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Identification of several members from the
ZIP family of metal ion transporters in Medicago truncatula
Several novel divalent metal transporters of the ZIP family have been characterized in Arabidopsis thaliana. Zrt1, Zrt2 and ZIP1-4 are probably Zn transporters, whereas Irt1 appears to play a role in Fe uptake. Because we are interested in metal homeostasis in legumes, we performed database searches on these genes and identified 24 ESTs in the model legume, Medicago truncatula, as probable homologs of the ZIP transporter family. Each of the ESTs was obtained and sequenced; six of them contained full-length sequences of genes sharing more than 70% homology with Arabidopsis ZIP family members. The genes from Medicago truncatula (MtZIPs) were aligned and a phylogenetic tree for the family was constructed. All of the proteins are predicted to have eight TM domains with a His rich region between domains III and IV. The expression pattern for each gene was studied by RT-PCR in roots and leaves from plants grown under different metal supplies. MtZIP5, MtZIP4 and MtZIP2 were upregulated under zinc deficiency in both roots and leaves. MtZIP5 was also upregulated in leaves under Mn deficiency and MtZIP4 under Zn and Mn toxicities. MtZIP3 was downregulated under Fe deficient conditions, while the expression of MtZIP7 was unchanged under all treatments. MtZIP2, MtZIP5 and MtZIP6 suppressed the growth defect of a Zn uptake-deficient yeast mutant, whereas MtZIP3 and MtZIP4 suppressed the growth defect of an Fe uptake-deficient yeast mutant. Phytoextraction of lead from soil and
effects of heavy metals on the properties of ion channels in plant
cell membranes Our work group is interested in the interaction of metal ions with
plants, both at macroscopic (whole plant) and microscopic (isolated
cells and biomolecules) levels. On the one hand, we are pursuing a
pre-applicative project for Phytoextraction of Lead from Soil (PhyLeS);
on the other hand, we are studying the mechanisms of ion transport
in plants, in particular ion channels that possibly play a key role
in plant growth and development. The project PhyLeS aims at verifying
the possibility of recovering soils contaminated by lead, using fast
growing and high yielding plants. The experimental field is adjacent
to a factory (now closed) that used to convert large amounts of lead
recovered from storage batteries. The average lead content in the
nearby fields ranges between 300 and 1000 mg/kg dry matter (i.e. well
above the limit permitted for land of both agronomic and industrial
interest). Both 'chelator-assisted' and 'continuous' phytoextraction
procedures were used: Brassica juncea, Helianthus annuus
and Zea mays are the plants utilised for the project. Metal
uptake by plants was stimulated by mild chelate treatment. We verified
that the treatment of contaminated soils increased lead absorption
tenfold in plant leaves. These values are not sufficient to support
an efficient phytoremediation of the soil in a reasonable period of
time; however, several parameters can be adjusted in order to improve
the system. Dealing with Ni: comparison between a
hyperaccumulator and a non-hyperaccumulator species of Alyssum
on serpentine soils Plants of Alyssum bertolonii and montanum collected from a serpentine site were analysed for Ni content. The aerial parts of A. bertolonii contained about 6000 mg g-1 dw of Ni as compared with 30 in A. montanum plants. Ni localisation in plant tissues was examined by SEM/EDX analyses of stem and root cross sections, and of leaf trichomes. Ni concentration in A. bertolonii tissues was significantly higher than in the corresponding A. montanum tissues (4-75 fold). Several other elements, such as Ca, P, Cu, Zn and Si, did not show any significantly different distribution. In A. bertolonii root parenchyma and stem epidermis had the highest Ni content. Conversely, in A. montanum it was not possible to show preferential sites for Ni storage. Moreover, in the leaf trichomes of A. bertolonii, high amounts of Ni could only be found in the inside of the trichome base, decreasing along branches; in roots, Ni content was negatively correlated with K content, even if this was significant only in xylem and periderm. When A. bertolonii plants were sown and grown for up to 7 years on Ni-free garden soil, all tissues showed considerably lower Ni levels, probably due to leaf shedding and biomass effect. Endemism vs invasibility in nickel hyperaccumulators Several species of nickel hyperaccumulators in the genus Alyssum are found on serpentine (ultramafic) soils throughout southern and eastern Europe and Asia, and some are endemic to serpentine substrates. This study examines the extent to which physiological factors restrict nickel hyperaccumulators to soil with high nickel concentrations. Two species of Alyssum were compared: (1) A. corsicum, a serpentine endemic with restricted distribution in Turkey and Corsica (Reeves 1992); and (2) A. murale, a widespread species on serpentine soils in southern Europe and Asia that also grows in non-serpentine areas in the Balkans (Brooks, 1998). Vegetative and reproductive characteristics of these two Alyssum species were compared in soil treatments with experimentally controlled nickel levels created by mixing nickel salts (1:1 nickel sulfate and nickel acetate) with fertilized sandy loam soil (pH 8.5) to produce a high nickel treatment (500 mg Ni/kg added), medium nickel treatment (50 mg Ni/kg added) and low nickel treatment (no nickel added). Both species germinated well without nickel and vegetative growth was not significantly different in high and low nickel soils. Nickel concentrations in vegetative and reproductive tissues were measured by ICP increase with soil nickel concentrations. Results of more than 2000 hand crosses indicate that both species are self-incompatible, the proportion of flowers that set seed is low, and seed production in high nickel soil is significantly greater than in medium nickel soil. Metallicolous and non-metallicolous populations
of Thlaspi caerulescens: a comparison of metal accumulation
capacity In continental Europe, the heavy metal hyperaccumulator Thlaspi caerulescens exists on both metalliferous and normal soil. We have compared the two types of population for growth, metal tolerance and accumulation capacity in controlled conditions with different forms of Zn. It appears that the two types of populations systematically differ in a number of traits. Non-metallicolous populations are only slightly less tolerant to Zn than metallicolous ones, this being apparent only with highly available Zn forms. Zn accumulation capacity is higher in non-metallicolous populations, this being apparently not due to a higher Zn mobilisation capacity. Within-population variation in Zn accumulation capacity is more restricted in metallicolous populations, suggesting that these have been subjected to directional selection in favour of a lower accumulation capacity. The possible implications of these findings to phytoremediation will be discussed. Heavy metal transport into root cells of
rice (Oryza sativa) Although essential heavy metal ions, such as Ni2+, are of major importance
in different enzymatic reactions, excess cellular levels of such metals
ares toxic to all living cells. Nickel is an important component in
many industry-originating soil contaminants. We are interested in
understanding the transport processes underlying the uptake of nickel
into the rice (Oryza sativa) root tip. Since rice is a staple
food for more than half the world's population, such knowledge could
serve in generating plants safe for consumption. To detect the time
course of Ni uptake into cells, we used fluorescence confocal microscopy
imaging of cells preloaded with the fluorescent heavy-metal indicator
Newport Green (Molecular Probes). Increased fluorescence could be
observed upon Ni application to root tips of whole 5-days old seedlings
of rice (Dongjin var.). To enable a detailed characterization of Ni
transport via the plasma membrane, we developed a method for the isolation
of root tip protoplasts. In 15-30 mm diameter protoplasts (attributable
to the elongation zone), Ni entry was linear with time in the order
of minutes. A carrot potassium channel with low susceptibility
towards metal ions Kdc1 is a potassium channel gene cloned from carrot roots that displays a peculiar sensitivity to heavy metals compared to other potassium channels. The characteristics of KDC1 currents were investigated by voltage-clamp method in Xenopus laevis oocytes coinjected with KDC1 and KAT1 (a potassium channel cloned from Arabidopsis thaliana) mRNA. The currents of the homomeric KAT1 channel are blocked by millimolar concentrations of Zn2+ added to the bath solution; on the contrary, the KDC1:KAT1 currents are enhanced by zinc. These effects might be due to the unusual amino acid composition of KDC1 in terms of histidine residues that are absent in the pore region, but present (four per subunit) in the proximity of the pore entrance. In particular, two histidine pairs H224-H225 and H161-H162 are present in the S3-S4 and S5-S6 channel linkers, respectively. Point mutations have been introduced to investigate the role of these histidines in the modulation of KDC1 currents by heavy metals. Histidines were mutated into alanines and mutated KDC1-mRNA was co-injected, together with KAT1 (ratio 1:1), in oocytes. No current increase was observed on the addition of Zn2 to oocytes co-injected with KAT1 and the KDC1-H224A mutant (i.e. Zn2+ inhibits the ionic current of the heteromeric-mutated channel, which behaves like the homomeric KAT1 channel). Conversely, the channel containing the KDC1-H225A mutation shows the same current enhancement as the wild-type heteromeric channel. These results were also confirmed by the co-injection of KAT1 with the double KDC1 mutant (H224A-H225A). Zn2+ was found to inhibit the potassium current in this experiment as well. On the contrary, the single mutations H161A or H162A do not show, on the addition of zinc, any inhibition of the current. Instead, Zn2+ induces a decrease of the current of the double mutant (H161A- H162A). This suggests that residue H224 alone and the two residues H161-H162 together may play a key role in the zinc-enhancement of the heteromeric KDC1:KAT1 current. Under the same conditions, nickel inhibition of the current was also reduced, but no current increase was observed. Channels like KDC1 could be at least partially responsible for the higher resistance of carrot cells in the presence of metals, and the insertion of metal-resistant K+ channel might participate in modulating plant tolerance to heavy metals. Identification and characterization of
a heavy metal transporting P-type ATPase from the heavy metal hyperaccumulating
plant species, Thlaspi caerulescens Thlaspi caerulescens is a Zn/Cd-hyperaccumulating plant species
that can accumulate and tolerate up to 30,000 ppm Zn and 4,000 ppm
Cd in the shoots without exhibiting toxicity symptoms. As part of
an overall program aimed at elucidating the molecular and physiological
mechanisms of heavy metal hyperaccumulation in T. caerulescens,
the research described here is focused on identification of genes
responsible for extreme Cd tolerance exhibited by this plant. We have
employed yeast functional complementation techniques to identify gene(s)
that play a role in Thlaspi Cd tolerance. We found that 100
mM Cd in the growth medium is lethal for our wild type yeast strain.
Subsequently, we complemented this yeast strain with a T. caerulescens
cDNA library in the yeast expression vector, pFL61, and have identified
a number of colonies of transformed yeast that grow well on this restrictive
level of Cd. After sequencing the transformants, this information
was used for sequence comparisons in GenBank, and several genes were
identified that may play a significant role in Cd tolerance mechanisms
in T. caerulescens. The effect of the pH of pH buffered nutrient
solutions on nickel hyperaccumulation by Alyssum corsicum and
Berkheya coddii It is hypothesized that plant hyperaccumulation of Ni evolved as
a defense mechanism against diseases and insects. Two hyperaccumulators,
Alyssum corsicum and Berkheya coddii, were compared
to Brassica oleracea grown in MES-HEPES buffered nutrient solutions
and maintained at four pH levels (5.6, 6.2, 6.8 and 7.5) and two Ni
concentrations (31.6 µM and 316 µM). This experiment followed
an earlier pot experiment comparing a Brockman serpentine soil with
two Ni-smelter contaminated soils where soil specific ion interactions
played a significant role in determining the effect of soil pH on
Ni hyperaccumulation. This study was designed to characterize the
effect of solution pH on plant uptake independent of soil ionic interactions.
A. corsicum and B. coddii showed hyperaccumulation of
Ni in shoots increased with solution pH. B. oleracea showed
signs of Ni-phytotoxicity stress at all pH levels except pH 7.5 where
Ni accumulation was significantly reduced. Characterization of a NifS-like Chloroplast
Protein from Arabidopsis thaliana - Implications for its Role
in Sulfur and Selenium Metabolism NifS-like proteins catalyze the formation of elemental S and alanine
from cysteine or of elemental Se and alanine from selenocysteine.
Cysteine desulfurase activity is required to produce the sulfur of
iron-sulfur clusters, while selenocysteine lyase activity is needed
for the incorporation of Se in selenoproteins. In plants, the chloroplast
is the location of (seleno)cysteine formation, as well as a location
of iron-sulfur cluster formation. The goal of these studies was to
identify and characterize chloroplast NifS-like proteins. Using selenocysteine
as a substrate it was found that 25-30% of the NifS activity in green
tissue in Arabidopsis thaliana is present in chloroplasts.
A cDNA encoding a putative chloroplast NifS-like protein, AtCpNifS,
was cloned and its chloroplast localization was confirmed using immunoblot
analysis and in vitro import. AtCpNIFS is expressed in all major tissue
types. The protein was expressed in E. coli and purified. The enzyme
contains a pyridoxal 5'-phosphate (PLP) cofactor and is a dimer. It
is a type II NifS-like protein, more similar to bacterial selenocysteine
lyases than to cysteine desulfurases. The enzyme is active on both
selenocysteine and cysteine, but has a much higher activity towards
the selenium substrate. This may reflect the need for regulation of
this enzyme in chloroplasts, being the site of cysteine synthesis,
or it may reflect a possible role in selenium metabolism in plants.
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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 |
