Journal of Experimental Botany, Vol. 57, No. 15, pp. 4033–4042, 2006
doi:10.1093/jxb/erl174 Advance Access publication 14 November, 2006
RESEARCH PAPER
Aluminium rhizotoxicity in maize grown in solutions with
Al3+ or Al(OH)4– as predominant solution Al species
A. Stass1, Y. Wang2, D. Eticha1 and W. J. Horst1,*
1
Institute of Plant Nutrition, University of Hannover, Herrenhaeuserstr. 2, D-30419 Hannover, Germany
2
College of Agriculture, Yangzhou University, Yangzhou 225009, PR China
Received 15 March 2006; Accepted 30 August 2006
Abstract
Introduction
3+
The rhizotoxicity of aluminium at low-pH with Al and at
high pH with AlðOHÞ
4 as the main Al species was
studied. Aluminium reduced root growth to similar levels
at pH 8.0 and pH 4.3, although the mononuclear Al concentration at pH 8.0 was three times lower than at pH 4.3.
Al contents of root apices were much higher at pH 8 than
at pH 4.3. Callose was induced only marginally at pH 8
and the formation was confined to the epidermis,
whereas it proceeded through the cortex with time at
pH 4.3. Well-documented genotypical differences in
callose formation and Al accumulation could not be
found at pH 8. The largest fraction of the root-tip Al was
recovered in the cell-wall fraction independent of the
solution pH. A sequential extraction of isolated cell walls
suggests that most of the cell-wall Al was precipitated
Al(OH)3 at pH 8.0. This can be explained by a drastic pH
reduction in the root apoplastic sap to 6.2, whereas at
bulk solution pH 4.3 it rose to 5.6. Al precipitation was
also confirmed by the microscopic localization of Al. At
pH 8, Al could mostly be found in the epidermis, but in
the apoplast of the outer cortex at pH 4.3. It is proposed
here that at pH 4.3, Al3+ inhibits root growth through
binding to sensitive binding sites in the apoplast of the
epidermis and the outer cortex. At pH 8, Al(OH)3 precipitation in the epidermis causes a mechanical barrier
thus impairing the root-growth control of the epidermis.
Key words: Al localization, aluminate, aluminium toxicity,
apoplast, callose, pH.
Aluminium toxicity has been well documented under acid
soil conditions, where Al3+ is the most abundant mononuclear Al species leading to rhizotoxicity in plants, and is
generally believed to be the most toxic form (see reviews by
Delhaize and Ryan, 1995; Matsumoto, 2000; Kochian et al.,
2005). However, Al toxicity is not just a plant growth and
yield-limiting factor on acid soils, Al toxicity has also been
reported in alkaline soils amended with alkaline fly ash
(Jones, 1961; Rees and Sidrak, 1955) and bauxite residue
(Fuller and Richardson, 1986). Also, in agreement with these
observations Al rhizotoxicity has clearly been demonstrated
in hydroponic culture with pH values adjusted to >8.0 (Fuller
and Richardson, 1986; Kinraide, 1990; Eleftheriou et al.,
1993; Ma et al., 2003). The aluminate ion (AlðOHÞ
4 ) is the
dominant Al species in alkaline Al solutions (Martin, 1988).
But it is not yet clear whether the aluminate ion is the toxic
Al species leading to Al rhizotoxicity in the alkaline pH
range. Eleftheriou et al. (1993) observed aluminate-induced
changes in morphology and ultrastructure of the roots of
Thinopyrum junceum grown in nutrient solution at pH 10. In
a more recent study Ma et al. (2003) presented evidence that
wheat plants were significantly inhibited in growth when Al
was present at a concentration of about 1 mg l1 in soil
solutions with a pH greater than 9. In his study addressing
the rhizotoxicity of the aluminate ion, Kinraide (1990) hypothesized that aluminate is non-toxic and that the inhibition
of root elongation by Al is attributable to the formation of the
metastable polynuclear hydroxy-aluminium Al13 complex
postulated to have formed in the free space of the roots.
Kopittke et al. (2004) also concluded that Al toxicity at
alkaline pH is mainly due to the formation of Al13, but they
could only show that Al13 forms in the bulk solution and not
in the root apoplast. Both studies are in agreement with the
* To whom correspondence should be addressed. E-mail: horst@pflern.uni-hannover.de
Abbreviations: CW, cell-wall material; EWS, ethanol wash-solution; FDA, fluorescein diacetate; PE, pachyman equivalents; SS, symplastic sap.
ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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4034 Stass et al.
conclusions drawn by Poléo and Hytterod (2003) investigating the effect of Al on Atlantic salmon in alkaline water
that the toxicity of the aluminate ion is low, particularly
lower than the corresponding toxicity of cationic Al
hydroxides.
Although much progress has been made recently, the
mechanisms of Al-induced inhibition of root elongation and
Al resistance are still not well understood. (Taylor, 1991;
Delhaize and Ryan, 1995; Kochian, 1995; Matsumoto, 2000;
Kochian et al., 2005). In particular, the relative importance
of symplastic versus apoplastic lesions of Al toxicity remains a matter of debate. Rengel (1996), and especially
Horst (1995), focused attention on the role of the apoplast
in Al toxicity with regard to short-term inhibition of root
elongation. This hypothesis is supported by experimental
evidence: root Al injury can be modulated by the negative
charge of the cell walls (Schmohl et al., 2000; Schmohl and
Horst, 2000), apoplastic flow of high molecular solutes is
inhibited by Al (Schmohl and Horst, 2002; Sivaguru et al.,
2006), and cell walls are the main sites of Al accumulation
(Marienfeld et al., 2000). In addition, Al-induced callose
formation, which is a very sensitive response of root apices
to short-term Al treatment (Wissemeier et al., 1987; Horst
et al., 1997; Sivaguru et al., 1999), can best be explained
by an interaction of cationic Al species with the plasma
membrane.
In the present study the focus of the experiments was on
the difference between low-pH and high-pH solutions on
Al uptake and distribution in the root apices, and on shortterm Al rhizotoxicity as reflected by inhibition of root
elongation and induction of callose formation.
Materials and methods
Seeds of an Al-sensitive maize cultivar ‘Lixis’ and an Al-resistant
cultivar ‘ATP-Y’ were germinated between moist filter-paper rolls for
3 d in the dark. The uniform seedlings were transferred to plastic pots
containing 18 l of culture solution with 500 lM CaCl2 and 8 lM
H3BO3. One day after transplanting, the pH of the nutrient solution
was adjusted stepwise to the target pH within 5 h. The plants were
then exposed to 0 lM and 50 lM AlCl3 for 2–8 h. Since the simple
culture solution was not well buffered, the H+ released by plants
grown at pH 8 and above decreased the pH of the solution considerably. Therefore the pH was kept constant within 0.2 pH units by
adding 0.1 M KOH manually. This manual adjustment proved to be
more reliable and effective than the available pH-stat device which
could not cope with the dramatic and rapid pH changes. With the low
pH treatment, the plants tended to increase the pH slightly, which was
then corrected by the addition of 0.1 M HCl. In order to compensate
for the K+ input at the high pH treatments by KOH addition for pH
adjustment, the plants at low solution pH treatments were supplied
with equal amounts of K+ by adding 0.1 M KCl.
At harvest, 1 cm root tips from the primary root were excised for Al
determination or frozen immediately in liquid nitrogen for callose
determination. For the measurement of root elongation, main roots
were marked 3 cm behind the tip at the beginning of the treatment.
All experiments were conducted in a growth chamber under the
controlled environmental conditions of a 16/8 h day/night cycle,
30/27 C day/night temperature, 75% relative air humidity, and
a photon flux density of 230 lmol m2 s1 photosynthetic active
radiation at plant level.
For the fractionation of Al in the root apices, 20 freshly excised
root tips from 20 seedlings were incubated in 3 ml of 0.1 mM HCl or
in 0.1 mM NaOH for 30 min, then rinsed with 2 ml of the same acid
or base solution. After the incubation the root apices were frozen
at –20 C overnight. Symplastic sap (SS) was recovered from the
frozen–thawed samples by centrifugation at 3000 g for 15 min at
4 C. The pellet was transferred to 2 ml Eppendorf vials and 1 ml of
95% ethanol was added. The sample was then homogenized with
a mixer mill (MM200, Retsch, Haan, Germany) at a speed of 30/s
for 3 min. After centrifugation, the supernatant and pellet were
separated, the pellet was washed again with ethanol followed by a
second centrifugation. The two supernatants were combined to give
the ethanol wash solution (EWS). Cell sap and EWS together
represented the symplastic fraction and the pellet represented the
cell-wall material (CW).
Al was extracted from the CW on a Millipore filtration unit by
a sequential procedure using solutions of 0.10, 0.25, 0.50, 1.0, and 10
mM KOH, each for 10 min. After the KOH solution was acidified by
HNO3, Al contents in the KOH solution were determined by ICPOES (Spektro Analytical Instruments, Kleve, Germany).
For callose determination, three root-tips were homogenized in 1 ml
1 M NaOH at a speed of 20/s for 2 min with a mixer mill (MM200,
Retsch, Haan, Germany). Callose was extracted for 20 min at 80 C in
a water bath and quantified according to Köhle et al. (1985), using
aniline blue as the colour reagent, with a fluorescence spectrophotometer (Hitachi f2000, Hitachi, Tokyo; excitation 400 nm, emission
485 nm). Callose localization was performed using free-hand sections, stained for 5 min with an aniline blue solution consisting of
0.1% aniline blue in 1 M glycine at pH 9.5.
Prior to the determination of the Al content fresh root tips were
washed with 1 ml water of the same pH as the treatment solution.
They were then digested in 500 ll ultrapure HNO3 overnight on a
rotary shaker (Heidolph, Reax 20, Germany). To finish the digestion,
samples were incubated in a water bath at 80 C for 20 min. Aluminium was measured by GFAAS (Unicam 939 QZ, Analytical
Technologies Inc., Cambridge, UK), at k¼302.2 nm and an injection
volume of 20 ll.
For the determination of the viability of the root tip cells, five root
tips were washed with 500 lM CaCl2 and then incubated in 500 ll
FDA (0.02% Stock in acetone, 1:100 diluted with 500 lM CaCl2) for
10 min. The FDA solution was discarded and the root tips homogenized in 500 lM CaCl2 in a mixer mill (MM200, Retsch, Haan,
Germany). After centrifugation for 5 min at 15 000 rpm (Heraeus
Biofuge primo R, Kendro Laboratory Products GmbH, Langenselbold, Germany) the supernatant was measured fluorometrically with
a Microplate Fluorescence Reader Flx 800 (Bio-Tek Instuments Inc.,
Winooski, Vermont, USA), at excitation k¼400/30 nm, emission
k¼485/40 nm and sensitivity 50.
After harvest, the culture solutions were immediately filtered
through a nitrocellulose membrane with 0.025 lm pore size in order
to eliminate precipitated Al. The filtered solutions were acidfied to
prevent new Al from precipitating. Al13 which might form under our
experimental conditions is supposed to be stable under acid conditions (Furrer et al., 1992). Total Al in the filtrate was determined
with ICP-OES (Spektro Analytical Instruments, Kleve, Germany)
and mononuclear Al concentrations were measured colorimetrically
according to Kerven et al. (1989).
For the apoplastic pH measurements, 30 root tips from primary
roots were excised at 4 C. Excised root tips from each treatment were
washed in precooled (4 C) solution (Al-free) with the same solution
pH as the treatment. The apoplastic sap of the root tips was collected
by centrifugation, according to the method described by Yu et al.
Al toxicity at low and high solution pH 4035
(1999) with some modifications. Briefly, root tips were arranged in a
filter unit (Nanosep MF, 0.45 lm, PALL, Ann Arbor, USA) with the
cut ends facing down. The root surface solution and the wash solution
retained between adhering root tips were collected by centrifugation
at 600 g for 5 min at 4 C. Thereafter, the apoplastic sap was collected
by centrifugation at 3000 g for 15 min at 4 C. The pH in the
apoplastic sap was measured with a microelectrode (MI129, ISFETpH-Electrode, Mettler Toledo GmbH, Giessen, Germany).
Aluminium localization in root cross-sections was performed by
morin staining. After 2 h Al treatment, root tips from each treatment
were excised and washed in Al-free solution with the same pH as the
treatment. Free-hand sections from the 1–5 mm zone behind the root
apex were stained with 25 lM morin pH 5.6 for 5 min at room
temperature. The sections were observed under a fluorescence
microscope with a filter set consisting of an excitation filter (BP
395–440) and a barrier filter (LP 470). Images were collected using
an AxioCam MRc (Carl Zeiss AG, Göttingen, Germany).
Statistical analysis was carried out using SAS version 8.1 (SAS,
2001).
Results
The determination of total and mononuclear Al in the
culture solution at different pH showed that at pH 4.3 and
pH 10 nearly all the nominally supplied Al was soluble and
this soluble Al was present as mononuclear Al. At a solution
pH of 8 nearly 60% of the Al supplied was precipitated, but
the Al which remained in solution could be determined as
mononuclear Al (Fig. 1)
At pH 10 and pH 9 (not shown) root growth of the control
plants (–Al) was inhibited compared with pH 4.3 after 8 h
of treatment. Plants grown at pH 4.3 and 8 showed the same
growth rate under control conditions, whereas root growth
was severely inhibited at pH 10 (Fig. 2). Al inhibited root
growth to a comparable degree by 37% and 54% at pH 4.3
and pH 8, respectively, although less mononuclear Al was
found in solution at pH 8. At pH 10, root growth was also
significantly, but less, affected by Al. This was mainly due
to the root growth depression of the controls at this elevated
pH level. All further experiments were, therefore, only
performed at pH 4.3 and pH 8.
Callose formation in the root tips was determined as
a sensitive indicator of Al injury in roots and of genotypic
differences in Al resistance (Fig. 3A). In root apices of
plants exposed to Al at pH 4.3 for 8 h, a significant increase
in callose content was found, particularly in the Al-sensitive
maize cultivar Lixis. At pH 8, callose formation was only
slightly enhanced with no differences between the cultivars.
This could have been due to an inhibition of callose synthase at high pH. However, callose could be induced by
digitonin, a known inducer of callose synthesis (Waldmann
et al., 1988), at pH 4.3 as well as at pH 8 (Fig. 4).
After 8 h of Al treatment, Al contents of the root apices of
plants grown in solution at pH 8 were much higher than at
pH 4.3 (Fig. 3B).This difference could be found when the
plants were treated with a nominal supply of 50 lM Al, and
was even more pronounced when plants were treated with
Fig. 1. Effect of solution pH on total and mononuclear Al concentrations
in the culture solution containing initially 500 lM CaCl2, 8 lM H3BO3,
and 50 lM AlCl3. Bars represent means 6SD, n¼3. No significant
differences between total soluble and mononuclear Al concentrations
were found (t test).
Fig. 2. Effect of Al on root elongation after 8 h treatment with 50 lM Al
at different solution pH. Bars represent means 6SD, n¼12. Columns
with different letters are significantly different, P <0.05 (Tukey test). ***
is significant at a¼0.001 (F test).
the same concentration of mononuclear Al (25 lM), which
was achieved with a nominal Al supply of 50 lM at pH 8
and 25 lM at pH 4.3.
In agreement with the Al-induced callose formation, Al
contents in the root apices of the Al-sensitive cultivar Lixis
were much higher than in ATP-Y at pH 4.3. This difference
disappeared at pH 8. The lack of genotypic differences in
Al resistance, reflected in Al-induced callose formation and
Al accumulation in root apices at pH 8, suggest a different
mechanism of Al rhizotoxicity in maize when grown under
acidic and alkaline conditions.
In order to characterize the binding stage of Al in the
root apices of plants grown at high compared with low-pH
4036 Stass et al.
Fig. 4. Effect of solution pH on digitonin-induced callose formation.
Plants were incubated for 2 h with 10 lM digitonin. Bars represent means
6SD, n¼3. Columns with different letters are significantly different,
P <0.05 (Tukey test). *** is significant at a¼0.001 (F test), ns¼nonsignificant.
Fig. 3. Effect of Al on callose (A) and Al contents (B) of the Al-sensitive
cultivar Lixis and the Al-resistant cultivar ATP-Y after 8 h treatment with
Al at different solution pH. Bars represent means 6SD, n¼3. Columns
with different letters denote significant differences between genotypes, P
<0.05 (Tukey test). *, ** and *** are significant at a¼0.05, 0.01, and
0.001, respectively (F test) for the 50 lM Al treatment.
solution, the root tips were submitted to a fractionated
extraction procedure. An initial washing step in acid or base
aimed at differentiating between ionically bound Al and
Al(OH)3 precipitates in the root apoplast. However, there
was no significant difference in Al distribution between
the root tips, which were acid or base washed with these
low concentrated acid and base solutions. Therefore,
the results were pooled for the presentation in Fig. 5. All
Al fractions reflected the difference in total Al content
between the plants treated with Al at acidic and alkaline pH:
the Al contents were higher at pH 8 (Fig. 5A). The largest
fraction of the root Al was recovered in the cell-wall fraction which represented 78% and 83% at pH 4.3 and pH 8,
respectively (Fig. 5B). When comparing plants treated with
Al at alkaline and acid pH, the statistical analysis of the
relative distribution of Al between different fractions revealed that, at pH 8 compared with pH 4.3, a significantly
lower percentage of Al was recovered in the wash solution
(9.161.0 versus 13.161.9) but a higher percentage in the
symplastic fraction (9.460.2 versus 6.760.8).
In order to characterize the nature of the main Al fraction
better, the cell-wall fraction, in the root tips grown at pH 4.3
and pH 8, isolated cell walls were subjected to a sequential
extraction procedure with increasing concentrations of KOH
(Fig. 6). Hardly any cell-wall Al could be solubilized by the
lowest KOH concentration of 0.10 mM, independently of
the pH (Fig. 6). The solubility of cell-wall Al was enhanced
with increasing KOH concentrations up to 10 mM for the
pH 8 treatment. However, the amount of hydroxyl ions
provided even with the highest KOH concentration (10 mM)
was not sufficient to dissolve all cell-wall Al. The amounts
of Al released from the cell walls of plants grown at pH 4.3
increased only up to 0.5 mM KOH and then remained
constant. The results indicate that, at pH 8, the majority of
cell-wall Al could be Al(OH)3, which readily dissolves in
higher concentrated KOH solutions.
Al speciation in aqueous solution is closely related to the
solution pH. Since Al is primarily localized in the root
apoplast of the root tips, the root apoplastic pH could be
a crucial factor in controlling the dominant Al species in the
root apoplast and thus at the outer face of the plasma membrane. The pH of the apoplastic sap of the root tips was
measured as affected by pH and Al treatments (Table 1).
In general, the apoplastic sap was acidic, independently of
the pH of the bulk solution. Under control conditions the
apoplastic pH was about 5.9 for both treatments. In roots
Al toxicity at low and high solution pH 4037
Fig. 6. Aluminium extractable from cell walls isolated from root apices
of maize. Cell walls of 10 root tips were sequentially extracted for 10 min
each with 4 ml of 0.10, 0.25, 0.5, 1.0, and 10 mM KOH. Plants were
cultured for 8 h at 50 lM AlCl3 at different solution pH. Bars represent
means 6SD, n¼3. Columns with different letters are significantly
different between pH treatments, P <0.05 (Tukey test). *** is significant
at a¼ 0.001 (F test), ns¼non-significant.
Table 1. Apoplastic pH (6SD, n¼3) of the root tips of maize as
affected by Al supply in culture solution kept constant at pH 4.3
and pH 8
pH-adapted plants were exposed to 0 or 50 lM AlCl3 for 2 h.
Fig. 5. Aluminium contents of different cellular fractions (A) and
percentage distribution of Al among the fractions (B) of root apices.
Plants were cultured for 8 h at 50 lM Al at different solution pH. Excised
root tips were washed with 0.1 mM HCl or 0.1 mM NaOH prior to the
subsequent fractionation procedure. Data were pooled because statistical
analysis did not reveal a difference between acid or base washingsolutions (WS). Bars represent means 6SD, n¼6. Columns with different
letters are significantly different within fractions, P <0.05 (Tukey test). *,
** and *** are significant at a¼0.05, 0.01, and 0.001, respectively (F
test), ns¼non-significant.
treated with Al the apoplastic pH was closer to the bulk
solution pH than under control conditions, even though the
plants were able to shift the pH considerably. This could
either be due to the buffering capacity of Al or the impairment of metabolic processes by Al. The difference between
bulk-solution and apoplastic pH needs to be considered in
the understanding of Al rhizotoxicity at high, but also at
low, bulk solution pH.
Aluminium in the root tips was localized using morin as
a stain for Al. Morin cannot stain Al bound to the cell wall
(Eticha et al., 2005a), but freshly precipitated, complexed
and soluble Al in the apoplast is stained, even at very low
concentrations.
Culture solution pH
Al supply (lM)
Apoplastic pH
4.3
0
50
0
50
5.9460.23
5.6460.17
5.9660.12
6.1860.13
8
The pattern of radial Al distribution differed between the
pH treatments as well as between root-tip zones (Fig. 7).
After 2 h Al treatment at pH 4.3, cell walls of the epidermis
and the first few cell layers of the cortex of the first 1–3 mm
of the root apex were fluorescent (Fig. 7A, B). After 8 h Al
treatment the whole cortex was fluorescent (data not shown),
while the central cylinder remained unstained, indicating
that the endodermis with its casparian strip represents an
effective barrier for radial Al movement. At pH 8 epidermal
and outer cortical cells were intensively fluorescent (Fig.
7D), within the cortex a blotchy distribution of Al could be
found (Fig. 7E), this Al seems to be located in the symplast.
At pH 4.3, in the elongation zone of the root tip (3–5 mm
behind the tip) large bright spots were dispersed over the
outer and middle cortex. This reflects the collapse of cell
clusters, probably due to mechanical stress imposed by
impaired cell elongation. The collapse of cell clusters at pH
4.3 is also reflected in the determination of the viability of
the root-tip cells. At pH 4.3 the viability of the root tips was
slightly lower than at pH 8 (Fig. 8).
4038 Stass et al.
Fig. 7. Localization of Al by staining with morin in cross-sections of maize root apices 1–3 mm (A, B, D, E) or 3–5 mm (C) behind the toot tip. pHadapted plants were exposed to 50 lM AlCl3 at pH 4.3 (A, B, C) or pH 8.0 (D, E) for 2 h. Scale bars¼100 lm.
Fig. 8. Effect of aluminium on the viability of root apices of maize
cultivated at different solution pH with 0 or 50 lM AlCl3 for 2 h or 8 h.
Bars represent means 6SD, n¼3. Columns with different letters are
significantly different within pH treatments, P <0.05 (Tukey test). +, *, **
are significant at a¼0.1, 0.05, and 0.01, respectively (F test).
Discussion
At nominal Al supplies of 50 lM, Al was equally toxic to
maize plants in both acid and alkaline solutions based on
inhibition of root elongation (Fig. 2). However, considering
higher Al contents in the root tips of plants grown in highpH solutions (Fig. 3B) the Al accumulated in the roots at
high solution pH appears to be less toxic than the Al accumulated at low pH. This is also indicated by results presented by Zavas et al. (1991), who studied the differential
response of two populations of Avena sterilis L. to Al
toxicity at pH 4.5 and 10.
There was a good correlation between mononuclear Al
concentration in the culture solution and root-growth
reduction of the maize plants in low-pH solutions (Blamey
et al., 1992). However, in our high-pH solutions, rootgrowth reduction could not be explained by mononuclear
Al concentration in the solution, especially at pH 8. Apart
from mononuclear inorganic Al-species, the formation of
polymeric Al species in the apoplast is possible. Among the
polymeric Al-species the formation of the metastable polynuclear hydroxy-aluminium Al13 complex is of special importance. It is supposed to be more toxic than Al3+ (Parker
et al., 1989; Kinraide, 1997). In order to maintain the
solution pH constant, addition of acid or base to the culture
solution is necessary because plants grown under low pH
and high pH conditions try to increase or decrease the
rhizosphere pH to optimal pH. Increasing the pH by base
injection may result in Al13 formation. In studying the
formation of Al13, Bertsch (1987) identified OH/Al ratio,
total Al concentration, base injection rate, and stirring rate
as important factors. He predicted that the high pH at the
point of base injection resulted in the significant formation
of the aluminate ion, which forms the central core of the
Al13 polymer. Kopittke et al. (2004) found enhanced Al13
formation in the bulk solution kept at pH 9.5 only after 3 d.
Al toxicity at low and high solution pH 4039
At the higher Al concentration of nominal 25 lM, Al13
could be measured even within the first day of treatment,
50 lM Al was supplied in the experiments presented here.
Due to the acidifying effect of the plant roots and the
necessary addition of KOH for maintaining the pH at 8.0,
precipitation and also polymerization of mononuclear Al
[AlðOHÞ
4 ] is very likely. Determination of the different
Al species in the nutrient solution (Fig. 1) showed that,
particularly at pH 8, more than 50% of the Al precipitated.
The soluble Al could be determined at all pH values tested
as mononuclear Al. This still does not exclude the formation of Al13, because Al13 can be toxic in extremely low
concentrations, which are even below the limit of detection
(Kopittke et al., 2004). Therefore, in agreement with the
conclusions by Kinraide (1990) and Kopittke et al. (2004),
the inhibition of root elongation at solution pH 8 might be
caused by Al13 in the experiments reported here also.
Whereas Kinraide (1990) attributed a major role to the
formation of Al13 in the root free space, Kopittke et al.
(2004) concluded from their work that Al13 formation in
the bulk solution and not in the apoplast was responsible
for the toxic effect at high pH. Al13 concentrations in the
bulk solution increased only after several days of cultivation and a transfer into fresh nutrient solution led to a
disappearance of the effects. Also the results presented here
do not support the formation of Al13 in the apoplast. As the
apoplastic pH in the root cortex at different bulk-solution
pH were similar (Table 1) the formation of Al13, can be
expected under both pH treatments. For the treatment at pH
4.3 this would mean that the presence of Al3+ and Al13,
should lead to a strong effect on root growth. At pH 8, the
Al content in the root tips was higher (Fig. 3B), but this Al
was mostly precipitated Al(OH)3 (Fig. 6). Therefore,
a smaller amount of Al was available for Al13 formation
in the cortex. In conclusion, if Al13 forms in the apoplast,
the formation should be stronger at pH 4.3 than at pH 8.
Since the same degree of root-growth inhibition at both pH
treatments (Fig. 2) was found, formation of Al13 in the
apoplast is unlikely to be the reason for the results presented here.
Although the formation of Al13 in the bulk solution and
particularly in the root apoplast cannot be ruled out, further
data are presented, which suggest a different mechanism of
Al toxicity involved under alkaline conditions: (i) the plants
were highly effective in maintaining the apoplastic pH in
the root apex independently of the external pH, (ii) at pH 8,
Al accumulated strongly in the root apices, (iii) the pattern
of Al distribution in the root apex and the effect of Al on the
structural integrity of the root tissue differed between the
pH regimes, (iv) Al toxicity at pH 8 was accompanied by
little callose formation, in contrast to pH 4.3 where callose
formation has been shown to be a sensitive indicator of Al
sensitivity in maize (Horst et al., 1997; Eticha et al.,
2005b), and (v) genotypical differences in Al resistance
disappeared at pH 8.
Large amounts of base were necessary to maintain a high
bulk-solution pH indicating that the activity of the roots led
to a release of protons into the bulk solution. From the
amount of base a proton-efflux rate of 347 nmol plant1
min1 was calculated. Whereas in the bulk solution the pH
was kept constant by the addition of KOH, a pH decrease at
the root surface, and even more in the root apoplast, can be
expected (Moore, 1999). Generally, H+ and OH– (or HCO
3)
released from the root would be rapidly neutralized in the
bulk solution. Depending on the release rate, the buffer
power of the bulk solution, and the rate of solution agitation,
a pH gradient between bulk solution, root surface,and root
apoplast can be substantial, even in hydroponic culture
(Cleland, 1976; Jacobs and Ray, 1976; Pilet et al., 1983;
Shabala et al., 1997; Felle, 1998; Kosegarten et al., 1999; Yu
et al., 2001).
Using microelectrodes Felle (1998) demonstrated that the
apoplastic pH of the root tip of maize was maintained
between 5.1–5.6 at different medium pH. An increase of
the bulk-solution pH from 4.3 to a root-surface pH 5.3 has
also been shown with the same maize cultivar used in this
study, cv. Lixis, by Kollmeier et al. (2000). Similar results were obtained by Kosegarten et al. (1999) using the
pH-dependent fluorescence ratio of fluorescein boronic
acid. Also, the modelling of the apoplastic pH below the
epidermis (external cortex) in wheat root apices revealed
a pH increase from the bulk solution pH of 4.3 to 5.83
(Kinraide et al., 2005).
These results are in agreement with measurements of the
pH in the apoplastic sap, recovered from the root tips by
centrifugation (Table 1). They clearly show that the plants
have been able strongly to decrease the pH in the apoplast,
in spite of rigorous control of the bulk-solution pH at 8.0.
Although the root apices after cutting have been kept at low
temperature during the recovery of the apoplastic sap, it
cannot be excluded that transport processes at the plasma
membrane could not be stopped immediately. Therefore,
the measured apoplastic pH in plants grown at pH 8.0 or at
pH 4.3 is probably slightly lower or higher, respectively,
than in vivo. It can be assumed that the severe root-growth
inhibition at pH 10, even of the control plants not treated
with Al, is due to the inability of the plants to acidify the
root tip apoplast to the acidic pH which is necessary for
cell elongation (Rayle and Cleland, 1992; Cosgrove, 1998)
and to avoid an increase in the cytosolic pH above the
optimum (Gerendás and Ratcliffe, 2000).
Based on the above discussion, a hypothesis explaining
possible reactions involved in Al toxicity in low and highpH solutions, is elaborated next. Since the Al speciation in
solution is pH-dependent, the pH gradient between the
medium and the root apoplast will influence the Al speciation. Under conditions of the solution pH of 4.3, Al3+
is the predominant Al species in the epidermal cells and
the first few cell layers of the cortex. Within the cortex
the plants maintain an apoplastic pH above 5 and, therefore,
4040 Stass et al.
Al-hydroxy species such as [Al(OH) 3 , AlðOHÞþ
2,
Al(OH)2+] will dominate. The release of Al from the cellwall material of plants cultivated at pH 4.3 by KOH (Fig. 6)
appears to reflect the release of Al from negative binding
sites through comparatively high K+ concentrations (Grauer
and Horst, 1992) and partial solubilization of cell-wall
pectins rather than the solubilization of Al(OH)3. As polycations are known to induce the induction of callose (Kauss
and Jeblick, 1985; Kauss et al., 1989), Al3+ as a trivalent
cation is possibly responsible for callose formation in the
epidermis and outer cell layers of the cortex (Fig. 9). In the
inner cortical cell layers the Al3+ concentration is rapidly
decreasing, and the dominating Al-hydroxy species, with
positive charges of 2–0 are not able to induce callose
formation. However, they are less firmly bound and thus
can be more easily stained by morin (Fig. 7) At these lower
Al concentrations the formation of Al13 is regarded to be
negligable (Bertsch, 1987).
At the high bulk-solution pH, the pH changes from pH 8 to
around pH 6 in the apoplast and protonation of AlðOHÞ
4
takes place, resulting particularly in the formation and
precipitation of Al(OH)3. The visualization of Al in crosssections of the roots with morin (Fig. 7) shows a strong Al
accumulation in the epidermis at pH 8, which is most likely
Al(OH)3, an Al species which can be stained with morin
(Eticha et al., 2005a). The precipitation of Al(OH)3 in the
epidermis is also corroborated by the fractionated extraction
of the cell walls (Fig. 6), where Al is solubilized particularly
at higher KOH supplies which readily solubilize freshly
precipitated Al(OH)3 as shown by parallel batch experiments
(not shown). The rapid precipitation of Al in the epidermis
suggests a pH of 6.3 where Al is least soluble. In the cortex,
the pH will be lower and, therefore, the Al-species distribution should be similar to that found at a bulk-solution pH
of 4.3. However, the formation of Al3+ is unlikely at the
measured apoplastic pH (Table 1).
Callose, as an indicator of Al stress, is induced by Al
exclusively in the epidermis at pH 8 (Fig. 9). This could be
due to the formation of the polycationic Al13 polymer at the
root surface where its formation is most likely because of
the pH gradient and the high solution Al concentration. In
line with Kinraide et al. (2005) who, based on theoretical
considerations as well as experimental evidence, came to
the conclusion that, at low pH, the cortex and not the
epidermis is the Al-responsive tissue, the callose formed in
the first few layers of the cortex at pH 4.3 (Fig. 9) is most
likely due to the stress imposed by Al3+. This area of maximum callose formation and, therefore, maximum Al stress
progesses further into the cortex with time (data not shown).
This is probably due to the inability of the cells to keep up
the high metabolic load of increasing the pH. The same can
be assumed for the roots treated at high pH. The area of
precipitated Al and callose formation will probably move
further into the cortex with time.
The precipitation of Al(OH)3 at bulk-solution pH 8 may
be regarded as a detoxification mechanism of rhizotoxic
mononuclear Al species. However, the precipitate may also
act as a physical block to the transport of nutrients and
other solutes into the apoplast necessary for root growth.
Ranathunge et al. (2005) investigating apoplastic bypass
flow in rice showed that precipitates of inorganic salts can
block the apoplasic pores in the root tips. It is suggested that
precipitates of Al(OH)3 in the epidermis, particularly in the
DTZ and EZ (7), inhibit cell-wall extension and thus root
growth. This proposition is supported by the role of the
epidermis in the control of growth (Hohl and Schopfer,
1992; Peters and Tomos, 2000). Even though the relevant
experiments have been performed with hypocotyls, it is assumed that growth in roots is controlled in the same way
(Swarup et al., 2005). A different mechanism of Al rhizotoxicity in maize when grown under alkaline conditions is
also suggested by the lack of well-documented genotypic
differences in Al resistance at low pH when grown at high
pH (Kollmeier et al., 2000; Collet et al., 2002; Fig. 3). It
appears that at pH 8.0 Al does not induce the release
of organic acid anions, or these are not effective in detoxifying Al.
In conclusion, it is proposed that root-growth inhibition
by Al at low and high solution pH is due to different
mechanisms. At pH 4.3 Al3+ inhibits growth through
Fig. 9. Localization of callose by staining with aniline blue in cross-sections of maize root apices 1–3 mm behind the toot tip. Plants were exposed to
50 lM AlCl3 at pH 4.3 or 8.0 for 2 h. Scale bars¼100 lm.
Al toxicity at low and high solution pH 4041
binding to the pectic matrix thus reducing the porosity of
the apoplast leading to a lower mobility of macromolecules
involved in cell-wall synthesis and signalling necessary for
root growth. At pH 8 the strong precipitation of Al(OH)3
causes a mechanical barrier in the epidermis thus impairing
the growth control of the epidermis, without changing the
potential for growth generated by the cortex.
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