Tree Physiology 24, 1267–1277
© 2004 Heron Publishing—Victoria, Canada
Responses of eucalypt species to aluminum: the possible involvement of
low molecular weight organic acids in the Al tolerance mechanism
I. R. SILVA,1,2 R. F. NOVAIS,1 G. N. JHAM,3 N. F. BARROS,1 F. O. GEBRIM,1 F. N. NUNES,1
J. C. L. NEVES1 and F. P. LEITE 4
1
Soil Science Department, Federal University of Viçosa, 36571-000 Viçosa, MG, Brazil
2
Corresponding author ([email protected])
3
Chemistry Department, Federal University of Viçosa, 36571-000 Viçosa, MG, Brazil
4
CENIBRA, Celulose Nippo-Brasileira S. A., Belo Oriente, MG, Brazil
Received September 8, 2003; accepted February 15, 2004; published online September 1, 2004
Summary Aluminum (Al) tolerance mechanisms in crop
plants have been extensively researched, but our understanding
of the physiological mechanisms underlying Al tolerance in
trees is still limited. To investigate Al tolerance in eucalypts,
seedlings of six species (Eucalyptus globulus Labill., Eucalyptus urophylla S.T. Blake, Eucalyptus dunnii Maiden, Eucalyptus saligna Sm., Eucalyptus cloeziana F. J. Muell. and Eucalyptus grandis w. Hill ex Maiden) and seedlings of six clones of
Eucalyptus species were grown for 10 days in nutrient solutions containing Al concentrations varying from 0 to 2.5 µM (0
to 648 µM Al3+ activities). Root elongation of most species was
inhibited only by high Al3+ activities. Low to intermediate Al3+
activities were beneficial to root elongation of all species and
clones. Among the species tested, E. globulus and E. urophylla
were more tolerant to Al toxicity, whereas E. grandis and
E. cloeziana were more susceptible to Al-induced damage. Although E. globulus seedlings were tolerant to Al toxicity, they
were highly sensitive to lanthanum (La), indicating that the tolerance mechanism is specific for Al. Fine roots accumulated
more Al and their elongation was inhibited more than that of
thick roots. In E. globulus, accumulation of Al in root tips increased linearly with increasing Al concentration in the nutrient solution. The majority of Al taken up was retained in the
root system, and the small amounts of Al translocated to the
shoot system were found mainly in older leaves. No more than
60% of the Al in the thick root tip was in an exchangeable form
in the apoplast that could be removed by sequential citrate
rinses. Gas chromatography/mass spectrometry and ion chromatography analyses indicated that root exposure to Al led to a
greater than 200% increase in malic acid concentration in the
root tips of all eucalypt species. The increase in malate concentration in response to Al treatment correlated with the degree of
Al tolerance of the species. A small increase in citric acid concentration was also observed in all species, but there were no
consistent changes in the concentrations of other organic acids
in response to Al treatment. In all eucalypt species, Al treatment induced the secretion of citric and malic acid in root
exudates, but no trend with respect to Al tolerance was ob-
served. Thus, although malate and citrate exudation by roots
may partially account for the overall high Al tolerance of these
eucalypt species, it appears that tolerance is mainly derived
from the internal detoxification of Al by complexation with
malic acid.
Keywords: aluminum resistance, aluminum toxicity, lanthanum, plantation trees, root exudates.
Introduction
About 30% of the world’s soils are acid and it is estimated that
humid tropical regions account for about 60% of those acid
soils. Aluminum (Al) toxicity is a major limiting factor for
crop production in acid soils. This is particularly important in
Brazil, where more than 50% of the soils are acid, creating the
potential for Al toxicity in surface and subsurface layers (von
Uexküll and Mutert 1995, Eswaran et al. 1997). High Al concentrations ([Al]) usually inhibit root growth, resulting in inefficient uptake of water and nutrients.
Plant species and genotypes within species differ in their
ability to tolerate Al, and there are several hypotheses to explain this tolerance (Taylor 1991, Foy 1992, Horst 1995, Rengel 1997, Ma et al. 2001, Ryan et al. 2001). Generally, tolerance mechanisms are separated into two broad classes (Taylor
1991, Kochian 1995): (1) prevention of Al uptake by roots;
and (2) internal detoxification of absorbed Al. One of the most
widely accepted mechanisms of Al tolerance involves the production and exudation of chelating substances, mainly low
molecular weight organic acids (OA) that form stable nontoxic complexes with Al, thereby detoxifying Al in the rhizosphere and plant cells (Delhaize and Ryan 1995, Kochian
1995, Jones 1998, Ma 2000, Ma et al. 2001, Ryan et al. 2001,
Silva et al. 2001, Kochian et al. 2002).
Although research on Al tolerance mechanisms has focused
on agricultural crops, it is known that some forest species are
tolerant to Al toxicity, and that the tolerance is usually greater
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SILVA ET AL.
than that of annual crops (Schaedle et al. 1989, Vale et al.
1996). Eucalypt species are known to be Al tolerant (Neves et
al. 1982a) and some may even benefit from the presence of Al
in the growth medium (Vale et al. 1984, Huang and Bachelard
1993). The ability to cope with high [Al] allows eucalypts to
thrive in acid soils containing Al, with little or no response to
liming (Neves et al. 1982b, Novais et al. 1990, Barros and
Novais 1996, Vale et al. 1996).
Despite efforts to elucidate possible mechanisms of Al tolerance in eucalypts, its physiological basis remains elusive.
The high Al tolerance of eucalypts is not related to the cation
exchange capacity (CEC) of the roots (Vale et al. 1996) or to
the ability of roots to induce changes in soil solution pH that
could decrease Al toxicity (Neves et al. 1982a, Vale et al.
1996). In other tree species, there is evidence that the concentration of low molecular weight OA in tissues and root exudation may be important in determining Al tolerance (Fujii 1997,
Ma et al. 1997b, Osawa et al. 1997). It has been suggested that
root secretion, and possibly root and shoot tissue concentrations of citric acid are associated with the capacity of Acacia
mangium Willd. to tolerate high [Al] (Osawa et al. 1997).
Complexation of Al with citric acid may protect against Al
damage, allowing plants to grow well in acid soils in the presence of high concentrations of exchangeable Al (Neves et al.
1982b, Vale et al. 1996).
The aim of this study was to evaluate the responses of several eucalypt species to Al in nutrient solution and to investigate whether Al tolerance is related to root tissue concentration and exudation of low molecular weight OA.
Materials and methods
Plant growth
Seeds of six eucalypt species (Eucalyptus globulus Labill., Eucalyptus urophylla S.T. Blake, Eucalyptus dunnii Maiden, Eucalyptus saligna Sm., Eucalyptus cloeziana F. J. Muell. and
Eucalyptus grandis Hill ex Maiden) were germinated on
washed sand in a greenhouse. The seedlings were grown on the
sand medium for about 4 weeks after emergence and were watered daily with a half-strength, complete nutrient solution as
described by Clark (1975), with minor modifications (hereafter modified Clark nutrient solution). The solution composition was: 1.26 mM Ca(NO3)2·4H2O, 0.65 mM KNO3,
0.25 mM KCl, 0.45 mM NH 4 NO3, 0.3 mM MgSO4·7H2O,
17.7 µM Ca(H2PO4)2·H2O, 3.5 µM MnCl2·4H2O, 9.5 µM
H3BO3, 1 µM ZnSO4·7H2O, 0.043 µM (NH4)6·Mo7O24·4H2O,
0.25 µM CuSO4·5H2O and 20 µM Fe-EDTA. Following the
4-week period in sand, seedlings were carefully removed,
thoroughly washed with deionized water and two to four
plants per replicate transferred to plastic trays filled with 10 l
of modified Clark nutrient solution. To avoid complexation of
Al by EDTA, Fe-EDTA was replaced with Fe(SO4)2. Nutrient
solutions were continuously aerated. The pH was adjusted
daily to 5.5 ± 0.3 through additions of 0.1 M HCl or 0.1 M
KOH. After 2 weeks in modified Clark nutrient solution under
greenhouse conditions, seedlings were transferred to similar
plastic trays in a growth chamber. Lighting was provided by
fluorescent lamps at a photon flux of 250 µmol m –2 s –1 for a
16-h photoperiod. Mean day/night temperature was 26 ± 2/19
± 1 °C. Seedlings were cultivated in modified Clark nutrient
solution, and the pH was gradually adjusted to 4.0 ± 0.1. After
3 to 4 days of acclimation, nutrient solutions were replaced
and the Al treatments were imposed.
In some experiments, seedlings of six commercial plantation clones of Eucalyptus spp. were used (Clones 129 and
7074 = E. grandis; Clone 1215 = E. grandis × E. urophylla hybrid; Clones 111, 57 and 965 are natural hybrids from Rio
Claro, Brazil, originated from open pollination and with unknown progenitors). Clonal plants were obtained by the minicutting technique. When the clonal plants were about 3 weeks
old, they were acclimated and subjected to Al treatments as described for seedlings.
Aluminum treatments
After acclimation in the growth chamber, primary roots were
measured and the Al treatments initiated. Root length was
remeasured 10 days after imposing the Al treatments. To compare root elongation among different eucalypt species, elongation (cm 10 days –1) of roots in Al-containing solutions was expressed as a percentage of that of roots growing in control (0
Al3+) solutions. The Al3+ activity in solution was estimated
with Geochem-PC software (Parker et al. 1995). In dose response experiments, Al3+ activities in solution were: 0, 63,
130, 256, 465 and 648 µM. Aluminum was added with vigorous stirring from a 1 M AlCl3 stock, freshly prepared on the
day of use. In treatment solutions that required upward pH adjustment, base was added slowly with continuous stirring of
the solution to avoid Al precipitation or polymerization. In the
experiment involving lanthanum (La), the estimated La3+ activities were 0, 155, 288, 501 and 682 µM.
Similar experiments were carried out to evaluate the differential response of root classes to Al, Al accumulation and distribution in the shoot and root systems, and the Al effects on
OA concentration and exudation by roots. All experiments had
a complete randomized design, with three to four replicates.
The root growth response to the Al doses experiment involving
six eucalypt species was carried out twice.
Analysis of total Al in whole roots and shoot
Seedlings were removed from the trays, and roots and shoot
were separated. Roots were rinsed three times in deionized
water and blotted on paper towel. The plant material was dried
at 75 °C to constant mass, ground in a Willey mill and wet-digested with concentrated nitric acid and perchloric acid. Aluminum in the acid extracts was identified by inductively
coupled plasma–atomic emission spectrometry (ICP–AES).
In some experiments, roots were separated into thin (< 1 mm
mean diameter) and thick (> 1 mm mean diameter) classes.
These roots were prepared and analyzed for Al as described
for the whole roots.
Analysis of total Al in root tips and leaves
After 10 days of growth in the presence of 0 or 648 µM Al3+,
about 30 thick root apices (about 5 mm in length) were excised
and immediately rinsed in ultra-pure water (Milli-Q) for
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ALUMINUM TOLERANCE IN EUCALYPTS
30 min to remove Al loosely bound to the root tip surface. Root
apices were then transferred to pre-weighed polypropylene
microcentrifuge tubes and weighed (± 0.1 mg). This material
was digested at room temperature with 500 µl of concentrated
HNO3 for 5 days, diluted to 1.5 ml with ultra-pure water,
passed through a 0.45-µm membrane filter and analyzed for Al
by ICP–AES. The same procedure was used to digest and analyze different root segments (0–5 and 5–30 mm) and young
and old leaves for Al. Leaves not fully opened that emerged
during the Al treatments were designated as young, whereas
leaves that fully opened at the beginning of the Al treatments
were designated as old.
Desorption of root tip Al
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previously. After 6 h, the solutions containing the root exudates were removed, lyophilized, reconstituted in 1 ml of ultra-pure water, passed through On Guard-Ag cartridges
(Dionex Corp., Sunnyvale, CA) to remove excess chloride,
and analyzed for OA by ion chromatography (Silva et al.
2001). Ion chromatography was used to analyze OA because
GC–MS required concentration of the solutions, which interfered with the derivatization of the samples, possibly because
of excess chloride (Kidd et al. 2001). Organic acids were identified based on retention times of known standards.
Results
Root growth response to solution Al
Ten days after exposure to 465 µM Al3+, E. globulus seedlings
were removed from trays, root tips (about 5 mm) were excised
and transferred to tissue processing wells (Electron Microscopy Science, Fort Washington, PA). The root tips were then
sequentially desorbed with ice-cold citrate in ultra-pure water
(0, 10, 50, 100 and 300 µM, pH 4.5) for 0–3, 3–6 or 6–9 h in a
horizontal rotary shaker (75 rpm). Root tips and the citrate
wash were analyzed for Al as described previously.
Analysis of organic acids in root apices
Following a 10-day exposure to solutions containing 0 or
465 µM Al3+, thick root apices (about 5-mm) from the six
eucalypt species were excised and immediately weighed. The
root tips were then macerated in 500 µl of ultra-pure water in a
mini-polytron and centrifuged at 16,000 g for 5 min. Supernatant was passed through a 0.45-µm filter and dried under
vacuum in a speed-vac sample concentrator. The dry residue
containing the water-soluble OA was derivatized with a mixture of 50 µl of BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide, Sigma, St. Louis, MO) and 150 µl of pyridine at 50 °C
for 30 min. Derivatized samples were passed again through a
0.45-µm filter and analyzed in a gas chromatograph (GC) with
detection of tetramethylsilane (TMS) derivatives by mass
spectrometry (MS). The GC–MS data were obtained with a
Shimadzu QP-5000 system (Shimadzu Scientific Instruments,
Columbia, MD) fitted with an auto sampler and a computerized data acquisition system. Capillary columns (30 m ×
0.25 mm; film thickness of 0.25 µm; DB-1) were purchased
from J&W Scientific (Palo Alto, CA). The GC oven temperature was increased from 80 to 250 °C at 10 °C min –1. Helium
was the carrier gas and electron ionization mass spectra
(70 eV) were recorded by scanning m/z values from 40 to 450.
For all analyses, the transfer line temperature was maintained
at 250 °C. The OA were identified based on retention times
and mass spectra.
Collection and analysis of organic acids in root exudates
Eucalypt seedlings grown for 10 days in the growth chamber
in solutions containing 0 or 465 µM Al3+ were transferred to
25-ml centrifuge tubes containing 500 µM CaCl2 (pH 4.0). After 30 min, the CaCl2 solution was replaced with solution lacking Al (control) or containing 465 µM Al3+. Seedlings were
kept in the growth chamber under the conditions described
The eucalypt seedlings generally showed remarkable tolerance to Al (Figure 1A). Root growth of all species was unaffected by up to 256 µM Al3+ in solution. Eucalyptus grandis
and E. cloeziana were more sensitive to Al toxicity than the
other species, showing reductions in root growth of more than
20% at Al3+ activities higher than 256 µM, whereas smaller decreases in root growth were observed in E. dunnii and
E. saligna. A small reduction in root growth in E. globulus and
E. urophylla was observed only at the highest Al3+ activity.
Low Al3+ activities had a positive effect on root elongation in
all species (Figure 1A).
The response pattern to Al of the clones was similar to that
of the six species (Figure 1B). Root elongation was either
stimulated or unaffected by low to intermediate Al activities
(Figure 1B). A decrease in root elongation was observed only
at an activity of 648 µM Al3+, but the decrease was usually less
than 20% for all clones, indicating that the clones were more
tolerant to Al than the seedlings (Figure 1B).
Specificity of tolerance to Al
To test the specificity of tolerance of eucalypts to Al, we used
La3+ as a proxy ion (Parker and Pedler 1998). The pattern and
magnitude of root and shoot growth differed between the Al
and La treatments. As in the previous experiments, plants were
highly tolerant to Al. In Clone 7074, root elongation was reduced by 20% only when Al3+ activity was 648 µM, whereas
root elongation was reduced by more than 90% by the lowest
La3+ activity (Figure 2A). The toxic effect of La was less pronounced on shoots than on roots. The mean reduction in plant
height by La3+ was about 50%, whereas there was no negative
effect of Al on plant height (Figure 2B).
Aluminum accumulation in the root tip
We selected the highly Al-tolerant E. globulus to evaluate the
extent of Al accumulation in the tips of thick roots. Root tip
[Al] increased linearly with Al3+ activity in solution, reaching
240 µg g –1 at the highest Al3+ activity (Figure 3). In root apices
of plants grown in control solutions, only trace amounts of Al
were detected in root tips (Figure 3).
Differential growth response and accumulation of Al by roots
and shoot
Although elongation of thick roots was unaffected by the
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Figure 1. Relative primary root
elongation of six eucalypt species (A) and clones (B) in response to a 10-day exposure to
increasing Al3+ activities. Bars
denote SE (n = 6 (A); n = 3 (B)).
range of [Al] in several eucalypt species and clones (Figures 1A and 1B), thin roots were visibly more damaged by Al
than thick roots. A comparison of the effects of high Al3+ activity (648 µM) on thick and thin roots of E. grandis and E. globulus indicated that thick roots of E. grandis were more
sensitive to Al toxicity than those of E. globulus and thin roots
of both species were more sensitive to Al toxicity than thick
roots (Figure 4).
Consistent with the differential elongation response to Al of
thin roots and thick roots, accumulation of Al in thin roots and
thick roots differed. Depending on the species, [Al] was about
2 to 10 times higher in thin roots than in thick roots, with the
greatest differences occurring in the more Al-sensitive species
E. cloeziana and E. grandis (Figure 5). In thick roots, [Al] was
slightly higher in E. globulus, E. urophylla and E. dunnii than
in E. cloeziana, E. saligna and E. grandis (Figure 5). Shoots
had much lower [Al] than roots. The distribution of Al in roots
and shoot was not uniform. Higher [Al] was found in the
0–5 mm root tip than in the more mature root segment (Figure 6). In the shoot, more Al accumulated in older leaves than
in younger leaves (Figure 6).
Partitioning of root tip Al
To determine if Al accumulated on the apoplast or in the symplast of cells in the root tip, root tips were sequentially rinsed
with the Al-chelating agent citric acid (Zhang and Taylor
1990). About 33% of Al in the root tip was loosely retained
and could be removed with an ultra-pure water wash. When
the concentration of citric acid was increased, there was a
greater proportion of Al desorbed from root tips, following a
Figure 2. Relative primary
root elongation (A) and
shoot height (B) of eucalypt Clone 7074 in response to a 10-day exposure to increasing Al3+ and
La3+ activities. Bars denote SE (n = 3).
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ALUMINUM TOLERANCE IN EUCALYPTS
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Figure 3. Aluminum accumulation in the primary root tips of E. globulus after a 48-h exposure to increasing Al3+ activities. Bars denote SE
(n = 3).
Figure 5. Aluminum accumulation in thick and thin root tips and shoot
of six plantation eucalypt species after a 10-day exposure to 465 µM
Al3+. Bars denote SE (n = 3).
Figure 4. Relative elongation of thick and thin roots of E. grandis and
E. globulus after a 10-day exposure to 648 µM Al3+. Bars denote SE
(n = 3).
biphasic time course, with the majority of Al being removed in
the first 3 h (Figure 7). However, even with sequential rinsing
for up to 9 h, about 40% of root tip Al was not desorbed (Figure 7).
Organic acid concentrations in the root tip
We used the more Al-tolerant E. globulus to evaluate the effect
of Al on the concentration of OA in the root tip. Cultivation of
seedlings in the presence of 465 µM Al3+ resulted in increased
production of certain organic compounds. Among the OAs,
malic acid showed the largest increase in concentration in root
tips of Al-treated plants, along with a greater than 100% increase in the concentration of water-soluble sugars (data not
shown). These sugars were identified based on spectrum similarity in a mass spectra library as mannose, rhamnose, erythrose, glucose, arabinose, xylopiranose, galactose and xylose.
The evaluation of OA in root tips was subsequently expanded to the other eucalypt species. Although there was some
variation among species, fumaric and malic acid were the
dominant root tip OAs in the absence of Al, with citric and
succinic acid present in lower concentrations (Figures 8A–F).
Because the TMS derivatives of trans-aconitic, and to a lesser
extent, oxalic acid, were somewhat unstable, quantification
was not attempted. When plants were exposed to Al, there was
a large increase in the concentration of malate in the root tips
of all species (Figures 8A–F). The greatest malate concentration was found in root tips of E. globulus (83 µg g –1, Figure 8A), whereas the lowest malate concentration was in root
tips of E. cloeziana (20.2 µg g –1, Figure 8F). Except for E. urophylla (Figure 8B), which was as tolerant to Al as E. globulus
and had a malate concentration lower than that of the moderately tolerant E. dunnii (Figure 8C), the concentration of root
tip malate in the presence of Al correlated closely with the degree of Al tolerance of the species. Also, the citric acid concentration of all species was higher in the presence of Al than
in its absence, but no close relationship to Al tolerance was observed. No clear trends were identified for the other OAs.
Root exudation of organic acids
In chromatograms of root exudates, only a few peaks were evi-
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Figure 6. Accumulation of Al in E. globulus exposed to 465 µM Al3+
for 10 days as a function of root segment and leaf age. Bars denote SE
(n = 3).
Figure 8. Concentrations of the dominant organic acids in thick root
tips from six plantation eucalypt species growing in the absence or
presence of 465 µM Al3+. Bars denote SE (n = 3).
Figure 7. Relative Al desorption from root tips of E. globulus as a
function of ice-cold citric acid (pH 4.5) concentration and rinsing period. Seedlings were exposed to 465 µM Al3+ for 10 days. Thick root
tips were then collected and rinsed with citrate. Removal is relative to
non-desorbed root tips from seedlings exposed to a similar Al treatment. Bars denote SE (n = 4).
dent as indicated by the profile for root exudates of the highly
Al-tolerant E. urophylla, collected in the absence (Figure 9A)
and presence (Figure 9B) of 465 µM Al3+. The Al treatment induced a large increase in malate exudation compared with the
control plants. Exudation of acetate, formate and oxalate was
also observed in this species, but was inconsistent across all
species (Figure 10). The induction of malate exudation by Al
was observed for all species, but the extent of induction was
species specific (Figure 10). For the species tested, citrate was
only secreted in the presence of Al (Figures 9B and 10). However, there was no clear trend of a greater exudation of either
citrate or malate in the presence of Al by the more Al-tolerant
species. Other OAs such as acetate, formate and oxalate were
also detected. In E. urophylla, the Al treatment increased root
exudation, but this pattern was inconsistent in the other species
(Figure 10). Citrate exudation was negligible (data not shown)
in plants not pre-exposed to Al for 10 days and whose exudates
were collected in the absence of Al, suggesting that secretion
of citrate was induced by Al.
Discussion
Growth response to Al
Schaedle et al. (1989) classified temperate trees into groups
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Figure 9. Chromatograms of
root exudates of E. urophylla
seedlings. Seedlings were
grown in control solution for
10 days and root exudates
were collected for 6 h in (A)
the absence (control; 0 µM Al)
or (B) the presence of 465 µM
Al3+. In both panels, a chromatogram of a mixed organic
acid standard is included for
comparison. Peaks: 1. acetate,
2. formate, 3. chloride, 4. succinate, 5. malate, 6. malonate/
tartarate, 7. fumarate, 8. oxalate, 9. citrate.
according to their degree of sensitivity to Al: (1) sensitive—toxicity is observed at [Al] < 200 µM; (2) intermediate—toxicity occurs at [Al] between 200 and 1000 µM;
and (3) tolerant—negative effects of Al are observed only if
[Al] > 1000 µM. Based on these categories, the majority of
eucalypt species and all the clones evaluated in the present
study would be classified as tolerant, as Al rhizotoxicity occurred only when Al3+ activity was greater than 465 µM
(1600 µM total Al), with the more sensitive species E. grandis
and E. cloeziana being placed in the intermediate tolerance
category. The high Al tolerance of eucalypt species and clones
corroborates earlier studies carried out in nutrient solutions or
acid soils (Neves et al. 1982a, 1982b, Huang and Bachelard
1993).
Based on root growth response, E. grandis and E. cloeziana
were the most sensitive to Al toxicity and E. globulus and
E. urophylla were the least sensitive. All of the clones appeared to be Al tolerant. Low to intermediate Al3+ activities
were beneficial to root growth (Figure 1). Previous studies
have reported a positive effect of Al on growth of eucalypts
and other species (Mullette 1975, Matsumoto et al. 1976, Keltjens and van Loenen 1989, Huang and Bachelard 1993, Watanabe et al. 1998b). Despite earlier suggestions of an essential
role for Al as a plant nutrient, the beneficial effect of Al, particularly on root growth, is now believed to be due to the alleviation of H+ toxicity at low pH (Kinraide 1991).
Compared with thick roots, thin, lateral roots were more
sensitive to Al (Figure 4). Thin roots accumulated more Al
(Figure 5), which may account for their higher sensitivity to
Al. Similar differentiation of root classes has been reported for
annual crops (Lazof et al. 1994, Ferrufino et al. 2000, Silva et
al. 2001), suggesting that cell differentiation and meristem formation processes are more sensitive to Al during the emergence of the lateral roots, or that the protection mechanism is
less active in lateral roots. The lower tolerance to Al of thin
roots compared with that of thick roots has practical implications. Thin roots typically comprise less than 5% of the mass
of the root system, but may contribute up to 90% of its length
and are responsible for water and nutrient acquisition (Vogt et
al. 1996). Furthermore, exploitation of short-rotation eucalypts leads to a significant reduction in exchangeable calcium
(Ca) and magnesium (Mg) and enhances exchangeable Al in
the soil over time (Leite 2001), increasing the exchangeable
Al:(Ca + Mg) ratio and potential Al toxicity (Raynal et al.
1990, Kinraide 1998, Sanzonowicz et al. 1998). Our results indicate that thin root growth may be hindered in Al-toxic acid
soils. On other hand, the high tolerance of thick roots allows
deeper growth where low Ca and Mg and high Al conditions
are prevalent. Deep rooting is essential, especially in the tropics, where eucalypt plantations frequently experience seasonal
droughts. On the surface soil under established eucalypt plantations, a thick layer of litter is deposited annually (Leite 2001)
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Figure 10. Organic acids (OA) secreted by intact roots of six eucalypt
species in the absence (control; 0 Al) or presence of 465 µM Al3+ during a 6-h period.
and the OA present in plant material could lead to the formation of Al–OA complexes (Shotyk and Sposito 1990, Kerven
et al. 1995, Hiradate et al. 1998). Because of the nontoxic nature of these Al–organic complexes (Harper et al. 1995), the
establishment of an active thin root network on the surface soil
is favorable (Gonçalves and Mello 2000, Neves 2000).
The high tolerance of eucalypts to Al was not observed in
plants grown in the presence of equivalent activities of the
toxic La3+ (Figure 2), indicating that the mechanism affording
protection was specific to Al. Lanthanum was not only rhizotoxic, but also reduced shoot growth, an effect not evident even
at high Al3+ activities (Figure 2).
Aluminum accumulation and partitioning in roots and shoot
Eucalypt shoots accumulated much lower amounts of Al than
roots (Figure 5). The restriction of Al translocation from roots
to shoots may provide a means of protecting the shoot from the
damaging effects of Al. The small proportion of Al transported
to the shoot was preferentially located in older leaves (Fig-
ure 6), probably carried in the transpiration stream (Shen and
Ma 2001, Watanabe et al. 2001). Increasing [Al] resulted in a
linear increase in [Al] in the root tip (0–5mm) of E. globulus
(Figure 3) and [Al] was higher in the apical 0–5 mm segment
than in the more mature 5–30 mm segment (Figure 6). The increased accumulation of Al in root tips with increasing Al3+
activities up to intermediate values was not paralleled by a decrease in root elongation (Figure 1), suggesting that the metal
was present in a nontoxic form. More than 60% of accumulated Al could be desorbed from the root tip of E. globulus
with a citrate wash. This Al could be inactive as a result of immobilization to the cell wall (Cuenca et al. 1990, Heim et al.
2000) or because it resides in the root tip mucilage layer as an
Al–OA complex (Henderson and Ownby 1991). When eucalypt seedlings were exposed to Al, there was an induction of
citric acid exudation by roots (Figure 9). This citrate may be
involved in the chelation and detoxification of Al in the
rhizosphere or root tip apoplast. The importance of OA exudation by root tips in Al tolerance has been demonstrated for several annual species (Miyasaka et al. 1991, Delhaize et al. 1993,
Pellet et al. 1995, Zheng et al. 1998b, Silva et al. 2001,
Kochian et al. 2002). The secretion of OA is confined to the
root tip (Pellet et al. 1995, Zheng et al. 1998a, Delhaize et al.
2001), the primary target for Al toxicity (Ryan et al. 1993,
Sivaguru and Horst 1998), and is induced by Al3+ but not La3+
(Ryan et al. 1995, Silva et al. 2001). Such a high degree of
specificity results from the inability of La3+ to activate the
plasma membrane channels mediating OA release to the root
apoplast (Ryan et al. 1997, Piñeros and Kochian 2001). Accordingly, wheat (Kinraide et al. 1992) and soybean genotypes
(Silva et al. 2001), which present species-specific degrees of
Al tolerance, were equally sensitive to La3+. This may be one
reason why E. globulus was highly tolerant to Al but extremely
sensitive to La3+ (Figure 2).
Organic acids in root tip and root exudates
About 40% of the Al in root tips could not be removed even
with a 300 µM citrate wash (Figure 7), suggesting that it was
irreversibly bound in the root apoplast or that it was retained in
a compartment inaccessible to the citrate rinse, possibly the
root symplast. Because intracellular Al can rapidly react with
several components and bring about irreversible damage to
normal cell function (Martin 1992, Kochian 1995, Rengel
1996, Haug and Vitorello 1997, Sivaguru et al. 2003), we speculate that this putative intracellular Al pool is compartmentalized in the vacuole (Vasquez et al. 1999) or detoxified by the
formation of Al–OA complexes (Ma 2000). Support for complexation with OA comes from our finding that when eucalypts seedlings were exposed to Al, there was a more than
200% increase in the concentration of malic acid in the root
tips, whereas the concentrations of succinic, fumaric and citric
acids decreased or remained largely unchanged (Figure 8).
The accumulation of soluble sugars in response to Al could
also serve as a source of energy and carbon skeletons for
malate synthesis.
Direct evidence for an internal Al detoxification mechanism
comes from studies with Al-accumulator plants that have tis-
TREE PHYSIOLOGY VOLUME 24, 2004
ALUMINUM TOLERANCE IN EUCALYPTS
sue [Al] frequently surpassing 10,000 µg g –1, but present no
symptoms of Al toxicity. Aluminum is not detrimental to these
plants because it occurs in citrate (Ma et al. 1997a) and oxalate
(Ma et al. 1998, Watanabe et al. 1998a) complexes. Based on
these studies and our own findings, we conclude that Al tolerance in eucalypts is achieved through a combination of an
Al-induced secretion of citric and malic acid and an increase in
malic acid concentration in the root tip. This corresponds with
the report that citric acid exudation and possibly the citric acid
concentration of roots and shoots are the underlying mechanisms of Al tolerance in A. mangium (Osawa et al. 1997). The
importance of OA in Al-tolerance mechanisms is underscored
by research reporting increased Al tolerance in naturally Alsensitive species through enhanced biosynthesis and exudation of OA by means of genetic engineering (Tesfaye et al.
2001, Anoop et al. 2003). However, the more Al-tolerant species such as E. globulus accumulated more Al in the root tissue
than the more Al-sensitive E. grandis (Figure 5). Furthermore,
there was no clear trend between the degree of Al-tolerance
(Figure 1) and the magnitude of malate and citrate secretion by
the species (Figure 8). These results appear to favor the hypothesis of internal detoxification of Al by malate. The absence of the clear correlation often observed between Al-tolerance and OA secretion in crop plants such as wheat (Delhaize
et al. 1993) may be a result of the overall higher degree of Al
tolerance of these eucalyptus species compared with crop species.
Although all the eucalypt species and clones tested were
more tolerant to Al than most crop plants, the higher sensitivity of E. cloeziana and E. grandis to high [Al] was evident.
This may partially explain why these species usually occur in
soils with relatively lower [Al] and higher concentrations of
exchangeable cations and phosphorus (P) than the other species investigated (Barros et al. 1990, Florence 1997). Some
eucalypt species may be more adapted than others to highly
leached soils low in P, Ca and Mg and high in Al (Neave et al.
1995). Such differential behavior of eucalypt species with respect to Al tolerance is not surprising given that, along with
climate changes, soil impoverishment played an important
role in the evolution and current geographic distribution of Eucalyptus spp. in Australia (Specht 1996, Florence 1997). Thus,
during the expansion of eucalypts from humid, temperate, nutrient-rich soils to drier, tropical, nutrient-poor regions (Specht
1996), enhanced synthesis and exudation of OA might have
been an important trait, as OA not only help alleviate Al toxicity, but also enhance P acquisition in nutrient-poor acid soils
(Jones 1998, Koyama et al. 2000, Ryan et al. 2001). Although
secretion of organic acids such as malate and citrate may contribute to the overall adaptation of eucalypt species to acid
Al-toxic soils, it does not seem to be the only mechanism underlying Al tolerance.
Acknowledgments
This research was partially funded by Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq) through a PostDoctoral fellowship to Ivo R. Silva (Grant No. 300543/00-0 and
PROFIX Grant No. 541090/01-1).
1275
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