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. 2006 Oct;142(2):742–749. doi: 10.1104/pp.106.085068

Detection and Quantification of Unbound Phytochelatin 2 in Plant Extracts of Brassica napus Grown with Different Levels of Mercury1

Santiago Iglesia-Turiño 1, Anna Febrero 1,*, Olga Jauregui 1, Cristina Caldelas 1, Jose Luis Araus 1, Jordi Bort 1
PMCID: PMC1586043  PMID: 16920879

Abstract

The mercury (Hg) accumulation mechanism was studied in rape (Brassica napus) plants grown under a Hg concentration gradient (0 μm–1,000 μm). Hg mainly accumulated in roots. Therefore, the presence of phytochelatins (PCs) was studied in the roots of the plants. The high stability of the PC-Hg multicomplexes (mPC-nHg) seems to be the main reason for the lack of previous Hg-PC characterization studies. We propose a modification of the method to detect and quantify unbound PC of Hg in plant extracts via high-performance liquid chromatography coupled to electrospray tandem mass spectrometry and inductively coupled plasma mass spectrometry in parallel. We separated the PC from the Hg by adding the chelating agent sodium 2,3-dimercaptopropanesulfonate monohydrate. We only detected the presence of PC after the addition of the chelating agent. Some multicomplexes mPC-nHg could be formed but, due to their large sizes, could not be detected. In this study, only PC2 was observed in plant samples. Hg accumulation was correlated with PC2 concentration (r2 = 0.98).

Mercury (Hg) pollution is a global environmental problem. The toxicity of inorganic Hg forms (for example, HgCl2) is at least in part explained by the element's great affinity for biomolecules containing sulfhydryl (SH) groups (Goyer, 2001), and by a lower affinity for phosphate, carboxyl, amide, and amine groups (World Health Organization, 1991).

Plants are capable of extracting a variety of metal ions from their growth substrates, including Hg. At present, at least 45 plant families with more than 400 plant species are known to include metal-accumulating species (Reeves and Baker, 2000; Guerinot and Salt, 2001). Many herbaceous species, including members of the Brassicaceae, also accumulate moderate amounts of various metals in their shoots (Dushenkov et al., 1995; Kumar et al., 1995). Kumar et al. (1995) tested many fast-growing Brassicas, including rape plants (Brassica napus), for their ability to tolerate and accumulate metals. Many studies have shown that plant roots accumulate Hg when they are exposed to Hg-contaminated soils (Lenka et al., 1992; Coquery and Welbourn, 1994; Bersenyi et al., 1999; Kalac and Svoboda, 2000). Laboratory studies have shown that plant roots absorb Hg from solution and that roots accumulate a much greater amount of Hg than do shoots (Cavallini et al., 1999).

The role of chelating agents in removing Hg from contaminated organs in adult animals has been extensively reviewed (Keith et al., 1997; Baum, 1999). meso-Dimercaptosuccinic acid (DMSA) and sodium 2,3-dimercaptopropanesulfonate monohydrate (DMPS) are water soluble. The effects of the water-soluble chelating agents DMPS and DMSA on the renal disposition of inorganic Hg have been studied in animals.

Treatment with DMPS has been found more effective than treatment with DMSA in reducing the renal burden of Hg in two groups of rats (Zalups, 1993). Common treatments to remove Hg2+ from contaminated sources are based on chelating agents (Keith et al., 1997; Baum, 1999; Nogueira et al., 2003). Keith et al. (1997) reported that the relative efficacies for the three agents were DMPS > DMSA > EDTA.

Phytochelatins (PCs) are a family of peptides with the general structure (γ-Glu-Cys)n-Gly (n = 2–11). PCs are synthesized enzymatically from glutathione by the constitutive enzyme PC synthase, which requires posttranslational activation (Grill et al., 1985, 1987, 1989; Zenk, 1996; Ha et al., 1999; Cobbett, 2000; Schat et al., 2002). Heavy metals do not bind directly to the enzyme to activate PC biosynthesis, but instead act as substrate ligands for a bisubstrate-substituted enzyme transpeptidation reaction in which free glutathione and its corresponding heavy metal thiolate are cosubstrates (Vatamaniuk et al., 2000, 2001, 2004).

PCs are rapidly induced in a wide range of plant species by heavy metal ions, such as As5+, Cd2+, Cu2+, Ag+, Hg2+, and Pb2+ (Zenk, 1996; Rauser, 1999; Cobbett, 2000; Goldsbrough, 2000; Schmoger et al., 2000; Hall, 2002; Raab et al., 2004).

Since the immobilized metals are less toxic than the free ions, PCs are considered part of the detoxifying mechanisms of higher plants (Grill et al., 1985; Zenk, 1996), algae (Gekeler et al., 1988), and some fungi (Kondo et al., 1985; Kneer and Zenk, 1992). In addition, it has been shown that a mutant having a defect in PC synthesis show enhanced sensitivity to cadmium (Cd) and arsenic (III) (Howden et al., 1995; Ha et al., 1999; Cobbett and Goldsbrough, 2002; Zhao et al., 2003).

There is strong evidence that PCs complex metal(loid) ions, for which they have a high affinity (Mehra et al., 1996a, 1996b; Leopold and Gunther, 1997; Cruz et al., 2001; Doreak and Krezel, 2003; Raab et al., 2004). PCs can form complexes with lead, silver, and Hg in vitro (Mehra et al., 1996b; Rauser, 1999). However, as far as we know, the only PC complexes identified in vivo are those with Cd, silver, copper, and arsenic ions. In fact, PCs were not detected in either sensitive or tolerant willow (Salix spp.) clones in recent reports for Hg (Wang and Greger, 2003) and other metals (Landberg and Greger, 2004).

Several analytical methods, such as chromatographic separation (gel filtration or HPLC) coupled with UV detection, flame atomic absorption spectrometry, radioactive labeling, and inductively coupled plasma-mass spectrometry (ICP-MS) or electrospray-MS (ESI-MS), have been used to analyze PCs and PC-metal complexes (Grill et al., 1985; Maitani et al., 1996; Vacchina et al., 1999, 2000; Fan et al., 2004). However, the above techniques may generate ambiguities concerning the identification of PCs. These drawbacks can be overcome by using ESI-tandem MS (ESI-MS/MS), which is a powerful technique for the analysis of proteins and peptides with a sensitivity of low femtomole 10−15 mol to high attomole 10−18 mol (Allen and Hutchens, 1992).

ESI-MS/MS has become a well-established tool for peptide identification and sequencing in biological samples (Hofstadler et al. 1996; Yates et al., 1996). It has been accepted by the U.S. Food and Drug Administration (Lee and Kerns, 1999; Stuart et al., 2003), by the U.S. Environmental Protection Agency, and by the European Union for characterization of proteins, peptides, drugs, and other substances (Richardson, 2002).

To date, no publication has reported the complex nature of PC-Hg in plant tissues. This is primarily because of limitations in technology, namely, the failure to use mild acid extraction procedures in parallel with metal-specific (ICP-MS) and organic-specific (ESI-MS) detection systems interfaced with a suitable chromatographic column and buffer condition. The method reported in this study is simple, reliable, and does not require a liquid-liquid or a solid-phase step. Furthermore, it does not require any derivatization step and only requires a few microliters of sample. Once properly described and tested, the method allows for reliable detection and characterization of PCs produced in rape plants in response to Hg stress.

RESULTS

Hg Content

The roots and shoots of rape plants were analyzed separately for Hg accumulation. Metal accumulation in rape plants was observed mainly in the roots (Table I). The accumulation of Hg in the roots was linearly related to Hg concentration in the growth medium (0–1,000 μL HgCl2). This clearly shows that the Hg accumulation in plant tissues was related to the Hg added to the culture medium. The total Hg concentrations detected in the tissues were significantly different for several Hg concentration levels in medium, the groups for roots being 0 to 125 μm HgCl2, 250 to 750 μm HgCl2, and 0 to 125 μm HgCl2, while for leaves the values ranged from 250 to 1,000 μm HgCl2.

Table I.

Total Hg content (μg Hg g dry weight plant−1) in root and shoot plant tissues extracts analyzed separately from rape plants treated with different HgCl2 concentrations

Total PC2 content (μm PC2 g fresh weight plant−1) in roots. Values are means of three replicates. Means followed by different letters are significantly different by Duncan test with P < 0.05. Relationships of μm PC2 g−1 fresh weight with μm HgCl2 g−1 dry weight in rape showed linear correlation r2 = 0.98, P < 0.05.

Treatment Shoots
Root
Root
Hg Content PC2 Content
μm HgCl2 μg Hg g dry weight plant−1 μg Hg g dry weight plant−1 μm PC2 g fresh weight plant−1
0 13.37a ± 3.15 1.63a ± 0.25 0.132a ± 0.040
50 26.56a ± 8.64 59.47a ± 16.76 1.417ab ± 0.644
125 37.60a ± 6.66 242.72a ± 26.60 2.246ab ± 0.517
250 129.34b ± 37.74 339.09ab ± 31.12 3.245ab ± 0.412
375 129.34b ± 37.74 399.63ab ± 56.64 4.461bc ± 0.589
750 114.12b ± 14.26 1,012.43b ± 338.95 7.830cd ± 0.960
1,000 115.07b ± 12.73 2,351.50c ± 407.66 14.705e ± 3.126
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In Vitro Chromatographic Analyses of Synthetic PCs and Hg Standard

Synthetic PC2, PC3, and PC4 chromatograms gave several retention times (spectra not shown). Hg was added to obtain PC-Hg retention time. We did not observe any signal chromatogram either for PC2, PC3, or PC4. The addition of the chelating agent DMPS revealed unbound PC in the chromatogram. In addition, new peaks also appeared in the chromatogram. We postulate that these peaks correspond to different mPC-nHg multicomplexes. In Figure 1A, we only show the chromatogram corresponding to PC2 because this is the only PC detected under in vivo conditions. The addition of the chelating agent DMPS (0.1 μm) revealed the signal for unbound PC2 (mass-to-charge ratio [m/z] 540) and PC2-Hg (m/z 739) were detected (Fig. 1B). The addition of chelating agent DMPS (1 μm) revealed the signals for both unbound PC2 (m/z 540) and 2PC2 + 3Hg (m/z 1,653; Fig. 1C).

Figure 1.

Figure 1.

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A, HPLC mass spectra chromatogram obtained for synthetic PC2 NH2-(γ-Glu-Cys)2-Gly-COOH (theoretical mean isotopic mass 540.1 D). A peak was detected at 540.9 m/z. Adding 10 μL of 0.1 m Hg to PC2, a peak corresponding to synthetic PC2-Hg was not detected (data not shown). B, The addition of 10 μL of chelating agent DMPS (10 mm) to the same sample used in A allowed detection of unbound PC and mPC2-nHg. HPLC chromatogram presented various peaks (540 PC2, 739 PC2 + Hg, 960 PC2 + 2Hg, 1,200 2PC2 + Hg, and 1,700 2PC2 + 3Hg). C, The addition of 10 μL of chelating agent DMPS (100 mm) to the same sample used in A allowed detection of unbound PC and 2PC2 + 3Hg.

Characterization of PCs by ESI-MS/MS

Characterization of PC standards in the HPLC-ESI-MS/MS system was done by injecting separately the individual PC2, PC3, and PC4 standards. The mass spectra in full-scan mode showed the protonated molecule ion [M + H]+, which allows the identification of each PC according to its m/z ratio. For a more complete identification of PCs, injection in the product ion scan mode was also done. The peptides were broken primarily at the amine bonds to produce a ladder of sequence ions. The charge can be retained on the amino terminus (type β-ion) or on the carboxy terminus (type γ-ion). Thus, a complete series made of ions from both types allows the determination of the amino acid sequence by subtraction of the masses of adjacent sequence ions.

Figure 2 shows the mass spectra obtained after the injection of the synthetic PC2 standard (Fig. 2A) and a sample (Fig. 2B) in product ion scan mode of m/z 540.1 produced by the fragmentation of the molecular peaks observed for each of the compounds investigated. Those were 130, 179, 233, 308, 336, 411, and 540 m/z. The image shows that there is a match between the ions obtained for the synthetic and for the natural sample. Of all the ions, two are characteristic of a PC2: the protonated molecule ion (540) and the γ-type fragment (411) with the greatest mass.

Figure 2.

Figure 2.

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Electrospray tandem mass spectra obtained after injection of PC2 standard or sample. A, Product ion spectra of PC2 standard, collision energy = 35 eV (CE 35 eV). B, Product ion spectra of PC2 sample extracted from root rape plants treated with 1,000 μm Hg (CE 35 eV). Product ion spectra provided excellent confirmation of unique species.

Performance Characteristics

Linearity

Standard solutions of PC2 were prepared over a range of 62.5 to 1,000 nm and injected in the HPLC-MS/MS system in multiple reaction monitoring (MRM) mode as described above. The peak areas were plotted against the corresponding concentration to obtain the calibration curve. The method was linear over the entire working range. The regression calibration curve (1/x2 weighting) was y = 2.21×104x − 23 (r2 = 0.9944). The residual analyses for this range of concentration were 100.0% ± 3.27 (mean ± sd).

Sensitivity

The limit of detection (LOD = 0.3 nm) was estimated as the concentration of PC2 that generated a peak with an area at least 3 times higher than the baseline noise.

Determination of PCs in Plant Extracts

The method developed was further applied to the characterization and determination of PCs in extracts of rape, a plant species reported to biosynthesize PCs when exposed to different levels (0 μm–1,000 μm) of Hg2+. The plant extracts were analyzed directly by HPLC-ESI-MS/MS, but only PC2 was detected. Two different chelating agents were then used to verify whether the results were similar for both chelators. The amount of unbound PC2 depends on the chelating agent used (Fig. 3). Under the Hg conditions of this study, DMPS was a better chelator than DMSA.

Figure 3.

Figure 3.

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Comparison of the results of the quantitative determination of unbound PC2 using different chelating agents (DMSA and DMPS) to release the Hg from the PC2. Rape plants are known to biosynthesize PCs when exposed to different levels of Hg2+ (0, 50, 125, 250, 375, 750, and 1,000 μm).

Once the PC2 data obtained with DMSA were discarded, it was observed that the PC2 concentration increased linearly and was related to the increased Hg concentration added to the nutrient solution (Fig. 3). Finally, the PC2 synthesis was correlated (r2 = 0.98) with accumulation of Hg (Table I).

DISCUSSION

Hg Accumulation

Hg accumulation in plants has been studied in several species. Pea (Pisum sativum) and spearmint (Mentha spicata) absorb Hg from solution, and roots accumulate much greater amounts of Hg than shoots (Beauford et al., 1977). Similar results have been found in Norway spruce (Picea abies; Godbold and Hütterman, 1988), willow (Wang and Greger, 2003), and aquatic plants (Coquery and Welbourn, 1994).

In our study, roots accumulated higher concentrations of Hg (Table I) than leaves, so roots were chosen to analyze PC accumulation/synthesis. The higher accumulation of metals in the roots than in the shoots might be possible because of a greater tolerance to toxic metals in the roots than in the shoots (Göthberg et al., 2004). In the results reported here, leaves accumulated more Hg than roots in plants cultured with no Hg. Uptake of Hg from the air via the stomata of untreated plants is a possible reason for the relatively high Hg concentrations in these plants in comparison to plants growing in a medium without Hg (Suckcharoen, 1978; Göthberg et al., 2002). Uptake of gaseous Hg via the stomata has been shown in laboratory studies (Browne and Fang, 1978; Du and Fang, 1983; Cavallini et al., 1999). Although the accumulation of Hg in aerial plant parts increased in direct proportion to Hg concentration in the culture medium, it was quite stable for higher concentrations. Rape extraction efficiency was low but constant for all treatments except for the highest concentration.

PC Detection

PCs are involved in the cellular detoxification mechanism due to their ability to form stable metal-PC complexes (Scarano and Morelli, 2002). For metals other than Cd, few studies demonstrate the formation of PC-metal complexes in vivo (Cobbett, 2000; Cobbett and Goldsbrough, 2002). Previous studies in willow had pointed out that there was no evidence that PCs were responsible for Hg tolerance because no PCs were detected in either sensitive or tolerant willow clones, as has also been found in the case of other heavy metals (Landberg and Greger, 2004). The same authors concluded that PCs were not involved in Hg extraction and storing from media.

To detect PCs, these had to be separated from Hg by adding the chelating agent DMPS. In both the in vivo and the in vitro experiments reported in this study, PCs were only detected after the addition of chelating agents. Hg bound to PC has a high stability through SH groups (−SH). The affinity constant for Hg binding to thiolate anions is approximately 1015 to 1020. In contrast, the affinity constants for Hg binding to oxygen- or nitrogen-containing ligands (e.g. carbonyl or amino groups) are about 10 orders of magnitude lower (Zalups, 2000). Each of the two SH groups of each PC2 can bind a Hg. Furthermore, every Hg can potentially be bound to an SH group from one PC2 molecule and to an SH group from another PC2 molecule, and this may be repeated a number of times, generating an mPC-nHg multicomplex, which should be heavy enough to be out of the resolution limits of the mass spectrometer chromatogram. This tight binding might explain why the mPC-nHg complex had not been detected in previous studies. A multicomplex mPC-nHg could have been formed but remained undetected due to its large size.

Unbound PC2 was obtained by binding Hg to a chelating agent. In agreement with previous studies, chelating agents DMPS and DMSA performed differently, DMPS having a stronger affinity for Hg (Keith et al., 1997). Only by using DMPS was Hg unbound from PC2, while DMSA was not able to release unbound PC2 (Fig. 3).

PC2, PC3, and PC4 were analyzed but only PC2 was detected (Fig. 2). Thus, only PC2 is involved in Hg phytochelation in rape. When the characterization was conducted on the 1,000 μm sample ESI-MS/MS, a similar pattern was observed (Fig. 2).

The roots of rape accumulate from 2- to 20-fold more Hg than do leaves. As only unbound PC2 appears in roots and as unbound PC2 is strongly linearly correlated with Hg concentration in growth media until 1,000 μm Cl2Hg, we conclude that PC2 is crucial in extracting Hg from rape. Furthermore, using DMPS as chelating agent, coupled with the ESI-MS/MS technique, has proven a useful and sensitive tool to determine PC content in Hg-treated plants by breaking Hg interactions with SH groups.

MATERIALS AND METHODS

Plant Material

Rape (Brassica napus) plants were grown under semihydroponic conditions, using half-strength Hoagland nutrient solution, with sand as a substrate. The sand was not in direct contact with the nutrient solution, but some cords allowed the movement of the solution up into the sand, so the advantages of both soil culture and hydroponic culture were present. The plants were grown under glasshouse conditions at the University of Barcelona. Three pots per treatment with three replicates (plants) per pot were used. Seeds germinated in sand. Seedlings were grown for 6 weeks. The Hg concentrations applied were 0, 50, 125, 250, 375, 750, and 1,000 μm HgCl2.

Analysis of Hg Content

Fresh weight was obtained from shoots and roots separately. Immediately after harvest, samples were frozen in liquid nitrogen and stored at −40°C. Later on, the aerial biomass of three plants per pot was ground together in liquid nitrogen. The samples were analyzed in duplicate with ICP-MS. After digestion, approximately 200 mg of fresh weight was digested in Teflon reactors using 3 mL of HNO3 and 3 mL of H2O2. Digestion was conducted overnight at 90°C. The next day, 20 mL of milliQ water was added to the samples, analyzing after adding a concentration of 2% rhodium. If the sample was too concentrated, it was diluted in 1% HNO3. The results are expressed in dry weight.

PC Analysis

Preparation of Root Tissue Extracts

The roots (one plant per pot) were harvested separately and immediately weighed, cut into small pieces, placed in plastic bags, and transferred to the laboratory in liquid nitrogen. In the laboratory, the contents of each bag were ground using a blender in liquid nitrogen. PCs were extracted from 0.5 g of fresh weight by adding 0.2 mL of chelating agent DMPS 1 m or chelating agent DMSA 1 m. The homogenates were vortexed for 0.5 min, adding 0.2 mL of 96% acetic acid. The homogenates were vortexed again for 0.5 min, and, after adding 0.6 mL of 60% perchloric acid, the homogenates were transferred to centrifuge tubes and centrifuged at 9,000g for 15 min (at 1°C). The supernatants were transferred to appropriately labeled Nalgene cryovials and stored at −80°C until use (Abdelrahim et al., 2003). Twenty-five microliters of the extract was injected in the HPLC-MS/MS system.

Synthesis of PC2, PC3, and PC4

The synthetic PC2 NH2-(γ-Glu-Cys)2-Gly-COOH (theoretical mean isotopic mass 540.1 D), PC3 NH2-(γ-Glu-Cys)3-Gly-COOH (theoretical mean isotopic mass 772.4 D), and PC4 NH2-(γ-Glu-Cys)4-Gly-COOH (theoretical mean isotopic mass 1,004.00 D) were synthesized and purified at the microchemical facility at the Servicio de Sintesis de Peptidos (Peptide Synthesis Service), Universidad de Barcelona, using tBOC chemistry as described (Lloyd-Williams et al., 1997).

Sample Preparation for HPLC

The first approach to the characterization of PCs by LC/MS was done in a single quadrupole mass spectrometer (Platform). Although all the studied PCs emitted a signal in full-scan mode, not enough sensitivity was attained for sample even in single ion monitoring (SIM) mode. Therefore, the study was finally performed using the triple quadrupole mass spectrometer API 3000 (Applied Biosystems PE Sciex). This instrument allows a more accurate characterization through product ion scan experiments and a more sensitive quantification through MRM experiments.

Different concentrations (2, 2.5, 3, and 3.5 μm) of synthetic PC2, PC3, and PC4 were used during preliminary assays for further detection of PCs in experimental samples. A chromatogram was obtained for each PC and for the mixture of synthetic PCs (PC2 + PC3 + PC4) as described by Abdelrahim et al. (2003). Hg (1 μm final concentration of Hg in this reaction) was added to obtain PC-Hg retention time. We did not observe any chromatogram signal for PC2, PC3, or PC4 after addition of Hg. Only the addition of the chelating agent DMPS (0.1 μm and 1 μm final concentrations of DMPS) allowed detection of unbound PC and mPC-nHg multicomplexes. An HPLC mass spectra chromatogram was obtained for each synthetic PC2, PC3, and PC4, and their mixture (PC2 + PC3 + PC4). Another chromatogram for the same synthetic PCs but adding Hg and DMPS was obtained. Only PC2 and (PC2 + Hg + DMPS) chromatogram spectra are shown (Fig. 1). Most PC2 is still complexed to Hg in 0.1 and 1 μm DMPS. DMPS was added to the sample in a concentration 2,000 times higher than the synthetic PC to saturate the entire PC. On the day of analysis, defrosted sample extracts were filtered, and 300 μL of each sample was transferred to vials for HPLC analysis and filtered through a 4 mm, 0.45 μm PTFE filter (Waters). A fraction of the purified PC2 was weighed and a standard solution of 0.24 mmol/L in 0.1% acetic acid was prepared. Acetic acid (0.1% v/v; 99%) was used as the medium to construct the standard curve.

HPLC Conditions

MS was performed on a VG Platform II (Fisons Instruments, VG Biotech,) quadrupole mass spectrometer equipped with an ESI source and using nitrogen as nebulizing gas (150 L h−1). Drying nitrogen was heated at 100°C and introduced into the capillary region at a flow-rate of 400 L h−1. The capillary voltage was held at +3.50 kV and the cone voltage at +50 V. For data acquisition in full-scan mode, the mass spectrometer was operated over a mass range of m/z 300 to 2,000 in the profile mode, at a cycle time of 2.00 s and at an interscan time of 0.20 s. Quantization was performed using the time-scheduled SIM mode at the masses given in Figure 1, with a dwell time of 100 ms.

A Luna C18 Phenomenex column (250 × 4.00 mm, i.d. 5 μm) equipped with a Securityguard C18 Phenomenex (4 × 3 mm i.d.) was used. Gradient elution was done with water with 0.1% acetic acid (solvent A) and acetonitrile with 20% solvent A (solvent B). Separation was achieved with a linear gradient of 2% solvent A to 100% solvent B at a flow rate of 1.0 mL/min, and a column temperature of 30°C split (1/3) of the flow rate was done.

LC-MS/MS System

An API 3000 triple quadrupole mass spectrometer (PE Sciex) was used to obtain the MS and MS/MS data. All the analyses were performed using the Turbo Ionspray source in positive ion mode. The following settings were used: capillary voltage +4,500 V, declustering potential +60V, nebulizer gas (N2) 10 (arbitrary units), curtain gas (N2) 12 (arbitrary units), collision gas (N2) 6 (arbitrary units), focusing potential +200 V, and entrance potential 10 V. Drying gas (N2) was heated to 300°C and introduced at a flow rate of 8,000 mL min−1. Full-scan data acquisition was performed scanning from m/z 300 to 2,000 amu in profile mode and using a cycle time of 2 s with a step size of 0.1 amu and a pause between each scan of 2 ms. Product ion scan experiments were done to confirm the identity of some of the compounds. In product ion scan experiments, MS/MS product ions were produced by collision-activated dissociation of selected precursor ions in the collision cell of the triple quadrupole mass spectrometer (Q1) and analyzed using the second analyzer of the instrument (Q3).

In this study, product ion scan experiments were performed by scanning Q between 100 and [M + H + 20]+ amu. The injection of the PC's standards in product ion scan mode of the [M + H]+ ion allowed us to construct an MRM method based on the characteristic transition for each compound.

Finally, quantification of plant extracts was done in MRM mode, monitoring the following characteristic transitions: 540.0/308.0 for PC2(CE 35), 772.0/643.0 for PC3(CE 45), and 1,004.0/697.0 for PC4(CE 55). The dwell time for each transition was fixed at 500 ms.

Statistical Methods

All statistical analyses were performed using the SPSS for Windows program (version 12.0). Figures were drawn by plotting data with Sigmaplot 6.1.

Acknowledgments

We thank Ricardo Simonneau and Josep Matas (Servei Camps Experimentals, Facultat de Biologia, Universitat de Barcelona) for helpful suggestions and support. Thanks to William Bain and Robin Rycroft for English corrections and Fagua Alvarez for comments. We also acknowledge the useful comments raised by the two anonymous referees.

1

This work was supported by the INCO European Project Mercury (ICA4–CT–2002–10055).

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Anna Febrero (annafebrero@ub.edu).

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