▪ Abstract Among the heavy metal-binding ligands in plant cells the phytochelatins (PCs) and metallothioneins (MTs) are the best characterized. PCs and MTs are different classes of cysteine-rich, heavy metal-binding protein molecules. PCs are enzymatically synthesized peptides, whereas MTs are gene-encoded polypeptides. Recently, genes encoding the enzyme PC synthase have been identified in plants and other species while the completion of the Arabidopsis genome sequence has allowed the identification of the entire suite of MT genes in a higher plant. Recent advances in understanding the regulation of PC biosynthesis and MT gene expression and the possible roles of PCs and MTs in heavy metal detoxification and homeostasis are reviewed.

PHYTOCHELATINS AND METALLOTHIONEINS: Roles in Heavy Metal Detoxification and Homeostasis

http://eportfolio.lib.ksu.edu.tw/user/G/9/G980N004/repository/PAPER/es10.pdf

Biochemical biomarkers in algae and marine pollution: a review.

Phytoremediation is an eco friendly approach for remediation of contaminated soil and water using plants. Phytoremediation is comprised of two components, one by the root colonizing microbes and the other by plants themselves, which degrade the toxic compounds to further non-toxic metabolites. Various compounds, viz. organic compounds, xenobiotics, pesticides and heavy metals, are among the contaminants that can be effectively remediated by plants. Plant cell cultures, hairy roots and algae have been studied for their ability to degrade a number of contaminants. They exhibit various enzymatic activities for degradation of xenobiotics, viz. dehalogenation, denitrification leading to breakdown of complex compounds to simple and non-toxic products. Plants and algae also have the ability to hyper accumulate various heavy metals by the action of phytochelatins and metallothioneins forming complexes with heavy metals and translocate them into vacuoles. Molecular cloning and expression of heavy metal accumulator genes and xenobiotic degrading enzyme coding genes resulted in enhanced remediation rates, which will be helpful in making the process for large-scale application to remediate vast areas of contaminated soils. A few companies worldwide are also working on this aspect of bioremediation, mainly by transgenic plants to replace expensive physical or chemical remediation techniques. Selection and testing multiple hyperaccumulator plants, protein engineering ofphytochelatin and membrane transporter genes and their expression would enhance the rate of phytoremediation, making this process a successful one for bioremediation of environmental contamination. Recent years have seen major investments in the R&D, which have also resulted in competition of filing patents by several companies for economic gains. The details of science & technology related to phytoremediation have been discussed with a focus on future trends and prospects of global relevance.

Phytoremediation—A Novel and Promising Approach for Environmental Clean-up

http://www.klinghardtacademy.com/images/stories/Heavy_Metal_Toxicity/Chlorella/chlorella%20chemosphere%20paper.pdf

Heavy metal detoxification in eukaryotic microalgae.

Background, aim, and scope
Trace elements (heavy metals and metalloids) are important environmental pollutants, and many of them are toxic even at very low concentrations. Pollution of the biosphere with trace elements has accelerated dramatically since the Industrial Revolution. Primary sources are the burning of fossil fuels, mining and smelting of metalliferous ores, municipal wastes, agrochemicals, and sewage. In addition, natural mineral deposits containing particularly large quantities of heavy metals are found in many regions. These areas often support characteristic plant species thriving in metal-enriched environments. Whereas many species avoid the uptake of heavy metals from these soils, some of them can accumulate significantly high concentrations of toxic metals, to levels which by far exceed the soil levels. The natural phenomenon of heavy metal tolerance has enhanced the interest of plant ecologists, plant physiologists, and plant biologists to investigate the physiology and genetics of metal tolerance in specialized hyperaccumulator plants such as Arabidopsis halleri and Thlaspi caerulescens. In this review, we describe recent advances in understanding the genetic and molecular basis of metal tolerance in plants with special reference to transcriptomics of heavy metal accumulator plants and the identification of functional genes implied in tolerance and detoxification.

Implications of metal accumulation mechanisms to phytoremediation

A cDNA library was constructed from macroalgae adapted to prolonged elevated environmental copper levels. To investigate the possible existence of a metallothionein (MT) gene, the library was screened with degenerate probes designed using plant MT cysteine-rich motifs. A gene was identified (1229 bp) with a putative open reading frame (204 bp) encoding a 67-amino-acid protein exhibiting several characteristic features of MT proteins, including 16 cysteine residues (24%) and only one aromatic residue. Although the protein sequence showed high identity with plant and invertebrate MTs, it contained a unique 'linker' region (14 amino acid residues) between the two putative metal-binding domains which contained no cysteine residues. This extended linker is larger than the tripeptide found in archetypal vertebrate MTs, but does not conform either with the 40-amino-acid linkers commonly found in plant MT sequences. An S-peptide Fucus MT fusion protein expressed in Escherichia coli exhibited a relative molecular mass of approximately 14 kDa. The recombinant fusion bound seven Cd ions, of which 50% were dissociated at pH 4.1. Under anaerobic conditions, the Cd ions were displaced by Cu(I), which associated with the protein at a ratio of 13:1. Laboratory exposure of F. vesiculosus to elevated copper resulted in induction of the MT gene. Thus this paper describes, for the first time, an MT gene identified from macroalgae which is induced by copper exposure and whose encoded protein product binds cadmium and copper.

Identification and characterization of a recombinant metallothionein protein from a marine alga, Fucus vesiculosus.

The macro-alga Fucus vesiculosus has a broad global and estuarine distribution and exhibits exceptional resistance to toxic metals, the molecular basis of which is poorly understood. To address this issue a cDNA library was constructed from an environmental isolate of F. vesiculosus growing in an area with chronic copper pollution. Characterisation of this library led to the identification of a cDNA encoding a protein known to be synthesised in response to toxicity, a full length 14-3-3 exhibiting a 71% identity to human/mouse epsilon isoform, 70–71% identity to yeast BMH1/2 and 95 and 71% identity to the Ectocarpus siliculosus 14-3-3 isoforms 1 and 2 respectively. Preliminary characterisation of the expression profile of the 14-3-3 indicated concentration- and time-dependent inductions on acute exposure of F. vesiculosus of copper (3–30 μg/l). Higher concentrations of copper (≥150 μg/l) did not elicit significant induction of the 14-3-3 gene compared with the control even though levels of both intracellular copper and the expression of a cytosolic metal chaperone, metallothionein, continued to rise. Analysis of gene expression within environmental isolates demonstrated up-regulation of the 14-3-3 gene associated with the known copper pollution gradient. Here we report for the first time, identification of a gene encoding a putative 14-3-3 protein in a multicellular alga and provide preliminary evidence to link the induction of this 14-3-3 gene to copper exposure in this alga. Interestingly, the threshold exposure profile may be associated with a decrease in the organism’s ability to control copper influx so that it perceives copper as a toxic response.

Induction of expression of a 14-3-3 gene in response to copper exposure in the marine alga, Fucus vesiculosus

This study has given the first indication that two commonly used environmental indicator species contain members of the metallothionein superfamily. This demonstrates that these species use similar proteins to higher life forms to adapt to heavy metal conditions. Future studies will investigate if the MT proteins are present in the specialised compartments containing high levels of sulphur and copper/cadmium or if other cysteine-rich proteins such as the phytochelatin family are involved. Previously reported functions of the MT proteins in metal homeostasis suggests that MT expression may be up-regulated as a consequence of the high metal concentration of the environment from which these organisms were collected. The use of the MT expression profiles, as sensitive indicators of the metal concentration of the environment, will be investigated.

Beate Nicolaus

Papers

Identification and characterization of a recombinant metallothionein protein from a marine alga, Fucus vesiculosus.

Induction of expression of a 14-3-3 gene in response to copper exposure in the marine alga, Fucus vesiculosus

Identification and characterisation of metallothioneins from environmental indicator species as potential biomonitors

Genetic mechanisms involved in the adaptation of marine algae to heavy metal pollution