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OsGLYII-2, a glutathione responsive rice glyoxalase II, functions in salinity adaptation by maintaining better photosynthesis efficiency and anti-oxidant pool. [1]

Annotated Information


Transgenic plants ectopically expressing OsGLYII-2 maintained physiological balance under salinity stress. (from reference [1]).

Glyoxalase II is the second enzyme of the glyoxalase pathway that converts SLG (product of the GLY I enzyme) to d-lactate with recycling of one molecule of reduced glutathione. Both GLY I and GLY II transcripts and proteins have been reported to be induced in response to various abiotic stresses in plants [2][3]. The plant genome contains multiple GLY II members, three in the case of rice and five in the case of Arabidopsis, based on their common Pfam ID (PF00753;)[3]. Moreover, it was observed that AtGlx2-1 and OsGLYII-1 show activity other than GLY II [4] [5]. Functional complementation of the yeast GLY II mutant by OsGLYII-2 indicated that OsGLYII-2 is an active GLY II enzyme. Moreover, expression of the OsGLYII-2 transcript has been reported to be up-regulated in response to salinity [3].

Various kinetic parameters of OsGLYII-2 were measured and compared with other reported GLY II from diverse species. The Km and kcat/Km values for OsGLYII-2 were found to be 254 μm and 2.00 × 106 m−1 sec−1 respectively, that is higher than prokaryotic and lower than eukaryotic GLY II, and quite comparable with higher eukaryotes. Activity of the enzymes is affected by their products or substrates for a prompt response on cellular need [6]. Since one of the product of GLY II i.e. GSH, is a signalling molecule [7] and plays a vital role in maintaining cellular redox status, it might influence GLY II activity also. Under normal physiological conditions, total GLY I activity is much higher than GLY II and occupies one molecule of GSH to form SLG. Additionally, the total glutathione in plants (~4.8 μmol g−1) is relatively lower as compared with animals (16–25 μmol g−1) [8]. In this context, the product inhibition of OsGLYII-2 gains importance and a tight correlation between OsGLYII-2 activity and cellular GSH level eventually leads to maintenance of redox status. As the level of MG increases during stress, more GSH will bind to MG to form SLG. Lower levels of GSH would increase the activity of OsGLYII-2 that will regenerate GSH from SLG and also fully detoxify MG to d-lactate.

Glyoxalase II is a member of the metallo-β-lactamase superfamily and requires a divalent cation for its activity [9]. Most members of this family appear to contain a binuclear metal centre, especially zinc or iron [10]. But the metal ion content might vary depending upon the composition of growth media, purification column and buffer composition [11]. Based on a metal content study of OsGLYII-2 using ICP-AES, OsGLYII-2 may contain a binuclear Zn/Fe centre, like the one in its Arabidopsis counterpart, AtGLYII-2 [12]. Some of the other Zn/Fe binuclear proteins have already been reported from different sources, such as kidney bean purple acid phosphatases and protein phosphatases I, 2A and 2B. Interestingly, none of Zn and/or Fe could reactivate the activity when added externally in the metal chelated form. A similar pattern of metal inhibition was observed in the case of the Zn-containing UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) enzyme [13]. The inability of Zn2+ to reactivate chelated OsGLYII-2, may be due to the presence of eight cysteine residues in the OsGLYII-2 protein that could form mis-metallation by thiolate ligation as observed previously [14]. Moreover, the activity of chelated OsGLYII-2 protein could be reactivated by incubation with Co2+ or Mn2+ because of their facile nature to insert into the active site cavity. Similar observations have been shown in the case of E. coli GLY II [14].

Methylglyoxal accumulates inside the cell under stress conditions [15] and glyoxalase enzymes protects the cells from the deleterious effects of MG. Since total GLY I activity is higher than GLY II, overexpression of GLY II provides better tolerance as compared with GLY I transgenic [16]. Previously, it has been shown that overexpression of OsGLYII-3 in tobacco, rice and Brassica enhanced tolerance against salinity [16][17][18]. These studies indicated towards the significant role of GLY II during stress that prompted us to investigate further the role of OsGLYII-2 in plants. Overexpression of GLY I leads to an increase in the enzyme activity of GLY II also [16], whereas the reverse was not observed in the case of OsGLYII-2-overexpressing tobacco transgenic plants, a finding which needs to be explored further. Transgenic plants showed significant tolerance towards dicarbonyl and salinity stresses, by resisting the accumulation of excess MG in the system.

Apart from the accumulation of MG, stress can disturb a number of other cellular processes, such as growth, photosynthesis, water balance and ion homeostasis etc. All these processes ultimately lead to a decrease in the total productivity and yield of plants. Photosynthesis machinery plays a central role to deal with the adverse conditions and was found to be affected first by stress [19]. During stress conditions, plants try to minimize water loss by closing their stomata. A low stomatal conductance leads to lower transpiration and sub-stomatal CO2 concentration that ultimately leads to a decrease in the rate of photosynthesis. Although the plants’ capability to capture light energy remains intact, this extra energy leads to the generation of excess ROS. GSH along with other components, played an important role to fight against the ROS [15]. OsGLYII-2-expressing transgenic tobacco plants can maintain physiologically favourable levels of all the important components of the photosynthesis machinery and thus are able to produce a yield with a minimum loss (~20%) under salinity stress. Transgenic plants could maintain a balance between osmotic loss and photosynthesis capability by fine tuning the stomatal conductance, transpiration rate, chlorophyll content, RWC and photosynthesis rate. However, WT plants were not able to maintain the balance that ultimately drives them towards senescence and death. Moreover, transgenic plants were able to maintain the anti-oxidant pool, by maintaining a GSH level that has been considered as an adaptation strategy for plants under stress [20].


Comparison of various growth and yield parameters of wild-type (WT) and OsGLYII-2 ectopically expressing tobacco plants, continuously grown under saline conditions. (from reference [1]).

As OsGLYII-2 transgenic plants showed better performance in dicarbonyl stress than WT, tolerance of these plants was further tested under salinity stress. For this, one set of 60-day-old transgenic plants (L1, L2, L4 and L5) along with WT were irrigated with saline water (150 mm NaCl) and another set of the same plants were irrigated with normal water (experimental control) in earthen pots until maturity. It was observed that transgenic plants were able to grow normally, flowered and produced seeds under salinity stress. However, WT plants showed stunted growth with only a few flowers and seeds. For the assessment of plant stress tolerance potential, different parameters such as plant height, fresh weight, number of pods and average weight of pods from all the plants grown under stress and control conditions were measured and compared. The transgenic plants were found to maintain higher biomass and yield than WT grown under saline conditions as well as control conditions. The number of pods in WT plants showed a 90% reduction under salinity stress while transgenic plants showed only a 30% decline in pod number under similar conditions as compared with their respective control counterparts.


Expression analysis and subcellular localization of OsGLYII-2. (from reference [1]).

The presence of multiple members of a family raises the question of their significance under various stages of plant development and tissues. To unravel the role of OsGLYII-2, its detailed expression pattern was analyzed based on publicly available microarray databases. OsGLYII-2 expression was checked at nine distinct developmental stages of rice; namely germination, seedling, tillering, stem elongation, booting, heading, flowering, and two stages of seed development (milk and dough) and data were analyzed. High transcript abundance of OsGLYII-2 was observed at all the developmental stages with slight upregulation during the late vegetative phase and a slight downregulation during the early reproductive stage. Moreover, OsGLYII-2 showed a medium to high level of expression in various tissues such as callus, seedling, leaf, flag leaf, shoot, root, inflorescence, panicle, anther, stigma, ovary, embryo and endosperm, except pollen, where its expression levels were relatively low. Apart from expression, localization of various members of the same family may also vary to perform the same job in different subcellular compartments. To check the in planta localization of the OsGLYII-2 protein, the corresponding cDNA was cloned into the pMBPII vector to make a OsGLYII-2:GFP (green fluorescent protein) fusion construct. This chimeric protein (OsGLYII-2–GFP) was expressed transiently in onion epidermal cells through particle bombardment and OsGLYII-2 was found to be localized in the cytosol as indicated through GFP visualization.


A binuclear metal centre is essential for OsGLYII-2 activity. (from reference [1]).

Previous studies have indicated the presence of varying amounts of metals bound to the active site of GLY II enzymes [21]. Thus, to know the active site residues and its coordination with metals, a homology-based structure of OsGLYII-2 was built based on the available crystal structure of human GLY II. According to the model, OsGLYII-2 is a monomeric protein consisting of two structural domains, a N-terminal domain (residues 1–171) with a two βββαβ topology and a C-terminal domain (172–258) with five folded α-helices. The structure is predominantly α-helical. Superimposition of the modelled and template structure indicated that the metal coordination residues are conserved between them and the residues of OsGLYII-2 that could form two metal-binding sites are His 54, His56, Asp58, His59, His112, Asp135 and His174. To study the exact metal content of OsGLYII-2, His-tag cleaved protein was analyzed by Inductively Coupled Plasma Atomic Emission spectroscopy (ICP-AES). The analysis indicated that there were ~1 mol of zinc, 0.9 mol of iron and 0.2 mol of manganese per mol of protein. Some other metals such as nickel, cadmium, cobalt, and copper were also found to be present in trace amounts. From this analysis, it can be inferred that OsGLYII-2 may contain a zinc/iron binuclear centre, and that is quite consistent with other GLY II. The role of bound metals of OsGLYII-2 was investigated further by measuring the activity after chelation of metals by EDTA. EDTA treatment resulted in a near complete loss of enzyme activity (~95% decrease as compared with the pre-EDTA treated form. After the removal of excess EDTA by dialysis, the metal chelated protein was incubated with different divalent ions to check their potential to reactivate the activity. As OsGLYII-2 has a Zn/Fe binuclear metal centre, GLY II activity of metal chelated OsGLYII-2 was expected to revert back by the addition of either Zn2+ or Fe2+ or both of them together. Interestingly, it was observed that the addition of zinc and/or iron was not able to reactivate the enzyme; whereas reconstitution of the protein with Co2+ resulted in a significant restoration of GLY II activity (73%) compared with the pre-EDTA treated form. These results indicated that Co2+ could reactivate the metal chelated form of a Zn/Fe-containing enzyme.

Labs working on this gene

1. Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India 2. Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India


<references> [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21]

Structured Information

Gene Name



Similar to Hydroxyacylglutathione hydrolase cytoplasmic (EC (Glyoxalase II) (Glx II)


NM_001056551.1 GI:115452830 GeneID:4332736


2956 bp


Oryza sativa Japonica Group Os03g0332400, complete gene.


Oryza sativa Japonica Group

 ORGANISM  Oryza sativa Japonica Group
           Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
           Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP
           clade; Ehrhartoideae; Oryzeae; Oryza.

Chromosome 3


Chromosome 3:12314136..12317091

Sequence Coding Region



GEO Profiles:Os03g0332400

Genome Context

<gbrowseImage1> name=NC_008396:12314136..12317091 source=RiceChromosome03 preset=GeneLocation </gbrowseImage1>

Gene Structure

<gbrowseImage2> name=NC_008396:12314136..12317091 source=RiceChromosome03 preset=GeneLocation </gbrowseImage2>

Coding Sequence


Protein Sequence


Gene Sequence


External Link(s)

NCBI Gene:Os03g0332400, RefSeq:Os03g0332400

  1. 1.0 1.1 1.2 1.3 1.4 1.5 Ghosh, A., Pareek, A., Sopory, S. K. and Singla-Pareek, S. L. (2014), A glutathione responsive rice glyoxalase II, OsGLYII-2, functions in salinity adaptation by maintaining better photosynthesis efficiency and anti-oxidant pool. The Plant Journal, 80: 93–105.
  2. 2.0 2.1 Hossain, M.A., Hossain, M.Z. and Fujita, M. (2009) Stress-induced changes of methylglyoxal level and glyoxalase I activity in pumpkin seedlings and cDNA cloning of glyoxalase I gene. Aust. J. Crop Sci. 3, 53–64.
  3. 3.0 3.1 3.2 3.3 Mustafiz, A., Singh, A., Pareek, A., Sopory, S.K. and Singla-Pareek, S.L. (2011) Genome-wide analysis of rice and Arabidopsis identifies two glyoxalase genes that are highly expressed in abiotic stresses. Funct. Integr. Genomics, 11, 293–305.
  4. 4.0 4.1 Limphong, P., McKinney, R.M., Adams, N.E., Bennett, B., Makaroff, C.A., Gunasekera, T. and Crowder, M.W. (2009) Human glyoxalase II contains an Fe(II)Zn(II) center but is active as a mononuclear Zn(II) enzyme. Biochemistry, 48, 5426–5434.
  5. 5.0 5.1 Kaur, C., Mustafiz, A., Sarkar, A.K., Ariyadasa, T.U., Singla-Pareek, S.L. and Sopory, S.K. (2014c) Expression of abiotic stress inducible ETHE1-like protein from rice is higher in roots and is regulated by calcium. Physiol. Plant. doi: 10.1111/ppl.12147.
  6. 6.0 6.1 Majumdar, R., Shao, L., Minocha, R., Long, S. and Minocha, S.C. (2013) Ornithine: the overlooked molecule in the regulation of polyamine metabolism3. Plant Cell Physiol. 54, 990–1004.
  7. 7.0 7.1 Ghanta, S. and Chattopadhyay, S. (2011) Glutathione as a signaling molecule: another challenge to pathogens. Plant Signal. Behav. 6, 783–788.
  8. 8.0 8.1 Newton, G., Arnold, K., Price, M., Sherrill, C., Delcardayre, S., Aharonowitz, Y., Cohen, G., Davies, J., Fahey, R. and Davis, C. (1996) Distribution of thiols in microorganisms: mycothiol is a major thiol in most actinomycetes. J. Bacteriol. 178, 1990–1995.
  9. 9.0 9.1 Crowder, M.W., Spencer, J. and Vila, A.J. (2006) Metallo-beta-lactamases: novel weaponry for antibiotic resistance in bacteria. Acc. Chem. Res. 39, 721–728.
  10. 10.0 10.1 Campos-Bermudez, V.A., Leite, N.R., Krog, R., Costa-Filho, A.J., Soncini, F.C., Oliva, G. and Vila, A.J. (2007) Biochemical and structural characterization of Salmonella typhimurium glyoxalase II: new insights into metal ion selectivity. Biochemistry, 46, 11069–11079.
  11. 11.0 11.1 Wenzel, N.F., Carenbauer, A.L., Pfiester, M.P., Schilling, O., Meyer-Klaucke, W., Makaroff, C.A. and Crowder, M.W. (2004) The binding of iron and zinc to glyoxalase II occurs exclusively as di-metal centers and is unique within the metallo-beta-lactamase family. J. Biol. Inorg. Chem. 9, 429–438.
  12. 12.0 12.1 Zang, T.M., Hollman, D.A., Crawford, P.A., Crowder, M.W. and Makaroff, C.A. (2001) Arabidopsis glyoxalase II contains a zinc/iron binuclear metal center that is essential for substrate binding and catalysis. J. Biol. Chem. 276, 4788–4795.
  13. 13.0 13.1 Jackman, J., Raetz, C. and Fierke, C. (1999) UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase of Escherichia coli is a zinc metalloenzyme. Biochemistry, 38, 1902–1911.
  14. 14.0 14.1 14.2 O'Young, J., Sukdeo, N. and Honek, J. (2007) Escherichia coli glyoxalase II is a binuclear zinc-dependent metalloenzyme. Arch. Biochem. Biophys. 459, 20–26.
  15. 15.0 15.1 15.2 Yadav, S.K., Singla-Pareek, S.L., Ray, M., Reddy, M.K. and Sopory, S.K. (2005) Methylglyoxal levels in plants under salinity stress are dependent on glyoxalase I and glutathione. Biochem. Biophys. Res. Commun. 337, 61–67.
  16. 16.0 16.1 16.2 16.3 Singla-Pareek, S.L., Reddy, M. and Sopory, S.K. (2003) Genetic engineering of the glyoxalase pathway in tobacco leads to enhanced salinity tolerance. Proc. Natl Acad. Sci. USA, 100, 14672–14677.
  17. 17.0 17.1 Singla-Pareek, S.L., Yadav, S.K., Pareek, A., Reddy, M. and Sopory, S.K. (2008) Enhancing salt tolerance in a crop plant by overexpression of glyoxalase II. Transgenic Res. 17, 171–180.
  18. 18.0 18.1 Saxena, M., Roy, S.D., Singla-Pareek, S.L., Sopory, S.K. and Bhalla-Sarin, N. (2011) Overexpression of the glyoxalase II gene leads to enhanced salinity tolerance in Brassica juncea. Open Plant Sci. J. 5, 23–28.
  19. 19.0 19.1 Tezara, W., Mitchell, V.J., Driscoll, S.D. and Lawlor, D.W. (1999) Water stress inhibits plant photosynthesis by decreasing coupling factor and ATP. Nature, 401, 914–917.
  20. 20.0 20.1 Tausz, M., Sircelj, H. and Grill, D. (2004) The glutathione system as a stress marker in plant ecophysiology: is a stress-response concept valid? J. Exp. Bot. 55, 1955–1962.
  21. 21.0 21.1 Sousa Silva, M., Gomes, R.A., Ferreira, A.E., Freire, P.A. and Cordeiro, C. (2013) The glyoxalase pathway: the first hundred years… and beyond. Biochem. J. 453, 1–15.