Characterization and functional validation of glyoxalase II from rice

Protein Expression and PuriWcation 51 (2007) 126–132 www.elsevier.com/locate/yprep

Characterization and functional validation of glyoxalase II from rice Sudesh Kumar Yadav a,1,2, Sneh L. Singla-Pareek a,¤,2, Manoj Kumar b, Ashwani Pareek b, Mukesh Saxena b, Neera B. Sarin b, S.K. Sopory a a

Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi 110 067, India b School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India Received 22 May 2006, and in revised form 6 July 2006 Available online 16 July 2006

Abstract Glyoxalase II, one of the enzymes of the glyoxalase pathway, cDNA cloned from rice (OsglyII) consists of 1623 nucleotides with an open reading frame of 1010 bp encoding a polypeptide of 336 amino acids and an estimated isoelectric point of 8.08. The recombinant protein puriWed from Escherichia coli using Ni–NTA aYnity chromatography showed molecular mass of »37 kDa. Catalytic parameters of the protein were determined using S-D-lactoylglutathione as a thioester substrate. The Km (61 M) and Kcat (301 s¡1) values were lower than those reported for Arabidopsis, human and yeast and showed pH optima at 7.2. The E. coli overexpressing OsglyII were able to grow on higher concentration of methylglyoxal. Transcript analysis in rice showed that OsglyII gene expression is stimulated within 15 min in response to various abiotic stresses as well as treatment with abscisic acid or salicylic acid. This multistress response of OsglyII gene documents its future utility in developing tolerance to various stresses in crop plants. © 2006 Elsevier Inc. All rights reserved. Keywords: Rice; Glyoxalase II; Characterization; Functional validation; Abiotic stresses; Abscisic acid

The glyoxalase pathway is a two step enzyme catalyzed reaction involving glyoxalase I and glyoxalase II enzyme. In the Wrst step of the pathway, glyoxalase I (glyI, lactoylglutathione lyase; EC 4.4.1.5)3 catalyzes the formation of S-D-lactoylglutathione (SLG) from methylglyoxal (MG) and glutathione (GSH) [1]. During the second step, SLG is hydrolyzed by glyoxalase II (glyII, hydroxyacylglutathione hydrolase; EC 3.1.2.6) and GSH is released leading to the formation of D-lactate [2,3]. During normal physiological conditions, MG is produced primarily through glycolysis at triose-phosphate step and also as a result of catabolism of amino acids and acetone [3,4]. Recently, we have reported that MG levels increase upon exposure of plants to various abiotic stresses

*

Corresponding author. Fax: +91 11 26162316. E-mail address: [email protected] (S.L. Singla-Pareek). 1 Present address: Biotechnology Division, Institute of Himalayan Bioresource Technology, Palampur 176061, India. 2 Both the authors have contributed equally to this work. 3 Abbreviations used: SLG, S-D-lactoylglutathione; MG, methylglyoxal; GSH, glutathione; glyII, glyoxalase II; glyI, glyoxalase I. 1046-5928/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2006.07.007

[5]. It is known that at higher concentrations, MG is harmful to the system as it is a potent cytotoxin and reacts with the major macromolecules like RNA, DNA and protein [6]. Apart from MG, pathway intermediate SLG (substrate for glyoxalase II) has also been shown to be cytotoxic at higher concentrations by inhibiting DNA synthesis [7]. Therefore, both the enzymes of glyoxalase system play a major role in chemical detoxiWcation of harmful metabolites [7,8]. Our recent studies on this pathway in plants have proven its role in providing salinity and heavy metal tolerance [9–11]. Our studies have also shown that the maintenance of glutathione homeostasis under stress condition is one of the key factor in conferring tolerance in plants overexpressing glyoxalase genes [5,12]. Despite functional analysis, the genomic and enzymatic studies on these two enzymes of the pathway are very limited. The glyI has been cloned and characterized from a few plants [9,13–16] and its role in stress tolerance has also been indicated [9,14,17]. The glyII has also been cloned from few organisms including humans [18,19], yeast [20], and African Trypanosomes [6]. In humans, both cytosolic and mitochondrial forms have been reported

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[18]. Detailed glyII enzyme puriWcation and characterization has been reported from Aloe vera and spinach [21,22] and in Arabidopsis thaliana [23,24], where Wve diVerent isoforms of glyII have been identiWed [23]. Three of these isoforms appears to be mitochondrial (GLX2-1, GLX2-4 and GLX25), while GLX2-2 is found to be cytosolic. Recently, glyII protein has also been characterized from Brassica juncea [25]. Biochemical characterization of glyII demonstrated that in all species, including yeast, human and Arabidopsis, the enzyme contains a highly conserved metal binding motif (THXHXDH). A similar motif was earlier reported in the metallo--lactamases family of enzymes that showed greater aYnity to Zn(II) [23,26–28]. Based on the structural similarity of glyII with metallo--lactamase, the glyII binuclear metal binding centre has been localized in Arabidopsis which was further shown to be essential for its substrate binding aYnity and catalytic activity [29]. We have earlier reported on the characterization and functional analysis of glyoxalase I in plants [9,10]. Molecular cloning of glyI from Brassica juncea, protein expression and puriWcation and also its overexpression in tobacco imparting tolerance to the transgenic plants during salt stress was the Wrst detailed study evincing the role of glyoxalase system in salinity tolerance in plants [9]. Following this, the overexpression of both the enzymes together, glyI and glyII, in tobacco plants has been shown to be better for generating stress tolerant plants [10]. However the detailed account on the molecular cloning and biochemical characterization of rice glyoxalase II (OsglyII) has not been attempted and in this report we present the data for the same. We observed the independent role of OsglyII in MG detoxiWcation upon its expression in Escherichia coli. For the Wrst time, it has been shown that OsglyII transcript levels increased not only with salt stress but with various other abiotic stresses as well. This documents the utility of OsglyII in developing tolerance to various stresses in crop plants. Materials and methods Cloning and sequence analysis of OsglyII The rice glyII cDNA (OsglyII) was cloned by PCR based approach from rice cDNA library [30]. Sequence analysis revealed one full length cDNA clone of OsglyII (Accession No. AY054407) based on homology with Accession No. U90927 [23]. For homology analysis, BLAST searches were conducted using GenBank. GlyII protein sequences were aligned using the ClustalW multiple alignment program (MacVector). Dendrogram was prepared from amino acid sequences between various glyII sequences already reported in the Data Bank using the MacVector. Heterologous expression and puriWcation of OsglyII The full length OsglyII cDNA was PCR ampliWed using forward primer with NdeI site 5⬘-GGGAATTCCATAT

127

GCGGATGCTGTCCAAGGC-3⬘ and reverse primer with BamHI site 5⬘-CGCGGATCCTTAAAAGTTATCCTTC GCTCGTC-3⬘ (the restriction site in both the primers is underlined) and cloned into NdeI and BamHI site of bacterial expression vector pET28a (Novagen). The expression plasmid was transformed into BL21 (DE3) pLysE E. coli cells and OsglyII protein was overexpressed using 0.5 mM IPTG for 4h. The recombinant native OsglyII protein was puriWed by Ni–NTA aYnity chromatography as per the manufacturer’s instructions (Invitrogen). The protein concentration was determined following the Bradford method [31]. The puriWed protein was checked by Western blotting using anti-His antibodies and used for raising polyclonal antibodies in rabbit [32] as well for its biochemical characterization. Determination of OsglyII activity and steady state kinetics The OsglyII activity was estimated following the method of Allen et al. [33]. The assay mixture contained 50 mM Tris–HCl (pH 7.2), 300 M SLG and 20–25 ng OsglyII protein. The rate of hydrolysis of SLG at 240 nm was read using a Cary IE UV–visible spectrophotometer ( D 3.1 mM¡1 cm¡1). The kinetic parameters (Km and Vmax) of the puriWed enzyme were determined by measuring the activity using diVerent concentrations of SLG (10–200 M) and observed values were plotted in double reciprocal plot. The optimum pH for its maximal activity was determined at a Wxed concentration of SLG (200 M) using buVers of diVerent pH (ammonium phosphate buVer, 4–6 pH and Tris–HCl, 6–10 pH). Stress inducibility of OsglyII transcript Seven day old seedlings of rice were subjected to various abiotic stresses (heat stress, 45 °C; cold stress, 4 °C; salt stress, 200 mM NaCl; desiccation stress, air drying at RT; Abscisic acid, 100 M; Salicylic acid, 100 M) for diVerent time points (15 min–2 h) according to the procedure described in Pareek et al. [32]. Total RNA was isolated from shoot tissues following the standard protocol [34]. Northern blot was prepared using 20 g total RNA and was probed with radiolabelled OsglyII cDNA. Hybridization was carried at 65 °C in 5£ SSC, 5£ Denhardt’s, 0.1% SDS and 100 g/ml denatured salmon sperm DNA for 16–18 h. Membrane was washed twice in 0.5£ SSC, 0.1% SDS and 0.1£ SSC, 0.1% SDS for 15 min each at 65 °C and scanned on Phosphorimager (Nikon). Results and discussion Molecular cloning and sequence comparison of OsglyII cDNA with other glyII in the data bank The sequence analysis of the cloned gene encoding for glyII enzyme of glyoxalase pathway from rice (OsglyII) revealed that the cDNA clone is 1623 bp long with an open reading frame of 1010 bp (representing the full length glyII

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systems tested in this study. Fig. 1a presents the multiple alignment of amino acid sequence of glyII from rice, Arabidopsis, yeast and human. The OsglyII shared 57% amino acid sequence identity with Arabidopsis, whereas the identity with human and yeast glyII was 30% and 24%, respectively. Certain positions of the aligned sequences have identical residues (Fig. 1a), e.g. the highly conserved metal binding motif (THHHXDH), involving a cluster of histidines centered around position 135 among plants and 60 among yeast and humans. About 55 residues away from the conserved histidine cluster (THHHXDH), another highly conserved sequence G/CHT is also present in all the sequences (Fig. 1a). Such conserved regions were also recognized previously in all other known glyII sequences [18,23,24].

coding sequence) and a 108 bp long 5⬘ UTR and 504 bp long 3⬘ UTR. The deduced amino acid sequence length for glyII was found to be 336 residues. The estimated molecular mass of the protein was »37 kDa with an isoelectric point of 8.08. The glyII from Arabidopsis and spinach show acidic pIs ranging from 4.5 to 6.2 [22,23]. However, the pI of animal glyII enzymes is also in the basic range similar to OsglyII [18,19]. This situation may be explained by the presence of more conserved basic amino acid regions in the OsglyII protein. The sequence conservation of glyII protein across various evolutionarily diverse genera was tested. An alignment of the deduced amino acid sequence of the OsglyII and several homologues revealed it to be conserved among the various a

Rice AY 054407 A. t. U90927 Ye ast Y10292 Hu man X90999

1 MRML S K A CS L V A S S - L P RCS S S A A P T I R GQ P S L L P S V R K E WL GK P L L Y GI GT L L V MP L RT L H GV G RMF G A GR F L C NMT S V S S S L Q I E L V P 89 1 MP V I S K A S S T T T N S S I P S CS R- - - - - I G GQ L CV WP GL R QL C L RK S L L Y GV MWL L S MP L K T L R GA R K T L K I T - HF C S I S N MP S S L K I E L V P 84 1 MQ V K S I K 7 1

MK V E V L P 7

* Rice AY 054407 90 C L Q D- - - - NY A Y I L HDV DT G T V GV V DP S E A T P I I N A L E K - R NQNL T Y I L NT H HH Y DHT GGN - - 85 C S K D- - - - NY A Y L L HDE DT G T V GV V DP S E A A P V I E A L S R- K NWNL T Y I L NT H HH DDHI GGN - - A. t. U90927 8 MRWE S GGV NY C Y L L S DS K NK K S WL I DP A E P P E V L P E L T E DE K I S V E A I V NT H HH Y DHA DGNA DI Ye ast Y10292 Hu man X90999 8 A L T D- - - - NY MY L V I DD E T K E A A I V DP V QP QK V V D A A R K - H GV K L T T V L T T H HH WDHA GGNE - -

- - - - L E L K A K Y GA K V I GS A K D RDRI P 167 - - - - A E L K E RY GA K V I GS A V D K DRI P 162 L K Y L K E K N P T S K V E V I GGS - - - K DC P 94 - - - - K L V K L E S GL K V Y GGD - - - DRI G 83

**

Rice AY 054407 A. t. U90927 Ye ast Y10292 Hu man X90999

168 G I D I T L S E GDT WMF A GH QV L V ME T P GHT S G HV CY H F - - P GS G- - - A I F T GDT L F S L S C GK L F E GT P QQMY S S L QK I I A - - - - - - L P DE T R 163 G I D I L L K DS DK WMF A GH E V R I L DT P GHT QG HI S F Y F - - P GS A - - - T I F T GDL I Y S L S C GT L S E GT P E QML S S L QK I V S - - - - - - L P DDT N 95 K V T I I P E NL K K L H L GDL E I T CI RT P CHT RD S I CY Y V K D P T T DE R- CI F T GDT L F T A GC GRF F E GT GE E MDI A L NN S I L E T V GR QN WS K T R 84 A L T HK I T HL S T L Q V GS L NV K CL A T P CHT S G HI CY F V S K P GG S E P P A V F T GDT L F V A GC GK F Y E GT A DE MCK A L L E V L GR - - - - - L P P DT R

Rice AY 054407 A. t. U90927 Ye ast Y10292 Hu man X90999

247 V Y C GHE Y T L S NS K F A L S I E P GNK D L Q EY A A N A A D L RK RNT P T V P T T I GRE K Q CN P F L RT S S P E I K NT L S I P - DHF D DA R V L E V V R RA K D N 242 I Y C GRE NT A G NL K F A L S V E P K NE T L Q SY A T R V A H L RS QGL P S I P T T V K V E K A CN P F L R I S S K DI R K S L S I P - DS A T E A E A L RR I Q RA RD R 184 V Y P GHE Y T S D NV K F V RK I Y P QV GE NK AL DE L E QF CS K HE V T A GRF T L K DE V E F N P F MRL E DP K V Q K A A G DT N NS WD RA Q I MDK L R A MK N R 169 V Y C GHE Y T I N NL K F A RHV E P GNA A I R EK L A WA K E K Y S I GE P T V P S T L A E E F T Y N P F MRV RE K T V Q QHA G E T - DP V T T MR A V R- - - RE K D Q

Rice AY 054407 A. t. U90927 Ye ast Y10292 Hu man X90999

336 F N 337 331 331 F 275 274 MN 2 5 5 F K MP R D N 2 6 1

b

246 241 183 168

335 330 273 254

0.401

Plasmodium glyII AF486285 0.367

Trypanosoma glyII AJ492819 0.386 0.004

Yeast glyII Y10292

0.016 0.296

Human glyII X90999 0.014

0.004

Pennisetum glyII AF508863

0.02 0.171

0.002

Rice glyII AY054407 0.148

0.025

Brassica glyII AY185202 0.203

Arabidopsis glyII U90927

Fig. 1. Comparison of OsglyII and representative glyoxalase II from other evolutionarily diverse organisms. (a) Alignment of the deduced amino acid sequences of OsglyII from rice (AY054407) with those from Arabidopsis thaliana (U90927), yeast (Y10292) and human (X90999). Amino acids are numbered from the beginning of the OsglyII sequence. Gaps are represented as ‘-’. The asterisk (¤) indicate the metal binding site (THHHXDH) conserved in all the aligned protein sequences and the double asterisk (¤¤) indicate the conserved third histidine residue as G/CHT sequence. (b) Phylogenetic relationship of OsglyII with that from other systems. The dendrogram was constructed using MacVector Clustal W alignment software. The sequences used for alignment are from diverse group of organism including Plasmodium, Trypanosoma, yeast, human and various plants. The accession number of each of the sequences is given along with the name of organism. Note that the plant glyII forms a close group and are divergent from the lower organisms.

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Further, a dendrogram was drawn by comparing the glyII amino acid sequences from various plant species reported in the data bank and also from evolutionarily diverse species such as humans, yeast, Trypanosoma and Plasmodium. This clustal analysis indicated that rice and Pennisetum glyII are closest in evolution (Fig. 1b), and rice, Arabidopsis, Pennisetum and Brassica form one close group which is diVerent from the group of Trypanosoma, humans and yeast. Interestingly, Plasmodium forms a distant divergent group. PuriWcation and characterization of OsglyII overexpressed in E. coli OsglyII cDNA was ampliWed and cloned into the NdeI and BamHI site of pET 28a vector for its expression in E. coli (Fig. 2a). The expression of OsglyII was induced by applying 0.5 mM IPTG for 4 h. The translation product of the entire OsglyII ORF resulted in at least 60% soluble product therefore only the soluble fraction was used further. The puriWcation was carried out by routine aYnity chromatography with immobilized Ni–NTA. The recombinant OsglyII protein so obtained was highly puriWed as it showed only single band of 39 kDa (fused with histidine tag) on the SDS–polyacrylamide gel stained with Coomassie brilliant blue R250. The protein was maximally recovered in the 2nd and 3rd eluted fractions (Fig. 2b). This

a

Gly I I

N d eI T7 Prom

b

UN

BamHI

MCS

I

T 7!

M

Kanamycin

F1 origin

1F

2F

3F

94 66 44 30

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puriWed protein was used for raising polyclonal antibodies in rabbit. The antibodies were found to be speciWc as protein blot (Western blot) of the puriWed protein showed only a single cross-reacting band when probed with anti-OsglyII antibodies (Fig. 2c). These antibodies also showed a major cross-reacting band of glyII in total protein extracts from leaves of glyoxalase overexpressing transgenic tobacco [10] and transgenic rice plants [unpublished results]. Before characterization of the OsglyII protein, it was crucial to check whether the overproduced protein is functionally active. For this we checked whether overproduction of OsglyII protein in bacteria can help it to tolerate toxic levels of methylglyoxal (a potent cytotoxin). This test was based on the reasoning that if the overproduced protein is functionally active, it should allow proliferation of bacteria on an otherwise lethal concentration of MG. For this, the OsglyII cDNA was functionally expressed in E. coli where it allowed proliferation of bacterial cells over a wide range of otherwise toxic MG concentration. The E. coli cells having recombinant and control plasmids were grown on media containing increasing concentrations of MG. Fig. 3 show that the growth of E. coli containing control plasmid was completely inhibited at a 10 mM concentration of MG while the cells overexpressing OsglyII showed an 80% survival (in number) even at 35 mM MG. In an earlier study with glyI overproducing E. coli, the recombinant plasmid containing bacterial cells were observed to tolerate up to 20 mM MG [9]. This shows that the overproduced glyI or glyII enzyme remains active in the heterologous system as well and glyII expression can lead to tolerance to higher concentrations of MG. Having established the functional activity of the OsglyII protein, the puriWed protein was used for further kinetic characterization. The steady state kinetics of OsglyII protein was carried out with SLG. The Km value for the enzyme was determined as 61 M from the double reciprocal plot (Fig. 4a). The kcat value was determined as 301 s¡1 for SLG. The calculated Kcat/Km value was 4.9 £ 106. All the kinetic parameters observed for OsglyII are of slightly

20 66

UN

I

M

1F

2F

3F

45 35 25 Fig. 2. Overexpression, puriWcation and characterization of recombinant OsglyII. (a) OsglyII cloned into the NdeI and BamHI site of pET 28a vector for the purpose of expression in E. coli. (b) An amount of 2 g of protein (puriWed by Ni–NTA aYnity column chromatography) was separated on 10% SDS–PAGE and stained with Coomassie blue. The band of puriWed OsglyII has been marked with an arrow. (c). Western blot of the respective samples as shown in Coomassie stained gel probed with antiOsglyII antibodies (1:5000 dilution), raised from the puriWed recombinant protein. Lanes: UN, uninduced; I, 0.5 mM IPTG induced fraction; M, low molecular mass markers; 1F, 2F, 3F represents 1st, 2nd and 3rd eluted fractions.

% Survival of Colonies

120

c

pET 28a+

100

pET 28a+ Osgly II

80 60 40 20 0 0

5

10

15

20

25

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35

Methylglyoxal (mM) Fig. 3. For methylglyoxal cytotoxicity test on OsglyII overexpressing bacteria, E.coli cells transformed with pET 28a and pET 28a + OsglyII vectors were plated on LB kanamycin medium supplemented with 1 mM IPTG and various concentrations of methylglyoxal (0–35 mM). The % survival denotes the number of colonies obtained.

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a

1/µmol/min/mg protein

Line-Weaver Burk Plot

a

-1 5

-2 0

-1 0

-1/Km (mM-1)

Glyoxalase II activity (µmol/min /mg protein)

b

C

0.015

15’

Cold 30’ 1h

Abscis ic acid 2h 15’ 30’ 1h 2h

Salicy lic acid 15’ 30’ 1h 2h

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-5

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1/S-D-Lactoylglutathione (mM -1 )

50 40 30 20 10 0 6

Heat 15’ 30’ 1h 2h

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Salinity 30’ 1h

2h

15’

0.02

80

4

Desiccation 30’ 1h 2h

0.025

0.005

-2 5

C 15’

6.5 6.75 7 7.25 7.5 8

9

10

pH Fig. 4. Steady state kinetics of the recombinant OsglyII puriWed from E. coli shown by Lineweaver–Burk plot obtained by plotting the inverse of OsglyII activity on Y-axis and inverse of substrate (SLG) concentration on X-axis. This plot was used for the determination of Km and Vmax of OsglyII (a). (b) pH optima curve of OsglyII follow a typical bell in shape. For the determination of OsglyII activity at 4–6 pH and 6–10 pH, 50 mM ammonium phosphate and 50 mM Tris–HCl buVers were used respectively keeping other things constant as described in Materials and methods.

lower magnitude than those reported for Arabidopsis, yeast (Glo2, cytosolic and Glo4, mitochondrial) and human [19,24,35]. This suggests that the OsglyII has high aYnity for its substrate, SLG. Besides kinetic analysis, pH dependence of OsglyII enzyme activity was also analyzed. The OsglyII showed higher enzyme activity in the pH range of 6.5–8.0, with pH optima for SLG at 7.2 (Fig. 4b). The glyII enzyme from Arabidopsis, yeast and humans also show maximum activity in the similar pH range [19,24,35]. DiVerential induction of rice glyII by various abiotic stresses In order to Wnd out the inducibility of OsglyII, rice seedlings were subjected to various stresses as detailed in materials and methods and the expression was monitored over a period of 2 h. Northern blot analysis employing OsglyII probe showed clear induction under desiccation, salinity, heat and cold stress (Fig. 5). Additionally, expression pattern of OsglyII was also analyzed in response to abscisic acid and salicylic acid. This analysis indicated the OsglyII to be inducible by the multiple stress agents tested in this study in a time-dependent and diVerential manner. This is a novel and important

Fig. 5. Induction of OsglyII transcript by multiple forms of stress. (a) Total RNA (20 g) isolated from shoots of rice (treated with diVerent stresses and for various time points as shown on top of the Wgure) was probed with radiolabelled OsglyII cDNA. Experiments were repeated three times using samples from independent treatments. C (Control), desiccation (air drying at RT), salinity stress (200 mM NaCl), heat stress (45 °C), cold stress (4 °C), abscisic acid (100 M), salicylic acid (100 M). (b) The lower panel shows ethidium bromide stained corresponding gel for equal loading of RNA used for blotting.

observation as in no other previous studies pertaining to characterization of glyII gene, this has been reported with respect to its stress regulation. All these stress agents resulted in the accumulation of transcript in a short duration of 15 min but beyond that the transcript accumulation was found to be diVerential, e.g. salinity and desiccation showed accumulation of transcript till 2 h whereas heat and cold stress showed a bell shaped curve with decreased transcript accumulation within 2 h. In contrast, OsglyII transcript accumulated signiWcantly within 15 min of exposure to ABA that remained high within the duration of 2 h. There was a progressive decline in the level of transcript as induced after 15 min of salicylic acid treatment. The rapid accumulation of the glyII transcript by various inducers clearly suggests towards the role of this gene in early stress response. In literature, the diVerential expression of at least two isoforms of glyII in Arabidopsis has been shown in diVerent tissues such as root, leaf, bud and Xower [23]. The other enzyme of the glyoxalase pathway, glyI, from tomato has been shown to be upregulated under salt and water stress [14] and in Brassica by salt, mannitol and zinc stress [9]. This suggests that both the enzymes of glyoxalase pathway become upregulated under diVerent stresses. However, the diVerential pattern of accumulation of transcripts under diVerent stresses warrants further investigation. The in silico analysis of the promoter region of OsglyII has indicated the presence of speciWc cis-regulatory sequences such as HSE, LTRE, ABRE and DRE (unpublished data) further supporting the multiple stress inducibility of this gene. In the glyoxalase pathway, which of the two enzymes govern the committing step is still not clear. Maintenance of MG levels during salinity stress in transgenic plants overexpressing glyI and glyII individually and together in the same plant has convincingly indicated that both the enzymes have a role in MG detoxiWcation even upon independent overexpression [5,12]. In other systems also, accumulation of MG in higher

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amounts has been reported to be toxic to the system [36,37]. Increasing level of MG can be regulated by overproduction of glyI during stress conditions [9]. However, production of SLG engages reduced glutathione (GSH) thus depleting it from the system whose optimum level is essential for various physiological processes [2,38,39]. Therefore, for keeping the cells under optimal physiological state, the maintenance of the level of glyII is also very essential. In view of the important role of glyII, its molecular study and functional study was an timely essence. So far, the role of glyoxalase pathway has been documented in enhancing tolerance towards salinity stress but here our results on the induction of transcript levels of OsglyII upon exposure to various stresses has deWnitely opened the new dimensions of its application. This can be suggested from the multistress inducibility of the glyII gene that its overexpression can lead to tolerance towards multiple forms of stress in plants. Acknowledgments 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 B.W. Porter, Kansas State University, USA, for the initial glyoxalase II clone. The Wnancial support by the Department of Biotechnology (DBT, New Delhi) Rice Network Project, International Foundation for Science, Sweden research grant to SLS-P, DBT Post-Doc fellowship to S.K.Y. and grants from the International Centre for Genetic Engineering and Biotechnology is duly acknowledged. References [1] P.J. Thornalley, The glyoxalase system in health and disease, Mol. Asp. Med. 14 (1993) 287–371. [2] S. Kumar, S.L. Singla-Pareek, M.K. Reddy, S.K. Sopory, Glutathione: biosynthesis, homeostasis and its role in abiotic stresses, J. Plant Biol. 30 (2003) 179–187. [3] S.K. Yadav, S.L. Singla-Pareek, M.K. Reddy, S.K. Sopory, Methylglyoxal detoxiWcation by glyoxalase system: a survival strategy during environmental stresses, Physiol. Mol. Biol. Plants 11 (2005) 1–11. [4] S.A. Phillips, P.J. Thornalley, The formation of methylglyoxal from triose phosphates. Investigation using a speciWc assay for methylglyoxal, Eur. J. Biochem. 212 (1993) 101–105. [5] S.K. Yadav, S.L. Singla-Pareek, M. Ray, M.K. Reddy, S.K. Sopory, Methylglyoxal levels in plants under salinity stress are dependent on glyoxalase I and glutathione, Biochem. Biophys. Res. Commun. 337 (2005) 61–67. [6] T. Irsch, R.L. Krauth-Siegel, Glyoxalase II of African trypanosomes is trypanothione-dependent, J. Biol. Chem. 279 (2004) 22209–22217. [7] P.J. Thornalley, Pharmacology of methylglyoxal: formation, modiWcation of proteins and nucleic acids, and enzymatic detoxiWcation–a role in pathogenesis and antiproliferative chemotherapy, Gen. Pharmacol. 27 (1996) 565–573. [8] P.J. Thornalley, The glyoxalase system: new developments towards functional characterization of a metabolic pathway fundamental to biological life, Biochem. J. 269 (1990) 1–11. [9] Veena, V.S. Reddy, S.K. Sopory, Glyoxalase I from Brassica juncea: molecular cloning, regulation and its over-expression confer tolerance in transgenic tobacco under stress, Plant J. 17 (1999) 385–395.

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[10] S.L. Singla-Pareek, M.K. Reddy, S.K. Sopory, Genetic engineering of the glyoxalase pathway in tobacco leads to enhanced salinity tolerance, Proc. Natl. Acad. Sci. USA 100 (2003) 14672–14677. [11] S.L. Singla-Pareek, S.K. Yadav, A. Pareek, M.K. Reddy, S.K. Sopory, Transgenic tobacco overexpressing glyoxalase pathway enzymes grow and set viable seeds in zinc spiked soils, Plant Physiol. 140 (2006) 613– 623. [12] S.K. Yadav, S.L. Singla-Pareek, M.K. Reddy, S.K. Sopory, Transgenic tobacco plants overexpressing glyoxalase enzymes resist an increase in methylglyoxal and maintain higher reduced glutathione levels under salinity stress, FEBS Lett. 579 (2005) 6265–6271. [13] R. Deswal, S.K. Sopory, PuriWcation and partial characterization of glyoxalase I from a higher plant, B. juncea, FEBS Lett. 282 (1991) 277–280. [14] J. Espartero, I. Sanchez-Aguayo, J.M. Pardo, Molecular characterization of glyoxalase I from a higher plant: upregulation by stress, Plant Mol. Biol. 29 (1995) 223–1233. [15] K.S. Johansen, I.I. Svendsen, S.K. Rasmussen, PuriWcation and cloning of the two domain glyoxalase I from wheat bran, Plant Sci. 155 (2000) 11–20. [16] M. Skipsey, C.J. Andrews, J.K. Townson, I. Jepson, R. Edwards, Cloning and characterization of glyoxalase I from soybean, Arch. Biochem. Biophys. 374 (2000) 261–268. [17] M. Jain, D. Choudhary, R.K. Kale, N. Bhalla-Sarin, Salt- and glyphosate-induced increase in glyoxalase I activity in cell lines of groundnut (Arachis hypogaea), Physiol. Plant. 114 (2002) 499–505. [18] P.A. Cordell, T.S. Futers, P.J. Grants, R.J. Pease, The Human hydroxyacylglutathione hydrolase (HAGH) gene encodes both cytosolic and mitochondrial forms of glyoxalase II, J. Biol. Chem. 279 (2004) 28653–28661. [19] M. Ridderstrom, F. Saccucci, U. Hellman, T. Bergman, G. Principato, B. Mannervik, Molecular cloning, heterologous expression and characterization of human glyoxalase II, J. Biol. Chem. 271 (1996) 319– 323. [20] A. Bito, M. Haider, I. Hadler, M. Breitenbach, IdentiWcation 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 (1997) 21509–21519. [21] S.J. Norton, V. Talesa, W.J. Yuan, G.B. Principato, Glyoxalase I and glyoxalase II from Aloe vera: puriWcation, characterization and comparison with animal glyoxalases, Biochem. Inter. 22 (1990) 411–418. [22] V. Talesa, G. Rosi, S. Contenti, C. Mangiabene, M. Lupattelli, S.J. Norton, E. Giovannini, G.B. Principato, Presence of glyoxalase II in mitochondria from spinach leaves: comparison with the enzyme from cytosol, Biochem. Inter. 22 (1990) 1115–1120. [23] M.K. Maiti, S. Krishnasamy, H.A. Owen, C.A. MakaroV, Molecular characterization of glyoxalase II from Arabidopsis thaliana, Plant Mol. Biol. 35 (1997) 471–481. [24] M. Ridderstrom, B. Mannervik, Molecular cloning and characterization of the thiolesterase glyoxalase II from Arabidopsis thaliana, Biochem. J. 322 (1997) 449–454. [25] M. Saxena, R. Bisht, S.D. Roy, S.K. Sopory, N. Bhalla-Sarin, Cloning and characterization of a mitochondrial glyoxalase II from Brassica juncea that is upregulated by NaCl, Zn, and ABA, Biochem. Biophys. Res. Commun. 336 (2005) 813–819. [26] N.O. Concha, B. Rasmussen, K. Bush, O. Herzberg, Crystal structure of the wide-spectrum binuclear zinc beta-lactamase from Bacteroides fragilis, Structure 4 (1996) 823–836. [27] M.W. Crowder, Z. Wang, S.L. Franklin, E.P. Zovinka, S.J. Benkovic, Characterization of the metal-binding sites of the beta-lactamase from Bacteroides fragilis, Biochemistry 35 (1996) 12126–12132. [28] M.W. Crowder, M.K. Maiti, L. Banovic, C.A. MakaroV, Glyoxalase II from A. thaliana requires Zn(II) for catalytic activity, FEBS Lett. 418 (1997) 351–354. [29] T.M. Zang, D.A. Hollman, P.A. Crawford, M.W. Crowder, C.A. MakaroV, Arabidopsis glyoxalase II contains a zinc/iron binuclear metal center that is essential for substrate binding and catalysis, J. Biol. Chem. 276 (2001) 4788–4795.

132

S.K. Yadav et al. / Protein Expression and PuriWcation 51 (2007) 126–132

[30] J.M. Chittoor, J.E. Leach, F.F. White, DiVerential induction of a peroxidase gene family during infection of rice by Xanthomonas oryzae pv. Oryzae, Mol. Plant Microbe Inter. 10 (1997) 861–871. [31] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 (1976) 248–254. [32] A. Pareek, S.L. Singla, A. Grover, Immunological evidence for accumulation of two high-molecular-weight (104 and 90 kDa) HSPs in response to diVerent stresses in rice and in response to high temperature stress in diverse plant genera, Plant Mol. Biol. 29 (1995) 293–301. [33] R.E. Allen, T.W. Lo, P.J. Thornalley, A simpliWed method for the puriWcation of human red blood cell glyoxalase I. Characteristics, immunoblotting, and inhibitor studies, J. Protein Chem. 12 (1993) 111–119. [34] P. Chomczynski, N. Sacchi, Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction, Anal. Biochem. 162 (1997) 156–159.

[35] A. Bito, M. Haider, P. Briza, P. Strasser, M. Breitenbach, Heterologous expression, puriWcation, and kinetic comparison of the cytoplasmic and mitochondrial glyoxalase II enzymes, Glo2p and Glo4p, from Saccharomyces cerevisiae, Protein Expres. Purif. 17 (1999) 456–464. [36] M.P. Kalapos, T. Garzo, F. Antonie, J. Mandl, Accumulation of S-Dlactoylglutathione and transient decrease of glutathione level caused by methylglyoxal load in isolated hepatocytes, Biochim. Biophys. Acta 1135 (1992) 159–164. [37] A.C. McLellan, S.A. Phillips, P.J. Thornalley, The assay of S-D-lactoylglutathione in biological systems, Anal. Biochem. 211 (1993) 37–43. [38] M.J. May, T. Vernoux, C. Leaver, M. Van Montagu, D. Inze, Glutathione homeostasis in plants: implications for environmental sensing and plant development, J. Expt. Bot. 49 (1998) 1102–1116. [39] G. Noctor, A.C.M. Arisi, L. Jouanin, K.J. Kunert, H. Rennenberg, C.H. Foyer, Glutathione: biosynthesis, metabolism and relationship to stress tolerance explored in transformed plants, J. Exp. Bot. 49 (1998) 623–647.