Journal of Experimental Botany, Vol. 52, No. 365, pp. 2291-2300,
December 1, 2001
© 2001 Oxford University Press
Original Papers |
Cellular compartmentation of nickel in the hyperaccumulators Alyssum lesbiacum, Alyssum bertolonii and Thlaspi goesingense
1 Universität Konstanz, Mathematisch-Naturwissenschaftliche Sektion, Fachbereich Biologie, Fach M665, D-78457 Konstanz, Germany
2 IACR-Rothamsted, Agriculture and Environment Division, Harpenden, Herts AL5 2JQ, UK
Received 26 February 2001; Accepted 8 August 2001
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Nickel uptake and cellular compartmentation were investigated in three Ni hyperaccumulators: Alyssum bertolonii (Desv), Alyssum lesbiacum (Candargy) and Thlaspi goesingense (Hálácsy). The three species showed similar hyperaccumulation of Ni, but T. goesingense was less tolerant to Ni than the two Alyssum species. An addition of 500 mg Ni kg-1 to a nutrient-rich growth medium significantly increased shoot biomass of all three species, suggesting that the Ni hyperaccumulators have a higher requirement for Ni than normal plants. Energy-dispersive X-ray microanalysis (EDXA) was performed on frozen-hydrated tissues of leaves (all species) and stems (Alyssum only). In all species analysed, Ni was distributed preferentially in the epidermal cells, most likely in the vacuoles, of the leaves and stems. In stems, there was a second peak of Ni in the boundary cells between the cortical parenchyma and the vascular cylinder. The non-glandular trichomes on the leaf surfaces of the two Alyssum species were highly enriched with Ca, but contained little Ni except in the base. In the leaves of T. goesingense, the large elongated epidermal cells contained more Ni than the cells of the stomatal complexes. The role of cellular compartmentation in Ni hyperaccumulation is discussed.
Key words: Nickel, hyperaccumulator, cellular compartmentation, Brassicaceae.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
In the late 1940s, it was reported that Alyssum bertolonii (Desv), growing in the Tuscany region of Italy, contained an extraordinarily high Ni content of about 10000 µg g-1 in the shoot dry matter (Minguzzi and Vergnano, 1948
). This was the discovery of the first Ni accumulator plant in the world, which was about 30 years before the term ‘hyperaccumulator’ was used to describe plants that accumulate more than 1000 µg Ni g-1 of dry weight in their shoots in the natural habitats (Brooks et al., 1977). Although there are hyperaccumulators for other heavy metals or metalloids, Ni hyperaccumulators are the most numerous, currently with a total number of 318 taxa distributed mainly in the tropical to warm temperature regions with Ni-rich ultramafic (serpentine) soils (Baker et al., 2000; Reeves and Baker, 2000).
Metal hyperaccumulators have received increased attention in recent years, due to the potential of using these plants for phytoremediation of metal-contaminated soils (McGrath, 1998; Salt et al., 1998) or phytomining (Brooks et al., 1998). Some of the Ni hyperaccumulators, e.g. A. bertolonii and Berkheya coddii, are particularly attractive for these purposes because of their relatively high biomass production (Robinson et al., 1997a, b). Progress has also been made in recent years in the understanding of the Ni hyperaccumulation phenomenon. It was found that exposing several Ni hyperaccumulator species of Alyssum to Ni elicited a large increase in the levels of histidine in the xylem sap (Krämer et al., 1996). The results suggest that complexation of Ni with histidine may be involved in the tolerance to Ni in roots and the delivery of Ni from roots to shoots. The histidine response may not be a universal feature associated with all Ni hyperaccumulators. For example, Persans et al. did not observe any Ni-inducible responses in terms of histidine concentrations in the roots, shoots and xylem sap of the Ni hyperaccumulator Thlaspi goesingense (Hálácsy), nor did they find any regulation by Ni of three cDNAs encoding the enzymes involved in the histidine biosynthetic pathway (Persans et al., 1999). In leaves, Ni appears to be associated mainly with organic acids, such as citrate (Lee et al., 1978; Homer et al., 1991; Sagner et al., 1998; Krämer et al., 2000), malate and malonate (Pancaro et al., 1978; Brooks et al., 1981).
Apart from complexation with organic ligands, cellular compartmentation is likely to be involved in the detoxification of metals in hyperaccumulator plants. In the Zn/Cd hyperaccumulator Thlaspi caerulescens, it has been shown that Zn and Cd are sequestered mainly in the vacuoles, particularly in the epidermal cells (Vázquez et al., 1994; Küpper et al., 1999). In the Ni hyperaccumulator T. goesingense, vacuolar sequestration appeared to be important too, although substantial cell wall binding was also implicated (Krämer et al., 2000). Cellular distribution of Ni in hyperaccumulator plants has been reported in only a few papers (Mesjasz-Przybylowicz et al., 1994; Heath et al., 1997; Krämer et al., 2000; Psaras et al., 2000), and the results appear to be contradictory. Therefore, the aim of this study was to investigate the cellular compartmentation of Ni in three species of Ni hyperaccumulators within the Brassicaceae family: Alyssum bertolonii, Alyssum lesbiacum and Thlaspi goesingense.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant culture
Alyssum bertolonii, Alyssum lesbiacum and Thlaspi goesingense were sown into vermiculite in trays. Sixteen days later, seedlings were transferred to plastic pots, each containing 500 g compost (John Innes No. 1, moisture content=27%). Three seedlings were planted in each pot. Because of a poor germination of A. lesbiacum in vermiculite, seeds of this species were later sown directly on to the pots containing compost. Each species was treated with four different Ni concentrations in the compost: control (no addition), 500, 2000 and 4000 mg kg-1. Nickel was applied as NiSO4 and mixed thoroughly with the compost before transplanting. Each treatment was replicated three times. Plants were grown in a greenhouse with the following conditions: 20/16 °C day/night temperature, natural sunlight supplemented with 1 kW SON-T lamps to maintain a minimum light intensity of 250 µmol m-1 s-1. A. bertolonii and T. goesingense were harvested 86 d after transplanting, and A. lesbiacum was harvested 64 d after emergence of seedlings.
Freeze-fracturing and energy dispersive X-ray analysis (EDXA)
EDXA analysis was performed on both Alyssum species growing in the 4000 mg Ni kg-1 treatment. Because T. goesingense failed to survive in the 4000 mg Ni kg-1 treatment, EDXA was done using plants from the 2000 mg Ni kg-1 treatment. For quantification of elements in each type of tissue, samples were taken from two or three different plants, two or three samples were taken from each plant, and two or three analyses were done per sample. For leaf and stem samples, mature but not senescent tissues were taken. When leaf surfaces were analysed, these were mounted flat on the surface of carbon stub using conductive carbon tabs. Cross-sections of plant tissue were prepared in the following way: a 0.5 cm wide strip of leaf was taken by two cuts perpendicular to the central vein or a piece about 0.5 cm long was cut out of the mature part of the stem. These pieces of tissue were mounted in a sample holder made of carbon. Samples were then rapidly (within 1 min after excision) frozen in melting nitrogen slush and transferred to a fracturing chamber cooled to -170 °C. To obtain cross-sections of leaves and stems, a sharp blade was used to cut through the cells rather than breaking the tissue at the cell surfaces. Thus the leaves and stems were cut in half, the lower of which stayed fixed in the sample holder and was used for analysis. Subsequently, the specimens were evaporatively coated with carbon to produce an electrically conductive surface and thus prevent charging under the electron beam. Carbon was used instead of metal coating to avoid interference with the elements measured.
EDXA was performed in a scanning electron microscope (SEM, Philips XL 40) on a cryostage (-160 °C to -180 °C), using an acceleration voltage of 30 kV and a working distance of 10 mm. The low temperature during analysis is essential to avoid evaporation of water during exposure of the sample to the electron beam. Tests have shown that even after continuously scanning an area of 200x200 µm for 8 h, at this temperature no visible or measurable damage occurred. Electron penetration depth (R) is
 | (001) |
is specific gravity of the absorber in g cm-3. With the chosen acceleration voltage, the maximum penetration depth (i.e. in an aqueous compartment like a vacuole) of the electron beam is about 15 µm. In materials with a higher specific gravity, the electron penetration depth is smaller (inversely proportional to specific gravity), so if, for example, a dense cell wall has a specific gravity of 2.5 g cm3, the maximum penetration depth would be decreased to about 6 µm. The horizontal resolution (determined mainly by the diameter of the electron beam) is less than 1 µm. Hence, differences in concentration, for example, between small organelles such as chloroplasts or mitochondria, and the cytoplasm, cannot be resolved. However, differences between the cell wall and the intercellular compartment can be measured by choosing cell walls that were approximately parallel to the electron beam. Nevertheless, cell wall data will always contain some counts from the neighbouring intracellular compartment, and if that compartment contains higher concentrations, this can lead to overestimation of element concentrations in the apoplast. Hence, measurements of intracellular concentrations are generally more accurate than those of cell walls.
The distribution of elements across a section of a sample was measured semi-quantitatively by displaying the count rate within a narrow spectrum window within its peak (0.6xpeak half width) along a transect line. A two-dimensional distribution pattern was also recorded by scanning an area of the specimen repeatedly for up to 2 h and integrating the counts for Ni, O, Ca, K, and S within their respective spectrum windows into dot-maps. In both line and area scans, the spectrum-analysis software did not allow for the separation of the element-specific X-rays (net peak counts) from the background counts. Therefore, these scans were performed only on those elements with a peak/background ratio greater than 2. The latter could be determined by recording an average EDXA spectrum of the region; this was done automatically during the recording of a dot-map. A dot-map of oxygen distribution was recorded before any further analysis of the specimen. Because of its low energy, the X-rays emitted by oxygen are most strongly affected by shading (caused by the sample holder or neighbouring cells) inside the sample, so that an oxygen dot-map clearly shows these shaded parts of the sample. The regions of specimen that suffered from shading effects were not used for quantitative analysis.
Spectra from 0–20 keV were collected at increments of 10 eV per channel with the electron beam focused on a rectangular area in the centre of selected cells. The spectra were analysed using the program Superquant (EDAX, San Francisco, USA), which separates the background from the element-specific peaks and fully deconvolutes the spectra for the correction of interference between elements. Thus, the analysis with this program yielded the net peak count rate and the peak/background ratio for each element, which were used for quantification. Quantification of EDXA data was done as described in detail (Küpper et al., 2000) using the oxygen K
line as an internal standard. Since the EDXA analysis of bulk-frozen tissues does not allow a solution or a suspension of an element to be distinguished, the term ‘concentration’ always refers to the apparent concentration of the element, meaning that not all of this is necessarily in soluble form.
Determination of element concentrations in whole plant tissues
At harvest, plant shoots were washed with deionized water, blotted dry, and oven-dried at 80 °C for 24 h before dry weights were determined. Dried samples were ground to <0.5 mm, and a 0.2 g subsample was digested with a mixture of HNO3 and HClO4 (Zhao et al., 1994). Concentrations of Ni and other elements in the digests were determined using inductively coupled plasma atomic emission spectrometry (ICP-AES; Fisons ARL Accuris).
Means and standard errors are presented. Where appropriate, analysis of variance or Student's t-test was performed.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant growth and Ni uptake
The compost used in this experiment is a nutrient-rich all-purpose growth medium. However, an addition of 500 mg Ni kg-1 to the growth medium increased plant growth of all three Ni hyperaccumulators significantly (P<0.01) compared to the control (Fig. 1a
). T. goesingense was clearly less tolerant to Ni than the two Alyssum species. The 4000 mg Ni kg-1 treatment was lethal to T. goesingense within a few weeks of exposure. The plants grown on 2000 mg Ni kg-1 also exhibited symptoms of phytotoxicity, and the shoot dry weight was decreased to about 50% of that in the control. In comparison, plants of both A. bertolonii and A. lesbiacum survived the highest Ni treatment, showing no obvious symptoms of phytotoxicity, although the shoot dry weights decreased. Shoot dry weights of A. lesbiacum were smaller than those of A. bertolonii, mainly because the former species was grown for a shorter period.
|
The concentrations of Ni in the shoots of all three species increased almost linearly with the increasing addition of Ni to the growth medium (Fig. 1b
). The patterns of Ni accumulation were remarkably similar in all three species. The highest Ni concentration obtained in this experiment was about 23000 mg kg-1 shoot dry weight in the two Alyssum species grown on the 4000 mg Ni kg-1 treatment, whereas for T. goesingense a maximum of 12400 mg Ni kg-1 shoot dry weight was obtained in the 2000 mg Ni kg-1 treatment.
Cellular distribution of Ni and other elements in leaves
Leaves of all species analysed had a rather similar pattern of Ni distribution in different cell types (Fig. 2). In all three species the Ni concentration was significantly higher (P<0.01) in the epidermal cells than in the mesophyll cells or various types of cells inside the vascular bundles (Fig. 2). No significant difference was observed between the upper and the lower epidermis (data not shown). The preferential distribution of Ni in the epidermal cells can be seen clearly in a dot-map of Ni recorded from a cross-section of a mature leaf of A. lesbiacum (Fig. 3a, b). Using a higher magnification, it can be seen that the Ni concentration appeared to be higher inside the epidermal cells than in the epidermal cell walls (Fig. 2c, d). In all three species, the concentrations of Ni were higher in the intracellular compartment than in the cell walls of the epidermal cells (Fig. 3). Due to the large variations between individual tissue samples, the differences in concentrations between cell walls and intracellular compartments were statistically significant in a t-test (P<0.05) only in A. lesbiacum. Therefore, line scans were performed to give a more direct comparison with the maximum possible spatial resolution, and these have consistently shown valleys of Ni counts in the cell walls and peaks in the intracellular compartments (Fig. 3c, d).
|
|
In contrast to Ni, the concentrations of Ca and Mg were higher in the epidermal cell walls than in the intracellular compartments (Fig. 2
). In both T. goesingense and A. lesbiacum, there was a significant and positive correlation between Ni and Mg in the mesophyll cells (n=13 and 12, r=0.72 and 0.96, respectively, P<0.01, data from all mesophyll cells analysed). The ratio of Mg to Ni was 4.0±1.2 and 4.5±1.3 for T. goesingense and A. lesbiacum, respectively. Potassium was abundant in most cell types, particularly in the vascular bundles. In the two Alyssum species, the S concentration was higher in the epidermal cells than in the mesophyll and vascular bundle cells, and there was a significant correlation between S and Ni concentrations in the intracellular compartments of different cells (for A. lesbiacum: n=32, r=0.72, P<0.001; for A. bertolonii: n=26, r=0.79, P<0.001; data from all cells analysed). In T. goesingense, the difference in S concentration between cell types was smaller, and the correlation between S and Ni was not significant. In the epidermal cells, cell walls appeared to have high S concentrations, which were comparable to the concentrations of Ca (Fig. 2).
Both Alyssum species have many trichomes on the leaf surfaces which are non-glandular and stellate in shape (Fig. 4a for A. lesbiacum). EDXA dot-map scans of leaf surfaces revealed that the trichomes contained little Ni compared to the other epidermal cells, whereas Ca was highly enriched in the trichomes (Fig. 4b, c). A further test was performed by immersing leaves in a solution of dimethylglyoxime (1% w/v in ethanol), which forms a red coloured precipitate with soluble Ni. Three hours later, leaves were dissected and observed by light microscopy. Some of the trichomes showed a pink colour in their basal part, possibly vacuoles (Fig. 4d), suggesting the presence of Ni.
|
There are no trichomes on the leaf surfaces of T. goesingense, but the size of epidermal cells varies widely (Fig. 5a
). Ni was preferentially accumulated in the large elongated epidermal cells, whereas the small cells around the stomatal complex and the guard cells contained lower Ni concentrations (Fig. 5b).
|
Cellular distribution of Ni and other elements in stems
Distributions of Ni and other elements in different cells in the stem were determined in the two Alyssum species, and both showed similar patterns (Fig. 6). Similar to the leaves, Ni concentrations were highest in the epidermis (Fig. 7). The concentrations of Ni in the intracellular compartment of epidermal cells were again higher than those measured in the epidermal cell walls (P<0.05 and P<0.01 for A. lesbiacum and A. bertolonni, respectively, in t-tests) (Fig. 6). An EDXA line scan along stem epidermal cells of A. bertolonii clearly shows that Ni was more enriched inside the cells than in the cell wall (Fig. 8). Ni concentrations in the stem epidermal cells (intracellular compartment) were around 120 mM in A. bertolonii and about 80 mM in A. lesbiacum, whereas the concentrations in the epidermal cell wall were 40–50 mM in both species (Fig. 6). In stems there was a second peak of Ni accumulation in the boundary cells between the vascular cylinder and the cortical parenchyma cells (Fig. 7). The concentrations of Ni in these boundary cells (intracellular compartment) were around 55–65 mM, compared to <10 mM in the bark parenchyma and vascular bundle cells (Fig. 6).
|
|
|
The concentrations of Ca and Mg were markedly higher in the epidermal cell walls than in the intracellular compartment of the epidermis, and were low in all other cell types in the stems (Fig. 6
). Potassium was again abundant in all cell types and tended to increase from epidermal cells to the vascular bundle cells. As in leaves, the patterns of S distribution in both Alyssum species were similar to those of Ni, with the intracellular compartment of epidermis and the boundary cells between bark parenchyma and vascular bundle containing more S than other cell types. In these cell types, there was also a significant (P<0.001) and positive correlation between Ni and S. Also, cell walls apparently contained high concentrations of S.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Growth of the Ni hyperaccumulators A. bertolonii, A. lesbiacum, and T. goesingense was enhanced by the addition of 500 mg Ni kg-1 as NiSO4 to the growth medium, an all-purpose nutrient-rich compost. It is unlikely that the positive effect was due to the addition of S, because of the abundance of available S in the compost. Although Ni is an essential nutrient, the requirement for Ni by normal plant species (non-hyperaccumulators) is so low that Ni deficiency has never been reported in field-grown crops (Marschner, 1995
). Even the small amount of Ni contained in seed is usually sufficient to sustain normal plant growth and development. To demonstrate that Ni is essential to higher plants, Brown et al. had to lower the content of Ni in the seeds of barley by growing plants for several generations with nutrient solution containing very high purity reagents that were further purified using an ion exchange column (Brown et al., 1987). The present study's results indicate that, compared to normal plant species, all three Ni hyperaccumulators tested have a much higher requirement for Ni. This is analogous to the higher requirement for Zn by the Zn hyperaccumulator Thlaspi caerulescens (Shen et al., 1997; Küpper et al., 1999). One beneficial effect of a highly elevated Ni concentration in the tissues of hyperaccumulator plants may be protection against pathogens and herbivores (Boyd, 1998). In other experiments (not reported here), it was also observed that fungal infections on the leaf surfaces of T. goesingense and the two Alyssum species were greatly reduced in the plants grown on +Ni treatments.
The three Ni hyperaccumulators studied all showed preferential accumulation in the epidermal cells. Mesophyll cells and the cells inside the vascular bundles in the leaves contained much less Ni than the epidermal cells. In the stems of A. lesbiacum and A. bertolonii, Ni was also preferentially accumulated in the epidermal cells, although there was a second peak of Ni accumulation in the boundary cells between the vascular cylinder and the cortical parenchyma cells. The function of these cells is not known. The Ni accumulation in leaf epidermal cells was not uniform. In T. gosingense, cells in the stomatal complexes, which are small in size, had less Ni than the other large and elongated epidermal cells. This differs from a report (Heath et al., 1997) that Ni was accumulated in the subsidiary cells within the stomatal complexes in the leaves of the Ni hyperaccumulator Thlaspi montanum.
Because vacuoles generally occupy the majority of cell space, particularly in epidermal cells, the Ni signal recorded in the central intracellular compartment of a cut cell essentially reflects the concentration of Ni in the vacuole. The EDXA data obtained from this study show that the majority of Ni in leaves and stems of the three hyperaccumulator species was located in the vacuoles, rather than in the cell wall. The pattern of cellular distribution of Ni seen in this study is in contrast to that of Al distribution in the mature leaves of tea (Camellia sinensis; Matsumoto et al., 1976; Memon et al., 1981), where Al was found to be located predominantly in the cell wall. The exclusive localization of Al in the cell wall of tea leaves was confirmed in the laboratory using the same EDXA technique (unpublished results). The results of this study disagree with the conclusion of Krämer et al., who reported that 67–73% of the Ni in the leaves of T. goesingense was associated with the cell wall (Krämer et al., 2000). There are two possible explanations for the difference. Firstly, Krämer et al. used young plants that were exposed to relatively low concentrations of Ni for short periods. The Ni concentrations in the leaves were far below 1000 mg kg-1, the concentration commonly used to define Ni hyperaccumulation. In contrast, the leaf samples used for the EDAX analysis in this study contained >10000 mg Ni kg-1 (dry weight), which is more typical of hyperaccumulator plants. Secondly, Krämer et al. used the techniques of cell fractionation and X-ray absorption spectroscopy to quantify cellular distribution of Ni. The accuracy of both techniques for quantifying in situ cellular distribution of elements, particularly in the cell wall, is not without doubt. Other studies with the Zn hyperaccumulator T. caerulescens indicate that Zn was located mainly in the vacuoles rather than in the cell wall (Vázquez et al., 1994; Küpper et al., 1999; Frey et al., 2000).
Previous studies showed inconsistent evidence regarding the localization of Ni in the trichomes in Alyssum species. Using a proton microprobe to examine dehydrated and fixed leaf specimens, Krämer et al. showed that Ni was sequestered to a considerable degree in the trichomes of A. lesbiacum (Krämer et al., 1997). In contrast, Psaras et al. found exclusion of Ni in the trichomes in dry leaves of eight Ni hyperaccumulators, including A. lesbiacum, which were obtained from plant materials kept in a herbarium (Psaras et al., 2000). In the present study, EDXA surface scans indicated exclusion of Ni from trichomes of A. bertolonii and A. lesbiacum. However, staining with dimethylglyoxime appeared to indicate the presence of Ni in the basal part of the trichomes, most likely in the vacuoles. The discrepancy may be caused by the different methods of sample preparation used. Dehydration and fixation prior to elemental mapping (Krämer et al., 1997) may lead to the redistribution of mobile ions. In the present study, EDXA was performed on hydrated tissues immediately after the specimens were frozen in melting nitrogen, which involved minimum sample preparation and was most likely to provide information of the in situ distribution of elements. One possible limitation of EDXA surface scans is that the electron beam may not penetrate the thick cell walls of trichomes, which may also be fortified with the deposition of calcite (Lanning, 1960). In the Zn hyperaccumulator Arabidopsis halleri, Zn was found to accumulate in the basal compartment of trichomes, most likely in the vacuoles (Küpper et al., 2000; Zhao et al., 2000).
Preferential accumulation of Ni in the leaf epidermis has previously been observed in the Ni hyperaccumulator Senecio coronatus (Mesjasz-Przybylowicz et al., 1994). Also, Zn was found to be sequestered preferentially in the vacuoles of leaf epidermal cells in the Zn hyperaccumulator Thlaspi caerulescens (Vazquez et al., 1996; Küpper et al., 1999), except the guard cells and subsidiary cells within the stomatal complexes (Frey et al., 2000). This pattern of metal accumulation may be seen as protecting both photosynthesis in the mesophyll cells and the function of stomata against metal toxicity. However, preferential accumulation of metals in the epidermis is apparently not a universal feature associated with all metal hyperaccumulators. For example, in the Zn/Cd hyperaccumulator Arabidopsis halleri, Zn and Cd were found to be stored mainly in the mesophyll vacuoles and in the basal compartment of leaf trichomes, but not in the other epidermal cells which tend to be very small in size (Küpper et al., 2000; Zhao et al., 2000). It is possible that the size of vacuoles is an important factor influencing metal accumulation. This hypothesis is supported by the observation that the concentration of Zn correlated positively with the size of epidermal cells in T. caerulescens (Küpper et al., 1999).
The reason for the apparent high concentrations of S in the epidermal cell walls is not clear. Because the cell wall is thinner than the intracellular compartment, it is more difficult to quantify element concentrations in the cell wall than inside the cell. It is possible that the counts measured in the cell wall may also include small amounts of the signal from intracellular contents. This will cause an overestimation of the cell wall concentrations if the intracellular concentrations are higher than in the neighbouring cell wall, as is the case for S and Ni (Figs 2, 6). If so, it follows that the Ni concentration in the epidermal cell walls could be overestimated. This actually reinforces the conclusion that Ni sequestration in the vacuoles is more dominant than binding in the cell walls.
The concentrations of Mg and Ni in the mesophyll cells of A. lesbiacum and T. goesingense were found to correlate positively. A similar relationship was observed between Mg and Cd in the mesophyll cells of A. halleri (Küpper et al., 2000). It is possible that increased Mg may be a defence response of the plants to resist the substitution of Mg in chlorophyll by Ni or Cd (Küpper et al., 1996). In the case of S, a cellular distribution pattern similar to that of Ni, and a positive correlation between S and Ni at the single cell level suggests two possibilities. First, sulphate may be an important counter ion for Ni2+ in the vacuoles. Indeed, the treatments in these experiments were created by supplying different concentrations of NiSO4. Second, S-containing ligands may be involved in complexing Ni. The second possibility is thought to be less likely, because organic ligands that have been identified to play a role in Ni complexation in Ni hyperaccumulator plants include organic acids (Lee et al., 1978; Brooks et al., 1981; Homer et al., 1991; Sagner et al., 1998; Krämer et al., 2000) and histidine (Krämer et al., 1996), but not S-containing ligands.
|
Acknowledgments |
|---|
We thank Chris Smith and Phil Jones for assistance in using the SEM and EDAX systems, and Sarah Dunham for help with setting up the plant experiments. We gratefully acknowledge financial support from DG XII of the European Commission for the PHYTOREM Project. IACR-Rothamsted receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom.
|
Notes |
|---|
3 To whom correspondence should be addressed. Fax: +44 1582 760981. E-mail: Steve.McGrath{at}bbsrc.ac.uk
|
Abbreviations |
|---|
EDXA, energy dispersive X-ray analysis; ICP-AES, inductively coupled plasma atomic emission spectroscopy; SEM, scanning electron microscope.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Baker AJM, McGrath SP, Reeves RD, Smith JAC. 2000. Metal hyperaccumulator plants: a review of the ecology and physiology of a biochemical resource for phytoremediation of metal-polluted soils. In: Terry N, Bañuelos G, eds. Phytoremediation of contaminated soil and water. Boca Raton, Florida, USA: Lewis Publishers, 85–107.
Boyd RS. 1998. Hyperaccumulation as a plant defence strategy. In: Brooks RR, ed. Plants that hyperaccumulate heavy metals. Wallingford, UK: CAB International, 181–201.
Brooks RR, Lee J, Reeves RD, Jaffre T. 1977. Detection of nickeliferous rocks by analysis of herbarium species of indicator plants. Journal of Geochemical Exploration 7, 49–57.
Brooks RR, Shaw S, Marfil AA. 1981. The chemical form and physiological function of nickel in some Iberian Alyssum species. Physiologia Plantarum 51, 167–170.
Brooks RR, Chambers MF, Nicks LJ, Robinson, BH. 1998. Phytomining. Trends in Plant Science 3, 359–362.
Brown PH, Welch RM, Cary EE. 1987. Nickel: a micronutrient essential for higher plants. Plant Physiology 85, 801–803.
Frey B, Keller C, Zierold K, Schulin R. 2000. Distribution of Zn in functionally different epidermal cells of the hyperaccumulator Thlaspi caerulescens. Plant, Cell and Environment 23, 675–687.
Heath SM, Southworth D, D'allura JA. 1997. Localization of nickel in epidermal subsidiary cells of leaves of Thlaspi monatum var. siskiyouense (Brassicaceae) using energy-dispersive X-ray microanalysis. International Journal of Plant Sciences 158, 184–188.
Homer FA, Reeves RD, Brooks RR, Baker AJM. 1991. Characterization of the nickel-rich extract from the nickel hyperaccumulator Dichapetalum gelonioides. Phytochemistry 30, 2141–2145.
Krämer U, Cotterhowells JD, Charnock JM, Baker AJM, Smith JAC. 1996. Free histidine as a metal chelator in plants that accumulate nickel. Nature 379, 635–638.
Krämer U, Grime GW, Smith JAC, Hawes CR, Baker AJM. 1997. Micro-PIXE as a technique for studying nickel localization in leaves of the hyperaccumulator plant Alyssum lesbiacum. Nuclear Instruments & Methods in Physics Research B 130, 346–350.
Krämer U, Pickering IJ, Prince RC, Raskin I, Salt DE. 2000. Subcellular localization and speciation of nickel in hyperaccumulator and non-accumulator Thlaspi species. Plant Physiology 122, 1343–1353.
Küpper H, Küpper F, Spiller M. 1996. Environmental relevance of heavy metal substituted chlorophylls using the example of water plants. Journal of Experimental Botany 47, 259–266.
Küpper H, Zhao FJ, McGrath SP. 1999. Cellular compartmentation of zinc in leaves of the hyperaccumulator Thlaspi caerulescens. Plant Physiology 119, 305–311.
Küpper H, Lombi E, Zhao FJ, McGrath SP. 2000. Cellular compartmentation of cadmium and zinc in relation to other elements in the hyperaccumulator Arabidopsis halleri. Planta212, 75–84.[Web of Science][Medline]
Lanning FC. 1960. Calcite in Lesquerella ovalifolia trichomes. Science 133, 380.
Lee J, Reeves RD, Brooks RR, Jaffré T. 1978. The relation between nickel and citric acid in some nickel-accumulating plants. Phytochemistry 17, 1033–1035.
Marschner H. 1995. Mineral nutrition of higher plants. London: Academic Press.
Matsumoto H, Hirasawa E, Morimura S, Takahashi E. 1976. Localization of aluminium in the tea leaf. Plant and Cell Physiology 17, 627–631.
McGrath SP. 1998. Phytoextraction for soil remediation. In: Brooks RR, ed. Plants that hyperaccumulate heavy metals. Wallingford, UK: CAB International, 261–287.
Memon AR, Chino M, Yatazawa M. 1981. Micro-distribution of aluminum and manganese in the tea leaf tissues as revealed by X-ray microanalyzer. Communication in Soil Science and Plant Aanlysis 12, 441–452.
Mesjasz-Przybylowicz J, Balkwill K, Przybylowicz WJ, Annegarn HJ. 1994. Proton microprobe and X-ray fluorescence investigations of nickel distribution in serpentine flora from South Africa. Nuclear Instruments & Methods in Physics Research B 89, 208–212.
Minguzzi C, Vergnano O. 1948. Il contenuto di nichel nelle ceneri di Alyssum bertolonii. Atti Societa Toscana Scienze Naturali 55, 49–74.
Pancaro L, Pelosi P, Vergnano Gambi O, Galoppini C. 1978. Ulteriori indagini sul rapporto tra nickel e acidi malico e malonico in Alyssum. Giornale Botanico Italiano 112, 141–146.
Persans MW, Yan XG, Patnoe J, Krämer U, Salt DE. 1999. Molecular dissection of the role of histidine in nickel hyperaccumulation in Thlaspi goesingense (Halacsy). Plant Physiology 121, 1117–1126.
Psaras GK, Constantinidis TH, Cotsopoulos B, Manetas Y. 2000. Relative abundance of nickel in the leaf epidermis of eight hyperaccumulators: evidence that the metal is excluded from both guard cells and trichomes. Annals of Botany 86, 73–78.
Reeves RD, Baker AJM. 2000. Metal-accumulating plants. In: Raskin I, Ensley BD, eds. Phytoremediation of toxic metals: using plants to clean up the environment. New York: John Wiley and Sons, 193–229.
Robinson BH, Brooks RR, Howes AW, Kirkman, JH, Gregg PEH. 1997b. The potential of the high-biomass nickel hyperaccumulator Berkheya coddii for phytoremediation and phytomining. Journal of Geochemical Exploration 60, 115–126.
Robinson BH, Chiarucci A, Brooks RR, Petit D, Kirkman JH, Gregg PEH, DeDominicis V. 1997a. The nickel hyperaccumulator plant Alyssum bertolonii as a potential agent for phytoremediation and phytomining of nickel. Journal of Geochemical Exploration 59, 75–86.
Salt DE, Smith RD, Raskin I. 1998. Phytoremediation. Annual Review of Plant Physiology and Plant Molecular Biology 49, 643–668.[Web of Science]
Sagner S, Kneer R, Wanner G, Cosson JP, Deus-Neumann B, Zenk MH. 1998. Hyperaccumulation, complexation and distribution of nickel in Sebertia acuminata. Phytochemistry 47, 339–347.[Web of Science][Medline]
Shen ZG, Zhao FJ, McGrath SP. 1997. Uptake and transport of zinc in the hyperaccumulator Thlaspi caerulescens and the non-hyperaccumulator Thlaspi ochroleucum. Plant, Cell and Environment 20, 898–906.
Vázquez MD, Poschenrieder C, Barceló J, Baker AJM, Hatton P, Cope GH. 1994. Compartmentation of zinc in roots and leaves of the zinc hyperaccumulator Thlaspi caerulescens J & C Presl. Botanica Acta 107, 243–250.
Zhao F, McGrath SP, Crosland AR. 1994. Comparison of three wet digestion methods for the determination of plant sulphur by inductively coupled plasma atomic emission spectroscopy (ICP-AES). Communications in Soil Science and Plant Analysis 25, 407–418.
Zhao FJ, Lombi E, Breedon T, McGrath SP. 2000. Zinc hyperaccumulation and cellular distribution in Arabidopsis halleri. Plant, Cell and Environment 23, 507–514.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  H. Kupper, B. Gotz, A. Mijovilovich, F. C. Kupper, and W. Meyer-Klaucke Complexation and Toxicity of Copper in Higher Plants. I. Characterization of Copper Accumulation, Speciation, and Toxicity in Crassula helmsii as a New Copper Accumulator Plant Physiology, October 1, 2009; 151(2): 702 - 714. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Mijovilovich, B. Leitenmaier, W. Meyer-Klaucke, P. M.H. Kroneck, B. Gotz, and H. Kupper Complexation and Toxicity of Copper in Higher Plants. II. Different Mechanisms for Copper versus Cadmium Detoxification in the Copper-Sensitive Cadmium/Zinc Hyperaccumulator Thlaspi caerulescens (Ganges Ecotype) Plant Physiology, October 1, 2009; 151(2): 715 - 731. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. L. Chaney, J. S. Angle, C. L. Broadhurst, C. A. Peters, R. V. Tappero, and D. L. Sparks Improved Understanding of Hyperaccumulation Yields Commercial Phytoextraction and Phytomining Technologies J. Environ. Qual., August 31, 2007; 36(5): 1429 - 1443. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Freeman, L. H. Zhang, M. A. Marcus, S. Fakra, S. P. McGrath, and E. A.H. Pilon-Smits Spatial Imaging, Speciation, and Quantification of Selenium in the Hyperaccumulator Plants Astragalus bisulcatus and Stanleya pinnata Plant Physiology, September 1, 2006; 142(1): 124 - 134. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. S. Sharma and K.-J. Dietz The significance of amino acids and amino acid-derived molecules in plant responses and adaptation to heavy metal stress J. Exp. Bot., March 1, 2006; 57(4): 711 - 726. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. A. Ingle, S. T. Mugford, J. D. Rees, M. M. Campbell, and J. A. C. Smith Constitutively High Expression of the Histidine Biosynthetic Pathway Contributes to Nickel Tolerance in Hyperaccumulator Plants PLANT CELL, July 1, 2005; 17(7): 2089 - 2106. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Punshon, A. Lanzirotti, S. Harper, P. M. Bertsch, and J. Burger Distribution and Speciation of Metals in Annual Rings of Black Willow J. Environ. Qual., June 7, 2005; 34(4): 1165 - 1173. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Freeman, D. Garcia, D. Kim, A. Hopf, and D. E. Salt Constitutively Elevated Salicylic Acid Signals Glutathione-Mediated Nickel Tolerance in Thlaspi Nickel Hyperaccumulators Plant Physiology, March 1, 2005; 137(3): 1082 - 1091. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Storey and R. A. Leigh Processes Modulating Calcium Distribution in Citrus Leaves. An Investigation Using X-Ray Microanalysis with Strontium as a Tracer Plant Physiology, November 1, 2004; 136(3): 3838 - 3848. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Freeman, M. W. Persans, K. Nieman, C. Albrecht, W. Peer, I. J. Pickering, and D. E. Salt Increased Glutathione Biosynthesis Plays a Role in Nickel Tolerance in Thlaspi Nickel Hyperaccumulators PLANT CELL, August 1, 2004; 16(8): 2176 - 2191. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Kupper, A. Mijovilovich, W. Meyer-Klaucke, and P. M.H. Kroneck Tissue- and Age-Dependent Differences in the Complexation of Cadmium and Zinc in the Cadmium/Zinc Hyperaccumulator Thlaspi caerulescens (Ganges Ecotype) Revealed by X-Ray Absorption Spectroscopy Plant Physiology, February 1, 2004; 134(2): 748 - 757. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Kindness, C. N. Sekaran, and J. Feldmann Two-Dimensional Mapping of Copper and Zinc in Liver Sections by Laser Ablation-Inductively Coupled Plasma Mass Spectrometry Clin. Chem., November 1, 2003; 49(11): 1916 - 1923. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Pineros and L. V. Kochian Differences in Whole-Cell and Single-Channel Ion Currents across the Plasma Membrane of Mesophyll Cells from Two Closely Related Thlaspi Species Plant Physiology, February 1, 2003; 131(2): 583 - 594. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||