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Short Talks The role of soil microorganisms in the uptake
of heavy metals by hyperaccumulators Uptake of heavy metals by hyperaccumulators is influenced either directly or indirectly by the presence of soil microbes within the rhizosphere. Most hyperaccumulators studied to date are not colonized by mycorrhizae, however a few exceptions have been reported. Vesicular mycorrhizae either increase or decrease metal uptake depending upon the initial metal content of soil. Bacteria within the rhizosphere of hyperaccumulators are quite different from bacteria lacking an association with a plant or found within the rhizosphere of non-hyperaccumulators. Generally, bacteria isolated from the rhizosphere of hyperaccumulators exhibit greater metal tolerance, reduced biodiversity, greater ability to produce acids and greater ability to solubilize metals. Reduced biodiversity has been observed both at a functional level as well as a genetic level. Individual bacteria that were both tolerant of Ni and able to solubilize this element were inoculated into a high Ni soil and planted to Alyssum murale. In greenhouse pot trials, the bacterial species increased Ni uptake into the plant by 33%. Uptake of other elements was not increased, including Co. This organism is currently being investigated for its ability to increase Ni uptake of plants grown under field conditions in Oregon. Mechanisms of As tolerance in Cytisus
striatus Enhanced arsenate tolerance was found in the mine population of the
shrub Cytisus striatus as compared to a nonmetallicolous population.
This increased tolerance is probably due to natural selection imposed
by the high As-concentrations on the mine site. The uptake of arsenate
was suppressible by phosphate, confirming that arsenate uptake is
mainly mediated by P-transporters. Our results suggest that the variation
in root growth response is determined solely by variation in uptake,
supporting the theory of Meharg and Macnair (1992) that enhanced As-tolerance
is entirely achieved by reducing uptake to a level that the internal
detoxification machinery can handle. When PC syntheses were inhibited,
both nonmetallicolous and tolerant plants exhibit a hypersensitive
response, showing that PC-detoxification is an essential mechanism
in the presence of arsenate. This study provided no evidence, however,
to support the involvement of PC-based detoxification in differential
As-tolerance as it was found for Holcus lanatus (Hartley-Whitaker
et al., 2001). After one week of exposure, the variation in PC-production
in C. striatus is governed by a variation in As accumulation.
However the PC chain length distribution between the two ecotypes
was clearly different, pointing to a possibly more effective detoxification
system in the mine population. Phytoextraction of Cd from contaminated soils
requiring remediation Ecotypic variation in the transport, compartmentation
and coordination of Cd between populations of the metal hyperaccumulator
Thlaspi caerulescens Thlaspi caerulescens is a hyperaccumulator of cadmium and zinc. While T. caerulescens populations from different areas are all found growing in soils with elevated heavy metals, the soil concentrations of Zn and Cd vary 10-fold or more. The hyperaccumulation of these metals by T. caerulescens also varies significantly, but is not related to the metal content of the soil. That is, the most efficient hyperaccumulators of a given metal do not necessarily originate on soils with the highest concentration of that metal. Our research is exploring the ecotypic variation in Cd hyperaccumulation between selected populations of T. caerulescens, searching for the underlying physiological, biochemical, and molecular basis of this variation. Transport data from radiotracer uptake studies will be presented, describing the variation in Cd transport observed between populations and its relation to the hyperaccumulation potential of each ecotype. In addition, results from subcellular compartmentation studies and X-ray spectroscopy analyses with selected populations will be presented describing the distribution and coordination of Cd in the different populations. By relating the pattern of accumulation and coordination to the extent of hyperaccumulation, the results will indicate the relative importance of specific biochemical and transport processes to metal hyperaccumulation and tolerance in this species. Evidence for Ni-proton antiport activity at
the vacuolar embrane of the hyperaccumulator Alyssum lesbiacum Hyperaccumulator plants are characterized by their ability to sequester high concentrations of metals in cell vacuoles, specially in their shoot tissues. As well as being partly responsible for the hyperaccumulator phenotype, accumulation of Ni in the cell vacuole must also be vital in such plants for metal-ion homeostasis in the cytoplasm. However, the transport mechanism by which metal ions are moved across the vacuolar membrane in these plants has not yet been elucidated. To investigate Ni transport at this membrane, isolated vacuoles were prepared from leaf mesophyll-cell protoplasts of the hyperaccumulator species Alyssum lesbiacum. Vacuoles were loaded with the Ni-sensitive fluorescent dye Newport Greenä and suitable conditions established for its use in ion-transport assays. Nickel uptake into individual isolated vacuoles was monitored in real time by confocal microscopy and was found to be stimulated by Mg/ATP. ATP-stimulated Ni uptake was abolished by bafilomycin and by dissipation of the transmembrane H+ gradient with ammonium sulphate. These observations are consistent with energization of Ni transport at the vacuolar membrane by Ni2+/H+ antiport driven by the activity of the H+-translocating V-ATPase. This transport mechanism would permit secondary active transport of Ni2+ into the vacuole against its electrochemical gradient and help to account for the high concentrations of Ni accumulated in leaf-cell vacuoles of A. lesbiacum. This work provides some further insight into the molecular basis of Ni tolerance in Alyssum and may aid in the identification of genes involved in Ni hyperaccumulation. Analysis of the heterogeneity of photosynthesis
in heavy metal-stressed plants by microscopic and macroscopic imaging
of chlorophyll fluorescence kinetics Macroscopic and microscopic fluorescence kinetic imaging were used to analyse the heterogeneity in photosynthetic response to heavy metal (Cd2+, Cu2+, Zn2+) treatment of plants that hyperaccumulate Zn and Cd (Arabidopsis halleri (= Cardaminopsis halleri) and Thlaspi caerulescens) as well as the taxonomically related non-accumulator plants Thlaspi fendleri and Thlaspi ochroleucum. Pronounced heterogeneity of photosynthetic characteristics occurred in leaves of a metal-resistant species and of resistant plants in a non-resistant species. A few mesophyll cells became partially inhibited while the majority survived intact, indicating selective metal sequestration in the former. In sensitive plants, as a rule, a non-localised, uniform, damage occurred all over the attacked leaves. If any heterogeneity was found it was a gradual transition from more affected to less affected cells. If differences in resistance were observed in one population then more resistant plants displayed a more pronounced pattern of microscopic heterogeneity. The correlation between resistance and heterogeneity documented in this paper complements earlier results on heterogeneity of heavy metal accumulation obtained by x-ray microanalysis. The heterogeneity appeared upon onset of heavy metal stress and lasted for weeks or months, before the plants either adapted or died. We conclude that in metal-resistant plants controlled heterogeneity of accumulation and damage represents an emergency defence mechanism. Additionally it was found that hyperaccumulator plants are as sensitive towards metals other than the accumulated ones as related non-accumulator species. Finding how and where plants bind the
metals: a combined EXAFS and SEM/EDX approach for lead EXAFS (Extended X-ray Absorption Fine Structure) spectroscopy was utilized to investigate the atomic environment of lead in tissues of walnut plants. EXAFS analyses were performed at the GILDA beamline inside the ESRF (Grenoble, France), operating at 6 GeV. Pb-LIII EXAFS spectra of powdered roots from walnut grown on lead nitrate supplemented soil were compared with spectra from lead salts and organic compounds, and of cellulose treated with lead nitrate. The latter highly resembled the one from walnut roots in peak position, number, phase in the imaginary part and intensity. According to fitting procedures, the most likely first nearest neighbors in Pb-treated cellulose matrix are oxygen atoms. Comparing with spectra from other Pb standards allowed us to rule out the presence of Pb salts or lead oxides. However by comparison with other lead organic compounds of simpler structures, it was possible to find out a certain degree of resemblance with Pb-salicylate and Pb-catechol. An analogous set of considerations held for root powder, in which therefore the cellulosic part of the plants seemed to play the major role in Pb storage. This conclusion was also supported by SEM/EDX data showing that Pb was concentrated in the outermost tissues, which are particularly rich in lignin and cellulose. Regulation of glutathione biosynthesis in
plants Glutathione (GSH) fills a broad range of functions in plants. In
addition to its role in normal metabolism it is essential for protecting
plants from heavy metals, oxidative stress and a range of xenobiotic
compounds. We are interested in determining how plants are able to
control the synthesis of glutathione so as to provide ample amounts
of the chemical for all of these different functions. When plants
are exposed to cadmium and copper, transcription of the genes for
glutathione biosynthesis is activated. At least in part this activation
proceeds through an as-1 cis-element and a TGA transcription factor.
Jasmonic acid also activates this same suite of genes by a parallel
mechanism. The presence of large amounts of these mRNAs, however,
does not necessarily mean that there will be elevated amounts of the
proteins. Our research suggests that the translation of these mRNA
molecules is strictly controlled and that protein synthesis only proceeds
when the ratio of GSH to GSSG drops due to oxidative stress. Finally,
g-glutamylcysteine synthetase activity is strongly controlled in vivo
by feedback inhibition, thus regulating the final amount of GSH present.
This combination of transcriptional, translational and post translational
controls allow plants to regulate glutathione levels in response to
a range of physiological and environmental parameters. Analysis of the mechanism and function of
the cation/H+ antiporter CAX2 in Mn2+ transport The vacuolar sequestration of metals is an important metal tolerance mechanism. The Arabidopsis thaliana vacuolar transporters CAX1 and CAX2 were originally identified as Ca2+/H+ antiporters. CAX2 has a much lower affinity for Ca2+ than CAX1 and can transport other metals. CAX2 has previously been shown to provide tolerance to Mn2+ stress when expressed in tobacco and increased transport of Ca2+, Cd2+ and Mn2+ was observed in membrane vesicles isolated from these plants. We have performed a structure/function study to identify domains on CAX2 that determine Mn2+ transport. As CAX1 cannot transport Mn2+, chimeras were made between CAX1 and CAX2, and various regions of the CAX2 sequence were identified that are essential for Mn2+ transport but do not disrupt Ca2+ transport. Furthermore, competition analysis indicated that the transport of other metals by CAX2 might also be determined by certain domains. Comparisons of full-length and N-terminal truncated variants of CAX2 found that the presence of the N-terminal tail prevents both Ca2+ and Mn2+ transport, suggesting a mechanism of regulation of metal transport. We are currently determining the possibility of functional overlap with CAX2 by studying two closely related genes, CAX5 and CAX6. We are also analyzing cax2 deletion mutant plants to determine the function and physiological importance of CAX2. Responses of herbivores to cadmium hyperaccumulation
in Thlaspi caerulescens The elemental defense hypothesis proposes that metal hyperaccumulation defends plants against herbivores through toxicity or deterrence. Research on this hypothesis has previously considered nickel and zinc. We have investigated the effects on herbivores of cadmium hyperaccumulated by Thlaspi caerulescens, spurred by the knowledge that some ecotypes of this plant hyperaccumulate much more cadmium than others. Using three herbivores, Schistocerca gregaria (generalist), Mamestra brassicae (oligophagous) and Pieris brassicae (crucifer specialist), we compared feeding preferences and survival on T. caerulescens plants with contrasting shoot cadmium concentrations resulting either from cultivation in substrates with differing cadmium availability, or from ecotypic differences between plants grown in a uniform substrate. In non-choice feeding tests, high-cadmium plants were toxic toward all herbivores. In preference tests, there was less feeding on high-cadmium plants. Behavioral studies suggested that this results from post-ingestive learned avoidance of cadmium rather than inherent distastefulness, but that specialists are more easily deterred than generalists. The high-cadmium ecotype was avoided, but also had lower acceptability when grown in cadmium-free medium, because of an unidentified plant property acting as a rapid deterrent. This emphasizes that elemental defenses do not operate in isolation, but in the context of the whole suite of plant physicochemical characteristics. An Azolla filter for heavy metal binding
and Nymphaea as a tool for sludge treatment The aquatic fern Azolla was found to take up heavy metals from polluted effluents and store the toxic ions in insoluble moieties in the cell wall and in the vacuole. Pectin, phytic acid and polyphosphates are involved in the binding of the heavy metal ions by ion-exchange. High affinity heavy metal binding was demonstrated in a process of biofiltration with Azolla biomass packed in columns. This process was applied to the treatment of industrial and mining waste, and in the ultrapurification of radioactive wastes. The effluents of these wastes were purified down to parts per million and even parts per trillion, and the metal binding capacity reached 4-10% w/w. This process is equivalent to or even better than ion-exchange with resins. The water lily Nymphaea was also introduced for detoxification of industrial sludges polluted with heavy metals. The metal ions were taken up by the roots in the sludge and by the shoots in the plant body. Epidermal gland cells were found to accumulate the heavy metals, which are bound to tannins, polyphenols and phytic acid. Nymphaea demonstrated high binding capacity of the toxic ions. This phytoremediation is a promising green technology for industrial waste treatment. AtNRAMP3 encodes a vacuolar metal transporter
that down regulates heavy metal accumulation under iron deficiency
in Arabidopsis The NRAMP gene family encodes integral membrane proteins mediating
the transport of heavy metals in bacteria, fungi, plants and animals.
NRAMP homologous genes have been identified in many plant species
including dicots (tomato, cotton, Medicago, Arabidopsis) and
grasses (rice, sorghum, maize). In the Arabidopsis genome,
six genes are predicted to encode proteins with high homology to NRAMPs,
AtNRAMP1 to 6. In addition, the ethylene insensitivity gene EIN2,
which functions in transduction of multiple stress signals, contains
an NRAMP homologous domain [3]. cDNAs correponding to Arabidopsis
NRAMP1,2,3 and 4 genes have been cloned and shown to encode metal
transporters. Comparative analysis of Arabidopsis
gene expression profiles in response to copper, zinc and iron deficiencies Virtually all organisms on earth depend on transition metals such as copper, iron and zinc for survival. Iron and copper are particularly important because they participate in vital electron transfer reactions, and are thus cofactors of many metabolic enzymes. Their ability to transfer electrons also renders them toxic when present in excess, so disturbances of their steady-state levels can have profound effects on cellular metabolism, growth and development. It is critical to maintain these metals in a narrow range between utility and toxicity. Zinc plays a critical role in transcriptional regulation of gene expression. It is a component of the zinc fingers found in many transcription factors and a catalytic cofactor of RNA polymerase. Although zinc is less toxic than copper and iron, its homeostasis must be controlled in a narrow range to assure proper growth and development. Organisms ranging from bacteria and plants to mammals have developed sophisticated mechanisms to control metal homeostasis. We now have the opportunity to analyze at a genomic scale the responses of organisms to perturbation in nutritional metals and better understand the mechanism involved in these responses. We have used Affymetrix DNA microarrays containing 8,300 genes to analyze gene expression profiles in roots and in leaves of Arabidopsis plants exposed to deficiencies in copper, iron and zinc, respectively. We will present a comparative analysis of these expression profiles with special emphasis on the genes involved in metal homeostasis, transcription factors and signal transduction.
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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: January 17, 2007 |
