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Plant Physiol. (1998) 116: 1063-1072
The Role of Iron-Deficiency Stress Responses in Stimulating
Heavy-Metal Transport in Plants1
Clara K. Cohen,
Tama C. Fox,
David F. Garvin, and
Leon V. Kochian*
United States Plant, Soil, and Nutrition Laboratory, United States
Department of Agriculture-Agricultural Research Service, Cornell
University, Ithaca, New York 14853 (C.K.C., D.F.G., L.V.K.); and Department of Biological Sciences, Dartmouth College, Hanover, New
Hampshire 03755 (T.C.F.)
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ABSTRACT |
Plant accumulation of Fe and other
metals can be enhanced under Fe deficiency. We investigated the
influence of Fe status on heavy-metal and divalent-cation uptake in
roots of pea (Pisum sativum L. cv Sparkle) seedlings
using Cd2+ uptake as a model system. Radiotracer techniques
were used to quantify unidirectional 109Cd influx into
roots of Fe-deficient and Fe-sufficient pea seedlings. The
concentration-dependent kinetics for 109Cd influx were
graphically complex and nonsaturating but could be resolved into a
linear component and a saturable component exhibiting Michaelis-Menten
kinetics. We demonstrated that the linear component was apoplastically
bound Cd2+ remaining in the root cell wall after
desorption, whereas the saturable component was transporter-mediated
Cd2+ influx across the root-cell plasma membrane. The
Cd2+ transport system in roots of both Fe-deficient and
Fe-sufficient seedlings exhibited similar Michaelis constant
values, 1.5 and 0.6 µm, respectively, for
saturable Cd2+ influx, whereas the maximum initial velocity
for Cd2+ uptake in Fe-deficient seedlings was nearly 7-fold
higher than that in Fe-grown seedlings. Investigations into the
mechanistic basis for this response demonstrated that
Fe-deficiency-induced stimulation of the plasma membrane
H+-ATPase did not play a role in the enhanced
Cd2+ uptake. Expression studies with the Fe2+
transporter cloned from Arabidopsis, IRT1, indicated
that Fe deficiency induced the expression of this transporter, which
might facilitate the transport of heavy-metal divalent cations such as
Cd2+ and Zn2+, in addition to Fe2+.
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INTRODUCTION |
Although abundant in the earth's crust, Fe predominates as
insoluble Fe(III) precipitates and is largely unavailable to plants, especially at neutral or alkaline pH. Plants use two distinct strategies to assimilate Fe from the environment. The grasses release
low-molecular-weight, high-affinity Fe(III)-chelating compounds called
phytosiderophores, which solubilize ferric Fe in the rhizosphere and
are recognized for uptake by specific membrane receptors (Römheld
and Marschner, 1986 ; Chaney, 1987; Bienfait, 1988). Fe uptake in the
dicots and the nongrass monocots is mediated by a plasma membrane-bound
ferric reductase that transfers electrons from intracellular NADH
(Buckhout et al., 1989) to Fe(III) chelates in the rhizosphere (Chaney
et al., 1972). The ferrous ions (Fe2+) released
from the chelates by this process are subsequently transported into the
cytoplasm via a separate transport protein (Kochian, 1991; Fox et al.,
1996).
When Fe deficient, dicot and nongrass monocots stimulate a number of
processes to enhance Fe accumulation from the soil. Fe deficiency
induces a 5- to 10-fold stimulation of ferric reductase activity
(Ambler et al., 1971; Chaney et al., 1972; Römheld and Marschner,
1979; Bienfait et al., 1983). Root-mediated acidification of the
rhizosphere is an additional strategy used by Fe-deficient plants to
enhance solubilization of Fe3+ from Fe hydroxides
(Venkat Raju and Marschner, 1972; Brown and Jones, 1974). Finally, root
Fe2+ influx is regulated by the Fe status of the
plant. Fox et al. (1996) found that Fe-deficient pea (Pisum
sativum L.) seedlings exhibit significantly higher rates of root
Fe2+ influx than Fe-sufficient seedlings. In
addition to these responses, which are usually linked specifically to
Fe accumulation, tissue concentrations of other mineral elements also
appear to be influenced by plant Fe status. Welch et al. (1993)
demonstrated that the shoot concentrations of many divalent cations,
including Cu, Mn, and Mg, increased in Fe-deficient pea seedlings.
Rodecap et al. (1994) also reported that Fe-deficient Arabidopsis
plants accumulated higher concentrations of Cd and Mg in racemes and
seeds compared with Fe-sufficient plants.
In this study we investigated the influence of Fe status in stimulating
heavy-metal uptake in pea using Cd uptake as a model system. Cd is a
common environmental contaminant introduced into soils through
anthropogenic activity. Cd contamination poses a serious hazard to
human health, and uptake into plants is the primary avenue through
which it can enter the food chain. Additionally, there has been
considerable interest in the use of terrestrial plants for the
remediation of surface soils contaminated with toxic heavy metals,
although little is understood about plant mechanisms of heavy-metal
hyperaccumulation.
We demonstrate that Fe deficiency elicits a large stimulation of Cd
influx into roots of pea seedlings. We investigated the physiological
basis of this enhanced Cd uptake through evaluation of Fe-deficiency
stress responses that might play a role in enhanced heavy-metal
absorption. These include induction of a divalent cation transporter,
induction of the plasma membrane H+-ATPase, and
induction of the plasma membrane ferric reductase. We found that Fe
deficiency induced the expression of a Fe-transporter gene, which might
facilitate the transport of heavy-metal divalent cations such as
Cd2+ and Zn2+, in addition
to Fe2+.
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MATERIALS AND METHODS |
Plant Material and Culture
At d 0, pea (Pisum sativum L. cv Sparkle) seeds were
allowed to imbibe overnight in aerated, distilled water. Seeds were
then placed between sheets of moistened filter paper in glass Petri dishes and germinated in the dark at 20°C. On d 3 seedling roots were
inserted through holes in black polyethylene seedling cups. The
seedling cups were inserted into the covers of black polyethylene pots
containing 5 L (five to seven plants per pot) of nutrient solution. The
polyethylene seedling cups were filled with black polyethylene beads to
prevent light exposure of the nutrient solution. In general, pea
seedlings were grown in a modified Johnson's nutrient solution
containing the following macronutrients in mm:
KNO3, 1.2;
Ca(NO3)2, 0.8;
NH4H2PO4,
0.1; and MgSO4, 0.2; and the following micronutrients in µm: KCl, 50;
H3BO3, 12.5;
MnSO4, 1; ZnSO4, 1; CuSO4, 0.4;
Na2MoO4, 0.1; and
NiSO4, 0.1. The solutions were supplemented with
10 µm Fe-EDDHA and 1 mm Mes buffer, adjusted
to pH 5.5 with KOH. Nutrient solutions were changed on d 9 and 12. Fe-deficient plants were prepared by growing the seedlings in solutions
without Fe from d 9 until d 14. Plants were grown in a
controlled-environment growth chamber with a 16-h, 20°C day and an
8-h, 15°C night regime and a photon flux density of 350 µmol
m 2 s1.
Arabidopsis thaliana (La-0) seeds were surface sterilized in
30% (v/v) bleach (5.25% sodium hypochlorite) and 0.01% Triton X-100
for 15 to 20 min and rinsed five times in sterile, 18-M water. Seeds
were vernalized overnight at 4°C, resuspended in a
low-gelling-temperature agarose solution (1%), and placed on polypropylene screens submerged in nutrient solution in sterile Magenta
Jars (Magenta Corp., Chicago, IL). Seedlings were grown in autoclaved
nutrient solution consisting of the following nutrients in
mm: KNO3, 2;
KH2PO4, 0.2;
MgSO4, 2;
(NH4)2SO4,
0.25; Ca(NO3)2, 1;
CaSO4, 1; and
K2SO4, 1; and the following
micronutrients in µm:
H3BO3, 70;
MnCl2, 14; CuSO4, 0.5;
ZnSO4, 1;
Na2MoO4, 0.2; NaCl, 10; and
CoCl2, 0.01. The nutrient solution was
supplemented with 10 g/L Suc, 1 mm Mes buffer, adjusted to
pH 5.5 with KOH, and 50 µm filter-sterilized Fe-EDDHA.
Fe-deficient seedlings were prepared by growing the seedlings in
solutions without Fe from d 16 until d 21. Sterile plants were grown in
an incubator at 20°C with a 16-h photoperiod at a light intensity of
60 µmol m2 s1.
Root 109Cd2+-Influx Experiments
Excised roots of 14-d-old pea seedlings were used for the
Cd2+-influx experiments. Root segments were
excised from the older portion of the root system and consisted of a 5- to 20-mm section of primary root with 2 to 10 attached, intact lateral
roots. Root segment fresh weights ranged from 0.1 to 0.4 g.
An uptake apparatus consisting of acrylic wells was used for the
Cd2+-influx experiments (Grusak et al., 1990).
Hollow acrylic cylinders were fitted with mesh-covered rubber stoppers
to allow vacuum withdrawal of solutions without disturbing the roots.
The uptake solutions were aerated with acrylic tubing placed near the
bottom of each well.
Root segments were gently inserted into each well using forceps and
permitted to recover from excision for at least 30 min in an aerated
pretreatment solution consisting of 5 mm Mes-Tris (pH 6.0),
1 mm KNO3, 0.8 mm
Ca(NO3)2, 0.2 mm MgSO4, 0.1 mm
K2SO4, and 0.2 mm NH4NO3. The
pretreatment solution was then evacuated and replaced with fresh uptake
solution of the same composition. Metabolic inhibitors, sulfhydryl
reagents, or treatment cations were added to the uptake solutions 30 min before the introduction of radiolabeled
109Cd. A 0.5-mL aliquot of the appropriate stock
Cd(NO3)2 solution was added
to each well immediately before addition of radiolabel to attain a
desired Cd concentration between 1 and 100 µm. The 20-min
uptake period was initiated by adding 50 µL of a radioactive stock
solution (109CdCl2 in 0.1 n HCl) to each uptake well to attain a final radioisotope concentration of either 0.0025 or 0.01 µCi/mL. One-milliliter aliquots of the uptake solution were removed at 1 and 19 min into the
uptake period to calculate an internal standard relating counts per
minute to total Cd and also to determine the amount of substrate (Cd)
depletion. After a 20-min uptake period, the uptake solution in the
wells was replaced with ice-cold desorption solution consisting of 5 mm Mes-Tris (pH 6.0), 5 mm
CaCl2, and 100 µm
Cd(NO3)2. After two 7.5-min
desorption periods (15 min total desorption time), root segments were
removed and blotted dry with paper towels. The section of primary root
on each root unit was removed, and the lateral roots were weighed.
Absorption of 109Cd into the lateral roots was
quantified via gamma detection using a counter (Auto-Gamma 5530, Packard, Downers Grove, IL).
Methanol:Chloroform Preparation of Root Cell Walls
To approximate the contribution of cell wall binding to Cd
absorption, root cell wall preparations that maintained the morphologic and geometric characteristics of intact roots were obtained. Root systems were immersed in methanol:chloroform (2:1, v/v) solutions for
3 d and rinsed in several changes of distilled water for 1 d.
This treatment has been shown to yield lipid-free cell wall preparations in maize while maintaining the structure and morphologic characteristics of an intact root (Hart et al., 1992).
Cd Accumulation in Roots and Shoots
Fe-deficient and Fe-sufficient plants were grown as described
above. On d 12, 0.2 µm
Cd(NO3)2 was added to the
growth solution. Tissue was harvested on d 14. Aerial portions of the
plant were rinsed in 18-M water, blotted dry, and placed in a bag to
dry. Root systems were desorbed in a solution containing 1 mm LaCl3 for 30 min, with the
desorption solution replaced with fresh solution every 5 min (Reid et
al., 1996). Dried tissue samples were wet digested in concentrated
HNO3 overnight at 100°C, then digested again
with HNO3 and HClO4 at
200°C. Samples were resuspended in 5% HNO3 and
analyzed via simultaneous inductively coupled argon-plasma emission
spectrometry (ICAP 61E trace analyzer, Themo-Jarrell Ashe, Franklin,
MA).
Measurement of Exofacial Reduced Sulfhydryl Groups
Reduced sulfhydryl groups on the root surface (presumably on the
plasma membrane surface and/or within the cell wall) of ± Fe-grown pea
seedlings were measured with the membrane-impermeant, sulfhydryl-reactive reagent DTNB using a procedure modified from that
of Welch and Norvell (1993). DTNB reacts with reduced sulfhydryl groups
to form a yellow nitromercaptobenzoic acid anion product. Intact root
systems were submerged in 100 mL of sulfhydryl reaction buffer (0.2 m Tris-HCl and 0.02 m Na-EDTA, adjusted to pH
8.2 with NaOH). The reaction buffer was purged with
N2 gas for 10 min before root submergence and
during the reaction period. Each reaction period was initiated by
adding 1 mL of a 10-mm DTNB solution to attain a final
concentration of 100 µm. After a 15-min reaction period,
absorbance values of aliquots of the assay solution were measured at
412 nm with a spectrophotometer (model DU 640, Beckman). The molar
concentration of the nitromercaptobenzoic acid anion product was
determined using a standard curve generated with fresh Cys stock
solutions. Controls for each treatment in which DTNB was added to the
solution after seedling removal were included to correct for soluble
reducing agents excreted from the roots during the assay.
Measurement of Root-Cell Em
Pea seedlings (14-15 d old) were secured in an acrylic chamber
mounted on the stage of a microscope (model BH-2, Olympus). Roots were
bathed in a solution identical to that used in the 109Cd-uptake experiments (5 mm
Mes-Tris [pH 6.0], 1 mm KNO3, 0.8 mm Ca(NO3)2,
0.2 mm MgSO4, 0.1 mm
K2SO4, and 0.2 mm NH4NO3).
Experiments with FC (10 µm) were performed in an
unbuffered solution that also contained 0.06% (v/v) methanol. Cells of
the root epidermis and cortex were impaled with microelectrodes at
several locations at least 2 to 3 cm from the root apex using a
hydraulically driven micromanipulator (model MO-204, Narashige USA,
Greenvale, NY) mounted on the microscope stage. Membrane potentials
were measured using a dual microprobe amplifier (model KS-700, World
Precision Instruments, Inc., Sarasota, FL). Microelectrodes (tip
diameter = 0.5 µm) were pulled from 1-mm-thick, single-barreled
borosilicate glass capillary tubes with an internal filling fiber
(World Precision Instruments), using a micropipette puller (model P-87,
Sutter Instruments, Inc., Novato, CA). Electrodes were filled with 3 m KCl solution, adjusted to pH 2.0 to minimize tip
potentials. A reference electrode (catalog no. 13-639-52, Fisher
Scientific) was placed in the chamber housing the seedling to complete
the electrical circuit.
Analysis of Fe-Transporter Expression
Polyadenylated mRNA from roots of ± Fe-grown Arabidopsis (21 d
old, 5 d without Fe) and pea (13 d old, 4 d without Fe) was isolated as described by Sambrook et al. (1989). Aliquots of each mRNA
preparation (1.9 µg for pea and 1.2 µg for Arabidopsis) were denatured, electrophoresed in a 1.2% agarose gel containing 1× Mops
and 2.2 m formaldehyde (Sambrook et al., 1989), and
transferred to a nylon membrane (HyBond N, Amersham). The mRNA was
subsequently cross-linked to the membrane by baking for 2 h at
80°C. The membrane was prehybridized at 60°C in 5× SSC, 5×
Denhardt's solution, and 0.5% (w/v) SDS plus 0.1 mg/mL denatured
salmon-sperm DNA and probed with a genomic clone of the Arabidopsis
IRT1 gene (Eide et al., 1996).
The genomic clone was isolated from an Arabidopsis (La-0) genomic
library using PCR. Primers were derived from the 20 nucleotides flanking the IRT1 coding region (forward primer,
5 -CAAATTCAGCACTTCTCATG-3; reverse primer,
5-TTCCGCAATATCTGGAGTAT-3). The resulting fragment was inserted into the TA cloning vector PCR2.1 (Invitrogen, Inc., San
Diego, CA) and introduced into competent DH5 Escherichia coli cells. Transformed colonies were screened for
 -galactosidase activity on Luria-Bertani medium plus
5-bromo-4-chloro-3-indolyl--d-galactoside and
ampicillin. Plasmids were isolated, purified (Qiagen, Chatsworth, CA),
and sequenced (ABI Prism, Perkin-Elmer). The IRT1 genomic clone was radiolabeled with 32P using the method
of Feinberg and Vogelstein (1984), denatured at 100°C for 10 min, and
hybridized to the membrane overnight at 60°C. After
hy-bridization, the nylon membrane was washed twice for 20 min in
2× SSC, 0.1% SDS at 60°C, and exposed to x-ray film at  80°C.
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RESULTS |
Cd2+-Uptake Experiments
The concentration-dependent kinetics of root
Cd2+ influx in Fe-sufficient and Fe-deficient pea
seedlings were graphically complex and nonsaturating. A least-squares
procedure was used to test the fit of the
Cd2+-influx data to two different kinetic models,
a three-parameter rectangular hyperbolic plus linear function (Eq. 1)
or a linear function (Eq. 2; SigmaPlot, Chicago, IL):
![J[<UP>Cd</UP><SUP>2+</SUP>]<UP> = </UP>k[<UP>Cd</UP><SUP>2+</SUP>]<UP> + </UP><FR><NU>V<SUB><UP>max</UP></SUB>[<UP>Cd</UP><SUP>2+</SUP>]</NU><DE>K<SUB><UP>m</UP></SUB><UP>+</UP>[<UP>Cd</UP><SUP><UP>2+</UP></SUP>]</DE></FR>](/content/vol116/issue3/fulltext/1063/img001.gif)
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(1)
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![J[<UP>Cd</UP><SUP>2+</SUP>]<UP> = </UP>k[<UP>Cd</UP><SUP>2+</SUP>]](/content/vol116/issue3/fulltext/1063/img002.gif)
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(2)
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An analysis to find the best fit was performed for each of the
treatments imposed. A kinetic model consisting of hyperbolic (saturable) and linear components was found to suitably describe Cd
influx in the Fe-deficient roots (r2 = 0.99). For Fe-sufficient roots, a linear function or a
linear-plus-saturable function described the kinetics of Cd influx
equally well (Fig. 1A;
r2 = 0.998); however, visual inspection of
the data for Fe-sufficient roots (Fig. 1A, inset) shows the
nonlinearity of the lower portion of the curve when the flux data for
concentrations < 1 µm Cd are included. When these
data are included, a linear-plus-hyperbolic equation describes the
fitted function slightly better (r2 = 0.999).
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| Figure 1.
A, Concentration-dependent kinetics of root
109Cd influx in ± Fe-grown pea seedlings. After a 30-min
recovery from excision in pretreatment solution, root segments were
exposed to radiolabeled uptake solutions for 20 min and then desorbed
for 15 min. The inset depicts the concentration-dependent kinetics of
Cd influx over the 0- to 5-µm concentration range in the
+Fe-grown pea seedlings. Values are means expressed as nmol
Cd2+ (g fresh weight [FW])1
h1 (for +Fe [ ], n = 3-6; for
Fe [ ], n = 4-6). Error bars represent se. B, The plots in A were mathematically dissected into
their linear and hyperbolic components to estimate the kinetic
parameters.
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The kinetic parameters (Km and
Vmax) for the saturable system (Fig. 1B)
were determined using the computer-generated fits of the data to
linear-plus-saturable equations. Saturable Cd2+
influx for Fe-deficient and Fe-sufficient plants exhibited similar Km values, 1.5 ± 0.6 and 0.6 ± 0.9 µm, respectively. The slopes of the linear components
were also similar, 6.7 ± 0.35 nmol (g fresh
weight)1 h1 µm1
for Fe-deficient roots and 5.3 ± 0.13 nmol (g fresh
weight)1 h1 µm1
for Fe-sufficient roots. However, the Fe-deficient plants exhibited a
nearly 7-fold higher Vmax for uptake
(236 ± 25 versus 34 ± 8 nmol [g fresh
weight]1 h1). Thus, Fe
deficiency stimulated a large increase in a high-affinity, saturable
Cd-uptake system but had relatively little effect on the slope of the
linear component. Cd2+ influx was also stimulated
in roots of Fe-deficient seedlings at low Cd2+
concentrations (0-750 nm; Fig.
2). This Fe-deficiency-induced high-affinity system also appears to transport Zn. In a preliminary study performed at 1 µm Zn, 65Zn
influx was nearly 3 times higher in Fe-deficient roots (70 ± 7 versus 25 ± 1 nmol 65Zn [g fresh
weight]1 h1).
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| Figure 2.
Concentration-dependent kinetics of root
109Cd influx in ± Fe-grown pea seedlings (0-0.75
µm range). After a 30-min recovery from excision in
pretreatment solution, root segments were placed in radiolabeled uptake
solutions for 20 min and then desorbed for 15 min. Values are means
expressed as nmol Cd2+ (g fresh weight
[FW])1 h1 (for +Fe [],
n = 6-9; for Fe [], n = 2-3). Error bars represent se.
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We tested the hypothesis that the saturable uptake represented
carrier-mediated transport across the root-cell plasma membrane, whereas the linear component reflected apoplastic binding of Cd in the
cell wall that was not removed during the desorption period. This model
has been demonstrated for the uptake of several other divalent cations,
including Zn2+ and putrescine, in roots of
Thlaspi caerulescens and maize, respectively (DiTomaso et
al., 1992; Lasat et al., 1996). This hypothesis was tested using
several different experimental approaches. First, we examined the
kinetics of Cd2+ uptake (or binding) with
morphologically intact root cell wall preparations
(methanol:chloroform-treated roots). A linear model was found to
adequately describe Cd binding to methanol:chloroform-treated root cell
wall preparations of both Fe-sufficient and Fe-deficient pea seedlings
(Fig. 3, A and B;
r2 = 0.98 for Fe and
r2 = 0.99 for +Fe roots). The slope of the
concentration-dependent Cd binding in the cell wall preparations was
significantly greater than that of the linear component for intact root
segments (Fe, 14 ± 0.7 versus 6.7 ± 0.3 nmol [g fresh
weight]1 h1
µm1 for intact roots; +Fe, 23 ± 0.9 versus 5.3 ± 0.13 nmol [g fresh weight]1
h1 µm1 for intact
roots). This difference in slope could be attributed to a greater
exposure of potential binding sites in the root cell wall preparations
resulting from removal of the root symplast.
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| Figure 3.
Concentration-dependent kinetics of
109Cd influx in intact and methanol:chloroform-treated
roots from +Fe-grown pea seedlings (A) and Fe-grown pea seedlings
(B). Excised roots were treated for 3 d in 2:1 (v/v)
methanol:chloroform to remove protoplast material and rinsed for 1 d in H2O. Root segments were then placed in radiolabeled
uptake solutions for 20 min and subsequently desorbed for 15 min.
Values are means expressed as nmol Cd2+ (g fresh weight
[FW])1 h1. For +Fe: intact roots (),
n = 3 to 6 and cell wall only (), n = 2 to 3; for Fe: intact roots () and cell
wall only (), n = 4 to 6. Error bars represent
se.
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We next investigated the influence of different ion-transport
inhibitors on the concentration-dependent kinetics of Cd uptake. LaCl3, a Ca2+-channel blocker, was
found to inhibit both kinetic transport components (Fig.
4). The best fit was a linear function
(r2 = 0.9) with a reduced slope (1.8 ± 0.01 versus 6.09 ± 0.45 nmol [g fresh weight]1
h1 µm1 for the control). This
result is consistent with a model in which La3+
may both displace Cd2+ from the cell wall,
decreasing the slope of the linear component, and compete with
Cd2+ for a plasma membrane transporter,
abolishing the saturable component. In
Cd2+-binding experiments with root cell wall
preparations, inclusion of 0.2 mm
La3+ in the uptake solution effectively inhibited
Cd2+ association with the cell wall (data not
shown).
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| Figure 4.
Concentration-dependent kinetics of
109Cd influx into roots of Fe-grown pea seedlings treated
with LaCl3, a Ca2+-channel blocker. After
excision, root segments were pretreated for 30 min in 0.2 mm LaCl3, and then placed in radiolabeled
uptake solution containing 0.2 mm LaCl3 for 20 min, and subsequently desorbed for 15 min. Values are means expressed
as nmol Cd2+ (g fresh weight [FW])1
h1 (n = 4). , Control roots; ,
+La3+. Error bars represent se.
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Treatment with the respiratory inhibitor KCN (0.5 mm; data
not shown) or the protonophore CCCP (20 µm; Fig.
5) specifically abolished the saturable
Cd2+-influx component but had no effect on the
linear component. A linear function was found to be the best-fit model
(r2 = 0.95 for CCCP-treated roots),
providing additional evidence that saturable Cd2+
uptake represents transporter-mediated Cd influx across the root-cell plasma membrane. Treatment with the same concentration of KCl (0.5 mm) was noninhibitory (data not shown), which indicates
that inhibition of the saturable component for
Cd2+ influx by KCN was not a consequence of
K+-induced depolarization of the transmembrane
electrical potential.
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| Figure 5.
A, Concentration-dependent kinetics of
109Cd influx in Fe-grown pea seedling roots treated with
the protonophore and metabolic inhibitor CCCP. After excision, root
segments were pretreated for 30 min in 20 µm CCCP, then
placed in radiolabeled uptake solution containing CCCP for 20 min, and
subsequently desorbed for 15 min. , CCCP; , +CCCP. B, The
concentration-dependent kinetics of root 109Cd influx for
CCCP-treated pea seedlings. The Cd2+-uptake kinetics for
CCCP-treated seedlings (solid lines) were dissected into linear and
hyperbolic components. , +CCCP. Values are means expressed as nmol
Cd2+ (g fresh weight [FW])1
h1 (n = 4). Error bars represent
se.
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The Mechanistic Basis of Enhanced Cd Uptake
As mentioned in the introduction, there are several possible
explanations for the Fe-deficiency-induced stimulation of heavy-metal transport observed here. These include (a) induction of the
plasma-membrane H+-ATPase, which could stimulate
cation uptake; (b) induction of a transporter (such as
IRT1), which could mediate Cd2+
influx; and (c) induction of the ferric reductase, which could play a
direct or indirect role in influencing ion transporters in the
root-cell plasma membrane. Experiments were conducted to investigate
all three of these scenarios.
Role of the Plasma Membrane H+-ATPase in Stimulating
Cd2+ Influx
We explored the possibility that Fe-deficiency-induced
H+-ATPase activity, resulting in increased
H+ extrusion and possible hyperpolarization of
the Em, played a role in stimulating Cd
influx. If enhanced proton efflux during Fe deficiency were involved in
stimulating Cd influx, we would expect FC, a fungal toxin that
stimulates P-type H+-ATPases, to stimulate Cd
uptake in +Fe-grown roots. In preliminary electrophysiological
experiments, we found that FC stimulated the pea root plasma membrane
H+-ATPase very rapidly, hyperpolarizing the
root-cell Em by approximately 40 mV within
30 min. However, we found no effect of FC on Cd influx in +Fe-grown pea
seedlings under either unbuffered or strongly buffered conditions,
which would presumably eliminate the effect of proton gradients and
allow us to look exclusively at the effect of
Em on Cd2+ influx
(Table I). We also investigated the role
of H+ gradients on enhanced
Cd2+ influx by using highly pH-buffered uptake
solutions to abolish H+ gradients in the
unstirred layer adjacent to the root. We had previously demonstrated
using pH microelectrodes that inclusion of 10 mm Mes-Tris
buffer abolished H+ gradients generated in this
unstirred layer (L.V. Kochian, unpublished results). Therefore, these
highly buffered solutions were used to abolish enhanced
H+ gradients established along roots of
Fe-grown plants. This treatment was found to have no effect on Cd
influx in Fe-deficient plants (Fig. 6).
We also compared measurements of root-cell membrane potentials in ± Fe-grown seedlings to determine if
H+-ATPase-mediated hyperpolarization of the
root-cell membrane potential was correlated with enhanced
Cd2+ influx, but we found no difference in
membrane potential values between Fe-deficient and Fe-sufficient pea
seedlings (102 ± 4 mV for +Fe-grown seedlings versus
95.5 ± 4 mV for Fe-grown seedlings; n = 21).
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Table I.
Effect of FC on Cd2+ accumulation in
+Fe-grown pea seedling roots
Root segments were pretreated for 30 min after excision in buffered (10 mm Mes-Tris, pH 6.0) or unbuffered solution with or without
10 µm FC, incubated in radiolabeled uptake solution
containing 0 or 10 mm buffer, 0 or 10 µm FC,
and 1 or 10 µm Cd(NO3)2 for 20 min, and desorbed for 15 min. Values are means (n = 4-5). Values in parentheses represent se.
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| Figure 6.
Concentration-dependent kinetics of root
109Cd influx in Fe-grown pea seedlings in buffered (10 mm Mes-Tris, pH 6.0) and unbuffered uptake solution. After
excision, root segments were pretreated for 30 min in buffered (10 mm; ) or unbuffered (0 mm; ) solutions, placed in fresh buffered or unbuffered radiolabeled uptake solution for
20 min, and subsequently desorbed for 15 min. Values are means expressed as nmol Cd2+ (g fresh weight
[FW])1 h1 (n = 4).
Error bars represent se.
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Role of Fe-Transporter Induction in Stimulating Cd2+
Influx
IRT1, a gene that encodes an Fe2+
transporter presumably involved in Fe acquisition in nongrass plants,
was recently cloned in Arabidopsis (Eide et al., 1996). We used PCR to
obtain an IRT1 genomic clone from Arabidopsis. This clone was sequenced
and found to be identical to the IRT1 cDNA (Eide et al.,
1996), with the addition of two introns. Northern-blot analysis with
the IRT1 genomic clone revealed hybridization to
polyadenylated RNA from Fe-deficient but not Fe-sufficient roots of
both Arabidopsis and pea seedlings (Fig.
7). Our results also corroborate the
findings of Eide et al. (1996) showing specificity of IRT1
expression to roots but not to shoots of Fe Arabidopsis (data not
shown). This result suggests that expression of the IRT1
homolog in pea is induced in an Fe-deficiency-dependent fashion as
observed in Arabidopsis, and this could account for the enhanced
ability of Fe-deficient pea seedling roots to absorb
Cd2+.
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| Figure 7.
Regulation of IRT1 mRNA levels by
Fe availability in Arabidopsis and pea seedlings. Polyadenylated RNA
from ± Fe-grown roots of Arabidopsis (1.2 µg) and pea (1.9 µg) was
probed with the Arabidopsis IRT1 genomic clone. RNA was
prepared from 21-d-old Arabidopsis plants grown for 5 d without Fe
or 13-d-old pea plants grown for 4 d without Fe.
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Role of the Plasma Membrane Ferric Reductase in Stimulating
Cd2+ Influx
We explored the possibility that redox modification of a divalent
cation carrier may be involved in stimulated uptake in Fe-grown plants via enhanced activity of the plasma membrane ferric reductase (Welch et al., 1993). One might expect that if a stimulated ferric reductase either directly or indirectly altered the redox status of
sulfhydryl groups that play a role in the functioning of a divalent
cation transporter, then Cd2+ influx via this
system would be sensitive to inhibition by sulfhydryl-binding compounds. However, we found that the enhanced
Cd2+ influx in roots of Fe-deficient seedlings
was unaffected by the inclusion of either permeant (0.3 mm
N-ethylmaleimide) or impermeant (2 mm
p-chloromercuribenzene sulfonic acid) sulfhydryl-modifying reagents in the uptake solution (data not shown). Sulfhydryl-containing reducing agents such as DTT and -mercaptoethanol were not used for
these experiments because they could potentially interfere with uptake
by binding to Cd2+.
In yeast it has been suggested that Fe deficiency increases the number
of reduced sulfhydryl groups on the outer face of the plasma membrane,
which might play a role in the uptake of Fe and other mineral ions
(Lesuisse and Labbe, 1992). We found that Fe deficiency elicited a 50%
increase in reduced sulfhydryls on the root surface (0.15 ± 0.004 µmol reduced sulfhydryl groups [g fresh weight]1 for
Fe-grown roots versus 0.10 ± 0.007 µmol
g1 fresh weight for +Fe-grown roots).
Cd Accumulation in Roots and Shoots
The concentrations of various metals in roots and shoots of
seedlings exposed to 0.2 µm
Cd(NO3)2 for 2 d are
presented in Table II. Enhanced Cd
accumulation was apparent in roots but not in shoots of Fe-deficient
seedlings. An approximately 2-fold greater Cd concentration was
observed in the roots of Fe-deficient plants relative to Fe-sufficient
plants. In shoots, however, Cd levels were lower in the Fe-deficient
plants relative to control plants. Zn followed the same pattern as Cd,
exhibiting enhanced accumulation only in roots of Fe-deficient plants
and slightly lower concentrations relative to the control plants in the
shoots. The concentrations of certain other metals, such as Cu and Mn,
increased in both roots and shoots of Fe-deficient plants relative to
control plants.
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|
Table II.
Tissue metal accumulation in ± Fe-grown pea
seedlings exposed to 0.2 µm Cd
Seedlings were grown hydroponically in modified Johnson's nutrient
solution. Fe was withheld from the growth solutions of Fe seedlings
on d 9. On d 12, 0.2 µm Cd(NO3)2
was added to the growth solution. Tissue was harvested on d 14. Values represent means (n = 3). Values in parentheses
represent se.
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DISCUSSION |
This study provides evidence for the induction of
transporter-mediated root Cd2+ influx in
Fe-deficient pea seedlings. The effect of Fe deficiency on the
concentration-dependent kinetics of root Cd2+
influx was a specific and significant stimulation of a saturable Cd2+ influx (Figs. 1 and 2). This result is
consistent with a model in which transporters are deployed at a greater
density on the plasma membrane under Fe-deficient conditions, or
existing transporters are modified to exhibit higher activity. Because
the kinetics for Cd2+ influx were complex and
appeared to be the sum of saturable and linear functions, a number of
different approaches were used to determine the mechanistic basis for
these two kinetic components of Cd2+ influx.
Several pieces of evidence indicated that the saturable system
represented true trans-plasma membrane Cd2+
influx. First, the selective abolition of the saturable component by
the metabolic inhibitors CCCP and KCN provides strong evidence that
saturable, transporter-mediated uptake is distinct from apoplastic Cd2+ binding and accounts for the differences in
Cd2+ influx between Fe-sufficient and
Fe-deficient plants. Also, La, a Ca2+-channel blocker that
also exhibits very high affinity for negatively charged sites on the
cell wall, partially inhibited both the saturable and the linear
components of Cd2+ influx. This result further
reinforces the existence of two discrete components of influx. We
would expect La to both interfere with transporter-mediated uptake and,
as a trivalent cation, to bind more effectively than
Cd2+ to cell wall surfaces. Finally, only a
linear kinetic phase of 109Cd2+ binding was
observed with morphologically intact root cell wall preparations. This
linear component was similar in root cell walls of Fe-sufficient and
Fe-deficient plants. Together, these results provide strong evidence
for Fe-deficiency-mediated induction of a saturable
Cd2+-transport system, with linear
Cd2+ "uptake" reflecting apoplastic
Cd2+ binding that remains after desorption. The
linear component of Cd2+ binding most likely
consists of ionic interaction of Cd2+ with
carboxyl and/or sulfhydryl groups contained in cell wall constituents
such as hemicelluloses and cell wall proteins. Longer desorption
periods might eventually remove this bound Cd2+
from the cell wall, but uptake and desorption periods must be kept
short to ensure that unidirectional influx, and not efflux, of
radiolabeled Cd is being measured. Although much of the
Cd2+ is only weakly bound to the cell wall by
electrostatic forces, a portion of the apoplastically bound
Cd2+ may be irreversibly bound, because
Cd2+ could form covalent bonds with sulfhydryl
and other functional groups within the cell wall.
Although roots of Fe-deficient plants exhibit enhanced Cd uptake during
both short-term radiotracer studies and long-term tissue-accumulation
experiments, shoots from Fe-deficient seedlings exhibited lower Cd
accumulation than control treatments in long-term experiments. When a
number of different micronutrient metals were measured in Fe-deficient
plants, Cd and Zn accumulation was found to be stimulated in roots but
not in shoots; however, Cu and Mn accumulation was stimulated in both
roots and shoots. We do not yet fully understand what governs the
differential distribution of metals in plant tissues. Nicotianamine, a
nonprotein amino acid thought to regulate root and shoot metal levels,
could be involved in mediating Cd partitioning to the different
tissues. In addition, variable phytochelatin affinity for specific
metals such as Cu or Cd could also determine how much of each metal is translocated to the shoot. For example, much of the Cd entering roots
of Fe-deficient plants could be sequestered in the vacuole as the
Cd-phytochelatin complex and would not be available for translocation
to the shoot.
Possible Mechanisms of Enhanced Cd2+ Uptake
We explored three potential scenarios that could explain the
observed induction of heavy-metal transport in Fe-deficient pea seedlings: induction of the proton pump, induction of a divalent cation
transporter, and induction of the ferric reductase. Our investigations
into the mechanistic basis for the Fe-deficiency-induced stimulation of
heavy-metal influx in pea point to the induction of a divalent cation
transporter as well as the possible involvement of sulfhydryl
modification.
We investigated the possibility that enhanced heavy-metal accumulation
in response to Fe deficiency involves the increased activity of the
plasma membrane H+ pump, either through
generation of a H+ gradient or through
hyperpolarization of the Em. To test
whether the H+ pump or consequences of the pump
play a key role in energizing heavy-metal absorption, we used buffers
to abolish the enhanced H+ gradient in
Fe-deficient plant roots, and FC to stimulate the H+-ATPase in Fe-sufficient plant roots. We also
compared measurements of root-cell membrane potentials in ± Fe-grown
pea seedlings to determine if H+-ATPase-mediated
hyperpolarization of the root-cell membrane potential could stimulate
Cd2+ influx. Neither treatment had any effect on
heavy-metal absorption, and membrane potentials were found to be the
same in ± Fe-grown seedlings. We concluded that the proton pump
does not appear to be involved in enhancing heavy-metal accumulation
under Fe deficiency.
We also investigated whether enhanced activity of the
Fe-deficiency-induced plasma membrane ferric reductase may stimulate heavy-metal accumulation, perhaps by modulating the redox state of a
divalent cation transporter. Modification of the redox status of
critical sulfhydryl groups involved in the gating of ion-channel proteins has been reported in the literature and is a potential mechanism for reductase-mediated regulation in absorption. Bertl and
Slayman (1990) described a cation channel in the yeast vacuolar membrane that required very high (and nonphysiological)
Ca2+ levels for activation (1 mm),
but was activated by much lower levels of Ca2+
when exposed to sulfhydryl-reducing agents such as DTT or
-mercaptoethanol. In addition, Lesuisse and Labbe (1992)
reported in yeast that Fe deficiency resulted in higher levels of
reduced sulfhydryl groups at the exofacial plasma membrane surface, as
well as higher concentrations of GSH within the cell. They speculated
that the yeast ferric reductase may play a general role in regulating
the redox status of cells, in addition to facilitating Fe uptake. Welch
et al. (1993) further hypothesized that the plasma membrane ferric
reductase may serve a regulatory function in gating channels involved
in divalent-cation influx, either directly, by affecting the redox
status of critical sulfhydryl groups in the membrane, or indirectly, by
increasing levels of GSH in the cell, which could in turn influence ion
channels. However, recent work with a recessive mutant in Arabidopsis,
frd1, in which ferric reductase activity is impaired,
suggests that on the contrary, the ferric reductase may not be involved
in regulating divalent cation influx. Under Fe deficiency,
frd1 exhibits accumulation of Mn, Cu, and Zn to levels even
higher than that of the Fe-deficient wild type (Yi and Guerinot, 1996).
It is clear that this area awaits further research.
In this study we used roots treated with sulfhydryl-modifying agents to
test whether redox modification is involved in stimulating metal
influx, and we also compared exofacial reduced sulfhydryl groups in
roots of ± Fe-grown pea seedlings. The significant increase in
reduced sulfhydryl groups on the root surface of Fe-grown pea
seedlings suggests that some type of sulfhydryl modification of plasma
membrane components may occur under Fe deficiency and may partially
account for the enhanced Cd influx.
Molecular studies of the recently cloned
Fe2+-transporter gene IRT1 from
Arabidopsis provide compelling support for the induction of a
divalent-cation transporter by plant Fe status (Eide et al., 1996).
IRT1 encodes a high-affinity Fe2+
transporter that functionally complements yeast mutants defective in
both high- and low-affinity Fe transport. IRT1 is highly
expressed under Fe deficiency and possibly transports other divalent
cations, including Cd, Co, Mn, and Zn. To determine whether the
IRT1 ortholog in pea is also induced by Fe deficiency, we
used an IRT1 genomic clone from Arabidopsis to probe root
poly(A+) mRNA from ± Fe-grown pea and
Arabidopsis. This clone hybridized to an approximately 1.3-kb
transcript from both pea and Arabidopsis roots that is induced under Fe
deficiency. This result suggests that induction of a Fe transporter
could account for enhanced uptake of divalent cations, including Cd, in
Fe-deficient plants.
IRT1 is a member of a family of closely related transporter
genes from a diverse group of eukaryotic organisms, including fungi,
yeast, nematodes, plants, and mammals. This family also includes the
yeast Zn-regulated transporters ZRT1 and ZRT2
(Zhao and Eide, 1996a, 1996b). It is interesting that, although
IRT1 is a functional Fe transporter in yeast mutants
defective in Fe transport, the predicted amino acid sequence of
IRT1 has no similarity to that of endogenous yeast Fe
transporters (Askwith et al., 1994; Dix et al., 1994; Eide et al.,
1996; Stearman et al., 1996). Yeast has two separate Fe-uptake
pathways, a high-affinity (Km = 0.15 µm) and a low-affinity (Km = 40 µm) Fe transporter. The FTR1 gene product,
a putative high-affinity transporter that is highly selective for
ferric Fe, interacts with the FET3 gene product, a
multicopper oxidase localized to the plasma membrane, to facilitate
high-affinity Fe3+ transport (Askwith et al.,
1994; Stearman et al., 1996). The FET4 gene in yeast is
required for low-affinity Fe uptake and encodes a ferrous transporter
that can also transport other metals such as Co2+
and Cd2+ (Dix et al., 1994). Yeast
fet3fet4 mutants defective in both high- and low-affinity
Fe-transport systems are extremely sensitive to Fe limitation but can
be functionally complemented with the Arabidopsis IRT1 gene.
The transporter activity we have characterized in the roots of
Fe-deficient pea seedlings exhibits high substrate affinity (0.9-1.5
µm) for Cd. As mentioned in "Results," this
Fe-induced system also effectively transports Zn. Based on our result
that Fe deficiency induces IRT1 expression and on our
preliminary results indicating that a pea IRT1 ortholog
complements yeast Fe- and Zn-transport mutants, we speculate that Cd
may be transported by a high-affinity Fe transporter. A future research
priority will be to test whether yeast Fe-transport mutants bearing the pea IRT1 ortholog have the ability to transport a range of
other heavy metals. This future work should more comprehensively
address the question of the transport specificity of this system; that is, in plants using a reductase-based mechanism of Fe uptake, is the
Fe2+ transporter specific for Fe or can it
transport other micronutrients and/or heavy metals? Our findings of a
correlation between enhanced Cd influx and induction of a high-affinity
Fe transporter under Fe deficiency suggest that the Fe transporter
might be a relatively nonspecific divalent-cation transporter. A more
thorough characterization of this transporter will have important
implications for both plant-micronutrient nutrition and heavy-metal
remediation of contaminated soils using plants.
| |
FOOTNOTES |
1
This work was supported by a grant from the U.S.
Department of Energy, Division of Energy Biosciences (Interagency
Agreement DE-A 102-95ER 21097) to L.V.K.
*
Corresponding author; e-mail lvk1{at}cornell.edu; fax
1-607-255-2459.
Received July 30, 1997;
accepted November 14, 1997.
| |
ABBREVIATIONS |
Abbreviations:
CCCP, carbonylcyanide
m-chlorophenylhydrazone.
DTNB, dinitrobenzoic acid.
EDDHA, N,N-ethylenebis-[(2-hydroxyphenyl)-Gly].
Em, plasma membrane electropotential.
FC, fusicoccin.
| |
ACKNOWLEDGMENTS |
We gratefully acknowledge the technical assistance provided by
Mike Rutzke, Jon Shaff, Heather Hill, and Ben Alexander.
| |
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Abstract
Introduction