Disorganized distribution of homogalacturonan epitopes in cell walls as one
possible mechanism for aluminium-induced root growth inhibition in maize
Ya Ying Li
1,†
, Jian Li Yang
2,†
, Yue Jiao Zhang
2
and Shao Jian Zheng
2,
*
1
Ministry of Education Key Laboratory for Environmental Remediation and Ecosystem Health, College of Environmental and
Resource Sciences, Zhejiang University, Hangzhou 310029, China and
2
State Key Laboratory of Plant Physiology and
Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China
Received: 20 February 2009 Returned for revision: 1 April 2009 Accepted: 14 April 2009 Published electronically: 30 May 2009
† Background and Aims Aluminium (Al) toxicity is one of the most severe limitations to crop production in acid
soils. Inhibition of root elongation is the primary symptom of Al toxicity. However, the underlying basis of the
process is unclear. Considering the multiple physiological and biochemical functions of pectin in plants, possible
involvement of homogalacturonan (HG), one of the pectic polysaccharide domains, was examined in connection
with root growth inhibition induced by Al.
† Methods An immunolabelling technique with antibodies specific to HG epitopes (JIM5, unesterified residues
flanked by methylesterifed residues; JIM7, methyl-esterified residues flanked by unesterified residues) was
used to visualize the distribution of different types of HG in cell walls of root apices of two maize cultivars differ-
ing in Al resistance.
† Key Results In the absence of Al, the JIM5 epitope was present around the cell wall with higher fluorescence
intensity at cell corners lining the intercellular spaces, and the JIM7 epitope was present throughout the cell wall.
However, treatment with 50 mM Al for 3 h produced 10 % root growth inhibition in both cultivars and caused the
disappearance of fluorescence in the middle lamella of both epitopes. Prolonged Al treatment (24 h) with 50 %
root growth inhibition in ‘B73’, an Al-sensitive cultivar, resulted in faint and irregular distribution of both epi-
topes. In ‘Nongda3138’, an Al-resistant cultivar, the distribution of HG epitopes was also restricted to the lining
of intercellular spaces when a 50 % inhibition to root growth was induced by Al (100 mM Al, 9 h). Altered dis-
tribution of both epitopes was also observed when of roots were exposed to 50 mM LaCl
3
for 24 h, resulting in
40 % inhibition of root growth.
† Conclusions Changes in HG distribution and root growth inhibition were highly correlated, indicating that Al-
induced perturbed distribution of HG epitopes is possibly involved in Al-induced inhibition of root growth in
maize.
Key words: Al toxicity, cell wall, homogalacturnonan, immunofluorescence, methylesterification, pectin.
INTRODUCTION
Aluminium (Al) toxicity is one of the most severe limitations
to crop production in acid soils around the world (Kochian,
1995; Matsumoto, 2000; Ma, 2007). Al primarily inhibits
root elongation, along with uptake of water and nutrients,
which ultimately results in the loss of production. However,
the primary causes leading to the inhibition of root elongation
are still unknown. One of the major controversies stems from
the discrepancy as to whether Al toxicity results from apoplas-
tic or symplastic lesions. When roots are exposed to Al, the
cell wall is the first site in contact with Al. Therefore, it is unli-
kely that Al toxicity occurs from symplastic lesions without
influencing cell wall functions. Several lines of evidence
support apoplastic lesions as primary factors in Al toxicity.
First, cell walls are the major sites of Al accumulation. For
example, in giant algal cells of Chara coralline,upto
99
.
9 % of the total cellular Al accumulated in the cell wall
(Taylor et al., 2000) and almost 90 % of the total Al was
associated with cell walls of cultured tobacco cells (Chang
et al., 1999). Secondly, binding of Al to cell walls affects
important properties like decreasing extensibility (Tabuchi
and Matsumoto, 2001; Ma et al., 2004). Furthermore, Al
also causes changes in cell wall polysaccharides. For
example, Al treatment resulted in the accumulation of pectin
and hemicellulose in both monocots (Tabuchi and
Matsumoto, 2001; Eticha et al., 2005; Yang et al., 2008)
and dicots (Van et al., 1994). Therefore, the cell wall is one
of the major targets of Al toxicity and plays a very important
role in Al-induced inhibition of root elongation.
Pectin is a major component of primary cell walls of all land
plants and plays many important roles in plant growth and
development including cell expansion (Willats et al., 2001a;
Pelloux et al., 2007). Al stress has been demonstrated to
increase cell wall pectin content in a number of plant
species (Van et al., 1994; Eticha et al., 2005; Yang et al.,
2008), which implies the possible involvement of pectin in
Al toxicity. Binding of Al to pectin may hamper the binding
of newly synthesized polysaccharides to cell wall materials
and result in disordered deposition of pectin and reduced cell
elongation. Furthermore, not only pectin content but also
structural features which are mainly modified by pectin methy-
lesterases (PME) are involved in its physiological and bio-
chemical functions. For example, Wen et al. (1999) showed
†
These two authors contributed equally to this work.
* For correspondence. E-mail sjzheng@zju.edu.cn
# The Author 2009. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
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Annals of Botany 104: 235–241, 2009
doi:10.1093/aob/mcp123, available online at www.aob.oxfordjournals.org

that the partial inhibition of PME by antisense RNA reduced
root elongation in transgenic pea hairy roots. Therefore, it
will be interesting to investigate whether the pectin structural
features are changed or not during the Al-induced root
growth inhibition process.
PMEs play a major role in pectin remodelling in muro and
belong to large multigene families. But overall changes in
PME activity do not reflect specific structural features of
pectin. Immunocytochemical approaches with anti-pectin anti-
body probes provided a powerful way to further our under-
standing of the structure/function relationships of pectin and
Al (Yang et al., 2008). This method was also adopted to inves-
tigate the relationship between cadmium-induced inhibition of
flax hypocotyl and changes in pectin structural features
(Douchiche et al., 2007). In the present study, changes in
homogalacturonan (HG) structures, one of the major pectic
polysaccharide domains, were investigated during the course
of Al-induced root growth inhibition in both Al-resistant and
Al-sensitive maize cultivars. Evidence is provided that disor-
ganized distribution of HG epitopes in the cell wall is associ-
ated with Al-induced root growth inhibition in maize.
MATERIALS AND METHODS
Plant material and growth conditions
Seeds of two maize (Zea mays L.) cultivars, ‘Nongda3138’ (Al
resistant) and ‘B73’ (Al sensitive) were soaked in de-ionized
water for 30 h and then transferred to an incubator at 25 8C
for germination in the dark for 2 d. Germinated seeds were
transferred to a plastic net tray floating in a plastic container
filled with 5 L of 0
.
5mM CaCl
2
solution at pH 4
.
5. The sol-
ution was renewed daily. The seedlings were grown for 3 d
in the growth chamber with a 14-h/26 8C day and a 10-h/
23 8C night regime, a light intensity of 250 mmol photon
m
22
s
21
and a relative humidity of 70 %.
Al treatments and growth measurements
A compartmental hydroponic screening system was adopted
to measure the effect of Al on root elongation (Yang et al.,
2005). In brief, uniform-sized seedlings were transplanted to
18-mL opaque glass test tubes containing 17 mL of treatment
solution. The seedlings were planted one to each tube. Al
resistance was examined by measuring root elongation of
primary roots of the 3-d-old seedlings, after they were grown
in 0
.
5mM CaCl
2
solution, pH 4
.
5, containing 0 or 50 mM
AlCl
3
. Root length was measured with a ruler before and
after treatments (3 h or 24 h). In another case, roots were
exposed to 0
.
5mM CaCl
2
solution (pH 4
.
5) containing 0 or
100 mM Al for 9 h.
Lanthanum (La) treatment
The protocol for La treatment was the same as used for Al
treatments. In brief, 3-d-old seedlings were subjected to a sol-
ution of 0
.
5mM CaCl
2
(pH 4
.
5) containing 0 or 50 mM LaCl
3
.
Root length was measured with a ruler before and after 24-h
treatments.
Monoclonal antibodies
Rat monoclonal antibodies, JIM5 and JIM7, were used.
They are kindly donated by Prof. Knox from the University
of Leeds. JIM5 is an antibody that specifically recognizes a
relatively low-methylesterified HG epitope, and JIM7 is an
antibody that specifically recognizes relatively a high-
methylesterified HG epitope. Detailed information on the gen-
eration of the antibodies and the epitopes recognized are given
in Clausen et al. (2003).
Immunolabelling of HG epitopes
After treatment, roots were hand-sectioned from 2 mm
behind the apex and directly collected into a fixation
solution containing 4 % paraformaldehyde in 50 mM PIPES
(1,4-piperazine-diethanesulphonic acid), 5 mM MgSO
4
and
5mM EGTA (ethylene glycol bis(b-amino-ethylether)-
N,N,N
0
,N
0
-tetraacetic acid), pH 6
.
9. After 1 h of fixation at
room temperature, the samples were washed repeatedly
with phosphate-buffered saline (PBS) and blocked with 0
.
2%
BSA in PBS for 30 min. Then the samples were incubated
with the monoclonal antibodies JIM5 and JIM7, diluted 1 : 10
in PBS containing 0
.
2 % BSA, for 2 h. Subsequently, the
samples were washed three times in PBS and incubated with
goat anti-rat IgG (whole molecule) FITC conjugate, diluted 1 :
50 in PBS containing 0
.
2 % BSA for 2 h at 37 8C. Then
samples were washed briefly with PBS three times and
mounted on glass slides and examined under the laser scanning
system LSM 510 confocal microscope (Zeiss).
Control samples (treated with secondary antibody instead of
primary antibody) were examined and, as no fluorescence was
observed, these images are not shown. To verify whether the
presence of Al interferes with immunofluorescence labelling,
a desorption experiment with 1 mM citric acid in 0
.
5mM
CaCl
2
solution (pH 4
.
5; see Fig. 6; þAl þ Cit) or with
0
.
5mM CaCl
2
solution (pH 4
.
5; see Fig. 6; þAl þ Ca) was
conducted with seedlings treated with 100 mM Al for 9 h.
Roots were placed in desorption solution which was replaced
once during a 30-min desorption period. After being washed
with de-ionized water three times, roots were used for haema-
toxylin staining according to Polle et al. (1978). In a parallel
experiment, roots were used for immunolabelling of
JIM7-recognized HG epitopes as described above.
Experimental design and images analysis
Ten seedlings were used per treatment, and about 30 sec-
tions were made from ten replications per treatment.
Experiments were independently repeated twice, and the data
shown are representative of two independent biological repli-
cates. Photoshop 7
.
0 (Adobe Systems) was used to compile
the fluorescence images.
RESULTS
Al impacts on root growth
The primary symptom of Al toxicity is the inhibition of root
elongation. In the present study, relative root elongation was
used to indicate the effect of Al on root growth. Root elongation
Li et al. — Al-induced alteration of HG distribution236