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Plant Physiol. (1999) 119: 435-444
Change in Apoplastic Aluminum during the Initial Growth Response
to Aluminum by Roots of a Tolerant Maize Variety1
María Dolores Vázquez,
Charlotte Poschenrieder,
Isabel Corrales, and
Juan Barceló*
Laboratorio de Fisiología Vegetal, Facultad de Ciencias,
Universidad Autónoma de Barcelona, E-08193 Bellaterra, Spain
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ABSTRACT |
Root elongation, hematoxylin
staining, and changes in the ultrastructure of root-tip cells of an
Al-tolerant maize variety (Zea mays L. C 525 M)
exposed to nutrient solutions with 20 µM Al (2.1 µM Al3+ activity) for 0, 4, and 24 h
were investigated in relation to the subcellular distribution of Al
using scanning transmission electron microscopy and energy-dispersive
x-ray microanalysis on samples fixed by different methods. Inhibition
of root-elongation rates, hematoxylin staining, cell wall thickening,
and disturbance of the distribution of pyroantimoniate-stainable
cations, mainly Ca, was observed only after 4 and not after 24 h
of exposure to Al. The occurrence of these transient, toxic Al effects
on root elongation and in cell walls was accompanied by the presence of solid Al-P deposits in the walls. Whereas no Al was detectable in cell
walls after 24 h, an increase of vacuolar Al was observed after
4 h of exposure. After 24 h, a higher amount of
electron-dense deposits containing Al and P or Si was observed in the
vacuoles. These results indicate that in this tropical maize variety,
tolerance mechanisms that cause a change in apoplastic Al must be
active. Our data support the hypothesis that in Al-tolerant plants, Al can rapidly cross the plasma membrane; these data clearly contradict the former conclusions that Al mainly accumulates in the apoplast and
enters the symplast only after severe cell damage has occurred.
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INTRODUCTION |
It is largely recognized that root tips are the primary site of
Al-induced injury in plants (Ryan et al., 1993 ). The accumulation of Al
in root tips has been found to be significantly correlated with
root-growth inhibition in maize (Zea mays L.) varieties
differing in Al tolerance (Llugany, 1994; Llugany et al., 1994). In
Al-sensitive maize plants an inhibition of root elongation has been
observed after only 30 min of exposure to Al (Llugany et al., 1995).
Such a short response time, in addition to the common belief (Kochian, 1995) that Al accumulates mainly in the apoplast and crosses the plasma
membrane slowly, has led to the hypothesis that Al-induced inhibition
of root elongation may be caused by toxicity mechanisms that occur in
the apoplast (Rengel, 1990, 1996; Horst, 1995) and that there is no
need for Al to enter the symplast to cause primary toxicity effects
(Rengel, 1992). However, investigations using the highly Al-sensitive
technique of secondary ion MS have shown that significant Al
concentrations accumulate in the symplast of root-tip cells of soybean
plants after only 30 min of exposure to Al (Lazof et al., 1994, 1996).
Recent experiments on giant algae (Chara corallina) cells,
where cell walls were separated from the cells by microsurgery, have
also shown that Al uptake across the plasmalemma may be linear and
occurs without delay (Rengel and Reid, 1997). These investigations
support the view that symplastic phytotoxicity mechanisms may also be
responsible for Al-induced inhibition of root elongation after short
exposure times (Kochian, 1995).
More information on the subcellular distribution of Al in root tips
would help to establish both the relative importance of apoplastic and
symplastic sites in the Al-toxicity syndrome and the role of Al
compartmentation in Al resistance or tolerance. Unfortunately,
ultrastructural investigations under environmentally realistic growth
conditions that relate the subcellular localization of Al in root tips
to root growth in Al-tolerant varieties are scarce (Delhaize et al.,
1993). Major difficulties for such an approach are the low sensitivity
of electron probe x-ray microanalysis for Al determination (Lazof et
al., 1994, 1997) and the poor visual distinction of subcellular
structures in freeze-dried samples, in combination with the extremely
low Al tissue concentrations, which have been shown to cause inhibition
of root elongation (Lazof et al., 1994, 1996).
Using a highly sensitive monitoring technique for
root growth, we have previously shown that 20 µM Al (2.1 µM Al3+ activity) causes a
significant decrease in the relative root-elongation rate in the
Al-tolerant maize var C 525 M after 112 min of exposure, whereas after
24 h the relative elongation rate did not differ from that of the
controls (Llugany et al., 1995). In this paper we report results on the
changes in the subcellular distribution of Al in root tips during the
initial root-growth response (0-24 h) of var C 525 M exposed to 20 µM Al (2.1 µM Al3+
activity). Hematoxylin staining, ultrastructural observations, and
EDXMA were performed on root tips after 0, 4, and 24 h of exposure
of plants to control or Al-containing nutrient solutions to detect a
possible relationship between changes in subcellular Al
compartmentation and ultrastructural alterations, which may explain
why, after a transient inhibition, the root-elongation rate recovers
during the initial 24 h of exposure to Al. EDXMA with scanning TEM
on glutaraldehyde-fixed, PA-stained, and freeze-substituted samples
were performed. Although these techniques only allow a semiquantitative
estimation of mineral contents, the better visual resolution obtained
results in more reliable data on the subcellular localization than
EDXMA with SEM on freeze-dried or frozen-hydrated bulk specimens (Van
Steveninck and Van Steveninck, 1991).
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MATERIALS AND METHODS |
Plant Material and Growth Conditions
Al-tolerant maize (Zea mays L. var C 525 M, Embrapa, Siete Lagoas, Brazil) seeds were
germinated in the dark at 25°C on filter paper moistened with 1 mM CaSO4. After 96 h,
uniform seedlings with a radicle length of 13.7 ± 0.9 cm were
transferred to plastic beakers (14-L capacity; 24 plants per beaker)
filled with continuously aerated nutrient solution (pH 4.3) of the
following composition (in µM): 500 Ca(NO3)2, 395 K2SO4, 5 KH2PO4, 100 MgSO4, 200 NH4NO3, 0.06 (NH4)6Mo7O24,
5 MnSO4, 0.38 ZnSO4, 0.16 CuSO4, 16 H3BO3, and 10 FeEDTA. After
72 h, the plants were transferred to treatment solutions of the
same composition and volume per plant. One-half of the plants received
solution supplemented with 20 µM Al as AlCl3. The pH of the control nutrient solutions
remained constant throughout the experiment (4.31 ± 0.01). In
Al-supplemented solutions pH values were 4.34 ± 0.02 and
4.13 ± 0.02 after 4 and 24 h, respectively. According to the
GEOCHEM speciation program (Parker et al., 1987), the activity of free
Al3+ in the treatment solution was 2.1 µM and all Al was in soluble form. The
concentrations of monomeric Al in the solution, analyzed by the
short-term pyrocatechol method (Kerven et al., 1989), was 13 µM.
The seedlings were grown in an environmentally controlled growth
chamber under the following conditions: 16 h of light/8 h of
darkness, day/night temperature 26°C/20°C, RH 70%, and PPFD 190 µmol m 2 s1.
Root Growth and Hematoxylin Staining
Seedling seminal root length (n  24 per
treatment and time sample) was measured with a ruler before the
transfer of the plants to nutrient solution, after the 72-h
pretreatment (0-h treatment), and after the 4- and 24-h treatments with
solutions containing 0 (control) or 20 µM Al.
Hematoxylin staining of whole roots was performed on 10 plants per
treatment and time sample (Polle et al., 1978).
Sample Fixation for Electron Microscopy and EDXMA
For EM studies, after a short (10 s) rinse with distilled water,
the tips (0-2 mm and the following 2-5 mm) from primary roots were
excised from control and Al-treated seedlings after 0, 4, and 24 h
of exposure to nutrient solutions. The samples were immediately fixed
by the different methods described below.
Some samples were fixed in 2.5% (w/v) glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2), but were not postfixed
with osmium. The fixed material was dehydrated in a graded alcohol series and embedded in Spurr's resin (Spurr, 1969). Some of the non-osmified, thin, longitudinal tip sections (near root halves) were
stained with saturated aqueous uranyl acetate, followed by Reynolds
lead citrate (Reynolds, 1963). Stained and unstained longitudinal
serial sections, between 0 and 1.5 mm from apex, were studied by
electron microscopy (model H-7000, Hitachi, Tokyo, Japan), and the
elemental distribution in cell walls and vacuoles was determined by
EDXMA on unstained, dry-cut sections.
Other sampled tips were treated with PA to retain easily diffusible
cations (Mentré and Escaig, 1988; Mentré and Halpern, 1988). The composition of the fixation mixture was 4% PA, 2%
paraformaldehyde, and 1% phenol (pH 7.8). After the treatment the
specimens were rinsed with distilled water, then dehydrated in graded
alcohol and embedded in Spurr's resin. Longitudinal tip sections were studied by light microscopy (Optiphot, Nikon) and electron microscopy. The elemental composition of PA precipitates was identified by EDXMA.
A third group of root tips was processed by freeze-substitution, as
previously described (Harvey, 1982; Vázquez et al., 1992). The
samples were cryofixed in pentane cooled with liquid nitrogen. The
freeze-substitution with acetone precooled with liquid nitrogen was
allowed to proceed for 1 week at  80°C in a deep freezer. The
specimens were gradually warmed to room temperature for 24 h and
then infiltrated with Spurr's resin. Transverse sections at
approximately 0.5 and 3 mm from the apex were stained for light and
electron microscopy (photographs not shown) as for the
glutaraldehyde-fixed sections described above. Corresponding unstained,
dry-cut sections were used for EDXMA.
EDXMA
Dry-cut sections of approximately 1.5 µm were mounted onto gold
grids. The microanalytical determinations were performed on an electron
microscope (model H-800, Hitachi) operated at 100 kV in the
scanning TEM mode using an energy-dispersive detector (Kevex, Valencia,
CA) and a Delta class 4460 analyzer (Kevex). The counts were made over
a 100-s period and spectra were recorded. Gaussian deconvolution was
applied to the results and, after background correction, the data were
expressed as the counts to second ratio. A variable number of samples
was used for each treatment and fixation method. For
glutaraldehyde-fixed samples or those treated with PA, n
values were as follows: control plants, n 4;
Al-treated plants, n 9. The n values for
freeze-substituted samples were 9 and 15 for control and Al-treated
plants, respectively. No Al signals were detectable by EDXMA in
electron-translucent cell areas and all data shown are from
electron-dense deposits. As usual in EDXMA studies, the SD
values of the results were high; therefore, the ranges were given in
addition to mean values ± SD. Blank resin was analyzed to
check for contaminants.
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RESULTS |
Maize seedlings exposed to Al for 4 h exhibited decreased
root-elongation rates, whereas after 24 h the rates had recovered to the control values of plants before the start of the Al treatment (Table I).
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Table I.
Root-elongation rates of maize seedlings
Plants were exposed to nutrient solutions containing 0 (control) or 20 µM Al for different times. The growth rate of the 0-h
time sample was determined during the 72-h pretreatment. Values are
means ± SE (n 24).
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Seminal roots of seedlings stained with hematoxylin are shown in Fig.
1. After 4 h of exposure to
Al-containing nutrient solution, root tips exhibited intense staining
(Fig. 1B), whereas after 24 h of Al exposure, no staining could be
observed (Fig. 1D) and the plants did not differ from controls (Fig. 1,
A and C).
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| Figure 1.
Seminal roots from maize plants stained with
hematoxylin. A and C, Control (0 µM Al) plants after 4 and 24 h, respectively. B and D, Plants treated with 20 µM Al containing nutrient solution for 4 and 24 h,
respectively.
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Ultrastructural alterations in cell walls of root-tip (1.5-mm) cells
were observed after 4 h of exposure to Al (Fig.
2). A thickening of tangential cell walls
occurred in the internal (third-sixth) cortex cells (Fig. 2, B and D).
Sections from glutaraldehyde-fixed specimens revealed cell walls with
electron-translucent areas and electron-dense zones near the
plasmalemma (Fig. 2B). In the corresponding sections that had been
stained with PA for visualizing otherwise soluble cations, a
significantly higher accumulation of PA-stained deposits was found at
the internal site of these cell walls in Al-treated plants (Fig. 2D)
than in controls (Fig. 2C). Al-treated plants also exhibited a higher
amount of PA-stained deposits inside of the cortex cells than control
plants.
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| Figure 2.
TEM images from longitudinal sections of root tips
of maize plants exposed for 4 h to control (0 µM Al)
(A and C) or 20 µM Al (B and D) in nutrient solution. A
and B, Non-osmified, glutaraldehyde-fixed samples. C and D, PA-stained
samples. Note thickening of cell walls (B) and higher amount of PA
precipitates at the internal site of cell walls (D) in samples from
Al-treated plants. Scale bars represent 0.5 µm in A and B, and 1.0 µm in C and D.
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The cortex cells exhibited numerous small vacuoles. In
glutaraldehyde-fixed samples from plants prior to the transfer to
treatment solutions (0 h) (Fig. 3A) and
from plants exposed to control (Fig. 3C) or Al-containing (Fig. 3E)
nutrient solution for 4 h, the small vacuoles were electron-lucent
and only small, peripherical, electron-dense deposits were detected in
some vacuoles of internal cortical cells (mainly the third to the
sixth) of each section (Fig. 3, C and E, arrows). In the corresponding
PA-stained sections abundant electron-opaque precipitates in the
central zone of the vacuoles were found (Fig. 3, B, D, and F).
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| Figure 3.
TEM images from longitudinal sections of root
tips of maize plants exposed for 0 (A and B) or 4 h (C-F) to
control (0 µM Al) (A-D) or 20 µM Al (E and
F) containing nutrient solution. A, C, and E, Glutaraldehyde-fixed
samples. B, D, and F, PA-stained samples. Note the abundance of
electron-translucent vacuoles with only some peripheric electron-dense
deposits (C and E, arrows) in conventionally fixed samples and the
abundance of electron-dense precipitates in the central part of
vacuoles from PA-stained samples (B, D, and F). All scale bars
represent 10 µm, except B, where bar is 1 µm.
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After 24 h of exposure to Al-containing nutrient solutions,
thickened cell walls similar to those observed after 4 h of
exposure were not detected (Fig. 4, B and
D). Root-tip vacuoles of plants exposed to Al for 24 h (Fig. 4D)
exhibited a considerably higher amount of electron-opaque aggregates
than those from the 4-h Al treatment (Fig. 3E). After 24 h, the
vacuolar deposits in Al-treated plants (Fig. 4D) were also more
abundant than in the corresponding control samples (Fig. 4C).
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| Figure 4.
TEM images from longitudinal sections of root tips
of maize plants exposed for 24 h to control (0 µM
Al) (A and C) or 20 µM Al in nutrient solution (B and D).
A and B, PA-stained samples. C and D, Non-osmified,
glutaraldehyde-fixed samples. Note the abundance of electron-dense
vacuolar deposits (arrows) in conventionally fixed samples from plants
exposed to Al (D) in comparison with the scarce presence of deposits in
controls (C). All scale bars represent 10 µm. Is, Intercellular
space.
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After 24 h of exposure to Al, but not after 4 h, abnormal,
irregular divisions of internal cortex cells were detected in
longitudinal sections of PA-stained samples (Fig.
5, B and D), whereas in the samples of
control plants all cell divisions occurred in regular planes (Fig. 5, A
and C).
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| Figure 5.
Light-microscopy images from longitudinal
sections (PA stained) of root tips of maize plants exposed for 24 h to control (0 µM Al) (A and C) or 20 µM
Al in nutrient solution (B and D). Controls (A and C) show
well-organized cortical cell lines with almost horizontal cell-division
planes. Al-exposed plants (B and D) exhibit irregular cell-division
planes in internal cortical cells. A and B, Scale bars represent 100 µm; C and D, scale bars represent 10 µm.
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Tables II and
III show relative contents (in counts per
second) of Al and selected mineral nutrients found by EDXMA in the electron-dense deposits of cell walls and vacuoles of control and
Al-treated plants after 0, 4, and 24 h of exposure. In
glutaraldehyde-fixed samples small amounts of Al were detected in
electron-dense deposits in the walls of the roots treated with 20 µM Al after only 4 h of exposure. P, S, Ca, K, and
Zn were also found in these deposits (Table II; Fig.
6B). However, Al was not detectable in
electron-dense areas of the walls after 24 h of exposure to Al
either in glutaraldehyde-fixed material or in samples prepared by
freeze-substitution or PA staining (Tables II and IV). Al was not
detected in the cell walls of controls at any sampling time (Table II;
Fig. 6A).
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Table II.
Relative contents of Al and selected mineral
nutrients in cell walls of maize root tips (0-1.5 mm)
Plants were exposed for different times to nutrient solutions
containing 0 (control) or 20 µM Al. If not indicated
otherwise, the values are from glutaraldehyde-fixed samples. Values are
given as means ± SD; n 4 for control and
n 9 for Al-treated plants.
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Table III.
Relative contents of Al and selected mineral
nutrients in vacuoles of root-tip (0-1.5 mm) cells of maize
Plants were exposed for different times to nutrient solutions
containing 0 (control) or 20 µM Al. All data are from
conventionally fixed samples. Values are given as ranges (parentheses)
and means ± SD; n 4 for control and
n 9 for Al-treated plants.
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| Figure 6.
Representative EDXMA spectra from electron-dense
deposits in cell walls (A-C) and vacuoles (D-H) of root-tip (0-1.5
mm) cells after 4-h (A, B, E, and F) and 24-h treatments (C, D, G, and
H). A, D, and E are from control plants and B, C, F, G, and H are from
Al-treated plants. All spectra are from glutaraldehyde-fixed samples,
except D and H, which are from PA-stained samples. All spectra are
printed at 1000 counts. Au is from the grid.
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Table IV.
Relative contents of Al and selected mineral
nutrients in cell walls and vacuoles of maize root-tip cells
(transverse sections 3 mm from apex)
Plants were exposed for 24 h to nutrient solutions with 0 (control) or 20 µM Al. All data are from
freeze-substituted samples. Values are given as ranges (parentheses)
and means ± SD; n 9 for control and
n 15 for Al-treated plants.
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Small amounts of Al were detected in electron-dense precipitates found
in the vacuoles of internal cortex cells of 1.5-mm tips of Al-treated
plants after the 4-h Al treatment (Table III; Fig. 6F). P, Zn, Ca, Mg,
and S were also present in these deposits. The few electron-dense
deposits in the vacuoles of control plants after 0 and 4 h of
exposure contained neither Al nor S, but exhibited counts for P, Mg,
Ca, and Zn (Table III; Fig. 6E), that were similar to those of
Al-treated plants (Fig. 6F). After 24 h of exposure to Al, higher
Al counts were found in the vacuoles (Table III; Fig. 6, G and H).
After 24 h in control solutions, small amounts of Al were also
detected in some of the few vacuolar deposits of controls (Table III).
These deposits contained P, Zn, Ca, S, and Mg.
Additional measurements on freeze-substituted samples were performed in
the more differentiated cells at 3 mm from the tip after 24 h
of treatment (Table IV). Al was
undetectable in electron-dense cell wall zones in both control and
Al-treated plants (Table IV). In these cell wall zones, similar counts
for P, S, K, and Ca were found in controls and Al-treated plants.
Counts for Si in walls were within the values of those in blank resin,
where 4.8 to 11 counts s1 Si were found. Traces
of Mg were only detected in some of the Al-treated samples. In a few of
the scarce, electron-dense deposits of the vacuoles of controls, Al was
detected (Table IV). In all deposits where the Al signal was
significant, P, K, and Ca were found, whereas signals for Mg and S were
only occasionally detected. Two types of electron-dense deposits were
found in the vacuoles of cortex cells at approximately 3 mm from
the tip in plants exposed to Al for 24 h (Table IV; Fig.
7, A and B). Deposits with a relatively high P content, either without or with low Al amounts (Table IV; Fig.
7B), and Si-rich deposits that contained high Al amounts in addition to
S, K, Ca, and Mg. These Si- and Al-rich deposits exhibited either low P
counts or did not contain P (Table IV; Fig. 7A).
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| Figure 7.
Representative EDXMA spectra from electron-dense
deposits in the vacuoles of root-tip cortex cells (3 mm) of plants
exposed to Al for 24 h. A, Al-containing deposit with high Si
content. B, Al-containing deposit with P, but without Si. All samples
were prepared by freeze-substitution. All spectra are printed at 1000 counts; Fe and Co are instrument contaminants; and Au is from the
grid.
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DISCUSSION |
The tropical maize variety used in this study, C 525 M, has been
found to be Al tolerant in both short-term (hours-days) nutrient solution studies (Llugany et al., 1995; Poschenrieder et al., 1995;
Calba and Jaillard, 1997; Horst et al., 1997) and long-term (weeks)
field experiments on acid soils (M.F. Guimaraes, C.H. The, C. Welcker,
Final Report CE-project TS 3-CT 92-0071, unpublished). Nonetheless,
after short-term exposure to nutrient solution with a low
Al3+ activity (2.1 µM), a decrease
of root elongation has already been observed in a previous
investigation (Llugany et al., 1995). However, as in the present study
(Table I), this inhibition was only transient, and after 24 h of
exposure, the elongation rates recovered to those of controls. As shown
by the analysis of nutrient solutions after the experiment, the
recovery of the root-elongation rate was neither due to an increase of
pH in the nutrient solution nor to depletion of Al during the
experiment.
Ultrastructural observations performed in this study also show that the
Al-tolerant maize var C 525 M is highly responsive to low Al
concentrations after 4 h of exposure, but is poorly affected after
24 h. Cell wall thickening and disturbance of apoplastic and
symplastic PA-stainable cations occurred only during the initial period
of the Al treatment (4 h), but not after longer (24 h) exposure times.
The swelling of cell walls is an early Al-toxicity symptom (Eleftheriou
et al., 1993), that seems to be related to a displacement of Ca from
the walls (Demarty et al., 1984). In our study, alterations of the
cation content in the cell walls of cortex cells of plants exposed for
4 h to Al could be observed in PA-stained samples. PA staining has
been shown to immobilize easily diffusible cations such as Ca and Na
(Mentre and Escaig, 1988; Mentré and Halpern, 1988). After 4 h of exposure, considerably higher amounts of PA precipitates were
found at the internal site of cell walls and inside of the cytoplasm of
cortex cells of Al-treated plants than in the controls. EDXMA showed
that the PA deposits had a high K and Ca content. This result suggests
that after short-term (4 h) exposure to Al, the cation homeostasis in
the apoplast was severely disturbed. The transient character of
Al-induced alterations of both cell wall ultrastructure and cation
homeostasis in our experimental plants was chronologically related to
the change in root-elongation rate.
There also was a clear coincidence in time between the Al-induced
inhibition of root-elongation rates and the detection of Al in the
apoplast of root tips by two different techniques: hematoxylin staining
and EDXMA. Hematoxylin is considered to stain Al-P deposits in root
tips that have been damaged by Al exposure (Ownby, 1993). In
Al-tolerant wheat a decrease of hematoxylin staining intensity after 6 to 24 h of Al exposure has been observed (Rincón and Gonzales, 1992). However, to our knowledge, this is the first time that
such a change in apoplastic Al in an Al-tolerant variety has been
confirmed by EDXMA. This change was related in time to a change in
root-elongation rates and ultrastructure.
The facts that Al caused damage in the apoplast and cation homeostasis,
decreasing root elongation only during the first hours of exposure, and
that the recovery of the plants occurred in coincidence with a change
in apoplastic Al, suggest that in maize var C 525 M, Al-tolerance or
-resistance mechanisms must have been activated that induced a change
in the speciation of apoplastic Al, making it undetectable for
hematoxylin and EDXMA. At present, there is considerable experimental
evidence indicating that increased exudation of organic acids from root
tips may play an important role in Al detoxification in Al-tolerant
maize and wheat (Pellet et al., 1995, 1997; Jorge and Arruda, 1997).
Ownby (1993) has shown that root tips of an Al-sensitive wheat variety
that stain intensely with hematoxylin exhibited no coloration when the
roots were rinsed with citrate before the staining procedure. We are
currently investigating whether exudation of organic acids was the
cause for the observed changes in apoplastic Al in the maize variety
used in this study. Our results on apoplastic Al, ultrastructural
alterations in the apoplast, and root-elongation rates support the
hypothesis that apoplastic Al can be toxic to plants (Rengel, 1992;
Horst 1995). However, because of the fact that after only 4 h of
exposure Al was detectable in root-tip vacuoles, we cannot exclude that
symplastic Al can also be responsible for the toxic effects observed
after 4 h of exposure.
Our EDXMA data showing a significant increase of vacuolar Al as soon as
4 h after the start of Al supply and only a transient occurrence
of insoluble Al deposits in the apoplast of root tips are in clear
contrast to those from several other studies. Marienfeld and Stelzer
(1993) and Marienfeld et al. (1995) have observed a high accumulation
of Al in root-tip cell walls and could not find Al inside cells unless
plants were exposed to Al for longer times. Lazof et al. (1997)
indicated that in only 6 out of 17 electron probe x-ray microanalysis
studies was Al detected inside of plant cells, whereas in all studies
Al was found mainly in cell walls. This apparent contradiction with our
results may be explained, at least in part, by clear differences in the
nutrient solutions employed, the exposure time, and the fact that most of these studies were performed with Al-sensitive plants exposed to
considerably higher Al concentrations.
That Al may enter root cells after short-term exposure to Al has
previously been demonstrated in investigations using secondary ion MS
(Lazof et al., 1994, 1996) or fluorescence microscopy on morin-stained
root tips (Tice et al., 1992). However, this is the first time to our
knowledge that Al has been shown to be present in root-tip vacuoles of
an Al-tolerant variety after a few hours of exposure. Tice et al.
(1992) also found most of the Al inside of the cells of root tips from
Al-tolerant wheat. In contrast, they could not detect Al in vacuoles.
However, it is not likely that morin would stain insoluble Al complexes
in vacuoles.
The Al-rich vacuolar deposits, found in root-tip cells between 0 and
1.5 mm from the apex of our experimental plants contained P, Ca, Zn,
and Mg (Table II; Fig. 6H), and their mineral composition was similar
to that reported for phytate (Mikus et al., 1992). Phytate or
polyphosphate deposits in the vacuoles may be involved in vacuolar
storage of Al in a way similar to that reported for Zn (Van Steveninck
et al., 1987). Compartmentational analysis of 32P
elution in root cortical cells of intact roots of Lolium
perenne exposed to nontoxic Al concentrations also suggests that
considerable amounts of a condensed Pi form may complex Al in
root-cortex vacuoles (Macklon and Sim, 1992).
According to our results, in the expanding cells at 3 mm from the apex
a second storage form for considerable Al amounts occurs in this
Al-tolerant maize variety. In this more mature root zone most of the
vacuolar Al of Al-treated plants was associated with high Si amounts
(Table III; Fig. 7A). The Al that was detectable occasionally in the
corresponding cells of control plants and that probably derived from
the seeds that had been formed on maternal plants growing on acid soils
in Brazil was exclusively found in P-rich deposits with a mineral
composition similar to phytate (Table III). There are several earlier
reports showing that the ameliorative effect of Si is not only due to
Al-Si interactions at the substrate level, but that Al-Si interactions
inside plants may play an important role in Al tolerance (Barceló
et al., 1993; Hodson and Evans, 1995; Corrales et al., 1997). It is
tentative to speculate that a preferential storage of Al in the form of Si-rich deposits in the vacuoles of expanding cortex cells after 24 h of exposure to Al would reduce the toxic effects of Al and may contribute to the enhancement of root elongation after an initial
transient growth reduction.
The relationship between vacuolar storage of Al in root-tip cells and
Al tolerance, however, remains unclear. Ernst (1998) states that
compartmentation into the vacuole is the principle of all hypotheses
explaining intracellular metal tolerance in plants. Genotype
differences in intracellular tolerance, however, cannot be explained
per se by the presence of metal deposits in vacuoles, but are probably
related to both the capacity of plants to form metal complexes that
would be less toxic to cell components during the transport to the
vacuole and the velocity of metal transport across the tonoplast.
In spite of the fact that the accumulation of Al increased in the small
vacuoles of root-tip cells, a toxic effect on the plane of cell
division occurred after 24 h of exposure to Al (Fig. 4D). This
result suggests that under our experimental conditions, not all Al was
efficiently detoxified by chelation and vacuolar storage, and that
there was sufficient Al remaining to interfere directly or indirectly,
perhaps by an interaction with phosphatidylinositol bisphosphate
(Kochian and Jones, 1997), with the direction of the
cytoskeletal-directed plane of cell division.
In conclusion, the fact that after 4 h of exposure to Al,
significant Al was detected in root-tip cell compartments, cell walls,
and vacuoles does not allow us to clarify the problem of the primary
site of Al toxicity: the apoplast or symplast. However, to our
knowledge, this is the first experiment performed with an Al-tolerant
maize variety that provides analytical data supporting a relation in
time between the lowering of insoluble, apoplastic Al, increased Al
accumulation in root-tip vacuoles, and a decrease of Al-toxicity
symptoms in the apoplast. Moreover, our results provide evidence for
the view that even in Al-tolerant maize Al enters rapidly into the
cells.
| |
FOOTNOTES |
1
This work was supported by the Research Council
of the European Union (contract nos. TS*CT922-0071 and ERBIC188CT-0063)
and by the Spanish National Research Council (contract no. DGICYT PB97-0163-C02-01).
*
Corresponding author; e-mail j_barcelo{at}cc.uab.es; fax
34-93-581-2003.
Received June 25, 1998;
accepted October 30, 1998.
| |
ABBREVIATIONS |
Abbreviations:
EDXMA, energy-dispersive x-ray microanalysis.
PA, pyroantimoniate.
SEM, scanning electron microscopy.
TEM, transmission electron microscopy.
| |
ACKNOWLEDGMENT |
The supply of seeds of var C 525 M from Embrapa is gratefully
acknowledged.
| |
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Top
Abstract
Introduction