PHYSIOLOGIA PLANTARUM 114: 499–505. 2002 Copyright C Physiologia Plantarum 2002

Printed in Denmark – all rights reserved ISSN 0031-9317

Salt- and glyphosate-induced increase in glyoxalase I activity in celllines of groundnut (Arachis hypogaea)

Mukesh Jaina,*, Dharamainder Choudharyb, Raosaheb K Kaleb and Neera Bhalla-Sarina

aPlant Developmental Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, IndiabFree Radical Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India*Corresponding author, e-mail: mukesh.jain/weizmann.ac.il

Received 15 March 2001; revised 18 September 2001

Glyoxalase I (EC 4.4.1.5) activity has long been associatedwith rapid cell proliferation, but experimental evidence isforthcoming, linking its role to stress tolerance as well. Pro-liferative callus cultures of groundnut (Arachis hypogaea L.cv. JL24) showed a 3.3-fold increase in glyoxalase I activityduring the logarithmic growth phase, correlating well with thedata on FW gain and mitotic index. Inhibition of cell divisiondecreased glyoxalase I activity and vice versa, thus furthercorroborating its role as a cell division marker enzyme. Celllines of A.hypogaea selected in the presence of high salt(NaCl) and herbicide (glyphosate) concentrations, yielded 4.2-to 4.5-fold and 3.9- to 4.6-fold elevated glyoxalase I activity,

Introduction

The glyoxalase system is comprised of two enzymes: gly-oxalase I (EC 4.4.1.5) and glyoxalase II (EC 3.1.2.6), anda catalytic amount of glutathione (GSH, g--glutamyl--cysteinyl-glycine) (Fig. 1). Glyoxalase I and II act co-ordinately to convert 2-oxoaldehydes into 2-hydroxyac-ids using reduced glutathione as a cofactor (Thornalley1993). Methylglyoxal, the primary physiological sub-strate for the glyoxalase I reaction, is produced spon-taneously under physiological conditions from the gly-colysis and photosynthesis intermediates glyceraldehy-de-3-phosphate (GAP) and dihydroxyacetone phosphate(DHAP) (Richard 1993). Furthermore, methylglyoxal isenzymatically produced as a by-product of the triose-phosphate isomerase reaction (Richard 1993) and fromDHAP by methylglyoxal synthetase, an enzyme reportedfrom a number of cell types, including plants (Deswalet al. 1993). Methylglyoxal is a potent cytotoxic com-pound that is known to arrest growth, react with DNA,

Abbreviations – DTNB, 5,5ƒ-dithiobis-2-nitrobenzoic acid; EPSP synthase, 5-enolpyruvyl shikimate-3-phosphate synthase; GSH/GSSG, glutathionereduced/oxidized form; LD50, inhibitor dose required for growth reduction by 50%; NADPH, b-nicotinamide adenine dinucleotide phosphate, reducedform; TCA cycle, tricarboxylic acid (Krebs) cycle.

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respectively, in a dose dependent manner reflective of the levelof stress tolerance. The stress-induced increase in enzyme ac-tivity was also accompanied by an increase in the glutathionecontent. Exogenous supplementation of glutathione could par-tially alleviate the growth inhibition of callus cultures inducedby methylglyoxal and -isoascorbic acid, but failed to recoverthe loss in glyoxalase I activity due to -isoascorbic acid.The adaptive significance of elevated glyoxalase I activity inmaintaining glutathione homeostasis has been discussed inview of our understanding on the role of glutathione in theintegration of cellular processes with plant growth and devel-opment under stress conditions.

RNA and proteins, and increase the incidence of sisterchromatid exchanges (reviewed by Kalapos 1999a).

The discovery of glyoxalase pathway and the realiza-tion of its extant and ubiquitous presence in all organ-isms, prompted a search for its biological function, thatfinally culminated in the ‘promine/retine theory of celldivision’ (Szent-Gyorgyi et al. 1967, reviewed by Kal-apos 1999b). Implicit to this theory, two antagonisticagents exist, a promoter called ‘promine (glyoxalase I)’,which eliminates a retarding compound designated ‘reti-ne (methylglyoxal)’, and thereby initiates cell prolifer-ation. Thus, conforming to the promine/retine theory, alarge body of evidence was accumulated from a melangeof experimental systems including both plants and ani-mals, corroborating glyoxalase I activity as a marker forcell growth and division (Paulus et al. 1993), though anexact causal relationship between glyoxalase activity andrapid cell proliferation has not yet been documented.

Fig. 1. The glyoxalase system: reactionscatalysed by glyoxalase I and glyoxalase II.

Rapidly proliferating cell lines were repeatedly shownto yield elevated glyoxalase I activity. For instance, inhuman colon carcinomas, the Northern blot analysisshowed a 12-fold increase of glyoxalase I transcript overnormal colon cells (Ranganathan et al. 1993). Con-versely, the inhibition of glyoxalase I and II, inducedgrowth arrest in tumour cell lines (Allen et al. 1993,Norton et al. 1993). Smits and Johnson (1981) foundan inverse correlation between methylglyoxal levels andglyoxalase I activity in Douglas fir (Pseudotsuga menzie-sii). In needles -the differentiated tissue, higher amountof methylglyoxal was present in comparison to the calliwhere no methylglyoxal was detected and active enzymewas present. Glyoxalase I activity was in good corre-lation with mitotic index in Pisum sativum roots (Rama-swamy et al. 1983), and FW, cell number and DNA syn-thesis data in cultured Datura innoxia calli (Ramaswamyet al. 1984). Similar results were obtained in Brassica sp.(Bagga et al. 1987, Sethi et al. 1988), Cocos nucifera(Basu et al. 1988), Corchorus capsularis (Seraj et al.1992) and Glycine max (Paulus et al. 1993). Treatmentsthat stimulate cell proliferation in callus cultures of Am-aranthus paniculatus, e.g. phytohormones (Chakravartyand Sopory 1990) and light (Chakravarty and Sopory1998), also increased glyoxalase I activity. Induction ofglyoxalase I activity was reported prior to the initiationof cell division cycle in Nicotiana tabacum mesophyllprotoplasts (Kalia et al. 1998) and synchronized suspen-sion cultured cells of Daucus carota (Ghosh et al. 1999).S--Lactoylglutathione, the product of glyoxalase I reac-tion, is believed to play an important role in cell cycleregulation by influencing the assembly of microtubules(Hooper et al. 1988) and an association of glyoxalase IIwith tubulin has been suggested (Norton et al. 1993).

The promine/retine theory has however, suffered criti-cism in view of the emerging evidence showing responsive-ness of glyoxalase I towards environmental stress con-ditions. In Lycopersicon esculentum, glyoxalase I cDNAwas isolated by differential screening of salt-inducedgenes and showed up-regulation in plants subjected toNaCl, mannitol and ABA stress (Espartero et al. 1995).Similarly in Brassica juncea, glyoxalase I expression waselevated in plants treated with NaCl, mannitol and ZnCl2,in a dose dependent manner. The over-expression of Bras-sica juncea glyoxalase I cDNA in transgenic Nicotiana ta-bacum conferred tolerance to salt stress (Veena et al.1999). Blomstedt et al. (1998) identified a clone by differ-ential screening of the cDNA library prepared from dehy-drated foliar tissue of the resurrection grass Sporobolusstapfianus, whose deduced amino acid sequence has

Physiol. Plant. 114, 2002500

strong homology to the enzyme glyoxalase I from severalspecies, including tomato. Interestingly, this putative gly-oxalase I cDNA showed 3-fold higher expression duringdesiccation over fully hydrated tissue and was also elev-ated in response to ABA application.

In the present communication, we report our resultson the stress-induced increase in glyoxalase I activity inNaCl and glyphosate tolerant cell lines of groundnut(Arachis hypogaea L. cv. JL24). The cell lines selectedon medium containing high salt or glyphosate, yieldedelevated enzyme activity reflective of the NaCl andglyphosate concentrations in the selective medium. Thisis also the first report on the effects of herbicide glyphos-ate on the activity of glyoxalase I. Finally, the signifi-cance of the glyoxalase system and the soundness of pro-mine/retine theory has been discussed in the light of re-cent findings on the regulation of cell cycle progressionand abiotic stress tolerance in higher plants. A criticalappraisal of the circumstantial evidence accumulated inthis study warrants a closer inspection on the role ofglyoxalase pathway in maintenance of the glutathionepool, with profound implications in regulation of celldivision, proliferation and stress tolerance.

Materials and methods

Plant material and biochemicals

Seeds of groundnut (Arachis hypogaea cv. JL24) wereobtained from ICRISAT (Patancheru, India). All thechemicals used were obtained from Sigma Chemical Co.,St. Louis, MO, USA, and were of analytical grade.

In vitro cultures

Shelled seeds of Arachis hypogaea cv. JL24 were surfacesterilized with 0.1% (w/v) HgCl2 and germinated on fil-ter paper bridges soaked in liquid MS medium (Mura-shige and Skoog 1962). The 14-day-old-plantlets wereused to obtain explants for the initiation of in vitro cul-tures. Leaf callus cultures were obtained on semisolidMS medium supplemented with 1 mg lª1 each of 6-ben-zylaminopurine and a-naphthalene acetic acid. Theselection and maintenance of glyphosate tolerant celllines of A. hypogaea has been described (Jain et al.1999). All the experiments were performed using GR2cell line maintained continuously on medium containing4.0–10.0 mM glyphosate. The salt tolerant cell lines wereisolated using a step-wise procedure for somaclone selec-tion (Jain et al. 2001). Presently, we have salt tolerant

cell lines of A. hypogaea growing in the presence of 50–200 mM NaCl. The callus cultures were transferred tofresh medium every 15 days, and maintained at 25 ∫2æC, under cool white fluorescent light at a photon flu-ence density of 12 W mª2 for 16 h day-1. For all experi-mental purposes, the callus cultures were harvested dur-ing the mid-logarithmic growth phase (day 12–13), un-less otherwise mentioned. The experiments wererepeated at least thrice with 5–6 replicates each.

Cell number and mitotic index determination

Aliquots of actively growing callus cultures were with-drawn at periodic intervals, fixed overnight in aceticacid: ethyl alcohol (3 : 1, v/v), and stained with 1% ace-toorcein: 1 M HCl (9 : 1, v/v). Mitotic index was deter-mined by counting the number of cells in late prophaseto telophase and expressed as percentage of total num-ber of cells.

Assay of glyoxalase I activity

Extraction and analysis of glyoxalase I activity was per-formed according to the procedure of Ramaswamy et al.(1983). Cell free homogenates were prepared at 0–4æC.Callus (1 g) was frozen and finely crushed in liquid nitro-gen, and extracted with 2 ml ice cold 0.1 M Na-phos-phate buffer (pH 7.0) containing 1 mM phenylmethylsul-phonylfluoride (PMSF) and 10% (w/v) polyvinylpyrrol-idone (PVP). The crude protein extract was filteredthrough four layers of cheese cloth and cleared by centri-fugation at 4æC at 10000 g for 20 min The reaction wasset in a final volume of 1 ml containing 100 mM Na-phosphate buffer (pH 7.5), 3.5 mM methylglyoxal, 1.7mM reduced glutathione (GSH) and 15.0 mMMgSO4¡7H2O. The reaction was incubated at 25æC for7 min before addition of 0.02 ml enzyme extract. Theformation of thioester was monitored at 240 nm. Theenzyme unit (IU) is defined as the amount of enzymecatalysing the formation of 1 mol of S--lactoylgluta-thione from methylglyoxal and reduced glutathione permin at 25æC. The protein was estimated according tothe procedure of Bradford (1976) using bovine serumalbumin (BSA) as the standard.

Assay of GSH/GSSG

Total glutathione was assayed by the GSSG reductase re-cycling assay as described by Anderson (1985). The reac-tion was set in a cuvette in a volume of 1 ml containing 700ml b-nicotinamide adenine dinucleotide phosphate(NADPH), 100 ml 5,5ƒ-dithiobis-2-nitrobenzoic acid(DTNB) and 175 ml water. NADPH (0.248 mg mlª1) andDTNB (6 mM) were both prepared in 143 mM Na-phos-phate buffer (pH 7.5) containing 6.3 mM Na4-EDTA. Thecrude extract (25 ml), as prepared for the glyoxalase I as-say, was added to the reaction mix, pre-warmed at 30æC ina jacketed water bath for 15 min. The reaction was in-itiated by adding 10 ml of GSSG reductase (Sigma Chemi-

Physiol. Plant. 114, 2002 501

Fig. 2. Correlation of glyoxalase I activity with cell proliferation incallus cultures of Arachis hypogaea cv. JL24. (a) FW increase (ex-pressed as percent of day 0) (b) glyoxalase I activity (IU mgª1 protein)and (c) mitotic index (%), were measured periodically from day 0–18,through the entire subculture cycle. Vertical bars are ∫ for 7 repli-cates.

cal Co., G3664, yeast enzyme) (EC 1.6.4.2) diluted to 266units mlª1 in Na-phosphate-EDTA buffer (pH 7.5). Oneunit of GSSG reductase reduces 1.0 mM of oxidized guta-thione min-1 at pH 7.6 at 25æC. The rate of stoichiometricformation of 5-thio-2 nitrobenzoic acid (TNB) fromDTNB was followed at 412 nm.

Results

Glyoxalase I activity and growth kinetics of Arachishypogaea cell lines

The leaf callus cultures of Arachis hypogaea were rou-tinely maintained on semisolid MS medium supple-mented with 1 mg lª1 each of 6-benzylaminopurine and

a-naphthalene acetic acid, and periodically transferredto fresh medium every 14 days. The mid-logarithmicgrowth phase reached by 12–13 days of the transfer andprogressed through stationary growth by 15–16 days(Fig. 2a). The specific activity of glyoxalase I was exam-ined in callus cultures of A. hypogaea through the entiresubculture cycle (Fig. 2b). There was a gradual increasein the activity of glyoxalase I with increasing FW of cal-lus through the logarithmic growth phase. Maximum,3.3-fold increase in enzyme activity was observed on day12, declining thereafter through the end of growth cycle.The elevation of glyoxalase I activity was also associatedwith a 14.5-fold rise in the mitotic index, reflective ofactive cell division responsible for the observed increasein callus FW (Fig. 2c). Glyoxalase I activity has earlierbeen correlated with FW gain and mitotic index in Pis-um sativum plants (Ramaswamy et al. 1983), Datura cal-lus (Ramaswamy et al. 1984), suspension cultured cellsof Glycine max (Paulus et al. 1993) and Daucus carota(Ghosh et al. 1999). Methylglyoxal reduced the FW in-crease and mitotic index with a concomitant reductionin glyoxalase I activity (measured on day 12), in a dosedependent manner. Similarly, -isoascorbic acid, a speci-fic inhibitor of glyoxalase I (Ramaswamy et al. 1984),reduced FW accumulation and cell division through in-hibition of enzyme activity. The data, summarized inTable 1, confirm the positive correlation between gly-oxalase I activity and proliferative potential of A. hypo-gaea callus cultures.

Elevated glyoxalase I activity in salt and glyphosatetolerant cell lines of Arachis hypogaea

The herbicide glyphosate elicits phytotoxicity by inhibit-ing 5-enolpyruvyl shikimate-3-phosphate (EPSP) syn-thase, the penultimate enzyme of common prechoris-mate trunk of shikimic acid pathway thus causing arrestof aromatic amino acid biosynthesis and secondary met-abolism. Isolation and biochemical characterization ofglyphosate tolerant, EPSP synthase over-expressing celllines of Arachis hypogaea has previous been published(Jain et al. 1999). Biochemical analyses of NaCl tolerantcell lines indicated active osmotic adjustments to main-tain cellular turgor, extrusion of Naπ ions with increase

Table 1. Inhibitory effects of methylglyoxal and -isoascorbic acid on growth, mitotic index and glyoxalase I activity in callus cultures ofArachis hypogaea cv. JL24. The callus growth is expressed as percent of fresh weight (FW) increase measured on day 12, with respect to day0. Mitotic index (%) and the enzyme activity (IU mgª1 protein) were also determined in the same samples on day 12. The values in parenthesesdenote the percent of control in each case.

FW increase Mitotic index Glyoxalase I activityTreatment (% of day 0) (%) (IU mgª1 protein)

Control 68.7 ∫ 0.7 (100) 76.6 ∫ 0.4 (100) 0.65 ∫ 0.02 (100)

Methylglyoxal5 mM 29.5 ∫ 0.2 (43) 34.3 ∫ 0.3 (45) 0.32 ∫ 0.02 (50)10 mM 21.6 ∫ 0.4 (32) 21.6 ∫ 0.4 (28) 0.26 ∫ 0.02 (39)

-Isoascorbic acid10 mM 55.3 ∫ 0.3 (80) 62.6 ∫ 0.3 (82) 0.55 ∫ 0.02 (84)50 mM 36.6 ∫ 0.2 (53) 37.2 ∫ 0.2 (49) 0.36 ∫ 0.03 (55)

Physiol. Plant. 114, 2002502

in salinity stress and accumulation of proline as the pri-mary compatible osmolyte (Jain et al. 2001). Given theassociation of glyoxalase I activity with rapid cell pro-liferation, and in order to gain an insight into the pro-posed role of glyoxalase I activity in abiotic stress toler-ance (Veena et al. 1999), the cell lines tolerant to 100–200 mM NaCl and 6.0–10.0 mM glyphosate were exam-ined for the enzyme activity (Table 2). Measurements ofFW increase through the entire culture cycle showed thatall the cell lines grew at rates comparable to the parentcell line (data not shown). In comparison to the stresssensitive parent cell line, which shows growth inhibitionin presence of salt (LD50 Ω 30 mM NaCl) as well asglyphosate (LD50 Ω 0.5 mM glyphosate), the specific ac-tivity of glyoxalase I was elevated by 4.2- to 4.5-fold inNaCl tolerant, and 3.9- to 4.6-fold in glyphosate toler-ant cell lines, respectively. The increase in enzyme activ-ity from day 3 (beginning of logarithmic growth phase)to day 12 (mid-logarithmic growth phase of culturecycle) was in accordance with earlier data (Fig. 2). Inter-estingly, the increase in specific activity of glyoxalase Ion day 12 (over day 3) was 45- to 55% higher in salttolerant, and 34- to 59% higher in glyphosate tolerantcell lines, respectively, as compared to the stress sensitiveparent cell line. Furthermore, the elevated status of gly-oxalase I activity in stress tolerant cell lines was alsoreflective of the level of stress tolerance. These resultsclearly indicate that besides having an association withrapid cell division and proliferation, glyoxalase systemalso offers a selective advantage in abiotic stress toler-ance, as was earlier reported in Brassica juncea, tobaccoand tomato (Espartero et al. 1995, Veena et al. 1999).

The tripeptide glutathione is the major low molecularweight thiol constituent of the cell with proposed rolesin the storage and transport of reduced sulphur, the syn-thesis of proteins and nucleic acids and as a modulatorof enzyme activity. Glutathione is of paramount import-ance for protection of the cell against oxidative damageresulting from a plethora of biotic and abiotic stress con-ditions and for the detoxification of xenobiotic com-pounds including herbicides and pesticides (May et al.1998). The salt tolerant cell lines of A. hypogaea showed1.2- to 1.4-fold higher glutathione content as comparedto the salt sensitive parent cell line. Similarly, the

glyphosate tolerant cell lines also yielded 1.4- to 1.5-foldelevated glutathione content, in comparison to the con-trols (Table 2). The increase in glutathione levels in thetolerant cell lines was strictly stress dependent, andshowed a pattern reminiscent of the glyoxalase I activity.Inhibition of glyoxalase I activity by 25 mM -isoascor-bic acid, resulted in decreased glutathione content (Table3). Although, the percent loss in enzyme activity wascomparable between the sensitive, salt tolerant andglyphosate tolerant cell lines (37–38%), the decline in theglutathione content was more significant in the stresssensitive parent cell line (34%) in comparison to the salttolerant and the glyphosate tolerant cell lines (21% and22%, respectively). This could be attributed to elevatedglutathione biosynthesis in NaCl and glyphosate toler-ant cell lines, implicit to the mechanisms for abioticstress tolerance in these lines. It has been suggested inone of our recent papers that glyphosate compromisesthe catalase and peroxidase activities through its inhibi-tory effect on the biosynthesis of 5-aminolevulinic acidand thus necessitates elevated glutathione content for ef-ficient scavenging of peroxides (Jain and Sarin 2001).Treatment with -isoascorbic acid also resulted in re-duced tolerance level, as evident by a lower LD50 dose,which measured a lower amount of NaCl or glyphosate

Table 2. Stress-induced increase in glyoxalase I activity and glutathione content in NaCl and glyphosate tolerant cell lines of Arachis hypogaeacv. JL24. The enzyme activity (IU mgª1 protein) was measured on day 3 and day 12, and glutathione content (mmol gª1 FW) on day 12 ofthe subculture cycle.

Glyoxalase I activity (IU mgª1 protein)Glutathione

Stress (day 3) (day 12) (m mol gª1 FW)

Stress sensitive cell line0.22 ∫ 0.02 0.63 ∫ 0.04 0.58 ∫ 0.02

Salt tolerant cell line100 mM NaCl 0.30 ∫ 0.04 1.25 ∫ 0.05 0.70 ∫ 0.02150 mM NaCl 0.35 ∫ 0.04 1.44 ∫ 0.04 0.74 ∫ 0.04200 mM NaCl 0.41 ∫ 0.04 1.85 ∫ 0.05 0.80 ∫ 0.02

Glyphosate tolerant cell line6.0 mM glyphosate 0.32 ∫ 0.03 1.24 ∫ 0.04 0.83 ∫ 0.038.0 mM glyphosate 0.34 ∫ 0.05 1.44 ∫ 0.05 0.86 ∫ 0.0210.0 mM glyphosate 0.36 ∫ 0.02 1.65 ∫ 0.05 0.88 ∫ 0.02

Table 3. Inhibitory effect of -isoascorbic acid on glyoxalase I activity and total glutathione content in callus cultures of Arachis hypogaeacv. JL24, with a concomitant reduction in the stress tolerance level. The stress sensitive parent cell line and cell lines tolerant to 150 mMNaCl and 4.0 mM glyphosate were grown in the presence of 25 mM -isoascorbic acid for 12 days and examined for the enzyme activity(IU mgª1 protein) and glutathione content (mmol gª1 FW). LD50 dose represents the amount of NaCl or glyphosate required to inhibit FWgain by 50% as compared to the unstressed controls. The values in parentheses denote the percent of control in each case.

Stress sensitive cell line Salt tolerant Glyphosate tolerant

Glyoxalase I activity (IU mgª1 protein)Control 0.66 ∫ 0.03 (100) 1.39 ∫ 0.04 (100) 1.15 ∫ 0.02 (100)-Isoascorbic acid 0.42 ∫ 0.02 (63.1) 0.87 ∫ 0.02 (62.1) 0.73 ∫ 0.03 (63.4)

Glutathione (mmol gª1 FW)Control 0.58 ∫ 0.02 (100) 0.74 ∫ 0.02 (100) 0.70 ∫ 0.02 (100)-Isoascorbic acid 0.38 ∫ 0.02 (66.3) 0.59 ∫ 0.04 (79.7) 0.54 ∫ 0.03 (77.8)

LD50 (NaCl)Control 30 mM 240 mM-Isoascorbic acid 19.5 mM 175 mM

LD50 (glyphosate)Control 0.5 mM 10.0 mM-Isoascorbic acid 0.3 mM 6.0 mM

Physiol. Plant. 114, 2002 503

needed to inhibit FW increase by 50% as compared tothe respective controls.

The methylglyoxal-induced growth inhibition could bepartially alleviated by exogenous supplementation ofglutathione (Fig. 3). Methylglyoxal (5 mM) decreasedFW gain of sensitive cell line by approx. 60% as com-pared to 52 and 53% in case of salt (150 mM NaCl)and herbicide (4.0 mM glyphosate) tolerant cell lines,respectively. Glutathione (25 mM) if supplementedalone, resulted in a 1.2-fold increase in the growth ofboth salt and glyphosate-tolerant, cell lines. The FWgain in case of the stress sensitive cell line was not sig-nificantly affected by exogenous supplementation of glu-tathione. Glutathione could reverse the growth inhibi-tory effects of methylglyoxal by about 20%, roughly toan equal extent in all the three cell lines. The inhibitionof glyoxalase I activity due to methylglyoxal treatmentwas also comparable (50 ∫ 1.08%) in all the three celllines. Furthermore, exogenous supplementation of glu-tathione was able to (partially) alleviate the growth inhi-bition caused by 25 mM -isoascorbic acid, but couldnot recover the deficit in the glyoxalase I activity causedby the enzyme inhibitor in callus cultures treated with-isoascorbic acid (data not shown). An obvious inter-pretation of the data presented above underlines the im-

portance of glyoxalase I catalysis in the recycling andmaintenance of the reduced glutathione pool with ameli-orative effects on stress induced growth inhibition.

Discussion

The salt and glyphosate tolerant cell lines of Arachis hypo-gaea offer an interesting experimental system for studyingthe role of glyoxalase I activity in rapidly proliferating cal-lus cultures, and at the same time, its association with abi-otic stress tolerance (in contrast to earlier studies whereintact seedlings were exposed to short-term stress treat-ments with an obvious decrease in growth parameters). Itis conceivable that elevated glyoxalase I activity is re-quired to bind and remove methylglyoxal, a toxic and un-avoidable by-product of triosephosphate metabolism pro-duced in ample amounts under normal physiological con-ditions. Increase in glyoxalase I activity during stresstolerance may also indicate active metabolic status of thecell, where the cell division and growth is compromised inorder to conserve energy for mobilization of resources to-wards stress tolerance and defense strategies. Analysis ofexpressed sequence tags from the cultured cells of Oryzasativa subjected to a wide range of stress conditions in-cluding 20% sucrose, salt, cold and nitrogen starvation,revealed a general and co-ordinated up-regulation of thegenes engaged in ATP-generating pathways (Umeda et al.1994).

Though the up-regulation of glyoxalase I activity inresponse to salinity stress has been reported earlier inplanta (Espartero et al. 1995, Veena et al. 1999), this isthe first report on salt stress-induced increase in gly-oxalase I activity in rapidly proliferating cell culturelines. Also, glyphosate-induced increase in glyoxalase Iactivity is being documented for the first time in anyexperimental system, and a plausible explanation fol-lows. The glyphosate tolerant cell lines of A. hypogaea

Fig. 3. Ameliorative effects of glutathione in reversal of methylgly-oxal-induced growth inhibition in callus cultures of Arachis hypo-gaea cv. JL24. The stress sensitive parent cell line and cell linestolerant to 150 mM NaCl and 4.0 mM glyphosate, were grown inpresence of 10 mM methylglyoxal and 25 mM glutathione, eitheralone or in combination. The values for the controls were 65.47 ∫0.76, 64.08 ∫ 0.89 and 66.99 ∫ 0.65, respectively, estimated as per-cent increase in FW on day 12. Vertical bars are ∫ for 5 repli-cates.

Physiol. Plant. 114, 2002504

used in the present investigation over-express EPSP syn-thase, which is still subject to inhibition by glyphosate(Jain et al. 1999). This could possibly mean that theseglyphosate tolerant cell lines suffer from a serious andwasteful loss of energy rich metabolites, erythrose-4-phosphate and phosphoenolpyruvate, into the shikimicacid pathway (Jain and Sarin 2000), and may thereforehave up-regulated energy generating pathways to com-pensate for the energy drain. This stems from the obser-vation that exogenous supplementation of TCA cycle in-termediates can partially alleviate the glyphosate-in-duced growth inhibition (Jain et al. 1999).

Methylglyoxal decreases the level of protein thiol andreduced glutathione (Kalapos 1999a). Reduced glutathi-one is essential for efficient scavenging of peroxides pro-duced as a result of cellular metabolism or during oxida-tive stress, and for the maintenance of other antioxidantslike tocopheroles and ascorbates. Besides detoxificationof methylglyoxal, high glyoxalase I activity is also instru-mental in recycling of glutathione that is spontaneouslytrapped by methylglyoxal to form hemithioacetal. Thusglyoxalase I catalysis may have significant contributiontowards maintaining the necessary redox poise for co-ord-inating the requirements for growth with mechanisms forsurvival under environmental stress conditions. The dis-covery of functions involving redox-regulated kinases(Link 1996), phosphatases (Haring et al. 1995) and tran-scription factors (Yang and Klessig 1996) in the transcrip-tion of plant genes during stress responses underline theubiquity and importance of redox couples in fine tuningof signal transduction cascades and reorganization ofgrowth and metabolic needs in commitment to the main-tenance of redox poise during growth under stress.

The celebrated association of elevated glyoxalase I ac-tivity with rapid cell proliferation may also mirror theredox needs of the cell. During root development in Ara-bidopsis thaliana, high levels of glutathione were associ-ated with epidermal and cortical initials, in contrast tothe cells of quiescent centre, which have an extended G1(Sanchez-Fernandez et al. 1997). Recently, the first en-zyme of glutathione biosynthesis, g-glutamylcysteinesynthetase, was shown to be essential for the establish-ment of an active post-embryonic meristem in the rootapex of Arabidopsis. Furthermore, an adequate amountof glutathione was a prerequisite for the G1/S transitionin the synchronized suspension cultures of N. tabacum(Vernoux et al. 2000). An increase in the cellular gluta-thione concentration was evident in the late S phase, be-ginning from an otherwise stable level till mid S phase.This study also provides support to the observed in-crease in the activity of glyoxalase I prior to G2/M tran-sition in N. tabacum protoplasts (Kalia et al. 1998) andDaucus carota suspension cultures (Ghosh et al. 1999).

The extant and ubiquitous glyoxalase pathway has re-mained preserved throughout evolutionary history forthe detoxification of a 2-oxaloaldehyde (methylglyoxal)ineradicably produced during glycolysis. But obviously,no functional role in the regulation of cell proliferationcan be assigned to glyoxalases, in absence of any experi-

mental data showing causal relationship between enzymeactivity and cell division. The elevated glyoxalase I activ-ity reported in actively proliferating tissue may simplymirror the need for active removal of methylglyoxal andfor maintenance of the redox status vital for entry intoand progression through the cell cycle. Glutathione ap-pears to be the prime link between the glyoxalase pathwayand the choice for rapid cell division (or differentiation)in response to the prevalent growth conditions.

Acknowledgements – This work was supported by the research grantno. 38(800)89-EMR II, from the Council of Scientific and IndustrialResearch, and grant no. 3–36/94(SRII) from the University GrantsCommission, India.

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