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University of Pennsylvania, Philadelphia, PA, USA   |
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The
evolution of zinc hyperaccumulation MR Macnair, S Huitson & S Taylor University of Exeter, UK Our work is concerned with the genetics and evolution of zinc hyperaccumulation, particularly in Arabidopsis halleri. This species grows on metal contaminated sites in Europe, and is a sister species to the model plant, A. thaliana. Our general approach is to use genetics to generate the variation to test adaptive hypotheses. We have crossed A. halleri to A. petraea, which is neither metal tolerant nor an accumulator. The F2 of this cross segregates for both zinc tolerance and zinc accumulation. We have been testing the adaptive hypotheses that accumulation increases tolerance or that it deters herbivores. Both hypotheses are not supported by our data. An alternative hypothesis would suggest that hyperaccumulation not directly related to the accumulation of zinc: the 'inadvertant uptake' hypothesis. This hypothesis will be discussed. Fundamental mechanics of phytochelatin biosynthesis Phytochelatins (PCs), (g-Glu-Cys)n-Xaa polymers derived from glutathione (GSH) and related thiol peptides by the action of phytochelatin synthases, play a pivotal role in heavy metal tolerance in plants, fungi and some animals by chelating metal ions and decreasing their free concentrations. Despite the importance of PCs for heavy metal tolerance, it is surprising how little was known about the mechanism of PC biosynthesis until quite recently. In our talk we will describe experiments aimed at the detailed analysis of this enzyme based on our facility for the molecular manipulation and purification of active heterologously expressed Arabidopsis thaliana PC synthase 1 (AtPCS1). The talk will center on two sets of findings. The first is the counter-intuitive discovery that free metal ions are not essential for catalysis. As exemplified by Cd2+- and Zn2+-dependent AtPCS1-mediated catalysis, the kinetics of PC synthesis approximate a substituted enzyme mechanism in which heavy metal GSH thiolate and free GSH act as g-Glu-Cys acceptor and donor. Indeed, as demonstrated by the facility of AtPCS1 for the net synthesis of S-alky-PCs from S-alkyl-glutathiones with biphasic kinetics in media devoid of metals, even heavy metal thiolates are dispensable for core catalysis. The second set of findings is the first unequivocal demonstration that PC synthase is a dipeptidyl, not a tripeptidyl, transferase that forms an acid-stable g-Glu-Cys intermediate during catalysis. These data and the kinetics of heavy metal-dependent PC synthesis from GSH implicate the formation, coincident with cleavage of the a-peptidyl Cys-Gly bond of the first substrate, of an enzyme g-Glu-Cys acyl intermediate, which in turn plays the role of activated donor for transpeptidation of the second substrate. It is probable therefore that the initial nucleophilic attack on the scissile bond of the first substrate is by an enzyme hydroxyl-derived oxyanion or thiol-derived thiolate anion and results in the formation of a g-Glu-Cys-enzyme oxyester or thioester, respectively. These fundamental findings have many physiological and biomaterials implications that will also be discussed. (This work is supported by a grant (MCB-0077838) from the National Science Foundation, USA) Metal-tolerant and metal-accumulating plants –
exploration and exploitation In spite of the considerable literature on the plants of metalliferous soils in many parts of the world, our knowledge in this field remains far from complete. Many former mine- and smelter waste sites have not been studied to examine the nature of the spontaneous recolonisation processes, and many areas of naturally occurring metal-rich soils have not been explored well enough to develop comprehensive inventories of the plant species, to understand their distribution and behaviour in relation to concentrations of particular metals in the soils, to define their endemism, to assess their potential utility for a wide range of applications (including site rehabilitation, in situ remediation, rhizofiltration and phytomining), and to address issues related to propagation and conservation of the more threatened and rare species. The hyperaccumulator plants are a special subset of the metal-tolerant species; for some applications, hyperaccumulators are most appropriate. We now have extensive lists of hyperaccumulators of elements such as Ni, Zn, Mn, Cu, Co, Se, Pb, Cd and As. Much of this knowledge has emerged only during the last 20 years, from field studies and analytical work. This paper describes recent discoveries and assesses critically the most promising applications and future exploration and conservation needs. Pathways of arsenic transport and detoxification
in prokaryotes and eukaryotes This presentation will focus on the mechanisms of arsenic transport and detoxification in Escherichia coli and Saccharomyces cerevisiae. In both As(V) is taken up by phosphate transporters, and aquaglyceroporins GlpF and Fps1p facilitate As(III) entry into cells. The first step in arsenate detoxification is biotransformation to As(III) by an arsenite reductase, ArsC or Acr2p. In both organisms, the next step involves extrusion of As(III) from the cytosol. In E. coli extrusion is catalyzed by the ArsAB ATPase. In yeast this is carried out by the arsenite carrier, Acr3p. In addition, Ycf1p, a homologue of the human MRP drug pump, transports glutathione conjugates of As(III) into the yeast vacuole. Identification of the routes of arsenic uptake and efflux in humans is of importance for understanding its action as a human carcinogen and as a chemotherapeutic drug in the treatment of leukemia. While at least one extrusion system has been identified, where arsenic is pumped into bile by MRP2 in the form of As(GS)3, the pathways for arsenite uptake are unknown. Recently we have shown that the mammalian aquaglyceroporins AQP7 and AQP9 facilitate arsenite uptake into cells, suggesting that this protein could be a pathway for arsenite uptake in humans. (Supported by NIH grants GM55425, GM52216 and ES10344) Molecular physiology of metal hyperaccumulation
in plants Certain plants, known as metal hyperaccumulators, have the extraordinary ability to accumulate in their natural habitat between 1,000-30,000 ppm foliar Cd, Ni, Se or Zn, depending on the species. A better understanding of the fundamental mechanisms involved in this hyperaccumulation process should allow the development of plants more ideally suited for phytoremediation of metal contaminated soils. This type of information could also lead to better human nutrition through micronutrient mineral enrichment in foodstuffs. Our laboratory has been taking both single gene and genome-wide approaches to uncover the molecular, physiological and biochemical mechanisms underlying Ni/Zn hyperaccumulation in various Thlaspi species and Se hyperaccumulation in Astragalus bisulcatus. This includes characterization of specific genes involved in vacuolar metal sequestration and genes involved in Se reduction and methylation. A large ICP-MS based metal-profiling project is also under way. We hope this will reveal more details about the way Arabidopsis thaliana, a close relative of the Thlaspi hyperaccumulators, controls the uptake and accumulation of nutrient and non-nutrient ions including Ca, Li, Na, P, Ni, Cr, K, Mg, Fe, Mn, Co, Mo, Cu, Zn, As, Se, Cd and Pb. Phytoremediation of heavy metals from the molecular
to the field level: possible roles for phytochelatin and other cellular
components Over the past 12 years, our laboratory has carried out a multidisciplinary program to investigate the phytoremediation of heavy metals and trace elements. We have conducted four wetland and two upland studies across the United States, as well as laboratory studies in ecophysiology, microbiology, plant–microbe interactions, plant biochemistry and the genetic engineering of plants to enhance their capacity for cadmium (Cd) and selenium (Se) phytoremediation. Our results show that constructed wetlands can be efficient in the removal of Se and heavy metals from agricultural and industrial wastewaters. We have demonstrated the importance of microbes in the uptake of zinc by the hyperaccumulator, Thlaspi caerulescens, and of microbes isolated from extreme environments (saline evaporation ponds) as a possible source of genes for enhancing phytoremediation. We have shown that overexpression of individual genes of enzymes of the phytochelatin synthesis pathway leads to improved tolerance of Indian mustard plants to Cd stress, and to increased uptake of Cd. These transgenic plants also enhance the tolerance and uptake of other trace elements, including arsenic. We are currently pursuing a microarray approach to locate other genes that are potentially involved in plant responses (regulation) to heavy metal stress. Phytochelatin-dependent heavy metal detoxification
in an animal, the nematode worm Caenorhabtidis elegans Involvement of phytochelatins (PCs) in heavy metal detoxification has generally been thought to be restricted to plants and some fungal species. Here we present results from the studies of the animal model, the nematode worm Caenorhabtidis elegans, establishing that the PC-dependent heavy metal detoxification pathway also plays a critical role in at least some animals. We demonstrate that CePCS1, the C. elegans homologue of the Arabidopsis thaliana and Schizosaccharomyces pombe phytochelatin synthases, AtPCS1 and SpPCS1, when heterologously expressed in Saccharomyces cerevisiae confers heavy metal tolerance concomitant with intracellular PC accumulation, and that cell free extracts of these transformants catalyze Cd2+-dependent PC synthesis from glutathione de novo. Evidence that CePCS1 participates directly in metal detoxification in the intact organism is provided by the finding that targeted suppression of ce-pcs-1 by the double-stranded RNA interference (RNAi) technique impairs heavy metal tolerance. When grown in the presence of Cd2+, ce-pcs-1 RNAi mutant worms show severe growth retardation, undergo developmental arrest and eventually die. Given that in both plants and S. pombe an important step in PC-dependent metal detoxification is the sequestration of metal-PC complexes in the vacuole, a process catalyzed by the half-molecule ABC transporter SpHMT1 in S. pombe, we are now defining the steps downstream of CePCS1 in C. elegans. A systematic search of the C. elegans genome database discloses 58 ORFs encoding half-molecule ABC transporters only one of which is topologically equivalent to and bears greater than 50% similarity to SpHMT1. Accordingly, when the cDNA corresponding to this gene is cloned and heterologously expressed in S. pombe hmt1- mutants it suppresses their Cd2+-hypersensitive phenotype. Moreover, and in strict equivalence to the results obtained with ce-pcs-1, suppression of ce-hmt-1 by RNAi yields Cd2+-hypersensitive worms (in fact, worms that are even more sensitive to Cd2+ toxicity than their ce-pcs-1 counterparts). Although PCs have never before been considered to participate in heavy metal detoxification in animals, the results presented here not only provide the first evidence of the involvement of PCs in heavy metal tolerance in C. elegans but also demonstrate that effective heavy metal detoxification is also contingent on CeHMT1. The discovery of PC- dependent pathway for heavy metal detoxification in the organisms other than plants together with the prominence of heavy metals as environmental toxins in many disease states including human cancers, might mean that processes of this type in animals will assume wide toxicological significance. (This work is supported by a grant (MCB-0077838) from the National Science Foundation, USA)
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Illustrations: Heavy Metal Plant cartoon by Sam
Day. Arabidopsis thaliana - the model plant (Philip Rea). Micrograph
of the leaf surface of the Ni-hyperaccumulator Alyssum lesbiacum (Ute
Kraemer). Arabidopsis halleri growing at the bottom of a heap of minewaste
(Ute Kraemer) Last updated: February 18, 2003 |
