**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 [61]. 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 [66].

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 [69]. 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**

as well. Nevertheless, little is known about how MTs and PCs may complement each other

The activation and function of PC synthase in animals came into light from studies on the nematode *C. elegans* [58], the flatworm *Schistosoma mansoni* [19, 59, 21], and Cionidae *Ciona intestinalis* [60]. The occurrence of PC synthase in animals suggests the occurrence, in these organisms, of a stress oxidative and metal detoxification system based on a class of mole‐ cules which was considered as the privilege of plants. The PC synthase gene has a wide phylogenetic distribution and can be found in species that cover almost all of the animal tree of life. But even though some members of particular taxonomic groups may contain PC synthase genes, there are also many species without these genes. Ron Elran et al. reported the regulation of GSH cycle genes in *Nematostella vectensis*, and an interesting finding was that PC synthase 1, which synthesizes the non-ribosomal formation of metal-binding PC, was upregulated after Hg and Cu treatments [15]. Phylogenetic analyses supported the hypothe‐ sis that PC synthase evolved independently in plants, cyanobacteria and green algae. Among the sequenced metazoan genomes, only a few contain a PC synthase gene. However, the reason for the scattered distribution of these genes remains unclear, considering that metazoans with PC synthase genes in their genomes do not share any physiological, behavioural or ecological features [60]. Just how (and if) PC in invertebrates complement the function of MTs remains to be elucidated, and the temporal, spatial and metal specificity of

**4. Methods for the assays of phytochelatins and phytochelatin synthase**

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 [61]. 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 [66].

for dealing with toxic metals [50].

400 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

the two systems are still unknown [6].

**4.1. Determination of phytochelatins**

**enzyme**

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. [72]. 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 [22]. 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 [73]. Electrochemical methods such as differential pulse voltammetry and highperformance liquid chromatography with electrochemical detection were used for determi‐ nation of Pt(IV) content, GSH levels, PC synthase activity in maize (*Zea mays*) and pea (*Pisum sativum*) plants treated with various doses of Pt(IV) [74].

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 (*Raphanus sativus*) roots. Furthermore, a few target transcripts including PC synthase 1 (PCS1), iron transporter protein and ABC transporter protein were involved in plant response to Cd stress [75]. In 2009, Amaro et al. reported the identification and characterization of a cDNA encoding a PC synthase homologous sequence from the ciliated protozoan *T. thermo‐ phila*, the first to be described in ciliates. A quantitative real-time PCR (qRT-PCR) expression analysis of PC synthase has been carried out under different metal stress conditions. Several experimental evidences suggest that this enzyme is biosynthetically inactive in PC formation, which makes it the first pseudo-PC synthase to be described in eukaryotes [76].
