Metal accumulation and tolerance mechanisms in Potamogeton crispus under single-metal exposure to Cd, Zn, Ni and Li: Implications for phytoremediation of metal-contaminated waters

Henk Schat.

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. Plants are susceptible to heavy metal toxicity and respond to avoid detrimental effects in a variety of different ways. The toxic dose depends on the type of ion, ion concentration, plant species, and stage of plant growth. Tolerance to metals is based on multiple mechanisms such as cell wall binding, active transport of ions into the vacuole, and formation of complexes with organic acids or peptides. One of the most important mechanisms for metal detoxification in plants appears to be chelation of metals by low-molecular-weight proteins such as metallothioneins and peptide ligands, the phytochelatins. For example, glutathione (GSH), a precursor of phytochelatin synthesis, plays a key role not only in metal detoxification but also in protecting plant cells from other environmental stresses including intrinsic oxidative stress reactions. In the last decade, tremendous developments in molecular biology and success of genomics have highly encouraged studies in molecular genetics, mainly transcriptomics, to identify functional genes implied in metal tolerance in plants, largely belonging to the metal homeostasis network. Analyzing the genetics of metal accumulation in these accumulator plants has been greatly enhanced through the wealth of tools and the resources developed for the study of the model plant Arabidopsis thaliana such as transcript profiling platforms, protein and metabolite profiling, tools depending on RNA interference (RNAi), and collections of insertion line mutants. To understand the genetics of metal accumulation and adaptation, the vast arsenal of resources developed in A. thaliana could be extended to one of its closest relatives that display the highest level of adaptation to high metal environments such as A. halleri and T. caerulescens. This review paper deals with the mechanisms of heavy metal accumulation and tolerance in plants. Detailed information has been provided for metal transporters, metal chelation, and oxidative stress in metal-tolerant plants. Advances in phytoremediation technologies and the importance of metal accumulator plants and strategies for exploring these immense and valuable genetic and biological resources for phytoremediation are discussed. A number of species within the Brassicaceae family have been identified as metal accumulators. To understand fully the genetics of metal accumulation, the vast genetic resources developed in A. thaliana must be extended to other metal accumulator species that display traits absent in this model species. A. thaliana microarray chips could be used to identify differentially expressed genes in metal accumulator plants in Brassicaceae. The integration of resources obtained from model and wild species of the Brassicaceae family will be of utmost importance, bringing most of the diverse fields of plant biology together such as functional genomics, population genetics, phylogenetics, and ecology. Further development of phytoremediation requires an integrated multidisciplinary research effort that combines plant biology, genetic engineering, soil chemistry, soil microbiology, as well as agricultural and environmental engineering.

Metal tolerance mechanisms in plants and microbe-mediated bioremediation

An ideal plant for environmental cleanup can be envisioned as one with high biomass production, combined with superior capacity for pollutant tolerance, accumulation, and/or degradation, depending on the type of pollutant and the phytoremediation technology of choice. With the use of genetic engineering, it is feasible to manipulate a plant's capacity to tolerate, accumulate, and/or metabolize pollutants, and thus to create the ideal plant for environmental cleanup. In this review, we focus on the design and creation of transgenic plants for phytoremediation of metals.Plant properties important for metal phytoremediation are metal tolerance and accumulation, which are determined by metal uptake, root-shoot translocation, intracellular sequestration, chemical modification, and general stress resistance.If we know which molecular mechanisms are involved in these tolerance and accumulation processes, and which genes control these mechanisms, we can manipulate them to our advantage. This review aims to give a succinct overview of plant metal tolerance and accumulation mechanisms, and to identify possible strategies for genetic engineering of plants for metal phytoremediation. An overview is presented of what has been achieved so far regarding the manipulation of plant metal metabolism. In fact, both enhanced metal tolerance and accumulation have been achieved by overproducing metal chelating molecules (citrate, phytochelatins, metallothioneins, phytosiderophores, ferritin) or by the overexpression of metal transporter proteins. Mercury volatilization and tolerance was achieved by introduction of a bacterial pathway. The typical increase in metal accumulation as the result of these genetic engineering approaches is 2-to 3-fold more metal per plant, which could potentially enhance phytoremediation efficiency by the same factor. As for the applicability of these transgenics for environmental cleanup, results from lab and greenhouse studies look promising for several of these transgenics, but field studies will be the ultimate test to establish their phytoremediation potential, their competitiveness, and risks associated with their use.

A number of review articles have appeared in the literature recently on metal accumulation by plants. However, reports on the tolerance, accumulation and detoxification of arsenic in plants, especially in terrestrial plants, are limited. In light of the strong needs for study of arsenic biogeochemistry and development of arsenic decontamination techniques, in this paper we. review arsenic uptake, accumulation, and detoxification mechanisms in terrestrial plants. In order to discuss the processes involved in arsenic accumulation, we will first provide a brief review on the general understanding about the mechanisms of metal tolerance in plants. We will also discuss the arsenic hyperaccumulators that were recently discovered.

This paper briefly reviews the progress in studies of wetland plants in terms of heavy metal pollution. The current research mainly includes the following areas: (1) metal uptake, translocation, and distributions in wetland plants and toxicological effects on wetland plants, (2) radial oxygen loss (ROL) of wetland plants and its effects on metal mobility in rhizosphere soils, (3) constitutional metal tolerance in wetland plants, and (4) mechanisms of metal tolerance by wetland plants. Although a number of accomplishments have been achieved, many issues still remain unanswered. The future research effort is likely to focus on the ROL of wetland plants affecting metal speciation and bioavailability in rhizosphere soils, and the development of rhizosphere management technologies to facilitate and improve practical applications of phytoremediation of metal-polluted soils.

Phytoremediation is a cost-effective, eco-friendly green technology exploiting plants with their natural uptake capabilities through root organization, along with the translocation, bioaggregation or detoxifying abilities to decontaminate the soil, water and groundwater from toxic pollutants. The plant approaches to cope with several xenobiotics include phytostabilization, phytoextraction, phytovolatilization, rhizofiltration, phytodegradation and phytostimulation. Some plants can survive under severe metal stress, and higher metal tolerance can be achieved either by reduced uptake or improved plant internal sequestration of metals, an outcome of interaction between specific plant genotype and its environment. Molecular genetics have led to better understanding of mechanisms of heavy metal tolerance or accumulation in plants, and genetic engineering has unlocked new gateways in phytoremediation strategies by producing elite plants with enhanced metal remediation capabilities. This article reviews the recent developments in phytoremediation of heavy metals addressing the genetic basis of metal hyperaccumulation and tolerance and a discussion on the likelihood of transgenic plants in phytoremediation of heavy metals.

Mycorrhizal fungi, obligate biotrophs, form mutualistic associations with plants and provide mainly phosphorus to plants. Mycorrhizal fungi colonize the roots of many plants growing on metal-contaminated soils and play an important role in metal tolerance and accumulation. Even though mycorrhizae are known to inhabit metal contaminated sites; the exact mechanism of colonization is unclear. For example, how mycorrhizal fungi tolerate and maintain homeostasis to toxic metals? Could metal tolerance be transferred to host plants? If so, how do mycorrhizal associations enhance metal accumulation in plants? Mycorrhiza possesses the same constitutive mechanisms as do the higher plants to circumvent metal toxicity. The adaptive tolerance is acquired by expressing genes that confer enhanced metal tolerance under stressed conditions. Various mechanisms adopted by mycorrhizal symbionts to overcome metal toxicity are highlighted. The metal detoxification mechanisms discussed here are likely to serve as a base for developing transgenic plants with abilities of increased metal tolerance and uptake, for decontamination and restoration of the metal polluted sites.KeywordsMycorrhizaHeavy metalsGlutathionesMetallothioneinsTransporter proteins

Effects of indole-3-acetic, kinetin and spermidine assisted with EDDS on metal accumulation and tolerance mechanisms in ramie (Boehmeria nivea (L.) Gaud.)

Approaches for enhanced phytoextraction of heavy metals

Owing to serious heavy metal pollution, much attention has been paid to its effects on soil-plant systems. The research of heavy metal tolerance and hyperaccumulation of higher plants has become a hot topic in the field of pollution ecology. With the development of molecular ecology, research on the mechanisms of heavy metal tolerance, detoxification and accumulation in higher plants has made progress in recent years. There are significant differences in the tolerance to and accumulation of heavy metals among higher plant species and genotypes. Root systems are the first entrance of heavy metal pollutants from the soil into plant. Root exudates reduce the availability and toxicity of metal pollutants and play an important role in ability for plants to absorb heavy metals. Almost all heavy metal ions enter root cells with the help of a metal transporter protein that are subsequently transported to the vacuole. The synthesis of PC in response to the stress caused by heavy metals is one of the adaptive responses common in higher plants. Heavy metal tolerant genotypes have higher levels of PC than non-tolerant genotypes under heavy metal stress. GSH is the substrate that synthesizes PC, which chelates the heavy metals. Heavy metal-PC chelatins are subsequently transported from the cytosol to the vacuole and heavy metal detoxification is thus achieved. MTs play the same role and in the same way as PC under heavy metal stress. The article reviews recent advances in understanding the role of root exudates, metal transporter proteins (MTs, PC and GSH), molecular mechanisms of heavy metal tolerance and hyperaccumulation in higher plants at the molecular level. Existing problems and major topics of future research were discussed.

Ericoid mycorrhizal plants dominate in harsh environments where nutrient-poor, acidic soil conditions result in a higher availability of potentially toxic metals. Although metal-tolerant plant species and ecotypes are known in the Ericaceae, metal tolerance in these plants has been mainly attributed to their association with ericoid mycorrhizal fungi. The mechanisms underlying plant protection by the fungal symbiont are poorly understood, whereas some insights have been achieved regarding the molecular mechanisms of heavy metal tolerance in the fungal symbiont. This review will briefly introduce the general features of heavy metal tolerance in mycorrhizal fungi and will then focus on the use of "omics" approaches and heterologous expression in model organisms to reveal the molecular bases of fungal response to heavy metals. Functional complementation in Saccharomyces cerevisiae has allowed the identification of several ericoid mycorrhizal fungi genes (i.e., antioxidant enzymes, metal transporters, and DNA damage repair proteins) that may contribute to metal tolerance in a metal-tolerant ericoid Oidiodendron maius isolate. Although a powerful system, the use of the yeast complementation assay to study metal tolerance in mycorrhizal symbioses has limitations. Thus, O. maius has been developed as a model system to study heavy metal tolerance mechanisms in mycorrhizal fungi, thanks to its high metal tolerance, easy handling and in vitro mycorrhization, stable genetic transformation, genomics, transcriptomic and proteomic resources.

To understand the possible role of the plant root associated fungi on metal tolerance, their role in the uptake of heavy metals and the potential transfer of these metal ions to the plant, three strains of dark septate endophytic (DSE) fungi were isolated from a waste smelter site in southwest China, and one strain was isolated from a non-contaminated site. According to molecular phylogenetic analysis of the ITS 1-5.8S rDNA-ITS 2 gene regions and morphological characteristics, one is identified as Exophiala pisciphila, and the other three are non-sporulating fungi under the experiment condition with the nearest phylogenetic affinities to the Thysanorea papuana strain EU041814. Tolerance and accumulation abilities of the three DSE strains for metals were investigated in liquid culture. Minimum inhibitory concentrations (MIC) of Pb, Zn, and Cd were determined. It was demonstrated that the tolerance of the DSE strains varied between metal species and strains. The E. pisciphila strain is able to accumulate lead and cadmium over 20% and 5% of dry weight of biomass, respectively. Partial of the sequestrated metals can be washed with CaCh. Morphological and enzyme activity changes taking place in the presence of excessive Pb, Cd, and/or Zn also indicate that the mechanism of heavy metal tolerance and accumulation of the DSE strains would be a complex process. The findings indicated promising tolerance and accumulation of the DSE strains with potential values in metal cycling and restoration of soil and water system.

Genome architecture of the heavy metal tolerant and accumulator Hirschfeldia incana: Insights from genome sequencing, assembly, and comparative analysis

Several hyperaccumulator plant species especially the species in Brassicaceae have been extensively investigated for their metal accumulation and detoxification. For example, Arabidopsis halleri, Noccaea caerulescens (formerly Thlaspi caerulescens), and Brassica nigra have enhanced our understanding of the physiological, molecular, and genetic basis of metal hyperaccumulation and associated hypertolerance. A number of regulatory mechanisms have been developed by metal hyperaccumulator plants for their survival in metal-polluted environment. In the last decade, with the development of advanced technologies most of the information about heavy metal stress in plants has been obtained through genome sequence, transcriptome, metabolome, and proteome studies. For example, through such techniques, it has been possible to identify numerous putative genes involved in the response to metal stress. A number of membrane transporter gene families have been found in accumulator plants such as ZIP (ZRT, IRT-like protein), NRAMP (natural resistance-associated macrophage protein), YSL (Yellow-stripe-like transporter), NAS (nicotinamine synthase), SAMS (S-adenosyl-methionine synthetase), FER (ferritin Fe (III) binding), CDF (cation diffusion facilitator), HMA (heavy metal ATPase), and IREG (iron-regulated transporter) families which are predicted to be involved in the cellular uptake and transport in plants. HMAs are particularly interesting and according to many recent studies they have been shown a key player in the metal hyperaccumulation. In this regard, we have analyzed the gene expression data of model crop plants in Brassicaceae family by searching several databases available online. The publicly available online resources for these plants from websites such as http://www.ncbi.nih.gov, http://www.tigr.org, http://www.brassica.info, and related sites were searched to collect nucleotide sequences that encode heavy metal ATPases and transporter protein homologues. The criterion observed in this research is that the sequences of different metal-induced genes have functional and evolutionary similarities among species. Our hypothesis is that the functionally related sequences of the genes from different species or organisms will be having conserved pattern or motif which will be possibly related to hyperaccumulation of heavy metals. Here, I will overview these findings and highlight their contribution to the field of plant metal homeostasis, and will discuss the emerging avenues of -omics technologies and their impact in understanding the mechanisms of metal accumulation and tolerance.