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Phytoremediation to phytochelatin – plant trace metal homeostasis

2003; Wiley; Volume: 158; Issue: 1 Linguagem: Inglês

10.1046/j.1469-8137.2003.00730.x

1469-8137

Ute Kraemer,

Plant Micronutrient Interactions and Effects

Plant trace metal homeostasis research has expanded rapidly over the past decade, a process driven by three cardinal developments. One of these was the introduction into the laboratory of naturally selected metal-tolerant plants such as Silene vulgaris (Van Hoof et al., 2001), Thlaspi caerulescens (Pence et al., 2000) and Alyssum lesbiacum (Krämer et al., 1996) for physiological, biochemical and molecular experiments. A second development was the idea that metal accumulating plants could be used for the decontamination of polluted soils – phytoremediation (R. L. Chaney, USDA-ARS, Beltsville, MD, USA). Finally, the discovery of the metal-detoxifying enzyme phytochelatin synthase in plants (Clemens et al., 1999; Ha et al., 1999; Vatamaniuk et al., 1999), which preceded its identification in an animal, Caenorhabditis elegans (O. Vatamaniuk, University of Pennsylvania, PA, USA), highlighted the power of the molecular genetic approach in uncovering the fundamental basis of metal detoxification. Where has all this research taken us? The development of phytoremediation technologies is continuing, involving transgenic (N. Terry; University of California, Berkeley) and nontransgenic approaches (E. Tel-Or, Hebrew University of Jerusalem, Israel). Several scientists reported difficulties encountered when attempting to transfer emerging technologies from the laboratory into the field. Access to contaminated experimental field sites and relevant information is often limited by legislative hurdles and regulatory uncertainties (A. Baker, University of Melbourne, Australia). Moreover, it has gradually become clear that the lack of a comprehensive understanding of the complex and tightly regulated metal homeostatic network in plants is still a major bottleneck in the development of phytoremediation technologies (Clemens et al., 2002). Therefore, some of the most promising phytoremediation strategies have involved the introduction into plants of microbial detoxification pathways for elements such as Hg (Bizily et al., 2000) and As (Dhankher et al., 2002). For example, Arabidopsis plants containing the Escherichia coli ArsC arsenate reductase (B. Rosen, Wayne State University, MI, USA) were hypersensitive to arsenate. This hypersensitivity was converted into a substantial arsenate hypertolerance and arsenate accumulation in transgenic lines expressing both EcArsC and a bacterial γ-glutamylcysteine synthetase (γ-ECS) gene (R. Meagher, University of Georgia, GA, USA) (Meharg, 2002). The accumulation of remarkably high concentrations of As is not new to the plant kingdom, because a number of ferns can accumulate even higher concentrations – up to 1000 ppm (F. Zhao, IACR Rothamsted, UK; Meharg & Hartley-Whitaker, 2002; Zhao et al., 2002). Along these lines, new metal hyperaccumulator species are still being discovered, such as a number of Ni hyperaccumulating Brassicaceae (R. Reeves, Massey University, New Zealand) and a population of Arabidopsis lyrata sp. capable of hyperaccumulating zinc (A. Murphy, Purdue University, IN, USA). This is beginning to provide researchers with a choice of model species and an opportunity to evaluate plants according to metals accumulated, extent of metal tolerance and accumulation, biomass production, as well as traits such as genome size and complexity, ease of genetic transformation, seed set, breeding system and availability of suitably related ecotypes or populations for the generation of crosses (D. E. Salt, Purdue University, IN, USA). Thus, new and more advantageous model species may be added to those that are already commonly used, namely Thlaspi caerulescens (Lombi et al., 2001; McGrath et al., 2001; S. Ebbs, Southern Illinois University, Carbondale) and Arabidopsis halleri (Baker & Whiting, 2002; Bert et al., 2002; Macnair, 2002). Meanwhile researchers are also interested in the driving force behind the evolution of metal hyperaccumulation. The hypothesis that hyperaccumulation may protect plants against herbivory is most actively tested (R. Boyd, Auburn University, AL, USA; J. Pollard, Furman University, Greenville, SC, USA). For a powerful genetic approach to metal hyperaccumulation and tolerance, segregating populations are already being studied. Crosses have been established between individuals from populations of T. caerulescens differing in the extent of metal tolerance and in specificity of metal accumulation (H. Schat, Vrije Universiteit Amsterdam, The Netherlands). All known ecotypes of T. caerulescens are hyperaccumulators and show a high degree of metal tolerance, restricting this approach to genes conferring only minor changes in tolerance, accumulation or metal selectivity. Similarly, an interspecific cross has been generated between A. halleri and a nontolerant, nonaccumulator population of the closely related A. lyrata ssp. petraea (M. Macnair, Exeter University, UK). Both parents are outcrossers, which complicates the genetic analysis. A large-scale expressed sequence tag (EST) sequencing project has been initiated for T. caerulescens, and microarrays are being generated (M. Aarts, Wageningen University, The Netherlands). Earlier work has identified several candidate genes involved in cellular metal uptake and sequestration of metal ions in T. caerulescens (L. V. Kochian, Cornell University, NY, USA). In A. halleri, expression profiling using oligonucleotide microarrays designed for Arabidopsis thaliana (U. Krämer, Max Planck Institute of Molecular Plant Physiology, Potsdam, Germany) and cDNA AFLP (S. Clemens, Leibniz Institute of Plant Biochemistry, Halle, Germany) have yielded numerous candidate genes simultaneously. In Thlaspi and Arabidopsis hyperaccumulators, these candidates include, among others, different members of the ZIP (ZRT-IRT related protein) and CDF (cation diffusion facilitator) families. The prominent role of Arabidopsis thaliana as a model organism is highlighted mainly by its suitability for genetic approaches and the availability of the full genomic sequence. In recent years micronutrient metals have been found to play vital roles in a growing number of processes, such as ethylene sensing (Rodriguez et al., 1999). Mary Lou Guerinot (Dartmouth College, NH, USA) reported the successful use of reverse genetics to demonstrate the role of the ZIP family member IRT1 in plant iron uptake (Vert et al., 2002), and the use of forward genetics to identify novel genes important in iron nutrition. Based on the physiological characterization of double-knockout mutants in two Nramp genes, S. Thomine (CNRS, Gif-sur-Yvette, France) suggested a role for Nramp proteins in the mobilization of vacuolar metal stores during germination and early seedling development. Henri Wintz (University of California, Berkeley, CA, USA) presented results of an expression profiling approach to identify transcripts which respond to micronutrient deficiency. Based on the phenotype of T-DNA knockout mutants, Chris Cobbett (University of Melbourne, Australia) proposed an involvement of the CPX-type ATPases HMA2 and HMA4 in zinc partitioning in Arabidopsis. However, we cannot yet clearly define the role of the Arabidopsis metallothioneins (P. Goldsbrough, Purdue University, IN, USA) or of a potential role in metal homeostasis of members of the CAX family of proteins related to the calcium/proton exchanger CAX1 (J. Pittman, Baylor College of Medicine, Houston, TX, USA). In a complementary, unbiased approach, mutants of Arabidopsis altered in elemental composition are being identified by employing high-throughput, inductively coupled plasma mass spectrometry (ICP-MS) analysis (D. Salt). When investigating the function of poorly characterized proteins it is often vital to reduce the complexity of the experimental system. Unicellular model systems are extremely powerful tools for addressing metal homeostasis, also because obtaining loss-of-function mutants is still much faster. This is exemplified by the work on the uptake and detoxification of As compounds in S. cerevisiae (B. Rosen), the ER zinc transporter Zhf from Schizosaccharomyces pombe (S. Clemens), and emerging work employing the unicellular green alga Chlamydomonas rheinhardtii (M. Hanikenne, University of Liege, Belgium). In recent years metabolic pathways and enzymes involved in metal homeostasis have been identified and characterized to some extent, most prominently the sulphur assimilation pathway and the enzyme phytochelatin synthase. Now it will be very important to elucidate, in detail, their regulation and biochemistry (P. Rea, University of Pennsylvania, PA, USA; Vatamaniuk et al., 2000). Another example was given by D. Oliver (Iowa State University, IA, USA). He reported that exposure to jasmonic acid or Cd was sufficient to effect an increase in γ-ECS transcript levels. However, an increase in γ-ECS protein levels was conditional on a further change in the glutathione oxidation state. In the Ni hyperaccumulator T. goesingense, serine acetyl transferase enzyme activity is elevated, and consequently tissue O-acetylserine levels are very high (D. Salt). In other metal hyperaccumulators, tissue concentrations of low-molecular-weight chelators, such as histidine (Krämer et al., 1996) or nicotianamine (S. Mari and M. Lebrun, XIII International Conference on Arabidopsis Research, 28 June to 2 July 2002, Sevilla, Spain), were found to be elevated. The genome of the hyperaccumulator A. halleri hosts several genes homologous to the ZAT gene of A. thaliana (U. Krämer), which is known to confer Zn tolerance when overexpressed (Van der Zaal et al., 1999). These gene copies differ by highly polymorphic overlapping in-frame deletions and expansions which overlap with a deletion proposed to result from alternative splicing of a homologue from the Ni hyperaccumulator Thlaspi goesingense (Persans et al., 2001). A detailed biochemical characterization of these and other proteins will be invaluable. In the Ni hyperaccumulator A. lesbiacum metal transport activities are being characterized across the membrane of isolated vacuoles (R. Ingle, University of Oxford, UK). Combined with the identification or generation of variants in expression of specific genes, novel techniques allowing us to localize metals and to determine their speciation and physiological effects at the suborgan level will be important tools. Some examples that were presented at the meeting are extended X-ray absorption fine structure (EXAFS) analysis, transmission electron microscopy combined with energy-dispersive X-ray (EDX) mapping (M. Marmiroli, University of Parma, Italy) and microscopic imaging of chlorophyll fluorescence (H. Küpper, Cornell University, Ithaca, NY, USA). With the engineering of a high-biomass, multimetal tolerant, metal-accumulating phytoremediator plant in mind, the modification of expression levels and/or introduction of multiple genes appears to be as daunting as it is indispensable. This insight may have triggered the search for hyperaccumulator genes with metalloregulatory functions (L. Kochian, S. Clemens). We should soon be able to shed light on some of the poorly understood phenomena observed in the field, such as the positive response of hyperaccumulator root growth to high, localized metal concentrations in the soil (A. Baker) or the role of the interaction of microorganisms with plant roots in modulating bioavailability of metals (J. S. Angle, University of Maryland, MD, USA). Whatever the prospects are of finding a 'master regulator', answering the questions as to how metals are sensed by higher plants, and how a signal is generated and transduced to result in a response to altered metal availability, will provide challenging and fascinating avenues for research in the years to come.