Mercury localization and speciation in plants grown hydroponically or in a natural environment.

Better understanding of mercury (Hg) accumulation, distribution, and speciation in plants is required to evaluate potential risks for the environment and to optimize phytostabilization strategies for Hg-contaminated soils. The behavior of Hg in alfalfa (Medicago sativa) plants grown under controlled conditions in a hydroponic system (30 µM HgCl2) was compared with that of naturally occurring Horehound (Marrubium vulgare) plants collected from a mining soil polluted with Hg (Almadenejos, Spain) to characterize common mechanisms of tolerance. Synchrotron X-ray Fluorescence microprobe (µ-SXRF) showed that Hg accumulated at the root apex of alfalfa and was distributed through the vascular system to the leaves. Transmission electron microscopy (TEM) implied association of Hg with cell walls, accompanied by their structural changes, in alfalfa roots. Extended X-ray absorption fine structure (EXAFS) determined that Hg was principally bound to biothiols and/or proteins in M. sativa roots, stems, and leaves. However, the major fraction of Hg detected in M. vulgare plants consisted of mineral species, possibly associated with soil components. Interestingly, the fraction of Hg bound to biothiols/proteins (i.e., metabolically processed Hg) in leaves of both plants (alfalfa and M. vulgare) was similar, in spite of the big difference in Hg accumulation in roots, suggesting that some tolerance mechanisms might be shared.

Imaging translocation and transformation of bioavailable selenium by Stanleya pinnata with X-ray microscopy.

Selenium hyperaccumulator Stanleya pinnata, Colorado ecotype, was supplied with water-soluble and biologically available selenate or selenite. Selenium distribution and tissue speciation were established using X-ray microscopy (micro-X-ray fluorescence and transmission X-ray microscopy) in two dimensions and three dimensions. The results indicate that S. pinnata tolerates, accumulates, and volatilizes significant concentrations of selenium when the inorganic form supplied is selenite and may possess novel metabolic capacity to differentiate, metabolize, and detoxify selenite concentrations surpassing field concentrations. The results also indicate that S. pinnata is a feasible candidate to detoxify selenium-polluted soil sites, especially locations with topsoil polluted with soluble and biologically available selenite.

Complexation of Hg with phytochelatins is important for plant Hg tolerance.

Three-week-old alfalfa (Medicago sativa), barley (Hordeum vulgare) and maize (Zea mays) were exposed for 7 d to 30 µm of mercury (HgCl(2) ) to characterize the Hg speciation in root, with no symptoms of being poisoned. The largest pool (99%) was associated with the particulate fraction, whereas the soluble fraction (SF) accounted for a minor proportion (<1%). Liquid chromatography coupled with electro-spray/time of flight mass spectrometry showed that Hg was bound to an array of phytochelatins (PCs) in root SF, which was particularly varied in alfalfa (eight ligands and five stoichiometries), a species that also accumulated homophytochelatins. Spatial localization of Hg in alfalfa roots by microprobe synchrotron X-ray fluorescence spectroscopy showed that most of the Hg co-localized with sulphur in the vascular cylinder. Extended X-ray Absorption Fine Structure (EXAFS) fingerprint fitting revealed that Hg was bound in vivo to organic-S compounds, i.e. biomolecules containing cysteine. Albeit a minor proportion of total Hg, Hg-PCs complexes in the SF might be important for tolerance to Hg, as was found with Arabidopsis thaliana mutants cad2-1 (with low glutathione content) and cad1-3 (unable to synthesize PCs) in comparison with wild type plants. Interestingly, high-performance liquid chromatography-electrospray ionization-time of flight analysis showed that none of these mutants accumulated Hg-biothiol complexes.

Phytoremediation of selenium using transgenic plants.

Selenium (Se) is a micronutrient for many organisms but also toxic at higher concentrations. Both selenium deficiency and toxicity are serious problems worldwide. Owing to the similarity of selenium to sulfur, plants readily take up and assimilate selenate via sulfur transporters and enzymes and can even volatilize selenium. Selenium accumulating or volatilizing plants may be used for phytoremediation of selenium pollution and as fortified foods. Several transgenic approaches have been used successfully to further enhance plant selenium accumulation, tolerance, and volatilization: upregulation of genes involved in sulfur/selenium assimilation and volatilization, methylation of selenocysteine, and conversion of selenocysteine to elemental Se. Lab and field trials with different transgenic plants have yielded promising results, showing up to ninefold higher levels of selenium accumulation and up to threefold faster volatilization rates.

Selenium volatiles as proxy to the metabolic pathways of selenium in genetically modified Brassica juncea.

In this study we demonstrate that the headspace selenium volatiles could be used as proxy to the metabolic pathways in the Se-accumulator plant Brassica juncea. The selenium metabolic pathways in wild type plants are compared to those of several genetically modified cultures. Complementary use of atomic and molecular mass spectrometric techniques also allowed for identification of yet unreported minor headspace Se-containing volatiles such as CH3SeSeSeCH3, CH3SeSSeCH3, and CH3SeCH2CH3. By combining the information resulting from this research with the previously known information about selenium metabolism in B. juncea, it is possible that a more efficacious phytoremediation tool can be constructed.

Transgenic Indian mustard overexpressing selenocysteine lyase or selenocysteine methyltransferase exhibit enhanced potential for selenium phytoremediation under field conditions.

Two new transgenic Indian mustard [Brassica juncea (L.) Czern.] lines were tested under field conditions for their ability to accumulate selenium (Se)from Se- and boron-contaminated saline sediment. The transgenic lines overexpress genes encoding the enzymes selenocysteine lyase (cpSL) and selenocysteine methyltransferase (SMT), respectively. In the first Spring planting, cpSL, SMT, and wildtype plants (WT) were compared, while SMT and WT were compared in a second, Fall planting. In the Spring planting, shoots of the cpSL transgenic plants accumulated 2-fold more Se (p < 0.01), had 1.8 times higher leaf Se concentrations (p < 0.01), and grew better on contaminated soil than WT. The SMT plants had a 1.7-fold higher leaf Se concentration than WT (p < 0.05). In the Fall planting, the SMT transgenic plants accumulated 1.6-fold more Se in their shoots than WT (p < 0.01) with Se concentrations being higher in both leaves and stems. These results conclusively demonstrate that cpSL and SMT transgenic lines have significantly greater Se phytoremediation potential than wildtype Indian mustard. Further, this study confirms the importance of field testing for evaluating future transgenic lines.

Overexpressing both ATP sulfurylase and selenocysteine methyltransferase enhances selenium phytoremediation traits in Indian mustard.

A major goal of our selenium (Se) phytoremediation research is to use genetic engineering to develop fast-growing plants with an increased ability to tolerate, accumulate, and volatilize Se. To this end we incorporated a gene (encoding selenocysteine methyltransferase, SMT) from the Se hyperaccumulator, Astragalus bisulcatus, into Indian mustard (LeDuc, D.L., Tarun, A.S., Montes-Bayón, M., Meija, J., Malit, M.F., Wu, C.P., AbdelSamie, M., Chiang, C.-Y., Tagmount, A., deSouza, M., Neuhierl, B., Böck, A., Caruso, J., Terry, N., 2004. Overexpression of selenocysteine methyltransferase in Arabidopsis and Indian mustard increases selenium tolerance and accumulation Plant Physiol. 135, 377-383.). The resulting transgenic plants successfully enhanced Se phytoremediation in that the plants tolerated and accumulated Se from selenite significantly better than wild type. However, the advantage conferred by the SMT enzyme was much less when Se was supplied as selenate. In order to enhance the phytoremediation of selenate, we developed double transgenic plants that overexpressed the gene encoding ATP sulfurylase (APS) in addition to SMT, i.e., APSxSMT. The results showed that there was a substantial improvement in Se accumulation from selenate (4 to 9 times increase) in transgenic plants overexpressing both APS and SMT.

Study of phytochelatins and other related thiols as complexing biomolecules of As and Cd in wild type and genetically modified Brassica juncea plants.

The accumulation of As and Cd in Brassica juncea plants and the formation of complexes of these elements with bioligands such as glutathione and/or phytochelatins (PCs) is studied. The genetic manipulation of these plants to induce higher As and Cd accumulation has been achieved by overexpressing the genes encoding for gamma-glutamyl cysteine synthetase (gamma-ECS) and glutathione synthetase (GS). These two enzymes are responsible for glutathione (GSH) formation in plants, which is the first step in the production of PCs. The biomass produced in both the wild type and the genetically modified plants, has been evaluated. Additionally, the total Cd and As concentration accumulated in the plant tissues was measured by inductively coupled plasma mass spectrometry (ICP-MS) after extraction. Speciation studies on the extracts were conducted using size exclusion liquid chromatography (SEC) coupled online with ICP-MS to monitor As, Cd and S. For further purification of the As fractions, reversed phase high performance liquid chromatography (RP-HPLC) was used. Structural elucidation of the PCs and other thiols, as well as their complexes with As and Cd, was performed by electrospray-quadrupole-time-of-flight (ESI-Q-TOF). In both the Cd and As exposed plants it was possible to observe the presence of oxidized PC2 ([M + H]+, m/z 538), GS-PC2(-Glu) ([M + H]+, m/z 716) as well as reduced GSH ([M + H]+, m/z 308) and oxidized glutathione (GSSG) ([M + H]+, m/z 613). However, only the GS plants exhibited the presence of As(GS)3 complex ([M + H]+, m/z 994) that was further confirmed by MS/MS. This species is reported for the first time in B. juncea plant tissues.

Phytoremediation of toxic trace elements in soil and water.

Toxic heavy metals and metalloids, such as cadmium, lead, mercury, arsenic, and selenium, are constantly released into the environment. There is an urgent need to develop low-cost, effective, and sustainable methods for their removal or detoxification. Plant-based approaches, such as phytoremediation, are relatively inexpensive since they are performed in situ and are solar-driven. In this review, we discuss specific advances in plant-based approaches for the remediation of contaminated water and soil. Dilute concentrations of trace element contaminants can be removed from large volumes of wastewater by constructed wetlands. We discuss the potential of constructed wetlands for use in remediating agricultural drainage water and industrial effluent, as well as concerns over their potential ecotoxicity. In upland ecosystems, plants may be used to accumulate metals/metalloids in their harvestable biomass (phytoextraction). Plants can also convert and release certain metals/metalloids in a volatile form (phytovolatilization). We discuss how genetic engineering has been used to develop plants with enhanced efficiencies for phytoextraction and phytovolatilization. For example, metal-hyperaccumulating plants and microbes with unique abilities to tolerate, accumulate, and detoxify metals and metalloids represent an important reservoir of unique genes that could be transferred to fast-growing plant species for enhanced phytoremediation. There is also a need to develop new strategies to improve the acceptability of using genetically engineered plants for phytoremediation.

Field trial of transgenic Indian mustard plants shows enhanced phytoremediation of selenium-contaminated sediment.

Three transgenic Indian mustard [Brassica juncea (L.) Czern.] lines were tested under field conditions for their ability to remove selenium (Se) from Se- and boron-contaminated saline sediment. The transgenic lines overexpressed genes encoding the enzymes adenosine triphosphate sulfurylase (APS), gamma-glutamyl-cysteine synthetase (ECS), and glutathione synthetase (GS), respectively. The APS, ECS, and GS transgenic plants accumulated 4.3, 2.8, and 2.3-fold more Se in their leaves than wild type, respectively (P < 0.05). GS plants significantly tolerated the contaminated soil better than wild type, attaining an aboveground biomass/area almost 80% of that of GS plants grown on clean soil, compared to 50% for wild type plants. This is the first report showing that plants genetically engineered for phytoremediation can perform successfully under field conditions.

Overexpression of selenocysteine methyltransferase in Arabidopsis and Indian mustard increases selenium tolerance and accumulation.

A major goal of phytoremediation is to transform fast-growing plants with genes from plant species that hyperaccumulate toxic trace elements. We overexpressed the gene encoding selenocysteine methyltransferase (SMT) from the selenium (Se) hyperaccumulator Astragalus bisulcatus in Arabidopsis and Indian mustard (Brassica juncea). SMT detoxifies selenocysteine by methylating it to methylselenocysteine, a nonprotein amino acid, thereby diminishing the toxic misincorporation of Se into protein. Our Indian mustard transgenic plants accumulated more Se in the form of methylselenocysteine than the wild type. SMT transgenic seedlings tolerated Se, particularly selenite, significantly better than the wild type, producing 3- to 7-fold greater biomass and 3-fold longer root lengths. Moreover, SMT plants had significantly increased Se accumulation and volatilization. This is the first study, to our knowledge, in which a fast-growing plant was genetically engineered to overexpress a gene from a hyperaccumulator in order to increase phytoremediation potential.

Speciation of volatile selenium species in plants using gas chromatography/inductively coupled plasma mass spectrometry.

Gas chromatography/inductively coupled plasma mass spectrometry (GC/ICP-MS) coupled with solid phase micro-extraction can provide a simple, extremely selective and sensitive technique for the analysis of volatile sulfur and selenium compounds in the headspace of growing plants. In this work, the technique was used to evaluate the volatilization of selenium in wild-type and genetically-modified Brassica juncea seedlings. By converting toxic inorganic selenium in the soil to less toxic, volatile organic selenium, B. juncea might be useful in bioremediation of selenium contaminated soil.