Search

Access to clean water, hygiene, and sanitation is becoming an increasingly pressing global demand, particularly owing to rapid population growth and urbanization. Phytoremediation utilizes a highly conserved phytochelatin in plants, which captures hazardous heavy metal ions from aquatic environments and sequesters them in vacuoles. Herein, we report the design of phytochelatin-inspired copolymers containing carboxylate and thiolate moieties. Titration calorimetry results indicate that the coexistence of both moieties is essential for the excellent Cd 2+ ion-capturing capacity of the copolymers. The obtained dissociation constant, K D  ~ 1 nM for Cd 2+ ion, is four-to-five orders of magnitude higher than that for peptides mimicking the sequence of endogenous phytochelatin. Furthermore, infrared and nuclear magnetic resonance spectroscopy results unravel the mechanism underlying complex formation at the molecular level. The grafting of 0.1 g bio-inspired copolymers onto silica microparticles and cellulose membranes helps concentrate the copolymer-coated microparticles in ≈3 mL volume to remove Cd 2+ ions from 0.3 L of water within 1 h to the drinking water level (<0.03 µM). The obtained results suggest that hyperconfinement of bio-inspired polymers in flow-through systems can be applied for the highly selective removal of harmful contaminants from the environmental water., (© 2024. The Author(s).)

An unprecedented and direct PS-MS (paper spray ionization mass spectrometry) method was proposed for the detection of native peptides, that is, glutathiones (GSHs), homoglutathiones (hGSHs), and phytochelatins (PCs), in basil (Ocimum basilicum L.) roots before and after cadmium exposure. The roots were submitted to cold maceration followed by sonication with formic acid as the extractor solvent for sample preparation. PS-MS was used to analyze such extracts in the positive mode, and the results allowed for the detection of several GSHs, hGSHs, and PCs. Some of these PCs were not distinguished in the control samples, that is, basil roots not exposed to cadmium. Other PCs were noticed in both types of roots, uncontaminated and cadmium-contaminated, but the intensities were higher in the former samples. Moreover, long-time exposure to cadmium stimulated the formation of some of these PCs and their cadmium complexes. The results, therefore, provided some crucial insights into the defense mechanism of plants against an external stress condition due to exposure to a toxic heavy metal. The present study represents a promising alternative to investigate other crucial physiological processes in plants submitted to assorted stress conditions., (© 2024 John Wiley & Sons Ltd.)

Arsenic (As) speciation analysis is scientifically relevant due to the pivotal role the As chemical form plays in toxicity, which, in turn, directly influences the effect it has on the environment. The objective of this study was to develop and optimize a method tailored for studying As compounds in plant samples. Different extraction procedures and HPLC methods were explored to assess their efficiency, determine mass balance, and improve the resolution of compounds in the chromatograms. Conventionally applied anion-exchange chromatography facilitated the separation of well-documented As compounds in the extracts corresponding to 19 to 82% of As present in extracts. To gain insight into compounds which remain undetectable by anion chromatography (18 to 81% of As in the extracts), but still possibly metabolically relevant, we explored an alternative chromatographic approach. The procedure of sample purification and preconcentration through solid-phase extraction, facilitating the detection of those minor As compounds, was developed. The system was further refined to achieve an online 2D-RP-HPLC system, which was employed to analyze the extracts more comprehensively with ICP and ESI MS. Using this newly developed method, As(III)-phytochelatins, along with other arseno-thio-compounds, were detected and identified in extracts derived from the tree roots of seedlings grown in the presence of As(III) and As(V), and a group of arseno lipids was detected in the roots of plants exposed to As(V).

Phytochelatins (PCs) are poly-Cys peptides containing a repeating γ-Glu-Cys motif synthesized in plants, algae, certain fungi, and worms by PC synthase from reduced glutathione. It has been shown that an excess of toxic metal ions induces their biosynthesis and that they are responsible for the detoxification process. Little is known about their participation in essential metal binding under nontoxic, basal conditions under which PC synthase is active. This study presents spectroscopic and thermodynamic interactions with the PC2-PC5 series, mainly focusing on the relations between Zn(II) complex stability and cellular Zn(II) availability. The investigations employed mass spectrometry, UV-vis spectroscopy, potentiometry, competition assays with zinc probes, and isothermal titration calorimetry (ITC). All peptides form ZnL complexes, while ZnL 2 was found only for PC2, containing two to four sulfur donors in the coordination sphere. Binuclear species typical of Cd(II)-PC complexes are not formed in the case of Zn(II). Results demonstrate that the affinity for Zn(II) increases linearly from PC2 to PC4, ranging from micro- to low-picomolar. Further elongation does not significantly increase the stability. Stability elevation is driven mainly by entropic factors related to the chelate effect and conformational restriction rather than enthalpic factors related to the increasing number of sulfur donors. The affinity of the investigated PCs falls within the range of exchangeable Zn(II) concentrations (hundreds of pM) observed in plants, supporting for the first time a role of PCs both in buffering and in muffling cytosolic Zn(II) concentrations under normal conditions, not exposed to zinc excess, where short PCs have been identified in numerous studies. Furthermore, we found that Cd(II)-PC complexes demonstrate significantly higher metal capacities due to the formation of polynuclear species, which are lacking for Zn(II), supporting the role of PCs in Cd(II) storage (detoxification) and Zn(II) buffering and muffling. Our results on phytochelatins' coordination chemistry and thermodynamics are important for zinc biology and understanding the molecular basis of cadmium toxicity, leaving room for future studies.

ATP-binding cassette transporter B6 (ABCB6), a protein essential for heme biosynthesis in mitochondria, also functions as a heavy metal efflux pump. Here, we present cryo-electron microscopy structures of human ABCB6 bound to a cadmium Cd(II) ion in the presence of antioxidant thiol peptides glutathione (GSH) and phytochelatin 2 (PC2) at resolutions of 3.2 and 3.1 Å, respectively. The overall folding of the two structures resembles the inward-facing apo state but with less separation between the two halves of the transporter. Two GSH molecules are symmetrically bound to the Cd(II) ion in a bent conformation, with the central cysteine protruding towards the metal. The N-terminal glutamate and C-terminal glycine of GSH do not directly interact with Cd(II) but contribute to neutralizing positive charges of the binding cavity by forming hydrogen bonds and van der Waals interactions with nearby residues. In the presence of PC2, Cd(II) binding to ABCB6 is similar to that observed with GSH, except that two cysteine residues of each PC2 molecule participate in Cd(II) coordination to form a tetrathiolate. Structural comparison of human ABCB6 and its homologous Atm-type transporters indicate that their distinct substrate specificity might be attributed to variations in the capping residues situated at the top of the substrate-binding cavity., (© 2024. The Author(s).)

Phytochelatins (PCs) are short Cys-rich peptides with repeating γ-Glu-Cys motifs found in plants, algae, certain fungi, and worms. Their biosynthesis has been found to be induced by heavy metals-both biogenic and toxic. Among all metal inducers, Cd(II) has been the most explored from a biological and chemical point of view. Although Cd(II)-induced PC biosynthesis has been widely examined, still little is known about the structure of Cd(II) complexes and their thermodynamic stability. Here, we systematically investigated glutathione (GSH) and PC2-PC6 systems, with regard to their complex stoichiometries and spectroscopic and thermodynamic properties. We paid special attention to the determination of stability constants using several complementary techniques. All peptides form CdL complexes, but CdL 2 was found for GSH, PC2, and partially for PC3. Moreover, binuclear species Cd x L y were identified for the series PC3-PC6 in an excess of Cd(II). Potentiometric and competition spectroscopic studies showed that the affinity of Cd(II) complexes increases from GSH to PC4 almost linearly from micromolar (log K 7.4 GSH = 5.93) to the femtomolar range (log K 7.4 PC4 = 13.39) and additional chain elongation does not increase the stability significantly. Data show that PCs form an efficient system which buffers free Cd(II) ions in the pico- to femtomolar range under cellular conditions, avoiding significant interference with Zn(II) complexes. Our study confirms that the favorable entropy change is the factor governing the elevation of phytochelatins' stability and illuminates the importance of the chelate effect in shifting the free Gibbs energy.

Phytochelatins (PyCs) are metal-binding compounds produced by plants. PyCs may reduce bioavailability of dietary toxic metals such as cadmium. However, the PyC concentrations in foods are unknown. The objective of this study was to analyze PyC contents in a subset of commonly consumed plant foods. Foods (20) across five groups were analyzed and PyCs quantified using liquid chromatography-mass spectrometry (LC-MS/MS). The impact of factors such as food processing were also explored. PyCs were in all 20 foods. Five PyC types were detected with PyC 2 -Gly, PyC 3 -Gly and PyC 2 -Ala at quantifiable concentrations. PyC 2 -Gly was found at the highest concentrations and most widely distributed. PyC 2 -Gly concentrations were highest in fruits and root vegetables. Foods with increased processing tended to have reduced PyC concentrations. This survey of commonly consumed plant foods in the United States demonstrates PyCs are widely distributed and provides a foundation for understanding their concentrations and impact in the human diet., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Elastin-like polypeptides (ELPs) are stimulus-responsive protein-based biopolymers that exhibit phase transition behavior. By joining them to synthetic phytochelatin (EC), EC-ELP fusion proteins with temperature sensitivity and metal-binding functionality were generated to remove heavy metal ions biologically. Three different EC domains (EC 10 , EC 20 , EC 30 ) were incorporated into the ELP, and the EC-ELP fusion proteins were expressed in E. coli. Their thermal properties and metal binding abilities were then investigated according to the EC length. In addition, the feasibility of reusing EC-ELPs and the cadmium ion binding affinity of reused EC-ELPs were explored., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Quantum dot (QD) biological imaging and sensing applications often require surface modification with single-stranded deoxyribonucleic acid (ssDNA) oligonucleotides. Furthermore, ssDNA conjugation can be leveraged for precision QD templating via higher-order DNA nanostructures to exploit emergent behaviors in photonic applications. Use of ssDNA-QDs across these platforms requires compact, controlled conjugation that engenders QD stability over a wide pH range and in solutions of high ionic strength. However, current ssDNA-QD conjugation approaches suffer from limitations, such as the requirement for thick coatings, low control over ssDNA labeling density, requirement of large amounts of ssDNA, or low colloidal or photostability, restraining implementation in many applications. Here, we combine thin, multidentate, phytochelatin-3 (PC3) QD passivation techniques with strain-promoted copper-free alkyne-azide click chemistry to yield functional ssDNA-QDs with high stability. This process was broadly applicable across QD sizes (i.e., λ em = 540, 560, 600 nm), ssDNA lengths (i.e., 10-16 base pairs, bps), and sequences (poly thymine, mixed bps). The resulting compact ssDNA-QDs displayed a fluorescence quenching efficiency of up to 89% by hybridization with complementary ssDNA-AuNPs. Furthermore, ssDNA-QDs were successfully incorporated with higher-order DNA origami nanostructure templates. Thus, this approach, combining PC3 passivation with click chemistry, generates ssDNA-PC3-QDs that enable emergent QD properties in DNA-based devices and applications.

In this work, AuPd core-shell nanoparticles (NPs) biosynthesized through Arabidopsis thaliana phytochelatin synthase-modified Escherichia coli (Au-Pd/AtPCS1-E. coli) with catalytic enhanced chemiluminescence (CL) and benzyl alcohol oxidation (BAO) was investigated. Such biosynthesis of AuPd core-shell NPs was obviously enhanced due to insertion of the gene sequence of Arabidopsis thaliana phytochelatin synthase (AtPCS1) to a plasmid vector (pET-28b) of Escherichia coli (E. coli). The obtained Arabidopsis thaliana phytochelatin synthase-modified Escherichia coli (AtPCS1-E. coli) could generate phytochelatins (PCs, (γ-Glu-Cys) n -Gly, n > 1) for efficient capture and enrichment of Au 3+ . The component and morphology of AuPd core-shell NPs were checked through X-ray diffraction (XRD), scanning electron microscope (SEM), transmission electron microscopy (TEM) and energy dispersive spectrometer (EDS). Catalytic CL (in H 2 O 2 -luminol system) and BAO (in H 2 O 2 -benzyl alcohol system) effect with different experimental conditions were examined, respectively. These results revealed that multifunctional PCs could effectively facilitate biosynthetic process of AuPd core-shell NPs with better distribution, higher yield and lower cost while stronger CL intensity and higher conversion could be obtained for further quantitative analysis and application., (Copyright © 2019. Published by Elsevier Inc.)

This work describes a high-yield extracellular biosynthesis of ZnS QDs via a unique molecular mediation mechanism driven by the mixed sulfate reducing bacteria (SRB). The mixed SRB have obtained the highest ever ZnS QD biosynthesis rate of 35.0-45.0 g/(L·month). The biogenic ZnS QDs with an average crystallite size (ACS) of 6.5 nm have greater PL activity and better uniformity than that of a chemical route. Peculiar extracellular proteins (EPs) with molecular weights of approximately 65 and 14 kDa specially adhere to the ZnS QDs, which cover extraordinarily high contents of acidic amino acids (14.0 mol % Glu and 13.0 mol % Asp) and of nonpolar amino acids (12.0 mol % Ala, 11.0 mol % Gly, and 7.0 mol % Phe), for novel molecular mediation. The vast amount of negative charges in Glu and Asp guides the strong absorption between the EPs and Zn 2+ via electrostatic attraction to reach a maximum absorption capacity of 745.9 mg/g within 2.0 h, motivating large and rapid nucleation as the first step of biosynthesis. Meanwhile, bridging and interlinkage occur inside the EPs or between the EPs via hydrophobic interactions dominated by the nonpolar amino acids, resulting in the formation of massive microcavities to control and restrict the growth of ZnS QDs as a template. The novel molecular mediation mechanism triggered by the peculiar EPs with an extraordinary amino acid composition and structure accounts for the high-yield biosynthesis of ZnS QDs. The mixed SRB have also successfully fabricated other metal sulfide QDs, including PbS, CuS, and CdS, through the novel molecular mediation.

To increase the platinum adsorption capacity of Escherichia coli (E. coli) biomass, we fused EC20 protein to the E. coli cell surface using an InaKN-based display system, which is the N-terminal region of ice nucleation protein that can be employed as a cell surface display motif. The media and culture conditions were optimized for EC20 (a phytochelatin analogue with 20 repeating units of glutamate and cysteine) expression and Pt (IV) biosorption. Furthermore, the adsorption process was elucidated from aspect of adsorption kinetics and equilibrium, and the characterization of blank and Pt-loaded cells were analyzed using SEM, AFM, TEM, FT-IR and XPS. Our study demonstrated that E. coli strain, which had InaKN-EC20 protein expressed on the cell surface, showed a great enhancement in Pt (IV) adsorption under optimized condition when comparing with that of original E. coli strain. The SEM-EDX analysis revealed that the cellular morphology has been changed in Pt-loaded cells, and the weight percent of platinum in the surface of E.coli increased substantially after displaying EC20 protein. Furthermore, intracellular platinum accumulation was detected in Pt-loaded EC20 cells since a clear peak of platinum exhibited, implying that cytoplasmic EC20 protein might also contribute to platinum accumulation. FTIR analysis revealed that the predominant functional groups in platinum adsorption were amine, carboxyl and phosphate groups., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Phytochelatins are widely known to chelate heavy metal in vacuole and decrease plant damage. Phytochelatin synthase gene (PCS), which is involved in phytochelatins synthesis, is commonly designated as a key gene for phytoremediation. In our study, we cloned three duplicated BnPCS genes from Brassica napus and transformed them into Arabidopsis thaliana AtPCS1 mutant cad1-3, respectively. Three transgene lines and cad1-3 were subjected to a cascade of concentrations of cadmium (Cd) treatment. Evaluation of morphological and physiological measurement results show that transgene lines possess higher Cd tolerance and resistance than A. thaliana mutant cad1-3. The analysis of PCs and Cd contents in root and shoot collectively indicated that transgenic plants promoted Cd accumulation and translocation. In conclusion, all the three BnPCS transgene lines enhanced Cd tolerance, accumulation and translocation, which could provide gene resources for phytoremediation.

Phytochelatins (PyCs) are a diverse set of plant compounds that chelate metals, protect against metal toxicity and function in metal homeostasis. PyCs are present in plants consumed as food by humans and could, in principle, impact absorption and utilization of essential and toxic metals such as selenium and cadmium, respectively. PyCs vary in terminal amino acid composition and chain length, exist in multiple oxidation states and reversibly bind multiple metals; consequently, PyCs include a large set of possible structures. Although individual PyC-metal complexes have been studied, no resource exists to characterize the diversity of PyCs and PyC-metal complexes. We used the scientific literature to develop a database of elemental formulas for polymer forms varying in chain length from 2 to 11 glutamyl-cysteine repeats. Using elemental formulas, we calculated monoisotopic masses using the most abundant isotopes of each element and calculated masses for complexes with 13 metals of nutritional and toxicological significance. The resulting phytochelatin database (PyCDB) contains 46 260 unique elemental formulas for PyC and PyC-metal complexes. The database is available online for download as well as for direct mass queries for mass spectrometry using an accurate mass annotation tool for user-selected PyC types, metals and adducts of interest. We performed studies of a commonly consumed food-onion-to validate the database and test utility of the tool. Onion samples were analyzed using ultra-high resolution mass spectrometry-based metabolomics. Mass spectral features were annotated using the PyCDB web tool and the R package, xMSannotator; annotated features were further validated by collision-induced dissociation mass spectrometry. The results establish use and a workflow for PyCDB as a resource for characterization of PyCs and PyC-metal complexes., (© The Author(s) 2019. Published by Oxford University Press.)

Phytochelatins or PCn, (γGlu-Cys) n -Gly, and their glutathione (GSH) precursor are thiol-rich peptides that play an important role in heavy metal detoxification in plants and microorganisms. Complex formation between Cd 2+ and Pb 2+ and GSH or PCn (n = 2, 4 and 6) are investigated by microcalorimetry, absorption spectrophotometry and T-jump kinetics. Complex formation with Pb 2+ or Cd 2+ is exothermic, and induces ligand metal charge transfer bands in UV absorption spectral range, which implies the formation of a coordination bond between the metal and the thiol groups of the phytochelatins. Absorption spectra and microcalorimetry experiments allow the determination of the affinity constants and the stoichiometry of the complexes. We show that the three PCn interact with Pb 2+ to form the 1:1 and 2:1 M:L complexes, with similar affinity constants (log K 11 Pb ∼4.6, log K 21 Pb ∼11.4). These affinities are independent of the number of thiols and are, moreover, lower than those determined for complex formation with Cd 2+ . On the other hand, with Cd 2+ , PC2-Cd, PC2-Cd 2 , (PC2) 3 -Cd 2 , PC4-Cd, PC4-Cd 2 , PC6-Cd, (PC6) 2 -Cd 3 and PC6-Cd 3 complexes are detected. Furthermore, for PC4-Cd, the 1:1 complex is the most stable: affinity constant (log K 11 Cd ∼7.5). Kinetic studies indicate that complex formation between Cd 2+ and GSH occurs in the ms range; direct rate constant k obs  = (6.8 ± 0.3) 10 6  M -1  s -1 and reverse rate constant k -obs  = 340 ± 210 s -1 . Thus, when encapsulated in a silica matrix, PCn can be good candidates for heavy metal detection., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Rationale: The identification and quantification of phytochelatins (PCs) and their derivatives are important to understand their roles in plant growth and development. A method couplling high-performance liquid chromatography with hybrid linear ion trap Orbitrap mass spectrometry (HPLC-LTQ/Orbitrap) was developed to screen PCs that have the same characteristic product ions. This approach was used for the fragmentation pattern analysis of glutathione (GSH) and PC standards, which allowed identification of the fragmentation pathways of their derivatives isolated from rice roots, stems and leaves., Methods: In this study, we developed a method to detect and identify PCs and their derivatives in rice based on HPLC/LTQ-Orbitrap. Spectrum interpretation and MS/MS fragmentation patterns of PCs provide sufficient information to discover the novel PC derivatives. This approach includes precursor ion scan and product ion scan to detect and character the novel PC derivatives., Results: Based on HCD-MS/MS fragmentation patterns, four PCs and 18 PC derivatives were identified. Among them, seven PC derivatives, i.e., iso-PC2 (Asn), iso-PC3 (Asn), iso-PC2 (Cys), des-γGlu-iso-PC3 (Ser), des-Cys-iso-PC2 (Glu), des-Cys-iso-PC3 (Glu) and des-Cys-iso-PC4 (Glu), have not been previously reported. This method was validated by profiling GSH, PCs and PC derivatives in rice. Preliminary results revealed that PCs and their derivatives, except GSH, are markedly induced by Cd treatment., Conclusions: The HPLC/LTQ-Orbitrap method was successfully developed for the identification of PCs and their derivatives. The C-terminal linked to Gly is replaced with Glu, Ser, Asn, Gln or Cys, thereby creating a family of chemicals that share several structural properties. This technique could be particularly useful for investigators studying plant metabolomics. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

A sensitive and robust high-performance liquid chromatography coupled with electrospray tandem mass spectrometry method for the identification and quantification of glutathione and phytochelatins from rice was developed. Homogenized samples were extracted with water containing 100 mM dithiothreitol, and solid-phase extraction using polymer anion exchange resin was employed for sample purification. Chromatography was performed on a polymeric column with acetonitrile and water containing 0.1% formic acid as the mobile phase at the flow rate of 300 μL/min. The limit of quantitation was 6-100 nM. This assay showed excellent linearity for both glutathione and phytochelatins over physiological normal ranges, with correlation coefficients (r) > 0.9976. Recoveries for four biothiols were within the range of 76-118%, within relative standard deviations less than 15%. The intraday precision (n = 7) was 2.1-13.3%, and the interday precision over 15 days was 4.3-15.2%. The optimized method was applied to analyze tissue samples from rice grown using nutrient solutions with three different cadmium concentrations (0, 50, and 100 μM). With increasing cadmium concentrations, the content of phytochelatin 2 and phytochelatin 3 in rice roots increased, in contrast to most phytochelatins, and the content of glutathione in rice stems and roots decreased significantly., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nanomaterials are of great interest for the development of electrochemical sensors. Multi-walled carbon nanotubes and graphene were used to modify the working electrode surface of different screen-printed carbon electrodes (SPCE) with the aim of improving the sensitivity of the SPCE and comparing it with the conventional glassy carbon electrode. To assay the usability of these sensors, a HPLC methodology with amperometric detection was developed to analyze several phytochelatins in plants of Hordeum vulgare and Glycine max treated with Hg(II) or Cd(II) giving detection limits in the low μmolL(-1) range. Phytochelatins are low molecular weight peptides with the general structure γ-(Glu-Cys)n-Gly (n=2-5) which are synthesized in plants in the presence of heavy metal ions. These compounds can chelate heavy metal ions by the formation of complexes which, are transported to the vacuoles, where the toxicity is not threatening. For this reason phytochelatins are essential in the detoxification of heavy metal ions in plants. The developed HPLC method uses a mobile phase of 1% of formic acid in water with KNO3 or NaCl (pH=2.00) and 1% of formic acid in acetonitrile. Electrochemical detection at different carbon-based electrodes was used. Among the sensors tested, the conventional glassy carbon electrode offers the best sensitivity although modification improves the sensitivity of the SPCE. Glutathione and several isoforms of phytochelatin two were found in plant extracts of both studied species., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Algae and aquatic macrophytes are capable of accumulating heavy metals up to concentrations several orders of magnitude higher than those existing in their surrounding environment. Investigation of mercury toxicology in microalgae is of great interest from ecological point of view, since they could be used as bioindicator to evaluate aquatic ecosystems affected by Hg pollution. In this study, we have performed an exposure experiment focused on the biological response of microalgae Chlorella sorokiniana, a unicellular model organism, to Hg-induced toxicity. The culture was exposed to different concentrations of this element for nine days, namely 0.5, 1, 5 and 10mg L(-1) of HgCl2 (as Hg). To achieve a better understanding of the biological mechanisms triggered by Hg-induced toxicity in this alga a metallomic approach based on SEC-ICP-ORS-MS was applied to survey biomarkers of biological response to mercury contamination in surface water. In addition, the combination of RP-HPLC-ICP-ORS-MS and RP-HPLC-ESI-QqQ-TOF-MS was applied to identify, for the first time, two Hg-binding phytochelatins in this aquatic organism, using cell extracts from microalgae exposed to inorganic mercury., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Metabolic engineering approaches are increasingly employed for environmental applications. Because phytochelatins (PC) protect plants from heavy metal toxicity, strategies directed at manipulating the biosynthesis of these peptides hold promise for the remediation of soils and groundwaters contaminated with heavy metals. Directed evolution of Arabidopsis thaliana phytochelatin synthase (AtPCS1) yields mutants that confer levels of cadmium tolerance and accumulation greater than expression of the wild-type enzyme in Saccharomyces cerevisiae, Arabidopsis, or Brassica juncea. Surprisingly, the AtPCS1 mutants that enhance cadmium tolerance and accumulation are catalytically less efficient than wild-type enzyme. Metabolite analyses indicate that transformation with AtPCS1, but not with the mutant variants, decreases the levels of the PC precursors, glutathione and γ-glutamylcysteine, upon exposure to cadmium. Selection of AtPCS1 variants with diminished catalytic activity alleviates depletion of these metabolites, which maintains redox homeostasis while supporting PC synthesis during cadmium exposure. These results emphasize the importance of metabolic context for pathway engineering and broaden the range of tools available for environmental remediation., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

A highly sensitive method was developed for the detection of phytochelatins (PCs) in rice by stable isotope labeling coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry (IL-LC-ESI-MS/MS) analysis. A pair of isotope-labeling reagents [ω-bromoacetonylquinolinium bromide (BQB) and BQB-d(7)] were used to label PCs in plant sample and standard PCs, respectively, and then combined prior to LC/MS analysis. The heavy labeled standards were used as the internal standards for quantitation to minimize the matrix and ion suppression effects in MS analysis. In addition, the ionization efficiency of PCs was greatly enhanced through the introduction of a permanent charged moiety of quaternary ammonium of BQB into PCs. The detection sensitivities of PCs upon BQB labeling improved by 14-750-fold, and therefore, PCs can be quantitated using only 5 mg of plant tissue. Furthermore, under cadmium (Cd) stress, we found that the contents of PCs in rice dramatically increased with the increased concentrations and treatment time of Cd. It was worth noting that PC5 was first identified and quantitated in rice tissues under Cd stress in the current study. Taken together, this IL-LC-ESI-MS/MS method demonstrated to be a promising strategy in detection of PCs in plants with high sensitivity and reliability.

Analysis of biothiols is still problematic, due to their high polarity, oxidation sensitivity and time-consuming sample preparation. In this paper, a direct, rapid and sensitive method was developed for simultaneous quantification of unbound cysteine (Cys), glutathione (GSH) and phytochelatins (PCs) in rice leaf, stem and root samples by hydrophilic interaction chromatography coupled with electrospray tandem mass spectrometry (HILIC-MS/MS). Homogenized samples were extracted with water containing 50mM dithiothreitol, without derivatization and further clean-up, and the extracts were injected directly onto an Xbridge Amide-HILIC column (3.5μm, 150mm×2.1mm i.d.). The best chromatographic separation and MS sensitivity was achieved using a linear gradient elution with 10mM aqueous ammonium formate and acetonitrile as the mobile phase. In MS/MS mode the detection limit (S/N≥3) of seven biothiols was 3-105nM. Good linearities were observed (r>0.995) with linear dynamic range at least over three orders of magnitude. Recoveries for most analytes were within the range of 77-128%, with relative standard deviations less than 18.2%. The intra-day precision (n=7) was 6.1-11.7%, and the inter-day precision over 15 d (n=15) was 8.5-16.3% for all biothiols. The optimized HILIC-MS/MS method was applied to study the influence of different cadmium (Cd) concentrations (0, 1 and 50μM) on contents of Cys, GSH and PC2-6 in rice tissue. With increasing Cd concentrations in nutrient solutions, contents of PC2-4 in rice roots increased but contents of Cys and GSH decreased. Contents of PC2-4 in both rice leafs and stems increased markedly at high dose Cd (50μM) treatment compared with controls, compared with low Cd concentrations (1μM). However, both PC5 and PC6 were not detected throughout the stress experiment., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Phytochelatins and glutathione (reduced (GSH) and oxidised (GSSG)) are important intracellular ligands involved in metal sequestration and detoxification in algae. Intracellular ratios of GSH:GSSG are sensitive indicators of metal stress in algae, and like phytochelatin production are influenced by metal speciation, concentration, exposure time and the biological species. This study investigated the effect of copper exposure on phytochelatin and glutathione content in two marine diatoms Phaeodactylum tricornutum and Ceratoneis closterium at various time intervals between 0.5 and 72h. Liberation of cellular glutathione and phytochelatins was optimised using freeze/thaw cycles and chemical extraction, respectively. Extracted phytochelatins were derivatised (by fluorescent tagging of thiol compounds), separated and quantified using HPLC with fluorescence detection. Glutathione ratios were determined using a commercially available kit, which uses the enzyme glutathione reductase to measure total and oxidised glutathione. Despite similarities in size and shape between the two diatoms, differences in internalised copper, phytochelatin production (both chain length and quantity) and reduced glutathione concentrations were observed. P. tricornutum maintained reduced glutathione at between 58 and 80% of total glutathione levels at all time points, which would indicate low cellular stress. In C. closterium reduced glutathione constituted <10% of total glutathione after 48h. P. tricornutum also produced more phytochelatins and phytochelatins of longer chain length than C. closterium despite the latter species internalising significantly more copper., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Phytochelatins (PC) are cystein-rich oligopeptides in plants for coordination with toxic metals and metalloids via their thiol groups. The composition, structure, and mass spectrometric fragmentation of arseno-PC (As-PC) with PC of different degree of oligomerization (PC2-PC5) in solution were studied using liquid chromatography coupled in parallel to inductively coupled plasma mass spectrometry and electrospray ionization quadrupole time-of-flight mass spectrometry. As-PC were detected from As(PC2) to As(PC5) with an increasing number of isomers that differ in the position of thiol groups bound to As. Thermodynamic modeling supported the identification process in case of these isomers. Mass spectrometric fragmentation of the As-PC does not follow the established pattern of peptides but is governed by the formation of series of As-containing annular cations, which coordinate to As via S, N, or O. Structure proposals for 30 As-PC fragment ions in the range m/z 147.92 to m/z 1290.18 are elaborated. Many of these fragment ions are characteristic to several As-PC and may be suited for a screening for As-PC in plant extracts. The mass spectrometric data offer the perspective for a future more sensitive determination of As-PC by means of liquid chromatography tandem mass spectrometry with multiple reaction monitoring., (Copyright © 2014 John Wiley & Sons, Ltd.)

An indoor channel system was colonised with fluvial biofilms to study the chronic effects of high Fe and SO4(2-) concentrations and acidic pH, the water chemistry in the surrounding streams of Aljustrel mining area (Alentejo, Portugal), and their contribution to community (in)tolerance to metal toxicity by short-term experiments with Cu and Zn. Biofilms were subjected to four different treatments during 8 weeks: high Fe and SO4(2-) concentrations (1 mg Fe l(-1)+ 700 mg SO4(2-) l(-1)) and acidic pH, high Fe and SO4(2-) at alkaline pH; lower Fe and SO4(2-) at acidic pH: and lower Fe and SO4(2-) concentrations at alkaline pH as negative control. During chronic exposure, acidic pH affected growth negatively, based on low values of algal biomass and the autotrophic index, high values of the antioxidant enzyme activities and low diversity diatom communities, dominated by acidophilic species (Pinnularia aljustrelica) in acidic treatments, being the effects more marked with high Fe and SO4(2-). Co-tolerance to metals (Cu and Zn) was also shown in biofilms from the acidic treatments, contrasting with the higher sensitivity observed in the alkaline treatments. We can conclude that the Aljustrel mining area acidic environment limits algal growth and exerts a strong selection pressure on the community composition which is in turn, more tolerant to metal exposure.

To evaluate plant response to Hg stress, glutathione, phytochelatins, and their Hg complexes were analyzed using HPLC with amperometric detection in samples of Asparagus acutifolius grown in the Almadén mining district (Ciudad Real, Spain), one of the most Hg-contaminated sites in the world. Soils of the Almadén mining district, and specifically from the Almadenejos zone, are highly contaminated, with some zones having values above 4,000 μg Hg g(-1) soil. Although soils have an extremely high concentration of mercury, generally less than 2% is available for plants, as is shown by various soil extractions simulating bioavailability. In plants, Hg concentration increases depending on the content of Hg in soils. In addition, Hg levels in roots are higher than in aerial parts, which is a strategy of plants for protecting their more sensitive aerial parts from the deleterious effects of metal stress. The total content of phytochelatins (PCs) and their complexes are directly related with the amount of mercury in soils. These findings highlight the important role of thiol compounds and their metal complexes in capturing and fixing Hg from soils, giving plants the capacity to deal with the heavy metal toxicity of polluted soils.

Bryophytes, a paraphyletic group which includes liverworts, mosses, and hornworts, have been stated as land plants that under metal stress (particularly cadmium) do not synthesize metal-binding peptides such as phytochelatins. Moreover, very little information is available to date regarding phytochelatin synthesis in charophytes, postulated to be the direct ancestors of land plants, or in lycophytes, namely very basal tracheophytes. In this study, it was hypothesized that basal land plants and charophytes have the capability to produce phytochelatins and possess constitutive and functional phytochelatin synthases. To verify this hypothesis, twelve bryophyte species (six liverworts, four mosses, and two hornworts), three charophytes, and two lycophyte species were exposed to 0-36 μM cadmium for 72 h, and then assayed for: (i) glutathione and phytochelatin quali-quantitative content by HPLC and mass spectrometry; (ii) the presence of putative phytochelatin synthases by western blotting; and (iii) in vitro activity of phytochelatin synthases. Of all the species tested, ten produced phytochelatins in vivo, while the other seven did not. The presence of a constitutively expressed and functional phytochelatin synthase was demonstrated in all the bryophyte lineages and in the lycophyte Selaginella denticulata, but not in the charophytes. Hence, current knowledge according to phytochelatins have been stated as being absent in bryophytes was therefore confuted by this work. It is argued that the capability to synthesize phytochelatins, as well as the presence of active phytochelatin synthases, are ancestral (plesiomorphic) characters for basal land plants.

Phytochelatins (PCs) are thiol-rich peptides, enzymatically synthesized by plants and algae under exposure to certain metals (Cd, Pb, Zn, Ag, As, Cu). Due to their ability to bind metal ions, they play an important role in the cellular detoxification, forming stable metal-PC complexes that minimize the intracellular deleterious effects of metals. The aim of the present work was to evaluate the efficiency of PC-Cd chelation in the freshwater diatom Nitzschia palea under 0, 0.1 and 0.2 mg Cd L(-1), which induced different levels of oxidative stress. This objective was accomplished by the isolation of PC-Cd complexes through size exclusion chromatography. Two peaks were identified, corresponding to high molecular weight (HMW) and low molecular weight (LMW) complexes. In each of the complexes, Cd was quantified by inductively coupled plasma-mass spectrometry, thiol composition was determined by HPLC analysis and the efficiency of Cd chelation calculated by -SH/Cd ratios in HMW and LMW complexes at both Cd concentrations. Results showed that the majority of intracellular Cd was complexed with PCs (75.2-91.2 %). PCs-binding efficiency in this diatom species was higher at HMW than at LMW complexes and enhanced with the increase of Cd concentration exposure. Our work evidenced the important role of PCs as the main intracellular tolerance mechanism in this species. The efficiency increase of Cd-PC binding is related to the increment of PCs synthesis and to the number of Cd ions coordinately bonded to -SH groups in LMW and HMW complexes.

Macrophytes bioaccumulate metals, the suggestion being made that they be considered for phytoremediation. However, a thorough understanding of the mechanisms of metal tolerance in these plants is necessary to allow full optimization of this approach. The present study was undertaken to gain insight into Hg and Cd accumulation and their effects in a representative macrophyte, Elodea nuttallii. Exposure to methyl-Hg (23 ng dm(-3)) had no significant effect while inorganic Hg (70 ng dm(-3)) and Cd (281 μg dm(-3)) affected root growth but did not affect shoots growth, photosynthesis, or antioxidant enzymes. Phytochelatins were confirmed as having a role in Cd tolerance in this plant while Hg tolerance seems to rely on different mechanisms. Histology and subcellular distribution revealed a localized increase in lignification, and an increased proportion of metal accumulation in cell wall over time. Proteomics further suggested that E. nuttallii was able to efficiently adapt its energy sources and the structure of its cells during Hg and Cd exposure. Storage in cell walls to protect cellular machinery is certainly predominant at environmental concentrations of metals in this plant resulting in a high tolerance highlighted by the absence of toxicity symptoms in shoots despite the significant accumulation of metals., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Phytochelatins are small cysteine-rich non-ribosomal peptides that chelate soft metal and metalloid ions, such as cadmium and arsenic. They are widely produced by plants and microbes; phytochelatin synthase genes are also present in animal species from several different phyla, but there is still little known about whether these genes are functional in animals, and if so, whether they are metal-responsive. We analysed phytochelatin production by direct chemical analysis in Lumbricus rubellus earthworms exposed to arsenic for a 28 day period, and found that arsenic clearly induced phytochelatin production in a dose-dependent manner. It was necessary to measure the phytochelatin metabolite concentrations directly, as there was no upregulation of phytochelatin synthase gene expression after 28 days: phytochelatin synthesis appears not to be transcriptionally regulated in animals. A further untargetted metabolomic analysis also found changes in metabolites associated with the transsulfuration pathway, which channels sulfur flux from methionine for phytochelatin synthesis. There was no evidence of biological transformation of arsenic (e.g. into methylated species) as a result of laboratory arsenic exposure. Finally, we compared wild populations of earthworms sampled from the field, and found that both arsenic-contaminated and cadmium-contaminated mine site worms had elevated phytochelatin concentrations.

Although arsenic (As) is a common pollutant worldwide, many questions about As metabolism in nonhyperaccumulator plants remain. Concentration- and tissue-dependent speciation and distribution of As was analyzed in the aquatic plant Ceratophyllum demersum to understand As metabolism in nonhyperaccumulator plants. Speciation was analyzed chromatographically (high-performance liquid chromatography-[inductively coupled plasma-mass spectrometry]-[electrospray ionization-mass spectrometry]) in whole-plant extracts and by tissue-resolution confocal x-ray absorption near-edge spectroscopy in intact shock-frozen hydrated leaves, which were also used for analyzing cellular element distribution through x-ray fluorescence. Chromatography revealed up to 20 As-containing species binding more than 60% of accumulated As. Of these, eight were identified as thiol-bound (phytochelatins [PCs], glutathione, and cysteine) species, including three newly identified complexes: Cys-As(III)-PC2, Cys-As-(GS)2, and GS-As(III)-desgly-PC2. Confocal x-ray absorption near-edge spectroscopy showed arsenate, arsenite, As-(GS)3, and As-PCs with varying ratios in various tissues. The epidermis of mature leaves contained the highest proportion of thiol (mostly PC)-bound As, while in younger leaves, a lower proportion of As was thiol bound. At higher As concentrations, the percentage of unbound arsenite increased in the vein and mesophyll of young mature leaves. At the same time, x-ray fluorescence showed an increase of total As in the vein and mesophyll but not in the epidermis of young mature leaves, while this was reversed for zinc distribution. Thus, As toxicity was correlated with a change in As distribution pattern and As species rather than a general increase in many tissues.

Size-exclusion chromatography (SEC) and capillary zone electrophoresis (CZE) coupled with inductively coupled plasma mass spectrometry were applied to characterize low, medium, and high molecular weight cadmium complexes with glutathione and phytochelatins (PCs). The dominant stoichiometry of the complexes formed in vitro was established as 1:1 using electrospray ionization mass spectrometry. Calculated molecular masses of Cd1L1 complexes were used for calibration of the SEC and CZE methods. The results showed a lower (2 kDa) SEC column exclusion limit for cadmium complexes compared with free peptides (10 kDa), and most of the high molecular weight cadmium species were eluted in the void volume of the column. Moreover, the CZE method based on the semiempirical model of Offord to elucidate peptide migration allowed us to show a high propensity of Cd-PC complexes for polymorphism on complexation, which was also observed for extracts of Arabidopsis thaliana treated with cadmium. All the information presented is vital for understanding the mechanism of metal deactivation in plants.

Ex- situ bismuth film on commercial screen-printed carbon electrodes (BiSPCE) has been used for the first time for the analysis of the complexation of Cd(2+) by thiol containing compounds as glutathione (GSH) and phytochelatin (γ-Glu-Cys)2-Gly (PC2). Reproducibility of data is quite satisfactory and allows their treatment by multivariate curve resolution by alternating least squares (MCR-ALS). Unitary voltammograms and concentration profiles provided by MCR-ALS confirm the formation of 1:1 and 1:2 Cd(2+):GSH and 1:2 Cd(2+):PC2 complexes. These results are in agreement with those previously obtained by mercury electrodes, and allow us to propose the use of BiSPCE for further studies on complexation., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In this study, in vitro formed Cd-phytochelatin (PC2) complexes were characterized using ion exchange chromatography (IEC) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The ratio of both studied compounds as well as experimental conditions were optimized. The highest yield of the complex was observed under an applied concentration of 100 µg·mL(-1) PC2 and 100 µg·mL(-1) of CdCl2. The data obtained show that IEC in combination with MALDI-TOF is a reliable and fast method for the determination of these complexes.

HPLC eluent systems employing acetonitrile and methanol were evaluated for the quantitation of glutathione (GSH) and phytochelatin (PC(n)), a family of peptides implicated in heavy-metal detoxification in higher plants. The detection system is based on the dequenching of copper(I)-bathocuproine disulfonate and is specific for soft-metal chelators. Although both elution systems yielded comparable analytical performance for each PC(n), the acetonitrile system had a lower sensitivity for GSH and a steadily increasing baseline. The inferior properties of the acetonitrile system may be due to complex formation between acetonitrile and Cu(I) ions. Both methods were applied to measure peptide levels in the primitive red alga Cyanidioschyzon merolae. Coefficients of variation (CVs) were less than 5%, except for GSH and PC(4) determinations in the acetonitrile system, in cases when CV values were found to be 8.8% and 6.3%, respectively. Recoveries were greater than 96%, except for GSH determination in the acetonitrile system, with a recovery of 84.4%; however, the concentration measured in the acetonitrile system did not differ from that measured in the methanol system at a significance level of 0.05., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Whilst there are a variety of methods available for the quantification of biothiols in sample extracts, each has their own inherent advantages and limitations. The ease with which thiols readily oxidise not only hinders their quantification but also alters the speciation profile. The challenge faced by the analyst is not only to preserve the speciation of the sample, but also to select a method which allows the retrieval of the desired information. Given that sulfur is not a chromophore and that it cannot easily be monitored by ICP-MS, a number of direct and indirect methods have been developed for this purpose. In order to assess these methods, they are compared in the context of the measurement of arsenic-phytochelatin complexes in plant extracts. The inherent instability of such complexes, along with the instabilities of reduced glutathione and phytochelatin species,necessitates a rapid and sensitive analytical protocol. Whilst being a specific example, the points raised and discussed in this review will also be applicable to the quantification of biothiols and thiol-metal(loid) species in a wide range of systems other than just the analysis of arsenic-phytochelatin species in plant extracts.

This study combines Tween 20-capped gold nanoparticles (Tween 20-AuNPs) with capillary electrophoresis (CE) for ultrasensitive detection of thiol-containing peptides, including glutathione (GSH), γ-glutamylcysteine (γ-GCS), and phytochelatin analogs. By forming AuS bonds, Tween 20-AuNPs can selectively extract and enrich these thiols from a complicated matrix. A Tween 20 capping layer not only suppresses nonspecific adsorption, but also enables NPs to disperse in a highly salinity solution. Dithiothreitol removes thiol-containing peptides from the NP surface through ligand exchange. The released peptides are selectively derivatized with o-phthaldialdehyde (OPA) to form tricyclic isoindole derivatives. Extraction efficiency of five thiol-containing peptides with Tween 20-AuNPs was highly reliable in the Tween 20-AuNP concentration, time of extraction and desorption thiols, and sample volume. After injecting a large sample volume, the OPA-derivatized peptides migrate against the electroosmotic flow (EOF) and enter the polyethylene oxide (PEO) zone. The sensitivity of these peptides was improved by stacking them at the boundary between the sample and PEO zones. As a result, limits of detection (LODs) for five peptides were down to 0.1-6 pM. Not only is the proposed method probably the first CE example for detecting dissolved thiols in seawater; it also has the lowest LODs for GSH, γ-GCS, and phytochelatins compared to other reported methods., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We reported an alternative strategy to reduce disulphide bonds in peptides with Ag-nanoparticle loaded nano-TiO(2) (Ag/TiO(2)) under UV irradiation. The feasibility of this strategy was adequately demonstrated using the model peptides oxidized glutathione, vasopressin and insulin, which contain various disulphide bonds, as well as by its application to the determination of Cd-induced phytochelatins in Phaeodactylum tricornutum.

Isothermal Titration Calorimetry (ITC) was used to study the binding of Cd(2+) by phytochelatins ((γGlu-Cys)(n)-Gly, PC(n); n = 1-5) and their selected fragments (Cys, Cys-Gly and γGlu-Cys) in order to understand the influence of the chain length on the complex stabilities and the origin of the enhanced affinities in Tris buffer at pH 7.5 and 8.5 and at 25 °C. Different complexes are formed with glutathione (GSH) and its fragments, Cys, Cys-Gly and γGlu-Cys, and their stabilities depend on the corresponding pK(a) value of the thiol group in the ligands. The stability of Cd-PC(n) complexes increases moving towards higher PC(2-5), as well as the complexing capacity expressed as the number of metal ions that can be bound by one ligand molecule. The affinity of Cd(2+) for the PC(n) can be described by the following GSH < PC(2) < PC(3)≤ PC(4)≤ PC(5) sequence. On the basis of these thermodynamic data it is possible to explain the abundance of certain Cd-PC(n) complexes found in nature. The comprehension of the thermodynamic rules that govern the interactions of Cd(2+) with PC(n) and their constituents is of great service in the research with real plant samples subjected to metal stress and in the development of new strategies of bio/phytoremediation.

Metal-binding thiols, involved in detoxification mechanisms in plant and other organism under heavy metal stress, are receiving more and more attentions, and various methods have been developed to determine related thiols such as cysteine (Cys), glutathione (GSH) and phytochelatins (PCs). In present study, an HPLC method was established for simultaneous determination of Cys GSH and PC(2-6) after treatment with disulfide reductant of tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and thiolyte reagent of monobromobimane (mBBr). The separation of thiol derivatives was performed on an Agilent Zorbax Eclipse XDB-C18 column (4.6 mm × 30 mm, 1.8 μm) with a linear gradient elution of 0.1% (v/v) trifluoroacetic acid (TFA)-acetonitrile (ACN) at 0.8 mL min(-1). The temperature of the column was maintained at 25°C. The excitation and emission wavelengths were set at 380 and 470 nm, respectively. The thiol derivatives were well separated in 19 min, and the total analysis time was 30 min. The established method was proved selective, specific and reproducible, and could be applicable to determine Cys, GSH and PC(2-6) and to evaluate their roles in detoxification mechanisms in Cd-treated Lolium perenne L. under ambient and elevated carbon dioxide (CO(2)). It was found that the total SH contents and proportions of thiols in roots and shoots were dependent on Cd concentration, whereas the total SH contents decreased and the proportions of thiols altered without significance at elevated CO(2) level., (Copyright © 2011 Elsevier B.V. All rights reserved.)

This study investigates the in vitro bioactivity of S-nitrosophytochelatins (SNOPCs), oligopeptide analogues of S-nitrosoglutathione (GSNO), and their mechanisms of nitric oxide (NO) delivery. SNOPCs were more potent than GSNO in inhibiting platelet aggregation and stimulating vasorelaxation. Their potency was related to the number of S-nitrosated moieties per mole compound. Transnitrosation reactions with cell membrane surface components were shown to be the primary mode of NO delivery to intracellular targets for SNOPCs, while delivery via γ-glutamyl transpeptidase was unique to GSNO. Due to rapid NO release, larger SNOPCs elicited a more transitory effect compared to smaller compounds. The duration of effect was influenced by compound molecular weight, NO release kinetics, ability to undergo transnitrosation, and incubation time with tissues. In summary, a new oligopeptide NO delivery system based on SNOPCs was shown to be biologically active and can be used to investigate the mechanisms of NO delivery to intracellular targets.

A simple and rapid methodology is optimised to analyse mixtures of different phytochelatins (PC(n), n=2-5) with Hg(II) by HPLC with amperometric detection as a first step towards the analysis of extracts of plants stressed with Hg(II). The separation was achieved in a C(18) column with a mobile phase of 0.1% trifluoroacetic acid (TFA) in water and 0.1% TFA in acetonitrile using gradient elution. Electrochemical detection with glassy carbon electrode and UV-vis detection were used in series. This methodology can clearly distinguish between the free peptides and their complexes and permits to study the evolution of the different complexes formed and predicts the possible interactions between the long chain phytochelatin complexes. ESI-MS is used as a complementary technique to find out the stoichiometries of such long chain phytochelatin complexes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Three-week-old alfalfa (Medicago sativa), barley (Hordeum vulgare) and maize (Zea mays) were exposed for 7 d to 30 µm of mercury (HgCl(2) ) to characterize the Hg speciation in root, with no symptoms of being poisoned. The largest pool (99%) was associated with the particulate fraction, whereas the soluble fraction (SF) accounted for a minor proportion (<1%). Liquid chromatography coupled with electro-spray/time of flight mass spectrometry showed that Hg was bound to an array of phytochelatins (PCs) in root SF, which was particularly varied in alfalfa (eight ligands and five stoichiometries), a species that also accumulated homophytochelatins. Spatial localization of Hg in alfalfa roots by microprobe synchrotron X-ray fluorescence spectroscopy showed that most of the Hg co-localized with sulphur in the vascular cylinder. Extended X-ray Absorption Fine Structure (EXAFS) fingerprint fitting revealed that Hg was bound in vivo to organic-S compounds, i.e. biomolecules containing cysteine. Albeit a minor proportion of total Hg, Hg-PCs complexes in the SF might be important for tolerance to Hg, as was found with Arabidopsis thaliana mutants cad2-1 (with low glutathione content) and cad1-3 (unable to synthesize PCs) in comparison with wild type plants. Interestingly, high-performance liquid chromatography-electrospray ionization-time of flight analysis showed that none of these mutants accumulated Hg-biothiol complexes., (© 2011 Blackwell Publishing Ltd.)

Multivariate curve resolution with alternating least squares (MCR-ALS) was applied to voltammetric data obtained in the analysis of the competitive binding of glutathione (GSH) and phytochelatins [(gammaGlu-Cys)(n)-Gly, PC(n), n=2-5] by Cd(2+). The displacements between ligands and chain length influence on the competitive binding of PC(n) toward Cd(2+) were investigated. The analysis of the resulting pure voltammograms and concentration profiles of the resolved components suggests that ligands containing more thiol groups are able to displace the shortest chain ligands from their metal complexes, whereas the opposite does not happen. However, when the length of the chain surpasses that of PC(3), the binding capacity of the molecule still increases (i.e., it can bind more metal ions), but the position and shape of the voltammetric signals practically rest unchanged. This suggests that at this level, the stability of metal binding could depend more on the nature of the binding sites separately than on the quantity of the sites (i.e., the chain length)., (2010 Elsevier Inc. All rights reserved.)

Quantitative phytochelatin (PC) analysis is, due to oxidation sensitivity of the PCs, matrix effects, and time consuming sample preparation, still a challenging analytical task. In this study, a rapid, simple, and sensitive method for precise determination of native PCs in crude extracts of the green alga Chlamydomonas reinhardtii was developed. Algae were exposed 48 h to 70 μM Cd. Coupling of ultra performance liquid chromatography and electrospray ionization tandem mass spectrometry with multi-reaction mode transitions for detection permitted the required short-time, high-resolution separation and detection specificity. Thus, under optimized chromatographic conditions, 10 thiol peptides were baseline-separated within 7 min. Relative detection limits in the nanomolar range in microliter sample volumes were achieved (corresponding to absolute detection limits at femtomole level). Next to glutathione (GSH), the most abundant cadmium-induced PCs in C. reinhardtii, namely CysGSH, PC(2), PC(3), CysPC(2), and CysPC(3), were quantified with high reproducibility at concentrations between 15 and 198 nmol g(-1) fresh weight. The biological variation of PC synthesis of nine independently grown alga cultures was determined to be on average 13.7%.

A new chemometric approach is put forward, dealing with the non-linear behaviour observed in the multivariate curve resolution (MCR) analysis of certain overlapping voltammetric signals obtained in titrations of metal complexes where pH is progressively changed. In such cases, non-reversible reduction signals move along the potential axis as a consequence of the involvement of H(+)-ions in the electrochemical process and cause a dramatic loss of linearity, which hinders accurate MCR analysis. The method proposed is based on the least-squares fitting of peak potential vs. pH datasets to parametric linear and sigmoid functions through the decomposition of the data matrix into both a concentration profile matrix and a unit signal matrix, in a similar way as in the alternating least-squares algorithm of MCR (ALS). Such calculations are carried out through several home-made Matlab programs which are freely available as Supplementary Material of the present work. The fitted parameters, along with the evolution of resolved concentrations and potential shifts with pH, provide valuable information on the complexation/reduction processes. The method is tested first on the relatively simple Cd(II)-NTA system and then applied to the study of the binding of Cd(II)-ions by glutathione (gamma-Glu-Cys-Gly, GSH) and the phytochelatin PC(2) ((gamma-Glu-Cys)(2)-Gly).

The accumulation and transport of lead in Brassica juncea and Sesuvium portulacastrum plants and the possible formation of complexes of this element with bioligands such as phytochelatins was studied in roots and shoots of plants exposed to different amounts of Pb(NO(3))(2). Speciation studies on the plant extracts were conducted using size exclusion liquid chromatography and ion pair liquid chromatography coupled to UV and ICP-MS to monitor lead and sulphur. The identification of the species separated by chromatography was performed by MALDI-TOF-MS. In both types of exposed plants it was possible to identify the presence of the phytochelatin isoform PC(3). The results obtained suggest that both types of plants can be useful in studies of phytoremediation but the ability of S. portulacastrum to accumulate and redistribute Pb from root to shoot is more effective than B. juncea., (Copyright 2010 Elsevier B.V. All rights reserved.)

Reversed-phase liquid chromatography (RPLC) and electrospray (ESI)-linear ion trap (LIT) mass spectrometry was applied to the direct characterization of in vivo S-nitrosylated (SNO) phytochelatins (PCs) expressed in cadmium-stressed Arabidopsis thaliana cells. Cys-nitrosylation is under discussion as in vivo redox-based post-translational modification of proteins and peptides in plants in which the -NO group is involved as signal molecule in different biological functions. The gas-phase ion chemistry of in vivo and in vitro generated SNO-PC(s) was compared with the aim of evaluating NO binding stability and improving MS knowledge about peptide nitrosation. Using RPLC separation and ESI-LIT-MS, mono-nitrosylated PCs were identified in in vivo cadmium treated A. thaliana cells without derivatization. The in vivo binding of the NO group to PC(2), PC(3) and PC(4) resulted to occur selectively on only one cystein residue. The fragmentation pathway energies of the in vitro GSNO-generated NO-PCs with respect to the in vivo NO-PCs were investigated, suggesting the presence of a different internal stability for these molecules. By carrying out MS(2) experiments on these quasi-symmetric peptides, the different stability degree of the NO group was demonstrated to be correlated with the PC chain length. In addition, the data obtained highlight a putative role of the adjacent Glu/Cys motif in the gas-phase stability of the NO-containing molecule., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Complexation of arsenite [As(III)] with phytochelatins (PCs) is an important mechanism employed by plants to detoxify As; how this complexation affects As mobility was little known. We used high-resolution inductively coupled plasma-mass spectrometry and accurate mass electrospray ionization-mass spectrometry coupled to HPLC to identify and quantify As(III)-thiol complexes and free thiol compounds in Arabidopsis (Arabidopsis thaliana) exposed to arsenate [As(V)]. As(V) was efficiently reduced to As(III) in roots. In wild-type roots, 69% of As was complexed as As(III)-PC4, As(III)-PC3, and As(III)-(PC2)2. Both the glutathione (GSH)-deficient mutant cad2-1 and the PC-deficient mutant cad1-3 were approximately 20 times more sensitive to As(V) than the wild type. In cad1-3 roots, only 8% of As was complexed with GSH as As(III)-(GS)3 and no As(III)-PCs were detected, while in cad2-1 roots, As(III)-PCs accounted for only 25% of the total As. The two mutants had a greater As mobility, with a significantly higher accumulation of As(III) in shoots and 4.5 to 12 times higher shoot-to-root As concentration ratio than the wild type. Roots also effluxed a substantial proportion of the As(V) taken up as As(III) to the external medium, and this efflux was larger in the two mutants. Furthermore, when wild-type plants were exposed to l-buthionine sulfoximine or deprived of sulfur, both As(III) efflux and root-to-shoot translocation were enhanced. The results indicate that complexation of As(III) with PCs in Arabidopsis roots decreases its mobility for both efflux to the external medium and for root-to-shoot translocation. Enhancing PC synthesis in roots may be an effective strategy to reduce As translocation to the edible organs of food crops.

Phytochelatins (PCs) are nonprotein peptides with the general structure (gamma-Glu-Cys)(n)-Gly (PC(n)), where n is greater than or equal to 2. They are synthesized through a reaction catalyzed by phytochelatin synthase (PCS) in the presence of metal cations and using the tripeptide glutathione (gamma-Glu-Cys-Gly) and/or previously synthesized PC(n) as the substrate. Here, a highly sensitive assay for PCS activity was devised, in which the dequenching of Cu(I)-bathocuproinedisulfonate complexes was used in the detection system of a reversed-phase high-performance liquid chromatograph. Using recombinant PCS from the higher plant Arabidopsis thaliana (rAtPCS1), this assay system was capable of determining PCS activity based on an amount of the enzyme preparation that was 100-fold less than that required for the 5,5'-dithiobis(2-nitrobenzoic acid) assay method. Although adsorption of the enzyme onto the reaction vessel hindered accurate activity determination, the inclusion of bovine serum albumin successfully resolved this issue. This method is a powerful tool for investigating PCS enzyme mechanisms with respect to the roles of metal ions., (Copyright 2009 Elsevier Inc. All rights reserved.)