Analysis of metal accumulation and Phytochelatin Synthase (PCS) gene expression in Saccharum spontaneum L. as regards its function for remediation of lead (Pb) contamination

Pollution caused by heavy metals, has become a serious problem. Adverse effects arising from the increased use of heavy metals in a variety of human activities lead to any environmental degradation. Lead (Pb) is one of most common contaminants in the environment and highly toxic. Pb is less mobile, so its compound tends to accumulate in soil and sediments. Definitely, efforts are needed to remove this contaminant in the environment. Saccharum spontaneum L. is a perennial grass which has potential to be used as an accumulator plant to clean up pollutants. The ability of this plant as metal accumulator was tested in this study. S.pontaneum plants were treated using Pb in the concentrations of 0 ppm (control), 100 ppm, 200 ppm, and 300 ppm for 8 weeks. The results showed that there was an increase in the percentage of relative accumulation of Pb in the treated plants. This also indicated that plant roots accumulated more Pb than shoots. Meanwhile, expression of Phytochelatin synthase (PCS) gene increased 1.3-to-3.5-fold inductions in roots by increasing concentration of Pb treatments for 24 h. PCS gene expression showed the higher induction in the roots than in the shoots of S.spontaneum plant under Pb treatments.

Phytochelatin Synthase in Heavy Metal Detoxification and Xenobiotic Metabolism

Phytochelatin synthase (PCS) is well-known for its role in heavy metal detoxification in plants, yeasts and non-vertebrate animals. It is a protease-like enzyme that catalyzes glutathione (GSH) to form phytochelatins (PCs), a group of Cys-rich and non-translational polypeptides with a high affinity to heavy metals. In addition, PCS also functions in xenobiotic metabolism by processing GS-conjugates in the cytosol. Because PCS is involved in GSH metabolism and the degradation of GS-conjugates, it is one of the important components in GSH homeostasis and GSH-mediated biodegradation. This chapter reviews the biochemical mechanism of PCS, how the enzyme activity is regulated, and its roles in heavy metal detoxification as well as GS-S-conjugate metabolism. This chapter also highlights the potential applications of PCS in the improvement of plant performance under combined stresses.

Overexpression of BnPCS1, a Novel Phytochelatin Synthase Gene From Ramie (Boehmeria nivea), Enhanced Cd Tolerance, Accumulation, and Translocation in Arabidopsis thaliana

Phytochelatins (PCs) play important roles in the detoxification of and tolerance to heavy metals in plants. The synthesis of PCs is catalyzed by phytochelatin synthase (PCS), which is activated by heavy metal ions. In this study, we isolated a PCS gene, BnPCS1, from the bast fiber crop ramie (Boehmeria nivea) using the RACE (rapid amplification of cDNA ends) method. The full-length BnPCS1 cDNA is 1,949 bp in length with a 1,518 bp open reading frame (ORF) that encodes a 505 amino acid protein. The deduced BnPCS1 protein has a conserved N-terminus containing the catalytic triad Cys58, His164, Asp182, and a flexible C-terminal region containing a C371C372QETC376VKC379 motif. The BnPCS1 promoter region contains several cis-acting elements involved in phytohormone or abiotic stress responses. Subcellular localization analysis indicates that the BnPCS1-GFP protein localizes to the nucleus and the cytoplasm. Real-time PCR assays show that the expression of BnPCS1 is significantly induced by cadmium (Cd) and the plant hormone abscisic acid (ABA). Overexpression lines of BnPCS1 exhibited better root growth and fresh weight, lower level of MDA and H2O2, and higher Cd accumulation and translocation factor compared to the WT under Cd stress. Taken together, these results could provide new gene resources for phytoremediation of Cd-contaminated soils.

Characterization of Brassica rapa metallothionein and phytochelatin synthase genes potentially involved in heavy metal detoxification

Brassica rapa is an important leafy vegetable that can potentially accumulate high concentrations of cadmium (Cd), posing a risk to human health. The aim of the present study was to identify cadmium detoxifying molecular mechanisms in B. rapa using a functional cloning strategy. A cDNA library constructed from roots of B. rapa plants treated with Cd was transformed into the Cd sensitive yeast mutant strain DTY167 that lacks the yeast cadmium factor (YCF1), and resistant yeast clones were selected on Cd containing media. Two hundred genes potentially conferring cadmium resistance were rescued from the surviving yeast clones and sequenced. Sequencing analysis revealed that genes encoding for metallothionein (MT)1, MT2a, MT2b and MT3, and phytochelatin synthase (PCS)1 and PCS2 accounted for 35.5%, 28.5%, 4%, 11.3%, 18.7% and 2%, respectively of the genes identified. MTs and PCSs expressing DTY167 cells showed resistance to Cd as well as to Zn. PCS1 expressing yeast cells were also more resistant to Pb compared to those expressing MTs or PCS2. RT-PCR results showed that Cd treatment strongly induced the expression levels of MTs in the root and shoot. Furthermore, the different MTs and PCSs exhibited tissue specific expression. The results indicate that MTs and PCS genes potentially play a central role in detoxifying Cd and other toxic metals in B. rapa.

Enhanced Extracellular Synthesis of Gold Nanoparticles by Soluble Extracts from Escherichia coli Transformed with Rhizobium tropici Phytochelatin Synthase Gene

Phytochelatins, the enzymatic products of phytochelatin synthase, play a principal role in protecting the plants from heavy metal and metalloid toxicity due to their ability to scavenge metal ions. In the present study, we investigated the capacity of soluble intracellular extracts from E. coli cells expressing R. tropici phytochelatin synthase to synthesize gold nanoparticle. We discovered that the reaction mediated by soluble extracts from the recombinant E. coli cells had a higher yield of gold nanoparticles, compared to that from the control cells. The compositional and morphological properties of the gold nanoparticles synthesized by the intracellular extracts from recombinant cells and control cells were similar. In addition, this extracellular nanoparticle synthesis method produced purer gold nanoparticles, avoiding the isolation of nanoparticles from cellular debris when whole cells are used to synthesize nanoparticles. Our results suggested that phytochelatins can improve the efficiency of gold nanoparticle synthesis mediated by bacterial soluble intracellular extracts, and the potential of extracellular nanoparticle synthesis platform for the production of nanoparticles in large quantity and pure form is worth further investigation.

Glutathione Restores Hg-Induced Morpho-Physiological Retardations by Inducing Phytochelatin and Oxidative Defense in Alfalfa

Mercury (Hg) is toxic to plants, but the effect of glutathione in Hg alleviation was never studied in alfalfa, an important forage crop. In this study, Hg toxicity showed morphological retardation, chlorophyll reduction, and PSII inefficiency, which was restored due to GSH supplementation in alfalfa plants treated with Hg. Results showed a significant increase of Hg, but Fe and S concentrations substantially decreased in root and shoot accompanied by the downregulation of Fe (MsIRT1) and S (MsSultr1;2 and MsSultr1;3) transporters in roots of Hg-toxic alfalfa. However, GSH caused a significant decrease of Hg in the shoot, while the root Hg level substantially increased, accompanied by the restoration of Fe and S status, relative to Hg-stressed alfalfa. The subcellular analysis showed a substantial deposition of Hg in the root cell wall accompanied by the increased GSH and PC and the upregulation of MsPCS1 and MsGSH1 genes in roots. It suggests the involvement of GSH in triggering PC accumulation, causing excess Hg bound to the cell wall of the root, thereby reducing Hg translocation in alfalfa. Bioinformatics analysis showed that the MsPCS1 protein demonstrated one common conserved motif linked to the phytochelatin synthase domain (CL0125) with MtPCS1 and AtMCS1 homologs. These in silico analysis further confirmed the detoxification role of MsPCS1 induced by GSH in Hg-toxic alfalfa. Additionally, GSH induces GSH and GR activity to counteract oxidative injuries provoked by Hg-induced H2O2 and lipid peroxidation. These findings may provide valuable knowledge to popularize GSH-derived fertilizer or to develop Hg-free alfalfa or other forage plants.