007/s11105-011-0363-9
  47. Prabu G, Kawar PG, Pagariya MC, Prasad DT (2011) Identification of water deficit stress upregulated genes in sugarcane. Plant Mol Biol Rep 29:291–304CrossRefGoogle Scholar
  48. Queval G, Thominet D, Vanacker H, Miginiac-Maslow M, Gakière B, Noctor G (2009) H2O2-Activated up-regulation of glutathione in Arabidopsis involves induction of genes encoding enzymes involved in cysteine synthesis in the chloroplast. Mol Plant 2:344–356PubMedCrossRefGoogle Scholar
  49. Reddy AR, Chaitanya KV, Vivekanandan M (2004) Drought induced responses of photosynthesis and antioxidant metabolism in higher plants. J Plant Physiol 161:1189–1202CrossRefGoogle Scholar
  50. Richman PG, Meister A (1975) Regulation of γ-glutamyl cysteine synthetase by non-allosteric feedback inhibition by glutathione. J Biol Chem 250:1422–1426PubMedGoogle Scholar
  51. Rivera-Becerril F, van Tuinen D, Martin-Laurent F, Metwally A, Dietz KJ, Gianinazzi S, Gianinazzi-Pearson V (2005) Molecular changes in Pisum sativum L. roots during arbuscular mycorrhiza buffering of cadmium stress. Mycorrhiza 16:51–60PubMedCrossRefGoogle Scholar
  52. Rüeggsegger A, Brunold C (1992) Effect of cadmium on y-glutamylcysteine synthesis in maize seedlings. Plant Physiol 99:428–433CrossRefGoogle Scholar
  53. Rüeggsegger A, Brunold C (1993) Localisation of y-glutamylcysteine synthetase and glutathione synthetase activity in maize seedlings. Plant Physiol 101:561–566Google Scholar
  54. Ruiz JM, Blumwald E (2002) Salinity-induced glutathione synthesis in Brassica napus. Planta 214:965–969PubMedCrossRefGoogle Scholar
  55. Sanchez-Fernandez R, Fricker M, Corben LB, White NS, Sheard N, Leaver CJ, Von-Montagu M, Inze D, May MJ (1997) Cell proliferation and hair tip growth in the Arabidopsis root are under mechanistically different forms of redox control. Proc Natl Acad Sci USA 94:2745–2750PubMedCrossRefGoogle Scholar
  56. Schafer HJ, Haag-Kerwer A, Rausch T (1998) cDNA cloning and expression analysis of genes encoding GSH synthesis in roots of the heavy metal accumulator Brassica juncea L.: evidence for Cd-induction of a putative mitochondrial γ-glutamylcysteine synthetase isoform. Plant Mol Biol 37:87–97PubMedCrossRefGoogle Scholar
  57. Sengupta D, Reddy AR (2011) Water deficit as a regulatory switch for legume root responses. Plant Signal Behav 6:914–917PubMedCrossRefGoogle Scholar
  58. Sengupta D, Kannan M, Reddy AR (2011) A root proteomics-based insight reveals dynamic regulation of root proteins under progressive drought stress and recovery in Vigna radiata (L.) Wilczek. Planta 233:1111–1127PubMedCrossRefGoogle Scholar
  59. Smirnoff N (1993) The role of active oxygen in the response of plants to water deficit and desiccation. New Phytol 125:27–58CrossRefGoogle Scholar
  60. Smith IK (1985) Stimulation of glutathione synthesis in photorespiring by catalase inhibitors. Plant Physiol 79:1044–1047PubMedCrossRefGoogle Scholar
  61. Su Z, Li X, Hao Z, Xie C, Li M, Weng J, Zhang D, Liang X, Wang Z, Gao J, Zhang S (2011) Association analysis of the nced and rab28 genes with phenotypic traits under water stress in maize. Plant Mol Biol Rep 29:714–722CrossRefGoogle Scholar
  62. Velikova V, Yordanov I, Edreva A (2000) Oxidative stress and some antioxidant systems in acid rain-treated bean plants. Plant Sci 151:59–66CrossRefGoogle Scholar
  63. Vernoux T, Wilson RC, Seeley KA, Reichheld JP, Muroy S, Brown S, Maughan SC, Cobbett CS, Montagu MV, Inze D, May MJ, Sung ZR (2000) The ROOT MERISTEMLESS1/CADMIUM SENSITIVE 2 gene defines a glutathione dependent pathway involved in initiation and maintenance of cell division during post embryonic cell development. Plant Cell 12:97–109PubMedGoogle Scholar
  64. Wu J, Qu T, Chen S, Zhao Z, An L (2009) Molecular cloning and characterization of a γ-glutamylcysteine synthetase gene from Chorispora bungeana. Protoplasma 235:27–36PubMedCrossRefGoogle Scholar
  65. Xiang C, Bertrand D (2000) Glutathione synthesis in Arabidopsis, multilevel controls coordinate responses to stress. In: Brunold C, Rennenberg H, De Kok LJ, Stulen I, Davidian JC (eds) Sulfur nutrition and sulphur assimilation in higher plants. Paul Haupt, Bern, pp 409–412Google Scholar
  66. Xiang C, Oliver DJ (1998) Glutathione metabolic genes coordinately respond to heavy metals and jasmonic acid in Arabidopsis. Plant Cell 10:1539–1550PubMedGoogle Scholar
  67. Xiang C, Werner BL, Christensen EM, Oliver DJ (2001) The biological function of glutathione revisited in Arabidopsis transgenic plants with altered glutathione levels. Plant Physiol 126:564–574PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Debashree Sengupta
    • 1
  • Golla Ramesh
    • 2
  • Shalini Mudalkar
    • 1
  • Koppolu Raja Rajesh Kumar
    • 1
  • Pulugurtha Bharadwaja Kirti
    • 1
  • Attipalli R. Reddy
    • 1
  1. 1.Department of Plant Sciences, School of Life SciencesUniversity of HyderabadHyderabadIndia
  2. 2.Division of Haematology and Oncology, School of MedicineUniversity of PennsylvaniaPhiladelphiaUSA

Personalised recommendations