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First published online December 12, 2008; 10.1104/pp.108.127472 Plant Physiology 149:938-948 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
Phytochelatin Synthesis Is Essential for the Detoxification of Excess Zinc and Contributes Significantly to the Accumulation of Zinc1,[W],[OA]Leibniz Institute of Plant Biochemistry, Department of Stress and Developmental Biology, 06120 Halle/Saale, Germany (P.T., D.P., C.B., S.C.); and University of Bayreuth, Department of Plant Physiology, 95440 Bayreuth, Germany (D.P., A.T., S.C.)
The synthesis of phytochelatins (PCs) is essential for the detoxification of nonessential metals and metalloids such as cadmium and arsenic in plants and a variety of other organisms. To our knowledge, no direct evidence for a role of PCs in essential metal homeostasis has been reported to date. Prompted by observations in Schizosaccharomyces pombe and Saccharomyces cerevisiae indicating a contribution of PC synthase expression to Zn2+ sequestration, we investigated a known PC-deficient Arabidopsis (Arabidopsis thaliana) mutant, cad1-3, and a newly isolated second strong allele, cad1-6, with respect to zinc (Zn) homeostasis. We found that in a medium with low cation content PC-deficient mutants show pronounced Zn2+ hypersensitivity. This phenotype is of comparable strength to the well-documented Cd2+ hypersensitivity of cad1 mutants. PC deficiency also results in significant reduction in root Zn accumulation. To be able to sensitively measure PC accumulation, we established an assay using capillary liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry of derivatized extracts. Plants grown under control conditions consistently showed PC2 accumulation. Analysis of plants treated with same-effect concentrations revealed that Zn2+-elicited PC2 accumulation in roots reached about 30% of the level of Cd2+-elicited PC2 accumulation. We conclude from these data that PC formation is essential for Zn2+ tolerance and provides driving force for the accumulation of Zn. This function might also help explain the mysterious occurrence of PC synthase genes throughout the plant kingdom and in a wide range of other organisms.
Both essential and nonessential metal ions can be toxic when present in excess. Zinc (Zn) ions, for instance, are used in biological systems as catalytic or structural components in a myriad of proteins (Frausto da Silva and Williams, 2001  ). In humans, about 10% of genes encode Zn-dependent proteins (Andreini et al., 2006) and it is reasonable to postulate similar numbers for plants. When the Zn-buffering capacity of a cell is exceeded, however, aberrant binding of Zn ions to thiols or other functional groups can occur, which disrupts the function of proteins. Also, Zn ions can displace other essential metal ions from their binding sites (Krämer and Clemens, 2005). Toxicity thresholds for Zn were found to range between 100 and 300 µg g–1 dry weight depending on plant species and physiological state (Marschner, 1995). Ions of the nonessential metal cadmium (Cd) have a high affinity for various functional groups in biological molecules, in particular thiols. When taken up into a cell they can inactivate proteins by uncontrolled binding or cause oxidative stress by depleting glutathione pools (Clemens, 2006a).
Because of the potential toxicity of metal ions, all living systems possess mechanisms to tightly regulate the distribution of metal ions and to minimize damage under conditions of excess metal supply (Eide, 2006
The synthesis of phytochelatins (PCs), glutathione-derived metal-binding peptides, represents a major detoxification mechanism for Cd and arsenic (As) ions in various species. More recently, PCs have also been implicated in long-distance transport of Cd in the phloem (Mendoza-Cozatl et al., 2008
S. pombe pcs  mutant cells unable to synthesize PCs are hypersensitive to Cd2+. Their Zn2+ tolerance is not significantly reduced even though Zn2+ exposure elicits PC synthesis in S. pombe wild-type cells (Clemens et al., 1999; Ha et al., 1999; data not shown). This is different when the pcs gene is knocked out in zhf mutant cells. Loss of Zhf (Zn homeostasis factor), an endoplasmic reticulum-localized transporter of the cation diffusion facilitator family, results in strong Zn2+ hypersensitivity (Clemens et al., 2002). Excess Zn can no longer be removed from the cytosol in zhf cells (Boch et al., 2008). When we compared the growth of the zhfpcs strain to zhf cells, we found a significant decrease in Zn2+ tolerance correlated with the loss of functional PCS (Fig. 1A
). In the presence of 100 µM Zn2+, zhf growth was reduced by about 50%, whereas zhfpcs cells showed a reduction in growth of about 80%. Furthermore, when exposed to a Zn2+ shock that completely inhibited growth (400 µM Zn2+ for 4 h), the survival rate of zhfpcs cells was significantly reduced as compared to zhf cells. Instead of 74% for zhf only 45% of the cells were able to form colonies (Fig. 1B). Almost identical observations were made for a zhfhmt1 strain that we generated. Growth inhibition in the presence of increasing Zn2+ concentrations as well as survival rates under very high Zn2+ exposure were very similar to those measured for zhfpcs cells (Fig. 1A; data not shown). Zn accumulation rates of zhf cells are strongly reduced compared to wild-type cells (Boch et al., 2008). This difference was not further aggravated by the loss of functional Pcs or Hmt1. After incubation for 2 h in the presence of 50 µM Zn2+ zhf cells contained 130 µg Zn/g dry weight (±16 µg/g dry weight), zhfpcs cells 120 µg Zn/g dry weight (±3 µg/g dry weight), and zhfhmt1 cells 112 µg Zn/g dry weight (±11 µg/g dry weight; n = 2–4).
Zn2+ exposure resulted in accumulation of PC2 in zhf cells. PC3 and other higher-order PCs were always near or below the detection limit when incubation times between 3 and 24 h were tested. Interestingly, even in the absence of elevated Zn2+ zhf cells accumulated PC2 to a clearly detectable level (Fig. 2A
). Comparison with Cd2+-treated cells showed that PC accumulation was far less pronounced in Zn2+-treated cells and more transient. Total PC content was up to 6-fold higher in cells that were grown in the presence of a Cd2+ concentration that was about as inhibitory as the Zn2+ concentration applied (please note that zhf mutant cells are more Cd2+ tolerant than wild-type cells; Clemens et al., 2002; Fig. 2B). Also, total PC levels returned to control levels already after 13 h of incubation.
Next, we tested the effects of PC formation on Zn2+ tolerance in cells not normally synthesizing PCs by expressing TaPCS1 in the Zn2+-hypersensitive zrc1 cot1 Saccharomyces cerevisiae strain, deficient in vacuolar Zn sequestration (MacDiarmid et al., 2000 ). When cells were grown in the presence of various Zn2+ concentrations, a slight but significant (P < 0.01 for 50, 100, 150, and 200 µM Zn2+) increase in Zn2+ tolerance was observed for the PC-producing cells. The 50% inhibition of initial activity value at which growth was inhibited by 50% was shifted from about 80 µM Zn2+ to about 160 µM Zn2+ (data not shown). Taken together, the data for Zn2+-sensitive yeast mutants indicated that PCs can contribute to the buffering of excess Zn2+ in the presence of a myriad of evolutionary conserved potential Zn2+-binding sites, i.e. they can effectively bind Zn2+ in vivo. Also, when more Zn is available in the cytosol under normal growth conditions due to a defect in Zn storage (Boch et al., 2008), there is elevated constitutive PC2 accumulation.
Prompted by the observations in S. pombe and S. cerevisiae, we decided to analyze Zn2+ sensitivity of PC-deficient Arabidopsis mutants. We chose for these experiments the AtPCS1 null mutant cad1-3 (Howden et al., 1995
Zn2+ sensitivity of Columbia-0 (Col-0), cad1-3, and cad1-6 seedlings was assayed on a medium with low cation content and an agar that possesses less cation exchange capacity than most. This allowed assessing Zn2+ tolerance at lower concentrations because of reduced interference by other cations that are present in vast excess in many plant media and because of reduced binding of Zn2+ to the agar. Seeds were germinated and grown on vertical plates containing modified one-tenth-strength Hoagland solution. Under control conditions wild-type and mutant seedlings grew equally well (Fig. 4A ). Surprisingly, we observed a pronounced Zn2+ hypersensitivity of cad1-3 and cad1-6 seedlings (Fig. 4C). The degree of increase in sensitivity was comparable to the long-known Cd2+ hypersensitivity of AtPCS1 mutants both when measuring root length and seedling weight as growth parameters. More than 200 seedlings each were analyzed in 10 independent experiments on control medium and at Cd2+ and Zn2+ concentrations close to the 50% inhibition of initial activity for Col-0 wild-type seedlings under our assay conditions (2 and 50 µM, respectively). We determined an average root growth inhibition for wild type of 57.6% at 2 µM Cd2+ and 43.2% at 50 µM Zn2+. The respective reduction in seedling weight was 59.5% and 42.4%, respectively (Fig. 4, D and E). The root growth inhibition measured for cad1-3 was 87.0% at 2 µM Cd2+ and 66.2% at 50 µM Zn2+, the seedling weight reduction was 91.0% at 2 µM Cd2+ and 77.0% at 50 µM Zn2+. Zn2+ hypersensitivity of cad1-6 was practically indistinguishable from that of cad1-3, while Cd2+ hypersensitivity was slightly less severe (Fig. 4B). Average root growth inhibition was 75.4% at 2 µM Cd2+ and 62.9% at Zn2+, average seedling weight reduction was 83.9% and 77.0%, respectively. The differences in growth between wild-type and mutant plants under conditions of Cd2+ or Zn2+ toxicity were in all cases highly significant, while growth in the presence of excess Cu2+ was not significantly different between Col and cad1-3 seedlings as reported earlier by Ha et al. (1999) . The same was found for cad1-6 (data not shown).
In earlier experiments with the cad1-3 mutant, no significant loss of Zn2+ tolerance had been observed (Ha et al., 1999 ). We therefore repeated our assays in a medium similar to the one that was used previously. One-half-strength Murashige and Skoog medium contains far higher concentrations of cations such as Ca2+ and Fe3+ and about 3-times higher Zn2+ concentrations have to be applied to cause the same reduction in root growth. In one-half-strength Murashige and Skoog medium prepared with Gelrite agar the Zn2+ hypersensitivity of cad1-3 and cad1-6 was indeed no longer detectable while Cd2+ hypersensitivity was still obvious (Fig. 5
).
PCs have more often been analyzed in metal-challenged plant cell cultures. There are fewer reports on PC detection in plants and in most of these studies Cd2+ ions were applied as the inducer of PC synthesis. This is at least partly due to the smaller amounts accumulating in plant tissues relative to cell cultures. To be able to detect and accurately quantify PCs even when accumulation rates are low, we established a PC2 assay using separation of extracts by capillary reversed-phase chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS). Thiols were labeled prior to chromatographic separation to obtain better resolution and higher sensitivity. As a chemically similar internal standard, we synthesized a PC2 derivative (H-βGlu-Cys-βGlu-Cys-Gly-NH2) (= PC2-NH2). Mass difference of this amide to PC2 with a free carboxyl group is 1 D. The two labeled compounds with theoretical masses of 920.29132 (PC2-bimane2, [M+H+]) and 919.30730 (PC2-NH2-bimane2, [M+H+]) were baseline separated with retention times of around 38.1 min (PC2-NH2-bimane2) and 40.5 min (PC2-bimane2; Fig. 6A ). They could be unequivocally identified based on their exact masses and CID-MSMS spectra (Fig. 6, B and C). Calibration curves were constructed for the two standards and used for quantification. The detection limit (signal-to-noise ratio of >5) was determined as 39 µg PC2/g dry weight when following the standard extraction and labeling protocol. Response was linear for at least 2 orders of magnitude.
Using this assay we compared PC2 levels in hydroponically grown plants 5 d after challenging them with same-effect concentrations of Cd2+ or Zn2+ (0.5 and 20 µM, respectively; please note that the concentrations are lower than on plates because there is no cation-binding agar present). In roots and leaves of wild-type plants, considerable amounts of PC2 were detectable even in control plants (Fig. 7 ). The measured concentrations were about 10-times higher than the detection limit (signal-to-noise ratio >5) in roots and 5-times higher than the detection limit in leaves. Zn2+ challenge resulted in a strong increase (about 6-fold) in PC2 levels that was about 34% of what was reached in the presence of a same-effect Cd2+ concentration. In leaves the difference was similar, with PC2 levels in Zn2+-treated plants that were about 28% of those in Cd2+-treated plants. The increase in leaf PC2 levels as compared to untreated controls was about 5-fold. No PC2 was detectable in cad1-3 under control conditions, while roots and leaves of cad1-6 showed almost wild-type level PC2 accumulation in the absence of metal excess. Remarkably, PC2 was detectable in roots and leaves of Cd2+-challenged cad1-3 plants (about 5% of the concentration in wild type) and also in the roots of Zn2+-treated cad1-3 plants (about 8% of the concentration in wild type). PC2 concentrations in cad1-6 were between 8% (in leaves of Zn2+-treated plants) and 26% (in roots of Cd2+-treated plants) of the level found in Col-0.
Because the uptake rates for a particular transition metal ion are positively correlated with the concentration of intracellular high-affinity binding sites for this ion (Frausto da Silva and Williams, 2001 ), we studied the effect of PC deficiency on accumulation and distribution of Cd and Zn. Wild type and the two mutant plant lines were grown hydroponically in one-tenth-strength Hoagland solution and after 5 weeks subjected to treatment with either 0.5 µM Cd2+ or 20 µM Zn2+ for 5 d. Roots and leaves were harvested separately and analyzed by atomic absorption spectroscopy. Cd was only detectable in treated plants. The roots of cad1-3 and cad1-6 accumulated less Cd than Col-0 (52%–58% of wild-type level). The difference was significant for cad1-6. In leaves, there was no significant difference. Also, roots of the AtPCS1 mutants accumulated only about half the amount of Zn found in wild-type plants: approximately 1.7 and 2.0 mg/g dry weight (cad1-3 and cad1-6, respectively) as compared to 3.6 mg/g dry weight (Fig. 8
). In leaves the reduction was less pronounced. Wild type accumulated about 1.1 mg/g dry weight and leaves of the cad1 mutants between 0.8 and 0.9 mg/g dry weight. In a separate set of experiments, we tested whether PCS overexpression leads to elevated Zn accumulation. The cad1-3 mutant was transformed with a 35S:AtPCS1-HA construct. Resulting transgenic lines were tested for AtPCS1 expression by western analysis using the AtPCS1 antiserum and an anti-HA monoclonal antibody. One line was selected that showed clear overexpression as compared to wild-type Col-0 (Supplemental Fig. S1; Fig. 1A). This line complemented the Cd2+ hypersensitivity phenotype (data not shown). However, when grown hydroponically the transgenic line did not accumulate more Cd or Zn than Col-0 plants. Both root and leaf accumulation rates were comparable while they were lower for cad1-3 as found before (Supplemental Fig. S1; Fig. 1, B and C).
The apparent contribution of PC synthesis to Zn accumulation at comparatively high external Zn2+ concentrations raises the question whether PC synthesis is also important for Zn2+ uptake under Zn2+-deplete conditions. We therefore tested growth of seedlings in the presence of EDTA and exposed 5-week-old hydroponically grown plants to Zn2+ deficiency. In none of these experiments was a phenotype of the cad1 mutants apparent.
The almost ubiquitous occurrence of PCS genes in the plant kingdom remains enigmatic as long as the proven functions of PCSs are confined to Cd, As, and mercury detoxification (Schat et al., 2002 ). Excess of these nonessential elements is a condition that during the course of evolution has been present only sporadically and in a very limited number of habitats. The question as to why the enzymes are so widely distributed and constitutively expressed had therefore been raised already soon after the discovery of PCs (Steffens, 1990) and was again emphasized a short while ago (Rea et al., 2004). At least two obvious hypotheses to explain PCS occurrence are being discussed. The more recent one states that PCSs have additional important functions in glutathione metabolism. Indeed, an additional catalytic activity for AtPCS1, the cleavage of Gly from GS conjugates, was demonstrated (Beck et al., 2003; Blum et al., 2007). This reaction is assumed to represent the first step in the turnover of GS conjugates in plants. The second hypothesis dates back to the early work on PC synthesis and proposes that PCs are involved in essential metal homeostasis (Steffens et al., 1986; Grill et al., 1988). However, no phenotypes had been described to date that would indicate a significantly reduced fitness of PC-deficient plants under conditions of either micronutrient excess or micronutrient deficiency (Howden et al., 1995; Ha et al., 1999; Cobbett and Goldsbrough, 2002). We were therefore surprised to observe a significant loss of Zn2+ tolerance in cad1-3 and the newly isolated cad1-6 allele. In fact, the hypersensitivity was comparable for Cd2+ and Zn2+ under our assay conditions and at concentrations that inhibited root growth of wild-type seedlings about half maximally. Appearance of the seedlings and data on seedling biomass indicated more pronounced toxic effects in root and leaf tissue. To determine why this phenotype had not been observed before, we repeated assays under conditions similar to the ones used previously, i.e. on Murashige and Skoog medium and typical plant tissue culture agar. Indeed, Zn2+ hypersensitivity was no longer observed while Cd2+ hypersensitivity was confirmed (Fig. 5). This demonstrates a clear influence of the growth medium on metal-related phenotypes and explains why Zn2+ hypersensitivity of PCS mutants has escaped notice so far. A detailed analysis of single media components to define the cause of this difference remains to be pursued.
In several early reports Zn2+ was seen as a weak inducer of PC synthesis in vivo. However, as Rauser (1990)
A small yet potentially interesting dissimilarity was observed between cad1-3 and cad1-6. Both mutant lines showed equal hypersensitivity toward Zn2+ (Fig. 4C) while cad1-6 was not quite as strongly affected by Cd2+ as cad1-3. Also, there was an increase in PC2 root levels in the presence of Cd2+ only in cad1-6, which fits the data obtained for the respective truncated version of AtPCS1 in S. pombe (Fig. 3B). We interpret these data as an in vivo confirmation for the hypothesis that the C-terminal portion of PCSs is important for activation of the proteins by a broader range of metal ions including Zn2+ (Ruotolo et al., 2004
A critical question with respect to the observed activation of PC synthesis by Zn2+ and the loss of Zn2+ tolerance in PC-deficient mutants is the formation of PC-Zn complexes. Direct evidence is lacking (Maitani et al., 1996
We conclude from our observations that PCs function as important chelators of excess Zn2+. This is supported by slightly elevated Zn2+ tolerance in Brassica juncea plants transformed with AtPCS1 (Gasic and Korban, 2007
Plant Growth
For metal tolerance assays seeds of Arabidopsis (Arabidopsis thaliana) wild-type Col-0 and the two cad1 mutants were surface sterilized (70% ethanol for 2 min, 10 min in 10% sodium hypochlorite) and rinsed several times with sterile water. Following stratification for 2 d at 4°C seeds were inoculated onto agar (Type A, Sigma) plates with one-tenth-strength Hoagland medium without microelements, 1% Suc. Cd2+ or Zn2+ were added to the medium as chloride salts. Plates were incubated vertically under long-day conditions (16-h light/8-h dark). After 12 d root length and seedling weight were measured. For the analysis of metal accumulation and PC2 accumulation plants were grown hydroponically as described (Weber et al., 2004
Seeds of an insertion line for AtPCS1 (At5g44070) were obtained from Syngenta (Garlic_650_C12.b.1a.Lb3Fa; Sessions et al., 2002
The AtPCS1-HA gene construct used for transformation was derived from a construct tested for activity in Schizosaccharomyces pombe (Cazalé and Clemens, 2001
The S. pombe strains employed in this study were the previously described mutant zhf
The Zn2+ concentration in the culture medium of Arabidopsis plants grown hydroponically was increased by 20 µM or Cd2+ was added to a concentration of 0.5 µM. After 5 d roots and leaves of treated and control plants were harvested. Root tissue was washed with Millipore water and 10 mM CaCl2 several times. Dried material was digested in a microwave (microPREPA/Terminal 320, MLS GmbH Mikrowellen Laborsysteme) in 4 mL 65% HNO3 and 1 mL 30% hydrogen peroxide. Zn content was determined using a Perkin Elmer Analyst 800 (Perkin Elmer) and an iCAP 6000 ICP-OES (Thermo). Zn content of S. pombe cells was analyzed as described (Boch et al., 2008
Root and leaf tissue of 5-week-old plants, treated for 5 d with either 20 µM Zn2+ or 0.5 µM Cd2+, was harvested, frozen in liquid nitrogen, and freeze dried. Thirty milligrams of lyophilized material were homogenized in 500 µL 0.1 M HCl spiked with 5.0 µL 10 mM PC2-NH2 (=26.9 µg) using zirconia beads and a bead beater (1 min, maximum power). Fifty microliters of the supernatant were incubated with 6 µL 20 mM Tris-(2-carboxyethyl)phosphine hydrochloride and 18 µL 4-(2-Hydroxyethyl)-piperazine-1-propanesulfonic acid/diethylenetriaminepentaacetic acid (EPPS/DTPA) buffer (200 mM EPPS, pH 9.2, 6.3 mM DTPA) for 30 min at ambient temperature in a brown eppendorf tube. Derivatization: After addition of 72 µL EPPS/DTPA buffer and 10 µL 10 mM monobromobimane (in CH3CN) the mixture was incubated for 30 min at 45°C. Through addition of 60 µL 1 M methanesulfonic acid the reaction was quenched. Before analysis by capillary liquid chromatography (CapLC) coupled to ESI-QTOF-MS the sample was filtered using a 0.45 µm PTFE syringe filter. Two microliters of labeled extract was injected onto a Phenomenex Luna C8 column (particle size 3 µm, pore size 100 Å, length 150 mm, i.d. 0.3 mm). Separation was performed using the following gradient system: solvent A = water/0.1% HCO2H; solvent B = CH3CN/0.1% HCO2H; 0 to 5 min 95% A, 5% B; 5 to 40 min linear from 5% B to 22% B, 40 to 50 min 95% B, 50 to 60 min 5% B. Flow rate was 5.5 µL/min. LC-ESI-QTOF mass spectra (positive ion mode) were recorded on an API QSTAR Pulsar Hybrid Quadrupole TOF instrument (Applied Biosystems). Ion spray voltage was +5.5 kV, detected mass range: 910 to 930 D, scan rate: 0.5 s–1, declustering potential 1: 50 V, declustering potential 2: 15 V. Each sample was analyzed twice, i.e. two extracts were run per sample. The signals for dialkylated PC2-NH2 (mass-to-charge ratio 919.0–919.5 [M+H]+) and dialkylated PC2 (mass-to-charge ratio 920.0–920.5 [M+H]+) were integrated. PC2 was quantified based on calibration curves recorded for PC2 and PC2-NH2 standards. PC2 and PC2-NH2 standards were synthesized on an Abimed Economy Peptide Synthesizer EPS 211 using N-
Statistical analysis was performed with SigmaStat 3.5 as indicated.
The following materials are available in the online version of this article.
The expert technical assistance of Jutta Elster (peptide synthesis), Regina Weiss (sequencing), and Marina Häussler at the IPB Halle, Germany, is gratefully acknowledged. We thank Dr. Edda V. Roepenack-Lahaye (IPB Halle, Germany) for help with the MS analysis, Dr. Gilles Peltier (CEA Cadarache, France) for the AtPCS1 antiserum, and Dr. Ute Krämer (University of Heidelberg) for the S. cerevisiae strain zrc1 cot1. Received August 1, 2008; accepted December 9, 2008; published December 12, 2008.
1 This work was supported by the Deutsche Forschungsgemeinschaft (grant no. SFB 363) and in part by the European Union through its Sixth Framework Programme for RTD (contract no. FOOD–CT–2006–016253). 
2 Present address: University of Dresden, 01307 Dresden, Germany. The author responsible for the 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: Stephan Clemens (stephan.clemens{at}uni-bayreuth.de).
[W] The online version of this article contains Web-only data.
[OA] Open access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.127472 * Corresponding author; e-mail stephan.clemens{at}uni-bayreuth.de.
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-Fmoc-L-Glu