Plant and Soil

, Volume 326, Issue 1, pp 171–185

Synthesis of phytochelatins in vetiver grass upon lead exposure in the presence of phosphorus

Authors

    • Environmental Geochemistry LaboratoryUniversity of Texas at San Antonio
  • Rupali Datta
    • Biological SciencesMichigan Technological University
  • Dibyendu Sarkar
    • Department of Earth and Environmental StudiesMontclair State University
  • Konstantinos C. Makris
    • The Cyprus International Institute for the Environment and Public Health in association with the Harvard School of Public Health
  • Conor P. Mullens
    • Department of ChemistryUniversity of Texas
  • Shivendra V. Sahi
    • Department of BiologyWestern Kentucky University
  • Stephan B. H. Bach
    • Department of ChemistryUniversity of Texas
Regular Article

DOI: 10.1007/s11104-009-9992-2

Cite this article as:
Andra, S.S., Datta, R., Sarkar, D. et al. Plant Soil (2010) 326: 171. doi:10.1007/s11104-009-9992-2
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Abstract

In a hydroponic setting, we investigated the possible role of phytochelatins (metal-binding peptides) in the lead (Pb) tolerance of vetiver grass (Vetiveria zizanioides L.). Pb was added to the nutrient medium at concentrations ranging from 0 to 1,200 mg L−1. Furthermore, we simulated the effect of soil phosphorus (P) on potentially plant available Pb by culturing vetiver grass in P-rich nutrient media. After 7 days of exposure to Pb, we evaluated the Pb uptake by vetiver grass. Results indicate that vetiver can accumulate Pb up to 3,000 mg kg−1 dry weight in roots with no toxicity. Formation of lead phosphate inhibited Pb uptake by vetiver, suggesting the need for an environmentally safe chelating agent in conjunction with phytoremediation to clean up soils contaminated with lead-based paint. Unambiguous characterization of phytochelatins (PCn) was possible using high pressure liquid chromatography coupled with electrospray ionization mass spectrometry (LC-ESMS). Vetiver shows qualitative and quantitative differences 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 confirmation of each PCn based on the amino acid sequence. Possible Pb-PC1 and Pb2-PC1 complexes were reported in vetiver root at the highest Pb concentration. The data from these experiments show that the most probable mechanism for Pb detoxification in vetiver is by synthesizing PCn and forming Pb–PCn complexes.

Keywords

HydroponicsLead-based paintLiquid chromatographyMass spectrometryPhytochelatinsPhytoremediationVetiver

Abbreviations

CID

Collision induced dissociation

ES-MS

Electrospray ionization mass spectrometry

GSH

Glutathione

HPLC

High-performance liquid chromatography

Pb

Lead

P

Phosphorus

PCn

Phytochelatins

SEM

Scanning electron microscopy

Introduction

Although past national public health efforts have reduced lead exposure significantly, lead poisoning remains the most common environmental health problem affecting American children (USEPA 2001). Currently, lead exposure occurs predominantly through ingestion of lead-contaminated household dust and soil in older housing containing lead-based paint. Despite the efforts made to reduce residential exposure to lead (including setting a maximum allowable lead content in paint of 0.06% in 1977), every city in the United States has a significant number of housing facilities that were built prior to the implementation of that policy (ATSDR 2000). Composite probe samples obtained from all over the United States (US) show that approximately 7% of US dwellings (6.46 million units) have soil lead (Pb) concentrations above US Environmental Protection Agency (EPA) and US Department of Housing and Urban Development (HUD) standards (400 mg kg−1 for bare play area soil and 1,200 mg kg−1 for bare soil in the rest of the yard) (Jacobs et al. 2002). In a previous study, total soil Pb concentrations in 20 different lead-based paint contaminated residential sites ranged between 36 mg Pb kg−1soil and 4,182 mg Pb kg−1soil, with mean and median values of 1,197 and 821 mg Pb kg−1soil, respectively (Andra et al. 2006). Children living in these houses are at the greatest risk of exposure, as crawling on the ground and playing in the backyard can result in ingestion and inhalation of soil- and dust-borne lead (USEPA 2001). The physiological and behavioral effects associated with Pb ingestion in children are well characterized (Landrigan 1991). Soil lead clean-up is traditionally done by soil removal for off-site disposal. This is extremely expensive and rather impractical for remediating residential properties. As a result, in situ remediation techniques, such as phytoremediation, are gaining attention as an environmentally safe and inexpensive alternative (Datta and Sarkar 2004).

Vetiver grass (Vetiveria zizanioides L.) is a native of the subtropics. Its ability to extract and accumulate higher levels of metals such as Zn and Cu is well documented (Chiu et al. 2005). Previous studies in our laboratory show the ability of vetiver grass to translocate up to 1,700 ± 28 and 3,350 ± 66 mg Pb kg−1 (dry weight) in shoot tissues, respectively, when exposed to 400 and 1,200 mg Pb L−1 in a hydroponic set up without phosphorus (Andra et al. 2009). Shoot tissue concentrations of Pb in vetiver seen from this experiment were greater than the specified criterion for a hyperaccumulator plant (Sahi et al. 2002). However, it is a noted fact that the plant available Pb fraction and phytoextraction efficiency are higher in a hydroponic set-up compared to in contaminated soils. Vetiver did not exhibit any phytotoxic symptoms such as growth retardation or chlorosis under these conditions. Vetiver is well adapted to environments ranging from aquatic to desert conditions, and is tolerant to frost, heat, acidic and alkaline conditions (Dalton et al. 1996). The additional virtues that make vetiver grass a suitable candidate for phytoremediation are high biomass, a dense root system and quick regeneration ability (Pichai et al. 2001). However, to include vetiver grass in a successful phytoremediation model to clean-up Pb-paint contaminated residential sites, it is essential to understand its Pb uptake potential and possible Pb tolerance mechanisms. Complete understanding of the Pb tolerance mechanisms in plants is still lacking to date (Piechalak et al. 2002). The role of metal-binding thiol peptides, typically known as ‘phytochelatins’, in the inactivation of certain metals including Pb is well established (Gwozdz et al. 1997). Phytochelatins (PCn), a class of metal-binding proteins with a typical three-amino-acid sequence of (γGlu-Cys)n-Gly (n = 2–11), are reported to bind metal ions in plants (Grill et al. 1989). These peptides are synthesized enzymatically from glutathione or its homologs due to the reaction catalyzed by phytochelatin synthase, which is an enzyme activated by heavy metals including Pb (Vatamaniuk et al. 2000). Because of the high content of cysteine, PCn are able to create complex compounds with toxic metals. These complexes are suggested to be transported into the vacuole by the ATP-binding-cassette-like transporters localized in the tonoplast (Salt and Rauser 1995), thus separating them from cell metabolism. Alternatively, certain ABC transporters, such as AtPDR12, induce Pb tolerance in Arabidopsis by transporting the toxic ions to the cell exterior (Lee et al. 2005). The term ‘PC1’ in this context refers to GSH and is represented by (γGlu-Cys)1-Gly (Rea et al. 2004; Wawrzynski et al. 2006; Chekmeneva et al. 2008). Liquid chromatography and mass spectrometry are the analytical techniques most commonly used for the separation and 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 considered a viable investigative method for phytoremediation studies prior to evaluating a plant’s performance in soils at the greenhouse and/or field level. Under natural conditions, Pb is strongly sorbed onto soil minerals and organic matter, making it the contaminant least amenable to phytoremediation. In general, the availability of Pb for plant uptake in soils depends on the levels of phosphorus, calcium, iron and aluminum hydroxides (Schmidt 2003; do Nascimento and Xing 2006; Nowack et al. 2006; Evangelou et al. 2007). Phosphorus in the form of phosphate is considered a potential inhibitor of Pb availability to plants due to the formation of stable lead phosphates in soils (Huang and Cunningham 1996; Cao et al. 2002). Although many lead phosphates have limited solubility, it is essential to clean-up Pb from contaminated soils because many of the soil-bound phases of Pb, despite being unavailable for plant uptake, are bioavailable to the human gastrointestinal system (Tang et al. 2004). Hence it is essential to mobilize Pb into the soluble pool in soils, making it available for plant uptake. Increased phytoextraction and/or phytostabilization of Pb will result in decreased total soil Pb levels, which correspond to reduced concentrations for human exposure and bioavailability.

We simulated a phosphorus-rich soil environment in a hydroponics study to investigate the ability of vetiver grass to remove soluble or dissolved Pb species, along with complexed or precipitated Pb forms. We hypothesized that application of phosphorus to the nutrient medium would reduce the plant-available Pb fraction and thereby Pb uptake capacity. Previous research has successfully shown that vetiver Pb uptake in a phosphorus-free hydroponic experiment was dramatic, as well as demonstrating induction of PCn and formation of Pb–PCn complexes as a plant biochemical response to elevated Pb concentrations (Andra et al. 2009). The present study aimed to (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 of phosphorus-containing nutrient medium.

Materials and methods

Hydroponic experiment

A hydroponic study was conducted in a temperature- and humidity-controlled environment in greenhouse facilities at the University of Texas at San Antonio. Vetiver grass was purchased from Horticultural Systems (Parrish, FL). Upon arrival, plants were thoroughly washed in tap water to remove adhering soil particles. Vetiver tillers were acclimatized for 30 days in plastic tanks (40 × 10 × 10 cm) containing 4 L nutrient medium that was continuously aerated with air circulation tubing. The nutrient solution used for this study was a modified Hoagland’s medium, prepared as described by Sahi et al. (2002). In brief, the composition was as follows, 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 dihydrogen 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 experimental conditions at which the hydroponic set-up was maintained were 16 h at 24 ± 2 °C/8 h at 20 ± 1 °C light/dark conditions. The measured light intensity at the shoot level was 250 μmol m−2 s−1, and the relative humidity was 60 ± 2%.

Following the initial 30-day vetiver acclimatization phase, fresh nutrient solutions were prepared with the same composition as mentioned above. Lead was spiked in the form of lead nitrate at concentrations of 0, 400, and 1,200 mg Pb L−1. We selected 400 and 1,200 mg Pb L−1 to reflect the worst-case scenario of US EPA and US HUD standards, where Pb is in the 100% soluble fraction. All the chemical solutions and nutrient media were prepared using deionized water. A completely randomized design with three replications was used for this experiment. Each replication consisted of three vetiver plants of similar biomass and height; a total of nine plants was exposed to each treatment. Vetiver tillers of similar biomass and height (45–50 cm high tillers and 28–32 g fresh weight per tank) were used for the Pb exposure experiment. The vetiver root system was sufficiently strong and long to keep all of the tops above the surface of the nutrient medium throughout the experimental period. Precaution was taken not to contaminate the above-ground parts of vetiver with Pb solution from spilling and splashing activities. The average pH of the Pb-spiked nutrient solutions remained constant (5.7 ± 0.4) during the experimental period. Vetiver was harvested after 7 days of Pb exposure. Plant material handling and digestion were carried out according to Carbonell et al. (1998). Vetiver tissues and nutrient solutions were analyzed for total Pb by atomic absorption spectroscopy (PerkinElmer AAnalyst 700, PerkinElmer Life and Analytical Sciences, Waltham, MA) in the flame mode using an air-acetylene gas mixture. Statistical analyses (descriptive statistics) were performed using JMP IN 5.1 (SAS, Cary, NC).

Speciation of Pb plays a major role in its availability for plant uptake. The ionic strengths and concentrations of each component in the nutrient solution were incorporated into chemical speciation modeling software prior to conducting the experiment. Visual MINTEQ version 2.53 (Gustafsson 2005) was used to identify Pb species and their activities to understand the distribution of soluble and precipitate Pb forms using published equilibrium constants (Kopittke et al. 2007).

Scanning electron microscopy

Scanning electron microscopy (SEM) was used to obtain Pb distribution patterns at an ultra-structural level, reveal the electron dense deposits of Pb and other elements, and identify the Pb compounds in the root and shoot tissues of vetiver grass following the protocol of Sahi et al. (2002). Frozen plant tissue samples were viewed uncoated in a JEOL 5400 LV SEM at 15 kV low vacuum mode using a backscattered electron detector. Elemental analysis of the vetiver tissues was carried out using the SEM-attached KEVEX Sigma energy dispersive X-ray spectrometer (SEM-EDS).

Plant tissue extraction and purification

Extraction and analytical procedures were adapted and modified from El-Zohri et al. (2005). Flash frozen plant samples were ground to fine powder with 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 tissue powder and the suspension was sonicated (0.5 s pulses, 400 W, 2 min) using a 40 kHz sonication bath (Branson 2510, Danbury, CT). Peptides were precipitated using 18 mmol L−1 HCl, incubated on ice for 10 min, centrifuged at 12,000 g for 10 min at 4°C (IEC Microlite RF refrigerated microcentrifuge, Thermo Electron Corporation, San Jose, CA), and filtered using 0.45 µm nylon membrane syringe filters (Fisherbrand, ThermoFisher Scientific, Waltham, MA). To enhance the recovery of low molecular weight peptides in the supernatant, we developed a two-step purification protocol by passing the filtrate in a series through microcon centrifugal filter devices [Chemical Abstract Number (CAS#) R7DN45102, Catalog# 42407, Millipore] followed by reversed-phase ZipTip pipette tips (CAS#, L7CN4514, Catalog# ZTC18MO96, Millipore). The composition of the solutions for sequential PCn purification and concentration consists of 100% acetonitrile for wetting the reversed-phase pipette tips, 0.5% trifluoroacetic acid (TFA) for equilibration and peptide binding onto the C 18 resin, 0.1% TFA to wash away contaminants and incompletely bound macromolecules, and 50% methanol in 0.1% formic acid (FA) for concentrated PCn elution. All the solutions were prepared in Milli-Q grade water (Millipore).

Analysis of phytochelatins

High-performance liquid chromatography coupled with electrospray mass spectrometry (LC-ESMS) was used to separate, identify, and quantify PCn in the vetiver tissues. Quantification of PCn was made possible using respective peptide standards ranging from PC1 to PC4 purchased from Sigma Genosys (Sigma-Aldrich, St. Louis, MO). Separation and identification were made possible using a Michrom Bioresources Magic HPLC system (Michrom Bioresources, Auburn, CA) and a Finnigan LCQ Duo ion trap mass spectrometer (Thermo Finnigan, San Jose, CA). Plant extracts (20 µL) were injected using an autosampler (Magic autosampler, Michrom Bioresources) on to a polymeric column (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 in acetonitrile (mobile phase B). Gradient mode was used to attain complete separation of the phytochelatins using the following three-step protocol: 0–20% B in the first 25 min, 20% B for next 10 min, followed by 20–100% B in the next 10 min. MS/MS analysis was performed to determine the fragment ions of each phytochelatin observed in the vetiver tissues. The normalized collision energies used for MS/MS were between 10 and 90%. The mass spectrometer conditions used 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 for this study was m/z 50–1,800.

Quantification of PCn was made possible using respective peptide standards ranging from PC1 to PC4 purchased from Sigma Genosys (Sigma-Aldrich). All standard solutions were prepared and diluted in 1:1 acetonitrile/ water solvent mixture. Separate stock solutions of 100 μg mL−1 of each phytochelatin were prepared and stored at −80°C. Aliquots of these solutions were mixed to obtain a 20 μg mL−1 mixed working 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, 10 μg mL−1 concentrations using the 20 μg mL−1 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.

Analysis of lead-phytochelatin complexes

Lead complexes with biomolecules such as peptides might interact with the stationary phase of the polymeric reversed phase column and result in instability, thus preventing them from appearance on the LC/MS scans. This problem was overcome by direct infusion of the filtered and concentrated vetiver tissue extracts into the mass spectrometer at 5 μL min−1 using a Hamilton (model #1750) (Hamilton, Reno, NV) syringe. Xcalibur software (Thermo Finnigan) was used to collect and analyze the data obtained from the full scan and MS/MS mode. Instrument settings were the same as used for the LC-ESMS experiments mentioned above. Lead-phytochelatin complexes were identified based on the simulations of the Pb isotope pattern obtained using Isotope Viewer in the Xcalibur software for all possible complexes, respectively.

Results

Lead uptake by vetiver grass

After 1 week of Pb exposure, in the presence of phosphorus, vetiver was able to accumulate up to between 2,500 and 3,100 mg Pb kg−1 dry weight in roots when treated with 400 or 1,200 mg Pb L−1 (Table 1). The added Pb was immobilized by phosphorus (Huang and Cunningham 1996; Cao et al. 2002) as a white precipitate of lead phosphate, making it less available for uptake by vetiver. The translocation of Pb from root to shoot tissues was limited. The shoot Pb concentrations of vetiver exposed to 400 and 1,200 mg Pb L−1 were 25 and 150 mg Pb kg−1, respectively. Immobilization of metals in roots is an exclusion mechanism exhibited by plants for metal toxicity tolerance (Baker et al. 1994). Pb translocation factor (TF) is a ratio of the Pb concentration in shoots to roots that indicates the metal translocation efficiency of any given plant species. Stoltz and Greger (2002) classified plant species with TF < 1 as ‘shoot metal excluders’ that are suitable for phytostabilization of metal-contaminated soils. In this study, the observed TF values were ≪ 1, indicating that vetiver accumulated Pb in root tissues. This observation is in accordance with the results of a study by Lai and Chen (2004) that considered vetiver as a Pb-phytostabilizer in contaminated soils. In contrast, vetiver was able to accumulate up to 3,350 and 19,800 mg Pb kg−1 dry weight of shoot and root tissue when exposed to 1,200 mg Pb L−1 in the absence of phosphorus (Table 1) (Andra et al. 2009).
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 different letters are significantly different using Tukey’s HSD at P < 0.001. Statistical analyses for Pb content in root and shoot compartments were 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

Pb precipitated out of solution upon addition to the nutrient medium, as evidenced by the formation of a white-colored substance and settling to the bottom of the tank. The clear solutions were analyzed for soluble Pb fraction before and after the 7-day phytoextraction period (Fig. 1). The dissolved Pb fraction was reduced by 93 and 86% in treatments following Pb addition at 400 and 1,200 mg L−1, respectively (Fig. 1). Soluble Pb levels were reduced significantly after the 7-day experimental period, while they remained constant in tanks with no vetiver grass. Operations with Visual MINTEQ for Pb speciation modeling in the nutrient media were based on the concentrations of solution anions (PO43−, NO3, SO42−, BO33−, and Cl), exchangeable cations (NH4+, Ca2+, Mg2+, and K+), metals (Pb, Fe, Zn, and Cu), and pH. Over 80% of the total Pb species consisted of insoluble chloropyromorphite [Pb5(PO4)3Cl]. The minor soluble Pb species observed were PbSO4, PbNO3+, PbCl+, and Pb3(BO3)2. In contrast, in a previous hydroponic study using phosphorus free nutrient medium (Andra et al. 2009), all the spiked Pb existed only as dissolved species with no precipitates.
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Fig. 1

Dissolved lead (Pb) concentrations in the hydroponic set-up measured at two time intervals. Values represent the mean (± SD) of three replicates. Means with different lower case letters are significantly different using Tukey’s HSD at P < 0.001

Scanning electron micrographs show differential distribution of Pb in root and shoot tissues of vetiver exposed to 1,200 mg Pb L−1. Lead accumulated mostly 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 different cross-sections at various locations of vetiver plants clearly demonstrates translocation to above-ground plant parts. Results from X-ray microanalysis (Fig. 2c) confirm the accumulation of phosphorus-containing Pb complexes in selected portions of vetiver root tissue (Fig. 2b). Further studies are needed to characterize specific Pb-phosphorus compounds using advanced spectroscopic techniques and to identify the biochemical/physiological mechanisms involved in their uptake by vetiver grass. Phosphorus signal was absent in the energy dispersive X-ray spectrum of a Pb deposit obtained from vetiver exposed to Pb in a phosphorus-free nutrient medium (Andra et al. 2009).
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Fig. 2

a, b Scanning electron micrographs of Pb deposits (red rectangles) in root tissue of vetiver grass exposed to 1,200 mg Pb L−1. a Cross-section of root showing Pb compounds in cortex and pith. b Magnified selected root tissue spot containing Pb and phosphorus. c Energy dispersive X-ray spectrum of the selected spot showing Pb and phosphorus signals

Induction of phytochelatins in vetiver grass

LC-ESMS was employed to analyze PCn. Separation was accomplished using a polymeric reversed phase column. The m/z of each PCn was compared with standards and with literature values (Vacchina et al. 1999). LC-ESMS obtained for the root (Fig. 3a) and shoot (Fig. 4a) tissues of vetiver grown in the absence of Pb show no PCn. Three possible PCn were observed 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 to 1,200 mg Pb L−1. Peaks of significant intensity with m/z values of 615.1, 921.8 and 1,228.4 were also observed in the ESMS scan of the vetiver root tissue (Fig. 3c) from the 1,200 mg Pb L−1 treatment. As confirmed using collision induced dissociation (CID), these peaks correspond to dimers, trimers, and tetramers of PC1 that resulted from gas-phase reactions and cluster formation during the electrospray process.
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Fig. 3

Liquid chromatography coupled with electrospray ionization mass spectrometry (LC-ESMS) spectra obtained for root tissue from vetiver exposed to Pb at a 0 mg L−1, b 400 mg L−1, and c 1,200 mg L−1

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Fig. 4

LC-ESMS spectra obtained for shoot tissue from vetiver exposed to Pb at a 0 mg L−1, b 400 mg L−1, and c 1,200 mg L−1

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Fig. 5

Selective magnification of the LC-ESMS spectra to represent the presence of certain phytochelatins (PCn), that are at trace levels 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

Unequivocal identification of each PCn was possible using CID at 70% normalized collision energy. The CID of the PCn standard mixture yielded a spectrum of fragment ions that retained charge either on the amino or carboxyl terminal of the respective peptide (Table 2). The fragment ions generated provide a strong reference database for confirming each PCn seen in the ESMS scans from vetiver root and shoot samples. Figure 6 depicts the CID of the root sample from vetiver exposed to 1,200 mg Pb L−1. Confirmation of the respective PCn was made by observing the largest daughter fragment: m/z 178.9 from PC1 with m/z 308.4 (Fig. 6a), m/z 410.9 from PC2 with m/z 540.4 (Fig. 6b), and m/z 643.0 from PC3 with m/z 772.1 (Fig. 6c). The loss of γGlu (m/z 129.0) from each PCn resulted in the largest fragment for each ion, respectively. Further confirmation was possible by observing many matching daughter ions from the root sample with those obtained from the PCn standards mixture. Similar confirmation was made for each PCn from the vetiver shoot tissue (data not shown). Induction of higher order PCn like PC4 was confirmed using CID, while the presence of PC5 was proposed based on theoretical and experimental isotopic patterns in vetiver root tissue obtained from 1,200 mg Pb L−1 treatment with no phosphorus (Andra et al. 2009).
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 daughter fragment 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%)

aPhytochelatin-1 [(γGlu-Cys)1-Gly]

bPhytochelatin-2 [(γGlu-Cys)2-Gly]

cPhytochelatin-3 [(γGlu-Cys)3-Gly]

dPhytochelatin-4 [(γGlu-Cys)4-Gly]

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Fig. 6a–c

Collision-induced dissociation (CID) of the protonated PCn obtained from LC-ESMS scans of root tissue of vetiver exposed to Pb at 1,200 mg L−1. a PC1, b PC2, c PC3

PC1 was also observed in vetiver tissues exposed to 0 mg Pb L −1. Because the signal intensities were weak during the ES-MS analyses, certain peaks of interest were magnified with Xcalibur software available with Finnigan LCQ Duo ion trap mass 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 from Pb-unspiked control treatments. However, they were present below instrument quantitation limits (< 0.3 μmol PCn kg−1 fresh wt) and hence represented as ‘trace’ quantities (Table 3). Similarly, PC4 was observed at a 250 magnification in vetiver root exposed to 1,200 mg Pb L−1 (Fig. 5c). Even at 1,000 factor magnification, neither PC3 nor PC4 was observed in vetiver shoot exposed to 1,200 mg Pb L−1 (Fig. 5d). In such cases, PCn quantities were denoted as ‘zero’ (Table 3).
Table 3

PCn concentrations in root and shoot tissues of vetiver grass exposed to different initial loads of Pb in nutrient solutions containing 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

To date, to the best of our knowledge, radio-active labeled and/or deuterated internal standards for PCn are unavailable. Matrix effects such as enhancement or suppression are possible, resulting in poor accuracy, low reproducibility, and erroneous quantification, especially in the case of peptides for which labeled standards are not available. Standard additions were performed for all PCn in root and shoot tissue extracts in order to determine matrix effects if any. Vetiver root or shoot extracts were spiked with individual PC standards in order to increase the background signal to 2–4 times that of the original signal for each peptide. Average concentrations for un-spiked plant samples determined from water-based calibration curves were statistically equivalent at the 95% confidence interval (CI) to those determined by standard additions (data not shown). Accuracy of each PC determination by the standard addition method indicated that there were no matrix effects and that PCn concentrations can be determined directly from calibration standards prepared in deionized water. Quantification of PCn in vetiver tissues was achieved by using the calibration curve obtained from the standards mixture. Vetiver grown in the absence of Pb shows no quantifiable amounts of PCn either in root or shoots. The sum of PCn concentrations in vetiver exposed to 1,200 mg Pb L−1 was 16.11 and 4.33 μmol kg−1 calculated on a fresh weight basis in root and shoot, respectively (Table 3).

Two prominent peaks observed at m/z 803.2 and 391.6 were present in all the treatments, irrespective of exposure to different Pb concentrations. CID on the m/z 803.2 complex yielded two distinct daughter fragments with m/z values of 413.1 (44%) and 391.6 (100%) (Fig. 7a). The major daughter fragment of m/z 803.2 is also the same as the second prominent and unidentified peak with m/z 391.6. CID on m/z 391.6 yielded daughter fragments that could possibly be cysteine (m/z 121.0, 75%) and aqueous γ-glutamic acid (m/z 149, 100%) (Fig. 7b). Based on the presence of cysteine, it could be suggested that the two unidentified peaks are thiols or phytochelatin-related peptides. Further work is required to confirm and gain detailed knowledge of these related peptides. Possible higher order phytochelatin-related peptides such as iso-PC5 (βAla) and des Gly-PC6 were observed in the root of vetiver grass grown in 1,200 mg Pb L−1 nutrient solution with no phosphorus (Andra et al. 2009).
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Fig. 7a,b

CID spectra for prominent and unidentified protonated molecules. am/z 803.2, bm/z 391.6. Cysteine and γ-glutamic acid, the principal amino acids of PCn, appear to be possible constituents of these complexes

Lead-phytochelatin complexes in vetiver grass

Tolerance in vetiver to Pb-induced stress was assumed to occur via the formation of Pb-PCn complexes. Direct infusion of plant tissue extracts resulted in the observation of certain Pb-PCn complexes. Figure 8a depicts the full ESMS scan obtained from injecting root samples obtained from vetiver exposed to 1,200 mg Pb L−1. PC1 (m/z 308.2) was the base peak in this sample. This was confirmed by CID yielding the largest fragment at m/z 178.7 that corresponds to a loss of γGlu from PC1. Two peaks with m/z 514.7 and 719.0 (Fig. 8a) show a typical Pb isotope pattern. Metal ions with natural isotope abundances enable 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 the assignment of m/z 514.7 for a PC1 complex with 1 Pb ion (Fig. 8b) and m/z 719.0 for a PC1 complex with 2 Pb ions (Fig. 8c). The expected isotope distribution patterns for Pb-PC1 and 2Pb-PC1 complex, 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, and PC6 were speculated in root tissues of vetiver exposed to Pb at 1,200 mg L−1 in the absence of phosphorus (Andra et al. 2009).
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Fig. 8

a Full-scan mass spectra obtained from direct infusion to see the possible Pb–PCn complexes in root tissue of vetiver exposed to Pb at 1,200 mg L−1. b Observed (left) and simulated (right) isotope signature for Pb–PC1 complex. c Observed (left) and simulated (right) isotope signature for 2Pb–PC1 complex

Discussion

Lead uptake by vetiver grass increased with increasing concentrations of Pb spiked in the nutrient medium. In the presence of P, there was an instantaneous visible Pb precipitation, resulting in white lead phosphate separating out of solution and settling at the bottom of the tanks. This confirms literature findings on Pb immobilization in soils by P, thereby reducing the soluble form of Pb, which is potentially plant available (Huang and Cunningham 1996; Cao et al. 2002). Vetiver accumulated significant (P < 0.001) low Pb levels of about 3,000 mg kg−1 dry tissue in the roots when exposed to 1,200 mg Pb L−1 in the presence of P compared to that of about 20,000 mg kg−1 root dry tissue 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 plant tissues, with increasing residual fractions of Pb resulting from P-containing amendments. Strong correlation (r > 0.95, n = 9) was observed between Pb concentration in the root and shoot tissues for any given treatment. In our study, the calculated translocation factor was ≪ 1, indicating that vetiver grass tends to store Pb in root tissues. It appears that vetiver is immobilizing Pb in roots as an exclusion strategy towards metal toxicity as indicated by Baker et al. (1994). The data supports the classification of vetiver grass as a potential Pb phytostabilization plant 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). Inactivation of free and toxic Pb ions is connected with synthesis of cysteine-rich thiol peptides known as phytochelatins (PCn) (Grill et al. 1989). Glutathione is the precursor for synthesis of these metal-binding peptides, which is catalyzed by phytochelatin synthase (PCS) in plants upon metal exposure (Vatamaniuk et al. 2000). We developed a two-step purification method for PCn concentration and characterization. We were able to show the presence of PCn ranging from PC1 to PC3 in vetiver roots upon exposure to 1,200 mg Pb L−1. PC3 was observed only in the root tissues owing to the high levels of Pb. The most abundant PCn in both root and shoot tissues of vetiver was PC1. This observation suggests that PC1 acts as a substrate for synthesizing higher order PCn with increasing Pb concentration in vetiver. In addition, the type and level of PCn shows a linear relationship with the Pb content in the vetiver tissue (Table 3). We propose that 1–2 Pb ions were possibly bound to PC1 in vetiver root at higher Pb levels. The current study provides a basic insight into the role of PCn synthesis towards Pb tolerance and detoxification in vetiver grass. Overexpression of genes that encodes glutathione synthetase (GS) (Zhu et al. 1999a) and γ-glutamylcysteine synthetase (γ-ECS) (Zhu et al. 1999b) enhanced cadmium uptake and tolerance in Indian mustard. Similar genetic manipulation of vetiver grass that has virtues like high biomass, fast growth, and adaptability to a wide range of soil and climatic conditions, could make it a more promising plant to clean up Pb from contaminated soils.

To conclude, the availability of Pb for plant uptake due to its interaction with P appears to be the biggest hurdle to achieving the clean up goal. Hence it is essential to use an environmentally safe chelating agent to mobilize Pb from bound forms to the soluble pool to enhance Pb uptake by vetiver. Greenhouse experiments have evaluated the performance of a chelant-aided phytoremediation model using vetiver grass to extract Pb from soils with varying physico-chemical properties (Andra 2008). We intend to validate the role of phytochelatins towards Pb tolerance in vetiver grown in a contaminated soil medium.

Acknowledgments

The research group from the University of Texas at San Antonio appreciates the funding support from the United States Department of Housing and Urban Development for this study. We thank Dr. Mohd Israr, Department of Biology, Western Kentucky University for help with SEM analysis.

Copyright information

© Springer Science+Business Media B.V. 2009