Abstract
The generation of excess reactive oxygen species (ROS) is one of the most common consequences of abiotic stress on plants. Glutathione reductase (GR, E.C. 1.6.4.2) and allied enzymes of the ascorbate–glutathione cycle play a crucial role to maintain the homeostatic redox balance in the cellular environment. GR plays an essential role in upholding the reduced glutathione pool under stress conditions. In the present study, a full-length GR cDNA and corresponding genomic clone was isolated from Pennisetum glaucum (L.) R. Br. The PgGR cDNA, encodes a 497-amino acid peptide with an estimated molecular mass of ~53.5 kDa. The PgGR peptide exhibits 54–89 % sequence homology with GR from other plants and is cytoplasmic in nature. The PgGR enzyme was purified to near homogeneity, the recombinant protein being relatively thermostable and displaying activity in a broad range of temperature, pH and substrate concentrations. The PgGR transcript level was differentially regulated by heat, cold, salinity and methyl viologen-induced oxidative stress. The heterologously expressed PgGR protein in E. coli showed an improved protection against metal- and methyl viologen-induced oxidative stress. Our overall finding underscores the role of PgGR gene that responds to multiple abiotic stresses and provides stress tolerance in the experimental model (E. coli) which can be potentially used for the improvement of crops under abiotic stress conditions.
Similar content being viewed by others
References
Suzuki N, Koussevitzky S, Mittler R, Mittler G (2012) ROS and redox signalling in the response of plants to abiotic stress. Plant Cell Environ 35:259–270
Bray CM, West CE (2005) DNA repair mechanisms in plants: crucial sensors and effectors for the maintenance of genome integrity. New Phytol 168:511–528
Foyer CH, Noctor G (2011) Ascorbate and glutathione: the heart of the redox hub. Plant Physiol 155:2–18
Sytar O, Kumar A, Latowski D, Kuczynska P, Strzałka K, Prasad MNV (2013) Heavy metals induced oxidative damage, defence reactions and de-toxification mechanisms in plants. Acta Physiol Plant 35:985–999
Lushchak VI (2012) Glutathione homeostasis and functions: potential targets for medical interventions. J Amino Acids 736837
Noctor G, Mhamdi A, Chaouch S, Han Y, Neukermans J, Marquez-Garcia B, Queval G, Foyer CH (2012) Glutathione in plants: an integrated overview. Plant Cell Environ 35:454–484
Gill SS, Anjum NA, Hasanuzzaman M, Gill R, Trivedi DK, Ahmad I, Pereira E, Tuteja N (2013) Glutathione and glutathione reductase: a boon in disguise for plant abiotic stress defense operations. Plant Physiol Biochem 70:204–212
Foyer CH, Lelandais M, Galap C, Kunert KJ (1991) Effects of elevated cytosolic glutathione reductase activity on the cellular glutathione pool and photosynthesis in leaves under normal and stress conditions. Plant Physiol 97:863–872
Guy CL, Carter JV (1984) Characterization of partially purified glutathione reductase from cold-hardened and non-hardened spinach leaf tissues. Cryobiology 21:454–464
Anderson J, Hess J, Chevone B (1990) Purification, characterization, and immunological properties for two isoforms of glutathione reductase from eastern white pine needles. Plant Physiol 94:1402–1409
Contour-Ansel D, Torres-Franklin ML, Cruz DE, Carvalho MH, D’Arcy-Lameta A, Zuily-Fodil Y (2006) Glutathione reductase in leaves of cowpea: cloning of two cDNAs, expression and enzymatic activity under progressive drought stress, desiccation and abscisic acid treatment. Ann Bot 98:1279–1287
Stevens R, Creissen G, Mullineaux PM (1997) Cloning and characterization of a cytosolic glutathione reductase cDNA from pea (Pisum sativum L.) and its expression in response to stress. Plant Mol Biol 35:641–654
Takeda T, Ishikawa T, Shigeoka S, Hirayama O, Mitsunaga T (1993) Purification and characterization of glutathione reductase from Chlamydomonas reinhardtii. Microbiology 139:2233–2238
Tang X, Webb MA (1994) Soybean root nodule cDNA encoding glutathione reductase. Plant Physiol 104:1081–1082
Kubo A, Sano T, Saji H, Tanaka K, Kondo N, Tanaka K (1993) Primary structure and properties of glutathione reductase from Arabidopsis thaliana. Plant Cell Physiol 34:1259–1266
Kaminaka H, Morita S, Nakajima M, Masmura T, Tanaka K (1998) Gene cloning and expression of cytosolic glutathione reductase in rice (Oryza sativa L.). Plant Cell Physiol 39:1269–1280
Lee H, Won SH, Lee BH, Park HD, Chung WI, Jo J (2002) Genomic cloning and characterization of glutathione reductase gene from Brassica campestris var. Pekinensis. Mol Cells 13:245–251
Lascano HR, Casano LM, Melchiorre MN, Trippi VS (2001) Biochemical and molecular characterization of wheat chloroplastic glutathione reductase. Biol Plant 44:509–516
Desingh R, Jutur PP, Reddy AR (2006) Salinity stress-induced changes in photosynthesis and antioxidative systems in three casuarina species. J Plant Biol 33:155–161
Kukreja S, Nandwal AS, Kumar N, Sharma SK, Sharma SK, Unvi V, Sharma PK (2005) Plant water status, H2O2 scavenging enzymes, ethylene evolution and membrane integrity of Cicerarietinum roots as affected by salinity. Biol Plant 49:305–308
Gueta-Dahan Y, Yaniv Z, Zilinskas BA, Ben-Hayyim G (1997) Salt and oxidative stress: similar and specific responses and their relation to salt tolerance in citrus. Planta 203:460–469
Hernandez JA, Jimenez A, Mullineaux P, Sevilla F (2000) Tolerance of pea (Pisum sativum L.) to long term salt stress is associated with induction of antioxidant defenses. Plant Cell Environ 23:853–862
Vaidyanathan H, Sivakumar P, Chakrabarty R, Thomas G (2003) Scavenging of reactive oxygen species in NaCl-stressed rice (Oryza sativa L.) differential responses in salt tolerant and sensitive varieties. Plant Sci 165:1411–1418
Khan F, Siddiqi TO, Mahmooduzzafar AA (2009) Morphological changes and antioxidant defence systems in soybean genotypes as affected by salt stress. J Plant Interact 4:295–306
Mittova V, Guy M, Tal M, Volokita M (2004) Salinity up-regulates the antioxidative system in root mitochondria and peroxisomes of the wild salt-tolerant tomato species Lycopersicon pennellii. J Exp Bot 55:1105–1113
Sairam RK, Srivastava GC, Agarwal S, Meena RC (2005) Differences in antioxidant activity in response to salinity stress in tolerant and susceptible wheat genotypes. Biol Plant 49:85–91
Sreenivasulu N, Grimm B, Wobus U, Weschke W (2000) Differential response of antioxidant compounds to salinity stress in salt-tolerant and salt-sensitive seedlings of foxtail millet (Setaria italica). Physiol Plant 109:435–442
Desingh R, Kanagaraj G (2007) Influence of salinity stress on photosynthesis and antioxidative systems in two cotton varieties. Gen Appl Plant Physiol 33:221–234
Smirnoff N, Colombe SV (1988) Drought influences the activity of enzymes of the chloroplast hydrogen peroxide scavenging system. J Exp Bot 39:1097–1108
Jiang M, Zhang J (2002) Water stress-induced abscisic acid accumulation triggers the increased generation of reactive oxygen species and up-regulates the activities of antioxidant enzymes in maize leaves. J Exp Bot 53:2401–2410
Pastori GM, Trippi VS (1992) Oxidative stress induces high rate of glutathione reductase synthesis in a drought resistant maize strain. Plant Cell Physiol 33:957–961
Sairam RK, Shukla DS, Saxena DC (1997) Stress-induced injury and antioxidant enzymes in relation to drought tolerance in wheat genotypes. Biol Plant 40:357–364
Selote DS, Chopra RK (2006) Drought acclimation confers oxidative stress tolerance by inducing co-ordinated antioxidant defense at cellular and subcellular level in leaves of wheat seedlings. Physiol Plant 127:494–506
Selote DS, Chopra RK (2004) Drought-induced spikelet sterility is associated with an inefficient antioxidant defense in rice panicles. Physiol Plant 121:462–471
Sharma P, Dubey RS (2004) Ascorbate peroxidase from rice seedlings: properties of enzyme isoforms, effects of stresses and protective roles of osmolytes. Plant Sci 67:541–550
Dhindsa RS (1991) Drought stress, enzymes of glutathione metabolism, oxidative injury and protein synthesis in Tortula ruralis. Plant Physiol 95:648–655
Kim DW, Shibato J, Agrawa GK, Fujihara S, Iwahashi H, Kim DH, Shim IS, Rakwal R (2007) Gene transcription in the leaves of rice undergoing salt-induced morphological changes (Oryza sativa L.). Mol Cells 24:45–59
Tsaia YC, Hongb CY, Liua LF, Kao CH (2005) Expression of ascorbate peroxidase and glutathione reductase in roots of rice seedlings in response to NaCl and H2O2. J Plant Physiol 162:291–299
Baek KH, Skinner DZ (2003) Alteration of antioxidant enzyme gene expression during cold acclimation of near-isogenic wheat lines. Plant Sci 165:1221–1227
Romero-Puertas MC, Corpas FJ, Sandalio LM, Leterrier M, Rodríguez-Serrano M, del Río LA, Palma JM (2006) Glutathione reductase from pea leaves: response to abiotic stress and characterization of the peroxisomal isozyme. New Phytol 170:43–52
Aono M, Kubo A, Saji H, Tanaka K, Kondo N (1993) Enhanced tolerance to photooxidative stress of transgenic Nicotiana tabacum with high chloroplastic glutathione reductase activity. Plant Cell Physiol 34:29–135
Ding S, Lu Q, Zhang Y, Yang Z, Wen X, Zhang L, Lu C (2009) Enhanced sensitivity to oxidative stress in transgenic tobacco plants with decreased glutathione reductase activity leads to a decrease in ascorbate pool and ascorbate redox state. Plant Mol Biol 69:577–592
Le Martret B, Poage M, Shiel K, Nugent GD, Dix PJ (2011) Tobacco chloroplast transformants expressing genes encoding dehydroascorbate reductase, glutathione reductase, and glutathione-S-transferase, exhibit altered anti-oxidant metabolism and improved abiotic stress tolerance. Plant Biotechnol J 9:661–673
Nakashima K, Shinwar ZK, Sakuma Y, Seki M, Miura S, Shinozaki K, Yamaguchi-Shinozaki K (2000) Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration- and high salinity-responsive gene expression. Plant Mol Biol 42:657–665
Bressan RA, Park HC, Orsini F, Oh D, Dassanayake M, Inan G, Yun G, Bohnert HJ, Maggio A (2013) Biotechnology for mechanisms that counteract salt stress in extremophile species: a genome-based view. Plant Biotech Rep 7:27–37
Hedimbi M, Ananias NK, Kandawa-schulz M (2012) Effects of storage conditions on viability, germination and sugar content of pearl millet (Pennisetum glaucum) grain. J Res Agric 1:088–092
Lopes MS, Araus JL, van Heerden PDR, Foyer CH (2011) Enhancing drought tolerance in C4 crops. J Exp Bot 62:3135–3153
Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159
Mishra RN, Ramesha A, Kaul T, Nair S, Sopory SK, Reddy MK (2005) A modified cDNA subtraction to identify differentially expressed genes from plants with universal application to other eukaryotes. Anal Biochem 345:149–157
Karan R, Singla-Pareek SL, Pareek A (2009) Histidine kinase and response regulator genes as they relate to salinity tolerance in rice. Funct Integr Genomics 9:411–417
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739
Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201
Sumathi K, Ananthalakshmi P, Roshan MN, Sekar K (2006) 3dSS: 3D structural superposition. Nucleic Acids Res 34:W128–W132
Jiang MY, Zhang JH (2001) Effect of abscisic acid on active oxygen species, antioxidative defence system and oxidative damage in leaves of maize seedlings. Plant Cell Physiol 42:1265–1273
Li Z, Liu X, Chu Y, Wang Y, Zhang Q, Zhou X (2011) Cloning and characterization of a 2-Cys peroxiredoxin in the pine wood nematode, Bursaphelenchus xylophilus, a putative genetic factor facilitating the infestation. Int J Biol Sci 7:823–836
Gomez KA, Gomez AA (1984) Statistical procedures for agricultural research. Wiley, New York
Jones DC, Ariza A, Chow WH, Oza SL, Fairlamb AH (2010) Comparative structural, kinetic and inhibitor studies of Trypanosoma brucei trypanothione reductase with T. cruzi. Mol Biochem Parasitol 169:12–19
Yousuf PY, Hakeem KUR, Chandna R, Ahmad P (2012) Role of glutathione reductase in plant abiotic stress. In: Ahmad P, Prasad MNV (eds) Abiotic stress responses in plants. Springer, New York, pp 149–158
Gadea J, Conejero V, Vera P (1999) Developmental regulation of a cytosolic ascorbate peroxidase gene from tomato plants. Mol Gen Genet 262:212–219
Chew O, Whelan J, Millar AH (2003) Molecular definition of the ascorbate-glutathione cycle in Arabidopsis mitochondria reveals dual targeting of antioxidant defenses in plants. J Biol Chem 278:46869–46877
Mahmood Q, Ahmad R, Kwak SS, Rashid A, Anjum NA (2010) Ascorbate and glutathione: protectors of plants in oxidative stress. In: Anjum NA et al (eds) Ascorbate–glutathione pathway and stress tolerance in plants. Springer, Dordrecht
Pang CH (2010) Wang BS (2010) Role of ascorbate peroxidase and glutathione reductase in ascorbate glutathione cycle and stress tolerance in plants. In: Umar S, Chan MT (eds) Anjum NA. Springer, Ascorbate glutathione pathway and stress tolerance in plants, pp 91–113
Turner B, Pollock CJ (1993) The effects of temperature and pH on the apparent Michaelis constant of glutathione reductase from maize (Zea mays L.). Plant Cell Environ 16:289–295
Hakam N, Simon JP (2005) Molecular forms and thermal and kinetic properties of purified glutathione reductase from two populations of barnyard grass (Echinochloa crus-galli (L.) Beauv.: Poaceae) from contrasting climatic regions in North America. Can J Bot 78:969–980
Stevens RG, Creissen GP, Mullineaux PM (2000) Characterization of pea cytosolic glutathione reductase expressed in transgenic tobacco. Planta 211:537–545
Arias DG, Marquez VE, Beccaria AJ, Guerrero SA, Iglesias AA (2010) Purification and characterization of a glutathione reductase from Phaeodactylum tricornutum. Protist 161:91–101
Liu ZJ, Zhang XL, Bai JG, Suo BX, Xu PL, Wang L (2009) Exogenous paraquat changes antioxidant enzyme activities and lipid peroxidation in drought-stressed cucumber leaves. Sci Hortic 121:138–143
Waraich EA, Ahmad R, Halim A, Aziz T (2012) Alleviation of temperature stress by nutrient management in crop plants: a review. J Soil Sci Plant Nutr 12:221–244
Szalai G, Kellős T, Galiba G, Kocsy G (2009) Glutathione as an antioxidant and regulatory molecule in plants under abiotic stress conditions. J Plant Growth Regul 28:66–80
Aono M, Kubo A, Saji H, Natori T, Tanaka K, Kondo N (1991) Resistance to active oxygen toxicity of transgenic Nicotiana tabacum that expresses the gene for glutathione reductase from Escherichia coli. Plant Cell Physiol 32:691–697
Park SH, Yi N, Kim YS, Jeong MH, Bang SW, Choi YD, Kim JK (2010) Analysis of five novel putative constitutive gene promoters in transgenic rice plants. J Exp Bot 61:2459–2467
Yi N, Kim YS, Jeong MH, Oh SJ, Jeong JS, Park SH, Jung H, Choi YD, Kim JK (2010) Functional analysis of six drought-inducible promoters in transgenic rice plants throughout all stages of plant growth. Planta 232:743–754
Acknowledgments
The authors are thankful to the ICGEB, New Delhi for providing support and facilities to carry out the research. The authors thank Prof. B. B. Panda, Department of Botany, Berhampur University, India, who read and helped for improvement of the manuscript. The award of Research Associateship by Department of Biotechnology (DBT) India, to Dr. V. M. M. Achary is acknowledged.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Achary, V.M.M., Reddy, C.S., Pandey, P. et al. Glutathione reductase a unique enzyme: molecular cloning, expression and biochemical characterization from the stress adapted C4 plant, Pennisetum glaucum (L.) R. Br. Mol Biol Rep 42, 947–962 (2015). https://doi.org/10.1007/s11033-014-3832-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11033-014-3832-z