Stephen D. Ebbs

Elevated expression of TcHMA3 plays a key role in the extreme Cd tolerance in a Cd-hyperaccumulating ecotype of Thlaspi caerulescens

Cadmium (Cd) is a highly toxic heavy metal for plants, but several unique Cd-hyperaccumulating plant species are able to accumulate this metal to extraordinary concentrations in the aboveground tissues without showing any toxic symptoms. However, the molecular mechanisms underlying this hypertolerance to Cd are poorly understood. Here we have isolated and functionally characterized an allelic gene, TcHMA3 (heavy metal ATPase 3) from two ecotypes (Ganges and Prayon) of Thlaspi caerulescens contrasting in Cd accumulation and tolerance. The TcHMA3 alleles from the higher (Ganges) and lower Cd-accumulating ecotype (Prayon) share 97.8% identity, and encode a P(1B)-type ATPase. There were no differences in the expression pattern, cell-specificity of protein localization and transport substrate-specificity of TcHMA3 between the two ecotypes. Both alleles were characterized by constitutive expression in the shoot and root, a tonoplast localization of the protein in all leaf cells and specific transport activity for Cd. The only difference between the two ecotypes was the expression level of TcHMA3: Ganges showed a sevenfold higher expression than Prayon, partly caused by a higher copy number. Furthermore, the expression level and localization of TcHMA3 were different from AtHMA3 expression in Arabidopsis. Overexpression of TcHMA3 in Arabidopsis significantly enhanced tolerance to Cd and slightly increased tolerance to Zn, but did not change Co or Pb tolerance. These results indicate that TcHMA3 is a tonoplast-localized transporter highly specific for Cd, which is responsible for sequestration of Cd into the leaf vacuoles, and that a higher expression of this gene is required for Cd hypertolerance in the Cd-hyperaccumulating ecotype of T. caerulescens.

Trans-generational impact of cerium oxide nanoparticles on tomato plants

Cerium oxide nanoparticles (CeO2-NPs) are increasingly used in polishing, engine enhancement agents and many other products. Even though the acute toxicity of CeO2-NPs to plants has been investigated, the long-term effects of CeO2-NPs in the environment are still unknown. The main objective of this study was to investigate whether the treatment of tomato plants with relatively low concentrations of CeO2-NPs (10 mg L(-1)) through their lifecycle would affect the seed quality and the development of second generation seedlings. The results indicated that second generation seedlings grown from seeds collected from treated parent plants with CeO2-NPs (treated second generation seedlings) were generally smaller and weaker, as indicated by their smaller biomass, lower water transpiration and slightly higher reactive oxygen species content. An interesting phenomenon noticed in the study was that the second generation seedlings grown from treated seeds developed extensive root hairs compared with the control second generation seedlings (seedlings grown from seeds collected from untreated parent plants) regardless of the treatment. Treated second generation seedlings also accumulate a higher amount of ceria than control second generation seedlings under the same treatment conditions even though such differences are not statistically significant.

Transport of Ferrocyanide by Two Eucalypt Species and Sorghum

The wastes from some industrial processes and the tailings from gold mining contain elevated concentrations of cyanide, which reacts with iron in the media to form iron cyanide complexes. This research examined the transport and possible metabolism of ferrocyanide by two native Australian trees, blue mallee and sugar gum, and by sorghum. Hydroponic studies using 15N-labeled ferrocyanide showed that both tree species transported ferrocyanide into roots and displayed significant increases in 15N enrichment and concentration with no evidence of phytotoxicity. A subsequent experiment with blue mallee and membrane-transport inhibitors showed that 15N enrichment was significantly inhibited in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone, suggesting that ferrocyanide uptake is mediated partly by H+-symporters. A study of the time dependence of 15N translocation showed a rapid equilibration of 15N from ferrocyanide in the root of blue mallee, accompanied by a slow increase in shoot 15N, suggestive of the metabolism of ferrocyanide in plant roots. A similar experiment with sorghum showed a more rapid translocation of 15N, suggesting that the transport and/or metabolism of ferrocyanide by roots of this species may differ. The results offer additional incentive for the use of these species as vegetative cover over cyanidation wastes and for cyanide phytoremediation.

The exchangeability and leachability of metals from select green roof growth substrates

Batch extraction and leaching studies were conducted with potential green roof substrates (e.g., Axis, Arklayte, coal bottom ash, Haydite, Lassenite, lava rock, and composted pine bark). The results indicated that these materials would not likely be sources of Cr, Cu, Fe, Ni, or Zn and that Lassenite would be considered a source of Mn if the leachate concentrations were compared to USEPA drinking water standards for these elements. Lassenite would not be a source of Mn if the data was compared to a USEPA standard for Mn toxicity to aquatic life. All of the substrates tested leached Cd and/or Pb concentrations that exceeded the USEPA water quality standards at least once during the 6-month leaching study, so these materials may be potential sources of Cd and Pb in green roof storm water runoff. The leaching of Cu, Cd, Fe, Mn, Pb, and Zn was differentially influenced by time and/or the presence of a single Sedum hybridum ‘immergrauch’ plant. The leaching of Cd, Cu, and Pb displayed complex, three-way interactions between main effects (substrate type and the presence or absence of a plant) and between leaching events. For all substrates except Lassenite, the presence of a S. hybridum plant decreased the leaching of Pb over time. The leaching of Cd was generally enhanced by plants for most substrates with time. Collectively the results suggest that changes in the biogeochemical conditions within green roof systems may alter metal solubility, decreasing the leaching of some elements and increasing the leaching of others.

Accumulation of zinc, copper, or cerium in carrot ( Daucus carota ) exposed to metal oxide nanoparticles and metal ions

The release of engineered nanoparticles (ENPs) into the environment has raised concerns about the potential risks to food safety and human health. There is a particular need to determine the extent of ENP uptake into plant foods. Belowground vegetables growing in direct contact with the growth substrate are likely to accumulate the highest concentration of ENPs. Carrot (Daucus carota) was grown in sand amended with ZnO, CuO, or CeO2 NPs or the same concentrations of Zn2+, Cu2+, or Ce4+. Treatment with ZnO or Zn2+ produced a concentration-dependent decrease in root and total biomass. Ionic Cu2+ and Ce4+ caused a greater reduction in shoot biomass as compared to the corresponding ENP treatments. Accumulation of Zn, Cu, or Ce in the taproot was restricted to the taproot periderm. Metal concentrations in the taproot periderm were higher for the ionic treatments than for the ENP treatments. Radial penetration of the metals into the taproot and subsequent translocation to shoots were also generally greater for plants receiving the ionic treatment than those receiving the ENP treatment. The distribution of the metals from the ENP treatments across the periderm, taproot, and shoots differed from that observed for the ionic treatments. Overall, the ENPs were no more toxic than the ionic treatments and showed reduced accumulation in the edible tissues of carrot. The results demonstrate that the understanding of ionic metal transport in plants may not accurately predict ENP transport and that an additional comparative study is needed for this and other crop plants.

Uptake and Accumulation of Bulk and Nanosized Cerium Oxide Particles and Ionic Cerium by Radish (Raphanus sativus L.)

The potential toxicity and accumulation of engineered nanomaterials (ENMs) in agricultural crops has become an area of great concern and intense investigation. Interestingly, although below-ground vegetables are most likely to accumulate the highest concentrations of ENMs, little work has been done investigating the potential uptake and accumulation of ENMs for this plant group. The overall objective of this study was to evaluate how different forms of cerium (bulk cerium oxide, cerium oxide nanoparticles, and the cerium ion) affected the growth of radish (Raphanus sativus L.) and accumulation of cerium in radish tissues. Ionic cerium (Ce(3+)) had a negative effect on radish growth at 10 mg CeCl3/L, whereas bulk cerium oxide (CeO2) enhanced plant biomass at the same concentration. Treatment with 10 mg/L cerium oxide nanoparticles (CeO2 NPs) had no significant effect on radish growth. Exposure to all forms of cerium resulted in the accumulation of this element in radish tissues, including the edible storage root. However, the accumulation patterns and their effect on plant growth and physiological processes varied with the characteristics of cerium. This study provides a critical frame of reference on the effects of CeO2 NPs versus their bulk and ionic counterparts on radish growth.

Cadmium sorption, influx, and efflux at the mesophyll layer of leaves from ecotypes of the Zn/Cd hyperaccumulator Thlaspi caerulescens

Differential sorption and transport characteristics of the leaf mesophyll layer of the Prayon and Ganges ecotypes of the hyperaccumulator Thlaspi caerulescens were examined. (109)Cd influx and efflux experiments were conducted with leaf sections, and X-ray absorption near edge structure (XANES) data were collected from leaves as a general comparison of in vivo cadmium (Cd) coordination. There were modest differences in cell wall sorption of Cd between ecotypes. There were obvious differences in time- and concentration-dependent Cd influx, including a greater V(MAX) for Prayon but a lower K(M) for Ganges for concentration-dependent Cd uptake and a notably greater Cd uptake by Ganges leaf sections at 1000 microm Cd. Leaf sections of Prayon had a greater Cd efflux than Ganges. The XANES spectra from the two ecotypes suggested differences in Cd coordination. The fundamental differences observed between the two ecotypes may reflect differential activity and/or expression of plasma membrane and tonoplast transporters. More detailed study of these transporters and the in vivo coordination of Cd are needed to determine the contribution of these processes to metal homeostasis and tolerance.

Nitrogen supply and cyanide concentration influence the enrichment of nitrogen from cyanide in wheat (Triticum aestivum L.) and sorghum (Sorghum bicolor L.).

Cyanide assimilation by the beta-cyanoalanine pathway produces asparagine, aspartate and ammonium, allowing cyanide to serve as alternate or supplemental source of nitrogen. Experiments with wheat and sorghum examined the enrichment of (15)N from cyanide as a function of external cyanide concentration in the presence or absence of nitrate and/or ammonium. Cyanogenic nitrogen became enriched in plant tissues following exposure to (15)N-cyanide concentrations from 5 to 200 microm, but when exposure occurred in the absence of nitrate and ammonium, (15)N enrichment increased significantly in sorghum shoots at solution cyanide concentrations of > or =50 microm and in wheat roots at 200 microm cyanide. In an experiment with sorghum using (13)C(15)N, there was also a significant difference in the tissue (13)C:(15)N ratio, suggestive of differential metabolism and transport of carbon and nitrogen under nitrogen-free conditions. A reciprocal (15)N labelling study using KC(15)N and (15)NH(4)(+) and wheat demonstrated an interaction between cyanide and ammonium in roots in which increasing solution ammonium concentrations decreased the enrichment from 100 microm cyanide. In contrast, with increasing solution cyanide concentrations there was an increase in the enrichment from ammonium. The results suggest increased transport and assimilation of cyanide in response to decreased nitrogen supply and perhaps to ammonium supply.

Transport of Cd and Zn to seeds of Indian mustard (Brassica juncea) during specific stages of plant growth and development.

The accumulation of excess Cd in the seeds of cereal and other crops compromises their commercial value and presents a potential risk to human health. Indian mustard [Brassica juncea (L.) Czern.] is a moderate accumulator of heavy metals such as Cd and Zn, and the seeds are consumed throughout the world, particularly in the Indian subcontinent. The study here examined the transport of Cd into Indian mustard plants and to seeds as a function of external Cd and the stage of the life cycle (vegetative growth, flowering and seed set) to identify critical developmental windows where transport from roots to seeds was the greatest. Plants were also treated simultaneously with Zn to determine if Zn fertilization mitigated the transport of Cd to seeds. Plants treated with Cd during the seed set accumulated the highest concentrations of Cd, exceeding 8 mg kg(-1) dry weight in some instances. Cadmium accumulated during vegetative growth was not highly redistributed to seeds. No effects of Zn were observed with regard to Cd redistribution to seeds. This may be because of the relatively small Zn : Cd ratios tested. However, the results suggest that if Zn fertilization is to be used to reduce the Cd accumulation in seeds of this species, that plants should be treated during the seed set stage. As the seeds of Indian mustard consistently accumulated Cd to concentrations that exceed acceptable limits for food crops, additional study of Cd redistribution in this species is warranted.

Alteration of selenium transport and volatilization in barley (Hordeum vulgare) by arsenic

Summary The investigation of interaction between arsenic and selenium indicated that effects on both selenate transport and Se metabolism were involved. Barley (Hordeum vulgare L.) seedlings were exposed to selenate in the presence of arsenate (0, 0.01, and 1.0 mg L –1 ), with Se uptake, translocation, and volatilization measured. Selenium uptake and translocation increased significantly for seedlings exposed to 1.0 mg As L –1 . Selenium volatilization was significantly reduced by treatment with either 0.01 or 0.1mg L –1 arsenate (P < 0.001). The potential implications of this interaction in terms of the Se content