Chelator profiling in Deschampsia cespitosa (L.) Beauv. Reveals a Ni reaction, which is distinct from the ABA and cytokinin associated response to Cd.

Plant hormones, including abscisic acid (ABA) and cytokinins (CKs), fluctuate as a result of excess metal exposure. Changes in hormonal concentration regulate plant growth and may also signal activation of metal chelators. The grass Deschampsia cespitosa was dosed with either Ni or Cd or pulsed with exogenous ABA. The roots were analyzed for ABA and CKs and for multiple potential metal chelators including: amino acids, nicotianamine (NA), and phytochelatins (PCs). They were quantified after 3 h and after 7 days, using LC-ESI MS/MS. The Ni treatment caused no measurable change in ABA or CK concentration; however, an increase in NA was documented. The Cd treatment resulted in a short-term ABA increase followed by a reduction in CKs and an increase in PC concentration. An exogenous ABA pulse in non-metal challenged plants induced changes in CKs and PCs that followed those of Cd treatment. Ni and Cd stress resulted in distinctly different detoxification responses. Since the reaction of CKs and putative metal chelators to Cd stress can be mimicked by an exogenous ABA pulse, it is suggested that ABA acts as a stress signal, resulting in reduced growth by way of decreased CK concentration and reduced metal toxicity through increased PC production.

Identification and quantification of glutathione and phytochelatins from Chlorella vulgaris by RP-HPLC ESI-MS/MS and oxygen-free extraction.

Phytochelatins are short, cysteine-containing, detoxification peptides produced by plants, algae, and fungi in response to heavy metal exposure. These peptides auto-oxidize easily. Current extraction protocols do not adequately address losses of phytochelatins because of their oxidation and the use of indirect methods for quantification. Method enhancements include the use of an argon environment during extraction to reduce auto-oxidation, the use of glycine-(13)C2-labeled glutathione as an internal standard, and an electrospray ionization source with a triple quadrupole mass spectrometer as a detector. The method-detection limits were 0.081 microM for glutathione, 0.440 microM for phytochelatin 2, and 0.120 microM for phytochelatin 3. These detection limits were comparable to similar studies and were not compromised incorporating these adjustments. The use of a labeled internal standard and an inert gaseous environment during sample preparation greatly improved calibration linearity and sensitivity. Furthermore, phytochelatin degradation was significantly reduced and more accurately tracked. Previous studies involving phytochelatin analyses have likely been subject to higher variability caused by this propensity for phytochelatins to degrade rapidly in air. The method adjustments were simple and cost-effective and allowed phytochelatin analyses to be performed for hours at a time with minimal auto-oxidation.

The quantification and distribution of pollution Pb at a woodland in rural south central Ontario, Canada.

Lead concentrations and Pb isotope ratios were measured in the forest floor, mineral soil and vegetation at a white pine and a sugar maple stand in a woodland in south central Ontario. Lead concentrations decreased and 206Pb/207Pb ratios increased with mineral soil depth reflecting the mixing of pollution and natural Pb sources. Lead concentrations and 206Pb/207Pb ratios at 20-30 cm depth were approximately 6-7 mg/kg and 1.31-1.32, respectively. Assuming an integrated 206Pb/207Pb ratio in deposition over time of 1.18, estimated from lichen measurements and published data for the region, approximately 65% of Pb in the surface (0-1 cm) mineral soil is from anthropogenic sources. Approximately 90% of pollution Pb is found in the 0-10 cm soil layer (Ah) and less than 3% of the pollution Pb is present in the forest biomass and mull-type forest floor combined. Despite low Pb concentrations in vegetation (<2.5 mg/kg), we estimate that between 65 and 100% of the Pb in vegetation and approximately 75% of the Pb in the forest floor is from pollution sources. In total, the pollution Pb burdens at the pine and maple stands are estimated to be 860 and 750 mg/m2, respectively.

Uptake of 207Pb and 111Cd through bark of mature sugar maple, white ash and white pine: a field experiment.

A field study was undertaken to determine whether 207Pb and 111Cd, applied to the exterior bark of sugar maple (Acer saccharum Marsh.), white ash (Fraxinus americana L.) and white pine (Pinus strobus L.), could enter xylem tissue. Stable isotope tracers (3 microg Pb ml(-1); 2 microg Cd ml(-1)) were applied separately to bark in simulated rainfall, acidified to pH 4.5, in multiple doses over a 4 month (July-October) period. Tree cores were extracted from the region of application in the following March, and Pb and Cd isotopes were measured in bark and the outer tree rings using inductively coupled plasma mass spectrometry. The majority of the applied stable isotope tracer recovered (over 94%) was present in bark tissue, although a small amount of each metal tracer entered the outer (1-3) tree rings in all trees. Despite high concentrations of excess 207Pb in bark (up to 50 microg g(-1)), the maximum concentration of excess 207Pb measured in tree rings was only around 50 ng g(-1), which represents less than 30% of the background Pb concentration in wood at the study site. High excess 111Cd concentrations in bark (up to 35 microg g(-1)) also resulted in small increases in 111Cd in wood (up to 50 ng g(-1)), but due to lower background Cd concentrations in wood, such increases more than doubled the amount of Cd in wood compared with background levels. However, at sites where such high bark Cd concentrations are found, uptake from Cd-contaminated soil would probably be much greater than found at our study site. It appears that Cd and Pb applied to bark can enter woody tissue, but that this route of uptake is likely to be a minor contributor to the metal burden in wood.

Historical changes in lead concentrations in tree-rings of sycamore, oak and Scots pine in north-west England.

Lead concentrations in tree rings of sycamore (Acer pseudoplatanus L.), oak (Quercus robur L.) and Scots pine (Pinus sylvestris L.) sampled at a parkland in north-west England were measured in wood formed since the mid-1800s. Concentrations of Pb in Scots pine and oak peaked in wood formed between 1900 and 1940, most likely because of Pb accumulation in heartwood, indicating that oak and Scots pine are unsuitable for monitoring temporal changes in Pb deposition at the study site. In contrast, Pb concentrations in sycamore, a species that has similar heartwood and sapwood chemistry, were relatively constant in wood formed between the mid-1800s and 1950. Lead concentrations decreased steadily in sycamore tree rings formed after the 1950s, and decreased more abruptly in wood formed after 1985. This sharp decrease in wood Pb cannot be due to decreases in soil Pb concentration. Stable Pb isotope analysis was used to further investigate Pb patterns in sycamore wood. Excess 206Pb/207Pb ratios in tree-rings of sycamore were relatively constant, approximately 1.17, in wood formed prior to the 1930s, but decreased steadily thereafter reaching a minimum value of approximately 1.16 in wood formed between 1975 and 1985 after which time 206Pb/207Pb ratios increased. This pattern is consistent with changes in Pb isotope ratios measured in peat, sediment and aerosol samples in the UK. However, the magnitude of the decrease in 206Pb/207Pb (largely due to gasoline Pb) is considerably lower than in other studies and our estimates indicate that less than 20% of the total Pb in sycamore wood measured since the mid-1800s is derived from gasoline emissions. A more likely explanation for the pattern of Pb observed in sycamore tree rings is that soil Pb accumulates within rings of the diffuse porous wood over a number of years. Such uptake patterns would result in lower Pb concentrations in the outer (more recently formed) tree rings, which coincide with recent reductions in Pb deposition in the UK. Overall, this study indicates that tree ring chemistry is unsuitable for monitoring historical changes in Pb deposition at the study site.

Nickel and zinc tolerance and co-tolerance in populations of Deschampsia cespitosa (L.) Beauv. subject to artificial selection.

The occurrence of co-tolerance to four heavy metals was investigated among seedlings selected from three different non-tolerant Canadian and German Deschampsia cespitosa (L.) Beauv. populations. Plants selected following screening of seedlings in solution containing Ni or Zn ions, subsequently showed enhanced tolerance to Ni and Zn, respectively. 'Nickel screened' seedlings exhibited no increased tolerance to Zn.'Zinc screened' seedlings, in contrast, had somewhat elevated tolerance to Ni in two of the three populations tested (Elm and Cypress Lake). Thus, co-tolerance may be a population-specific rather than a species-specific response. Individual Elm plants screened for Ni response did not have elevated Zn tolerance, whereas'Zn screened' seedlings did show elevated Ni tolerance. This confirms the result from the population tolerance tests. We conclude that co-tolerance is not a reciprocal process. While the selection for one metal may confer elevated tolerance to a second, selection for the second may not confer tolerance to the first.

Occurrence of heavy metal tolerance and co-tolerance in Deschampsia cespitosa (L.) Beauv. from European and Canadian populations.

By sampling soils and plants from a variety of metal-contaminated sites in Canada and Europe, heavy metal tolerance and co-tolerance have been investigated in the grass Deschampsia cespitosa L. (Beauv.). Soils were analyzed for water extractable Ca, as well as for their heavy metal content (Cu, Ni, Zn, Pb, Cd, Co), electrical conductivity, pH and organic matter. Canadian plant material was collected from non-contaminated areas, as well as sites contaminated by copper and nickel (Sudbury) and nickel and cobalt (Cobalt). European plants were sampled from reference sites and from zinc and lead contaminated locations (Bleikuhle, Pochsand/Germany). Using cloned material the 'in parallel' root elongation technique was employed to determine tolerance indices to individual metals (Cu, Ni, Zn, Pb). Among the Canadian plants, patterns of tolerances matched patterns of soil contamination. Thus, at the Cobalt sites, Cu was not present in elevated amounts, and the plants did not exhibit Cu tolerance. Also, evidence suggests that the close correlation of Cu and Ni tolerance among the Sudbury plants was not the result of Cu-Ni co-tolerance but occurred because ions of both metals were present in elevated amounts at these sites. In addition to the tolerances expected on soil chemistry, plants taken from metal contaminated sites exhibited enhanced tolerance also to metal ions not elevated in the soils of their origin. This is interpreted as general, non-specific, low level co-tolerance. Thus, most Cobalt and Sudbury plants exhibited incidental tolerance at a higher level to Zn and Pb. Correlation analysis revealed very close relations both between Zn and Pb, and especially between Ni and Zn, supporting findings from previous studies. It was concluded that among the Canadian populations tested in this study, Ni tolerance conferred Zn tolerance. Among the European plants examined, the expected tolerances to Zn and Pb were found. However, whereas some control plants from non-contaminated sites exhibited relatively high tolerances to these metal ions, the tolerance of plants from mines was lower than expected. The Zn and Pb tolerant mine populations had no tolerance to Cu and only very low tolerance to Ni, suggesting that the enhanced Zn tolerance associated with Ni tolerance in the Canadian plants is not necessarily reversible, i.e. Zn tolerance does not necessarily confer Ni tolerance.

Arsenic, cobalt and nickel tolerances in two populations of Silene vulgaris (Moench) Garcke from Ontario, Canada.

A population of Silene vulgaris (Moench) Garcke collected from mine tailings near the town of Cobalt, Ontario was compared in its metal tolerance with a population collected at an uncontaminated site (Baymouth, Ontario). Seedlings of the Cobalt population were tolerant to elevated levels of arsenic, cobalt and nickel, both compared with the Baymouth population and with other literature reports for this species. However, the tolerance indices in the Cobalt plants for these toxic elements did not correlate well with their concentration in water extractions in the mine soils. Indices of arsenic tolerance, based on the inhibition of root growth in 12 d experiments, showed a similar response to that in a 28 d experiment for both populations. The latter however, appeared to be a more accurate indicator of As tolerance in S. vulgaris, because the longer-term tolerance indices had lower standard deviations and higher probabilities for tolerance than the short-term tolerance indices. Reduced uptake of As occurred in the roots of tolerant individuals compared with non-tolerant individuals at a low As treatment. At a higher arsenic exposure, however, arsenic entered the roots of tolerant individuals. A partial exclusion or reduced translocation of arsenic to the shoots then appeared to operate in the shoots of tolerant individuals, i.e. arsenic levels were higher in shoots of non-tolerant plants. At the highest arsenic exposure, where the individuals from the Cobalt population were no longer tolerant, this exclusion pattern broke down. From a preliminary selection experiment, some arsenic tolerant individuals occurred in the Baymouth population. This suggests that under circumstances of arsenic contamination this population also has the potential to develop a tolerance to high arsenic exposure.

Crystal occurrence and wax disruption on leaf surfaces of cabbage treated with simulated acid rain.

Epicuticular waxes on leaves of Brassica oleracea L. (cabbage) were studied using scanning electron microscopy after a single treatment with simulated rain of pH 5.6, 3.0 or 2.5 which was either sprayed on to plants in an exposure chamber or applied as droplets with a micropipette. Treatments with acidified rain caused serious structural degradation of the wax crystals. The alteration of crystalline wax structures was similar for leaves treated with nitric acid solutions, but less severe, than for leaves treated with sulphuric acid solutions. With both H2 SO4 , and HNOa -derived rain solutions numerous gypsum (CaSO4 ) crystals were found in and near lesions on the leaves treated with rain of pH 3.0 and 2.5. The crystals probably resulted from damage of cuticular membranes by acidic rain which significantly altered their permeability to ions in the area of lesions. Crystalline leaf waxes may be an important target for acidic pollutants, and the physiological consequences of their degradation are discussed.