Structure and function of metal chelators produced by plants: the case for organic acids, amino acids, phytin, and metallothioneins.

Plants produce a range of ligands for cadmium (Cd), copper (Cu), nickel (Ni), and zinc (Zn). Cd- and Zn-citrate complexes are prevalent in leaves, even though malate is more abundant. In the xylem sap moving from roots to leaves, citrate and histidine are the principal ligands for Cu, Ni, and Zn. Phosphorus-rich globular bodies in young roots are probably Zn-phytate. Metallothioneins (MTs) are cysteine (Cys)-rich ligands. Plants produce class II MTs (MT-IIs) which differ from the archetypal mammalian MT-I in the location and number of Cys. The Ec protein from wheat embryos has Cys in three domains, binds Zn, and disappears with seedling development. The first 59 amino acids have been sequenced for the protein. Fifty-eight genes for MT-IIs, from a range of plants and tissues, predict proteins with Cys in two domains. Most of the predicted proteins have not been isolated, and their metal binding is poorly documented. Three protein bands, corresponding to six MT genes, have been isolated from Arabidopsis, and the amino acids sequenced for nine fragments. The MT-IIIs are atypical, nontranslationally synthesized polypeptides with variously repeating gamma-glutamylcysteine units. Of the five families known, those with carboxy-terminal glycine are the most widespread among plants, algae, and certain yeasts. A heterogeneous grouping of these molecules form Cd-binding complexes with tetrahedral coordination and a Cd-sulfur interatomic distance of 2.52 A. One complex is cytosolic, the dominant one is vacuolar. Together, they can bind a large proportion of cellular Cd; other ligands may also function. Little is known about the counterpart situation for Cu and Zn.

X-ray absorption spectroscopy of cadmium phytochelatin and model systems.

Higher plants, algae and some yeasts respond to potentially toxic heavy metals such as cadmium by synthesizing phytochelatins and related cysteine-rich polypeptides. We have used X-ray absorption spectroscopy to study the nature of cadmium binding in such peptides isolated from maize (Zea mays) exposed to low levels of cadmium, and in two synthetic cadmium-peptide complexes, Cd-(gamma-Glu-Cys)3Gly and Cd-(alpha-Glu-Cys)3Gly. We have used the synthetic ions [Cd(SPh)4]2-, [Cd4(SPh)10]2- and [S4Cd10(SPh)16]4-as crystallographically defined models for the cadmium site. The Cd K-edge extended X-ray absorption fine structure (EXAFS) data, together with the Cd K, LI, LII and LIII near-edge spectra, reveal a predominantly tetrahedral coordination of cadmium by sulfur in both the phytochelatin and synthetic peptide complexes. In particular, the Cd LIII-edge lacks a peak at 3534.9 e V which was found to be prominent for oxygen- or nitrogen-coordinated species. The Cd-S distance in the phytochelatin complex is 2.54 A. The Cd K-edge EXAFS does not show any isolated, well-defined Cd-Cd interactions; however, contrary to the conclusion of previous work, their absence is not necessarily indicative of isolated cadmium-thiolate ligation. Evidence from other studies suggests that high static disorder, combined with a large vibrational component, serve to effectively wash out this contribution to the EXAFS. The sulfur K-edge, moreover, shows a low-energy feature both in the phytochelatin and in the synthetic cadmium-peptide complexes which is consistent with sulfide bound in a cluster with cadmium as found for [S4Cd10(SPh)16]4-. This feature strongly suggests the presence of a polynuclear cadmium cluster in maize phytochelatin.

Retention of cadmium in roots of maize seedlings. Role of complexation by phytochelatins and related thiol peptides.

Cd from roots of maize was partitioned in seedlings exposed to 3 microM CdSO4 for 1 to 7 d. Most of the root Cd (92-94%) was buffer soluble and provided the classical metal-induced cysteine-rich, high-molecular-weight Cd-binding complex. This complex, however, bound only part of the Cd within the roots, from 19% after 1 d of exposure to 59% by d 7. Three families of peptides formed the Cd-binding complex: (gamma-glutamic acid-cysteine)n-glycine [(gamma-Glu-Cys)n-Gly], or phytochelatins, (gamma-Glu-Cys)n, and (gamma-Glu-Cys)n-Glu. The monothiols gamma-Glu-Cys-Gly (glutathione), gamma-Glu-Cys, and gamma-Glu-Cys-Glu were absent from the complex. The n2 oligomers of any peptide were the least concentrated, whereas the n3 and n4 oligomers increased in the complex with exposure to Cd. By d 7, 75% of (gamma-Glu-Cys)4-Gly, 80% of (gamma-Glu-Cys)4, and 73% of (gamma-Glu-Cys)3-Glu were complexed with Cd. The peptide thiol:Cd molar ratio for the complexes was 1.01 +/- 0.07, as if the minimal amount of thiol was used to bind Cd. Acid-labile sulfide occurred in the complexes from d 1 onward at the low S2-;Cd molar ratio of 0.18 +/- 0.02.

MgATP-Dependent Transport of Phytochelatins Across the Tonoplast of Oat Roots.

In Cd-exposed oat (Avena sativa) roots Cd was found to be associated primarily with the phytochelatin ([gamma]-glutamylcysteinyl)3-glutamic acid [([gamma]EC)3G], with a peptide to Cd ratio of 1:3 (cysteine to Cd ratio of 1:1), even though both ([gamma]EC)2G and ([gamma]EC)3G were present in the roots. Phytochelatins are known to accumulate in the vacuoles of plant cells on exposure to Cd, but the mechanism is not clear. Here we present evidence for the transport of the phytochelatins ([gamma]EC)3G and ([gamma]EC)2G as well as the Cd complex Cd-([gamma]EC)3G across the tonoplast of oat roots. Transport of ([gamma]EC)3G had a Km, for MgATP of 0.18 mM and a Vmax of 0.7 to 1 nmol mg-1 protein min-1. Transport of ([gamma]EC)3G was also energized by MgGTP and to a lesser extent MgUTP and was highly sensitive to orthovanadate, with a 50%-inhibitory concentration of 0.9 [mu]M. The Cd complex Cd-([gamma]EC)3G and ([gamma]EC)2G were also transported in a MgATP-dependent, vanadate-sensitive manner. Therefore, this process is a candidate for the transport of both phytochelatins, and Cd as its peptide complex, from the cytoplasm into the vacuole.

Three families of thiol peptides are induced by cadmium in maize.

Phytochelatins ((gamma GluCys)nGly) are synthesized from glutathione by plants exposed to metals like Cd2+, Cu2+ and Zn2+. An intracellular complex formed by phytochelatins with Cd2+ and sulfide is thought to detoxify the metal possibly by sequestration in the vacuole. It was found that maize seedlings exposed to Cd2+ produced phytochelatins and two additional families of cysteine-containing peptides, (gamma GluCys)n and (gamma GluCys)nGlu. All thiol peptides with n = 2 and 3 were purified and their structure characterized by tandem mass spectrometry. For maize plants exposed to Cd2+ for 7 days, phytochelatins were synthesized preferentially in the first 24 h whereas the amounts of (gamma GluCys)n and (gamma GluCys)nGlu were the highest thereafter. This was probably due to an initial large pool of glutathione available in control plants compared with a dearth of gamma GluCys and no detectable gamma GluCysGlu. The (gamma GluCys)nGlu peptides were induced exclusively by Cd2+ as they were below the detection limit in control seedlings that contained low amounts of phytochelatins and (gamma GluCys)n. Since the Cys moiety of the peptides is essential for binding Cd2+, a role for accumulated (gamma GluCys)n and (gamma GluCys)nGlu in detoxifying Cd2+ in plants must be considered.

gamma-Glutamylcysteinylglutamic acid--a new homologue of glutathione in maize seedlings exposed to cadmium.

Exposure of plants to Cd induces the appearance of several thiols based on glutathione and known as class III metallothioneins (or phytochelatins). A new tripeptide with the structure gamma-GluCysGlu accumulated in roots and shoots of Cd-exposed maize seedlings. This thiol was purified and identified by tandem mass spectrometry. The fragmentation pattern of the maize tripeptide was identical to that of the synthetic compound. Like glutathione, this new tripeptide may serve as a precursor for longer-chain peptides involved in metal detoxification through the formation of Cd-binding complexes.

Alteration of thiol pools in roots and shoots of maize seedlings exposed to cadmium : adaptation and developmental cost.

Roots of intact 5-day-old maize (Zea mays L.) seedlings were exposed to 3 micromolar Cd during a 7-day period. Cysteine, gamma-glutamylcysteine, glutathione (GSH), and Cd-induced acid-soluble thiols (ASTs), including phytochelatins, were quantified in roots and shoots. Adaptation to Cd and its cost to seedling development were evaluated by measuring Cd content, tissue fresh weight, and rate of root elongation. Roots contained 60 to 67% of the Cd in the seedlings between 4 and 7 days of exposure. Exposure to Cd decreased the fresh weight gain in roots from day 4 onward without affecting the shoots. Between days 1.5 and 3.5 of Cd treatment, roots elongated more slowly than controls; however, their growth rate recovered thereafter and exceeded that of controls. Exposure to Cd did not appreciably affect the concentration of cysteine in the seedlings. However, the initial low concentration of gamma-glutamylcysteine increased (after a lag of 6 hours in roots and 2 days in shoots), reaching a plateau by day 6 at 28.5 nanomoles per gram of fresh weight in roots and by day 5 at 19.1 nanomoles per gram of fresh weight in shoots. During the first 9 hours of Cd exposure, the concentration of GSH in roots decreased dramatically (at 31.6 nanomoles per gram of fresh weight per hour) and thereafter decreased more slowly than in controls. The depletion of GSH in the roots (366 nanomoles per gram of fresh weight) matched the synthesis of ASTs (349 nanomoles per gram of fresh weight) during the first 48 hours. The concentration of ASTs in roots increased steadily thereafter to reach 662.2 nanomoles per gram of fresh weight by 6 days of Cd exposure. In shoots, Cd had little influence on the concentration of GSH, but ASTs still accumulated to 173.3 nanomoles per gram fresh weight after 5 days. The molar ratio of thiols in ASTs to Cd increased to a maximum of 10.24 in roots after 4 hours and of 4.25 in shoots after 2 days of Cd exposure. After 4 days, the ratio reached a plateau of approximately 2 in roots and between 2 and 3 in shoots, as if a steady state of Cd chelation had been achieved in both organs. The plateau coincided with recovered root elongation or an adaptation to Cd. The reduced fresh weight gain of the roots during this time, however, indicated that the synthesis of Cd-induced thiols was at a cost to root development.

Cysteine, gamma-Glutamylcysteine, and Glutathione Levels in Maize Seedlings : Distribution and Translocation in Normal and Cadmium-Exposed Plants.

The levels of cysteine (Cys), gamma-glutamylcysteine (gammaEC), and glutathione (GSH) were measured in the endosperms, scutella, roots, and shoots of maize (Zea mays L.) seedlings. GSH was the major thiol in roots, shoots, and scutella, Cys predominated in endosperms. The endosperm, scutellum, and functional phloem translocation were required for maintenance of GSH pools in roots and shoots of 6-day-old seedlings. Exposure of roots to 3 micromolar Cd, besides causing a decline in GSH, caused an accumulation of gammaEC, as if the activity of GSH synthetase was reduced in vivo. [(35)S]Cys injected into endosperms of seedlings was partly metabolized to [(35)S]sulfate. The scutella absorbed both [(35)S]sulfate and [(35)S]Cys and transformed 68 to 87% of the radioactivity into [(35)S]GSH. [(35)S]GSH was translocated to roots and shoots in proportion to the tissue fresh weight. Taken together, the data supported the hypothesis that Cys from the endosperm is absorbed by the scutellum and used to synthesize GSH for transfer through the phloem to the root and shoot. The estimated flux of GSH to the roots was 35 to 60 nanomoles per gram per hour, which totally accounted for the small gain in GSH in roots between days 6 and 7. For Cd-treated roots the GSH influx was similar, yet the GSH pool did not recover to control levels within 24 hours. The estimated flux of GSH to the entire shoot was like that to the roots; however, it was low (11-13 nanomoles per gram per hour) to the first leaf and high (76-135 nanomoles per gram per hour) to the second and younger leaves.

Changes in metal-binding peptides due to acclimation to cadmium transferred between ramets of Salvinia minima.

Different ramets of a clonal plant exploiting a patchily metal-contaminated habitat may be exposed to different levels of toxic metals. This study investigated whether the exposure of older ("parent") ramets to Cd affected the levels of metal-binding peptides and essential elements in younger ("daughter") ramets which were not exposed to ambient Cd. Pre-treatment of parent ramets of Salvinia minima with 50 µg Cd·l-1 increased the levels of thiols and phytochelatins (PCs), decreased Mg and increased Cu, Zn and S in daughter ramets growing in a Cd-free medium. Acclimation of parents to lower Cd levels (10 and 25 µg Cd·l-1) increased thiols and decreased cysteine and glutathione in daughters, but did not increase PCs. Parental acclimation to all Cd concentrations tested reduced PC production in daughter ramets during subsequent Cd exposure. Daughter ramets from parents pre-treated with 25 µg Cd·l-1 were more Cd tolerant than controls. Although the tolerant daughters contained 35% more thiols than controls, fractionation of tissue Cd showed no difference between the control and tolerant ramets in the fraction of Cd bound by thiol compounds. Thus, while the acclimation of parent plants to Cd increased levels of metal-binding peptides and thiols in daughter ramets, the relationship between these compounds and the Cd tolerance of daughters is unclear.

Compartmental efflux analysis and removal of extracellular cadmium from roots.

Profiles of (109)Cd efflux from roots into three solutions were determined for young intact plants of Agrostis gigantea and maize. The solutions were (a) nutrient culture medium containing 3 micromolar Cd at room temperature, (b) ice-cold 5 millimolar CaCl(2), and (c) ice-cold 5 millimolar PbCl(2). Efflux profiles were clearly resolved into three easily discernible components having fast, medium, and slow exchange rates. These results were unexpected for the situation where some intracellular Cd was present both as extractable Cd-binding peptide and in electron-dense granules within the cytoplasm and the vacuoles. Adding a fourth compartment to the curve-fitting model produced a splitting of the fast exchanging component. Use of these efflux kinetics to estimate Cd fluxes through membranes was inappropriate. However, they were useful in determining optimal washing times for the removal of extracellular Cd. A 10 minute wash in ice-cold 5 millimolar CaCl(2) is recommended for this purpose for Agrostis and maize roots.

The Cd-binding protein from tomato compared to those of other vascular plants.

Cd-binding protein from tomato roots was partially purified and characterized. The Cd-protein complex was eluted as a single Cd peak from QAE-Sephadex A-25 and purified further on Sephadex G-75 in 1 M KC1 buffer. Circular dichroism measurements showed positive Cotton bands at 232 and 273 nm and a negative band at 253 nm, indicative of Cd-thiolate coordination. The major amino acids were Cys (25.6%), Glx (53.3%), Asx (5.4%) and Gly (12.8%) with no aromatic residues detected. The Cd:Cys ratio was 1:2.4. The material had an apparent molecular mass of 3,000 daltons on gel filtration through Sephadex G-50 fine in 1 M KC1 buffer. The tomato protein resembled the Cd-binding proteins isolated from black bentgrass, maize and cabbage. The presence of Cd-thiolate coordination and the high content of Cys are critical features typical of metallothioneins. The abundance of Glx creates a highly anionic protein which is unlike animal metallothioneins. It is proposed that the Cd-binding proteins from vascular plants be designated phytometallothioneins.

Partial characterization of cadmium-binding protein from roots of tomato.

Cd-binding protein was extracted from tomato roots and purified on QAE-Sephadex A-25 and on Sephadex G-75 in 1 molar KCl buffer. The protein preparation was light brown and contained predominantly Cd and small amounts of Zn and Cu. Polyacrylamide gel electrophoresis at pH 6.9 removed the brown material from protein which now bound mostly Cd and some Cu. The apparent molecular weight was 3,100 daltons in high ionic strength medium (1 molar KCl buffer) and 21,500 daltons at low ionic strength. Ionic strength also affected the apparent molecular weight of the Cd-binding protein in crude root extracts. The protein contained 26% cysteine, 53% glutamic acid/glutamine, and 2.8 gram atoms (Cd+Zn+Cu)/mole. The (Cd+Zn+Cu):cysteine ratio was 1:2.3. Circular dichroism measurements indicated Cd-thiolate coordination. The tomato Cd-binding protein was more similar to phytochelatins than to animal metallothioneins.

Isolation and Partial Purification of Cadmium-Binding Protein from Roots of the Grass Agrostis gigantea.

A cadmium-binding protein was isolated from roots of the grass Agrostis gigantea Roth. Heat-stable proteins were chromatographed on the anion exchanger QAE-Sephadex A-25. The major cadmium fraction was purified further by gel filtration on Sephadex G-75 in 1 molar KCl buffer. The resulting protein preparation was light brown, had an apparent molecular weight of 3700, contained 29% cysteine and close to 4 gram atoms cadmium/mole. The cadmium:cysteine ratio was 1:2.7. Spectroscopic measurements indicated cadmium-thiolate coordination. The roots produced the metallothionein-like protein when they were exposed to cadmium for 7 days.

Estimating Metallothionein in Small Root Samples of Agrostis gigantea and Zea mays Exposed to Cadmium.

An established procedure for enrichment of Cu-binding protein was adapted to metallothionein in roots exposed to Cd. Metallothionein in Agrostis gigantea and maize was quantitated by the amount of Cd in prepurified protein fractions. Differential pulse polarography, an independent estimate of protein, was a reliable predictor of Cd content only in metallothionein fractions of Agrostis roots exposed to 3 mmol m(-3) Cd or less and not in maize. Up to 34 % of the Cd in roots of Agrostis plants was associated with metallothionein. The Cd in metallothionein of maize increased 9 fold as the root Cd content tripled during four days of exposure to 3 mmol m(-3) CdSO(4).

Partial Purification and Characterization of Copper-binding Protein from Roots of Agrostis gigantea Roth.

A Cu-binding protein was isolated from roots of the grass Agrostis gigantea Roth. Heat stable proteins were chromatographed on the strongly basic anion exchanger QAE-Sephadex A-25. Dark brown pigments were retained by the ion exchanger during elution of Cu-binding protein. Further purification was achieved by gel filtration on Bio-Gel P-6 in buffer containing 1 kmol m(-1) KCl. The resulting protein was a light blue powder; had an apparent molecular weight of 1700; contained 11% cys, 14% asp, 27% glu and 0.25g atoms Cu per mol; the Cu : cysteine ratio was 1:6. No Cu(I) typical of Cu(I)-thiolate bonds of Cu-thionein was found despite taking precautions to prevent Cu oxidation.

Vein Loading in Seedlings of Phaseolus vulgaris Exposed to Excess Cobalt, Nickel, and Zinc.

Vein loading in unifoliate leaves of white bean seedlings exposed to excess Co(2+), Ni(2+), or Zn(2+) for 1 to 4 days was studied by incubating leaf discs in [(14)C]sucrose. The discs from plants exposed to metal exhibited an increased total uptake of radiosucrose but reduced vein loading. Differences between treatments due to infiltration of disc margins were eliminated by analyzing 7-millimeter discs cut from the center of incubated 14-millimeter discs. Uptake of radiosucrose was greater particularly in discs from seedlings exposed to excess Ni(2+) and Zn(2+). The effect increased as exposure of the seedlings to metal increased up to 4 days. Autoradiographs showed vein loading in control leaf tissues with most of the radiosucrose accumulating in minor veins and little remaining in the mesophyll. In discs from metal-treated plants, most of the sucrose remained in the mesophyll without accumulating preferentially in the minor veins. This effect was evident within 24 hours of exposure to excess metal and intensified with longer exposures to metal. The inhibition of vein loading was also evident in situ. Both the preferential accumulation of sucrose into the minor veins of control tissues and the accumulation into mesophyll of metal exposed tissues were sensitive to 2,4-dinitrophenol and the blockage of sulfhydryl groups. It is concluded that the inhibition of vein loading contributes markedly to the observed toxicological effects of reduced photoassimilate export and of accumulation of carbohydrates in fully expanded leaves of bean seedlings exposed to excess metal ions.