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Plant Physiol. (1998) 118: 875-883
Altered Zn Compartmentation in the Root Symplasm and Stimulated
Zn Absorption into the Leaf as Mechanisms Involved in Zn
Hyperaccumulation in Thlaspi caerulescens
Mitch M. Lasat,
Alan J.M. Baker, and
Leon V. Kochian*
United States Plant, Soil, and Nutrition Laboratory, United States
Department of Agriculture-Agricultural Research Station, Cornell
University, Ithaca, New York 14853 (M.M.L., L.V.K.); and Department of
Animal and Plant Sciences, University of Sheffield, Sheffield, United
Kingdom S10 2TN (A.J.M.B.)
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ABSTRACT |
We investigated Zn compartmentation
in the root, Zn transport into the xylem, and Zn absorption into leaf
cells in Thlaspi caerulescens, a Zn-hyperaccumulator
species, and compared them with those of a related nonaccumulator
species, Thlaspi arvense. Zn-compartmental
analysis conducted with roots of the two species indicated that a
significant fraction of symplasmic Zn was stored in the root vacuole of
T. arvense, and presumably became unavailable for
loading into the xylem and subsequent translocation to the shoot. In
T. caerulescens, however, a smaller fraction of the absorbed Zn was stored in the root vacuole and was readily transported back into the cytoplasm. We conclude that in T. caerulescens, Zn absorbed by roots is readily available for
loading into the xylem. This is supported by analysis of xylem exudate
collected from detopped Thlaspi species seedlings. When
seedlings of the two species were grown on either low (1 µM) or high (50 µM) Zn, xylem sap of
T. caerulescens contained approximately 5-fold more Zn
than that of T. arvense. This increase was not
correlated with a stimulated production of any particular organic or
amino acid. The capacity of Thlaspi species cells to
absorb 65Zn was studied in leaf sections and leaf
protoplasts. At low external Zn levels (10 and 100 µM),
there was no difference in leaf Zn uptake between the two
Thlaspi species. However, at 1 mM
Zn2+, 2.2-fold more Zn accumulated in leaf sections of
T. caerulescens. These findings indicate that altered
tonoplast Zn transport in root cells and stimulated Zn uptake in leaf
cells play a role in the dramatic Zn hyperaccumulation expressed in
T. caerulescens.
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INTRODUCTION |
Phytoextraction is an emerging technology that involves the use of
vascular plants to remediate soils contaminated with heavy metals
and/or radionuclides (Nanda Kumar et al., 1995 ). This approach is based on the ability of higher plants to absorb contaminants from
the soil and translocate them to their shoots. The identification of
several metal-hyperaccumulator plant species (Baker and Brooks, 1989;
Baker et al., 1998) demonstrates that the genetic potential exists for
successful phytoremediation of contaminated soils. One of the
best-known metal hyperaccumulators is Thlaspi caerulescens J&C Presl, which has been reported to have a great potential for extraction of Zn and Cd from metalliferous soils (Reeves and Brooks, 1983; Chaney, 1993; Baker et al., 1994; Brown et al., 1994, 1995a, 1995b). Recently, Brown et al. (1995b) reported that from a hydroponic medium, T. caerulescens accumulated more than 25,000 µg Zn
g 1 before symptoms of Zn toxicity (i.e. shoot
biomass reduction) occurred.
Although T. caerulescens has the ability to transfer high
levels of Zn and other metals from the soil into the shoot, the use of
this species for commercial remediation of contaminated soils is
severely limited by its small size and slow growth (Ebbs et al., 1997).
The transfer of Zn-hyperaccumulating properties from T. caerulescens into a high-biomass-producing plant has been suggested as a potential avenue for making phytoremediation a commercial technology (Brown et al., 1995a). Progress in this area,
however, is hindered by a lack of understanding of the basic physiological mechanisms involved in Zn uptake into roots and translocation to aboveground tissues.
In a previous study we reported that Zn influx into root symplasm of
Zn-hyperaccumulator and -nonaccumulator species of Thlaspi was mediated by a saturable component with similar affinities for Zn
(Lasat et al., 1996). However, the maximum capacity for Zn transport
across the plasma membrane into the cytosol was 4.5-fold greater in the
Zn-accumulator T. caerulescens compared with the Zn-nonaccumulator Thlaspi arvense L. These results indicate
that one characteristic of T. caerulescens is a greater
capacity for Zn absorption from soil solution into root cells. Enhanced
Zn uptake into the root symplasm of T. caerulescens was also
associated with a greater capacity for Zn translocation to the shoot
(Lasat et al., 1996). For example, after 96 h of
exposure to a 65Zn-labeled uptake solution,
10-fold more 65Zn accumulated in the shoots of
T. caerulescens compared with T. arvense, and
1.2-fold more 65Zn accumulated in the roots of
T. arvense compared with T. caerulescens. These
results suggest that in addition to Zn entry into the root symplasm,
other Zn-transport sites are altered in T. caerulescens, contributing to the dramatic increase in Zn translocation and storage
in the shoot.
We used hydroponically grown T. arvense and T. caerulescens seedlings to investigate several mechanisms possibly
involved in Zn hyperaccumulation: (a) stimulated Zn transport from the root symplasm into the xylem sap; (b) xylem accumulation of
low-Mr organic ligands possibly involved in
Zn complexation and transport to the shoot; and (c) enhanced Zn
transport into leaf cells.
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MATERIALS AND METHODS |
Plant Material
Seeds of Thlaspi arvense were obtained from the
Crucifer Genetics Cooperative (University of Wisconsin, Madison).
Thlaspi caerulescens seeds were obtained from plants grown
near a Zn/Cd smelter in Prayon, Belgium (Vázquez et al.,
1994). Seeds were grown hydroponically in 5-L black plastic tubs
as described previously (Lasat et al., 1996). To obtain seedlings of
similar size and developmental status, T. caerulescens
seedlings were used when they were 5 to 10 d older than T. arvense seedlings. Seedlings were detopped for collection of xylem
sap and grown in a nutrient solution with either a control level of
Zn2+ (1 µM) or supplemented with 50 µM Zn2+ (as
ZnCl2).
Short-Term 65Zn-Efflux (Compartmentation) Studies
Seedlings of 40-d-old T. arvense and 45-d-old T. caerulescens were incubated with roots in Plexiglas uptake wells
filled with 85 mL of the aerated uptake solution containing 2 mM Mes-Tris buffer (pH 6.0), 0.5 mM
CaCl2, and 20 µM
65Zn2+ (1.4 µCi
L1) (as ZnCl2). After
24 h the radioactive uptake solution was removed, roots were
quickly rinsed with deionized water, and wells were refilled with an
efflux solution that was identical to the radioisotope-loading solution
except that it lacked 65Zn (2 mM
Mes-Tris buffer, pH 6.0, 0.5 mM
CaCl2, and 20 µM
Zn2+). At various time intervals, a 1-mL aliquot
of the efflux solution was collected and 65Zn was
measured with a gamma counter (model 5530, Packard Instruments, Downers
Grove, IL). After the removal of each aliquot, wells were drained and
refilled with fresh efflux solution. After 6 h roots were excised,
blotted, and weighed, and root 65Zn content was
determined. The efflux of
65Zn2+ from roots was
determined by monitoring the appearance of 65Zn
in the efflux solution over time.
Long-Term 65Zn-Efflux Studies
Roots of intact T. arvense and T. caerulescens seedlings were incubated for 24 h in a plastic
container filled with 800 mL of the aerated uptake solution containing
20 µM 65Zn2+
(1.4 µCi L1). After 24 h the radioactive
solution was removed, roots were rinsed with deionized water, and the
container was refilled with nonradiolabeled efflux solution. At various
time intervals up to 46 h, four T. arvense and four
T. caerulescens seedlings were harvested and roots were
separated from shoots, blotted, and weighed, and root and shoot
65Zn was measured. After removal of each set of
seedlings at the specific time intervals the efflux solution was
replaced with fresh solution. Efflux from roots was assessed by
determining the 65Zn remaining in roots over
time.
Xylem Sap Collection and Analysis
Fifty-day-old T. arvense and 55-d-old T. caerulescens seedlings grown for the entire period in a nutrient
solution containing 1 µM Zn2+ or
transferred 10 d before the experiment to a similar nutrient solution supplemented with 50 µM
Zn2+ were detopped just below the leaf rosette
with a razor blade. The entire excised root system and remaining stem
material were used for collection of xylem exudate. The excised stem
surface was gently wiped, and a Pasteur pipette tip was sealed onto the cut end of the stem by applying a thin layer of high-vacuum grease (Dow-Corning Corp., Midland, MI). Xylem sap exuded into the pipette for
the subsequent 24-h period was collected, frozen in liquid N2, and stored at  70°C until analyzed. Zn
content of the xylem sap was analyzed with an inductively coupled
plasma emission spectrometer (model ICAP 61E, Thermo-Jarrell Ash,
Waltham, MA).
To analyze organic acids in the xylem sap, we used an
ion-chromatography system (model 300, Dionex, Sunnyvale, CA) with an ion-exchange analytical column (4 mm, model AS11, Dionex) and an
eluent gradient of NaOH in 18% high-purity methanol. Organic acids
were detected by determination of electrical conductivity. Identification and quantification of sample components were achieved by
comparison of retention times in standard solutions and peak integration to fit into standard curves. The detection limit was 10 µM.
To analyze amino acids, xylem sap was dried under a vacuum and
hydrolyzed with constant boiling in HCl at 116°C for 24 h. Hydrolyzed samples were dried under a vacuum, redissolved in 1:3 (v/v)
20 mM HCl:AccQ-Fluor borate buffer (Waters), and
fluorescently derivatized with 1 volume of AccQ-Fluor for 10 min at
55°C. Samples were chromatographed using a liquid-chromatography
system (Waters) with a C18 column (4 µm,
Waters) according to the directions of the manufacturer. Derivatized
amino acids were identified via fluorescence detection using an
excitation wavelength of 250 nm and an emission wavelength of 395 nm.
For most amino acids the detection limit was 10 µM; for
Cys the detection limit was 5 µM.
65Zn2+ Uptake in Leaf Sections
Leaves were gently abraded with Carborundum powder (320-grit,
Fisher Scientific) to facilitate infiltration of the radioactive uptake
solution, thoroughly rinsed with deionized water to remove any adhering
powder, and cut into 10- to 20-mm2 sections. Leaf
material was then immersed in an aerated uptake solution containing 10, 100, or 1000 µM
65ZnCl2 (1.4 µCi
L1), 2 mM Mes-Tris buffer, pH 6.0, and 0.5 mM CaCl2. At different time
intervals up to 48 h,
65Zn2+ uptake was
terminated by replacing radioactive solution with desorption solution
containing 5 mM CaCl2, 5 mM Mes-Tris, pH 6.0, and 100 µM
ZnCl2. After 15 min of desorption to remove
apoplastic Zn, leaf sections were harvested,
blotted, and weighed, and leaf Zn content was
measured via gamma detection.
65Zn Uptake in Leaf Protoplasts
Protoplast Isolation
Leaves (4 g fresh weight) were gently abraded with Carborundum
powder, rinsed thoroughly with deionized water to remove adhering powder, and chopped into fine pieces using a razor blade. Chopped tissues were suspended in 25 mL of a cell wall-digesting medium composed of 600 mM mannitol, 3 mM Mes-Tris
buffer, pH 5.5, 2 mM CaCl2, 1 mM DTT, 0.7 mM
KH2PO4, 0.1% (w/v) BSA,
1% (w/v) Cellulysin (Calbiochem), and 0.1% (w/v) Pectolyase (Sigma).
The tissues were incubated at 20°C in the dark for 30 to 45 min. The
resulting suspension was filtered through four layers of Miracloth
(Calbiochem), and protoplasts were pelleted by centrifugation at
60g for 5 min at 2°C. The supernatant was discarded and
the pellet resuspended in protoplast buffer (600 mM
mannitol, 3 mM Mes-Tris buffer [pH 5.5], 2 mM
CaCl2, 1 mM DTT, 0.7 mM
KH2PO4, and 0.1% [w/v]
BSA).
Protoplasts were washed in 20 mL of the protoplast buffer and
centrifuged at 60g for 5 min, and then the supernatant was
discarded and the pellet resuspended in the residual liquid. This step
was repeated three times. After the last centrifugation, the pellet was
resuspended in 1 mL of preuptake solution consisting of 500 mM mannitol, 50 mM Suc, 3 mM
Mes-Tris (pH 5.5), 0.05 mM CaCl2, 1 mM DTT, and 0.7 mM
KH2PO4. The number of
protoplasts was determined with a hemocytometer. The protoplast stock
was kept in a test tube on ice until the uptake experiments were
performed.
Determination of Protoplast Viability
The percentage of viable protoplasts in the stock was determined
by staining with FDA used at a final concentration of 36 µM from a stock of 7.2 mM FDA dissolved in
acetone (final acetone concentration was 0.5%, v/v). After 10 min of
incubation in FDA, protoplasts were inspected using a fluorescence
microscope (model IMT-2, Olympus). Protoplasts showing bright
fluorescence were counted as viable. Protoplast viability ranged
between 70% and 80%.
65Zn-Uptake Experiments with Protoplasts
The time course of 65Zn uptake was initiated
by the addition of 900 µL of the protoplast stock to a 1.5-mL
Eppendorf microcentrifuge tube with 100 µL of the preuptake solution
containing 0.1, 1.0, or 10 mM
65ZnCl2 (0.5 µCi
mL1). Therefore, the
65Zn2+ concentration to
which protoplasts were exposed was 10, 100, or 1000 µM (protoplast volume did not interfere with this
dilution, because it represented less than 5% of the stock volume).
The final protoplast concentration in the uptake solution was 2 × 107 protoplasts mL1. At
different time intervals up to 12 min, a 100-µL aliquot of the
radioactive protoplast suspension was removed and placed on the top of
a discontinuous gradient consisting of (from the top to the bottom of a
1.5-mL microcentrifuge tube) 50 µL of 10% (v/v) HClO4 on top of 400 µL of silicon oil (550 Fluid, Dow-Corning) with a specific density of 1.06 at 25°C.
The microcentrifuge tubes were immediately centrifuged using a
bench-top microcentrifuge (model 5415, Brinkmann Instruments) at high
speed for 1 min to pellet the protoplasts through the silicon oil
layer. After centrifugation, tubes were immersed in liquid
N2. Tips containing the frozen protoplast pellet
were cut off and placed in counting vials. The uptake of Zn2+ into protoplasts was
quantified via gamma detection.
To determine whether Zn accumulation in protoplasts was caused by
65Zn2+ movement across the
plasma membrane into the cytosol or by the binding of Zn to negatively
charged sites associated with the external face of the plasma membrane,
two different experiments were carried out. First, we conducted a study
to investigate the time course of Zn uptake in intact versus ruptured
T. arvense protoplasts. To rupture the plasma membrane,
T. arvense protoplasts were frozen in liquid
N2 and then thawed at room temperature. Microscopic inspection confirmed that after this treatment the protoplast stock had been entirely ruptured. The stock of ruptured and
intact protoplasts was used to conduct a time-course study of
65Zn2+ uptake from a 10 µM Zn solution. In the second experiment, we investigated
the effect of the protonophore and metabolic inhibitor CCCP on the
time-dependent kinetics of 65Zn uptake in
T. caerulescens protoplasts. In this experiment, 10 µM CCCP was added to the protoplast stock 30 min before
the beginning of the experiment. The time-course study was conducted in
an uptake solution containing 10 or 100 µM
65Zn2+, as described above.
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RESULTS |
Efflux Compartmentation Studies
To investigate the subcellular compartmentation of
65Zn2+ in roots, we
conducted a short-term (6 h) study on the time-dependent kinetics of
65Zn efflux from T. caerulescens and
T. arvense roots. Plots representing a first-order kinetic
transformation of Zn efflux (log 65Zn remaining
in the root as a function of time) could be dissected into three linear
phases representing Zn efflux from three compartments in series: the
vacuole, cytoplasm, and cell wall (Fig.
1). The straight line drawn through data
representing the slowest-exchanging phase (180-360 min) was
interpreted to represent 65Zn efflux from the
vacuole (Fig. 1A). From the slope of this line we estimated the
half-time for 65Zn efflux from the vacuole (Table
I). The y-axis intercept of this line was used to calculate the distribution of
65Zn in root cells at the termination of the
radioisotope-loading period (Table I).
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| Figure 1.
Short-term efflux of 65Zn from roots
of T. arvense and T. caerulescens
seedlings. After a 24-h incubation in an uptake solution containing 20 µM 65Zn2+, roots were rinsed in
deionized water, and, to initiate 65Zn efflux, the
radioactive uptake solution was replaced with an identical, nonlabeled
solution containing 20 µM Zn2+. Efflux of
65Zn from roots into the external solution was subsequently
monitored for a 6-h period. Lines represent regressions of the linear
portion of each curve extrapolated to the y axis. The
curve shown in B was derived by subtracting the linear component in A
from the data points in A. The curve in C was similarly derived from
the curve in B. Data points in A represent means ± SE
of four replicates.
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Table I.
Intracellular 65Zn2+
compartmentation and half-time (t1/2) for
65Zn2+ efflux from different root compartments
from T. arvense and T. caerulescens seedlings
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Subtraction of the linear component from total efflux data (Fig. 1A)
yielded a second curve, which was analyzed similarly and was
interpreted to represent 65Zn efflux from the
cytoplasm (30-60 min) and cell wall (0-15 min) (Fig. 1B). Efflux from
the cell wall (Fig. 1C) was obtained after subtracting the linear phase
associated with the cytoplasmic efflux from the data points plotted in
Figure 1B. Translocation of 65Zn to the shoots
did not interfere with the compartmentation analysis, in that it
represented only 0.7% and 2.5% of the total radioactive Zn
accumulated in roots of T. arvense and T. caerulescens, respectively. At the end of a 24-h
radioisotope-loading period, comparable amounts of
65Zn2+ had accumulated in
the roots of the two Thlaspi species (first pair of data
points in Fig. 1A).
As shown in Table I, Zn compartmentation in the root cell wall and
cytoplasm were similar in T. arvense and T. caerulescens seedlings; however, approximately 2.4-fold more
65Zn was accumulated in the root vacuole of
T. arvense compared with T. caerulescens.
Although the rates of 65Zn efflux from the cell
wall and cytoplasm were similar in T. arvense and T. caerulescens, the half-time for radioactive Zn efflux from the
vacuole was nearly 50% shorter in T. caerulescens, indicating that Zn efflux from the T. caerulescens root
vacuole was almost twice as rapid as in T. arvense (Table
I). In a long-term Zn efflux experiment,
approximately 6-fold more 65Zn remained in
T. arvense roots compared with T. caerulescens at
the end of the 46-h washout period (Fig.
2A); however, at the end of the same
period, 6-fold more 65Zn was translocated from
the root to the shoot of T. caerulescens (Fig. 2B).
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| Figure 2.
Long-term efflux of 65Zn from roots
(A) and 65Zn translocation to shoots (B) of T. arvense and T. caerulescens seedlings. Bundles
of four T. arvense and T. caerulescens
seedlings were immersed with roots in a 20 µM
65Zn2+ uptake solution. After a 24-h loading
period, roots were rinsed in deionized water, and the radioactive
uptake solution was replaced with an identical, nonlabeled solution
containing 20 µM Zn2+. At different time
intervals up to 46 h, one bundle of each Thlaspi
species was harvested, roots were excised, blotted, and weighed, and
gamma activity was measured. Data points represent means ± SE of four replicates. wt, Weight.
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Analysis of Xylem Exudates
Relatively large volumes of xylem sap (approximately 5 mL from 15 to 20 plants) were collected from both Thlaspi species
during the 24-h period after detopping. Regardless of the Zn
concentration in the nutrient solution, significantly more Zn was
accumulated in the xylem sap of T. caerulescens compared
with T. arvense. For example, from the hydroponic growth
solution containing 50 µM Zn2+,
approximately 5-fold more Zn was found in the xylem sap of T. caerulescens compared with T. arvense (524 versus 100 µM Zn; Table II). A
comparable ratio was found when the two Thlaspi species were
grown in 1 µM Zn2+ (54 versus 15 µM; Table II). Both species accumulated Zn in the xylem
to significantly higher concentrations when the Zn level in the growth
solution was increased from 1 to 50 µM.
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Table II.
Concentrations of Zn, organic acids, and amino
acids in the xylem sap of T. arvense and T. caerulescens grown for the
entire 55-d period in a nutrient solution containing 1 µM
Zn2+ or for the last 10 d in the solution supplemented
with 50 µM Zn2+
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The possibility that a significant amount of Zn moving to the T. caerulescens shoot was complexed or associated with one or more
low-Mr organic ligands was investigated by
analyzing the xylem sap for organic and amino acids. We could not
detect the presence of aconitate, citrate, fumarate, pyruvate, or
succinate anions in the xylem sap of either Thlaspi species
(Table II). The predominant organic acids in the xylem sap of both
Thlaspi species were acetate and malate. Acetate was the
only organic-acid anion that showed an increase in response to elevated
Zn exposure. When the Zn level in the growth solution was
increased from 1 to 50 µM, xylem acetate levels
increased approximately 3-fold in T. arvense and almost
5-fold in T. caerulescens.
Analysis of the xylem sap amino acid content revealed few significant
differences between T. arvense and T. caerulescens (Table II). The major difference was in Glu, which
was 3- to 4.5-fold greater in T. caerulescens compared with
T. arvense. However, the concentration of Glu did not
increase in the xylem sap of plants grown in the high-Zn solution. His
was not detected in the xylem sap of T. caerulescens.
Uptake of 65Zn2+ into Leaf Tissues
After an initial rapid phase the time course of Zn accumulation
into leaf sections from the uptake solutions containing from 10 to 1000 µM 65Zn2+ was
approximately linear for up to 48 h. We speculate that the initial
rapid phase involves Zn movement into the apoplast through the residual
cuticle and cut edges of the leaf sections (Fig. 3). At the lower Zn concentrations (10 and 100 µM), there were no detectable differences in leaf
Zn accumulation between the two Thlaspi species (Fig. 3, A
and B). However, at the higher Zn concentration (1000 µM), which is probably more representative of the Zn
concentration in the xylem sap of T. caerulescens,
approximately 2.5-fold more Zn was accumulated in T. caerulescens leaf sections by the end of the 48-h uptake period
(Fig. 3C).
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| Figure 3.
Time course of 65Zn accumulation in
leaf sections of the two Thlaspi species. Leaves of
T. arvense and T. caerulescens seedlings
were cut into 10- to 20-mm2 sections and immersed in an
uptake solution containing 2 mM Mes-Tris buffer, pH 6.0, 0.5 mM CaCl2 and 10 µM (A), 100 µM (B), or 1000 µM (C)
65ZnCl2. After exposures for up to 48 h,
the radioactive uptake solution was replaced with a solution consisting
of 5 mM Mes-Tris, pH 6.0, 5 mM
CaCl2, and 100 µM ZnCl2 and
allowed to desorb for 15 min. Leaf sections were then harvested,
blotted, and weighed, and the gamma activity was measured. Data points
represent means ± SE of four replicates.
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Leaf Zn transport was also studied at the cellular level using
protoplasts isolated from leaves of the two Thlaspi species. The time dependence of 65Zn uptake was studied in
uptake solutions containing 10, 100, and 1000 µM
65Zn2+ (Fig.
4). Zn uptake from a solution containing
10 or 100 µM
65Zn2+ was similar in
protoplasts isolated from T. arvense and T. caerulescens leaves. At a high Zn2+
concentration (1000 µM), there was a small tendency for
higher Zn uptake in T. caerulescens protoplasts, but this
difference was considerably less than that obtained with leaf sections.
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| Figure 4.
Time course of 65Zn accumulation in
protoplasts isolated from the two Thlaspi species.
Protoplasts isolated from T. arvense and T. caerulescens leaves were suspended in an uptake buffer
containing 10 µM (A), 100 µM (B), or 1000 µM (C) 65ZnCl2. At different time
intervals up to 12 min, an aliquot of the uptake suspension was
collected and placed on top of a discontinuous gradient consisting of
50 µL of 10% (v/v) HClO4 on top of 400 µL of silicon
oil, and the protoplasts were pelleted by centrifugation. The tube was
then frozen in liquid N2 and the tip containing the pellet
was cut and placed in a vial, and the gamma activity was measured. For
the experiments with CCCP, protoplasts were exposed to 10 µM CCCP for 30 min before the uptake experiment.
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Exposure of protoplasts to the protonophore and metabolic inhibitor
CCCP (10 µM) elicited a large (>50%) inhibition of
65Zn2+ uptake into T. caerulescens protoplasts (Fig. 4, A and B). Uptake of
65Zn2+ into
liquid-N2-ruptured protoplasts saturated rapidly
(in less than 1 min) and very little 65Zn
subsequently accumulated. At the end of a 12-min uptake period, approximately 2.2-fold more
65Zn2+ had accumulated in
intact compared with ruptured protoplasts (Fig.
5).
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| Figure 5.
Time course of 65Zn uptake in intact
and ruptured T. arvense protoplasts. Protoplasts were
ruptured by freezing in liquid N2. The uptake solution
contained 10 µM 65Zn2+. At
different time intervals up to 12 min, an aliquot of the uptake
suspension was removed and placed on top of a discontinuous gradient
consisting of 50 µL of 10% (v/v) HClO4 on top of 400 µL of silicon oil, and the protoplasts were pelleted by
centrifugation. The tube was then frozen in liquid N2 and
the tip containing the pellet was cut and placed in a vial, and the
gamma activity was measured.
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DISCUSSION |
The mechanism of Zn hyperaccumulation in leaf cells of T. caerulescens is not clearly understood. It is likely that a number of steps are involved in the Zn2+ movement from
the rhizosphere to the shoots, including (a) Zn2+
influx across the root cell plasma membrane; (b) radial Zn movement across the root; (c) loading into xylem vessels in the stelar region of
the root; (d) translocation from the root to the shoot; (e) absorption
of Zn from the xylem into leaf cells; and (f) transport across the leaf
cell tonoplast. Additionally, because Zn tolerance plays a major role
in hyperaccumulation, Zn detoxification involving a number of different
organic ligands most likely plays a part in the overall response.
We have previously shown that despite a small
Zn2+ influx into root cells, more radioactive Zn
was accumulated in T. arvense roots than in T. caerulescens (Lasat et al., 1996). However, after a 96-h period,
65Zn translocation to the shoot was 10- to
20-fold higher in T. caerulescens compared with T. arvense. These findings suggest that Zn compartmentation is
fundamentally different in the roots of the two Thlaspi
species, and that certain processes associated with Zn loading into the
xylem are also stimulated in T. caerulescens.
We conducted a radiotracer efflux (compartmental) analysis to
investigate indirectly 65Zn compartmentation in
the cytoplasm and vacuole of the two Thlaspi species. We
have used this approach previously in several different studies to
investigate the root compartmentation of a number of monovalent and
divalent cations (Kochian and Lucas, 1982; Hart et al., 1992; DiTomaso
et al., 1993; Lasat et al., 1997) and are very familiar with its
limitations. This technique was developed to study ion compartmentation
in single giant algal cells, in which the ion content of the individual
compartments (vacuole, cytoplasm, and cell wall) can be measured
directly (MacRobbie, 1971). Several researchers have questioned the
extension of compartmental analysis to complex, multicellular higher
plant organs such as roots. For example, it has been argued that
radiolabeled ions may be slowly released from the cell wall-binding
sites (Spanswick and Williams, 1965; Jorgenson, 1966), chemically
bound within the cytoplasm (Robinson and Jackson, 1986), or
compartmentalized in other organelles such as plastids (Cheeseman,
1986). However, in compartmental studies with oat coleoptiles (Pierce
and Higinbotham, 1970) and carrot root tissues (Cram, 1968), the
authors provided strong arguments and analyses that allowed them to
validate this technique for the application of the
three-compartment-in-series model to plant organs such as roots and
leaves. For a detailed examination of the application of this technique
for ion compartmentation in roots, see Kochian and Lucas (1982).
The application of radiotracer efflux analysis to roots only allows for
an indirect and semiquantitative estimate of ion fluxes and loading in
the vacuole, cytoplasm, and cell wall. Unfortunately, no other
experimental method is currently available to investigate ion
compartmentation in a semiquantitative manner. Moreover, this technique
has been used to provide valuable information on the efflux and
compartmentation of several ions such as K+
(Kochian and Lucas, 1982), Na+ (Pierce and
Higinbotham, 1970), NH4+
(Macklon et al., 1990), Cd2+ (Rauser, 1987),
Cu2+ (Thornton, 1991), Zn2+
(Santa Maria and Cogliatti, 1988), and Co2+
(Macklon and Sim, 1987).
At the end of the 24-h 65Zn-loading period,
65Zn2+ had accumulated to
comparable levels in roots of T. arvense and T. caerulescens (Figs. 1A and 2A). The Zn-compartmentation data
summarized in Table I suggest that similar amounts of Zn were stored in
the root apoplast of the two Thlaspi species, which argues
against sequestration of Zn in the root cell wall of T. arvense as a mechanism of Zn tolerance, as suggested by Peterson
(1969) for Agrostis tenuis. Zn levels were also similar in
the T. arvense and T. caerulescens root cell
cytoplasm (Fig. 1B; Table I); however, there were significant differences in the degree of Zn vacuolar accumulation and the rate of
Zn efflux back out of the vacuole. As shown in Table I, almost 2.5-fold
more Zn was stored in the vacuoles of T. arvense roots, and
Zn efflux out of the vacuole was about 2-fold faster in T. caerulescens. These findings suggest that Zn is sequestered in the
root vacuole of T. arvense, possibly being made unavailable for loading into the xylem. Because vacuolar efflux was slower in
T. arvense, the difference in root residual Zn between
T. arvense and T. caerulescens should increase
after longer efflux periods. In support of this, after a 46-h period,
approximately 6-fold more 65Zn remained in the
root of T. arvense compared with T. caerulescens (Fig. 2A).
Although Zn flux into T. caerulescens roots was
significantly greater (Lasat et al., 1996), cytoplasmic Zn content was
similar in the two Thlaspi species (Fig. 1A), presumably
because of a greater Zn throughput across the T. caerulescens root and into the xylem. This was supported by a
5-fold increase in Zn concentration in the xylem sap of T. caerulescens compared with T. arvense. From these data
we cannot determine whether Zn transport involved in the loading of Zn
from xylem parenchyma into xylem vessels was stimulated in T. caerulescens. It is possible that the elevated Zn in the xylem sap
of T. caerulescens was attributable solely to the enhanced
Zn influx into the root and to altered root compartmentation, which
should facilitate radial Zn movement across the root to the xylem.
An important aspect of Zn hyperaccumulation in T. caerulescens might be the production of
low-Mr organic ligands that can complex
Zn2+ in the plant cell cytoplasm, rendering it
nontoxic. It is also likely that organic ligands help facilitate the
long-distance transport of Zn in the xylem (White et al., 1981) and
might be instrumental in helping sequester Zn in the leaf cell vacuole. To investigate the possible role of organic ligands in long-distance Zn
transport in the xylem and in Zn reabsorption in leaves, we analyzed
the composition of xylem sap isolated from both Thlaspi species in plants grown at low and high Zn levels. Our results showed a
significant increase in the level of acetic acid in the xylem sap of
T. caerulescens compared with T. arvense.
However, because acetate is a weak ligand for
Zn2+ (Smith and Martell, 1989), this increase may
not directly facilitate Zn translocation from root to shoot, but,
rather, might be involved in the maintenance of cation-anion balance in
response to the high level of Zn2+ transported in
the xylem sap of T. caerulescens. In support of this, the
addition of acetate to the uptake medium at concentrations as high as 4 mM did not stimulate
65Zn2+ accumulation in leaf
sections of either Thlaspi species (results not shown).
We also investigated the possibility that amino acids are involved in
long-distance transport of Zn, because His production and accumulation
in the xylem was shown to be involved in Ni hyperaccumulation in shoots
of Alyssum (Krämer et al., 1996). In the present
study, however, His was not detected in the xylem sap of T. caerulescens, but was found in moderate levels in the xylem of
T. arvense. Glu was found to be the most abundant amino acid
in the xylem sap of both species of Thlaspi, but Glu
concentrations were 3- to 5-fold higher in the T. caerulescens xylem sap. However, the Glu concentration of the
xylem sap did not increase in response to increasing Zn concentrations
in the xylem, and addition of Glu at concentrations as high as 500 µM did not stimulate
65Zn2+ uptake in leaf
sections of either Thlaspi species (results not shown),
suggesting that Glu does not facilitate reabsorption of xylem-borne Zn
into leaf cells.
Because one of the major storage sites for Zn is the leaf vacuole
(Vázquez et al., 1994), we investigated the possibility that Zn
influx across the leaf cell plasma membrane was stimulated in T. caerulescens. We found that at lower Zn concentrations (10 and 100 µM), there were no differences in
65Zn uptake into leaf sections or protoplasts
isolated from the two Thlaspi species. However, when the Zn
concentration was increased to 1 mM,
65Zn accumulation was significantly stimulated in
leaves of T. caerulescens. This finding is interesting, for
it suggests that Zn transport is stimulated at both the root cell and
leaf cell plasma membrane in the hyperaccumulator species. If this is
the case, then the Zn transporter has different kinetic properties in
roots versus shoots, because stimulated root Zn2+
influx was observed in T. caerulescens at external
Zn2+ concentrations as low as 1 µM
(Lasat et al., 1996), whereas in leaves it was observed at much higher
Zn2+ concentrations (1 mM). It should
also be noted that enhanced Zn transport into leaf cells could reflect
a stimulated transport system operating in the leaf cell tonoplast,
which could effectively move cytoplasmic Zn into the vacuole.
To avoid the confounding effect of the cuticle and the cell wall,
accumulation of 65Zn2+ in
leaf tissues was also investigated using protoplasts isolated from
leaves of the two Thlaspi species. The uptake curves were biphasic, with an initial rapid linear component followed by a slower
one. We previously found similar biphasic, time-dependent kinetics for
the uptake of another divalent cation, methyl viologen, in maize
protoplasts (Hart et al., 1993). Those findings were consistent with
the initial, rapid phase being a combination of true uptake and
considerable cation binding to the negatively charged sites on the
outer face of the plasma membrane and the second, slower phase being
influx across the plasma membrane.
To determine whether this interpretation applied to the uptake curves
shown in Figure 4, we conducted uptake studies in the presence of CCCP,
an anionic protonophore and metabolic inhibitor (Heyter and Prichard,
1962). Exposure to CCCP has also been shown to dramatically depolarize
the electrical potential across the plasma membrane of plant cells
(DiTomaso et al., 1992), which could inhibit the electrical driving
force for divalent cation uptake. Our data (Fig. 4, A and B) indicate
that the CCCP treatment strongly inhibited
65Zn2+ transport into
protoplasts, indicating that we were measuring primarily true
transmembrane Zn2+ transport into
Thlaspi protoplasts.
65Zn2+ binding to the
plasma membrane was also investigated in
liquid-N2-ruptured protoplasts (Fig. 5), and
indicated that Zn binding to the plasma membrane was rapid, saturating
within 1 to 2 min, whereas Zn accumulation into intact protoplasts
continued for at least 10 min.
It is interesting to note that although we observed a significant
increase in Zn accumulation in cells from leaf segments of T. caerulescens compared with T. arvense (at high
exogenous Zn concentrations), these Zn-transport differences
disappeared in isolated leaf protoplasts. This suggests that either an
intact leaf is required to express the enhanced Zn uptake in T. caerulescens, or that the artificial conditions associated with
protoplasts are such that they do not accurately reflect the physiology
of leaf cells in an intact leaf. An estimate of "true Zn influx" of
6 pmol Zn 106
protoplasts1 h1 was
obtained by subtracting the amount of
65Zn2+ binding in ruptured
protoplasts from the Zn accumulation measured in intact protoplasts.
Our calculations indicate that this rate was at least 2 orders of
magnitude lower than the rate of Zn uptake into intact leaf cells (leaf
sections). Therefore, under the altered physiological conditions the
identification of Zn-transport differences in protoplasts isolated from
the two Thlaspi species might be compromised and should be
viewed with some caution.
In summary, the results of this study indicate that altered tonoplast
Zn transport in the root and stimulated Zn uptake in the leaf play a
role in the dramatic Zn hyperaccumulation expressed in T. caerulescens.
| |
FOOTNOTES |
*
Corresponding author; e-mail lvk1{at}cornell.edu; fax
1-607-255-1132.
Received March 12, 1998;
accepted August 4, 1998.
| |
ABBREVIATIONS |
Abbreviations:
CCCP, carbonyl cyanide
m-chlorophenylhydrazone.
FDA, fluorescein diacetate.
| |
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Top
Abstract
Introduction