Analysis of phytochelatin complexes in the lead tolerant vetiver grass [Vetiveria zizanioides (L.)] using liquid chromatography and mass spectrometry

Environmental Pollution 157 (2009) 2173–2183

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Analysis of phytochelatin complexes in the lead tolerant vetiver grass [Vetiveria zizanioides (L.)] using liquid chromatography and mass spectrometry Syam S. Andra a, *, Rupali Datta b, Dibyendu Sarkar c, Sumathi K.M. Saminathan a, Conor P. Mullens d, Stephan B.H. Bach d a

Environmental Geochemistry Laboratory, University of Texas at San Antonio, One UTSA Circle, San Antonio, TX, USA Biological Sciences, Michigan Technological University, Houghton, MI, USA Department of Earth and Environmental Studies, Montclair State University, Montclair, NJ, USA d Department of Chemistry, University of Texas at San Antonio, San Antonio, TX, USA b c

Chelated lead in conjunction with phytochelatins synthesis and complexation reduces stress in the lead tolerant vetiver grass.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 September 2008 Received in revised form 4 February 2009 Accepted 5 February 2009

Ethylenediamene tetraacetic acid (EDTA) has been used to mobilize soil lead (Pb) and enhance plant uptake for phytoremediation. Chelant bound Pb is considered less toxic compared to free Pb ions and hence might induce less stress on plants. Characterization of possible Pb complexes with phytochelatins (PCn, metal-binding peptides) and EDTA in plant tissues will enhance our understanding of Pb tolerance mechanisms. In a previous study, we showed that vetiver grass (Vetiveria zizanioides L.) can accumulate up to 19,800 and 3350 mg Pb kg1 dry weight in root and shoot tissues, respectively; in a hydroponics set-up. Following the basic incubation study, a greenhouse experiment was conducted to elucidate the efficiency of vetiver grass (with or without EDTA) in remediating Pb-contaminated soils from actual residential sites where Pb-based paints were used. The levels of total thiols, PCn, and catalase (an antioxidant enzyme) were measured in vetiver root and shoot following chelant-assisted phytostabilization. In the presence of 15 mM kg 1 EDTA, vetiver accumulated 4460 and 480 mg Pb kg1 dry root and shoot tissue, respectively; that are 15- and 24-fold higher compared to those in untreated controls. Despite higher Pb concentrations in the plant tissues, the amount of total thiols and catalase activity in EDTA treated vetiver tissues was comparable to chelant unamended controls, indicating lowered Pb toxicity by chelation with EDTA. The identification of glutathione (referred as PC1) (m/z 308.2), along with chelated complexes like Pb–EDTA (m/z 498.8) and PC1–Pb–EDTA (m/z 805.3) in vetiver root tissue using electrospray tandem mass spectrometry (ES-MS) highlights the possible role of such species towards Pb tolerance in vetiver grass. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Catalase EDTA Glutathione Lead-based paint Liquid chromatography Mass spectrometry Phytochelatins Phytoremediation Thiols Vetiver

1. Introduction The amount of lead (Pb) entering the residential soil environment through weathering, chipping, scraping, sanding and sand blasting of structures bearing lead-based paint is a major concern in the United States. Though a maximum allowable limit of Pb content in paint was set at 0.06% in 1977, there exists a significant number of housing facilities in every city in the US that were built prior to the implementation of this policy (USEPA, 2001). Both children and

Abbreviations: CID, collision induced dissociation; ES-MS, electrospray ionization mass spectrometry; EDTA, ethylenediamene tetraacetic acid; GSH, glutathione; HPLC, high performance liquid chromatography; Pb, lead; LMWT, low molecular weight thiols; PCn, phytochelatins. * Corresponding author. Tel.: þ1 210 458 5443; fax: þ1 210 458 4469. E-mail address: [email protected] (S.S. Andra). 0269-7491/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2009.02.014

adults are susceptible to health effects from lead exposure, although the typical exposure pathways and effects are somewhat different. Children who reside in pre-1977 housing facilities (and especially those in inner cities built before 1950) are at greatest risk (ATSDR, 2000), because natural curiosity and the tendency to explore leaves children open to Pb health risks from soil ingestion that adults can more easily avoid. Toxicological effects of Pb in children such as impaired development of hematologic, skeletal and nervous systems, are well characterized (Landrigan, 1991). As a result, a great deal of effort has been focused on developing remediation methods for the clean-up of Pb and its products in residential sites. Given the limitations of physical reclamation measures in reducing human health risk from exposure to Pb-paint-contaminated residential soils, phytoremediation is an attractive option for clean-up of such sites (Datta and Sarkar, 2004). This technique is environment-friendly, inexpensive, and visually

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unobtrusive. Since Pb is held strongly by soil minerals and organic matter, it is a difficult metal for phytoremediation. It is essential to take out Pb from the contaminated soils because many of the soil bound phases of Pb, despite being unavailable for plant uptake, are bioavailable to the human gastrointestinal system. In order to increase plant uptake, various studies were done to mobilize Pb from soils using a range of compounds such as chelants, the most prominent and effective being ethylenediamene tetraacetic acid (EDTA) (Schmidt, 2003; do Nascimento and Xing, 2006; Nowack et al., 2006; Evangelou et al., 2007). Vetiver grass (Vetiveria zizanioides L.) is a tall (1–2 m), fastgrowing, perennial grass, with a long (3–4 m), massive and complex root system, which can penetrate to the deeper layers of soil (Pichai et al., 2001). Vetiver grass was shown to tolerate high concentrations of a range of heavy metals with no effects on growth and development (Roongtanakiat and Chairoj, 2002). Previous studies in our laboratory show the ability of V. zizanioides to accumulate up to 19,800  2400 and 3350  66 mg Pb kg1 dry weight in root and shoot tissues, respectively; with no phytotoxic symptoms such as growth retardation and chlorosis, under hydroponics set-up conditions (Andra et al., in press). Vetiver fulfills the requirements of an accumulator plant with virtues like fast growth, high biomass, extensive root system, ease of harvest, and ability to accumulate high levels of Pb concentrations in its harvestable parts. Vetiver meets the specifications for a Pb accumulator given by Sahi et al. (2002) based on shoot tissue concentrations. Phytoremediation is not likely to become a viable technology unless the fundamental biochemical mechanisms controlling the uptake and detoxification of Pb are properly understood in vetiver grass. This led us to question the tolerance mechanisms behind Pb hyperaccumulation by this grass. We hypothesize that Pb tolerance in vetiver could result from either (1) complexation of Pb with phytochelatins (PCn), which are low molecular weight peptides that complex with heavy metals (Leopold et al., 1999), and/or (2) enhanced production of antioxidants (both enzymatic and nonenzymatic) that detoxify free reactive oxygen species produced in response to Pb (Piechalak et al., 2002; Ruley et al., 2004). Phytochelatins are thiol peptides which are synthesized enzymatically from glutathione (GSH) or its homologs due to the reaction catalyzed by phytochelatin synthase (PCS) an enzyme activated by heavy metal ions including Pb. Phytochelatins are made up of three amino acids with a typical sequence of (gGlu– Cys)n–Gly (n ¼ 2–11) (Grill et al., 1989). The term ‘PC1’ in this context refers to GSH and is represented by (gGlu–Cys)1–Gly (Rea et al., 2004; Wawrzynski et al., 2006; Chekmeneva et al., 2008). Owing to the high content of cysteine, PCn are able to create complexes with Pb ions. The complexed Pb ions might be transported into the vacuole and there be separated from interfering with the regular cellular activities. Vetiver is able to tolerate Pb up to 1200 mg L1 grown hydroponically with no phytotoxicity. Structural determination of PCn is essential for understanding their Pb-binding properties. The coupling of liquid chromatography with electrospray mass spectrometry (HPLC-ES-MS) and tandem mass spectrometry (MS/MS) have proven to be powerful techniques for separating and identifying phytochelatins in plant tissues. HPLC and MS techniques have also been applied to characterize phytochelatins in plants exposed to Pb ions (Gupta et al., 1995; Leopold and Gunther, 1997; Leopold et al., 1999; Mishra et al., 2006; Kozka et al., 2006; Figueroa et al., 2007). However, no studies are available regarding phytochelatin and lead complexes in plant tissues. We are able to characterize PCn ranging from PC1 to PC4 and PCn bound to one or two Pb ions using HPLC-ES-MS in vetiver exposed to 1200 mg L1 Pb in a hydroponic set-up (Andra et al., in press). Free radical production was observed using electron paramagnetic resonance (EPR) in lupine plants upon Pb exposure at

350 mg L1 (Rucinska et al., 1999). In addition to phytochelatins, plants generate antioxidants to reduce oxidative stress from free radicals generated in the presence of metals. Low molecular weight thiols constitute non-enzymatic antioxidants and include glutathione, and ascorbate (Hofgen et al., 2001) which have been shown to be induced in terrestrial plants exposed to Pb (Gupta et al., 1995; Piechalak et al., 2002, 2003; Ali et al., 2003; Sun et al., 2005; Kozka et al., 2006; de la Rosa et al., 2007; Wierzbicka et al., 2007). Primary enzymatic antioxidants in terrestrial plants are superoxide dismutase, peroxidase and catalase (Gratao et al., 2005) and have been shown to be up regulated in the presence of Pb (Gupta et al., 1995; Ruley et al., 2004; Mishra et al., 2006; de la Rosa et al., 2007; Qureshi et al., 2007). Plants grown in EDTA applied Pb-contaminated media have shown lowered antioxidant activities compared to chelant untreated controls, which relate to lowered Pb toxicity. It is assumed that chelant bound Pb forms are less toxic and more available for plant uptake compared to free Pb ions in soil solution. No studies are reported on the response of phytochelatins, thiols and antioxidative enzymes in vetiver grass exposed to Pb and EDTA in soils. The present study focuses on complex formations between PCn, Pb and EDTA. Formation of these complexes is speculated to yield Pb tolerance in vetiver grass. One aim of this study was to better understand the responses of low molecular weight thiols and antioxidants during Pb phytostabilization by vetiver grass. Beyond its fundamental interest, however, this study deals with induction of phytochelatins and related complexes upon Pb exposure. Our main objective was to identify possible PCn, PCn–Pb, and PCn–Pb–EDTA complexes in root and shoot compartments of vetiver grass, using HPLC-ES-MS. The overall purpose of this study was to study vetiver’s ability to accumulate Pb in EDTA treated contaminated soils, followed by understanding certain Pb tolerance responses such as production of low molecular weight thiols, antioxidant enzymes, and phytochelatins. The long-term goal of this study is to understand the biochemical mechanisms towards Pb tolerance, enhance such abilities further using genetic engineering (Zhu et al., 1999a,b), and incorporate vetiver grass in an environmentally friendly and cost-effective phytoremediation model. The information from this study will begin to characterize biochemical plant responses to Pb in vetiver which is absolutely critical in developing an efficient phytoremediation method for Pb-paint-contaminated residential soils using vetiver grass. 2. Materials and methods 2.1. Soil and plant analysis For the evaluation of Pb in soils, 20 Pb-based-paint-contaminated residential sites were selected in two metropolitan cities viz., San Antonio, TX and Baltimore, MD. All soil samples were collected in triplicate from surface horizons (0–6 inches), air-dried, and sieved for 2 mm particle size. Soil pH, texture (clay, sand and silt %), electrical conductivity (EC), total Ca, Mg and P, oxalate-extractable Fe and Al, and soil organic matter (SOM %) were characterized by following the standard protocols outlined in the Soil Science Society of America Handbooks for Chemical and Mineralogical Analysis (Sparks, 1996). The EPA 3050B acid-digestion method was used to determine total Pb concentrations in the soils (USEPA, 1996). An inductively coupled plasma mass spectrometer (ICP-MS), Perkin Elmer (Elan 9000 model, Perkin Elmer Life and Analytical Sciences Inc., Waltham, MA, USA), was used for elemental determinations. QA/QC measures were carefully considered and recoveries of 90–110% of spikes and external standards were considered acceptable. Based on the results, the two most appropriate soils (one high pH and another low pH, having otherwise similar conditions, including similar lead contents) were selected for the greenhouse study to evaluate Pb stress responses in vetiver grass. Greenhouse Pb phytostabilization studies were carried out in packed soil columns made of PVC (1500 high  600 diameter). Each column was filled with 7 inches of play sand, followed by 6 inches of Pb-paint-contaminated residential soil. Vetiver grass was grown in the columns for a period of 2 months. The columns were maintained at 70% water holding capacity. EDTA was used in the form of a Na2-EDTA salt

S.S. Andra et al. / Environmental Pollution 157 (2009) 2173–2183 (CAS no. 6381-92-6, catalog no. BP120-500, Sigma–Aldrich, St. Louis, MO, USA) and added at four different levels (0, 5, 10, and 15 mM kg1 soil). Ten days after the addition of the chelant, plants were harvested, washed thoroughly using tap water and 0.01 mol L1 HNO3 (de la Rosa et al., 2007), rinsed with deionized water, separated into root and shoot compartments, and analyzed for Pb uptake following the procedure by Carbonell et al., (1998).

2.2. Thiols assay

Baltimore

5000

San Antonio

4000

BC B

BC

3000 2000 C C 1000

The catalase (CAT) assay kit was obtained from Cayman Chemical (catalog no. 707002, Cayman Chemical Co., Ann Arbor, MI, USA) and the assays were conducted according to their instructions. The kit consists of assay buffer, sample buffer, formaldehyde standard, catalase, potassium hydroxide, methanol, hydrogen peroxide, Purpald and potassium periodate. To the standard wells, 100 mL of dilute assay buffer, 30 mL of methanol and 20 mL of formaldehyde standards were added. To the positive control wells (bovine liver CAT), 100 mL of diluted assay buffer, 30 mL of methanol and 20 mL of diluted CAT (control) were added to two wells. To the sample wells, 100 mL of diluted assay buffer, 30 mL of methanol and 20 mL of sample were added. Reaction was initiated by adding 20 mL of hydrogen peroxide to all the wells being used. The plate was covered with a plate cover and incubated on a shaker for 20 min at room temperature. Immediately 30 mL of potassium hydroxide was added to all wells to terminate the reaction and then 30 mL of Purpald was added to each well. The plate was covered and incubated for 10 min at room temperature on a shaker. Potassium periodate (10 mL) was added to all wells and the covered plate was incubated for 5 min at room temperature on a shaker. Finally, the CAT activity was determined at 540 nm in a microplate reader and compared with the controls.

2.4. Phytochelatins analysis For the determination of phytochelatins and related Pb-binding peptides using high performance liquid chromatography–electrospray mass spectrometry (HPLCES-MS), the modified procedure described by El-Zohri et al. (2005) was used. Phytochelatin assays of vetiver tissues were performed by freezing the plant material in liquid nitrogen followed by grinding with a mortar and pestle. The plant sample

600

A

6000

2.3. Catalase assay

D D

Shoot Tissue Pb (mg kg–1)

A

Root Tissue Pb (mg kg–1)

Total thiols in plant tissues were analyzed following the procedure given by Hartley-Whitaker et al. (2001). Fresh plant tissues, both root and shoot, were immediately frozen in liquid nitrogen (196  C) for 10 min and stored at 80  C for analysis of thiols. Frozen plant tissues were ground in liquid nitrogen and homogenized with 0.02 M EDTA at a rate of 10 mL g1 fresh tissue. Homogenates were centrifuged at 12,000 g at 4  C for 10 min and the supernatant (0.5 mL) was transferred to a polyethylene test tube and mixed with 0.2 M Tris buffer (pH 8.2, 1.5 mL), 0.01 M DTNB (100 mL), and methanol (7.9 mL). The reaction was allowed to develop for 10 min at room temperature before the absorbance was measured. A sample blank (without adding DTNB) and a reagent blank (without sample) was prepared and measured in the same manner. A UV–vis spectrophotometer (Benchmark plus microplate spectrophotometer, BioRad Life Science Research, Hercules, CA, USA) was used to quantify total thiols in plant samples. The absorbance at 412 nm was measured within 5 min after adding DTNB. Reduced glutathione (CAS no. 70-18-8, catalog no. G4251-5G, Sigma chemicals, Sigma–Aldrich, St. Louis, MO, USA) in the concentration range of 5–20 mmol used as standard. Thiol concentrations were calculated by using the extinction coefficient obtained from the standard curve.

0

5

10

Baltimore San Antonio

1000 750

C D

500

B

C C

E 250

300 d

100

e e 0

450

5

a

10

Baltimore

375

15

a

San Antonio

300 b

b

b

225

c c

150

d

75 0

5

10

0

15

EDTA concentration (mM kg–1) 200

A

Baltimore San Antonio

1000

A

B

750

C

B

500

D

250

D

E

5

10

15

EDTA concentration (mM kg–1)

Shoot CAT Activity (nmol min–1 g–1)

Root CAT Activity (nmol min–1 g–1)

d

200

EDTA concentration (mM kg–1)

0

1250

a c c

15

Shoot LMWT (mmol kg–1)

Root LMWT (mmol kg–1)

A A

0

C

San Antonio

400

0

0

1250

a

Baltimore 500

EDTA concentration (mM kg–1)

B

2175

150

Baltimore San Antonio

a

b c

cd e

100

d

cd

e 50

0

0 0

5

10

15

EDTA concentration (mM kg–1)

0

5

10

15

EDTA concentration (mM kg–1)

Fig. 1. Lead uptake and responses of tolerance mechanisms in vetiver grass grown in San Antonio, TX and Baltimore, MD soils. Left and right side figures represent data from root and shoot tissues for: (A) Lead accumulation (upper panel); (B) levels of low molecular weight thiols (middle); and (C) catalase activities (lower).

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(1 g of tissue powder) was mixed with 3 mL cold (4  C) aqueous dithiothreitol (DTT), an antioxidant, solution and the suspension was sonicated using 0.5 s pulses at a power of 400 W for 2 min in a 40 kHz Branson 2510 (Dambury, CT, USA) sonication bath. The cell debris was subsequently sedimented by centrifugation at 12,000 rpm at 4  C for 10 min (IEC Microlite RF refrigerated microcentrifuge, Thermo Electron Corporation, San Jose, CA, USA). The supernatant (1 mL) was filtered through a 0.45 mm nylon membrane syringe filter (Fisherbrand, ThermoFisher Scientific, Waltham, MA, USA). Two-step purification and concentration was carried out using: (1) microcon centrifugal filter devices (CAS no. R7DN45102, catalog no. 42407, Millipore Inc., Billerica, MA, USA); and (2) reversed-phase ZipTip pipette tips (CAS no., L7CN4514, catalog no. ZTC18MO96, Millipore Inc., Billerica, MA, USA). The concentrated plant samples (0.5 mL) were used for qualitative analysis of PCn, PCn– Pb and PCn–Pb–EDTA complexes. LC/MS/MS analysis was conducted using a Michrom Bioresources Magic HPLC system (Michrom Bioresources, Inc., Auburn, CA, USA) and a Finnigan LCQ Duo ion trap mass spectrometer (Thermo Finnigan, San Jose, CA, USA). Volumes of 20 mL of the plant extract were injected with an autosampler (Magic autosampler, Michrom Bioresources, Inc., Auburn, CA, USA), and eluted from a polymeric column (PLRS, 5 m, 100 Å) (Polymer Laboratories, Varian Inc., Amherst, MA, USA). The plant samples were eluted using 0.1% formic acid (FA) and 0.01% trifluoroacetic acid (TFA) in water (mobile phase A) and 0.1% FA and 0.01% TFA in acetonitrile (mobile phase B). A threestep gradient was used consisting of 0–20% B in the first 25 min, 20% B for the next 10 min, followed by 20–100% B in the next 10 min was used. The MS/MS analysis was accomplished using an automated data acquisition procedure, in which a cyclic series of different scan modes was performed. The data acquisition was conducted using the full-scan mode to obtain the most intense peak (signal > 1.5  105 counts) as the precursor ion, followed by MS/MS to determine the structural fragment ions of the precursor ion. This enables the determination of the amino acid sequence of each of the PCn, and detects the number of Pb ions complexed with respective PCn. For quantification of PCn, respective standards ranging from PC1 to PC5 were obtained from Anaspec Inc. (Anaspec Inc., San Jose, CA, USA). All standard solutions were prepared and diluted in 1:1 acetonitrile:water solvent mixture. Separate stock solutions of 100 mg mL1 of each phytochelatin were prepared and stored at 80  C. Aliquots of these solutions were mixed to obtain a 10 mg mL1 mixed working standard stock solution that was stored at 20  C. Six-point calibration curves of mixed PCn analytes were prepared daily at 1, 10, 100, 250, 500, and 1000 mg mL1 concentrations using the 10 mg mL1 stock solution. The final volume was bought up to 0.5 mL using 1:1 acetonitrile:water solvent mixture and stored at 4  C. Calibration curves were used for quantifying phytochelatins in the experimental plant samples. 2.5. Chelated lead complexes analysis Interactions of metal complexes with the polymeric reversed phase stationary phase of the column and the ion pairing agent in the mobile phase could change their chemical nature. This would result in the instability and/or decreased ionization efficiency of metal complexes and potentially inhibit their appearance in the LC/MS scans. Hence, we relied on direct infusion of the filtered and concentrated plant samples into the mass spectrometer at 5 mL min1 using a Hamilton (model no. 1750) (Hamilton, Co., Reno, NV, USA) syringe. All data were collected and analyzed using the Xcalibur software (Thermo Finnigan) in full-scan and MS/MS mode. The settings of the mass spectrometer were the same as used for HPLC-ES-MS set-up. The relative collision energy used for CID was uncalibrated and in arbitrary units. Normalized collision energies (NCE) between 10 and 90% were used. The MS conditions routinely used were: source voltage, 5 kV; capillary temperature, 225  C;

100

Relative Abundance (

)

(PC2+H)+1 540.9 (PC3+H)+1 772.7 (PC4+H)+1 1004.7

(PC1+H)+1 308.8

50

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 mass spectrometer was m/z 50–1800. Simulations of the Pb isotope patterns were done using Isotope Viewer in Xcalibur. To ensure optimal detection limits and reproducibility, the sample cone of the mass spectrometer was cleaned daily according to the manufacturer’s instructions.

3. Results and discussion 3.1. Lead uptake by vetiver The uptake and accumulation of Pb under different EDTA concentrations measured at the end of the chelant exposure is shown in Fig. 1A. Lead accumulation in vetiver tissues was linear and increased significantly with increasing concentrations of EDTA in both the Baltimore and San Antonio soils. Vetiver was able to accumulate 290  120 and 20  2 mg Pb kg1 dry weight in root and shoot tissues, respectively, when grown in Baltimore soils with 0 mM kg1 EDTA. At 15 mM kg1 EDTA application, vetiver accumulated 4460  1550 and 480  60 mg Pb kg1 dry weight in root and shoot tissues, respectively. Similarly, measured Pb concentrations were 270  20 and 10  20 mg Pb kg1 dry weight in root and shoot tissues of the vetiver treated with 0 mM kg1 EDTA in San Antonio soils, respectively, and 3120  60 and 330  80 mg Pb kg1 dry weight in root and shoot tissues of the vetiver treated with 15 mM kg1 EDTA. These results suggest that Pb translocation to the above ground parts is increased by EDTA application to the soils. These findings support the hypothesis that chelant bound Pb forms are readily available for plant uptake and translocation as shown in other studies (Huang and Cunningham, 1996; Blaylock et al., 1997; Huang et al., 1997; Wu et al., 1999). Baker et al., 1994 suggested that immobilization of metals in roots is an exclusion strategy of plants towards metal toxicity. We calculated a Pb translocation factor (TF), the ratio of Pb concentration in shoot to root, in order to know the translocation efficiency of vetiver. Stoltz and Greger (2002) classified plant species with TF less than one as ‘shoot metal excluders’, suitable for phytostabilization of metal-contaminated soils. In our study, the calculated TF was less than one in both soils, indicating vetiver grass tends to store Pb in root tissues. This supports the findings by Lai and Chen (2004) who considered vetiver grass as a potential Pb phytostabilization plant when grown in soils with 1000 mg kg1 Pb. Morphological phytotoxic symptoms such as chlorosis, necrosis and/or stunting were not observed in vetiver grown in any of the study treatments. This observation supports the earlier findings by Ruley et al. (2004) that chelant bound Pb forms are less toxic to plants compared to free Pb ions in the rhizosphere. Pb uptake by vetiver is governed by the soil properties. Pb is more available for vetiver uptake in acidic soil of Baltimore, while the carbonates rich San Antonio soil binds Pb making it unavailable for plant uptake. The soil properties of the two study soils are given in Table S1 (Supplementary Information).

Table 1 Concentrations of PC1 on a fresh weight basis in root and shoot tissues of vetiver plants grown in Pb-based paint-contaminated residential soils and exposed to different levels of EDTA.

(PC5+H)+1 1236.6

Treatment (mM EDTA kg1 soil)

0 100

300

500

700

900

1100

1300

1500

m/z Fig. 2. Electrospray mass spectrum obtained for the phytochelatin standards mixture with 1 mg mL1 of each PCn.

0 5 10 15

Baltimore, MD

San Antonio, TX

mg PC1 kg1 fresh wt

mg PC1 kg1 fresh wt

Root

Shoot

Root

Shoot

50.3  6.1 6.1  2.3 8.4  3.5 14.8  1.9

Trace 1.1  0.5 2.9  0.2 4.2  1.7

22.4  4.6 1.9  0.7 2.4  0.4 3.7  1.1

Trace 0.8  0.3 1.2  0.5 2.5  0.7

100

A

(PC1) 308.1

Relative Abundance (%)

Relative Abundance (%)

S.S. Andra et al. / Environmental Pollution 157 (2009) 2173–2183

50 614.6 179.1

332.4

550.7 391.0

162.1 290.1 0

200

400

802.8

600

800

1000

100

B

2177

(PC1) 307.9

50

614.4 289.9

332.4

178.9 161.9 0

200

600

Relative Abundance (%)

Relative Abundance (%)

550.8 50

(PC1) 308.1

391.3

121.8

0

802.9

522.5 200

614.2 600

400

800

1000

m/z

332.4

C

802.8

400

m/z 100

550.7

391.0

800

1000

100

D

(PC1) 307.9

332.4 614.4

50 391.0

550.7 802.8

0

200

400

m/z

600

800

1000

m/z

Fig. 3. LC-ES-MS spectra for the possible PCn complexes in vetiver plants grown in Baltimore soil; (A) root 0 mM kg1 EDTA; (B) root 15 mM kg1 EDTA; (C) shoot 0 mM kg1 EDTA; and (D) shoot 15 mM kg1 EDTA.

Relative Abundance (%)

Fig. 1B shows the effect of Pb, in the presence and absence of EDTA, on the concentrations of total thiols in root and shoot of vetiver grown in the two soils. As expected, root thiol levels in vetiver grown in soils with no EDTA addition are significantly higher compared to the chelant added treatments. With increasing concentration of applied EDTA, there is an increase in thiol concentrations resulting from a higher Pb uptake and accumulation. Concentrations of total thiols in response to Pb stress reached 1040  70 and 340  60 mmol thiols kg1 fresh weight in root and

100

A

(PC1) 308.8

50 615.4

1229.2 1537.1

922.3 0

200

600

1000

1400

1800

shoot tissues of the vetiver treated with 0 mM kg1 EDTA in Baltimore soils, respectively, and 920  15 and 350  30 mmol thiols kg1 fresh weight in root and shoot tissues of the vetiver treated with 15 mM kg1 EDTA. Similarly, measured total thiol concentrations were 850  300 and 220  30 mmol thiols kg1 fresh weight in root and shoot tissues of the vetiver treated with 0 mM kg1 EDTA in San Antonio soils, respectively, and 220  30 and 150  20 mmol thiols kg1 fresh weight in root and shoot tissues of the vetiver treated with 15 mM kg1 EDTA. Despite the significantly higher Pb levels in both the root and shoot tissues from vetiver treated with EDTA compared to the chelant unamended controls,

Relative Abundance (%)

3.2. Total thiol levels

100

B

(Pb-EDTA) 498.8

(PC1) 308.2

(PC1-Pb-EDTA) 805.4

50 130.7

615.5

1112.6 0

200

600

C

163.0 392.4

50

0

(PC1) 308.5

200

513.6

600

1000

m/z

1000

1400

1800

m/z

Relative Abundance (%)

Relative Abundance (%)

m/z 100

995.2

1400

1800

100

50

D

(Pb-EDTA) 499.2

(PC1) 308.1 392.5

0

200

996.8 520.9

600

812.7 1000

1400

1800

m/z

Fig. 4. Full-scan ES-MS spectra obtained using direct infusion approach for the possible chelated Pb and PCn complexes in vetiver plants grown in Baltimore soil; (A) root 0 mM kg1 EDTA; (B) root 15 mM kg1 EDTA; (C) shoot 0 mM kg1 EDTA; and (D) shoot 15 mM kg1 EDTA.

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Relative Abundance ( )

A 100

178.9

m/z 179.0 γGlu--Cys--Gly

50 232.9 130.3

0

80

100

120

140

160

180

200

220

240

260

280

300

320

m/z

Relative Abundance ( )

B

100

366.2

O m/z 366.0

O N

HO

O 50

O

N

+ Pb OH

384.0

338.3 247.3

0

240

453.1

419.4

260

280

300

320

340

360

380

400

420

440

460

480

500

m/z

C

805.3

498.9

100

Relative Abundance ( )

(PC1-Pb-EDTA + 1H)+1

(Pb-EDTA + 1H)+1 m/z 499.0 PC1--Pb--EDTA - (PC1) 50 (PC1 + 1H)+1 307.7

0

300

400

- (Pb-EDTA)

500

600

700

800

900

1000

m/z Fig. 5. Fragment spectra of protonated phytochelatins: (A) PC1 (upper); (B) Pb–EDTA (middle); and (C) PC1-Pb–EDTA (lower) from MS/MS experiments on the root tissue of vetiver grass exposed to EDTA at 15 mM kg1.

the concentrations of thiols are significantly lower compared to the latter. A similar trend was observed in the thiol levels of vetiver grown in the two soils, however the differences in thiol levels is related to tissue Pb concentrations which in turn are influenced by the differences in soil properties. de La Rosa et al. (2007) reports a significant reduction in thiol concentrations of Salsola kali roots grown in a medium containing Pb and EDTA compared to plants grown in media with Pb alone and controls with no Pb. Findings from this study are in accordance with the reported one and support the hypothesis that EDTA bound Pb forms induce lower stress levels and hence a reduced response of the defense mechanisms. However, to the best of our knowledge, no study was conducted to relate thiol responses to the possible metal-binding peptide and chelated Pb complexes in plants. 3.3. Catalase activities Fig. 1C shows the effect of Pb, in the presence and absence of EDTA, on catalase activities in root and shoot tissues of vetiver

grown in the two soils. The responses of catalase towards chelated Pb are similar to that of total thiols. With increasing concentrations of EDTA added to the soils there is an increase in Pb uptake and thereby a related increase in the catalase activities. However tissue activities of catalase were greater in vetiver grown in EDTA untreated controls compared to EDTA treatments. Catalase activity upon Pb exposure was up to 900  7 and 140  10 nmol min1 g1 fresh weight in root and shoot tissues of the vetiver treated with 0 mM kg1 EDTA in Baltimore soils, respectively, and 850  8 and 120  30 nmol min1 g1 fresh weight in root and shoot tissues of the vetiver treated with 15 mM kg1 EDTA. Similarly, measured catalase activities were 570  60 and 380  75 nmol min1 g1 fresh weight in root and shoot tissues of the vetiver treated with 0 mM kg1 EDTA in San Antonio soils, respectively, and 100  4 and 80  2 nmol min1 g1 fresh weight in root and shoot tissues of the vetiver treated with 15 mM kg1 EDTA. Similar patterns in antioxidant responses to Pb and chelated Pb have been reported in a Pb hyperaccumulator, Sesbania drummondii (Ruley et al., 2004).

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Fig. 6. Isotopic pattern of chelated Pb and PCn complexes identified in the root of vetiver grass exposed to EDTA at 15 mM kg1 using ESMS: (A) PC1 (upper); (B) Pb–EDTA (middle); and (C) PC1-Pb–EDTA (lower). Left and right side figures represent spectra from experimental and theoretical isotope pattern of the respective complexes.

3.4. Phytochelatins 3.4.1. Calibration of phytochelatins standards Fig. S1 (Supplementary Information) shows the reconstructed ion chromatograms (RIC) obtained from the phytochelatins (PCn) standards mixture (n ¼ 1–5). The retention time of each phytochelatin on the polymeric column depends on its molecular mass. PC1 with the lowest mass (m/z ¼ 307) eluted first at 13.3 min while

PC5 with an m/z of 1236 eluted at 17.4 min. PC2, PC3 and PC4 eluted in between at 15.2, 16.4 and 17.0 min, respectively. Full MS of the PCn standard mixture (n ¼ 1–5) with respective m/z values are shown in Fig. 2. The differences in the ion counts observed for the same concentration of the different PCn in the solution is attributed to the differences in the extent of protonation of each PCn, which is peptide specific (Chassaigne et al., 2001). Collision induced dissociation (CID) of the PCn results in bond cleavage. This gives rise to

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Fig. 7. Isotopic pattern of possible Pb, EDTA, and PCn complexes observed in the root of vetiver grass exposed to EDTA at 15 mM kg1 using ESMS: (A) 2PC1–Pb (upper); (B) PC12Pb–EDTA (middle); and (C) 2PC1-Pb–EDTA (lower). Left and right side figures represent spectra from experimental and theoretical isotope pattern of the respective complexes.

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daughter fragments either retaining charge on the amino or carboxyl terminal fragments, represented as ‘b’ and ‘y’ type ions, respectively (Vacchina et al., 1999). These charged fragments not only give structural information about the peptide based on the amino acid sequence, but also aids in identifying the metal-binding amino acid of the peptide. Normalized collision energy of 70% was used for the MS/MS step on the PCn standards mixture. The presence of the largest y-fragment/mono-protonated precursor pairs for the corresponding PCn (m/z 179.0/308.8 for PC1, m/z 411.0/540.9 for PC2, m/z 643.0/772.7 for PC3, m/z 874.9/1004.7 for PC4, and m/z 1106.9/1236.6 for PC5) from the mass spectra clearly demonstrates the presence of the respective PCn in the standards mixture (data not shown). The common loss of 129.8 m/z unit from each PCn to yield the largest daughter fragment corresponds to a g-glutamic acid. Peptides of interest from vetiver tissues samples were subjected to CID followed by matching of the daughter fragments for identifying respective PCn. 3.4.2. Identification and quantification of phytochelatins in vetiver tissues Chromatograms from the HPLC were obtained from EDTA treated and untreated vetiver plants grown in the two soils. PC1 alone was observed in both root and shoot tissues exposed to 15 mM EDTA. In chelant untreated plants, PC1 was seen in the root tissues while it was below detection limits in shoot tissue (Table 1). LC-ES-MS spectra obtained from these treatments are shown in Fig. 3. These show the clear presence of PC1 as demonstrated by the peak at m/z 308. CID on the PC1 yielded three distinct daughter fragments with m/z values of 232.9 (26%), 178.9 (100%) and 130.3 (29%) (Fig. 5A). In the CID spectra for PC1 obtained for all the plant samples from different treatments under study, both b and y type ions are frequent and abundant enough for identifying the parent ion as PC1. The m/z values of both b and y type fragment ions matched with the theoretical values given by Vacchina et al. (1999). PC1 in the treatments was quantified using the area under the curve from the calibration curve and expressed as mg PC1 kg1 plant tissue on a fresh weight basis (Table 1). In EDTA untreated vetiver plants, the concentration of PC1 was a maximum at 55.5 mg kg1 in root tissue compared to all other treatments with EDTA. The concentration of PC1 in the root tissue of vetiver grown in 15 mM EDTA treatments was 11.5 mg kg1, five times lower compared to the levels in root tissue of vetiver grown in the absence of EDTA (Table 1). These findings support the hypothesis that chelant bound Pb forms are less toxic and induce lower stress in plants compared to the free Pb ions. The concentrations of PC1 were higher in vetiver grown in Baltimore soil compared to the San Antonio soil (Table 1). A peak with an m/z value of 615.4, 922.3, 1229.2 and 1537.1 could be a dimer, trimer, tetramer and pentamer of PC1, respectively (Fig. 4A). Observing polymers of PC1 could be due to cold cluster formation in the gas phase during the electrospray process. This is a well known phenomenon and was further confirmed by doing CID on these clusters. 3.5. Possible phytochelatin–lead–EDTA complexes Confirming the presence of PC1 in vetiver tissues upon exposure to Pb leads to the next logical step, identifying possible PC1–Pb complexes. However, we were unable to observe any Pb compounds using LC-ES-MS. We hypothesize that the HPLC conditions for separating phytochelatins might not be favorable for retaining PCn–Pb complexes intact. Hence we tried to inject the plant samples directly into the mass spectrometer following the extraction procedure as given in Section 2. ES mass spectra of root and shoot tissues from vetiver grown in Baltimore soil in the presence and absence of EDTA are shown in Fig. 4. Only the peaks

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with a relative intensity >5.0% were subjected for MS/MS. As shown in Fig. 4B, root tissue from vetiver treated with EDTA shows two new peaks at m/z 498.8 and 805.4 in addition to the peak at 308.2 (PC1). Pb exhibits a characteristic natural abundance isotope pattern [204Pb (1.4%), 206Pb (24.1%), 207Pb (22.1%), and 208Pb (52.4%)] which assists in identifying the metal in a compound. It also helps in determining the number of metal ions in the complex (Bach et al., 2005, 2007; Polec-Pawlak et al., 2007). The isotope signature of Pb enabled us to identify the possible Pb complexes by comparing the experimental obtained pattern with the theoretical pattern. In Fig. 6B, the isotopic peaks around 499.4 fit the isotope distribution pattern (right-side figure) calculated with the Xcalibur Isotope Viewer for a Pb–EDTA complex with a molecular formula of [C10H15O8N2Pb1]. As shown in Fig. 6C, the isotopic peaks of 805.4 are attributed to a phytochelatin complex of PC1–Pb–EDTA with a molecular formula of [C20H31O14N5S1Pb1]. CID experiments were carried out first on PC1, then on PC1–Pb, and PC1–Pb–EDTA complexes. In each case, CID spectra were recorded at several collision energies up to the energy required to totally dissociate the parent. Fig. 5A–C display CID spectra for PC1, PC1–Pb, and PC1– Pb–EDTA, respectively. Peaks observed at m/z 828.2, 1010.4 and 1112.7 correspond to the possible mono-protonated forms of 2PC1– Pb, PC1–2Pb–EDTA and 2PC1–Pb–EDTA, respectively (Fig. 7A–C). Further work is required to confirm and gain more detailed knowledge of the binding of Pb with EDTA and phytochelatins. This should include improved purification of the plant samples to further isolate the PCn–Pb bio-molecules of interest intact therefore enabling a more in-depth study by MS/MS. We also intend to incubate PCn standards with Pb and EDTA at different molar ratios. The ES-MS data generated from these aqueous solutions might help to identify the number of Pb ions complexed with both phytochelatins and EDTA. The results from this comprehensive and comparative study will give a better understanding, and perhaps illuminate the nature and stoichiometry of complexes that could occur in vetiver tissues in response to chelant bound Pb.

4. Conclusions Exciting breakthroughs have been made during the past few years in understanding the molecular basis of heavy metal detoxification in plants. In order for these breakthroughs to be translated into practical and cost-effective solutions for phytoremediation of lead, it is important to understand the physiological and biochemical basis of lead detoxification in plants such as vetiver, which has all of the ideal characteristics necessary for use in the remediation of lead-contaminated soils. The current study provides the mechanistic background behind the enhanced tolerance of vetiver grass towards chelated-Pb phytostabilization. These greenhouse-scale experiments attempt to validate the data obtained in a previous hydroponic study (Andra et al., in press) on the production of PCn and their role in Pb detoxification by vetiver when grown in contaminated soils. The present study focuses on complexes formed between PCn, Pb and EDTA. Formation of these complexes was speculated to yield Pb tolerance in vetiver grass. The primary objective of this study was to better understand the responses of low molecular weight thiols and antioxidants during the Pb extraction by vetiver grass. In addition, characterization of phytochelatins and related complexes that were speculated to sustain normal plant growth despite high tissue Pb concentrations was deemed necessary. The presence of PC1–Pb–EDTA complex from our ES-MS study, accompanied with the literature findings, support our hypotheses that chelant bound Pb forms are less toxic to plants. Our assumption is that similar complexes might be available in the plant tissues, however, it is not clear yet whether

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such complexes were dissociated during extraction and/or are unstable in the solution. Because some studies showed that the chelant bound Pb forms are less toxic and easy for plant uptake, we paid particular attention to the nature of complex formations between PCn, Pb and EDTA. Future studies aim at using inductively coupled plasma mass spectrometry (ICP-MS) as a preliminary step for detecting element specific molecules/complexes (Navaza et al., 2006). An appropriate cleaning and pre-concentration procedure will be used to enhance characterization ability for PCn–metal– chelant complexes in plants using ES-MS. Different tolerance mechanism responses were reported here according to the applied chelant concentration, and the nature and combination of the Pb complexes. In conclusion, the present study shows the potential of vetiver grass inclusion in developing a successful phytoremediation model for remediating Pb-paint-contaminated residential sites. Acknowledgments The authors from the University of Texas at San Antonio would like to thank the United States Department of Housing and Urban Development for funding this study. Appendix A. Supplemental material Supplementary information for this manuscript can be downloaded at doi: 10.1016/j.envpol.2009.02.014. References Ali, M.B., Vajpayee, P., Tripathi, R.D., Rai, U.N., Singh, S.N., Singh, S.P., 2003. Phytoremediation of lead, nickel, and copper by Salix acmophylla Boiss.: role of antioxidant enzymes and antioxidant substances. Bull. Environ. Contam. Toxicol 70, 462–469. Andra, S.S., Datta, R., Sarkar, D., Makris, K.C., Mullens, C.P., Sahi, S.V., Bach, S.B.H., in press. Induction of lead-binding phytochelatins in vetiver grass [Vetiveria zizanioides (L.)]. J. Environ. Qual. (in press). ATSDR (The Agency for Toxic Substances and Disease Registry), 2000. Lead toxicity: in case studies in environmental medicine, publication no.: ATSDR-HE-CS2001-0001. Bach, S.B.H., Green, C.E., Nagore, L.I., Sepeda, T.G., Merrill, G.N., 2007. Complexes of dichloro(ethylenediamine)palladium(II) observed from aqueous solutions by electrospray mass spectrometry. J. Am. Soc. Mass Spectrom 18, 769–777. Bach, S.B.H., Sepeda, T.G., Merrill, G.N., Walmsley, J.A., 2005. Complexes of dibromo (ethylenediamine)palladium(II) observed from aqueous solutions by electrospray mass spectrometry. J. Am. Soc. Mass Spectrom 16, 1461–1469. Baker, A.J.M., McGrath, S.P., Sidoli, C.M.D., Reeves, R.S., 1994. The possibility of insitu heavy metal decontamination of polluted soils using crops of metal-accumulating plants. Resour. Conservat. Recycl 11, 41–49. Blaylock, M.J., Salt, D.E., Dushenkov, S., Zakharova, O., Gussman, C., Kapulink, Y., Ensley, B.D., Raskin, I., 1997. Enhanced accumulation of Pb in Indian mustard by soil-applied chelating agents. Environ. Sci. Technol 31, 860–865. Carbonell, A.A., Aarabi, M.A., DeLaune, R.D., Gambrell, R.P., Patrick Jr., W.H., 1998. Arsenic in wetland vegetation: availability, phytotoxicity, uptake and effects on plant growth and nutrition. Sci. Total Environ 217, 189–199. Chassaigne, H., Vacchina, V., Kutchan, T.M., Zenk, M.H., 2001. Identification of phytochelatin-related peptides in maize seedlings exposed to cadmium and obtained enzymatically in vitro. Phytochemistry 56, 657–668. ˜ o, C., Esteban, M., 2008. TherChekmeneva, E., Prohens, R., Dı´az-Cruz, J.M., Arin modynamics of Cd2þ and Zn2þ binding by the phytochelatin (g-Glu–Cys)4–Gly and its precursor glutathione. Anal. Biochem 375, 82–89. Datta, R., Sarkar, D., 2004. Effective integration of soil chemistry and plant molecular biology in phytoremediation of metals: an overview. Environ. Geosci 11, 53–63. de la Rosa, G., Peralta-Videa, J.R., Cruz-Jimenez, G., Duarte-Gardea, M., MartinezMartinez, A., Cano-Aguilera, I., Sharma, N.C., Sahi, S.V., Gardea-Torresdey, J.L., 2007. Role of ethylenediaminetetraacetic acid on lead uptake and translocation by tumbleweed (Salsola kali L.). Environ. Toxicol. Chem. 26, 1033–1039. do Nascimento, C.W.A., Xing, B.S., 2006. Phytoextraction: a review on enhanced metal availability and plant accumulation. Sci. Agric 63, 299–311. El-Zohri, M.H.A., Cabala, R., Frank, H., 2005. Quantification of phytochelatins in plants by reversed-phase HPLC-ESI-MS-MS. Anal. Biochem 382, 1871–1876. Evangelou, M.W.H., Ebel, M., Schaeffer, A., 2007. Chelate assisted phytoextraction of heavy metals from soil. Effect, mechanism, toxicity, and fate of chelating agents. Chemosphere 68, 989–1003.

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