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First published online March 4, 2004; 10.1104/pp.103.033506 Plant Physiology 134:1113-1122 (2004) © 2004 American Society of Plant Biologists The Nature of Arsenic-Phytochelatin Complexes in Holcus lanatus and Pteris cretica1Department of Chemistry, University of Aberdeen, Meston Building, Meston Walk, Aberdeen AB24 3UE, United Kingdom (A.R., J.F.); and School of Biological Sciences, University of Aberdeen, Cruickshank Building, St. Machar Drive, Aberdeen, AB24 3UU, United Kingdom (A.A.M.).
We have developed a method to extract and separate phytochelatins (PCs)—metal(loid) complexes using parallel metal(loid)-specific (inductively coupled plasma-mass spectrometry) and organic-specific (electrospray ionization-mass spectrometry) detection systems—and use it here to ascertain the nature of arsenic (As)-PC complexes in plant extracts. This study is the first unequivocal report, to our knowledge, of PC complex coordination chemistry in plant extracts for any metal or metalloid ion. The As-tolerant grass Holcus lanatus and the As hyperaccumulator Pteris cretica were used as model plants. In an in vitro experiment using a mixture of reduced glutathione (GS), PC2, and PC3, As preferred the formation of the arsenite [As(III)]-PC3 complex over GS-As(III)-PC2, As(III)-(GS)3, As(III)-PC2, or As(III)-(PC2)2 (GS: glutathione bound to arsenic via sulphur of cysteine). In H. lanatus, the As(III)-PC3 complex was the dominant complex, although reduced glutathione, PC2, and PC3 were found in the extract. P. cretica only synthesizes PC2 and forms dominantly the GS-As(III)-PC2 complex. This is the first evidence, to our knowledge, for the existence of mixed glutathione-PC-metal(loid) complexes in plant tissues or in vitro. In both plant species, As is dominantly in non-bound inorganic forms, with 13% being present in PC complexes for H. lanatus and 1% in P. cretica.
Phytochelatins (PCs) are induced in a wide range of plant species by the oxy-anions arsenate [As(V)] and selenate and a range of cations such as Ag+, Cd2+, Cu2+, Hg2+, and Pb2+ (Grill et al., 1985  ). They are synthesized from reduced glutathione (GSH) by the transpeptidation of  -glutamyl-cysteinyl dipeptides through the action of the constitutive enzyme PC synthase (Schmöger et al., 2000; Vatamaniuk et al., 2000). PCs have the common structure (-Glu-Cys)nGly, where n = 2 - 11, although PC2 and PC3 are the most common (Cobbett, 2000; Cobbett and Goldsbrough, 2002). There is strong evidence that PCs complex metal(loid) ions, for which they have high affinity, in plant extracts (Mehra et al., 1996a, 1996b; Bae and Mehra, 1997; Leopold and Gunther, 1997; Leopold et al., 1998; Pickering et al., 1999; Satofuka et al., 2001; Cruz et al., 2002; Doreak and Krezel, 2003).
Little is known of the nature of metal(loid) ion-PC complexes in planta. Preliminary experiments with cadmium and copper using HPLC using chromatography with metal-specific detectors (coupled to inductively coupled plasma [ICP]-mass spectrometry [MS], UV detection, or off-line atomic absorption spectrometry; Maitani et al., 1996
More detailed studies that give an indication of the complexes actually formed have been conducted with purified PCs in vitro. Pickering et al. (1999
PCs have a high affinity for As(III), not As(V). As is mainly taken into terrestrial plants as As(V) or As(III) (Abedin et al., 2002
To date, no publication has reported intact metal-(loid)-PC speciation in plant extracts to identify the exact nature of As-PC species present in plant tissues. This has been primarily because of limitations in technology, namely the failure to use mild acid extraction procedures and the use of parallel metal-specific (ICP-MS) and organic-specific (ESI-MS) detection systems interfaced with a suitable chromatographic column and buffer condition. Presented here are the first unequivocal speciations, to our knowledge, of metal(loid)-PC complexes using HPLC-(ICP-MS)-(ESI-MS). As-PC complexes were extracted from the As-tolerant grass H. lanatus (Meharg and Macnair, 1992
Chromatography and Spectra of PCs and As-PC Complexes Produced in Vitro
Chromatographic and detector conditions have been explored and optimized in a study of, arsenicglutathion, As(III)-GS3 complex formed in vitro (Raab et al., 2004 Optimization of chromatography and detection of As-PC complexes by HPLC-(ICP-MS)-(ESI-MS) was performed using a mixed GSH, PC2, and PC3 solution in 1% (v/v) formic acid. Spectra were first obtained for the PCs not reacted with As(III). The mixture contained 36% glutathione, 50% PC2, and 14% PC3 (percentage on molar basis calculated from -SH equivalents). PC2, PC3, and GSH gave strong signals at their [M + H]+ masses of 540, 772, and 308 (spectra not shown). The oxidized forms PC2 (two S-S bridges) and oxidized glutathione (GSSG) were detectable at mass-to-charge ratio (m/z) 538, which is [M + 2H]2+, and m/z 613 [M + H]+ and 307 [GSSG + 2H]2+, respectively. None of the oxidized forms of PC3 were detectable (spectra not shown). PC2 oxidized at one S by GSH was also detectable at m/z 845 and 423 [M + 2H]2+. The mixed glutathione-PC solution was then reacted with a surplus of As(III) in the presence of 1% (v/v) formic acid. On adding As(III) to the PC solution, the following complex species could be formed theoretically: As(III)-GS3, As(III)-(PC2)2, GS-As(III)-PC2, As(III)-PC3, As(III)-PC2, and GS-As(III)-PC3 (GS: glutathione bound to arsenic via sulphur of cysteine). The molecular masses expected from these compounds are shown in Table I. The complexes actually detected were As(III)-(PC)2, GS-As(III)-PC2, As(III)-PC3, and As(III)-PC2, showing a preference for the formation of the double charged ion [M + 2H]2+ under the acidic conditions investigated. As(III)-(PC2)2 showed a main signal at m/z 576 [M + 2H]+2 and the expected [M + H]+ signal at m/z 1,151 as minor signal. Signals at m/z 919 [M + H]+ and dominant signal at m/z 460 [M + 2H]2+ were from GS-As(III)-PC2. As(III)-PC3, the complex formed in the greatest quantity, showed a strong signal at m/z 844 [M + H]+ and a minor one at m/z 422.5 [M + 2H]2+, respectively. The amount of As(III)-PC2 formed was very small and resulted in a signal at m/z 630. Corresponding to [PC2-As(OH)+H]+, no double charged form of this molecule was detectable. The spectra are shown in Figure 1.
Good separation for GSH, PC2, PC3, their oxidized forms and the As(III)-containing complexes was achieved as shown by the ESI-MS and ICP-MS data. Besides reduced GSH, PC2, and PC3, the sample contained some oxidized GSSG, oxidized (PC2)2, and a mixed complex of PC2 and one GSH (Fig. 2).
The ICP-MS data showed that besides unbound As(III), six other As-containing species were present in the solution. Combination of the results from ICP-MS (elemental information) and ESI-MS (molecular information) showed that As(III)-PC3 is the dominantly formed As(III)-PC complex, despite the higher availability of PC2 in the mixture. As(III)-(PC2)2 and GS-As(III)-PC2 complexes were formed at about the same rate and identified by their molecular ions and the bound As (Fig. 3). A small amount of As(III)-PC2 was detectable as well. It was not yet possible to identify two other As-containing species in this solution detected by ICP-MS because there were no corresponding detectable signals for the ESI-MS, due to the lower sensitivity of ESI-MS compared with ICP-MS. One of these unknowns might be GS-As(III)-PC3. As(III)-(GS)3 did not form in the solution as shown by the lack of the [M + H]+ peak, despite the fact that about 20% of the thiol groups in the solution are contributed by GSH. The separation of synthesized As(III)-(GS)3 using the same chromatographic conditions showed that the complex would be separable if present in solution and would show a signal in ESI-MS and ICP-MS at 16.8 min (Fig. 4).
An intact turf of As(V)-tolerant H. lanatus was obtained from As mine spoil and maintained in a greenhouse for 2 years before experiments commenced. The extract of shoots clipped from the turf contained about 2.5 µg As g sample fresh weight-1, when extracted in 1% (v/v) formic acid for 24 h at 1°C. This is approximately 70% of the total As present in the H. lanatus leaves (Table II). Ninety-six percent of the As present in the extracts eluted from the column. GSH, PC3, and PC2 were all present in the extract as determined by ESI-MS by comparison of the retention times and ESI-MS spectra with the purified standard mixture (Fig. 5). No attempts were made to quantify the species via the ESI-MS signals. The As trace of the ICP-MS showed that there are four organic-bound As species beside unbound inorganic As present in the H. lanatus extracts. The As(III)-PC3 complex was identified positively using ESI-MS at m/z 844 [M + H]+ and 422.5 [M + 2H]2+ and ICP-MS m/z 75 (As; Fig. 6). The GS-As(III)-PC2 complex was identified mainly by comparison of the retention times of the As species measured by ICP-MS because the ESI-MS peak was too small to provide a reliable mass spectrum. Whether As(III)-(PC2)2 is present in H. lanatus or not is uncertain because the retention time of the As peak and the retention times of ESI-MS signals of m/z 1,151 and 576 did not fit with the retention times of the standard.
For quantification of the As complexes, the ICP-MS data were used based on the ICP-MS response to sodium dimethylarsinic acid [DMA(V)] assuming that every As species gives the same sensitivity. The As concentrations of the different species were determined in two plants and showed that most of the extractable As is in the inorganic form (approximately 78% = 1.5 µg As g fresh weight plant-1), about 10% of the As is bound in the PC3 complex (approximately 230 ng As g-1), and about 3% is present in the GS-As(III)-PC2 complex (approximately 60 ng As g-1). The rest is present in two species not identified yet, one eluting shortly after inorganic As (approximately 3%) and the other later than As(III)-PC3 but before As(III)-(PC2)2 (approximately 5%; Table III). Previous experiments have shown that small amounts of PC4 are synthesized by H. lanatus (Hartley-Whitaker et al., 2001
Leaves including spores and stems were taken from P. cretica growing on soil containing 100 mg kg-1 As for over 1 year. Tissues were extracted as for H. lanatus. The total As concentration in the extracts was about 150 µg As g fresh weight-1 of the plant, with inorganic As contributing about 99% of the total As. The chromatographic recovery was 99% of the total extractable As (Table II). The main As-PC complex in P. cretica is the GS-As(III)-PC2 complex (approximately 36 ng As g-1) and two minor species As not identified yet (Table III). No signal at the retention time of As(III)-(PC2)2 was detectable with the ICP-MS. The presences or absences of the complexes were confirmed by the ESI-MS data of the same separation. The ESI-MS data of the same measurements showed that the P. cretica extract contained large amounts of oxidized and reduced GSH but that there was no As species bound in the form of the As(III)-(GS)3 complex (Figs. 7 and 8).
Repeated runs of P. cretica extracts were made (10 in total), all showing the same result. We have only presented two representative chromatographic runs.
Samples of both model plants were used to study the stability of the As-PC complexes in solution and in the intact plant during storage. The extracted complexes are stable for about 3 d in 1% (v/v) formic acid when stored at 1°C; after 4 weeks, the complexes were no longer detectable whether they were stored at 1°C or -20°C. Storage of the intact plant material at -20°C for 4 weeks before powdering and extraction also destroyed the As-PC complexes (data not shown). The stability of synthesized As(III)-GS3 complex during this kind of extraction procedure was found to be approximately 50%. As-PC complexes are more stable than the As(III)-GS3 complex in 1% (v/v) formic acid. No changes in signal intensity were observed in the partially purified standard after storing it for up to 2 months at 1°C.
We have developed a method that enables the detection and quantification of individual As-PC complexes in plant extracts using HPLC coupled to ESI-MS and ICP-MS in parallel, which enabled us to provide the first report, to our knowledge, of how a metal(loid) is complexed by PCs in plant extracts. The separation of purified As-PC complexes and plant extracts showed that As-PC complexes in different forms, until now more deduced than definitively detected (Sneller et al., 1999 , Pickering et al., 2000; Schmöger et al., 2000), do exist. Combining a separation method developed for the measurement of As(III)-(GS)3 (Raab et al., 2004) with the low detection limit of an ICP-MS and the molecular information delivered by an ESI-MS, the unequivocal identification of As(III)-(PC2)2, GS-As(III)-PC2, As(III)-PC3, and As(III)-PC2 in the standard preparation and in extracts of As-containing plants were obtained, revealing for the first time, to our knowledge, the stoichiometry of As-PC complexes and the presence of mixed metal(oid)-GS-PC2 complexes.
The few studies that had investigated previously the nature of As-PC complexes mainly used preparative scale size exclusion chromatography to separate the inorganic As from the "organic" As with the use of off-line separate detection of the PCs, and As (Schmöger et al., 2000
The current study using HPLC with parallel ICP-MS and ESI-MS detection, which enables the separation of a mixture of GSH, PC2 and PC3 complexes with As(III), showed that the complex formation is not only driven by the availability of the ligand but by kinetic and steric considerations. The formation of As(III)-PC3 was preferred over the formation of the As(III)-(PC2)2 complex by a factor of 2.5 in a mixture of purified PC2 and PC3, where PC2 was present at higher concentrations (3.6:1 PC2:PC3 in -SH equivalents). The mixed complex of GS-As(III)-PC2 was about one-half as likely to be formed as the As(III)-(PC2)2 complex. The amount of unbound PC3 in the presence of As(III) was also very small compared with PC2. Although we have observed As(III)-(GS)3 complexes in vitro (Raab et al., 2004
It is possible that As associated with PCs dissociates during the plant extraction procedure or that the reverse happens: on cellular disruption, As-PC complexes form. Any technique investigating the nature of plant complexes that is chromatographically based will have similar uncertainties. Because the complexes were stable for 24 h, and our observations in vitro fit well with what is observed in plant extracts, the extraction procedure shows what complexes are present in the plant, rather than their absolute concentrations. Extraction was conducted under acidic conditions (pH 2.0) for which it is known that As-PC complexes are more stable. At cytoplasmic and vacuolar pHs, speciation may differ. The only approach used to study As-PC complexes in intact plant tissues that gives molecular information is XAS. XAS can only predict the atomic environment in which the As exists, i.e. the atoms to which it is bound or coordinated. It cannot give information concerning the actual chemical formulae of the complexes. When used to investigate As speciation in Brassica juncea, XAS showed that virtually all the As was trihedrally coordinated with S, presumably as PC complexes (Pickering et al., 2000
P. cretica, an As hyperaccumulator (Meharg 2003
The only previous study attempting to speciate As-PC complexes was that of Schmöger et al. (2000
The results of this study show that the As-tolerant grass H. lanatus and the As-hyperaccumulating fern P. cretica (and related As-hyperaccumulating ferns such as P. vittata) have evolved different mechanisms for coping with high internal levels of As. H. lanatus excludes As from its roots via suppression of As(V) uptake from soils (Meharg and Macnair, 1992
The PC complexation in P. cretica contrasts strongly with H. lanatus, where in the fern only 1% of the As is PC complexed and only PC2 is synthesized. Although synthesis of PC2 is induced on As exposure in hyperaccumulating ferns (Zhao et al., 2003
The low quantities of PCs produced on As exposure in P. vittata (Zhao et al., 2003 In conclusion, we have developed an HPLC-(ICP-MS)-(ESI-MS) method that has provided, to our knowledge, the first unequivocal speciation in plant extracts of metal(loid)-PCs. We applied the method to the study of As in tolerant and hyperaccumulating plants. The same basic methodologies could be applied to other elements to understand their PC speciation. The technique has shown for the first time, to our knowledge, that glutathione forms mixed complexes with PCs in plant tissues and that As(III)-PC3 is the most stable complex out of the possible complexes that PC2, PC3, and GSH theoretically allow.
Plant Culture and Extraction
An intact turf of As(V)-tolerant Holcus lanatus was obtained from As mine spoil and maintained in a greenhouse for 2 years before experiments commenced. Pteris cretica Mayii, an As hyperaccumulator (Meharg, 2003 For both plants, fresh cut leaves were ground within 2 h after harvest under liquid nitrogen and then extracted with 1% (v/v) formic acid. The grounded leaf suspension was shaken and than stored for 12 h at 1°C, filtered (0.45-µm syringe filter, Sulpelco, Dorset, UK), and analyzed for the As species at once. In another experiment, the intact plants were stored at -20°C for 1 month before extraction, or the extracts were stored at -20°C or 1°C.
All reagents used were of analytical grade or better quality. Deionized water (18 m
The HP1100 HPLC system (Agilent Technologies, Stockport, Cheshire, UK) with cooled auto-sampler and a Peltier controlled column compartment was used throughout the experiments. The column compartment was set to 30°C, and the auto-sampler was cooled to 4°C. The HPLC parameters used throughout the experiments were 1 mL min-1 flow for the mobile phase and 100 µL of sample volume. A Spherisorb S5 ODS2 column (250 x 4.6 mm) reverse-phase column (Waters, Elstree, Hertfordshire, UK) was used throughout the study because it had been shown to be suitable for separation of As(III)-glutathione complexes (Raab et al., 2004 The HP1100 series LC/MSD instrument (Agilent Technologies, Stockport, Cheshire, UK) was used as a molecule-specific detector for post-column detection of the As-PC complexes by their molecular peaks, [M + H]+ or [M + 2H] 2+. The MSD was used in the positive ionization mode from m/z 120 to m/z 1,400 or in the single ion mode with the API-electrospray head. The settings chosen were: capillary voltage of 4,000 V, nebulizer pressure of 40 psi, drying gas flow of 12 L min-1 at 350°C, quadrupole temperature 100°C, and fragmenter voltage of 100 V for positive ionization mode. The mass calibration was controlled regularly and, when necessary, optimized using the calibration solution supplied by Agilent (m/z 112–2,233). An ICP-MS 7500 (Agilent Technologies) was used for element-specific detection of As. The instrument was equipped with a microconcentric nebulizer (flow rate < 100 µL min-1), a Peltier cooled spray chamber, and oxygen as additional plasma gas. The instrument was used in the soft extraction mode with 2% (v/v) oxygen. The instrument settings were checked daily for As sensitivity and optimized when necessary. The elements monitored were As (m/z 75), tellurium (m/z 130), S (m/z 34), sulfur oxide (m/z 48), copper (m/z 63 and 65), zinc (m/z 64), and Cd (m/z 112).
Henk Schat, Vrije Universiteit, Amsterdam generously supplied the PC mixture used in this study. Received September 17, 2003; returned for revision October 19, 2003; accepted December 4, 2003.
Article, publication date, and citation information can be found at http://www.plantphysiol.org/cgi/doi/10.1104/pp.103.033506.
1 This work was supported by the Biotechnology and Biological Science Research Council (grant no. I/REI 18479) and by the College of Engineering and Physical Sciences, Aberdeen, Aberdeenshire, UK. * Corresponding author; e-mail a.meharg{at}abdn.ac.uk; fax 44–0–1224–272703.
Abedin MJ, Feldmann J, Meharg AA (2002) Uptake kinetics of arsenic species in rice (Oryza sativa L.) plants. Plant Physiol 128: 1120-1128 Bae W, Mehra RK (1997) Metal-binding characteristics of a phytochelatin analog (Glu-Cys)(2)Gly. J Inorg Biochem 68: 201-210[CrossRef]
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Wang J, Zhao F-J, Meharg AA, Raab A, Feldmann J, McGrath SP (2002) Mechanisms of arsenic hyperaccumulation in Pteris vittata: uptake kinetics, interactions with phosphate, and arsenic speciation. Plant Physiol 130: 1552-1561 Zhao FJ, Wang JR, Barker JHA, Schat H, Bleeker PM, McGrath SP (2003) The role of phytochelatins in arsenic tolerance in the hyperaccumulator Pteris vittata. New Phytol 159: 403-410[CrossRef] This article has been cited by other articles:
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ABSTRACT
RESULTS
Elga Ltd., High Wycomb, Bucks, UK) was used throughout the experiments. As arsenic trioxide (As2O3) (BDH, Leics, UK), sodium arsenate (Na2HAsO3-7 H2O; BDH) and DMA(V) (Strem, Newburyport, MA) were used for the preparation of standard solutions and for the synthesis of the complexes. For the preparation of the mobile phase solutions, formic acid (100% [v/v] p.a., BDH) and methanol (p.a., Fisher, Loughborough, Leicestershire, UK) were used. A mixture of PCs, containing 36% (v/v) GSH, 49.7% (v/v) PC2, and 14.3% (v/v) PC3 (percentage calculated on a molar basis), used as a standard was a gift from Henk Schat (Vrije Universiteit, Amsterdam). The lyophilized PC mixture was dissolve in 1% (v/v) formic acid, and a portion was mixed with a surplus of As(III). Two plant samples were extracted and prepared for H. lanatus and 10 samples for P. cretica. Each extract was run in duplicate through the chromatographic procedure. 
