REGULAR ARTICLE

Synthesis of phytochelatins in vetiver grass upon leadexposure in the presence of phosphorus

Syam S. Andra & Rupali Datta & Dibyendu Sarkar &

Konstantinos C. Makris & Conor P. Mullens &

Shivendra V. Sahi & Stephan B. H. Bach

Received: 10 August 2008 /Accepted: 2 April 2009 /Published online: 28 April 2009# Springer Science + Business Media B.V. 2009

Abstract In a hydroponic setting, we investigatedthe possible role of phytochelatins (metal-bindingpeptides) in the lead (Pb) tolerance of vetiver grass(Vetiveria zizanioides L.). Pb was added to thenutrient medium at concentrations ranging from 0 to1,200 mg L−1. Furthermore, we simulated the effectof soil phosphorus (P) on potentially plant availablePb by culturing vetiver grass in P-rich nutrient media.After 7 days of exposure to Pb, we evaluated the Pbuptake by vetiver grass. Results indicate that vetivercan accumulate Pb up to 3,000 mg kg−1 dry weight inroots with no toxicity. Formation of lead phosphateinhibited Pb uptake by vetiver, suggesting the needfor an environmentally safe chelating agent inconjunction with phytoremediation to clean up soils

contaminated with lead-based paint. Unambiguouscharacterization of phytochelatins (PCn) was possibleusing high pressure liquid chromatography coupledwith electrospray ionization mass spectrometry (LC-ESMS). Vetiver shows qualitative and quantitativedifferences in PCn synthesis between root and shoot.In root tissue from vetiver exposed to 1,200 mg Pb L-1,phytochelatins ranged from PC1 to PC3. Collision-induced dissociation of the parent ion allowed confir-mation of each PCn based on the amino acid sequence.Possible Pb-PC1 and Pb2-PC1 complexes were reportedin vetiver root at the highest Pb concentration. The datafrom these experiments show that the most probablemechanism for Pb detoxification in vetiver is bysynthesizing PCn and forming Pb–PCn complexes.

Plant Soil (2010) 326:171–185DOI 10.1007/s11104-009-9992-2

Responsible Editor: Fangjie J. Zhao.

S. S. Andra (*)Environmental Geochemistry Laboratory,University of Texas at San Antonio,One UTSA Circle,San Antonio, TX 78249-0663, USAe-mail: syam.andra@gmail.com

R. DattaBiological Sciences, Michigan Technological University,Houghton, MI, USA

D. SarkarDepartment of Earth and Environmental Studies,Montclair State University,Montclair, NJ, USA

K. C. MakrisThe Cyprus International Institutefor the Environment and Public Health in associationwith the Harvard School of Public Health,Nicosia, Cyprus

C. P. Mullens : S. B. H. BachDepartment of Chemistry, University of Texas,San Antonio, TX, USA

S. V. SahiDepartment of Biology,Western Kentucky University,Bowling Green, KY, USA

Keywords Hydroponics . Lead-based paint . Liquidchromatography .Mass spectrometry . Phytochelatins .

Phytoremediation . Vetiver

AbbreviationsCID Collision induced dissociationES-MS Electrospray ionization mass spectrometryGSH GlutathioneHPLC High-performance liquid chromatographyPb LeadP PhosphorusPCn PhytochelatinsSEM Scanning electron microscopy

Introduction

Although past national public health efforts havereduced lead exposure significantly, lead poisoningremains the most common environmental healthproblem affecting American children (USEPA 2001).Currently, lead exposure occurs predominantlythrough ingestion of lead-contaminated householddust and soil in older housing containing lead-basedpaint. Despite the efforts made to reduce residentialexposure to lead (including setting a maximumallowable lead content in paint of 0.06% in 1977),every city in the United States has a significantnumber of housing facilities that were built prior tothe implementation of that policy (ATSDR 2000).Composite probe samples obtained from all over theUnited States (US) show that approximately 7% ofUS dwellings (6.46 million units) have soil lead (Pb)concentrations above US Environmental ProtectionAgency (EPA) and US Department of Housing andUrban Development (HUD) standards (400 mg kg−1

for bare play area soil and 1,200 mg kg−1 for bare soilin the rest of the yard) (Jacobs et al. 2002). In aprevious study, total soil Pb concentrations in 20different lead-based paint contaminated residentialsites ranged between 36 mg Pb kg−1soil and4,182 mg Pb kg−1soil, with mean and median valuesof 1,197 and 821 mg Pb kg−1soil, respectively (Andraet al. 2006). Children living in these houses are at thegreatest risk of exposure, as crawling on the groundand playing in the backyard can result in ingestionand inhalation of soil- and dust-borne lead (USEPA2001). The physiological and behavioral effects

associated with Pb ingestion in children are wellcharacterized (Landrigan 1991). Soil lead clean-up istraditionally done by soil removal for off-site disposal.This is extremely expensive and rather impractical forremediating residential properties. As a result, in situremediation techniques, such as phytoremediation, aregaining attention as an environmentally safe andinexpensive alternative (Datta and Sarkar 2004).

Vetiver grass (Vetiveria zizanioides L.) is a nativeof the subtropics. Its ability to extract and accumulatehigher levels of metals such as Zn and Cu is welldocumented (Chiu et al. 2005). Previous studies inour laboratory show the ability of vetiver grass totranslocate up to 1,700±28 and 3,350±66 mg Pb kg−1

(dry weight) in shoot tissues, respectively, whenexposed to 400 and 1,200 mg Pb L−1 in a hydroponicset up without phosphorus (Andra et al. 2009). Shoottissue concentrations of Pb in vetiver seen from thisexperiment were greater than the specified criterionfor a hyperaccumulator plant (Sahi et al. 2002).However, it is a noted fact that the plant availablePb fraction and phytoextraction efficiency are higherin a hydroponic set-up compared to in contaminatedsoils. Vetiver did not exhibit any phytotoxic symp-toms such as growth retardation or chlorosis underthese conditions. Vetiver is well adapted to environ-ments ranging from aquatic to desert conditions, andis tolerant to frost, heat, acidic and alkaline conditions(Dalton et al. 1996). The additional virtues that makevetiver grass a suitable candidate for phytoremedia-tion are high biomass, a dense root system and quickregeneration ability (Pichai et al. 2001). However, toinclude vetiver grass in a successful phytoremediationmodel to clean-up Pb-paint contaminated residentialsites, it is essential to understand its Pb uptakepotential and possible Pb tolerance mechanisms.Complete understanding of the Pb tolerance mecha-nisms in plants is still lacking to date (Piechalak et al.2002). The role of metal-binding thiol peptides,typically known as ‘phytochelatins’, in the inactiva-tion of certain metals including Pb is well established(Gwozdz et al. 1997). Phytochelatins (PCn), a class ofmetal-binding proteins with a typical three-amino-acid sequence of (γGlu-Cys)n-Gly (n=2–11), arereported to bind metal ions in plants (Grill et al.1989). These peptides are synthesized enzymaticallyfrom glutathione or its homologs due to the reactioncatalyzed by phytochelatin synthase, which is anenzyme activated by heavy metals including Pb

172 Plant Soil (2010) 326:171–185

(Vatamaniuk et al. 2000). Because of the high contentof cysteine, PCn are able to create complex com-pounds with toxic metals. These complexes aresuggested to be transported into the vacuole by theATP-binding-cassette-like transporters localized in thetonoplast (Salt and Rauser 1995), thus separatingthem from cell metabolism. Alternatively, certainABC transporters, such as AtPDR12, induce Pbtolerance in Arabidopsis by transporting the toxicions to the cell exterior (Lee et al. 2005). The term‘PC1’ in this context refers to GSH and is representedby (γGlu-Cys)1-Gly (Rea et al. 2004; Wawrzynski etal. 2006; Chekmeneva et al. 2008). Liquid chroma-tography and mass spectrometry are the analyticaltechniques most commonly used for the separationand characterization of PCn (Gupta et al. 1995;Leopold and Gunther 1997; Leopold et al. 1999;Mishra et al. 2006; Kozka et al. 2006; Figueroa et al.2007).

Hydroponic (nutrient solution) studies are consid-ered a viable investigative method for phytoremedia-tion studies prior to evaluating a plant’s performancein soils at the greenhouse and/or field level. Undernatural conditions, Pb is strongly sorbed onto soilminerals and organic matter, making it the contami-nant least amenable to phytoremediation. In general,the availability of Pb for plant uptake in soils dependson the levels of phosphorus, calcium, iron andaluminum hydroxides (Schmidt 2003; do Nascimentoand Xing 2006; Nowack et al. 2006; Evangelou et al.2007). Phosphorus in the form of phosphate isconsidered a potential inhibitor of Pb availability toplants due to the formation of stable lead phosphatesin soils (Huang and Cunningham 1996; Cao et al.2002). Although many lead phosphates have limitedsolubility, it is essential to clean-up Pb from contam-inated soils because many of the soil-bound phases ofPb, despite being unavailable for plant uptake, arebioavailable to the human gastrointestinal system(Tang et al. 2004). Hence it is essential to mobilizePb into the soluble pool in soils, making it availablefor plant uptake. Increased phytoextraction and/orphytostabilization of Pb will result in decreased totalsoil Pb levels, which correspond to reduced concen-trations for human exposure and bioavailability.

We simulated a phosphorus-rich soil environmentin a hydroponics study to investigate the ability ofvetiver grass to remove soluble or dissolved Pbspecies, along with complexed or precipitated Pb

forms. We hypothesized that application of phospho-rus to the nutrient medium would reduce the plant-available Pb fraction and thereby Pb uptake capacity.Previous research has successfully shown that vetiverPb uptake in a phosphorus-free hydroponic experi-ment was dramatic, as well as demonstrating induc-tion of PCn and formation of Pb–PCn complexes as aplant biochemical response to elevated Pb concen-trations (Andra et al. 2009). The present study aimedto (1) study Pb uptake by vetiver, (2) characterize PCn

responses to Pb stress, and (3) identify possible Pb–PCn complexes in vetiver tissues in the presence ofphosphorus-containing nutrient medium.

Materials and methods

Hydroponic experiment

A hydroponic study was conducted in a temperature-and humidity-controlled environment in greenhousefacilities at the University of Texas at San Antonio.Vetiver grass was purchased from Horticultural Systems(Parrish, FL). Upon arrival, plants were thoroughlywashed in tap water to remove adhering soil particles.Vetiver tillers were acclimatized for 30 days in plastictanks (40×10×10 cm) containing 4 L nutrient mediumthat was continuously aerated with air circulationtubing. The nutrient solution used for this study was amodified Hoagland’s medium, prepared as described bySahi et al. (2002). In brief, the composition was asfollows, with salts obtained from Sigma Chemicals(Sigma-Aldrich, St. Louis, MO): 3,960 μmol L−1

calcium nitrate, 2,967 μmol L−1 potassium nitrate,2,521 μmol L−1 magnesium chloride, 1,249 μmol L−1

ammonium nitrate, 367 μmol L−1 potassium dihydro-gen phosphate, 49 μmol L−1 boric acid, 16 μmol L−1

manganese chloride, 9 μmol L−1 ferric tartrate,1.2 μmol L−1 zinc sulfate, 0.6 μmol L−1 cupric sulfate,and 0.1 μmol L−1 molybdenum trioxide. The experi-mental conditions at which the hydroponic set-up wasmaintained were 16 h at 24±2°C/8 h at 20±1°C light/dark conditions. The measured light intensity at theshoot level was 250 μmol m−2s−1, and the relativehumidity was 60±2%.

Following the initial 30-day vetiver acclimatizationphase, fresh nutrient solutions were prepared with thesame composition as mentioned above. Lead wasspiked in the form of lead nitrate at concentrations of

Plant Soil (2010) 326:171–185 173

0, 400, and 1,200 mg Pb L−1. We selected 400 and1,200 mg Pb L−1 to reflect the worst-case scenario ofUS EPA and US HUD standards, where Pb is in the100% soluble fraction. All the chemical solutions andnutrient media were prepared using deionized water.A completely randomized design with three replica-tions was used for this experiment. Each replicationconsisted of three vetiver plants of similar biomassand height; a total of nine plants was exposed to eachtreatment. Vetiver tillers of similar biomass and height(45–50 cm high tillers and 28–32 g fresh weight pertank) were used for the Pb exposure experiment. Thevetiver root system was sufficiently strong and long tokeep all of the tops above the surface of the nutrientmedium throughout the experimental period. Precau-tion was taken not to contaminate the above-groundparts of vetiver with Pb solution from spilling andsplashing activities. The average pH of the Pb-spikednutrient solutions remained constant (5.7±0.4) duringthe experimental period. Vetiver was harvested after7 days of Pb exposure. Plant material handling anddigestion were carried out according to Carbonell etal. (1998). Vetiver tissues and nutrient solutions wereanalyzed for total Pb by atomic absorption spectros-copy (PerkinElmer AAnalyst 700, PerkinElmer Lifeand Analytical Sciences, Waltham, MA) in the flamemode using an air-acetylene gas mixture. Statisticalanalyses (descriptive statistics) were performed usingJMP IN 5.1 (SAS, Cary, NC).

Speciation of Pb plays a major role in itsavailability for plant uptake. The ionic strengths andconcentrations of each component in the nutrientsolution were incorporated into chemical speciationmodeling software prior to conducting the experi-ment. Visual MINTEQ version 2.53 (Gustafsson2005) was used to identify Pb species and theiractivities to understand the distribution of soluble andprecipitate Pb forms using published equilibriumconstants (Kopittke et al. 2007).

Scanning electron microscopy

Scanning electron microscopy (SEM) was used toobtain Pb distribution patterns at an ultra-structurallevel, reveal the electron dense deposits of Pb andother elements, and identify the Pb compounds in theroot and shoot tissues of vetiver grass following theprotocol of Sahi et al. (2002). Frozen plant tissuesamples were viewed uncoated in a JEOL 5400 LV

SEM at 15 kV low vacuum mode using a back-scattered electron detector. Elemental analysis of thevetiver tissues was carried out using the SEM-attached KEVEX Sigma energy dispersive X-rayspectrometer (SEM-EDS).

Plant tissue extraction and purification

Extraction and analytical procedures were adaptedand modified from El-Zohri et al. (2005). Flashfrozen plant samples were ground to fine powderwith liquid nitrogen. The buffer used in PCn

extraction was 5 mmol L−1 dithiothreitol (DTT)prepared in Milli-Q grade water (Millipore, Billerica,MA) with no pH adjustment. Aqueous DTT (3 mL,4°C), an antioxidant, was mixed with 1 g plant tissuepowder and the suspension was sonicated (0.5 spulses, 400 W, 2 min) using a 40 kHz sonicationbath (Branson 2510, Danbury, CT). Peptides wereprecipitated using 18 mmol L−1 HCl, incubated onice for 10 min, centrifuged at 12,000 g for 10 min at4°C (IEC Microlite RF refrigerated microcentrifuge,Thermo Electron Corporation, San Jose, CA), andfiltered using 0.45 µm nylon membrane syringefilters (Fisherbrand, ThermoFisher Scientific, Waltham,MA). To enhance the recovery of low molecularweight peptides in the supernatant, we developed atwo-step purification protocol by passing the filtratein a series through microcon centrifugal filterdevices [Chemical Abstract Number (CAS#)R7DN45102, Catalog# 42407, Millipore] followedby reversed-phase ZipTip pipette tips (CAS#,L7CN4514, Catalog# ZTC18MO96, Millipore).The composition of the solutions for sequentialPCn purification and concentration consists of 100%acetonitrile for wetting the reversed-phase pipettetips, 0.5% trifluoroacetic acid (TFA) for equilibra-tion and peptide binding onto the C 18 resin, 0.1%TFA to wash away contaminants and incompletelybound macromolecules, and 50% methanol in 0.1%formic acid (FA) for concentrated PCn elution. Allthe solutions were prepared in Milli-Q grade water(Millipore).

Analysis of phytochelatins

High-performance liquid chromatography coupled withelectrospray mass spectrometry (LC-ESMS) was usedto separate, identify, and quantify PCn in the vetiver

174 Plant Soil (2010) 326:171–185

tissues. Quantification of PCn was made possible usingrespective peptide standards ranging from PC1 to PC4

purchased from Sigma Genosys (Sigma-Aldrich, St.Louis, MO). Separation and identification were madepossible using a Michrom Bioresources Magic HPLCsystem (Michrom Bioresources, Auburn, CA) and aFinnigan LCQ Duo ion trap mass spectrometer(Thermo Finnigan, San Jose, CA). Plant extracts(20 µL) were injected using an autosampler (Magicautosampler, Michrom Bioresources) on to a polymericcolumn (PLRS, 5 µ) (Polymer Laboratories, Varian,Amherst, MA). The elution solvents consisted of 0.1%FA and 0.01% trifluoroacetic acid TFA in water(mobile phase A) and 0.1% FA and 0.01% TFA inacetonitrile (mobile phase B). Gradient mode was usedto attain complete separation of the phytochelatinsusing the following three-step protocol: 0–20% B inthe first 25 min, 20% B for next 10 min, followed by20–100% B in the next 10 min. MS/MS analysis wasperformed to determine the fragment ions of eachphytochelatin observed in the vetiver tissues. Thenormalized collision energies used for MS/MS werebetween 10 and 90%. The mass spectrometer conditionsused were: source voltage, 5 kV; capillary temperature,225°C; capillary voltage, 5 V; sheath gas flow rate, 40(arbitrary units), and the auxiliary gas flow rate, 20(arbitrary units). The scan range of the instrument forthis study was m/z 50–1,800.

Quantification of PCn was made possible usingrespective peptide standards ranging from PC1 to PC4

purchased from Sigma Genosys (Sigma-Aldrich). Allstandard solutions were prepared and diluted in 1:1acetonitrile/ water solvent mixture. Separate stocksolutions of 100 μg mL−1 of each phytochelatin wereprepared and stored at −80°C. Aliquots of thesesolutions were mixed to obtain a 20 μg mL−1 mixedworking standard stock solution that was stored at−20°C. Six-point calibration curves of mixed PCn

analytes were prepared daily at 0.1, 0.5, 1.0, 2.5, 5.0,10μgmL−1 concentrations using the 20 μg mL−1 stocksolution. The final volume was bought up to 0.5 mLusing 1:1 acetonitrile/ water solvent mixture and storedat −4°C. Calibration curves were used for quantifyingphytochelatins in the experimental plant samples.

Analysis of lead-phytochelatin complexes

Lead complexes with biomolecules such as peptidesmight interact with the stationary phase of the

polymeric reversed phase column and result ininstability, thus preventing them from appearanceon the LC/MS scans. This problem was overcomeby direct infusion of the filtered and concentratedvetiver tissue extracts into the mass spectrometer at5 μL min−1 using a Hamilton (model #1750)(Hamilton, Reno, NV) syringe. Xcalibur software(Thermo Finnigan) was used to collect and analyzethe data obtained from the full scan and MS/MSmode. Instrument settings were the same as used forthe LC-ESMS experiments mentioned above. Lead-phytochelatin complexes were identified based onthe simulations of the Pb isotope pattern obtainedusing Isotope Viewer in the Xcalibur software for allpossible complexes, respectively.

Results

Lead uptake by vetiver grass

After 1 week of Pb exposure, in the presence ofphosphorus, vetiver was able to accumulate up tobetween 2,500 and 3,100 mg Pb kg−1 dry weight inroots when treated with 400 or 1,200 mg Pb L−1

(Table 1). The added Pb was immobilized byphosphorus (Huang and Cunningham 1996; Cao etal. 2002) as a white precipitate of lead phosphate,making it less available for uptake by vetiver. Thetranslocation of Pb from root to shoot tissues waslimited. The shoot Pb concentrations of vetiverexposed to 400 and 1,200 mg Pb L−1 were 25 and150 mg Pb kg−1, respectively. Immobilization ofmetals in roots is an exclusion mechanism exhibitedby plants for metal toxicity tolerance (Baker et al.1994). Pb translocation factor (TF) is a ratio of thePb concentration in shoots to roots that indicates themetal translocation efficiency of any given plantspecies. Stoltz and Greger (2002) classified plantspecies with TF<1 as ‘shoot metal excluders’that are suitable for phytostabilization of metal-contaminated soils. In this study, the observed TFvalues were ≪ 1, indicating that vetiver accumulatedPb in root tissues. This observation is in accordancewith the results of a study by Lai and Chen (2004)that considered vetiver as a Pb-phytostabilizer incontaminated soils. In contrast, vetiver was able toaccumulate up to 3,350 and 19,800 mg Pb kg−1 dryweight of shoot and root tissue when exposed to

Plant Soil (2010) 326:171–185 175

1,200 mg Pb L−1 in the absence of phosphorus(Table 1) (Andra et al. 2009).

Pb precipitated out of solution upon addition to thenutrient medium, as evidenced by the formation of awhite-colored substance and settling to the bottom of thetank. The clear solutions were analyzed for soluble Pbfraction before and after the 7-day phytoextractionperiod (Fig. 1). The dissolved Pb fraction was reducedby 93 and 86% in treatments following Pb addition at400 and 1,200 mg L−1, respectively (Fig. 1). SolublePb levels were reduced significantly after the 7-dayexperimental period, while they remained constant intanks with no vetiver grass. Operations with VisualMINTEQ for Pb speciation modeling in the nutrientmedia were based on the concentrations of solutionanions (PO4

3−, NO3−, SO4

2−, BO33−, and Cl−), ex-

changeable cations (NH4+, Ca2+, Mg2+, and K+),

metals (Pb, Fe, Zn, and Cu), and pH. Over 80% ofthe total Pb species consisted of insoluble chloropyr-omorphite [Pb5(PO4)3Cl]. The minor soluble Pbspecies observed were PbSO4, PbNO3

+, PbCl+, andPb3(BO3)2. In contrast, in a previous hydroponic studyusing phosphorus free nutrient medium (Andra et al.2009), all the spiked Pb existed only as dissolvedspecies with no precipitates.

Scanning electron micrographs show differentialdistribution of Pb in root and shoot tissues of vetiverexposed to 1,200 mg Pb L−1. Lead accumulatedmostly in the cortex and pith regions in root (Fig. 2a)and vascular bundles in shoot (data not shown).Localization of Pb in xylem obtained from differentcross-sections at various locations of vetiver plantsclearly demonstrates translocation to above-groundplant parts. Results from X-ray microanalysis(Fig. 2c) confirm the accumulation of phosphorus-

containing Pb complexes in selected portions ofvetiver root tissue (Fig. 2b). Further studies areneeded to characterize specific Pb-phosphorus com-pounds using advanced spectroscopic techniques andto identify the biochemical/physiological mechanismsinvolved in their uptake by vetiver grass. Phosphorussignal was absent in the energy dispersive X-rayspectrum of a Pb deposit obtained from vetiverexposed to Pb in a phosphorus-free nutrient medium(Andra et al. 2009).

Induction of phytochelatins in vetiver grass

LC-ESMS was employed to analyze PCn. Separationwas accomplished using a polymeric reversed phase

0

50

100

150

200

Control 400 1200

Added Pb (mg L-1)

InitialFinal

d

b

a

c cd

Fig. 1 Dissolved lead (Pb) concentrations in the hydroponicset-up measured at two time intervals. Values represent themean (± SD) of three replicates. Means with different lowercase letters are significantly different using Tukey’s HSD atP < 0.001

Table 1 Lead (Pb) accumulation in vetiver grass after a week’s exposure to Pb in a hydroponic experiment. Numbers represent mean± standard deviation of three independent replications, with three plants in each nutrient chamber (n = 9). Values labeled with differentletters are significantly different using Tukey’s HSD at P < 0.001. Statistical analyses for Pb content in root and shoot compartmentswere done separately

Treatment Total lead uptake by vetiver (mg Pb kg−1 dry wt)

With phosphorus Without phosphorus(Andra et al. 2009)

Root Shoot Root Shoot

0 mg L−1 Pb 0 ± 0 B 0 ± 0 b 0 ± 0 C 0 ± 0 c

400 mg L−1 Pb 2,500 ± 252 A 25 ± 6 b 13,240 ± 1,773 B 1,700 ± 28 b

1,200 mg L−1 Pb 3,100 ± 110 A 150 ± 4 a 19,800 ± 2,400 A 3,350 ± 66 a

Table 1 Lead (Pb) accumulation in vetiver grass after a week’sexposure to Pb in a hydroponic experiment. Numbers representmean ± standard deviation of three independent replications,with three plants in each nutrient chamber (n = 9). Values labeled

with different letters are significantly different using Tukey’sHSD at P < 0.001. Statistical analyses for Pb content in root andshoot compartments were done separately

176 Plant Soil (2010) 326:171–185

column. The m/z of each PCn was compared withstandards and with literature values (Vacchina et al.1999). LC-ESMS obtained for the root (Fig. 3a) andshoot (Fig. 4a) tissues of vetiver grown in the absenceof Pb show no PCn. Three possible PCn wereobserved in the positive mode of ESMS scans,ranging from PC1 to PC3 in root (Fig. 3c) and PC1

to PC2 in shoot (Fig. 4c) tissues of vetiver exposed to1,200 mg Pb L−1. Peaks of significant intensity withm/z values of 615.1, 921.8 and 1,228.4 were alsoobserved in the ESMS scan of the vetiver root tissue(Fig. 3c) from the 1,200 mg Pb L−1 treatment. Asconfirmed using collision induced dissociation (CID),these peaks correspond to dimers, trimers, andtetramers of PC1 that resulted from gas-phase reac-tions and cluster formation during the electrosprayprocess.

Unequivocal identification of each PCn was possi-ble using CID at 70% normalized collision energy.The CID of the PCn standard mixture yielded aspectrum of fragment ions that retained charge eitheron the amino or carboxyl terminal of the respectivepeptide (Table 2). The fragment ions generatedprovide a strong reference database for confirmingeach PCn seen in the ESMS scans from vetiver rootand shoot samples. Figure 6 depicts the CID of theroot sample from vetiver exposed to 1,200 mg Pb L−1.Confirmation of the respective PCn was made byobserving the largest daughter fragment: m/z 178.9from PC1 with m/z 308.4 (Fig. 6a), m/z 410.9 fromPC2 with m/z 540.4 (Fig. 6b), and m/z 643.0 fromPC3 with m/z 772.1 (Fig. 6c). The loss of γGlu (m/z129.0) from each PCn resulted in the largest frag-ment for each ion, respectively. Further confirma-

0 1 2 3 4 5 Energy (keV)

Inte

nsit

y

a

b c

Fig. 2 a, b Scanningelectron micrographs of Pbdeposits (red rectangles) inroot tissue of vetiver grassexposed to 1,200 mg PbL−1. a Cross-section of rootshowing Pb compounds incortex and pith. b Magnifiedselected root tissue spotcontaining Pb and phospho-rus. c Energy dispersiveX-ray spectrum of theselected spot showing Pband phosphorus signals

Plant Soil (2010) 326:171–185 177

tion was possible by observing many matchingdaughter ions from the root sample with thoseobtained from the PCn standards mixture. Similarconfirmation was made for each PCn from the vetivershoot tissue (data not shown). Induction of higherorder PCn like PC4 was confirmed using CID, whilethe presence of PC5 was proposed based on theoret-ical and experimental isotopic patterns in vetiver roottissue obtained from 1,200 mg Pb L−1 treatment withno phosphorus (Andra et al. 2009).

PC1 was also observed in vetiver tissues exposedto 0 mg Pb L −1. Because the signal intensitieswere weak during the ES-MS analyses, certainpeaks of interest were magnified with Xcalibursoftware available with Finnigan LCQ Duo ion trapmass spectrometer (Thermo Finnigan, San Jose,CA) (Fig. 5). A magnification factor of 50 and 100

is required to show the presence of PC1 in root(Fig. 5a) and shoot (Fig. 5b) tissues of vetiver fromPb-unspiked control treatments. However, theywere present below instrument quantitation limits(< 0.3 μmol PCn kg−1 fresh wt) and hencerepresented as ‘trace’ quantities (Table 3). Similarly,PC4 was observed at a 250 magnification in vetiverroot exposed to 1,200 mg Pb L−1 (Fig. 5c). Even at1,000 factor magnification, neither PC3 nor PC4 wasobserved in vetiver shoot exposed to 1,200 mg PbL−1 (Fig. 5d). In such cases, PCn quantities weredenoted as ‘zero’ (Table 3).

To date, to the best of our knowledge, radio-activelabeled and/or deuterated internal standards for PCn

are unavailable. Matrix effects such as enhancementor suppression are possible, resulting in poor accuracy,low reproducibility, and erroneous quantification,

Rel

ativ

e A

bund

ance

(%

)

200 600 1000 1400m/z

0

50

100803.2

391.5 (PC1 + 1H)+

308.3

`

Rel

ativ

e A

bund

ance

(%

)

0

50

(PC2 +1H)+

540.4 (PC3+1H)+

772.1

100

308.4 (PC1 + 1H)+

200 600 1400 m/z

391.5

615.1

921.8

1228.4

1000

Rel

ativ

e A

bund

ance

(%

)

200 600 1000 1400m/z

0

50

100803.2

391.6

a

b

c

Fig. 3 Liquid chromatog-raphy coupled with electro-spray ionization massspectrometry (LC-ESMS)spectra obtained for roottissue from vetiver exposedto Pb at a 0 mg L−1, b 400mg L−1, and c 1,200 mg L−1

178 Plant Soil (2010) 326:171–185

especially in the case of peptides for which labeledstandards are not available. Standard additions wereperformed for all PCn in root and shoot tissueextracts in order to determine matrix effects if any.Vetiver root or shoot extracts were spiked withindividual PC standards in order to increase thebackground signal to 2–4 times that of the originalsignal for each peptide. Average concentrations forun-spiked plant samples determined from water-based calibration curves were statistically equivalentat the 95% confidence interval (CI) to thosedetermined by standard additions (data not shown).Accuracy of each PC determination by the standardaddition method indicated that there were no matrixeffects and that PCn concentrations can be deter-mined directly from calibration standards prepared in

deionized water. Quantification of PCn in vetivertissues was achieved by using the calibration curveobtained from the standards mixture. Vetiver grownin the absence of Pb shows no quantifiable amountsof PCn either in root or shoots. The sum of PCn

concentrations in vetiver exposed to 1,200 mg PbL−1 was 16.11 and 4.33 μmol kg−1 calculated on afresh weight basis in root and shoot, respectively(Table 3).

Two prominent peaks observed at m/z 803.2 and391.6 were present in all the treatments, irrespectiveof exposure to different Pb concentrations. CID on them/z 803.2 complex yielded two distinct daughterfragments with m/z values of 413.1 (44%) and 391.6(100%) (Fig. 7a). The major daughter fragment of m/z803.2 is also the same as the second prominent and

Rel

ativ

e A

bund

ance

(%

)

200 600 1000 1400m/z

0

50

100803.2

391.5 (PC1 + 1H)+

308.4

(PC2 + 1H)+

540.4

Rel

ativ

e A

bund

ance

(%

)

200 600 1000 1400m/z

0

50

100803.2

1167.3 391.6

m/z

200 600 1000 14000

50

100803.1

391.7

Rel

ativ

e A

bund

ance

(%

)

a

b

c

Fig. 4 LC-ESMS spectraobtained for shoot tissuefrom vetiver exposed to Pbat a 0 mg L−1, b 400 mgL−1, and c 1,200 mg L−1

Plant Soil (2010) 326:171–185 179

unidentified peak with m/z 391.6. CID on m/z 391.6yielded daughter fragments that could possibly becysteine (m/z 121.0, 75%) and aqueous γ-glutamicacid (m/z 149, 100%) (Fig. 7b). Based on the presenceof cysteine, it could be suggested that the twounidentified peaks are thiols or phytochelatin-related

peptides. Further work is required to confirm and gaindetailed knowledge of these related peptides. Possiblehigher order phytochelatin-related peptides such asiso-PC5 (βAla) and des Gly-PC6 were observed in theroot of vetiver grass grown in 1,200 mg Pb L−1 nutrientsolution with no phosphorus (Andra et al. 2009).

Rel

ativ

e A

bund

ance

(%

)

800

X1000

200 400 600 1000 1200 1400 1600m/z

0

50

100803.2

391.5

308.4 540.4

X1000

(PC1 + 1H)+

(PC2 + 1H)+

Rel

ativ

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bund

ance

(%

)

800

200 400 600 1000 1200 1400 1600m/z

0

50

100308.4

615.1

391.5 921.8

1228.4540.4

772.11005.5

x250

(PC2 + 1H)+ (PC3 + 1H)+

(PC1+ 1H)+

(PC4 + 1H)+

m/z

Rel

ativ

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bund

ance

(%

)

200 400 600 800 1000 1200 1400 16000

50

803.2

1167.3391.6

308.2x100

(PC1 + 1H)+

100

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(%

)

x50

200 400 600 800 1000 1200 1400 1600m/z

0

50

803.1

391.5 308.2

(PC1 + 1H)+

100 a b

c d

Fig. 5 Selective magnification of the LC-ESMS spectra to represent the presence of certain phytochelatins (PCn), that are at tracelevels for instrumental quantitation: a root 0 mg Pb L−1, b shoot 0 mg Pb L−1, c root 1,200 mg Pb L−1, and d shoot 1,200 mg Pb L−1

Table 2 Collision-induced dissociation (CID) of the phytochelatins (PCn) standards mixture with 2.50 μg mL−1 of each PC1, PC2,PC3, and PC4. The values in parenthesis indicate the mass over charge ratio (m/z) and percent relative abundance of each daughterfragment obtained from the MS/MS

PC1a PC2b PC3c PC4d

[PC1] (308.5, 4%),[PC1 - Gly] (233.3,7%), [PC1 - γGlu](179.3, 100%)

[PC2] (541.4, 6%), [PC2 - Gly](466.2, 9%), [PC2 - γGlu] (411.6,100%), [PC2 - γGlu - Cys] (309.0,16%), [PC2 - γGlu - Cys - Gly](233.0, 11%), [PC2 - 2γGlu - Cys](178.8, 5%)

[PC3] (773.4, 19%), [PC3 - Gly](698.0, 17%), [PC3 - γGlu] (644.0,100%), [PC3 - Cys - Gly] (595.4,5%), [PC3 - γGlu - Cys] (541.7,12%), [PC3 - γGlu - Cys - Gly](466.1, 14%), [PC3 - 2γGlu - Cys](412.1, 15%) [PC3 - 2γGlu - 2Cys](308.9, 9%), [PC3 - 2γGlu - 2Cys -Gly] (233.2, 4%)

[PC4] (1005.9, 56%), [PC4 - Gly](929.7, 11%), [PC4 - γGlu] (876.7,100%), [PC4 - γGlu - Cys] (773.6,27%), [PC4 - γGlu - Cys - Gly](698.9, 15%), [PC4 - 2γGlu - Cys](643.8, 8%) [PC4 - γGlu - 2Cys -Gly] (595.9, 8%), [PC4 - 2γGlu -2Cys] (540.7, 36%), [PC4 - 2γGlu- 2Cys - Gly] (466.3, 7%), [PC4 -3γGlu - 2Cys] (412.1, 12%) [PC4 -3γGlu - 3Cys] (308.3, 7%)

a Phytochelatin-1 [(γGlu-Cys)1-Gly]b Phytochelatin-2 [(γGlu-Cys)2-Gly]c Phytochelatin-3 [(γGlu-Cys)3-Gly]d Phytochelatin-4 [(γGlu-Cys)4-Gly]

Table 2 Collision-induced dissociation (CID) of the phytoche-latins (PCn) standards mixture with 2.50 μg mL−1 of each PC1,PC2, PC3, and PC4. The values in parenthesis indicate the mass

over charge ratio (m/z) and percent relative abundance of eachdaughter fragment obtained from the MS/MS

180 Plant Soil (2010) 326:171–185

Lead-phytochelatin complexes in vetiver grass

Tolerance in vetiver to Pb-induced stress was assumedto occur via the formation of Pb-PCn complexes.Direct infusion of plant tissue extracts resulted in the

observation of certain Pb-PCn complexes. Figure 8adepicts the full ESMS scan obtained from injectingroot samples obtained from vetiver exposed to1,200 mg Pb L−1. PC1 (m/z 308.2) was the base peakin this sample. This was confirmed by CID yielding

Table 3 PCn concentrations in root and shoot tissues of vetiver grass exposed to different initial loads of Pb in nutrient solutionscontaining phosphorus. Calculations are on a fresh weight basis. Values are mean (± standard deviation) of three replicates

Treatment (Root tissue, μmol kg−1 fresh wt) (Shoot tissue, μmol kg−1 fresh wt)

PC1 PC2 PC3 PC4 PC1 PC2 PC3 PC4

0 mg L−1 Pb Trace 0.00 0.00 0.00 Trace 0.00 0.00 0.00

400 mg L−1 Pb 5.91 ± 1.6 Trace 0.00 0.00 Trace Trace 0.00 0.00

1,200 mg L−1 Pb 11.70 ± 3.9 3.43 ± 1.2 0.98 ± 0.5 Trace 2.79 ± 0.7 1.54 ± 0.8 0.00 0.00

Rel

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250 350 450 550 650 750m/z

0

50

100

(PC3 - γGlu - Cys)540.2 (PC3 - Gly)

696.8 (PC3 - PC1)

465.2 (PC3 - 2γGlu - Cys)411.3 (PC3 - Cys - Gly)

594.1

(PC3 - 2γGlu - 2Cys) 308.1

(PC3 - PC1 - γGlu - Cys)233.3

643.0 (PC3 - γGlu)

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140 180 220 300 340 380 420 460 500 540m/z

0

50

100

(PC2 - Gly)464.8

(PC2 - γGlu - Cys)308.2 (PC2 - PC1)

233.1 (PC2 - 2γGlu - Cys)178.9

410.9 (PC2 - γGlu)

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100 140 180 220 260 300m/z

0

50

100

(PC1 - Gly) 232.9

178.9 (PC1 - γGlu)

260

a

b

c

Fig. 6a–c Collision-induceddissociation (CID) of theprotonated PCn obtainedfrom LC-ESMS scans ofroot tissue of vetiver exposedto Pb at 1,200 mg L−1. aPC1, b PC2, c PC3

Plant Soil (2010) 326:171–185 181

the largest fragment at m/z 178.7 that corresponds toa loss of γGlu from PC1. Two peaks with m/z 514.7and 719.0 (Fig. 8a) show a typical Pb isotopepattern. Metal ions with natural isotope abundancesenable them to be identified in associated complexes(Bach et al. 2005, 2007; Polec-Pawlak et al. 2007).The isotope of lead [204Pb (1.4%), 206Pb (24.1%),207Pb (22.1%), and 208Pb (52.4%)] assisted in theassignment of m/z 514.7 for a PC1 complex with 1Pb ion (Fig. 8b) and m/z 719.0 for a PC1 complexwith 2 Pb ions (Fig. 8c). The expected isotopedistribution patterns for Pb-PC1 and 2Pb-PC1 com-plex, as calculated using Xcalibur Isotope Viewer,are shown on the right side of Fig. 8b and c,respectively. Similarly, based on isotopic patterns,certain possible Pb complexes with PC2, PC4, andPC6 were speculated in root tissues of vetiverexposed to Pb at 1,200 mg L−1 in the absence ofphosphorus (Andra et al. 2009).

Discussion

Lead uptake by vetiver grass increased withincreasing concentrations of Pb spiked in thenutrient medium. In the presence of P, there wasan instantaneous visible Pb precipitation, resultingin white lead phosphate separating out of solutionand settling at the bottom of the tanks. Thisconfirms literature findings on Pb immobilizationin soils by P, thereby reducing the soluble form ofPb, which is potentially plant available (Huang andCunningham 1996; Cao et al. 2002). Vetiver accu-mulated significant (P<0.001) low Pb levels ofabout 3,000 mg kg−1 dry tissue in the roots whenexposed to 1,200 mg Pb L−1 in the presence of Pcompared to that of about 20,000 mg kg−1 root drytissue when grown in P-free nutrient medium(Table 1) (Andra et al. 2009). A study by Zhu et al.(2004) supports our finding of reduced Pb levels in

(Cys)

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120 160 200 240 280 320 360 400m/z

0

50

100149.0

121.0

391.6

242.12(Cys)

(γ Glu + H2O)

803.2

Rel

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)

300 400 500 600 700 800m/z

0

50

100

413.1

391.6a

b

Fig. 7a,b CID spectra forprominent and unidentifiedprotonated molecules. a m/z803.2, b m/z 391.6. Cyste-ine and γ-glutamic acid, theprincipal amino acids ofPCn, appear to be possibleconstituents of thesecomplexes

182 Plant Soil (2010) 326:171–185

plant tissues, with increasing residual fractions of Pbresulting from P-containing amendments. Strongcorrelation (r>0.95, n=9) was observed betweenPb concentration in the root and shoot tissues for anygiven treatment. In our study, the calculated translo-cation factor was ≪ 1, indicating that vetiver grasstends to store Pb in root tissues. It appears thatvetiver is immobilizing Pb in roots as an exclusionstrategy towards metal toxicity as indicated by Bakeret al. (1994). The data supports the classification ofvetiver grass as a potential Pb phytostabilizationplant when grown in soils with 1,000 mg Pb kg−1

soil (Lai and Chen 2004).The mechanisms behind plant Pb tolerance are

poorly understood (Piechalak et al. 2002). Inactiva-tion of free and toxic Pb ions is connected with

synthesis of cysteine-rich thiol peptides known asphytochelatins (PCn) (Grill et al. 1989). Glutathione isthe precursor for synthesis of these metal-bindingpeptides, which is catalyzed by phytochelatin synthase(PCS) in plants upon metal exposure (Vatamaniuk etal. 2000). We developed a two-step purificationmethod for PCn concentration and characterization.We were able to show the presence of PCn rangingfrom PC1 to PC3 in vetiver roots upon exposure to1,200 mg Pb L−1. PC3 was observed only in the roottissues owing to the high levels of Pb. The mostabundant PCn in both root and shoot tissues of vetiverwas PC1. This observation suggests that PC1 acts as asubstrate for synthesizing higher order PCn withincreasing Pb concentration in vetiver. In addition,the type and level of PCn shows a linear relationship

200 400 600 800 1000 1200 1400 1600 1800m/z

0

50

100

(PC1 + 1H)+

308.2

(PC1 + 1Pb + 1H)+

514.7

(PC1 + 2Pb + 1H)+

719.0

Rel

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(%

)

a

b

c

Fig. 8 a Full-scan massspectra obtained from directinfusion to see the possiblePb–PCn complexes in roottissue of vetiver exposed toPb at 1,200 mg L−1. bObserved (left) and simulated(right) isotope signature forPb–PC1 complex. cObserved (left) and simulated(right) isotope signature for2Pb–PC1 complex

Plant Soil (2010) 326:171–185 183

with the Pb content in the vetiver tissue (Table 3).We propose that 1–2 Pb ions were possibly bound toPC1 in vetiver root at higher Pb levels. The currentstudy provides a basic insight into the role of PCn

synthesis towards Pb tolerance and detoxification invetiver grass. Overexpression of genes that encodesglutathione synthetase (GS) (Zhu et al. 1999a) andγ-glutamylcysteine synthetase (γ-ECS) (Zhu et al.1999b) enhanced cadmium uptake and tolerance inIndian mustard. Similar genetic manipulation of vetivergrass that has virtues like high biomass, fast growth,and adaptability to a wide range of soil and climaticconditions, could make it a more promising plant toclean up Pb from contaminated soils.

To conclude, the availability of Pb for plant uptakedue to its interaction with P appears to be the biggesthurdle to achieving the clean up goal. Hence it isessential to use an environmentally safe chelatingagent to mobilize Pb from bound forms to the solublepool to enhance Pb uptake by vetiver. Greenhouseexperiments have evaluated the performance of achelant-aided phytoremediation model using vetivergrass to extract Pb from soils with varying physico-chemical properties (Andra 2008). We intend tovalidate the role of phytochelatins towards Pb tolerancein vetiver grown in a contaminated soil medium.

Acknowledgments The research group from the Universityof Texas at San Antonio appreciates the funding support fromthe United States Department of Housing and Urban Develop-ment for this study. We thank Dr. Mohd Israr, Department ofBiology, Western Kentucky University for help with SEManalysis.

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