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Plant Physiol. (1999) 119: 305-312
Cellular Compartmentation of Zinc in Leaves of the
Hyperaccumulator Thlaspi caerulescens1
Hendrik Küpper,
Fang Jie Zhao, and
Steve P. McGrath*
Universität Konstanz, Fakultät Biologie, Postfach 5560, 78434 Konstanz, Germany (H.K.); and Soil Science Department,
IACR-Rothamsted, Harpenden, Hertfordshire AL5 2JQ, United Kingdom
(F.J.Z., S.P.M.)
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ABSTRACT |
Cellular compartmentation of Zn in the leaves of the
hyperaccumulator Thlaspi caerulescens was investigated
using energy-dispersive x-ray microanalysis and single-cell sap
extraction. Energy-dispersive x-ray microanalysis of frozen, hydrated
leaf tissues showed greatly enhanced Zn accumulation in the epidermis
compared with the mesophyll cells. The relative Zn concentration in the
epidermal cells correlated linearly with cell length in both young and
mature leaves, suggesting that vacuolation of epidermal cells may
promote the preferential Zn accumulation. The results from single-cell
sap sampling showed that the Zn concentrations in the epidermal
vacuolar sap were 5 to 6.5 times higher than those in the
mesophyll sap and reached an average of 385 mM in plants
with 20,000 µg Zn g 1 dry weight of shoots. Even when
the growth medium contained no elevated Zn, preferential Zn
accumulation in the epidermal vacuoles was still evident. The
concentrations of K, Cl, P, and Ca in the epidermal sap generally
decreased with increasing Zn. There was no evidence of association of
Zn with either P or S. The present study demonstrates that Zn is
sequestered in a soluble form predominantly in the epidermal vacuoles
in T. caerulescens leaves and that mesophyll cells are
able to tolerate up to at least 60 mM Zn in their sap.
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INTRODUCTION |
Different mechanisms have been proposed to explain the tolerance
of plants to toxic heavy metals (Baker and Walker, 1990 ; Verkleij and
Schat, 1990). Some tolerant plant species, the so-called "excluders," use exclusion mechanisms by which uptake and/or
root-to-shoot transport of heavy metals are restricted. Other tolerant
plant species are able to cope with elevated concentrations of toxic metals inside of their tissues through production of metal-binding compounds, cellular and subcellular compartmentation, or alterations of
metabolism.
An extreme strategy for metal tolerance that is in sharp contrast to
metal exclusion is "hyperaccumulation," a term that was originally
used by Brooks et al. (1977) to describe plants that can accumulate
more than 1,000 µg Ni g1 dry weight in their
aerial parts. Approximately 400 taxa of terrestrial plants have been
identified as hyperaccumulators of various heavy metals, with about 300 being Ni hyperaccumulators (Baker and Brooks 1989; Brooks, 1998). Only
16 species of Zn hyperaccumulators, which are defined as being able to
accumulate more than 10,000 µg Zn g1 in the
aboveground parts on a dry weight basis in their natural habitat
(Brooks, 1998), have been reported. Thlaspi caerulescens J. & C. Presl (Brassicaceae) is the best-known example of a Zn/Cd hyperaccumulator. Under hydroponic culture conditions T. caerulescens can accumulate up to 25,000 to 30,000 µg Zn
g1 dry weight in the shoots without showing any
toxicity symptoms or reduction in growth (Brown et al., 1996a; Shen et
al., 1997). Recently, there has been a surge of interest in the
phenomenon of heavy-metal hyperaccumulation because this property may
be exploited in the remediation of heavy-metal-polluted soils through phytoextraction and phytomining (McGrath et al., 1993; Brown et al.,
1995b; Robinson et al., 1997).
The mechanisms for metal hyperaccumulation are not fully understood,
and this is particularly true in the case of the Zn/Cd hyperaccumulators. To cope with the consequence of hyperaccumulation, plants must also be hypertolerant to the heavy metals that accumulate. Recent studies comparing the different populations of T. caerulescens have shown that hyperaccumulation of Zn is a
constitutive property, although the traits are probably separate from
those for tolerance (Baker et al., 1994; Meerts and Van Isacker, 1997).
Compared with the nonaccumulating species, T. caerulescens
possesses an enhanced capacity to take up Zn and transport it from
roots to shoots (Baker et al., 1994; Brown et al., 1995a; Shen et al.,
1997). Lasat et al. (1996) found that roots of T. caerulescens and the nonaccumulator Thlaspi arvense had
similar apparent Km values for
Zn2+, but that the
Vmax in the former was 4.5-fold higher than
that in the latter species, indicating that the hyperaccumulator
T. caerulescens possessed more
Zn2+-transport sites in the plasma membranes of
root cells. Shen et al. (1997) showed that T. caerulescens
was much more effective in exporting the Zn that was accumulated
previously in roots to the shoots than an intermediate accumulator
species, Thlaspi ochrolucum. Organic acids such as malic
acid have been suggested to play a key role in shuttling Zn from
cytoplasm to vacuoles (Mathys, 1977). However, the
low affinity of malate to chelate Zn (stability constant pK = 3.5 at infinite dilution) does not favor this
hypothesis. Moreover, high concentrations of malate found in the shoot
tissues of T. caerulescens appear to be a constitutive
property (Tolrà et al., 1996; Shen et al., 1997).
The extraordinary tolerance of hyperaccumulator plants must also
involve compartmentation of toxic metals at the cellular and
subcellular levels. Vázquez et al. (1992, 1994) studied
localization of Zn in the root and leaf tissues of T. caerulescens using EDXMA. They compared two methods of sample
preparation and found that Na2S fixation was not
suitable for preventing the loss of metal ions from the samples. Using
cryofixation and freeze substitution, they showed that Zn accumulated
mainly in the vacuoles as electron-dense deposits. Many vacuoles of
leaf-epidermal and subepidermal cells contained globular crystals that
were very rich in Zn. However, it is not known whether the Zn-rich,
globular crystal deposits occur inside of the leaf vacuoles in vivo or
if they are artifacts caused by sample preparation. Also, the technique
used by Vázquez et al. (1992, 1994) allows only semiquantitative
determination of Zn concentrations.
In this study we used two techniques to investigate cellular
compartmentation of Zn in the leaves of T. caerulescens. The first utilized EDXMA of frozen, hydrated tissue to survey the distribution patterns of Zn and other elements across different leaf
cells. The second method involved sampling sap from single cells using
microcapillaries, followed by fully quantitative determination of Zn
and other elements using EDXMA.
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MATERIALS AND METHODS |
Plant Culture
Seeds of Thlaspi caerulescens (from the population at
Prayon, Belgium) were sown on a mixture of perlite and vermiculite
moistened with deionized water. Three weeks later, seedlings were
transplanted to plastic pots each filled with 500 g of John Innes
II compost (John Innes Centre, Norwich, UK). Four plants were grown in
each pot. The total concentration of Zn in the unamended compost was 88 µg g1. There were eight Zn treatments,
ranging from 0 to 4000 µg g1 compost, and
each treatment was replicated in three pots. Zinc was added as
ZnSO4 solution to the compost and mixed
thoroughly before potting. Plants were grown for 6 weeks inside of a
growth room with the following conditions: 16-h daylength with a photon flux density of 350 µmol m1
s1 supplied by fluorescent tubes and a
20°C/16°C day/night temperature.
EDXMA of Frozen, Hydrated Leaf Tissues
Mature and young leaves from the plants grown on 4000 µg Zn
g1 were used. The optimum conditions for sample
preparation and EDXMA of frozen, hydrated plant tissues were critically
reviewed by Van Stevenink and Van Steveninck (1991). A small section
from the middle of young or mature leaves was excised, mounted in
stainless steel stubs, and rapidly frozen in liquid nitrogen slush. The sample was transferred to a preparation chamber cooled at  180°C and
fractured with a liquid nitrogen-cooled scalpel blade just above the
level of the stub to reveal the surface of the cells. Ice was removed
from the cell surface by exposing the sample to a high vacuum at
85°C for 2 min. After this etching process, the sample was recooled
to 180°C and evaporatively coated with carbon to produce an
electrically conductive surface. Carbon was used instead of a metal
coating to avoid interference on the elements measured. The specimen
was then transferred to a liquid- nitrogen-cooled stage (180°C)
inside of the SEM (model XL 40, Philips, Eindhoven, The Netherlands).
EDXMA was performed in the SEM using an acceleration voltage of 30 kV,
a takeoff angle of 45o, and a working distance
(sample to final lens of the SEM) of 10 mm. Spectra from 0 to 20 keV
were collected at increments of 10 eV per channel with the electron
beam focused on a rectangular area in the center of selected cells. The
background and element-specific peak spectra were analyzed using the
program Superquant (EDAX, San Francisco, CA), which fully deconvolutes
the spectra and allows the corrections for interference between
elements. Epidermal and mesophyll cells were randomly selected for
EDXMA of element-specific spectra. To remove the effects of variation
of surface topography between the different selected cells on the
efficiency of EDXMA rate counting, counts (peak minus
background) of Zn and other elements were normalized on a molar basis
with the Ca counts. This was because the distribution of Ca appeared to
be relatively homogenous, as revealed by EDXMA dot maps (see below).
Normalization based on Ca resulted in a smaller variation between
replicated determinations of the same cell type compared with that
based on K.
In addition, the distribution of Zn across different cells from the
upper to the lower epidermis of fractured, frozen, hydrated leaf tissue
was measured semiquantitatively by collecting and analyzing the Zn
spectrum within a narrow spectrum window (±20 eV) around its peak. 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 Zn, Ca, and K 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 (peaks)
from the background counts. Therefore, area scans were performed only
on the elements with an average peak-to-background ratio greater than
2, including Zn, K, and Ca.
Extraction of Sap from Single Cells and Determination of Solute
Concentrations
Plants grown in pots with additions of 0, 1000, and 4000 µg Zn
g1 were used. Mature leaves were selected from T. caerulescens rosettes. Glass microcapillaries with tip outside
diameters of 5 and 2 µm were used to extract sap from single
epidermal and mesophyll cells, respectively. Sampling was carried out
according to the method described by Tomos et al. (1994). To extract
sap from mesophyll cells, the epidermis was stripped off physically and
the sample was washed briefly with deionized water to remove solutes
from destroyed epidermal cells. The same leaves were used for
extraction of both epidermal and mesophyll sap. The extracted sap
samples were transferred under water-saturated paraffin oil from the
sampling microcapillary to a copper-finder grid coated with a 2%
solution of Pioloform (Agar Aids, Stanstead, UK). To avoid changes in
concentration due to evaporation, samples were stored for no longer
than 30 min. The sap samples were then transferred from the storage to the analysis grid with a constriction pipette (approximately 10 pL). An
equal volume of the internal RbF standard in mannitol was added to the
sample to give a final concentration of 20 mM RbF
and 100 mM mannitol. Samples were then dried
according to the method described by Tomos et al. (1994) and analyzed
directly, i.e. without coating, using EDXMA in the SEM.
Calibrations were established using multi-element standards containing
equal molar concentrations of Zn, Ca, Cd, Cl, K, Mg, and P, as well as
the internal standard RbF (Tomos et al., 1994). A reliable
quantification (±10%) within the concentration range of 1 to 100 mM was achieved for the above elements with this method. The sap samples were usually diluted by 1:4 or 1:8 using constriction pipettes. Determination of S in the sap samples was not possible because the Pioloform used in the sample preparation contains S.
Determination of Zn Concentrations in Whole Shoots of
T. caerulescens
Plants were harvested after growing on different treatments for 6 weeks. Shoots were cut, weighed, washed with deionized water, blotted
dry, and frozen in liquid nitrogen. The samples were lyophilized for
72 h and dry weights were determined. Dried shoot samples were
ground and a 0.2-g subsample was digested with a mixture of
HNO3 and HClO4 (Zhao et
al., 1994). Concentrations of Zn and other elements in the digests were
determined using inductively coupled atomic emission spectrometry
(Fisons-ARL Accuris, Ecublens, Switzerland).
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RESULTS |
Plant Growth and Zn Uptake
Additions of Zn to the compost that already contained a sufficient
amount of Zn for the growth of normal plant species increased the dry
weight of T. caerulescens shoots significantly (P < 0.01; Fig. 1), indicating a higher
requirement for Zn in this species. Maximum shoot dry weight was
obtained with the addition of 2,000 µg Zn g1
and was 82% greater than that from the control (without Zn addition). Shoot dry weight was lower than the maximum with the addition of 4,000 µg Zn g1 but was still significantly higher
than that of the control. The concentration of Zn in the shoot dry
matter increased from 509 µg g1 in the
control to 20,010 µg g1 in the highest Zn
treatment (Fig. 1).
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| Figure 1.
Effects of Zn additions on mean shoot dry weight
(DW) and mean total Zn concentration in T. caerulescens.
Bars represent ±SE (n = 3).
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The mean concentrations of K and Ca in the shoots of T. caerulescens were 4% and 2%, respectively, based on dry weight.
Both K and Ca decreased generally with increasing Zn, with the effect being most pronounced at the highest Zn addition (data not shown). The
concentration of P decreased significantly only at the highest Zn
addition, whereas the additions of Zn as ZnSO4
beyond 1000 µg Zn g1 increased the
concentration of S in the shoots. The concentrations of the
micronutrients Fe, Cu, and Mn in the shoots were all well above
deficiency thresholds (Marschner, 1995).
EDXMA of Frozen, Hydrated Leaf Tissues
Both the EDXMA line and area scans revealed that Zn accumulated
predominantly in the epidermal cells in the mature leaves of T. caerulescens (Fig. 2, a and b). The mesophyll
cells, in contrast, appeared to have much lower concentrations of Zn.
The Zn signal was considerably smaller in the young than in the mature leaves. Nevertheless, the accumulation of Zn in the epidermis was still
apparent in the young leaves, particularly in those with enlarged
epidermal cells (Fig. 2, c and d). Distribution of K and Ca was much
more even across the leaf section (Fig. 2, e and f).
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| Figure 2.
Distribution of Zn, K, and Ca across different
leaf cells of T. caerulescens as revealed by EDXMA of
frozen, hydrated tissues. Plants were grown on 4000 µg Zn
g1 for 6 weeks. a, Intensity of the Zn K line across a
mature leaf section. The measurement took place at the solid straight
line. b, Dot map of Zn distribution in a mature leaf section. c,
Intensity of the Zn K line across a young leaf section. d, Dot map
of Zn distribution in a young leaf section. e, Dot map of K
distribution in a mature leaf section. f, Dot map of Ca distribution in
a mature leaf section. Arrows indicate epidermal cells. UE, Upper
epidermis; LE, lower epidermis.
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Figure 3 shows the relative
concentrations of Zn, K, Mg, S, and Cl in the different cell types of
young and mature leaves from the plants given 4000 µg Zn
g1. The relative concentration of Zn was 5- to
7-fold higher in the epidermal cells of mature leaves than in the
mesophyll cells. In young leaves the difference was about 2 to 4 times
higher. There were no consistent differences between the upper and
lower epidermis or between the upper and lower mesophyll cells. In
general, the variability of the relative concentration of Zn in the
epidermis of young leaves was larger than in the mature leaves. The
variation of the relative Zn concentration in the epidermal cells
appeared to be associated with the variation in the cell size. The
correlation between the relative Zn concentrations in epidermal cells
and their length was highly significant (Fig.
4), and the relationship appeared to be
linear and similar for both young and mature leaves.
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| Figure 3.
Mean relative concentrations (based on Ca) of Zn,
Mg, P, S, Cl, and K in different types of cells of mature leaves and
young leaves of T. caerulescens. Plants were grown on
4000 µg Zn g1 for 6 weeks. Frozen, hydrated leaf
tissues were prepared for EDXMA spectrum analysis on central rectangles
of randomly selected cells. Relative concentrations were calculated
using the following formula:
The slopes were obtained from the multi-element
calibrations. Bars represent SEs (n = 4-7).
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| Figure 4.
Relationship between cell length and relative Zn
concentration in the epidermal cells in frozen, hydrated leaf tissues
of T. caerulescens grown on 4000 µg Zn
g1.
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In contrast to the distribution pattern of Zn, higher relative
concentrations of K were observed in the mesophyll cells than in the
epidermis in both young and mature leaves (Fig. 3). The relative
concentrations of both P and S were greater in the young than in the
mature leaves, respectively, and in the former, the mesophyll cells
had greater relative concentrations of P and S than the epidermal
cells (Fig. 3), a pattern that was the opposite of that for Zn.
Concentrations of Zn and Other Solutes in Single-Cell Sap
Determination of the single-cell sap samples showed that
the epidermis contained much greater concentrations of Zn than the mesophyll cells (Fig. 5). The mean
concentration of Zn in the epidermal saps extracted from the mature
leaves of T. caerulescens grown on the 4000 µg Zn
g1 treatment was 385 mM,
equal to 2.5% (w/v) of soluble Zn. Even when no Zn was added to the
compost, the epidermal cells still accumulated considerable
concentrations of Zn in the sap, whereas the concentrations of Zn in
the sap from mesophyll cells were barely detectable. In the 1000 and
4000 µg Zn g1 treatments, the concentrations
of Zn in the epidermal sap were 5 to 6.5 times greater than those in
the mesophyll sap.
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| Figure 5.
Concentrations of Zn in the single-cell saps
extracted from epidermal and mesophyll cells of mature leaves of
T. caerulescens. Plants were grown on 0, 1000, or 4000 µg Zn g1 for 6 weeks. Bars represent SEs
(n = 3-5).
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Concentrations of K, Ca, Mg, P, and Cl are shown in Table
I. In the epidermal sap extracted from
the plants treated with 4000 µg Zn g1, Zn was
by far the most abundant element. In this treatment the concentrations
of K, Ca, Mg, and P were lower in the epidermal than in the mesophyll
sap, whereas the concentrations of Cl were similar. Different patterns
for these elements were observed in the 0 and 1000 µg Zn
g1 treatments, with epidermal sap having higher
concentrations of these elements than the mesophyll sap. In general,
the concentrations of K, Cl, P, and Ca in the epidermal sap decreased
with increasing Zn, whereas the concentrations in the mesophyll sap
were less affected by the Zn treatments.
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Table I.
Concentrations of K, Ca, Mg, P, and Cl in the
single-cell sap extracted from the epidermal and mesophyll cells of
mature leaves of T. caerulescens
The results are presented as means ± SE
(n = 3-5).
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DISCUSSION |
The two techniques used in this study are complimentary. EDXMA on
frozen, hydrated samples provides information about the in vivo
distribution of Zn and other elements in adjacent cells but is only
semiquantitative and relatively insensitive (Lazof and Läuchli,
1991; Van Steveninck and Van Steveninck, 1991), whereas the
single-cell-extraction technique allows quantitative determination of
the sap composition. Using both techniques, we have demonstrated
conclusively that the epidermal cells of the leaves of the
hyperaccumulator T. caerulescens accumulated much higher
concentrations of Zn than the mesophyll cells. The preferential accumulation of Zn in the epidermis was evident even when the growth
medium contained no elevated concentration of Zn. Vázquez et al.
(1994) also found accumulation of Zn in the epidermal cells of T. caerulescens leaves. In contrast to their results, we did not
observe preferential accumulation of Zn in the subepidermal cells.
Preferential distribution of Ni in the epidermal cells or trichomes has
been observed in the leaves of Ni hyperaccumulator plants
(Mesjasz-Przybylowicz et al., 1994; Krämer et al., 1997).
Using malate dehydrogenase as the cytoplasmic marker enzyme, Fricke et
al. (1994) showed that the epidermal sap extracted from barley leaves
was completely vacuolar in origin, whereas extracts from mesophyll
cells also contained cytoplasmic constituents. The difference was
explained by a much larger proportion in terms of intracellular volume
of vacuole in the epidermal cells (approximately 99%) than in the
mesophyll cells (about 60%). Thus, at least in the epidermis, the
concentrations of Zn and other elements in the single-cell saps may
truly represent those in the vacuolar sap. The total width of the upper
and lower epidermal cells was about 15% of the thickness of the mature
leaves in T. caerulescens. When the same proportions of
vacuole volume in the cell volume as those for barley leaves was used,
it is estimated that more than 60% of the Zn accumulated by T. caerulescens leaves was present in the epidermal vacuoles.
Therefore, epidermal vacuoles are an important location for Zn
sequestration in T. caerulescens leaves.
Vázquez et al. (1992, 1994) performed EDXMA of T. caerulescens leaf tissues by transmission electron microscopy. The
advantage of using transmission electron microscopy is the capability
of investigating the distribution pattern at the subcellular level; however, this technique requires removal of tissue water through lengthy sample preparation, which can then cause redistribution of ions
and other artifacts (Van Steveninck and Van Steveninck, 1991). One
possible artifact reported by Vázquez et al. (1994) is that
inside the vacuoles, Zn appeared to be present in deposits as globular
crystals. In contrast, our results obtained with single-cell-sap extraction indicate that Zn is present in soluble forms. We also showed
in a previous study that more than 80% of the Zn accumulated in the
leaves of T. caerulescens was water soluble (Zhao et al., 1998).
The decrease in the concentrations of K, Ca, Mg, and Cl in epidermal
sap in response to increasing Zn concentration was probably a result of
osmotic adjustment. The concentrations of Ca in both epidermal and
mesophyll saps extracted from the mature leaves of T. caerulescens were much greater than those reported for barley (Leigh and Tomos, 1993; Fricke et al., 1994). This is not surprising because of the high total concentration of Ca in the shoots of T. caerulescens. The charge balance in the single-cell saps could not
be calculated because major anions such as sulfate and nitrate were not
determined. It is also not possible to calculate the speciation of Zn
in the vacuolar saps, because organic solutes were not determined in
this study, and the species of P and S were also unknown.
In the 4000 µg Zn g1 treatment, which
produced a large enrichment of Zn in the epidermis, both P and S were
preferentially distributed in the mesophyll cells. Vázquez et al.
(1994) also observed a low P signal in the epidermal vacuoles of
T. caerulescens. Furthermore, there is little evidence that
coprecipitation of Zn with P occurs in the leaves of T. caerulescens (Zhao et al., 1998). To avoid coprecipitation of P
with Zn, which could induce P deficiency, T. caerulescens
must be able to maintain a low Pi concentration or physically separate
Zn from Pi in different leaf cells or different subcellular
compartments. In the case of S, glucosinolates have been suggested to
play a role in chelating Zn (Mathys, 1977), despite the lack of any
evidence that Zn-glucosinolate complexes exist. The different
distribution patterns of Zn and S in the leaf cells of T. caerulescens indicate that S-containing compounds are unlikely to
play an important role in the sequestration of Zn in vacuoles.
The mechanisms involved in the preferential accumulation of Zn in the
epidermal vacuoles in T. caerulescens leaves are not known.
Several possible pathways have been proposed to explain the transfer of
ions and water from xylem to leaf cells in cereal leaves (Leigh and
Tomos, 1993). Different selectivity of transport at various interfaces
among xylem, vein extensions, mesophyll cells, and epidermis are
possibly involved, resulting in the asymmetric distribution of ions and
other solutes. It is also possible that the tonoplast of the epidermis
of T. caerulescens leaves may have a higher capacity for the
transport of Zn into vacuoles than that of the mesophyll cells. The
close relationship between cell size and the Zn concentration (relative
to Ca) in the epidermal cells is also very interesting and indicates
that large epidermal cells were particularly enriched with Zn. This
relationship suggests that vacuolation in the epidermal cells may be an
important driving force for the preferential Zn sequestration in
T. caerulescens leaves.
The ability of T. caerulescens leaves to sequester Zn
preferentially in the epidermal vacuoles is probably an important
aspect of the hypertolerance of this species to Zn. Preferential
distribution of Zn in the epidermis helps to protect mesophyll cells
from the buildup and toxicity of Zn and maintain the functionality of
mesophyll cells over a wide range of Zn concentrations in the leaves.
Still, mesophyll cells of T. caerulescens leaves must also
be able to cope with concentrations of Zn that are much higher than
what might be found in most plant species (probably <1
mM). The concentration of Zn in the mesophyll sap
of T. caerulescens leaves reached 60 mM when the total concentration of Zn in the
shoot dry weight was about 20,000 µg g1, a
concentration that is considerably lower than the reported toxic
threshold for this species (Brown et al., 1995a; Shen et al., 1997).
Excess Zn in mesophyll cells is also likely to be sequestered in the
vacuoles.
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FOOTNOTES |
1
IACR-Rothamsted receives grant-aided support
from the Biotechnology and Biological Sciences Research Council of the
United Kingdom.
*
Corresponding author; e-mail steve.mcgrath{at}bbsrc.ac.uk; fax
44-1582-760981.
Received July 7, 1998;
accepted October 14, 1998.
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ABBREVIATIONS |
Abbreviations:
EDXMA, energy-dispersive X-ray microanalysis.
RbF, rubidium fluoride.
SEM, scanning electron microscope.
TEM, transmission electron microscope.
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ACKNOWLEDGMENTS |
We thank Miss Tara Breedon and Miss Sarah Dunham for assistance
in setting up the pot experiment, Mr. Chris Smith and Dr. Phil Jones
for assistance in using the SEM and EDXMA systems, Mrs. Janice Proud
and Dr. Tracey Cuin for the help in using micropipettes, and Dr. Deri
Tomos for providing some of the constriction pipettes used.
| |
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