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Cloning of an ABC transporter gene involved
in cadmium tolerance in the unicellular green alga Chlamydomonas
reinhardtii Insertional mutagenesis has been used to induce Chlamydomonas
mutants hypersensitive to heavy metals. Out of 28 mutants isolated,
six were only sensitive to cadmium while five were only sensitive
to copper. The seventeen other mutants were pleiotropic and displayed
sensitivity to several (2 to 7) of the following agents: cadmium,
copper, lead, hydrogen peroxide, tert-butylhydroperoxide, paraquat,
UVC and light. Five mutations determining Cd-hypersensitivity were
found to be allelic (CDS1 gene). One of the cds1 mutants was submitted
to atomic absorption spectroscopy and gel filtration chromatography
analyses: it accumulated cadmium at the same rate as the wild type
but produced higher amounts of phytochelatins. Cloning of the CDS1
gene was undertaken using plasmid rescue in E. coli and screening
of a Chlamydomonas BAC library with the rescued probe. A BAC
clone and two derived subclones complementing the cds1 mutation were
isolated. A single gene sharing similarities with ABC transporters
was identified in these subclones. Metal-specificity of the histidine response
in hyperaccumulator plants As a response to exposure to Ni, hyperaccumulator plants in the genus Alyssum export increased amounts of the amino acid histidine from the roots to the shoot in the xylem. Previous experimental evidence has implicated this histidine response in both tolerance to Ni at the cellular level (through coordination of the metal ion) and generation of the hyperaccumulator phenotype (by facilitating Ni translocation to the shoot). In the present work, we have investigated whether the histidine response occurs in other taxa of Ni-hyperaccumulating plants and whether other metals can also generate this response. To examine the relationship between metal exposure and histidine production, xylem sap was collected as root-pressure exudate from plants exposed to a range of metal concentrations. Exposure to Ni increased xylem histidine concentrations in all species of Ni-hyperaccumulator plants tested, which included five species of Brassicaceae (Thlaspi caerulescens, T. cariense, T. goesingense, T. montanum var. siskiyouense and Streptanthus polygaloides) and one species of Asteraceae (Berkheya coddii). There was also a significant increase in xylem histidine in response to Co in T. caerulescens, as previously noted for Alyssum spp. In contrast, there was no response of xylem histidine to Zn in the Zn-hyperaccumulators Thlaspi caerulescens or Arabidopsis halleri, or to Mn in the Mn-hyperaccumulator Grevillea exul var. exul (Proteaceae). Thus, the histidine response appears to be widespread in Ni-hyperaccumulator plants, but restricted in its metal specificity to Ni and Co. Characterization of zinc tolerance genes in
the zinc/cadmium hyperaccumulator Thlaspi caerulescens Thlaspi caerulescens, a heavy metal hyperaccumulating plant
species, accumulates up to 30,000 ppm zinc in the above ground biomass
without exhibiting toxicity symptoms. Previous work in our lab has
shown that altered regulation of micronutrient uptake, transport and
sequestration in this species plays a key role in the hyperaccumulation
phenotype. Thus, T. caerulescens is an excellent model system
to study mechanisms of micronutrient homeostasis and extreme metal
tolerance. Additionally, as a member of the Brassicaceae, the rich
genomic resources of Arabidopsis thaliana are readily accessible
for comparative studies. For these reasons, our lab is examining the
mechanisms of zinc uptake and tolerance in T. caerulescens
to further provide insights into plant mineral nutrition and the use
of plants in phytoremediation efforts. While previous research has
shown increased uptake and transport of zinc from the roots to foliar
tissue in T. caerulescens relative to non-accumulating species,
the mechanism of sequestration and tolerance of these elevated metal
concentrations within the plant are still unknown. We are examining
the molecular basis for this metal tolerance through a screen based
on functional complementation in yeast. Yeast were transformed with
a T. caerulescens expression library, screened for growth on
high levels of zinc, and putative zinc tolerance genes were isolated
and identified. From the genes identified in the screen, we have narrowed
our focus to four putative zinc tolerance genes including a 14-3-3
protein, a putative protein kinase, a vesicle related protein and
a putative DNA binding protein. Current research efforts are focused
on characterizing their activity both in T. caerulescens and
A. thaliana. Analysis of accumulation and distribution
of heavy metals in ecosystems of the Eastern Carpathian Mountains
(Ukraine) Contamination of soil, surface water and groundwater with heavy metals
and radionuclides poses an increasing environmental problem in Eastern
Europe and worldwide. In the present study we are analyzing the dynamics
of contamination with heavy metals of the Eastern Carpathian Mountains
(Chornohora massif, Ukraine), territories of which are the least subjected
to anthropogenic disturbance. Evaluation of the present status of
the pollution of the Chornohora massif is based on study of the elemental
composition and concentration of heavy metals in the topsoil and plant
nappe. Assessment of the atmospheric pollution of the Chornohora massif
is based on the analysis of thaw snow, mosses and lichens. The values
for plant nappe are compared to those obtained from herbarium samples
that were collected from the same territories at the beginning of
the 20th century. Molecular aspects of the adaptive zinc
tolerance of the ectomycorrhizal fungus Suillus luteus Zinc is an essential micronutrient for life. As a structural component of the zinc finger motifs found in many transcription factors and as a catalytic co-factor for RNA polymerase, zinc is required for gene transcription. Excess zinc, however, can be detrimental to cells. Industrial activities have led to large-scale contamination of the environment with toxic heavy metals. The further spread of these heavy metals remains a permanent and current problem. Phytostabilization and, in the future, phytoremediation can offer a solution to this problem. In this project we want to find the genes that are responsible for the zinc tolerance of an ectomycorrhizal fungus, Suillus luteus, found in a zinc-contaminated area in Lommel-Maatheide (Belgium) (Colpaert et al., 2000). Understanding the molecular mechanism used by this fungus to cope with excess Zn can help us in the phytostabilization of contaminated areas. It is already clear that metal tolerant ectomycorrhizal fungi can help host plants to survive in this situation. We want to find the genes by using a cDNA-library (constructed in the pYES2 plasmid with a galactose-promoter) of S. luteus, express this library in a zinc sensitive yeast strain (zrc1D) and screen for surviving clones on plates with high zinc concentrations. After checking the selected cDNAs in yeast by making use of the galactose promoter to switch the gene on or off, we want to transform a non-tolerant isolate of S. luteus with the tolerance-cDNA to check if the cDNA is really responsible for the zinc-tolerance. Eventually, we will perform some localization studies using a Green Fluorescent Protein (GFP) construct in yeast as well as in S. luteus. Arabidopsis mutants exhibiting increased tolerance
to arsenate One proposed approach towards the remediation of arsenic is phytoremediation, the use of plants to remove and detoxify arsenic from contaminated sites. While native plants have been identified in contaminated regions with increased tolerance to toxic metals, the genetic and molecular mechanisms which confer arsenic tolerance remain largely unknown. To elucidate some of the mechanisms involved in arsenic detoxification, we developed a genetic screen using the model plant Arabidopsis thaliana. From this screen we identified a number of mutants which exhibit a significantly increased ability to grow in the presence of toxic arsenate concentrations. The strongest of these mutants, ars1, can grow on levels of arsenate which completely inhibit growth of wild type seeds. ars1 accumulates as much arsenic at the whole plant level as compared to wild type plants, suggesting that ars1 plants have an increased ability to detoxify arsenate. Phytochelatins, small metal binding peptides, are currently believed to be the primary mechanism of arsenic detoxification in plants. However, ars1 produces phytochelatin levels similar to wild type plants, and the mutation does not map to the known phytochelatin synthase genes. Furthermore, ars1 plants do not show resistance to arsenite or other toxic metals such as cadmium and chromium. These data suggest that Ars1 functions upstream of arsenite chelation by phytochelatins. Progress in the genetic, physiological and biochemical characterization of ars1 will be presented, along with models suggesting that altered arsenate biotransformation could be responsible for the ars1 phenotype. Ectopic expression of bacterial genes encoding
glutathione synthetic enzymes increases the capacity of Arabidopsis
thaliana AtPCS1 transgenics for PC biosynthesis and accumulation.
Analysis of tissue distribution of expression and subcellular localization
of AtPCS1 fusions It has been established that phytochelatin (PC) synthases, as exemplified
by Arabidopsis thaliana PC synthase 1 (AtPCS1), are capable
of conferring tolerance to heavy metals such as cadmium, mercury and
arsenic. On this basis and from what is known of the ligand requirements
and core catalytic capabilities of these synthases, many investigators
have suspected that transgenic plants with markedly increased levels
of AtPCS1 expression will exhibit an enhanced capacity to tolerate
and accumulate heavy metals. Indeed, we (and we expect others) have
shown this to be the case but have been disappointed by how moderate
the increases in PC biosynthesis are despite the large increases in
AtPCS1 transcript levels. Working from the hypothesis that PC biosynthesis
is at least in part upstream-limited by the availability of the immediate
and/or next to immediate precursors, glutathione (GSH) and g-glutamylcysteine
(g-EC), of PC biosynthesis, we have attempted to overcome this limitation
by engineering (stacking) the biosynthetic genes for these thiols
in addition to those for PCs. Specifically we have stacked the two
GSH synthetic genes concerned in series with AtPCS1 by the ectopic
expression of Escherichia coli g-glutamylcysteine synthase
(g-ECS) or glutathione synthase (GSH2) together with AtPCS1. The properties
of the various transgenic lines generated indicate simple additive
interactions between the enzymes concerned. Analyses of the AtPCS1
single transgenic lines demonstrate that overexpression of this gene
increases PC biosynthesis and the capacity for Cd2+ accumulation by
1.5- and 2.0-fold, respectively. Similar analyses of the AtPCS1 and
g-ECS or GSH2 double transgenic lines demonstrate an additive augmentation
of PC biosynthetic capacity such that plants overexpressing either
one of the ATP-dependent precursor biosynthetic enzymes accumulate
1.5-fold and 2.0-fold more PCs than AtPCS1 single transgenics and
wild-type controls, respectively. Gene stacking strategies of this
type may prove instrumental for some phytoremediation applications. Analyses of the subcellular localization of ectopically expressed AtPCS1-GFP fusions by a combination of cell fractionation and Western analysis of whole tissues and by fluorescence microscopy of isolated protoplasts reveal that although fusion protein predominantly localizes to the soluble fraction, some is also associated with the membrane fraction. The bearing this might have on processes downstream of PC biosynthesis and the properties of the Caenorhabditis elegans ortholog of AtPCS1 will be discussed. Objective environmental risk assessment
methods for genetically modified hyperaccumulators The risk of genetic pollution from genetically modified hyperaccumulators
is a cause for concern. This is especially true when gene flow may
occur between crop plants such as canola and closely related GM hyperaccumulators,
which may transfer heavy metal accumulating genes to crop plants resulting
in contamination of food crops. Such events pose human health, environmental
and economic risks. Human health and environmental risks arise because
gene flow from GM hyperaccumulators offers one possible pathway for
heavy metals to enter the food chain. Economic risks arise from potential
crop contamination leading to the rejection of the crop by trading
partners or government regulators. To date most risk assessments have
been subjective. This work addresses this issue by introducing a structured
risk assessment paradigm.
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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 |
