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Acquisition of Cd and Zn by the hyperaccumulator
plant Thlaspi caerulescens: role of chemical and spatial availability
Acid soils might adversely affect plant growth in different ways,
such as by increased availability of Al and Mn, but also of toxic
heavy metals like Cd. The rhizosphere has been the focus of intensive
research for many years because of its importance for the transfer
of mineral nutrients from soil to roots. Recently, the role of the
rhizosphere has also been recognized for the uptake of pollutants
by plants. Nowadays it is well established that acidification of the
rhizosphere mediates transformation processes resulting in a higher
chemical availability with a possible subsequent higher plant uptake.
But more recently, it has been shown that rhizosphere acidification
is not involved in heavy metal uptake of the hyperaccumulating plant
Thlaspi caerulescens (McGrath et al., 2001). Effect of arsenate treatment on growth and
arsenic accumulation in four high biomass members of the Brassicaceae The ability of four members of the Brassicaceae, Brassica juncea, Brassica carinata, Brassica nigra and Crambe abyssinica, to tolerate and accumulate arsenic was examined. Plants grown hydroponically were treated with 10 or 20 mg/L arsenate for two weeks. Plant growth, development of toxicity symptoms and accumulation of arsenic were examined. All four species exhibited a reduction in growth relative to controls, but lacked severe toxicity symptoms. The average arsenic accumulation in leaves ranged from 15 µg/dr g (B. carinata) to 82 µg/dry g (C. abyssinica) after a two-week treatment with 10 ppm arsenate. Older leaves accumulated more arsenic than younger leaves, with values reaching 130 µg/dry g in C. abyssinica. C. abyssinica shows a greater potential than B. juncea for use in the phytoremediation of arsenic. Responses of Populus deltoides x P. nigra
(P. x euramericana) I-214 to high concentrations of zinc. Implications
for phytoremediation Phytoremediation is an emerging technology that uses various plants
to remove, degrade, contain or immobilize contaminants in soil, sediments
and water. Metal-tolerant, hyperaccumulating plants can be found in
naturally occurring metal-rich sites, but these plants are not ideal
for phytoremediation since they are usually small in size and have
a slow growth rate and a low biomass production. On the other hand,
trees have a rapid growth rate and produce a high biomass. Besides,
trees have a profuse root system, for the exploration of a large volume
of contaminated substrate, and a high transpiration flux, a key variable
to determine the rate of metal uptake for phytoremediation designs.
The full-scale feasibility and cost-effectiveness of phytoremediation
requires an understanding of physiological and biochemical bases of
heavy metal tolerance, accumulation and translocation in plants. Cd tolerance and hyperaccumulation in Arabidopsis
halleri: genetic basis and identification of genes using a cDNA-AFLP
approach The genetic basis of Cd tolerance and hyperaccumulation was investigated in Arabidopsis halleri (Bert et al., in press). The study was conducted in hydroponic culture with a backcross progeny, derived from a cross between A. halleri and a non tolerant and non accumulating related species Arabidopsis lyrata ssp. petraea, as well as with the parents of the backcross. The backcross progeny segregates for both cadmium (Cd) tolerance and accumulation. The results support the following: (i) Cd tolerance is a more complex character than zinc (Zn) tolerance in A. halleri, and may be governed by more than one major gene; (ii) Cd accumulation is a complex character; (iii) Cd tolerance and Cd accumulation are independent characters; (iv) Cd and Zn tolerances are related characters; (v) Cd and Zn are co-accumulated. Published results only concerned Cd accumulation at 10 µM. Another study was conducted at 100 µM Cd and concerned the most tolerant progenies. Moreover, the interactions between Cd and Zn, Ca, Fe or Mg were studied in the backcross progeny. These new results will be presented and discussed. At the same time, analysis of the backcross progeny has allowed the selection of extreme genotypes. Comparison of the transcript patterns of the most and less Cd tolerant plants by cDNA-AFLP (Amplified Fragment Length Polymorphism) is in progress. This method may enable the identification of genes involved in cadmium tolerance in A. halleri. In vitro and in situ analysis of the effects
of heavy metals on Azobacter spp. and Rhizobium spp. Recycling of industrial wastes, particularly heavy metal salts, is one of the most crucial problems of the contemporary world. Special attention is paid to the study of how such pollutants affect microorganisms, which are an important structural element of modern agro-ecosystems. In the present study we have examined, in vitro and in situ, the effect of heavy metal salts such as CuSO4, Ni(NO3)2·6H2O, Co(NO3)2·6H2O, Sr(NO3)2, and CsNO3 on the growth of the nitrogen-fixing bacteria Rhizobium meliloti 425a, Rhizobium loti 1801, Rhizobium leguminosarum 250a and Azotobacter chroococcum. In so doing, we determined that the toxicity of the metals tested fell in the rank order copper > nickel > cobalt > cesium > strontium in all four cases. Among the symbiotic nitrogen fixing bacteria tested, R. loti strain 1801 is the most resistant to Co, Ni and Sr nitrates. R. leguminosarum strain 250a manifests tolerance only to Cs+. Co2+ and Cu2+ are lethal, even when applied at the lowest concentrations. R. meliloti strain 425a is moderately sensitive to heavy metals. The A. chroococcum strain manifests the lowest level of resistance to heavy metal salts. In R. leguminosarum 250a toxic metals increased mutation rates and decreased the production of exopolysaccharide. Long-term cultivation of R. loti 1801 in the presence of Co(NO3)2·6H2O results in the appearance of non-typical pigmentation. The resistance to Co2+ and appearance of pigmentation of this strain is genetically determined. We have isolated a small-size plasmid DNA and demonstrated its involvement in R. loti resistance to Co2+. We have also demonstrated extracellular accumulation of Co2+ in this strain. The results obtained under laboratory conditions are confirmed by the data gathered from the field experiments. The only exception is the higher resistance of A. chroococcum to heavy metals compared to those obtained in situ. Engineered tolerance and hyperaccumulation
of arsenic in plants: combining arsenate reductase and g-glutamylcysteine
synthetase We have developed a genetics-based phytoremediation strategy for arsenic where the oxyanion arsenate is transported above ground, reduced to arsenite, and sequestered in thiol-peptide complexes. The E. coli arsC gene encodes arsenate reductase (ArsC), which catalyzes the glutathione coupled electrochemical reduction of arsenate to the more toxic arsenite. Arabidopsis plants transformed with the arsC gene expressed from a light-induced soybean rubisco promoter (SRS1p) strongly express ArsC protein in leaves, but not roots, and were consequently arsenate hypersensitive. Arabidopsis plants expressing the E. coli gene encoding g-glutamylcysteine synthetase (g-ECS) from a strong constitutive actin promoter (ACT2p) were moderately tolerant to arsenic compared to wild-type. However, plants expressing SRS1p/ArsC and ACT2p/g-ECS together showed significantly greater arsenic tolerance than g-ECS or wild-type plants. When grown on arsenic, these plants accumulated 4- to 17-fold greater fresh shoot weight and accumulated 2- to 3-fold more arsenic per gram of tissue than wild-type or plants expressing g-ECS or ArsC alone. This arsenic remediation strategy should be applicable to a wide variety of plant species. We are in the process of engineering plants with higher biomass for arsenic phytoremediation and these results will be discussed. Characterisation of ZAT-like AhCDF1 genes
encoding putative vacuolar zinc transporters in A. halleri Arabidopsis halleri is a Cd/Zn-tolerant Zn-hyperaccumulator
closely related to Arabidopsis thaliana. The A. halleri
CDF1 genes encode putative metal transporters, orthologous to the
previously characterized ZAT (Van der Zaal et al., 1999), here named
AtCDF1. We have cloned a number of AhCDF1 cDNAs, each between 95 and
98% identical to AtCDF1 at the nucleotide level. The AtCDF1 protein
has been proposed to act in Zn detoxification (Van der Zaal et al.,
1999), and is a member of the cation diffusion facilitator (CDF) family
of bacterial, yeast and mammalian metal transporters (Paulsen and
Saier, 1997). CDF proteins share a common topology with six predicted
membrane spanning domains. Because of its potential ability to bind
Zn2+ ions, the histidine-rich region in the cytoplasmic loop between
membrane-spanning domains IV and V is of particular interest. Regulation of metal uptake in Arabidopsis Iron, an essential nutrient, is not readily available to plants due to its low solubility. In addition, iron is toxic in excess. Consequently, plants must carefully regulate iron uptake. We now have in hand genes that encode ferric chelate reductase (FRO2) and a ferrous iron transporter (IRT1) in Arabidopsis. IRT1 is the major transporter responsible for high affinity iron uptake from the soil; it also transports zinc, manganese and cadmium. FRO2 is known to reduce both iron and copper at the root surface. Transgenic plants engineered to overexpress IRT1 revealed that IRT1 is subject to post-transcriptional regulation by iron and zinc; IRT1 protein accumulates only in iron-deficient roots of 35S-IRT1 plants. Similar experiments with FRO2 revealed that it is also subject to post-transcriptional regulation by iron. These results demonstrate that the targeted overexpression of either IRT1 or FRO2 may not be sufficient to confer dominant gain-of-function enhancement of metal accumulation either for the purpose of phytoremediation or fortification of crop foods. 35S-IRT1 plants show enhanced sensitivity to cadmium only under iron-deficiency conditions. The enhanced sensitivity of the 35S-IRT1 plants to cadmium has allowed the identification of cadmium resistant mutants. Several of these mutants display alterations in IRT1 protein accumulation and presumably define factors involved in the regulation of IRT1 accumulation. Site-directed mutagenesis of IRT1 is also yielding insight into amino acid residues important for IRT1 protein accumulation. Comparative analysis of tolerance to low-zinc
stress in food crops Zn efficiency (ZE) is the ability of plants to maintain high yield under low Zn and its mechanisms are not well understood. In this study, Zn efficient bean and wheat genotypes were grown on chelate-buffered solution with low (0.1 pM), sufficient (150 pM) and high (1 mM) Zn2+ activities. The results showed that there was no correlation between ZE and Zn uptake, translocation to the shoot, shoot Zn accumulation, or Zn compartmentation in the leaf apoplasm, cytoplasm and vacuole. Biochemical Zn utlization was investigated by looking at the expression of genes encoding the activity of the key Zn requiring enzymes CuZn superoxide dismutase (CuZnSOD) and carbonic anhydrase (CA), as well as activities of the respective enzymes in Zn efficient and inefficient wheat lines grown under low and sufficient levels of Zn. We found that CuZnSOD and CA enzyme activities were significantly higher in Zn efficient wheat lines compared with Zn inefficient lines under Zn deficiency conditions. Furthermore, these higher activities were associated with higher levels of mRNA expression, at least for CuZnSOD. These findings suggest that biochemical utilization of Zn may play a role in ZE. The current status of this project and further detailed results will be presented. The property of plant roots to affect soil
geochemistry and release of heavy metals In this study the capacity of plant roots to influence the geochemistry of soil and release of metals, especially by release of organic acid and change in pH, was investigated. The aim was to find out how plants can regulate the metal uptake by influencing the geochemistry, if differences in root mechanisms occur between plants with different properties to accumulate low and high metal levels, and if the mechanisms vary due to the metal and concentration of the metal in the soil. Various clones of Salix were used since clones with specific properties related to heavy metal uptake exist. Therefore, two clones of each with the following traits were used: low and high cadmium (Cd) uptake, respectively; low and high copper (Cu) uptake, respectively; and low and high zinc (Zn) uptake, respectively. Plants were cultivated in nutrient medium for 3 months and thereafter transferred to a rhizobox-like system, in which the root mass was connected to the soil via a nylon net (25 µm). The soil was of compost type either untreated or spiked with Cu, Zn or Cd. After 48 hours treatment, exudation solution was collected from the rhizosphere, and analyses were made of organic acids by ion cromatography, peptides by HPLC, Cu, Cd and Zn by AAS and pH. The organic acids found (citric acid, formic acid, succinic acid, malic acid) were also used in an extraction study with untreated compost to find out how these acids lower the pH and release of metals compared with extraction in solely water or HNO3 of the same concentration (1 M). The results showed that plant roots release both organic acids (especially succinic acids) and in some cases hydrophilic peptides, and change the pH of the rhizosphere. The organic acids and the changed pH seem to influence the release of metal ions from the soil of the rhizosphere separately. According to the extraction study, the release of Cu seems to be the one most influenced by the pH, where more Cu is released the lower the pH. On the other hand, the Zn release was most affected by organic acids (but none specifically). The release of Cd seemed to be both influenced by pH and by the organic acids, and the release was more significant the more carboxyl groups the molecule had (i.e. the more H+ which could be released from each molecule). The mechanisms which plants used and which differed between clones were found in the low accumulating clones. Compared with high accumulating clones, clones with low Cd accumulation had a higher pH in the rhizosphere, which decreased the release of Cd from the colloids and thus also the uptake. At higher external Cd levels (spiked soil) a higher level of hydrophilic peptides was released from the roots, probably trapping the Cd-ions decreasing the uptake by plant roots. This, however, did not change the level of Cd in the soil solution. Clones with low Cu accumulation increased the pH of the rhizosphere when the Cu level of the surrounding soil increased (spiked soil), in this way the resorption to soil particles increased and the release from soil particles of Cu decreased. Release of organic acids was used by clones with low Zn accumulating properties to decrease the Zn uptake by roots. This, however, did not influence the soluble Zn level of the rhizosphere, which was probably a result of the organic acids binding to Zn and the complex staying in the soil solution. In the presence of higher Zn content in the soil, the mechanism of the low Zn accumulator changed towards increased pH of the rhizosphere, which diminished the release of Zn from the colloids and thereby the uptake of Zn.
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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 |
