Research Article Effect of Chromium(VI) Toxicity on ...downloads.hindawi.com/archive/2014/784036.pdfResearch Article Effect of Chromium(VI) Toxicity on Enzymes of Nitrogen Metabolism

Research ArticleEffect of Chromium(VI) Toxicity on Enzymes of NitrogenMetabolism in Clusterbean (Cyamopsis tetragonoloba L.)

Punesh Sangwan,1 Vinod Kumar,2 and U. N. Joshi1

1 Department of Biochemistry, CCS Haryana Agricultural University, Hisar 125001, India2Department of Biochemistry, G. B. Pant University of Agriculture and Technology, Pantnagar 263145, India

Correspondence should be addressed to Punesh Sangwan; [email protected]

Received 5 December 2013; Accepted 6 February 2014; Published 18 March 2014

Academic Editor: J. Paul G. Malthouse

Copyright © 2014 Punesh Sangwan et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Heavy metals are the intrinsic component of the environment with both essential and nonessential types. Their excessive levelspose a threat to plant growth and yield. Also, some heavy metals are toxic to plants even at very low concentrations. Thepresent investigation (a pot experiment) was conducted to determine the affects of varying chromium(VI) levels (0.0, 0.5, 1.0,2.0, and 4.0mg chromium(VI) kg−1 soil in the form of potassium dichromate) on the key enzymes of nitrogen metabolism inclusterbean. Chromium treatment adversely affect nitrogenase, nitrate reductase, nitrite reductase, glutamine synthetase, andglutamate dehydrogenase in various plant organs at different growth stages as specific enzyme activity of these enzymes decreasedwith an increase in chromium(VI) levels from 0 to 2.0mg chromium(VI) kg−1 soil and 4.0mg chromium(VI) kg−1 soil was foundto be lethal to clusterbean plants. In general, the enzyme activity increased with advancement of growth to reach maximum atflowering stage and thereafter decreased at grain filling stage.

1. Introduction

Excessive levels of heavy metals in agricultural lands con-stitute an increasingly serious threat not only for intactplant growth and yield but also for the environment andhuman health [1]. The toxicity of plants due to heavy metals,particularly on agricultural economic crops, presents a chal-lenge to plant scientists concerned with yield and quality incrop production [2]. These metals retard farming efficiencyand destroy the health of the plants and animals [3]. Someheavy metals such as chromium (Cr), lead (Pb), mercury(Hg), and cadmium (Cd), especially in large amounts, couldaffect growth and productivity of plants [4]. They are usuallyaccumulated due to unplanned municipal waste disposal,mining, use of extensive pesticides, and chemical fertilizers[5].

Nowadays, contamination of the environment by Cr hasbecome amajor concern and its toxicity to plants depends onits valence state with Cr(VI) being highly toxic and mobilethan Cr(III) [6]. Cr(III) occurs naturally in the environmentand is an essential nutrient, whereas Cr(VI) is generally

produced by industrial processes [7]. It is found in all partsof the environment including air, water, rocks, and soil[5]. The anthropogenic sources of Cr input are electroplat-ing, mining processing, wood preservation, iron and steelproduction, pigment manufacture, power station industry,leather tanning, textile preservation, textile printing, paintand porcelainmanufacturing, and during combustion of coaland petroleum [8, 9]. Significant quantities of Cr are alsoadded to soil by the disposal of fly ash as well as phosphaticfertilizer application [10].

There are a number of factors which influence plantgrowth, among them nitrogen is one of the most essentialelements and its availability in soil is one of the key factorsfor determining plant growth and productivity [10]. Nitrogenis considered to be a vital macronutrient for plants andhas a role in metabolism [11]. It is incorporated in planttissues as nucleotides, nucleic acids, coenzymes, vitamins,pigments, alkaloids, amines, and other compounds [12]. Itis structural component of wide array of vital biomolecules,such as amino acids, proteins, nucleotides, porphyrins, sev-eral coenzymes, chlorophylls, vitamins, and glycosides. On

Hindawi Publishing CorporationEnzyme ResearchVolume 2014, Article ID 784036, 9 pageshttp://dx.doi.org/10.1155/2014/784036

2 Enzyme Research

average, proteins and nucleic acids contain about 15% and13% nitrogen, respectively [12]. Hence, the maintenance ofproper N-metabolism of plant can lead to proper growthand development of plant. The nitrogen metabolism andenzymology are related to the selection of high yielding crops.It has been shown that enzymes of nitrogen metabolism areseverely affected by different metals and thus reduced cropyields. The nitrogen metabolism is of central importanceunder stressful conditions [13].

According to Dixit et al. [14], Cr is toxic to plants andinterferes with several metabolic processes as exhibited byreduced seed germination or early seedling development,induced chlorosis in young leaves, reduced pigment content,damaged root cells, impaired photosynthesis, altered enzy-matic function, stunted growth, and plant death [15]. In India,Cr(VI) contamination is a major problem around variousindustries using Cr compounds, which causes considerablenegative impact on crop production [16].

Clusterbean (Cyamopsis tetragonoloba L.) has been grownin India since ancient times for vegetables and fodder pur-poses. It is an important kharif legume commonly known asguar and is cultivated throughout India for its edible pods. Itsgreen pods are used as vegetable, seeds as source of industrialgum, and green plants as fodder and for soil manuringpurposes. It has recently assumed great industrial importancedue to the presence of gum, that is, galactomannan, in itsendosperm which constitutes 25–35% of whole seeds andis highly mucilaginous. Gum is used extensively in paper,mining, explosive, food, pharmaceuticals, cosmetics, textiles,and oil industries [17]. In Haryana (a major guar producingstate), Sonepat, Panipat, Dharuhera, Gurgaon, Yamunanagar,Faridabad, and Shahabad are the main industrial areas,where poor plant growth of field crops has been observed.Contamination of soils by heavy metals as a result of human,agricultural, and industrial activities is a major cause ofpoor plant growth [18]. However, not much work has beenreported on the effect of Cr(VI) on guar. Considering theabove facts and the importance of nitrogen in plant growth,the present study was carried out to explore the effect ofCr(VI) on nitrogen metabolism in clusterbean which will bebeneficial to the understanding of Cr(VI) induced changes inplant nitrogen metabolism.

2. Material and Methods

2.1. Chemicals, Reagents, and Soil. The chemicals andreagents used during the present investigation were of ana-lytical grade. A nutrient deficient loamy sand soil from theRegional Research Station, Gangwa block of Hisar district,was used in the present study. The characteristics of soil werepH (1 : 2) 8.50; organic carbon, 0.22%; N, 4.0mg kg−1 soil;P, 13.0mg kg−1 soil; K, 163mg kg−1 soil; Zn2+, 0.61mg kg−1soil; Fe2+, 0.9mg kg−1 soil; Cu2+, 0.18mg kg−1 soil; Mn2+,3.6mg kg−1 soil; EC, 1.5; CaCO

3

3.5%; and Cr2+, 0.01mg kg−1soil; texture—sandy loam.

2.2. Plant Growth and Environmental Conditions. Seeds ofclusterbean (Cyamopsis tetragonoloba (L.) Taub.) cv HG 2–20

were procured from Forage Section, Department of Geneticsand Plant Breeding, C.C.S. Haryana Agricultural University,Hisar, and raised in pots filledwith 5 kg of sandy loam soil in anaturally lit net house.The temperature and relative humidityduring the experiment ranged from 11.0 to 35.6∘C and 34.5to 95.2%, respectively. The light intensity ranged from 36100to 84000 lux. The pots were lined with polythene bags andthe soil in each pot was treated with different levels of Crin the form of potassium dichromate at concentrations of0.0, 0.5, 1.0, 2.0, and 4.0mg kg−1 soil. The seeds were surfacesterilized with mercuric chloride and after proper washingwith distilled water, they were inoculated with Rhizobiumculture. An equal amount of nutrient solution was suppliedat weekly intervals to each pot.The plants were irrigated withequal quantities of tap water as and when required. Plantsamples from each treatment were collected at vegetative (30DAS), flowering (50 DAS), and grain filling stages (65 DAS).

2.3. Enzyme ActivityMeasurement. Specific activity in leaves,shoot, and root at different growth stages was measured bystandard methods. All observations were measured up to2.0mg kg−1 soil because plants treated withmore than 2.0mgCr(VI) kg−1 soil concentration did not survive 20 days aftersowing.

2.3.1. Sample Extraction for Nitrate Reductase and NitriteReductase. One gram of plant tissue was hand homogenizedin 10mL cold phosphate buffer (0.15M, pH 7.5) contain-ing 1mM cysteine and 1% (w/v) casein in a previouslychilled mortar using acid-washed sand as an abrasive. Thehomogenate was centrifuged at 10,000×g for 30min at 4∘C.The resultant supernatant was taken as the enzyme extractand stored in a refrigerator for enzyme assays and solubleprotein estimation.

2.3.2. Sample Extraction for Glutamine Synthetase, GlutamateDehydrogenase, and Glutamate Synthase. One g of planttissue was hand homogenized in 10mL of cold phosphatebuffer (0.1M, pH 7.6) containing 2% polyvinylpyrrolidone,1% 𝛽-mercaptoethanol, and 10mM dithiothreitol in previ-ously chilled mortar using acid-washed sand as an abrasivematerial [19]. The homogenate was centrifuged at 10,000×gfor 30min at 4∘C.The resultant supernatant was taken as theenzyme extract and stored in a refrigerator for enzyme assaysand soluble protein estimation.

2.3.3. Nitrogenase (E.C.1.7.99.2). Nitrogenase activity in nod-ules was measured by acetylene reduction assay as describedby Hardy et al. [20].The roots were separated from the shootsand blotted to remove excess moisture and intact noduleswere placed in test tubes (75mL). A subaseal was placedon the mouth of tubes. An atmosphere of 10% acetylenewas created and the tubes were incubated for 2 h at roomtemperature (28∘C).The ethylene so produced was measuredby Gas Chromatograph (Hewlett Packard Model 5730) usinga dual flame ionizing detector. Nitrogen at a flow rate of38mLmin−1 was used as carrier gas and hydrogen at theflow rate of 25mLmin−1 was used as fuel gas. Temperature

Enzyme Research 3

of oven, detector, and injection port was set at 65, 150, and100∘C, respectively. Standard ethylene (110 ppm) was used forquantification of data. Specific nitrogenase activity expressedas 𝜇MC

2

H4

produced g−1 fresh weight of nodules h−1.

2.3.4. Nitrate Reductase (E.C.1.6.6.1). Nitrate reductase (NR)was assayed by the method suggested by Hageman andFlesher [21]. The assay mixture in a final volume of 2mLcontained (𝜇M) phosphate buffer (pH 7.5), 200; KNO

3

, 20;and NADH, 0.4. The enzymatic reaction was initiated bythe addition of 0.5mL of enzyme extract. A blank withoutNADHwas also run simultaneously. After incubation at 30∘Cfor 15min, the reaction was terminated by rapidly adding0.1mL of 1M zinc acetate and 1.9mL of 70% ethanol. Thecontents were mixed thoroughly and centrifuged at 3000×gfor 15min. Two milliliters of supernatant was then removedand transferred to another test tube. One milliliter of 1% sul-phanilamide reagent (prepared in 1N HCl) followed by 1mLof 0.02%N-1-naphthyl ethylene diamine dihydrochloridewasadded. After 30min, the absorbance of the violet color wasmeasured at 540 nm on a spectrophotometer (Systronics 118).The enzyme activity has been expressed as 𝜇M of nitriteproduced h−1mg−1 protein.

2.3.5. Nitrite Reductase (E.C.1.7.7.1). Nitrite reductase (NiR)activity was measured following the method of Sawhneyand Naik [22]. The assay mixture in a final volume of 2mLcontained (𝜇M) phosphate buffer (pH 7.5), 100; NaNO

2

, 1.0;and methylviologen, 0.4 and 0.5mL of enzyme extract. Thereaction was started with the addition of 0.1mL of sodiumdithionite solution (prepared by dissolving 10mg sodiumdithionite in 10mL of 0.29M sodium bicarbonate). In aseparate tube, sodium bicarbonate without sodium dithionitewas run simultaneously as a blank. After incubation for30min at 30∘C, the reaction was stopped by shaking vig-orously. A 0.1mL volume of the supernatant was aliquotedinto a test-tube and added to this was 1.9mL of distilledwater and 1mL of 1% sulphanilamide reagent (prepared in1N HCl) followed by 1mL of 0.02% N-1-naphthyl ethylenediamine dihydrochloride. After 30min, the absorbance ofviolet color wasmeasured at 540 nm on a spectrophotometer.The enzyme activity has been expressed as 𝜇M of nitritereduced h−1mg−1 protein.

2.3.6. Glutamine Synthetase (E.C.6.3.1.2). The activity of glu-tamine synthetase (GS) was assayed by the method of O’Nealand Joy [23]. The reaction mixture (4mL) contained 0.1MTris-maleate buffer (pH 7.5); 1M hydroxylamine; 100mMglutamate; 10mM ATP; 1M MgSO

4

; and 0.2mL of dilutedenzyme extract. The reaction was started by adding hydroxy-lamine and themixture was incubated for 20min at 30∘C.Thereactionwas stopped by the addition of 1mL of FeCl

3

reagent,prepared by mixing equal volumes of 10% FeCl

3

⋅6H2

O in0.2M HCl, 24% TCA, and 5% HCl. After 10min, the proteinprecipitate was removed by centrifugation. Absorbance ofthe supernatant was taken at 540 nm and hydroxamic acid

concentration was computed using 𝛾-glutamyl monohydrox-amate (𝛾-GMH) as standard. The results were expressed as𝜇M of 𝛾-GMH formed h−1mg−1 protein.

2.3.7. Glutamate Synthase (E.C.1.4.1.14). Glutamate synthase(GOGAT) activity was measured using the method of Singhand Srivastava [24]. Briefly, the assay mixture contained0.4mL 20mM L-glutamine, 0.4mL 5mM 2-oxoglutarate,1mM EDTA (added in assay buffer), 0.1mL 100mM KCI,0.6mL 1mM NADH, and 0.5mL of the enzyme preparationin a final volume of 3.0mL prepared with 25mM sodiumphosphate (pH 7.5). The reaction was started by adding L-glutamine immediately following the enzyme preparation.The decrease in absorbance was recorded for 5min at 340 nmon a spectrophotometer.The amount of NADH oxidized wascalculated from a standard curve of NADH.The results wereexpressed as 𝜇MNADH oxidized h−1mg−1 protein.

2.3.8. Glutamate Dehydrogenase (E.C.1.4.1.4). Glutamatedehydrogenase (GDH) activity was measured following themethod of Boland et al. [19]. In brief, the assaymixture (2mL)contained 14mM 2-oxoglutarate; 80mM imidazole-HCl(pH 7.9); 200mM ammonium acetate; 60mMNADH; 2mMADP; and 0.1mL enzyme extract. Rate of the reaction wasfollowed by recording the change in absorbance at 340 nm ona spectrophotometer. Background rates were also measuredin the absence of ammonium acetate. The enzyme activitywas expressed as 𝜇MNAD+ formed h−1mg−1 protein.

2.4. Protein Estimation. The soluble protein in the enzymeextract was determined by the method of Lowry et al. [25]followed by itsprecipitation by 20% TCA, centrifugation,and dissolving residue in 0.1 N sodium hydroxide (NaOH)solution.

2.5. Statistical Analysis. A two-factorial ANOVA in completerandomized block design was used to confirm the validity ofthe data using the OPSTAT software available on CCSHAUwebsite homepage (http://hau.ernet.in/opstat.html). The val-ues used in graphs are mean of three replicates and are shownas ±standard error.

3. Results and Discussion

3.1. Nitrogenase. Nitrogen is one of the major constituents ofbiological systems. In nature, highly specialized nitrogenaseenzymes are expressed by soil bacteria and perform animportant function of nitrogen fixation by converting atmo-spheric nitrogen into ammonia under mild temperature andpressure conditions [26]. In the present study, the gradual andsignificant decrease in specific nodule nitrogenase activity(SNA) (𝜇M ethylene produced g−1 fresh weight of nodulesh−1 × 102) was observed at different stages of growth innodules with increasing doses of Cr(VI) concentration from0.0 to 2.0mg Cr(VI) kg−1 soil (Figure 1). A similar decreasein nitrogen fixation in response to Cr was observed in

4 Enzyme Research

Pisum sativum [27]. At 30 DAS, SNA decreased by 1.3-, 1.9-, and 3.2-fold with respect to control at 0.5, 1.0, and 2.0mgCr(VI) kg−1 soil, respectively. Maximum decline in SNA was69% at 65 DAS with 2.0mg Cr(VI) kg−1 soil. Specific nodulenitrogenase activity decreased by 16.5, 35.4, and 65.4% withrespect to control at 0.5, 1.0, and 2.0mg Cr(VI) kg−1 soilat 50 DAS, respectively. In control as well as Cr treatedplants, SNA increased abruptly from day 30 onwards toevince maximum value at day 50 and after that declined at65 DAS. According to Balestrasse et al. [28], high cadmiumconcentration or salt stress resulted in oxidative stress withincreased thiobarbituric acid reactive substances content anddecreased leghemoglobin level and, consequently, decreasedSNA. Decreased SNA may also be due to the effect ofheavy metal ions on O

2

uptake by bacteroid which couldresult in inhibition of acetylene reduction activity. Bacteroidrespiration provides the energy and reducing power thatnitrogenase needs for efficient nitrogen fixation [28]. Undera high concentration of metals, the decreased SNA mightalso be due to decreased nodule number and their biomass[29, 30], induced nodule senescence [28], altered noduleultrastructure [31], and/or altered plant growth-promotingactivities of microorganisms [34].

3.2. Nitrate Reductase. NR is the first enzyme in the processof nitrate reduction and plays an important role in nitro-gen assimilation by catalyzing the reduction of nitrate tonitrite. Nitrate is the common source of nitrogen availableto plants. Once nitrate is being absorbed by plants it maybe reduced in roots, stored in the vacuoles, or transferredto the shoots before being processed. The conversion ofnitrate into ammonia is brought about by successive andregulated action of NR and NiR [32]. It is affected by plantdevelopment stages and plant parts such as roots and topsand environmental conditions [18]. Cr application in soil wasfound to have negative effect on the NR specific activity (𝜇Mof nitrite produced h−1mg−1 protein). The specific activitywas decreased by 70.1, 71.9, and 74.9% in leaves, 57.6, 55.9,and 59.2% in stem, and 63.4, 57.3, and 65.0% in root of2.0mg Cr(VI) kg−1 soil treated plants over control at 30,50, and 65 DAS, respectively (Figure 2). In each treatment,NR specific activity in various plant parts increased withplant age, attaining the maximum value at 50 DAS, anddecreased thereafter at 65 DAS. Root had higher specificactivity followed by stem and leaves.

Cr-induced toxicity resulting in reduced activity of NRwas also reported in sorghum and Indian mustard [18, 33].Study on wheat and Indian mustard revealed that lowerdoses of Cr had stimulatory effects, while higher doseshad inhibitory effects on NR activity [33, 34]. A significantincrease in NR activity was observed corresponding to Crconcentration inBrassica juncea [35]. However, in the presentstudy, no stimulatory effect of low Cr(VI) concentration wasobserved. The results are in conformity with the observationof Kumar and Joshi [18], where NR specific activity decreasedwith increase in Cr(VI) concentration. It is suggested thata negative influence of Cr on NR specific activity mightbe due to a reduced supply of NADH which might result

0

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0mg Cr(VI)/kg soil0.5mg Cr(VI)/kg soil

1mg Cr(VI)/kg soil2mg Cr(VI)/kg soil

Nitr

ogen

ase a

ctiv

ity (𝜇

mol

es et

hyle

nepr

oduc

edg−

1fre

sh w

eigh

t of n

odul

esh−

1)

Figure 1: Effect of Cr(VI) on specific nitrogenase activity in nodulesof clusterbean plants at different stages of growth.

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Leaves Stem Root Leaves Stem Root Leaves Stem Root30 50 65



(DAS)

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ate

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ctas

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tivity

(𝜇m

oles

of n

itrite

pro

duce

dh−

1m

g−1 pr

otei

n)

Figure 2: Effect of Cr(VI) on nitrate reductase activity in cluster-bean plant parts (leaves, stem, and root) at different stages of growth.

from disorganization of chloroplasts, reduced rate of pho-tosynthesis, respiration, NADH oxidation, or reduction inNO3

− supply to the site of the enzyme as a consequence ofwater stress induced by the metal and direct effect of heavymetals on protein synthesis [36]. Similar results have beenobserved by Rai et al. [37], where Cr toxicity resulted in

Enzyme Research 5

0

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(DAS)

Nitr

ite re

duct

ase a

ctiv

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mol

esof

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ite re

duce

d h−

1m

g−1

prot

ein)

Figure 3: Effect of Cr(VI) on nitrite reductase activity in clusterbeanplant parts (leaves, stem, and root) at different stages of growth.

the reduction of nitrate reductase activity through impairedsubstrate utilization in Ocimum tenuiflorum.

3.3. Nitrite Reductase. NiR catalyzes the reduction of nitriteto ammonium in the second step of the nitrate assimilationpathway. A decrease in NiR specific activity (𝜇M of nitritereduced h−1mg−1 protein) was observed in leaves, stem, androots of clusterbean plant treated with Cr(VI) in comparisonto the control (Figure 3). Similar to nitrate reductase, specificactivity of nitrite reductase also decreased with increasinglevel of Cr(VI) at different stages of growth. The maximumdecrease in NiR specific activity as compared to control was56.0, 57.6, and 54.0% in leaves, 56.7, 53.6, and 56.6% in stem,and 64.1, 61.3, and 55.7% in root with 2.0mg Cr(VI) kg−1 soiltreated plants at 30, 50, and 65 DAS, respectively. In everytreatment, NiR activity increased tomaximum at 50 DAS anddecreased thereafter at 65 DAS (Figure 3).

These findings are in agreement with previous reports[18]. The observed decrease in NiR activity may be due toeither of reduced carbon fixation, NO

3

− uptake by roots andlow NO

3

− translocation in the xylem which may lead tosubsequent limitation in reducing power and/or low NO

3

−

availability to plants [38]. Gajewska and Skłodowska [39]have shown that a decline in NiR activity was associated withthe reduced availability of NO

2

− ions, which are consideredto have originated primarily from the NR-catalyzed NO

3

−

reduction. Moreover, it has been shown that the accumula-tion of NH

4

+ within a cell in higher concentrations altersintracellular pH and osmotic balance, inhibits ATP synthesisand secondary growth, and causes nutrient deficiency and

chlorosis [40]. Therefore, under 2.0mg Cr(VI) kg−1 soiltreatment, the increase in NH

4

+ content may be linkedwith decreased growth of clusterbean plants. The observeddecrease in NiR activity may either be due to reducedcarbon fixation or low nitrate translocation in the xylemand, subsequently, low nitrate availability in shoot or reduceduptake of nitrate by roots or limitation in reducing power [18].

3.4. Glutamine Synthetase. Ammonia is primarily assimi-lated through glutamine synthetase via GS/GOGAT pathway.Inorganic nitrogen could be assimilated by plants into theforms of glutamine and glutamic acid. GS is a key enzyme fornitrogen assimilation, which regulates nitrogen metabolism[41]. It is involved in the assimilation of ammonia derivedeither from nitrate reduction, N

2

fixation, photo respiration,or asparagine breakdown. In the present study, the specificactivity of GSwas progressively decreased in leaves, stem, androot of clusterbean plants treated with increasing dosages ofCr(VI).The gradual decrease inGS specific activity (𝜇Mof 𝛾-GMH formed h−1mg−1 protein) was observed at all stagesof growth. Maximum decline in enzyme specific activity wasrecorded at 2.0mg Cr(VI) kg−1 soil at all stages of growth.Compared to control, a 59.6, 56.7, and 65.7% decrease inits specific activity was observed at 50 DAS in leaves, stem,and root of plants treated with 2.0mg Cr(VI) kg−1 soil,respectively. The enzyme specific activity increased fromvegetative to flowering stage and then declined in differentplant parts at the grain filling stage (i.e., 65 DAS) (Figure 4).

Inhibition of GS activity has also been described in barleyseedlings [42]. The decreased availability of ATP and Mg2+ions, which act as cofactors in various metabolic processes,may be the possible reason for the observed decrease inGS activity. Moreover, decreased activity in GS may alsobe responsible for increased NH

4

+ content as it has beenshown that accumulation of high concentrations of NH

4

+

within a cell leads to nutrient deficiency and chlorosis, altersintracellular pH and osmotic balance, inhibits ATP synthesis,and eventually inhibits secondary growth [40].Thus, it mightbe possible that supplementation of Cr led to an increasein NH

4

+ content and a decrease in GS activity. Severalstudies indicated degradation of nitrogenous compoundsunder heavy metal stress [36, 39].

3.5. Glutamate Synthase. The key enzyme involved in thede novo synthesis of glutamate is glutamate synthase, alsoknown as glutamine: 2-oxoglutarate aminotransferase. Theacidic amino acid is formed by the action of glutamate syn-thase, utilizing glutamine and 2-oxoglutarate. The reactionis a reductant-driven transfer of the amide amino groupof glutamine to 2-oxoglutarate to yield two molecules ofglutamate [43]. The specific activity of GOGAT (𝜇M NADHoxidized h−1mg−1 protein) in various plant parts decreasedsignificantly with increasing concentration of Cr(VI) at allstages of growth (Figure 5). Amongst the Cr(VI) levels,2.0mg kg−1 soil had a maximum adverse effect on its specificactivity. At this concentration, the enzyme specific activitydecreased by 70.0, 60.36, and 64.9% in leaves, 61.9, 64.3,and 58.5% in stem, and 54.9, 55.0, and 56.5% in roots as

6 Enzyme Research

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tam

ine s

ynth

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e act

ivity

(𝜇m

oles

of𝛾

-GM

H fo

rmed

h−1

mg−

1pr

otei

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Figure 4: Effect of Cr(VI) on glutamine synthetase activity inclusterbean plant parts (leaves, stem, and root) at different stages ofgrowth.

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(DAS)

Glu

tam

ate s

ynth

ase a

ctiv

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mol

es o

fN

AD

H o

xidi

zed

h−1

mg−

1pr

otei

n)

Figure 5: Effect of Cr(VI) on GOGAT activity in clusterbean plantparts (leaves, stem, and root) at different stages of growth.

compared to control at 30, 50, and 65 DAS, respectively.The specific activity increased continuously in leaves, stem,and root up to 50 DAS and decreased thereafter at 65 DASin each individual treatment (Figure 5). It is suggested thatCr may also induce premature senescence of plants throughan increased level of NH

4

+ either through rapid reductionof NO

3

− or enhanced proteolysis. Moreover, it has been

shown that accumulation of NH4

+ within a cell in higherconcentrations leads to nutrient deficiency and chlorosis,alters intracellular pH and osmotic balance, inhibits ATPsynthesis, and eventually inhibits secondary growth [40].

3.6. Glutamate Dehydrogenase. In addition to GS andGOGAT, another enzyme potentially involved in ammoniametabolism is GDH. It can catalyze the reductive amina-tion of 2-oxoglutarate and the reverse catabolic reactionof oxidative deamination of glutamate. It is located in themitochondrial matrix where it is mainly responsible for glu-tamate catabolism under carbon and N-limiting conditions[44]. Glutamate dehydrogenase is present in the leaves, roots,and nodules in abundance [19]. Robinson et al. [45] havesuggested that GDH activity is altered in response to shiftsin carbon rather than nitrogen metabolism. Increasing theconcentration of Cr(VI) from 0.0 to 2.0mg Cr(VI) kg−1 soilresulted in a decrease in GDH specific activity (𝜇M NAD+formed h−1mg−1 protein) at different stages of growth indifferent plant parts. The specific activity decreased by 65.1,60.5, and 68.4% in leaves, 55.6, 51.9, and 52.2% in stem, and62.7, 60.9, and 59.5% in root as compared to the control at30, 50, and 65 DAS, respectively, in 2.0mg Cr(VI) kg−1 soiltreated plants. In individual treatment, maximum enzymespecific activity was observed at 50 DAS followed by adecrease at 65 DAS. The stem exhibited higher GDH specificactivity than leaves and root (Figure 6).

In the present study, results are in agreement with previ-ous observations regarding Cr(VI) induced GDH inhibitionin leaves, stem, and root of sorghum [18]. Burzynski andBuczek [46] studied the influence of Cu2+, Cd2+, Pb2+, andFe2+ at various pH conditions (5.0, 6.0, and 7.0) on the uptakeand assimilation of ammonia by cucumber seedlings andfound that GS and NADH-GDH were also inhibited after 1 hof plant exposure to these metals. Joshi et al. [47] reportedthat the activity of GDH was increased in different plantparts of cowpea with Cr(VI) concentration up to 1 ppm andthen decreased with further increase in it till 6 ppm. Duringthe present study, GDH activity also decreased under Cr(VI)treatments. Heavy metals like Ni and Al treatments alsoimpaired the nitrate assimilation process in rice seedlings byinhibiting the activities of key nitrogen assimilatory enzymes,that is, NR, GS, and GDH [48]. Evidence showed thatGDH is not involved in ammonia assimilation, which occurssolely via the GS/GOGAT cycle. It was hypothesized thatthe primary role of GDH is the catabolism of glutamateto provide carbon skeletons for the TCA cycle functionunder conditions of carbon limitation, and consequently, thisenzyme fulfills an important anapleurotic function linkingcarbon and nitrogen metabolism in higher plants [49].

4. Conclusion

In conclusion, our results indicate that Cr(VI) applicationadversely affected nitrogen metabolism by inhibiting theactivity of these enzymes and growth of clusterbean plants,possibly as a result of its interference with photosyntheticpigments and key enzymes of nitrogen metabolism. The

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ate d

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1pr

otei

n)




(DAS)

Figure 6: Effect of Cr(VI) on glutamate dehydrogenaseactivity in clusterbean plant parts (leaves, stem, and root) at different stages of growth.

increased accumulation of Cr in plant parts negativelyaffected nitrogen metabolism. It may have further implica-tions in designing and conducting similar studies as well asunderstanding the detailedmechanism of this toxicity and itsremoval thereafter.

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper.

Acknowledgments

The authors gratefully acknowledge the Head, Departmentof Biochemistry and Director Research, CCSHAU, Hisarfor providing the necessary infrastructural facility. The leadauthor is grateful to the Indian Council of AgricultureResearch (ICAR) for providing financial assistance in theform of ICAR-SRF.

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