Varied environmental compartments (such as soil and water) potentially contaminated with different metals/metalloids can impact the health of both plants and animals/humans. Trace amounts of Cu, Mn, Mo, Ni and Zn are beneficial for higher plants, whereas, Cr, Cu, Co, Mn, Mo, Se, V and Zn are known as the micronutrient metal/metalloids for animals/humans. However, elevated levels of the metals/metalloids can cause severe toxic consequences in both plants and animals/humans. Common in plants and animals/humans, phytochelatins (PCs), the principal non-protein, S-rich, thiolate peptides, protect (through different mechanisms) cellular functions and metal/metalloid homeostasis by performing their chelation and/or detoxification. With the major aim of broadening the current knowledge on the subject, this chapter (a) overviews PCs’ role and modulation separately in metal/metalloid-exposed plants and animals/humans; (b) discusses major methods for determination of PCs and bioassays for enzymes involved in PC synthesis; (c) evaluates the connection of PCs with bionanoparticles; and finally (d) highlights so far unexplored aspects in the present context.
Anthropogenic activities have caused the release of a wide range of hazardous metals/metalloids (hereafter termed as ‘metal/s’) into the environment. In particular, increasing emissions of metals such as Cd, Hg and As into the environment pose an acute problem for all organisms. Metals, unlike organic contaminants, are not degradable and remain persistent in soils [1–3]. Once taken up, these metals can bring severe toxic consequences in cells due to their chemical similarity to replace the metals necessary for cellular functions. Nevertheless, metals at toxic levels have the capability to interact with several vital cellular biomolecules such as nuclear proteins and DNA, leading to excessive augmentation of reactive oxygen species (ROS) [4–6]. In addition, these metals generate ROS which in turn can cause neurotoxicity, hepatotoxicity and nephrotoxicity in humans and animals [7, 8]. Notably, higher plants, algae, certain yeasts and animals are equipped with a repertoire of mechanisms to counteract metal toxicity. The key elements of these are chelation of metals by forming phytochelatins (PCs) and related cysteine-rich polypeptides [9–11]. PCs are produced from glutamine, cysteine and glycine and the process is catalysed by PC synthases known as γ-glutamylcysteine (γ-Glu-Cys) dipeptidyl transpeptidases [12, 11]. PCs have been identified in a wide variety of plant species, microorganisms and invertebrates. They are structurally related to glutathione (GSH) and were presumed to be the products of a biosynthetic pathway. Numerous physiological, biochemical and genetic studies have confirmed that GSH is the substrate for PC biosynthesis [13, 14]. The general structure of PCs is (γ-Glu-Cys)n-Gly, with increasing repetitions of the dipeptide Glu-Cys, where
In the light of recent literature, the PCs’ role and modulation are overviewed separately in metal-exposed plants and animals/humans and major methods for the determination of PCs and the bioassays for enzymes involved in PC synthesis are discussed hereunder. Additionally, connection of PCs with bionanoparticles is evaluated, and finally, major aspects so far unexplored in the present context are briefly highlighted.
2. Phytochelatins in metal/metalloid-exposed plants
Contamination by metals can be considered as one of the most critical threats to soil and water resources as well as to human health [25, 26]. In fact, the contamination of soils with toxic metals has often resulted from human activities, especially those related to accelerated rate of industrialization, intensive agriculture and extensive mining. Metal belongs to group of non-biodegradable, persistent inorganic chemical having cytotoxic, genotoxic and mutagenic effects on humans or animals and plants through influencing and tainting food chains, soil, irrigation or potable water and aquifers [27, 28, 6]. Chelation and sequestration of metals by particular ligands are the major mechanisms employed by plants to deal with metal stress. The two best-characterized metal-binding ligands in plant cells are the PC and metallothioneins (MTs) [29–33, 6, 34].
Figure 2 shows the scheme of metal-detoxification by PCs in a plant cell. PC, which has a higher affinity for Cd, is formed by the polymerization of 2–11 γ-EC moieties via PC synthase. Several studies confirm that in plants, both GSH and PC synthesis are increased after exposure to Cd and other metals [12, 35–41]. In Figure 3, we show both general functions of the PC and a model of complex between Cd+2 ion and one molecule of PC.
Gonzalez-Mendoza et al. showed that PC synthase gene (in coordination with the expression of metallothionein gene) is present in
Naturally hyperaccumulating plants do not overproduce PCs as a part of their mechanism against toxic metals. This appears to be an inducible rather than a constitutive mechanism, observed especially in metal non-tolerant plants . Some reports have argued against the roles of PC in some metal-tolerant plants based on the effects of buthionine-S-sulphoximine and PCs/metal concentrations . Several studies on plants overexpressing γ-glutamyl-cysteine synthetase or transgenic plants expressing bacterial γ-glutamyl-cysteine synthetase evaluated its effect on metal tolerance based on the assumption that higher levels of GSH and PCs will lead to more efficient metal sequestration .
3. Phytochelatins in metal/metalloid-exposed animals
As mentioned also above, PC proteins have been broadly described and characterized in plants, yeasts, algae, fungi and bacteria . However, PC synthase genes are also present in animal species from several phyla. PC synthesis appears not to be transcriptionally regulated in animals . Nevertheless, originally thought to be found only in plants and yeast, PC synthase genes have since been found in species that span almost the whole animal tree of life. Notably, PC synthase genes are found in species from several other metazoan phyla, including Annelida, Cnidaria, Echinodermata, Chordata and Mollusca (both Gastropoda and Bivalvia classes) [51, 52].
Several phyla of the Metazoa contain one or more species harbouring PC synthase homologous sequences: the Cnidaria (
The activation and function of PC synthase in animals came into light from studies on the nematode
4. Methods for the assays of phytochelatins and phytochelatin synthase enzyme
4.1. Determination of phytochelatins
We briefly discuss herein different methods for the detection and quantification of PC. Additionally, we are giving an overview of the methods used for determination of PC, comprising a broad range of electrochemical as well as spectrometric methods, which have been optimized and even hyphenated with different separation methods to detect PC. Recently, Wood et al. showed the analytical methodology for quantification of PC and their metal(loid) complexes . The classical approach to the analysis of PC is reversed phase HPLC with post-column derivatization of the sulphydryl groups and spectrophotometric detection, but the detection is not specific to PC. The use of an analytical technique is able to detect compounds, specifically mass spectrometry. Independent studies showed a sensitive method for determining PCs by HPLC with fluorescence detection [62, 63]. A simple sensitive method for the identification, sequencing and quantitative determination of PCs in plants by electrospray tandem mass spectrometry (ESI MS-MS) was showed for different studies [64, 65]. Other study showed the combination of three processes for identification PC: (1) easy sample preparation including thiol reduction, (2) rapid and high-resolution separation using ultra-performance liquid chromatography (UPLC) and (3) specific and sensitive ESI-MS/MS detection using multi-reaction mode (MRM) transitions in alga’s extract . Nevertheless, in vitro formed Cd–PC2 complexes were characterized using ion exchange chromatography (IEC), flow injection analysis/high-performance liquid chromatography with CoulArray or Coulochem electrochemical detector and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry [67, 68]. Zitka et al. optimized high-performance liquid chromatography coupled with electrochemical detector for determination of PC2 . Many studies showed the determination of cysteine, reduced and oxidized glutathione and PC in different species of plants using high-performance liquid chromatography with electrochemical detection [70, 71].
4.2. Bioassays for phytochelatin synthase activity
The methods for identification and quantification of PC synthase are multidisciplinary, among themselves, comprising a broad range of molecular biology, electrochemical and spectrometric methods. HPLC coupled with electrochemical detector has been suggested as a new tool for the determination of PC synthase activity. The optimized procedure was subsequently used for studying PC synthase activity in the tobacco BY-2 cells treated with different concentrations of Cd(II) ions and the results were in good agreement with Nakazawa et al. . Other study in animals showed that HPLC-LC system coupled to a single quadrupole LC–MS equipped with ESI was a sensitive method for PC synthase activity . A highly sensitive assay for PC synthase activity was devised, where, the dequenching of Cu(I)-bathocuproinedisulphonate complexes was used in the detection system of a reversed-phase high-performance liquid chromatography. The present assay method is a sensitive tool that can be used to investigate this issue and would allow for the determination of PC synthase activity using 10–100-fold less protein . Electrochemical methods such as differential pulse voltammetry and high-performance liquid chromatography with electrochemical detection were used for determination of Pt(IV) content, GSH levels, PC synthase activity in maize (
Other methods required for the identification and characterization of PC synthase are, for example, the novel technology of molecular biology. Xu et al. showed a study that represents the first transcriptome-based analysis of miRNAs and their targets responsive to Cd stress in radish (
5. Phytochelatins in connection with bionanoparticles
The connection of nanoparticles and PC has two faces: on one hand, the biosynthesis of nanoparticles and on the other hand, the protection of stress caused by the damage of any harmful nanoparticles. An
The concept of phytoremediation of contaminated soils has been increasingly supported by research in recent years. The identification of PC synthase genes from plants and other organisms is a significant breakthrough that will lead to a better understanding of the regulation of a critical step in PC biosynthesis. Many studies showed the mechanisms of chelation of metals–PC in plants in recent years. Chelation and sequestration of metals by particular ligands are also mechanisms used by plants to deal with metal stress. The two best-characterized metal-binding ligands in plant cells are the PCs and MTs. While the role played by PC synthase enzymes and PCs in animals still remains to be fully explored, there is increasing evidence that PC synthase genes are likely to be found in many important animal groups and that PCs may well turn out to be important players in metal ion detoxification in many of these species. It will be of interest in the future to see whether different animal species coordinate PC and MT responses to potentially toxic elements and if this is different for different metal ions.
Research of MAMR, ZH, OZ, VA and RK was financially supported by NANOSEMED KAN208130801. NAA and EP gratefully acknowledge the financial supports received from both FCT (Government of Portugal) through contract (SFRH/BPD/84671/2012) and from the Aveiro University Research Institute/CESAM (UID/AMB/50017/2013).