Distribution of Zn in functionally different leaf epidermal cells of the hyperaccumulator Thlaspi caerulescens

Authors


Correspondence: B.Frey. Fax: +41 1739 22 15; E-mail: beat.frey@wsl.ch

ABSTRACT

The aim of this study was to show the potential of Thlaspi caerulescens in the cleaning-up of a moderately Zn -contaminated soil and to elucidate tolerance mechanisms at the cellular and subcellular level for the detoxification of the accumulated metal within the leaf. Measured Zn concentrations in shoots were high and reached a maximum value of 83 mmol kg1 dry mass, whereas total concentrations of Zn in the roots were lower (up to 13 mmol kg1). In order to visualize and quantify Zn at the subcellular level in roots and leaves, ultrathin cryosections were analysed using energy-dispersive X-ray micro-analysis. Elemental maps of ultrathin cryosections showed that T. caerulescens mainly accumulated Zn in the vacuoles of epidermal leaf cells and Zn was almost absent from the vacuoles of the cells from the stomatal complex, thereby protecting the guard and subsidiary cells from high Zn concentrations. Observed patterns of Zn distribution between the functionally different epidermal cells were the same in both the upper and lower epidermis, and were independent of the total Zn content of the plant. Zinc stored in vacuoles was evenly distributed and no Zn-containing crystals or deposits were observed. From the elemental maps there was no indication that P, S or Cl was associated with the high Zn concentrations in the vacuoles. In addition, Zn also accumulated in high concentrations in both the cell walls of epidermal cells and in the mesophyll cells, indicating that apoplastic compartmentation is another important mechanism involved in zinc tolerance in the leaves of T. caerulescens.

INTRODUCTION

Human activities such as industrial production release high loads of heavy metals into the biosphere. Zinc is the heavy metal found in the greatest concentrations in the majority of waste products in modern industrialized communities ( Boardman & McGuire 1990). In the past many research projects in agriculture and forestry have focused on the zinc tolerance of plants in order to overcome the adverse growth conditions in Zn-polluted soils. It is known that plant species have evolved basic strategies of metal tolerance ( Baker 1987). Certain wild plants and to a lesser extent crop plants are able to accumulate large amounts of heavy metals in their aerial parts ( Brooks 1998). For example, the hyperaccumulator Thlaspi caerulescens has the ability to accumulate up to more than 153 mmol kg−1 (or 10000 mg kg−1) of Zn in its foliar dry matter without showing any toxicity symptoms in certain conditions ( Baker & Brooks 1989). This characteristic may be exploited for the cleaning of zinc-contaminated soils, and may be of particular interest for agricultural soils as it allows restoration of soil fertility and the possible re-use of the land ( Baker et al. 1994 ; Robinson et al. 1997 ; McGrath 1998). Continuous extraction of Zn from the soil, leading to high metal accumulation in the plant shoots, requires tolerance mechanisms at the cellular and subcellular level for the detoxification of the accumulated metal within the leaf, such as effective sequestration and compartmentation ( Baker 1987). Further development of soil remediation by metal hyperaccumulator plants depends on the acquisition of more information about these mechanisms.

Energy dispersive X-ray micro-analysis (EDXMA) is a versatile technique for estimating the spatial distribution of chemical elements in cells ( Zierold & Hagler 1989). Vázquez et al. (1992 , 1994), using EDXMA at room temperature, proposed an involvement of vacuolar Zn sequestration in Zn tolerance where Zn was localized mainly as globular crystals inside the epidermal vacuoles of leaves ( Vázquez et al. 1994 ). However, room temperature methods for EDXMA cause redistribution and loss of elements ( Zierold & Steinbrecht 1987; Orlovich & Ashford 1993), and this lowers the reliability of the data from this study. Large losses of loosely bound Zn from roots during tissue preparation by conventional fixation for electron microscopy have been documented by Davies, Davies & Francis (1991). The study of Zn localization in freeze-fractures of T. caerulescens leaves under low temperature can partly overcome these difficulties of redistribution and loss of mobile elements ( Küpper, Zhao & McGrath 1999). Cryofixation is fast enough to retain the original distributions of inorganic elements sufficiently for micro-analytical studies ( Zierold & Steinbrecht 1987). However, the freeze-fracture method of leaves has not previously produced good results in studies on element distribution at the subcellular level. EDXMA of freeze-fractures gives only a qualitative estimation of the distributed elements with a low spatial resolution ( Stelzer & Lehmann 1993). Cryo-ultramicrotomy of high-pressure-frozen, biological tissue prior to EDXMA offers the possibility to obtain quantitative estimations of elements at the subcellular level. This method (without the interference of chemical fixatives and without ice crystal damage) allows new opportunities to localize elements under conditions closely resembling the natural state. This technique of preparing ultrathin cryosections (100 nm thick) for EDXMA has been used on animal tissue ( Zierold 1988) and on root tissue ( Frey et al. 1997 ). To our knowledge, EDXMA of ultrathin cryosections has never been applied to plant leaves. Plant leaves with their air-filled intercellular spaces and their differentiated tissues have still proved to be difficult to freeze uniformly, which is a major prerequisite for obtaining ultrathin cryosections of plant material ( Michel, Hillmann & Müller 1991).

Here we report, for the first time, EDXMA on high-pressure-frozen and cryosectioned leaves and roots of Thlaspi caerulescens, in order to obtain information about the localization of Zn in T. caerulescens and to improve our understanding of the mechanisms involved in the Zn tolerance of this species. Particular attention was paid to the quantitative estimation of Zn between different epidermal cells (epidermal cells and cells from the stomatal complex) and to the characterization of Zn-associated elements. We used a moderately Zn-polluted soil and it was not our objective to assess tolerance of T. caerulescens to Zn at the Zn hyperaccumulation level. Zn tolerance has been studied previously in artifical hydroponics culture ( Brown et al. 1995a ; Shen, Zhao & McGrath 1997; Lasat, Baker & Kochian 1998).

MATERIALS AND METHODS

Soil characteristics

Two different agricultural soils were selected for the experiment. Both were contaminated by atmospheric depositions of industrial Cu and Zn emissions, one by a steel smelter at Giornico (Canton Ticino, 46°23′40″N 8°53′37″E), and the other by a brass smelter at Dornach (Canton Solothurn, 47°28′47″N 7°36′42″E). Table 1 gives the heavy metal concentrations (extracted with 2 mol dm−3 HNO3 and 0·1 mol dm−3 NaNO3), pH and other selected properties of the two soils.

Table 1.  Chemical and physical properties of the soils used in the pots
 GiornicoDornach
  1. CEC, cation exchange capacity

Sand (g kg−1) 725182
Silt (g kg−1) 206500
Clay (g kg−1) 69318
pH (CaCl2)4·57·6
N total (g kg−1) 0·4310·3
C org (g kg−1) 2844
CaCO3 (g kg−1) 0137
CEC (mmol kg−1) 127519
Cd HNO3 (μmol kg−1) 8·918·7
Cd NaNO3 (μmol kg−1) 0·30·02
Cu HNO3 (mmol kg−1) 0·36·7
Cu NaNO3 (μmol kg−1) 2·29·9
Zn HNO3 (mmol kg−1) 4·39·9
Zn NaNO3 initial (μmol kg−1) 2491·24
Zn NaNO3 after harvest of
Thlaspi (μmol kg−1)
780·32

Plant material and experimental conditions

Seeds of Thlaspi caerulescens J. & C. Presl (Brassicaceae) (Prayon population) were sown in a commercial compost under controlled conditions (climate chamber, light 16 h, temperature day/night: 20/16 °C) and irrigated with deionized water. Plastic pots were prepared in replicate with 250 g fresh weight of the two soils after they had been passed through a 0·63 cm mesh sieve. A top dressing of fertilizer was applied to all pots when the seedlings were 2 months old, and 4 days later the seedlings were transplanted from the compost to the experimental pots (one plant per pot). Control pots were also set up with no plants. The pots were placed in a greenhouse (light 16 h, temperature day/night: 20–25/16 °C) and maintained at field capacity with deionized water.

Harvest and total element concentration

A minimum of three pots of each soil, together with seedlings, were collected 6, 12 and 24 weeks after transplantation, together with soil samples in the control pots. The plants were quickly rinsed with deionized water. The roots were separated from the soil, washed with deionized water for 5 min with 20 mmol dm−3 ethylenediaminetetraacetic acid (EDTA, C10H14N2Na2O8·2H2O) to remove the metals adsorbed on the roots. Finally, the roots were quickly rinsed three times with distilled water. All plant parts were dried at 60 °C (leaves) or 80 °C (roots) and the dry weights were determined. The samples were then ground (> 1 mm stainless steel sieve) and subsamples of 500 mg were microwave-digested (Lavis ETHOS, EM-2, MLS GmbH, Leutkirch, Germany) with a mixture of 5 cm3 HNO3 65% (v/v) and 1 cm3 H2O2 30% (v/v). Distilled water was added to bring the volume up to 25 cm3 and Cd, Zn and Cu were measured in the final solution as well as in the EDTA root-washing solutions.

The initial soils and samples from all pots were dried at 40 °C, and 2 mm-sieved samples were analysed for 2 mol dm−3 HNO3 and 0·1 mol dm−3 NaNO3-extractable heavy metal concentrations ( FAC 1989) and the pH (10 mmol dm−3 CaCl2) was measured. Copper and Zn were measured by flame atomic absorption spectrometry (Flame-AAS; VarianSpectrAA 400, Zug, Switzerland), Cd by graphite furnace atomic absorption spectrometry (GF-AAS; Varian SpectrAA 300) and the other major elements by inductively coupled plasma atomic emission spectroscopy (ICP-AES; Perkin Elmer Plasma 2000, Perkin Elmer Europe, Rotkreuz, Switzerland).

Cryopreparation for micro-analytical studies

Elemental cell composition of experimental plants was determined from subsamples of root and leaf material. Twenty-week-old plants, which were representative of nearly mature plants before flowering, were taken from both soils. The fresh roots were washed and blotted dry. The fine roots were cut in small pieces (length 3–4 mm) using a razor blade. Mature leaves were cut to small pieces (approximately 4 mm × 4 mm). For the analysis of freeze-fractures, the roots and leaf samples were mounted vertically on a scanning electron microscope (SEM) stub using a cryo-adhesive (BioRad, Munich, Germany) and the samples were rapidly frozen by plunging into liquid propane. In addition to transverse sections of the leaf, abaxial and adaxial epidermal tissues were analysed. In this case the tissue was mounted horizontally on an SEM stub using the same cryo-adhesive and plunge frozen. High-pressure freezing was a major prerequisite for obtaining ultrathin cryosections of the plant material ( Michel et al. 1991 ). Subsamples of fine roots and leaves were cut (approximately 200 μm in length), transferred into 1-hexadecene and were kept under a mild vacuum to replace intercellular gases and then mounted in aluminium support platelets which were high-pressure frozen in a high-pressure freezer (HPF 010; BAL-TEC, Balzers, Liechtenstein). The purpose of hexadecene is to exclude any gas bubbles that would collapse when subjected to high pressure. For each soil type five different roots and leaves were frozen for the analysis of freeze-fractures and cryosections.

Analysis in the cryo scanning electron microscrope

Micro-analysis was performed in a Philips SEM 515 (Philips, Eindoven, The Netherlands) equipped with an SEM cryo unit (SCU 020, Bal-Tec, Balzers, Liechtenstein) and a Tracor Northern energy dispersive X-ray analysis system (Noran Instruments Inc., Middleton, WI, USA) interfaced with a Voyager software package as previously described ( Brunner, Frey & Riesen 1996; Frey et al. 1996 ). Electron-induced X-rays were detected by a Si(Li) spectrometer detector (Tracor Northern 30 mm2 Microtrace) with an ultra-thin beryllium window. The microscope was operated at an acceleration voltage of 20 kV with a beam current of 80 nA and the stage-tilt was adjusted to obtain a take-off angle of 44°. Working distance was 12 mm. The temperature on the SEM cold stage was kept below − 120 °C. Single-spot analyses of selected cells in the roots and leaves and elemental maps in the leaves were carried out to determine the distribution of sulphur, calcium and zinc. X-ray data for elemental maps in element-specific energy windows were collected with a spatial resolution of 128 × 128 pixels with a dwell time of 0·1 s per pixel. Leaf surfaces were observed at 12 kV.

Analysis of ultrathin cryosections in the scanning transmission electron microscopy

Details of preparation and X-ray micro-analysis have been described previously ( Frey et al. 1997 ). The high-pressure-frozen samples were removed from the sandwich in the microtome chamber cooled to 160 °C. Approximately 100 nm thick cryosections were cut dry by means of a Reichert FC4 Ultracut cryo-ultramicrotome (Reichert, Vienna, Austria) at a temperature below − 120 °C. The sections were cryotransferred and freeze-dried in a Siemens Elmiskop ST 100 F scanning transmission electron microscope (STEM) (Siemens, Berlin, Germany) equipped with a cold stage. They were imaged and analysed at − 135 °C using a 100 kV electron beam of 1·4 nA. EDXMA was carried out by means of a Si(Li) detector (Nuclear Semiconductors, Menlo Park, CA, USA) combined with a Link AN10000 system (multichannel analyser) (Link Systems, High Wycombe, Buckinghamshire, UK). X-ray spectra were processed for quantitative evaluation according to the peak-to-continuum method using the Link Quantem FLS program. Spectra were collected within 100 s live time either in a spot mode or in a scanning mode over the area of interest (subcellular compartments such as cell walls, cytoplasm, vacuole and organelles). Quantitative analysis was carried out according to Zierold (1988). The dry mass portion, d, of analysed compartments was determined by measuring the electron optical darkfield intensity of freeze-dried cryosections by STEM. The electron optical darkfield intensity measured by an annular electron detector, was found to be linearly related with the dry mass portion, d, of the irradiated area in the freeze-dried cryosection. The value of d is related to the water portion, w, by the equation d + w = 1. Element contents, cd, obtained by X-ray micro-analysis in terms of mmol kg−1 dry mass can be converted to cw (mmol kg−1 water) by the equation cw = cdd/(1 −d). The dry mass portion, d, of the analysed compartments had the following values: d (cell wall) = 0·5 ± 0·05; d (vacuole) = 0·2 ± 0·03; d (cytoplasm) = 0·25 ± 0·03; d (chloroplast) = 0·25 ± 0·02. The relative water portions are w (cell wall) = 0·5, w (vacuole) = 0·8, w (cytoplasm) = 0·75, w (chloroplast) = 0·75, respectively. The detection limit for Zn accounted for 5–7 mmol kg−1 dry mass or 2 mmol l−1 water (depending on the dry mass portion of the corresponding cell compartments). Elemental maps were obtained by scanning the electron beam in 10 frames of 128 × 128 pixels with a dwell time of 10 ms and collecting X-rays in element-specific energy windows. Differences in element distribution were presented qualitatively by a pseudocolour scale. Mass thickness variation were corrected by normalizing the element maps to the continuum X-ray map.

RESULTS

Plant growth and total elemental concentrations in roots and leaves

Zinc concentrations in dry matter were always higher in Thlaspi caerulescens grown on Giornico than on Dornach soil in all cases (statistically significant at the 5% level of probability) except for shoots after 12 weeks ( Table 2). For both soils, the Zn concentrations in shoots were higher after 6 weeks than after 12 and 24 weeks, whereas they were higher in roots after 24 weeks than after 6 weeks. Concentrations in shoots were, however, always higher than in the roots, with a maximum of 83 mmol kg−1 dry mass in the shoots of plants grown on Giornico soil, demonstrating that the efficient translocation of Zn from roots to shoots is typical of this plant ( Table 2). For both soils, Zn concentrations in shoots were high, but did not reach the 1% Zn in dry matter required to term the process hyperaccumulation. Thlaspi caerulescens leaves showed no visible symptoms of metal-induced toxicity, although plants from the Giornico soil displayed purple pigmentation on some leaves. Total Zn concentrations in plants from the Giornico soil for micro-analytical studies (see below) were found to be 63 mmol kg−1 dry mass in the shoots and 15 mmol kg−1 dry mass in the roots. The Zn concentrations in plants from the Dornach soil were 20 mmol kg−1 dry mass in the shoots and 2 mmol kg−1 dry mass in the roots.

Table 2.  Total concentrations of Zn and other elements (mmol kg−1 dry wt; mean ± SD) in roots and shoots of Thlaspi caerulescens during the experiment
 SoilCaMgPFeZnCu†Cd†
  1. Dornach

  2. 760 ± 80b

  3. 84 ± 18b

  4. 35 ± 3a

  5. 5 ± 2a

  6. 28 ± 10b

  7. 236 ± 256a

  8. 39 ± 23a

  9. a, bDifferent letters indicate significant differences (P < 0·05) between the two soils within the same harvest time, by Student’s t-test.

  10. ‡Concentrations for Cu and Cd given in μmol kg−1 dry wt.

  11. n.d. not determined.

Roots
After 6 weeks
 Giornico33 ± 2a23 ± 2a92 ± 19a10 ± 3a4 ± 1a315 ± 126a5 ± 1a
 Dornach90 ± 26b65 ± 28b83 ± 6a6 ± 4a2 ± 0b315 ± 141a3 ± 1a
After 12 weeks
 Giornico56 ± 10a56 ± 3a66 ± 1a25 ± 13a8 ± 2a441 ± 94a10 ± 2a
 Dornach93 ± 14b75 ± 5b65 ± 5a22 ± 8a3 ± 1b1338 ± 205b6 ± 2b
After 24 weeks
 Giornico76 ± 47a80 ± 15a56 ± 6a26 ± 13a13 ± 6a283 ± 94an.d.
 Dornach139 ± 19a110 ± 15a40 ± 9a9 ± 4a2 ± 1b1998 ± 1730an.d.
Shoots
After 6 weeks
 Giornico614 ± 66a145 ± 28a121 ± 16a2 ± 1a83 ± 4a94 ± 16a55 ± 6a
 Dornach988 ± 65b158 ± 20a105 ± 7a2 ± 1a49 ± 13b236 ± 31b29 ± 5b
After 12 weeks
 Giornico371 ± 32a63 ± 2a43 ± 3a3 ± 1a36 ± 13a63 ± 16a28 ± 6a
 Dornach645 ± 202b85 ± 14b49 ± 9a4 ± 1b24 ± 8a409 ± 346a16 ± 5b
After 24 weeks
 Giornico343 ± 50a55 ± 6a34 ± 6a3 ± 1a52 ± 10a126 ± 63a35 ± 9a

In contrast with Zn, Cu concentrations were highest in plants grown on Dornach soil, reflecting the high Cu concentration in this soil, whereas higher Cd concentrations were found in plants grown on Giornico soil (total Cd in the soil was very low but the NaNO3-extractable fraction was higher than in the Dornach soil) than on Dornach soil. The trends were similar to those for Zn, with higher Cd concentrations in shoots than in roots, whereas Cu concentrations were higher in roots than in shoots ( Table 2). The Cu and Cd concentrations in the plants were always too low to be detected by EDXMA, therefore no further studies were made on these elements.

Major element concentrations varied according to soil and harvest ( Table 2). Concentrations of P and Fe were similar in shoots regardless of whether they were grown on Dornach or Giornico soil. Major element concentrations in individual plants used for ultrathin cryosections were within the range found for the replicates (i.e. Ca, 290 mmol kg−1; Mg, 48 mmol kg−1; P, 32 mmol kg−1 and Fe, 3 mmol kg−1 in the shoots of Thlaspi grown on Giornico soil). On one hand Zn concentrations were higher in Thlaspi grown on Giornico soil and on the other hand we observed the same major element distribution within the plant (apart from higher Ca concentrations in plants grown on Dornach soil) and the same evolution over time whatever the soil used. As a consequence we focused more on the results obtained for Thlaspi grown on Giornico soil which were considered to be representative of both soils.

Distribution of Zn and other elements in freeze-fractures of leaves

Identification of elemental composition depends on avoidance of ion migration prior to analysis. Therefore, leaves of T. caerulescens were cryofixed and analysed under low temperature. Figure 1 shows an elemental map of Zn, Ca and S across a freeze-fracture of a leaf of T. caerulescens, providing a qualitative image of element distribution across a large portion of the tissue surface. This represents a major advantage over the use of X-ray micro-analysis of individual cells or cell compartments, at least for a preliminary study. The map shows that Zn accumulated mainly in the epidermal cells and was almost absent from the mesophyll cells and the vein. Single spot analyses of epidermal cells showed a high Zn accumulation in elongated cells and a distinctly lower accumulation of Zn in smaller cells identified as cells from the stomatal complex. The patterns of Zn distribution were similar for both the Dornach and the Giornico soils. From structural observations of the leaf surface, it was possible to identify two morphologically different cell types in the epidermis: epidermal cells covered with epicuticular wax crystals and cells from the stomatal complex which were much smaller and with no (or distinctly fewer) wax crystals ( Fig. 2). Stomata were observed on both the upper and lower surfaces. The Zn accumulated predominantly in the epidermal cells, whereas cells with low amounts of Zn were mainly cells from the stomatal complex. Calcium did not show a homogeneous distribution across the leaf section. It was found in epidermal cells and in some large mesophyll cells and was almost absent from the veins. Sulphur was relatively homogeneously distributed over the cells but with a distinctly higher content in the veins ( Fig. 1). Although the concentrations of Zn in the leaves were lower on Dornach soil than on Giornico soil, the distribution pattern of Zn and other relevant elements in the leaves was similar in both soils and therefore samples from the Dornach soil were not studied further on ultrathin cryosections.

Figure 1.

Elemental mapping of freeze-fracture of Thlaspi caerulescens leaf section (origin Giornico soil). Scanning electron microscope image and the corresponding elemental maps for S, Ca and Zn of tissue section. Zinc accumulated predominantly in the epidermal cells (EC) and was almost absent from the cells of the stomatal complex (SC). Calcium was abundant in the epidermal and mesophyll (MC) cells, but the distribution was not homogeneous. Sulphur was higher in the vein (V) compared to the epidermal and mesophyll cells. Bar = 50 μm.

Figure 2.

Scanning electron micrograph of the adaxial leaf surface of Thlaspi caerulescens (origin Giornico soil). The two characteristic types of cells analysed for the Zn distribution: elongated epidermal cell (EC) and the smaller cells from the stomatal complex (SC). Bar = 50 μm.

Structural aspects of ultrathin cryosections

EDXMA of freeze-fractures gives only a qualitative estimation of the distributed elements with a low spatial resolution. To obtain quantitative estimations of elements at the subcellular level ultrathin cryosections of high-pressure-frozen roots and leaves of T. caerulescens were used. The freeze-dried and unstained cryosections were translucent, exhibiting structural details in the dark-field STEM. Section thickness was approximately 100 nm. The sections clearly showed the cell wall, the cytoplasm, the vacuoles and smaller organelles in the individual cells ( Fig. 3a & b). This fact made it possible to perform EDXMA measurements on ultrathin cryosections at the subcellular level. Good structural preservation of the subcellular compartments without the formation of damaging ice crystals was observed in the cryosections of leaf cells ( Fig. 3a & b). Membranes such as the plasma membrane and the vacuolar membrane were smooth and organelles appeared turgid and were not shrunken. The matrix of the vacuoles was homogeneous and free of electron-opaque deposits. Spherical to slightly elliptical chloroplasts were recognized in the mesophyll cells but not in epidermal cells ( Fig. 3b).

Figure 3.

STEM image of a high-pressure-frozen, freeze-dried ultrathin (100 nm) cryosection of a leaf of Thlaspi caerulescens derived from a metal-polluted soil (Giornico). (a) The unstained cryosection shows the arrangement of the cells from the stomatal complex (G = guard cell and S = subsidiary cell) interspaced between epidermal cells (E). Epidermal ridges were recognized on the surface of the epidermis. Chloroplasts were located in the mesophyll cells (M). Bar = 10 μm (b) The unstained cryosection shows good structural preservation of subcellular compartments such as the vacuoles, cell wall and organelles. The cell wall complex with the middle lamella is visible. Bar = 5 μm.

Distribution of Zn and other elements in ultrathin cryosections of leaves

The elemental maps showed that Zn accumulated preferentially in the vacuoles of epidermal cells ( Fig. 4) and was almost absent from the cell vacuoles of the stomatal complex ( Fig. 5). High concentrations of K as shown in Fig. 5, indicate guard cells. Remarkable amounts of Zn were also detected in the cell walls of the epidermal cells and mesophyll cells and presumably also in the intercellular spaces between epidermal and mesophyll cell walls ( Fig. 5). Zinc was homogeneously distributed over the vacuoles. The corresponding STEM image reveals a uniform matrix throughout the vacuole and a small rim of the cytoplasm as shown in Fig. 4. Interestingly, Zn is less evident in these small rims indicating that the cytoplasm contained little or almost no Zn compared with the cell wall and the vacuole. Calcium was not homogenously distributed across epidermal and mesophyll cells. It was abundant in the vacuoles and in the cell walls (apoplast) of epidermal cells, but was almost completely absent from the vacuoles of the guard cells ( Fig. 5). Calcium also clearly accumulated in the epidermal ridges ( Fig. 4) and in high concentrations in the vacuoles of mesophyll cells ( Fig. 5). In other words, high concentrations of Zn in the vacuoles and the cell walls (apoplast) of epidermal cells were always associated with high concentrations of Ca. On the contrary, there was no evidence of any association between Zn and P or S.

Figure 4.

Elemental maps of the high-pressure-frozen and freeze-dried cryosection shown in Fig. 3a at a higher magnification. The STEM micrograph (bright/dark field image) and the corresponding element distribution maps for Zn, P, Cl, K, Ca. The colour scale indicates X-ray intensity from low (black, blue) to high (red, white). The following cells were identified: epidermal cell (E), subsidiary cells (S). Bar = 3 μm.

Figure 5.

Elemental maps of the high-pressure-frozen and freeze-dried cryosection shown in Fig. 3a at a higher magnification. The STEM micrograph (bright/dark field image) and the corresponding element distribution maps for Zn, P, Cl, K, Ca. The colour scale indicates X-ray intensity from low (black, blue) to high (red, white). The following cells were identified: Subsidiary cell (S), guard cell (G) and mesophyll cell (M). Bar = 3 μm.

Quantitative EDXMA micro-analysis in ultrathin cryosections of leaves

Similar patterns of element distribution in leaf cells were found by spot analyses. Tables 3 and 4 list the quantitative determinations of elements in various cell compartments in the leaves. The highest accumulation of Zn was up to 1136 mmol kg−1 dry mass in the vacuoles of epidermal cells ( Table 3). Distinctly lower concentrations (46 mmol kg−1 dry mass) were found in the cellular vacuoles of the stomatal complex. Zinc not only accumulated in the vacuoles of epidermal cells, but was also found in high concentrations in the walls (apoplast) of these cells ( Table 3). Taking into account that the dry mass in the cell walls (d = 0·5) was higher than in the vacuoles (d = 0·18), the difference in Zn concentration in terms of cell water in cell walls and in vacuoles of epidermal cells at 177 and 249 mmol kg−1 water, respectively, was smaller (for conversion, see MATERIALS AND METHODS section). This indicates that the cell wall (apoplast besides the vacuole) was also a preferential site for Zn accumulation in the leaves. High concentrations of Ca and Cl were found in the epidermal vacuoles ( Table 3). Interestingly, high concentrations of Zn in the vacuoles correspond to low concentrations of K (up to 28 mmol kg−1 dry mass). There was no evidence of any association of Zn with either P or S in the vacuoles of epidermal cells. Organelles, presumably mitochondria, showed a similar pattern of elemental distribution throughout all types of epidermal cells; they were rich in K, Ca, P and S but relatively low in Zn.

Table 3.  Element content (mmol kg−1 dry wt; mean ± SD, n = 6–8) of epidermal subcellular compartments in ultrathin (100 nm) cryosections of Thlaspi caerulescens leaves. The two characteristic types of cells analysed (as shown in Fig. 2): epidermal cells and the cells from the stomatal complex (including guard and subsidiary cells). Data are derived from STEM-EDXMA
 Epidermal cellsGuard cellsSubsidiary cells
 Cell wallVacuoleOrganelleCell wallVacuoleOrganelleCell wallVacuoleOrganelle
  1. Element contents obtained in terms of mmol kg−1 dry mass can be converted to mmol kg−1 water; see ‘Materials and Methods’. Detection limit for Zn accounted for 5–7 mmol kg−1 dry mass.

Mg54 ± 8196 ± 4290 ± 831 ± 1122 ± 664 ± 2344 ± 13124 ± 4188 ± 15
P15 ± 32 ± 1363 ± 5113 ± 535 ± 14431 ± 718 ± 412 ± 6399 ± 75
S66 ± 4243 ± 15219 ± 4345 ± 1747 ± 28158 ± 8351 ± 2088 ± 25199 ± 51
Cl68 ± 23489 ± 20177 ± 2429 ± 1194 ± 37102 ± 4537 ± 8261 ± 90124 ± 37
K170 ± 3328 ± 13296 ± 44125 ± 40310 ± 68324 ± 57103 ± 32152 ± 49302 ± 63
Ca447 ± 54812 ± 74259 ± 26467 ± 6221 ± 688 ± 53520 ± 46649 ± 288224 ± 29
Zn177 ± 361136 ± 13049 ± 11129 ± 3122 ± 139 ± 4147 ± 5546 ± 1937 ± 10
Table 4.  Element content (mmol kg−1 dry wt; mean ± SD, n = 6–8) of mesophyll cell compartments in ultrathin (100 nm) cryosections of Thlaspi caerulescens. Data are derived from STEM-EDXMA
 Cell wallVacuoleChloroplast
  1. Element contents obtained in terms of mmol kg−1 dry mass can

  2. be converted to mmol kg−1 water; see ‘Materials and Methods’. Detection limit for Zn accounted for 5–7 mmol kg−1 dry mass.

  3. n.d. not detected.

Mg47 ± 1011 ± 1055 ± 14
P16 ± 440 ± 1374 ± 11
S43 ± 8244 ± 6186 ± 21
Cl49 ± 17154 ± 5013 ± 1
K131 ± 5774 ± 22128 ± 40
Ca438 ± 701347 ± 31419 ± 3
Zn143 ± 325 ± 2n.d.

In mesophyll cells, Zn was found in the cell walls in similar concentrations to those in the cell walls of the epidermal cells, whereas Zn was near the detection limit in vacuoles and below the detection limit in chloroplasts ( Table 4). This means that distinctly less Zn had accumulated in the vacuoles of mesophyll cells compared with the vacuoles of epidermal cells ( Table 3 versus Table 4). Calcium was by far the most abundant element in the mesophyll cells, with highest concentrations in the vacuoles and with somewhat lower concentrations in the cell walls. However, Ca concentrations in the cell were in the same range in both the cell wall of mesophyll and epidermal cells ( Table 3 versus Table 4). Interestingly, the concentrations of sulphur in the vacuoles of mesophyll cells were slightly higher than in the vacuoles of the epidermal cells ( Table 3 versus Table 4) whereas the concentrations of S were strongly reduced in comparison with root cortical cells ( Table 3 versus Table 5).

Table 5.  Element content (mmol kg−1 dry wt; mean ± SD, n = 6–8) of cortical cell compartments in ultrathin (100 nm) cryosections of Thlaspi caerulescens roots. Data are derived from STEM-EDXMA
 Cell wallCytoplasmVacuole
  1. Element contents obtained in terms of mmol kg−1 dry mass can

  2. be converted to mmol kg−1 water; see ‘Materials and Methods’. Detection limit for Zn accounted for 5–7 mmol kg−1 dry mass.

  3. n.d. not detected.

Mg46 ± 1424 ± 8226 ± 70
P19 ± 17422 ± 10521 ± 9
S58 ± 7145 ± 361214 ± 294
Cl13 ± 134 ± 1245 ± 18
K207 ± 84300 ± 88544 ± 190
Ca436 ± 1057 ± 5124 ± 35
Zn9 ± 4n.d.n.d.

Quantitative EDXMA micro-analysis in ultrathin cryosections of roots

The elemental concentrations of cortical root cells are compiled in Table 5. The concentrations of Zn in the roots were in general low compared to Zn in epidermal cells of the leaves ( Table 5 versus Table 3). Zinc was only detectable in the cell walls of cortical cells. In the samples from the Dornach soil Zn was below the detection limit in the individual root cells (data not shown). Calcium was abundant in the cell walls, whereas K was distributed homogeneously over the root section, and the concentrations of Cl were low in the vacuoles. Surprisingly, high concentrations of S were found in the vacuoles of root cortical cells as compared to the vacuoles in the leaves ( Table 5 versus Tables 3 and 4). The apparent accumulation of S within root cortical cells was confirmed in measurements taken from freeze-fractures, where S was high in root cortical cells and in the stele (data not shown).

DISCUSSION

Total element concentrations and uptake by Thlaspi caerulescens

Thlaspi caerulescens always had a higher biomass when grown in Giornico soil (acidic) than in Dornach soil (calcareous) although this species is usually defined as a calcareous soil indicator ( Lauber & Wagner 1996). Combined with higher Zn concentrations, which were also present in plants, the resulting Zn uptake increased when the Zn bioavailable pool was larger, as was found by Brown et al. (1994) . Indeed, the uptake was not related to total (HNO3-extractable) Zn concentrations in T. caerulescens; the total quantities of Zn extracted by this plant from the Giornico soil were twice as high as those removed from the Dornach soil, although the total (HNO3-extractable) Zn concentrations in the Giornico soil were only half those in the Dornach soil. As a consequence, Thlaspi caerulescens removed 10% of the initial total Zn in the Giornico soil in 24 weeks instead of less than 2% in the Dornach soil. This amount was larger than the amount predicted by extraction with NaNO3; T. caerulescens was able to remove only twice the equivalent of the NaNO3-extractable Zn present in the soil of Giornico, whereas in Dornach soil it could extract the equivalent of 100 times the NaNO3-extractable Zn. Thus, even though the plants were grown in pots, that is in a confined environment which might have enhanced the extractability, this result raises the question of the significance of NaNO3 extraction as a tool to assess metal availability to T. caerulescens ( Gupta & Aten 1993) and, as a consequence, the type of metal pool which is depleted by T. caerulescens. Brown et al. (1994) suggested that when Zn concentrations in soil solutions are limiting (which is the case for Dornach), an active mechanism could be responsible for the discrepancy between uptake and indices of Zn bioavailability in the soil. Such a mechanism may occur in the Dornach soil, in which NaNO3-extractable Zn is very low.

Cadmium concentrations in the T. caerulescens tissue were always very low, although T. caerulescens has also been reported to hyperaccumulate Cd ( Robinson et al. 1998 ). These relatively low Cd concentrations are related to the soil concentrations, which were below 19 μmol kg−1 of HNO3-extractable Cd. The Cd concentrations, however, were in the same range as that found by Brown et al. (1995b) originating from soils with similar Cd concentrations. The relatively low Cd concentrations might also be related to the Thlaspi genotype itself ( Meerts & Van Isacker 1997).

At the plant level, the concentrations of Zn and other elements in T. caerulescens grown on both Dornach and Giornico soils showed changes with time. In the shoots, all elements except Fe and Cu became diluted with time, whereas in the roots some elements such as Zn, Ca, Mg, Fe, Cu and Cd accumulated. A similar decrease over time has also been described by Brown et al. (1995b) for Mg and Ca in leaves of T. caerulescens grown in sludge-amended soil, and by Rascio (1977) in field conditions. However, in pot experiments lasting 6 months, Meerts & Van Isacker (1997) measured concentrations of major elements which were in general higher than those found by Brown et al. (1995b) as well as higher than those detected in the present experiment. These differences may be related to variations in plant populations, age of the plants or decrease in supply of bioavailable elements to the plants.

The Zn concentrations, although not above 1% in the dry matter, were within the range obtained by McGrath et al. (1993) as well as that measured by Brown et al. (1995b) and Meerts & Van Isacker (1997) in Thlaspi populations originating from Prayon and Plombières. They were higher than those measured by Ebbs et al. (1997) in Thlaspi grown in soil with higher Zn and Cd concentrations than the Dornach and Giornico soils. This indicates that our samples have Zn concentrations commonly observed for T. caerulescens in a wide range of soil conditions.

Elemental distribution at the subcellular level

Our micro-analytical data on high-pressure-frozen and cryosectioned leaves of T. caerulescens show for the first time that the cells from the stomatal complex contained distinctly lower zinc concentrations in the vacuoles compared with the vacuoles of epidermal cells. Zinc was mainly localized in elongated epidermal cells in the upper and lower epidermis of the leaves. Preferential accumulation of Zn in the epidermal cells was also observed in the study of Küpper et al. (1999) , with T. caerulescens containing total Zn concentrations of up to 306 mmol kg−1 dry mass in the leaves and in the study of Vázquez et al. (1994) containing total Zn concentrations of up to 92 mmol kg−1 dry mass in the leaves. Küpper et al. (1999) reported that the relative Zn concentration in epidermal cells correlated linearly with the cell length, suggesting that vacuolation of epidermal cells promoted the Zn accumulation within the cells. However, the authors did not distinguish between different cell compartments (vacuoles versus cell walls) and between epidermal cells and cells from the stomatal complex in the freeze-fractures.

Lowering the Zn content in the cells of the stomatal complex indicates that these cells have little Zn tolerance and may have membrane properties distinct from other epidermal cells. Identification of guard cells in the cryosections was facilitated by the high concentrations of K in comparison with the adjacent cells ( Marschner 1995) and the intracellular structures as well as the abundance of starch granules (observed at higher magnification) were similar to the structures of guard cells shown by Vázquez et al. (1994) . Guard cells and subsidiary cells are known to have a special function in stomatal regulation. Stomatal movements are mediated by the accumulation and release of ions (K+, balanced by Cl and malate from starch) in and from the vacuoles of the guard cells ( Hite & Outlaw 1994). The specific ion-transport properties of cells from the stomatal complex may be able to partly exclude zinc from the stomata, thus making this potentially toxic metal unavailable for interaction with metabolically active cellular compartments. A similar mechanism to protect chloroplasts from the toxicity of Zn (exclusion of Zn in chloroplasts) could occur in the mesophyll cells. Nickel showed a different pattern of distribution in the epidermal cells of leaves of T. montanum var. siskiyouense ( Heath, Southworth & D’Allura 1997). Compartmentation of Ni was found in subsidiary cells that surround guard cells but not in guard cells or in epidermal cells. This difference might be related to the different behaviour of Ni as opposed to Zn, the different Thlaspi species or the different preparation procedure used for EDXMA. Measurements were carried out on chemically fixed leaf surfaces and not in freeze-fractures or in cryosections.

Vázquez et al. (1994) , using EDXMA at room temperatures, localized Zn in electron-dense deposits in the vacuoles of epidermal and subepidermal cells of the leaves of T. caerulescens. In our study, however, Zn stored in the vacuoles of the leaf epidermis was evenly distributed and no Zn-containing crystals or deposits were observed in high-pressure-frozen, cryosectioned plant material. High concentrations of Zn were often accompanied by high concentrations of Ca in the epidermal cells, and Ca was the most abundant element in the vacuoles of mesophyll cells. The role of the relatively high Ca concentrations in vacuoles of epidermal and of mesophyll cells in T. caerulescens is not clear. Accumulation of Ca in leaves seems to be plant-family specific, and the Ca concentration in the soil solution may influence the relative distribution of Ca between mesophyll and epidermal cells ( Dietz et al. 1992 ). The high concentrations of cations in the vacuoles raises the question of which anions counterbalance the high Zn and Ca concentrations inside the vacuoles. Neither Cl, S nor P were present in appreciable quantities, and therefore anions of these elements alone would not have been sufficient to balance the high concentrations of cations. There was also no evidence that Zn was precipitated with inorganic P as suggested as a Zn detoxification mechanism in the root vacuoles of Deschampsia caespitosa ( Van Steveninck et al. 1987 ). Recently, Salt et al. (1999) determined the ligand environment of Zn in different tissues of T. caerulescens using X-ray absorption spectroscopy. They demonstrated that organic acids are involved in xylem transport and Zn storage in shoots and the Zn coordination in the shoots was dominated by citrate with smaller contributions from hydrated zinc, histidine and oxalate binding.

Vacuolar Zn sequestration in epidermal cells, in the present study, was not the only tolerance mechanism in leaves of T. caerulescens. The high levels of Zn binding to epidermal cell walls and intercellular spaces between epidermal and mesophyll cell walls in this study are in good agreement with the findings of Vázquez et al. (1994) , indicating that the accumulation of Zn in the cell wall and intercellular spaces of epidermal cells is an important tolerance mechanism for Zn in T. caerulescens. Whether the Zn located in the intercellular spaces is an artefact or not is unclear from this study. The leaf intercellular spaces would normally be air-filled. Here, however, it was necessary to replace intercellular gases by 1-hexadecene before high-pressure freezing. Gas bubbles in the intercellular spaces would collapse when subjected to high pressure ( Michel et al. 1991 ). Another source of artefacts can be introduced by cryofixation. Jeffree et al. (1987) demonstrated that water may be displaced from cells of Phaseolus vulgaris into intercellular spaces prepared by conventional freezing. However, extracellular deposits on the surfaces of cells have not been observed in high-pressure-frozen material.

High Zn accumulation in the leaves is partly due to the binding of the cationic Zn to the anionic sites in the cell wall. Similarly, Salt et al. (1999) , found a substantial fraction of Zn in the leaves of T. caerulescens to be complexed to the cell wall, suggesting that cell wall binding sites have a high affinity for Zn, thereby lowering potentially toxic-free Zn ions in the cytoplasm. Zinc exudation as metal-rich viscous droplets of leaves was recently proposed as a Zn tolerance mechanism (Leblanc, published in Brooks 1998). Using SEM-EDXMA he observed small hemispherical bodies containing Zn and S at the surface of some leaves of T. caerulescens. In the present study no metal-rich droplets were observed. Both the adaxial and the abaxial leaf surfaces were covered with epicuticular wax crystals only, suggesting that these structures are a constitutive property of the T. caerulescens species used in this experiment.

In the roots, both the cell walls and the vacuoles of epidermal and cortical cells represent sites for the localization of Zn ( Vázquez et al. 1992 , 1994). Salt et al. (1999) found that the majority of the intracellular Zn in roots of T. caerulescens was coordinated with histidine, with the rest complexed to the cell wall. In our study, binding of Zn to negatively charged sites associated with the cell wall contributed substantially to the binding of Zn accumulated in roots. However, the binding property of root cell walls seems unlikely to be significant, since most of the Zn was transported into the shoot (accumulation five times higher than in roots). In contrast to Vázquez et al. (1992) , we did not find that Zn had accumulated in the root vacuoles of T. caerulescens. However, in their study the total Zn concentrations in roots reached 60 mmol kg−1 dry mass, four times higher than in our study. The apparent accumulation of S in the vacuoles of root cortical cells, in the stele and the veins of the leaves raises the question of whether sulphur is involved in Zn tolerance and hyperaccumulation. Phytochelatins are important S-containing chelates known to detoxify higher plants from certain heavy metals. However, a key role of phytochelatins in Zn tolerance mechanism is questionable ( Rauser 1990). From this study, binding of Zn to sulphhydryl compounds can probably be rejected since there was no evidence of association between Zn and S.

From our results we conclude that T. caerulescens has a specific aptitude for taking up Zn. This is expressed by the high general Zn uptake in leaves whatever the level of soil contamination and also the ability to remove forms of Zn more tightly bound to the soils than those extractable with NaN03 (0·1 mol dm−3). Zinc accumulation in leaves was limited to peripheral epidermal cells. Observed patterns of Zn distribution were the same regardless of the inital Zn content of the soil or the total Zn content of the plant. Both the cell walls and the vacuoles were involved in the Zn tolerance mechanisms to protect metabolically active cellular compartments from toxic Zn concentrations. Vacuolar Zn sequestration was predominantly restricted to epidermal cells, whereas guard and subsidiary cells were protected from exposure to deleterious concentrations of this metal. It would be rewarding to study the element distribution over a full growing season in order to determine the re-allocation of elements within the plant and the mechanisms involved in the process. Further elucidation of the specifity of induced Zn-binding processes in functionally different epidermal cells remains a challenge.

ACKNOWLEDGMENTS

We thank S. Dongard, Dortmund for excellent technical assistance in preparing the ultrathin cryosections for X-ray micro-analysis and P. Walther, ETH Zurich for the help in using the high-pressure freezer. We are grateful to A. Baker for providing the Thlaspi seeds, and to P. Christie of The Queen’s University of Belfast and M. J. Sieber for helpful comments on the manuscript.

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