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Plant Physiology and Biochemistry 62 (2013) 63e69

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Plant Physiology and Biochemistry

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Research article

Inhibition of 5-aminolevulinic acid dehydratase by mercury in excised greeningmaize leaf segments

Priyanka Gupta, Meeta Jain, Juliana Sarangthem, Rekha Gadre*

School of Biochemistry, Devi Ahilya University, Takshashila Campus, Khandwa Road, Indore 452 017, Madhya Pradesh, India

a r t i c l e i n f o

Article history:Received 18 May 2012Accepted 26 October 2012Available online 7 November 2012

Keywords:5-Aminolevulinic acid dehydrataseMaize leavesMercury effectsZea mays

Abbreviations: d-ALA, d-aminolevulinic acid; d-Adehydratase; DTNB, 5,50 dithio bis 2-nitrobenzoic acreduced glutathione; LA, levulinic acid; NR,porphobilinogen.* Corresponding author. Tel.: þ91 731 2460276.

E-mail address: rekhagadre29@gmail.com (R. Gad

0981-9428/$ e see front matter � 2012 Elsevier Mashttp://dx.doi.org/10.1016/j.plaphy.2012.10.008

a b s t r a c t

Mercury (Hg), a potent metallic toxicant, is known for having inhibitory effect on chlorophyll biosyn-thesis. In vivo supply of HgCl2 inhibited 5-aminolevulinic acid dehydratase (ALAD, EC 4.2.1.24) activity inexcised greening maize (Zea mays) leaf segments. The inhibition caused by Hg was alleviated by additionof KNO3. Amongst the nutrients and metabolites tested, NH4Cl and sucrose increased the inhibitory effectof Hg on enzyme activity, while glutamine and glutathione decreased it. The inhibitors, levulinic acid and5,50 dithio bis 2-nitrobenzoic acid, also reduced the % inhibition of enzyme activity caused by Hg supply.In vitro inclusion of Hg during assay of the enzyme preparations obtained from the tissue treated withoutHg (�Hg enzyme) and with Hg (þHg enzyme) caused the inhibition of �Hg enzyme but activationof þHg enzyme. Almost similar trend was observed for the in vitro inclusion of Hg in the presence oflevulinic acid. It is suggested that two forms of enzyme exist in Hg-treated tissue, i.e. the usual Mgdependent form and an unusual Hg modified form. Kinetic studies for the two enzymes, �Hg enzymeand þHg enzyme, involving the effect of varying concentrations of d-aminolevulinic acid yielded distinctapparent Km and apparent Vmax values being 532 mM and 118 units g�1 fr. wt., respectively, for �Hgenzyme and 347 mM and 52 units g�1 fr. wt., respectively, for þHg enzyme indicating that þHg enzymehas higher affinity for d-aminolevulinic acid but lower activity as compared to the �Hg enzyme.

� 2012 Elsevier Masson SAS. All rights reserved.

1. Introduction

Heavy metals when present in excess in the environment causetoxic effects on plant growth and metabolism. These are known tointerferewith various photosynthetic functions [1]. Mercury (Hg) isone of the most toxic heavy metals, which is released into theatmosphere from industrial wastes, burning of fossil fuels and useof agrochemicals [2]. Hg toxicity has been reported to be associatedwith decline in the chlorophyll content [3e5] and photosyntheticefficiency [6,7]. It reduced the levels of chlorophyll precursors,protoporphyrin, Mg protoporphyrin and protochlorophyllide inbajra seedlings [5]. Its interactionwith protochlorophyllide has alsobeen shown in wheat leaves [8]. It substitutes the central Mg of thechlorophyll molecule [9,10]. Mercury has also been reported toinhibit the enzymes involved in chlorophyll biosynthesis, such as,d-aminolevulinic acid dehydratase in radish leaves [11], and in

LAD, d-aminolevulinic acidid; DTT, dithiothreitol; GSH,nitrate reductase; PBG,

re).

son SAS. All rights reserved.

mung bean [12] and bajra [13] seedlings, porphobilinogen deami-nase in mung bean seedlings [14], protochlorophyllide oxidore-ductase in dark grown wheat leaves [8,15], and in isolatedprolamellar bodies of dark grown wheat leaves [16].

Chlorophyll biosynthesis is a vital physiological process inplants, which is regulated at several steps [17e20]. Earlier steps ofthe pathway are common with biosynthesis of other tetrapyrrolederivatives like heme, phytochromes, phycobilins etc. d-Amino-levulinic acid, the universal precursor of the pathway, is synthe-sized by two routes, the Shemin pathway and the Beale pathway[21]. The Beale’s pathway involves the synthesis of ALA from C-5compounds, like glutamate or 2-oxoglutarate [22]. ALAD, one of theregulatory enzymes of the pathway, catalyzes the asymmetriccondensation of two molecules of ALA leading to the formation ofthe basic unit of tetrapyrroles, the porphobilinogen. ALADs fromdifferent sources are metalloenzymes that utilize a variety ofdivalent and monovalent cations [23]. The plant enzymes requireMg2þ for enzymatic activity. The metal chelators, such as, EDTA, ordivalent heavy metal ions inhibit enzyme activity [24,25]. Further,the reactive sulfhydryl groups of enzyme appear to be important inthe binding of the metal ion to the enzyme [26]. The present studyhas been carried out to understand the mechanism of inhibition ofALAD activity by Hg. To analyze the mechanism behind the

Fig. 1. XeY scatter for Hg treatment and ALAD activity.

P. Gupta et al. / Plant Physiology and Biochemistry 62 (2013) 63e6964

inhibitory effect of Hg on ALAD activity, the effect of Hg was studiedon the activity and other related parameters of enzyme in theabsence and presence of nitrogenous compounds. In addition, toobserve possible interactions with other plant processes the effectof some key metabolites, nutrients and inhibitors on enzymeactivity was also investigated in the absence and presence of Hg. Tocharacterize the enzyme of Hg untreated and treated tissue, theactivities of these preparations were assayed with in vitro inclusionof Hg and with varying concentrations of ALA.

2. Results

2.1. Effect of mercury supply on ALAD activity, ALA content, totalchlorophyll and Hg content

Supply of 0.01e0.05 mM HgCl2 to excised maize leaf segmentsduring greening for 24 h in continuous light of intensity 30 W m�2

inhibited ALAD activity substantially, while 0.001 mM HgCl2increased it slightly (Table 1). Correlation analyses, performed withMicrosoft excel XY scatter, yielded R2 value of 0.731 indicatinga very good correlation between Hg treatment and ALAD activity(Fig. 1). Hg supply at 0.02 mM decreased the ALA content and thechlorophyll content also, but the effect was more prominent for theformer (Table 1). Mercury content of the leaf segments treated with0.0, 0.001 and 0.02 mM HgCl2 were 20, 66 and 465 ng g�1 dry wt.,respectively. Correlation analyzed from Microsoft excel XY scatteryielded R2 value of 0.997 indicating a perfect correlation (Fig. 2).In vitro inclusion of 185 and 370 mM HgCl2 during assay of ALADactivity of preparations obtained from leaf segments treatedwithout Hg (�Hg enzyme) and with 0.02 mM Hg (þHg enzyme)decreased the activity of �Hg enzyme with a more pronouncedeffect at lower concentration while it increased the activity of þHgenzyme (Fig. 3).

2.2. Effect of mercury supply on ALAD activity and NR activity in thepresence of nitrate

When etiolated leaf segments were incubated with 0.001e0.1 mM HgCl2 in the presence of 10 mM KNO3, the ALAD activitydecreased gradually with increasing concentrations of Hg (Table 2).In the presence of nitrate R2 value of 0.959 was obtained by per-forming XY scatter for Hg treatment and ALAD activity (Fig. 4a). Atthe high concentration used, the relative level of activity wasmaintained higher in the presence of nitrate than in its absence(compare Tables 1 and 2). Supply of 0.02 and 0.05 mM HgCl2inhibited significantly nitrate reductase activity also (Table 2).Perfect correlation with R2 value of 0.995 for Hg treatment andnitrate reductase activity also resulted (Fig. 4b). In vitro inclusion of185 and 370 mM HgCl2 during the assay of ALAD activity of prepa-rations obtained from leaf segments treated with 10 mM KNO3 andcontaining no Hg (�HgþN enzyme) and 0.05 mM Hg (þHgþN

Table 1d-Aminolevulinic acid dehydratase activity, aminolevulinic acid content and totalchlorophyll content in excised etiolated maize leaf segments during greening. Leafsegments from dark grown maize seedlings were treated with ¼ strength Hoag-land’s solution containing desired concentrations of HgCl2 in continuous light ofintensity 30 W m�2 for 24 h. Values relative to control are given in parentheses.

HgCl2 conc.,mM

ALAD activity,units g�1 fr. wt.

ALA content,n mol g�1 fr. wt.

Total chlorophylls,mg g�1 fr. wt.

0.000 52 � 3 (100) 30 � 8 (100) 309 � 31 (100)0.001 60 � 8 (115)0.01 45 � 4 (87)0.02 35 � 3 (67)c 11 � 4 (37)a 168 � 16 (54)b

0.05 32 � 3 (62)c

Level of significanceea: p < 0.05, b: p < 0.01, c: p < 0.001.

enzyme), decreased the activity of �HgþN enzyme with a morepronounced effect at lower concentration, while Hg inclusionincreased the activity of þHgþN enzyme (Fig. 3).

2.3. Effect of nutrients and metabolites on inhibition of ALADactivity by mercury

Key compounds, such as, ammonium (a nutrient exerting toxiceffects), glutamine (serving as a regulator of nitrogen assimilation),sucrose (a photosynthate), glutathione (involved in maintainingthiol groups of enzymes), of the different pathways, were used tostudy their effect on the inhibition of ALAD activity caused by Hg.Thus, incubation of greening leaf segments with 10 mM NH4Clinhibited the ALAD activity in the absence as well as presence of Hgwith the inhibition being more substantial for the latter, hence,increased the % inhibition of ALAD activity due to Hg supply from33% in control to 68% in the presence of NH4Cl (Table 3). When leafsegments were treated with 5 mM glutamine, the enzyme activitydecreased marginally in the absence of Hg while it increased in thepresence of Hg, thus the % inhibition of enzyme activity by Hg waslowered to 14% in the presence of glutamine (Table 3). Supply of5 mM sucrose decreased the ALAD activity only in the presence ofHg, so the % inhibition by Hg was increased to 44% in the presenceof sucrose (Table 3). Incubation of leaf segments with 5 mM GSHdecreased the enzyme activity in the absence of Hg and the %inhibition of activity by Hg was slightly lower (Table 3).

2.4. Effect of inhibitors on the inhibition of ALAD activity bymercury

Supply of levulinic acid, a competitive inhibitor of ALAD, at10 mM and 20 mM decreased the ALAD activity in a concentration

Fig. 2. XeY scatter for Hg treatment and Hg content of the leaf segments.

Fig. 3. Effect of in vitro inclusion of Hg on ALAD activity of the enzyme preparations obtained from leaf segments treated without Hg (�Hg enzyme) or with 0.02 mM Hg (þHgenzyme) or treated in the presence of 10 mM KNO3 without Hg (�HgþN enzyme) or with 0.05 mM Hg (þHgþN enzyme) in continuous light of intensity 30 W m�2 for 24 h. Valuesrelative to their respective controls are given.

P. Gupta et al. / Plant Physiology and Biochemistry 62 (2013) 63e69 65

dependentmanner in the absence as well as presence of Hg, but thedecrease was more substantial for the former (Table 4). Thus, the %inhibition of activity by Hg was prominantly reduced to 23% and 8%with 10 and 20mMLA, respectively (Table 4). Treatmentwith 1mMDTNB, an eSH blocking reagent, decreased the ALAD activity in theabsence of Hg, while it remained almost unaffected in the presenceof Hg, hence, decreased the % inhibition caused by Hg to 28%(Table 4). Effect of in vitro inclusion of HgCl2 along with DTNB or LAon ALAD activity of preparations obtained from leaf segmentstreated without Hg (�Hg enzyme) and with 0.02 mM Hg (þHgenzyme) was also studied. In the presence of 2 mM LA, the activityof �Hg enzyme was reduced and that of þHg enzyme wasincreased slightly with the addition of 185 and 370 mM HgCl2(Fig. 5). With 0.1 mMDTNB, the activity of�Hg enzymewas loweredslightly due to Hg addition, while it remained almost unaffectedfor þHg enzyme (Fig. 5).

2.5. Effect of varying concentrations of ALA on ALAD activity

Effect of varying concentrations of ALA on the ALAD activity ofthe enzyme preparations obtained from leaf segments treatedwithout Hg (�Hg enzyme) and with Hg (þHg enzyme) wasanalyzed. Increasing concentrations of ALA from 44 to 660 mMincreased the enzyme activity progressively in both �Hg and þHgenzyme preparations, with a higher level beingmaintained for�Hgenzyme (Fig. 6a). Further, the increase in activity was morepronounced at higher concentrations in both the cases. Doublereciprocal plots yielded apparent KM and apparent VMAx valuesfor �Hg enzyme and þHg enzyme (Fig. 6b). Thus, �Hg and þHg

Table 2d-Aminolevulinic acid dehydratase and nitrate reductase activity in the presence ofKNO3 and Hg in excised etiolated maize leaf segments during greening. Leafsegments from dark grown maize seedlings were treated with ¼ strength Hoag-land’s solution containing different concentrations of HgCl2 in the presence of10 mM KNO3 in continuous light of intensity 30 W m�2 for 24 h. Values relative tocontrol are given in parentheses.

HgCl2 conc.,mM

ALAD activity,units g�1 fr. wt.

NR activity, n molesNO2 formed h�1 g�1 fr. wt.

0.00 68 � 4 (100) 1105 � 38 (100)0.001 64 � 3 (94)0.01 67 � 5 (98)0.02 58 � 5 (85)a 913 � 45 (83)b

0.05 51 � 4 (75)b 541 � 32 (49)c

0.10 38 � 6 (56)c

Level of significanceea: p < 0.05, b: p < 0.01, c: p < 0.001.

enzymes gave apparent KM values of 532 mM and 347 mM, respec-tively and apparent VMAx values of 118 and 52 units g�1 fr. wt.,respectively.

3. Discussion

The results demonstrate a concentration dependent decrease inALAD activity by in vivo supply of 0.01e0.05 mM HgCl2 in greeningmaize leaf segments, while 0.001 mM HgCl2 slightly increased theactivity (Table 1). R2 value of 0.731 between Hg treatment and ALADactivity indicated a very good correlation (Fig. 1). Further, verystrong correlation for Hg treatment and Hg content of leafsegments results with R2 value being 0.997 (Fig. 2) showing anaccumulation of Hg in the treated tissue. A specific stimulation of

Fig. 4. a. XeY scatter for Hg treatment and ALAD activity in the presence of nitrate. b.XeY scatter for Hg treatment and nitrate reductase activity in the presence of nitrate.

Table 3Effect of some nutrients and metabolites on the inhibition of d-aminolevulinic aciddehydratase activity by Hg in excised etiolatedmaize leaf segments during greening.Leaf segments from dark grown maize seedlings were treated with ¼ strengthHoagland’s solution containing the desired compounds either in the absence orpresence of 0.02mMHgCl2 in continuous light of intensity 30Wm�2 for 24 h. Valuesrelative to control are given in parentheses.

Metaboliteincluded

ALAD activity, units g�1 fr. wt. % Inhibitionby Hg�Hg þHg (0.02 mM)

None 52 � 3 (100) 35 � 3 (100)c 33NH4Cl, 10 mM 40 � 4 (77) 13 � 2 (37)c 68Glutamine, 5 mM 50 � 11 (96) 43 � 10 (123) 14Sucrose, 5 mM 52 � 7 (100) 29 � 5 (83)c 44Glutathione, 5 mM 48 � 10 (92) 36 � 8 (103) 25

Level of significanceea: p < 0.05, b: p < 0.01, c: p < 0.001.

Table 4Effect of inhibitors on inhibition of d-aminolevulinic acid dehydratase activity by Hgin excised etiolated maize leaf segments during greening. Leaf segments from darkgrownmaize seedlings were treatedwith ¼ strength Hoagland’s solution containingthe desired compounds either in the absence or presence of 0.02 mM HgCl2 incontinuous light for 24 h. Values relative to control are given in parentheses.

Inhibitorincluded

ALAD activity, units g�1 fr. Wt. % Inhibitionby Hg�Hg þHg (0.02 mM)

None 52 � 3 (100) 35 � 3 (100)c 33LA, 10 mM 35 � 4 (67) 27 � 3 (77) 23LA, 20 mM 24 � 3 (46) 22 � 3 (63) 08DTNB, 1 mM 47 � 4 (87) 34 � 3 (100)a 28

Level of significanceea: p < 0.05, b: p < 0.01, c: p < 0.001.

P. Gupta et al. / Plant Physiology and Biochemistry 62 (2013) 63e6966

chlorophyll synthesis and photosynthetic activities in maize andbean seedlings at low concentrations of a stressor, such as Cd, hasbeen reported in the literature [31]. In detached bean leaves bothCd and Pb stimulated chlorophyll accumulation and photosyntheticactivity and increased the level of cytokinin [32]. Further, in barleyseedlings such stimulatory effect on chlorophyll synthesis andphotosynthetic activity along with delayed senescence andincreased amounts of cytokinins have been observed [33].However, at higher concentrations mercury supply causeda significant decline in the total chlorophyll content and ALAaccumulation (Table 1). Amongst these, the ALA accumulation wasmost severely affected, suggesting that the ALA availability islimited due to Hg supply. On the other hand, Hg had no significanteffect on ALA formation but influenced ALAD activity [12,13]. In thesame study, Hg inhibited ALAD activity in vitro [12,13]. In thepresent system, two different types of effects resulted for in vitro

Fig. 5. Effect of in vitro inclusion of Hg in the presence of 2 mM LA or 0.1 mM DTNB on ALAD(�Hg enzyme) or with 0.02 mM Hg (þHg enzyme) in continuous light of intensity 30 W m

inclusion of mercury during enzyme assay causing inhibitionof �Hg enzyme but activation of þHg enzyme (Fig. 3). Similarly,differential sensitivity of �Hg and þHg enzymes is observed in thepresence of LA and DTNB (Fig. 5). Such differential response of theenzyme from Hg untreated and treated tissue suggests the possi-bility of coexistence of two forms of the enzyme in Hg-treatedtissue, the usual Mg dependent form and the other Hg-substituted form. Inhibition of enzyme activity by in vivo supplyof Hg affects the usual Mg-form while Hg-form is activated byin vitro addition of Hg.

The experiments suggest a protective role of nitrate againstinhibition of ALAD by Hg, as the inhibitory effect of Hg in thepresence of nitrate is observed at higher concentration ascompared to the inhibitory effect in the absence of nitrate (compareTables 1 and 2). Further, in vitro inclusion of mercury to þHgand þHgþN enzymes maintained a relatively high activity withlatter (Fig. 3). Although the nitrate reductase activity was moreseverely inhibited than ALAD activity by mercury at high concen-tration, the correlation analyses yielded R2 values being above 0.9with ALAD as well as NRA (Fig. 4a and b). Thus, the inhibitory effectof Hg on ALAD seems to be correlated with NO3

� assimilation andthe supply of NO3

� may reduce the uptake of Hg and/or increasethe mobilization of Hg towards vacuoles to exert its protectiveeffect. Vacuoles have been suggested to be the sites for accumula-tion of heavy metals [34,35].

Varying effects of nutrients and metabolites, such as, NH4Cl,glutamine, sucrose and GSH were observed in relation to inhibitionof ALAD activity by Hg. Thus, the % inhibition of ALAD activity by Hgwas greatly increased by NH4

þ as it lowered the enzyme activitymore strongly in presence of Hg (Table 3). This suggests that Hgtoxicity and NH4

þ-toxicity have a synergistic effect on ALADactivity. Glutamine, an overall regulator of N-metabolism, causeda significant lowering of the % inhibition of ALAD activity bymercury (Table 3). Glutaminemay overcome the inhibitory effect ofHg on ALAD by forming Hg-glutamine complex and therebyreducing the effective concentration of Hg. Moreover, glutamateand 2-oxoglutarate being the precursors of ALA in higher plants,glutamine supply under the condition of Hg toxicity may maintainthe supply of C5 compounds for ALA formation. The exogenoussupply of sucrose, a photosynthate and a carbon source, decreasedthe enzyme activity in presence of Hg, thereby increasing % inhi-bition of ALAD caused by Hg. ALAD has been reported to havereactiveeSH groups [36,37]. The % inhibition of ALAD activity by Hgwas reduced slightly due to supply of GSH and DTNB, as the activityremained almost same in the presence of Hg (Tables 3 and 4). Thus,it is likely that Hg may block reactive eSH groups of the enzyme

activity of the enzyme preparations obtained from leaf segments treated without Hg�2 for 24 h. Values relative to their respective controls are given.

Fig. 6. Effect of inclusion of varying concentrations of ALA during assay of ALADactivity of the enzyme preparations obtained from (�Hg enzyme) or with 0.02 mM Hg(þHg enzyme) in continuous light of intensity 30 W m�2 for 24 h. a) Direct plot. b)Double reciprocal plot. Symbols used: open circles (B——B); �Hg enzyme, closedcircles (C——C); þHg enzyme.

Fig. 7. Summary of mode of action of Hg on ALAD activity.

P. Gupta et al. / Plant Physiology and Biochemistry 62 (2013) 63e69 67

and hence the effect of these compounds on enzyme activity is notexerted in the presence of Hg.

ALAD is a substrate-modulated enzyme [38]. The % inhibition ofALAD activity by Hg is strongly reduced by LA, a competitiveinhibitor of the enzyme, due to lesser sensitivity in the presence ofHg (Table 4). Counteracting effects of Hg and LA suggests that Hgmay reduce LA binding to the enzyme or vice versa. On the otherhand, the observed inhibition of ALAD activity by Hg at varyingconcentration of ALA indicates thatþHg enzyme has higher affinityfor ALA but lower activity than �Hg enzyme, as indicated by lowerapparent Km and lower apparent Vmax for þHg enzyme (Fig. 6b).Allosteric Mg of E.coli ALAD causes reduction in Km for substrateand an increase in Vmax and Mg and substrate also cooperativelyenhance the stability of the octameric quaternary structure [39]. Inthe present study, with Hg-treated enzyme it is likely that due to Hgbinding at allosteric site where usually Mg is bound, the affinity forsubstrate is increased but the enzyme activity is reduced.

Here it is worth discussing the results of molecular modelingrevealing structural differences and characterization of enzymeobtained from expression of artificial genes. It has been reportedthat all ALAD contain an active site metal binding sequence, eithercysteine rich or aspartate rich that dictates a requirement for Znand Mg [23,40], respectively. These also correlate with distinctactive site basic residues, being arginine for cysteine rich sequenceand lysine for aspartate rich sequence. Mostly, ALADwas also foundto contain binding determinants for allosteric Mg [41]. Maximalbinding of three Mg (II) per subunit has been shown, two beingfunctionally distinct type, i.e. catalytic and allosteric and the thirdbeing nonphysiological and inhibitory [42]. Involvement of reactiveor allosteric (Cys) residues forming intermolecular disulfide linkage

to construct octameric enzyme has also been demonstrated [42].Hence, it is possible that treatment with Hg replaces one or more oftheMg atoms and/or causes modification of reactive residues of theenzyme causing inhibition. The possible modes of action of Hg onALAD activity have been summarized in (Fig. 7). Two such modesproposed are (1) Binding of Hg at allosteric Mg site to causeincrease in affinity of the enzyme for ALA, which is supported bylower apparent Km ofþHg enzyme (Fig. 6b) and lesser sensitivity ofthe enzyme for LA in the presence of Hg (Table 4). (2) Hg causingdissociation of the enzyme by disrupting eSeSe interactionleading to reduced activity, which is reflected by lower apparentVmax with þHg enzyme (Fig. 6b) and no effect of GSH and DTNB onthe enzyme activity in the presence of Hg (Tables 3 and 4).

4. Conclusion

In vivo supply of Hg inhibited ALAD activity and the inhibitionwas alleviated by KNO3. The inhibitory effect of Hg on enzymeactivity was increased by NH4Cl and sucrose, but decreased byglutamine, glutathione, levulinic acid and 5,50 dithio bis 2-nitrobenzoic acid. In vitro inclusion of Hg during assay of the �Hgenzyme and þHg enzyme caused the inhibition of former butactivation of later. It is suggested that two forms of enzyme exist inHg-treated tissue, i.e. the usual Mg dependent form and an unusualHg modified form. Kinetic studies yielded distinct apparent Km andapparent Vmax values eHg enzyme and þHg enzyme indicatingthat þHg enzyme has higher affinity for d-aminolevulinic acid butlower activity as compared to the �Hg enzyme. The proposed

P. Gupta et al. / Plant Physiology and Biochemistry 62 (2013) 63e6968

modes of action of Hg on ALAD activity are the binding of Hg atallosteric Mg site to cause increase in affinity of the enzyme for ALAand Hg causing dissociation of the enzyme by disrupting eSeSeinteraction leading to reduced activity.

5. Materials and methods

5.1. Plant material, growth conditions and treatments

Seeds of Zea mays (L.) cv. GSF-2 were surface sterilized with 0.1%HgCl2 for 1e2 min and then rinsed thoroughly with distilled water.The seedlings were grown in plastic pots containing acid washedsand in continuous dark at 26 � 2 �C for 7e8 days and watered onalternate days with ½ strength Hoagland’s nutrient medium notcontaining any inorganic nitrogen (eN Hoagland). For variousanalyses primary leaves from uniformly grown seedlings were cutinto pieces of 0.5 * 0.5 cm2 and were treated with ¼ strengthHoagland’s nutrient medium (pH 6) containing desired compoundsin continuous light of intensity 30 W m�2 at 25 � 2 �C for 24 hinside “Newtronic” Growth Chamber model NEC 216.

5.2. Analytical procedures

ALAD activity was assayed colorimetrically by estimating theamount of porphobilinogen formed by using Ehrlich reagent.Extraction and assay of ALAD were carried out according to theprocedure described in Jain and Gadre [27]. The enzyme wasextracted with ice-cold 50 mM TriseHCl buffer (pH 8.2) containing10 mMDTT. The ratio of material to extraction mediumwas 1:4 (w/v). The supernatant obtained after centrifugation at 15,000� g for30 min in cold (0e4 �C) using ‘Sorvall RC 5B plus’ was used forenzyme assay. One ml of enzyme was incubated with 0.27 ml of1 mg ml�1 ALA, 1.35 ml of 50 mM TriseHCl buffer (pH 8.2) con-taining 10mMDTTand 0.08ml of 0.02MMgCl2. After incubation at37 �C for 1 h the reaction was stopped by adding 0.3 ml of 3 M TCAand it was centrifuged at 5000� g for 10 min. For PBG estimationthe supernatant was mixed with Ehrlich reagent (prepared freshlyby dissolving 1.0 g of 4-dimethyl aminobenzaldehyde in 30.0 ml ofglacial acetic acid and 8.0 ml of 70% PCA and then made up to50.0 ml with glacial acetic acid) in a ratio of 1:1 (v/v) and theabsorbance was measured at 553 nm after 15 min against zero timecontrol. One unit of enzyme activity is defined as 1 nmol of PBGformed h�1.

The ALA content was determined according to the method ofTewari and Tripathy [28]. The treated leaf segments were incubatedin 50 mM phosphate buffer (pH 6.0) containing 60 mM levulinicacid for 3 h in light and then thoroughly rinsed with distilled waterand homogenized by using 1.0 ml of 1 M Na-acetate buffer (pH 4.6).After centrifugation for 10 min, ALA of the supernatant wascondensed into PBG using ethylacetoacetate. For this 0.7 mlsupernatant, 0.8 ml distilled water and 0.1 ml ethylacetoacetatemixture was kept in a boiling water bath for 10 min. After cooling,an equal volume of Ehrlich reagent was added and the coloredcomplex formed was read for absorbance at 553 nm. Amount ofPBG formed was calculated using a standard curve of ALA.

In vivo nitrate reductase activity was assayed by measuring thenitrite production by the method of Srivastava [29]. The assaymixture formed of 0.1 M phosphate buffer (pH 7.4), 25% iso-propanol, 0.2 M KNO3 containing the segments of the treatedmaterial was incubated in dark for 30 min. An aliquot of it was thentreated with 1% sulphanilamide prepared in 1N HCl and 0.02%naphthyl ethylene diamine and measured for absorbance at540 nm. Chlorophyll was extracted from the treated leaf segmentsusing 80% acetone and quantified from its extinction at 663 nm and645 nm according to the method of Strain and Svec [30].

Mercury content of the treated material was estimated by usingan atomic absorption spectrophotometer after acid digestion of thedried material.

5.3. Statistical analysis

Data presented in the paper are average of at least four inde-pendent experiments with�S.E. Significance of difference obtainedfor various treatments was tested by Student’s t test.

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