Annals of Botany 111: 69 77, 2013

doi:10.1093/aob/mcs234, available online at www.aob.oxfordjournals.org

Aluminium tolerance in rice is antagonistic with nitrate preference

and synergistic with ammonium preference
Xue Qiang Zhao1, Shi Wei Guo2, Fumie Shinmachi3, Michio Sunairi4, Akira Noguchi5, Isao Hasegawa5
and Ren Fang Shen1,*
1

State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing
210008, China, 2Institute of Foods Crop, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China, 3Department of
Bioresource Science, Junior College, 4Department of Applied Biological Sciences and 5Department of Chemistry and Life
Science, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan
* For correspondence. E-mail rfshen@issas.ac.cn
Received: 24 June 2012 Returned for revision: 17 August 2012 Accepted: 24 September 2012 Published electronically: 31 October 2012

Key words: Aluminium, ammonium, correlation, Indica, Japonica, nitrate, rice.

IN T RO DU C T IO N
Aluminium (Al) is the most abundant metallic element in the
Earths crust (Yaroshevsky, 2006). Currently, there is no evidence that Al is essential for plant growth, although it is beneficial for some plant species (Pilon-Smits et al., 2009). Most
Al in soil is present as harmless mineral forms. However, as
soils become acidic, potentially toxic ionic forms of Al dissolve into the soil solution, inhibiting root growth and function. Consequently, Al toxicity may become the primary
factor limiting crop production in acidic soils, which account
for 50 % of the worlds potentially arable lands (Kochian
et al., 2005), whereas this toxicity is often lacking in neutral
to calcareous soils.
Nitrogen (N) is the most abundant mineral nutrient element
in plants, and plays an essential role in plant growth and development. Despite its relatively high concentrations and important functions in plants, N is present in very low concentrations
in soil (Yaroshevsky, 2006). Ammonium (NH+
4 ) and nitrate
(NO2
3 ) are the two main inorganic N sources available for
plant uptake. In the field, inorganic N occurs predominantly as
2
.
.
.
NH+
4 in soils of pH 4 06 0 and as NO3 in soils of pH 6 0
8.0 (McGrath and Rorison, 1982). Therefore, a supply of
NH+
4 -N becomes a critical factor for the survival of plants in
acidic soils, whereas NO2
3 -N does so in neutral to calcareous
soils.

Thus, soil Al and N are two major pH-linked factors in plant

nutrition. Acidic soils may be dominated chemically not only
by Al but also by NH+
4 , and neutral to calcareous soils, although lacking potentially toxic concentrations of Al, have
higher concentrations of NO2
3 (Rorison, 1985). Soil characteristics can greatly influence the distribution of plant species or
genotypes. Different plants vary greatly in their ability to
2
absorb and utilize NH+
4 - and NO3 -N (Haynes and Goh,
1978) and in their tolerance to excess Al (Foy, 1988; Ma,
2005). Rice (Oryza sativa) is one of the worlds most important crops, supplying food for nearly half its population, especially in Asia. About 13 % of global rice production occurs in
acidic soils (von Uexkull and Mutert, 1995). On the whole,
rice plants are the most Al-tolerant among small cereal crop
species (Foy, 1988; Famoso et al., 2010). At the same time,
because NH+
4 is the predominant N species in anaerobic agricultural soils, in particular in paddy fields, it has been generally accepted that NH+
4 is the most important source of N for rice
plants, despite some reports that partial addition of NO2
3 to
growth media can improve rice growth (Fan et al., 2005;
Duan et al., 2007).
There are extensive reports on the responses of plants to N
sources and Al toxicity, but N utilization and Al toxicity are
often studied separately. Given the parallel association of Al
and inorganic N forms with soil pH, plants may have

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Background and Aims Acidic soils are dominated chemically by more ammonium and more available, so more
potentially toxic, aluminium compared with neutral to calcareous soils, which are characterized by more nitrate
and less available, so less toxic, aluminium. However, it is not known whether aluminium tolerance and nitrogen
source preference are linked in plants.
Methods This question was investigated by comparing the responses of 30 rice (Oryza sativa) varieties (15
subsp. japonica cultivars and 15 subsp. indica cultivars) to aluminium, various ammonium/nitrate ratios and
their combinations under acidic solution conditions.
Key Results indica rice plants were generally found to be aluminium-sensitive and nitrate-preferring, while japonica cultivars were aluminium-tolerant and relatively ammonium-preferring. Aluminium tolerance of different
rice varieties was significantly negatively correlated with their nitrate preference. Furthermore, aluminium
enhanced ammonium-fed rice growth but inhibited nitrate-fed rice growth.
Conclusions The results suggest that aluminium tolerance in rice is antagonistic with nitrate preference and synergistic with ammonium preference under acidic solution conditions. A schematic diagram summarizing the
interactions of aluminium and nitrogen in soilplant ecosystems is presented and provides a new basis for the
integrated management of acidic soils.

70

Zhao et al. Relationship of aluminium and nitrogen in rice

evolved mechanisms that link Al stress and NH+

4 -N nutrition
as a result of ecological adaptation and natural selection.
Thus, we hypothesized that: (1) Al-tolerant rice plants prefer
2
NH+
4 -N while Al-sensitive rice plants prefer NO3 -N, and Al
tolerance and N source preference may be linked in rice; and
(2) Al can synergistically interact with NH+
4 but antagonistically with NO2
3 in their effects on rice growth. The present
study was conducted in an attempt to test these hypotheses
using various rice varieties supplied with various N and Al
sources. The results obtained demonstrate coordination of inorganic N and Al in rice grown under acidic conditions.
M AT E R IA L S A ND M E T HO DS
Plant materials and growth conditions

Root elongation measurement

The roots of 2-d-old seedlings were exposed to 0.5 mM

CaCl2 ( pH 4.5) with or without 50 mM Al for 24 h. The root
length of each seedling was measured with a ruler before
and after the treatments. Thirty rice varieties (Table 1) were
used for root elongation measurement, and ten roots of each
variety were measured. Relative root elongation was calculated
as: (root elongation with Al treatment)/(root elongation
without Al) 100. Because different rice cultivars vary
TA B L E 1. Rice varieties used: Nos. 1 15 are japonica, and Nos.
16 30 are indica
No.

Name

No.

Name

No.

Name

1
2
3
4
5
6
7
8
9
10

Nanjing55108
Nanjing43
Nipponbare
Nanjing41
Koshihikari
Suhuxiangjing
2723
Nanjing42
Louming
Yandao8

11
12
13
14
15
16
17
18
19
20

Zhendao2
Wuyujing14
Zaofeng9
86you8
Wuyujing7
Yangdao6
Liangyoupei9
Jin04-12
Zhongxian98
Teyou559

21
22
23
24
25
26
27
28
29
30

Yangfuxian2
IIyou838
Zhenhui084
Yueyou277
Youmi7
Minghui70
IR26
Shengtai1
Nanjing16
Kasalath

N source preference evaluation

The 2-d-old seedlings were first grown in an N-free modified

Kimura B and Arnon solution as described previously (Zhao
et al., 2009). After 4 d of growth, uniform seedlings were
selected and transplanted to a plastic pot containing 20 L of
nutrient solution. The N sources were supplied as NH+
4 and
+
2
NO2
3 at various NH4 /NO3 ratios (100 : 0, 75 : 25, 50 : 50, 25
: 75 and 0 : 100) at an N concentration of 2 mM. After 24 d,
roots and shoots were harvested separately, dried in a
forced-air oven at 80 8C to constant weight, and weighed.
Thirty rice varieties (Table 1) were used for N source preference evaluation, and there were two experimental replicates
for each variety. Relative root dry weight was then calculated
as follows: (root dry weight of rice seedlings at a specific
2
NH+
4 /NO3 ratio)/(root dry weight of rice seedlings at the
2
NH+
/NO
4
3 ratio of 100 : 0) 100; or (root dry weight of
2
rice seedlings at a specific NH+
4 /NO3 ratio)/(root dry weight
+
2
of rice seedlings at the NH4 /NO3 ratio of 0 : 100) 100.
Relative shoot dry weight was calculated in the same way,
using shoots instead of roots. Because different rice cultivars
vary greatly in the absolute biomass dry weight at the NH+
4/
NO2
3 ratio of 100 : 0 or 0 : 100, the relative dry weight was
used here to compare the difference in N source preference
of different rice cultivars.
The interaction between Al and N

The 2-d-old seedlings were first grown in an N-free modified

Kimura B and Arnon solution as described previously (Zhao
et al., 2009). After 4 d of growth, uniform seedlings were
then selected and exposed to nutrient solutions with or
2
without 50 mM Al at various NH+
4 /NO3 ratios (100 : 0, 75 :
25, 50 : 50, 25 : 75 and 0 : 100) at an N level of 2 mM for 24
d. During harvest, the roots of seedlings were washed with
deionized water. Their roots and shoots were then harvested
separately and dried in a forced-air oven at 80 8C to constant
weight, weighed and ground for analysis of total N and Al concentrations in the plant tissues. In this experiment, ten rice varieties (Nos. 1, 2, 3, 5, 15, 16, 27, 28, 29, 30) (Table 1) were
randomly chosen to investigate the interaction between Al
and N, and there were three experimental replicates for each
variety. Relative root dry weight increment was calculated
as: (root dry weight of rice seedlings with Al treatment
root dry weight of seedlings without Al treatment)/(root dry
weight of seedlings without Al treatment) 100. Relative
shoot dry weight increment was calculated using shoots
instead of roots. Relative biomass dry weight increment was
used here to compare the effects of Al on rice growth at differ2
ent NH+
4 /NO3 ratios.
During the rice culture period, NH+
4 concentrations in the
2
solution with NO2
3 treatment and NO3 concentrations in the
+
solution with NH4 treatment were negligible, so it can be
assumed that N was absorbed by rice plants in the forms
supplied.

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Fifteen genetically diverse varieties each of two rice subspecies

(Oryza sativa subsp. japonica and O. sativa subsp. indica) were
used (Table 1). Plants were grown in a controlled-environment
growth chamber with day/night temperatures of 28 + 1/20 +
1 8C, a day length of 14 h, light intensity of 50 klux, and relative
humidity of 65 + 5 %. Seeds were surface-sterilized with 10 %
H2O2 for 10 min, washed with deionized water several times,
soaked for 24 h in deionized water in the dark, and then germinated for 24 h between filter papers soaked with deionized
water. Subsequently, seedlings were transferred to a framed
net floating on 0.5 mM CaCl2 ( pH 4.5) and grown for 2 d
before use.
2
The Al3+, NH+
4 and NO3 were applied as AlCl3.6H2O,
NH4Cl and NaNO3, respectively. At the outset of all experiments, the initial pH of the solutions was adjusted to 4.5 by
the addition of 0.1 M HCl. The culture solutions were renewed
every day in order to reduce the effects of pH changes on rice
2
growth due to the uptake of NH+
4 and NO3 .

greatly in the absolute root elongation under no Al conditions,

the relative root elongation was used here to compare the
difference in Al tolerance of different rice cultivars.

Zhao et al. Relationship of aluminium and nitrogen in rice

Analysis of total N and Al concentrations in plant tissue

Statistical analysis

R E S U LT S
Comparison of Al tolerance between the japonica and indica
subspecies

The most direct and obvious effect of Al toxicity on plant

growth is the inhibition of root elongation after only several
minutes or hours by micromolar concentrations of Al.
Therefore, relative root elongation is considered to be the parameter most directly related to Al tolerance in plants (You
et al., 2005). Here, relative root elongation was also used to
compare Al tolerance between the japonica and indica subspecies. The relative root elongation of the japonica varietal group
was significantly higher than that of the indica group (Fig. 1),
indicating that the former was more tolerant to Al than the
latter.
Comparison of N source preference between the japonica and
indica subspecies
2
Moderate NH+
4 - and NO3 -N concentrations generally have
small short-term influences on the root elongation of plants,
so changes in the root elongation of rice seedlings are not suitable for assessing N source preference in rice. It is somewhat
difficult to compare the N source preference among different
plant species and cultivars within the same species because
it is not easy to find a uniform evaluating index as with the
relative root elongation in evaluating Al tolerance.
Relative root dry weight and relative shoot dry weight were
employed to compare the N source preference between japonica and indica subspecies by assigning the dry weight at the
2
NH+
4 /NO3 ratio of 100 : 0 a value of 100. The results
showed that the relative root dry weight and relative shoot

75

50

25

japonica

indica

F I G . 1. Comparison of Al tolerance in the japonica and indica varietal groups

using relative root elongation of rice seedlings. Two-day-old rice seedlings
were exposed to 0.5 mM CaCl2 (pH 4.5) with or without 50 mM Al in the
absence of N supply for 24 h. Data are means + s.d. (n 15) for 15 japonica
varieties or 15 indica varieties as shown in Table 1. The method used to calculate relative root elongation is described in the Materials and Methods.
*Indicates a statistically significant difference between japonica and indica
(P , 0.05 by independent-samples t-test).

dry weight of the indica varietal group were significantly

higher than those of the japonica varietal group at NH+
4/
NO2
3 ratios of 75 : 25, 50 : 50, 25 : 75 and 0 : 100 (Fig. 2A,
2
B). When the dry weight of seedlings at the NH+
4 /NO3 ratio
of 0 : 100 was assigned a value of 100, the relative root dry
weight and relative shoot dry weight of the japonica varietal
group were significantly higher than those of the indica var2
ietal group at the NH+
4 /NO3 ratio of only 100 : 0, but there
was no significant difference in the relative root dry weight
and relative shoot dry weight between japonica varietal
2
group and indica varietal group at the NH+
4 /NO3 ratios of
75 : 25, 50 : 50 and 25 : 75 (Fig. 2C, D). These results demonstrated that the indica group performed better in the growth
medium containing NO2
than did the japonica group,
3
whereas the latter grew better in the medium supplied solely
with NH+
4 -N than did the former.
2
The different NH+
4 /NO3 ratios markedly affected the growth
of the indica group, and the relative root dry weight and relative shoot dry weight of the indica varietal group gradually
increased from 100 : 0 to 25 : 75 and then significantly
decreased at 0 : 100 (Fig. 2), suggesting the indica group pre2
2
+
ferred certain ratios of NH+
4 and NO3 to single NO3 or NH4
2
although it showed better growth with single NO3 than single
+
2
NH+
4 . However, different NH4 /NO3 ratios had much less influence on the relative root dry weight and relative shoot dry
weight of the japonica group (Fig. 2). Therefore, growth of
the indica group was affected more by differences in NH+
4/
NO2
3 ratios than the japonica group.

Correlation between Al tolerance and N source preference

Simple linear correlation analysis between Al tolerance and

N source preference showed that, when the dry weight at the
2
NH+
4 /NO3 ratio of 100 : 0 was assigned a value of 100, relative
root elongation of rice plants was significantly negatively correlated with both relative root dry weight and relative shoot dry

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The pots used for rice growth were randomly placed in the
growth chamber after rice seedlings were transplanted, and the
positions of all pots were changed every day during the subsequent growth period in order to reduce the influence of possible light non-homogeneity. A mean comparison was made
according to independent-samples t-test (two-range comparison) or Tukeys test (multi-range comparison) using the
Statistical Analysis System (SPSS 18.0).

100
Relative root elongation (%)

A total of 100 500 mg of the ground samples were digested

with 5 mL of concentrated HNO3 to analyse root and shoot Al
concentrations as described by Shen (2008). Al contents in the
digested solutions were determined after appropriate dilution
by inductively coupled plasma-atomic emission spectroscopy
(ICP-AES; IRIS-Advantage, Thermo Elemental, Franklin,
MA, USA). For the analysis of N concentrations in roots and
shoots, 100 200 mg of the ground samples was digested
with 5-mL aliquots of a mixture of concentrated H2SO4 and
30 % H2O2, added to the digestion solution occasionally
until the solution became clear. The total Kjeldahl N concentrations in the digested solutions were determined after appropriate dilution via a continuous flow analyser (AutoAnalyzer 3,
Bran & Luebbe, Norderstedt, Germany).

71

72

Zhao et al. Relationship of aluminium and nitrogen in rice

250

A
a

japonica
indica

200

ab
bc

cd

150
de de
100

de

de
e

50

Relative shoot dry weight (%)

Relative root dry weight (%)

250

abc

abc

bcd

de de

100

50

Relative shoot dry weight (%)

Relative root dry weight (%)

b b

100

50

200
a
150

ab

ab

ab

ab

ab

c
c

100

50

0
100 : 0

75 : 25

50 : 50

Ratio of

NH+4

25 : 75
:

0 : 100

NO3

100 : 0

75 : 25

50 : 50

Ratio of

NH+4

25 : 75
:

0 : 100

NO3

F I G . 2. Comparison of N source preference in the japonica and indica varietal groups using relative root dry weight (A, C) and relative shoot dry weight (B, D).
2
Six-day-old rice seedlings were grown in nutrient solutions with various NH+
4 /NO3 ratios (as indicated) at an N concentration of 2 mM in the absence of Al
2
2
/NO
ratio
of
100
:
0
was
assigned
a value of 100, whereas in (C, D) dry weight at the NH+
treatment for 24 d. (A, B) Dry weight at the NH+
4
3
4 /NO3 ratio of
0 : 100 was assigned a value of 100. Data are means + s.d. (n 15) for 15 japonica varieties and 15 indica varieties, as shown in Table 1. The method to calculate relative root dry weight and relative shoot dry weight is described in the Materials and Methods. Different letters above the column indicate statistically
significant differences among different columns (P , 0.05 by Tukeys test).

2
weight of rice plants grown at NH+
4 /NO3 ratios of 75 : 25, 50 :
50, 25 : 75 and 0 : 100 (Fig. 3A H), indicating that Al tolerance is significantly negatively correlated with a preference
of rice seedlings for NO2
3 -N. Nevertheless, when the dry
2
weight of seedlings supplied with N at the NH+
4 /NO3 ratio
of 0 : 100 was assigned a value of 100, relative root elongation
of rice plants was significantly positively correlated with both
relative root dry weight and relative shoot dry weight of rice
2
plants grown at the NH+
4 /NO3 ratio of 100 : 0 (Fig. 3I, M)
but not at ratios of 75 : 25, 50 : 50 and 25 : 75 (Fig. 3J L,
N P), indicating that Al tolerance was positively correlated
with a preference of rice seedlings for NH+
4 -N only at the
2
NH+
4 /NO3 ratio of 100 : 0.

Interaction between Al and N source supply

2
After comparing Al tolerance and NH+
4 or NO3 preference
among the 30 rice varieties, ten rice varieties, including five
japonica (Nos. 1, 2, 3, 5 and 15 in Table 1) and five indica
(Nos. 16, 27, 28, 29 and 30 in Table 1) varieties, were

randomly chosen for further study of the interaction between

Al and inorganic N.
The relative dry weight increment of biomass was used to
evaluate the effects of Al on rice growth at different NH+
4/
+
+
2
NO2
3 ratios. When supplied with only NH4 -N (NH4 /NO3
ratio of 100 : 0), the values of relative root dry weight increment and relative shoot dry weight increment of both japonica
and indica varietal group were positive, but they were negative
2
when only nitrate was supplied (NH+
4 /NO3 ratio of 0 : 100)
(Fig. 4A, B), indicating that rice growth was severely inhibited
by Al when supplied with only NO2
3 -N but improved by Al
when supplied with only NH+
4 -N. Aluminium addition did
2
not generally affect rice growth at other NH+
4 /NO3 ratios
(75 : 25, 50 : 50 and 25 : 75; data not shown).
Regardless of japonica or indica group, nitrate-fed rice
seedlings accumulated about 30 times more Al in roots than
plants supplied with NH+
4 -N (Fig. 4C). The shoot Al concentration of the japonica varietal group was significantly higher
2
at NH+
4 /NO3 ratio of 0 : 100 than 100 : 0 while that of indica
was not (Fig. 4D). The Al concentrations of roots at other

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ab
cd cd

a
a

150

250

200

150

200

0
250

NH4+ : NO3 = 75 : 25
R 2 = 049*

250

NH4+ : NO3 = 25 : 75
R 2 = 051*

NH4+ : NO3 = 0 : 100

R 2 = 050*

200
150
100
indica
japonica

50
0

NH4+ : NO3 = 75 : 25
R 2 = 043*

250

NH4+ : NO3 = 50 : 50
R 2 = 060*

NH4+ : NO3 = 25 : 75
R 2 = 060*

NH4+ : NO3 = 0 : 100

R 2 = 050*

NH4+ : NO3 = 100 : 0

R 2 = 059*

NH4+ : NO3 = 75 : 25
R 2 = 000

NH4+ : NO3 = 50 : 50
R 2 = 005

NH4+ : NO3 = 25 : 75
R 2 = 013

NH4+ : NO3 = 100 : 0

R 2 = 050*

NH4+ : NO3 = 75 : 25
R 2 = 014

NH4+ : NO3 = 50 : 50
R 2 = 003

NH4+ : NO3 = 25 : 75
R 2 = 000

200
150
100
50
0
250

Relative root dry weight (%)

NH4+ : NO3 = 50 : 50
R 2 = 056*

73

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Relative shoot dry weight (%)

Relative root dry weight (%)

Zhao et al. Relationship of aluminium and nitrogen in rice

200
150
100
50

Relative shoot dry weight (%)

0
250

200
150
100
50
0
0

25

50

75

100

Relative root elongation (%)

25

50

75

100

Relative root elongation (%)

25

50

75

100

Relative root elongation (%)

25

50

75

100

Relative root elongation (%)

F I G . 3. Correlation of Al tolerance and N source preference across 30 genetically diverse rice varieties. The concentration of Al was 50 mM (Fig. 1). The concentration of N was 2 mM (Fig. 2). Correlation analysis between relative root elongation and relative root dry weight (AD, IL) or relative shoot dry weigh (E H, MP)
2
was done at NH+
4 /NO3 ratios of 100 : 0, 75 : 25, 50 : 50, 25 : 75 and 0 : 100, as indicated on the graphs, using 30 rice varieties as shown in Table 1. (AH) Dry weight
2
2
/NO
ratio
of 100 : 0 was assigned a value of 100; (IP) dry weight at the NH+
at the NH+
4
3
4 /NO3 ratio of 0 : 100 was assigned a value of 100. Each data point represents
the value for each rice variety of 15 japonica varieties and 15 indica varieties (indicated in the key in A), as shown in Table 1. The methods to calculate relative root
elongation, relative root dry weight and relative shoot dry weight are described in the Materials and Methods. *Significant correlations between Al tolerance and N
source preference among the different rice varieties at the 1 % level (n 30).

74

Zhao et al. Relationship of aluminium and nitrogen in rice

2
NH+
4 /NO3 ratios (75 : 25, 50 : 50 and 25 : 75) increased as
2
NO3 -N ratios increased in the growth medium (data not
shown). These results indicated that Al accumulation in roots
+
was greatly increased by NO2
3 in comparison with NH4 .
No significant differences in the N concentrations of roots
and shoots were observed in the japonica and indica groups
between Al and +Al treatments, or with different NH+
4/
NO2
ratios
(data
not
shown).
Nevertheless,
because
Al
3
increased the dry weights of NH+
4 -fed rice seedlings but
decreased those of NO2
3 -fed rice seedlings (Fig. 4A, B), Al
increased N uptake (N concentration dry weight) of
2
NH+
4 -fed rice seedlings while it decreased it in NO3 -fed seedlings (calculated results not shown).

NH4+ : NO3 = 100 : 0

Relative root dry weight increment (%)

80

NH4+ : NO3 = 0 : 100

a
60
40
b

20
0
20

bc

40
60

D IS C US S IO N

60
a

40
a

20
0
20

40

Root Al concentration (mg kg1, 103)

60
20

a
15
b
10

5
c

c
0
120

Shoot Al concentration (mg kg1)

100

a
ab

Rice is the most Al-tolerant among small cereal crop species

(Foy, 1988; Famoso et al., 2010). In this study, we have
further demonstrated that japonica is more tolerant to Al toxicity than the indica group (Fig. 1), consistent with previous
reports (Ma et al., 2002; Watanabe and Okada, 2005; Chen
and Shen, 2008; Yang et al., 2008; Famoso et al., 2010).
Famoso et al. (2010) hypothesized that, over the course of evolution, Oryza experienced a dramatic shift in its position
within the range of plant responses to Al, which led to dramatic genetic change and enhanced Al tolerance. The possibility
of Al toxicity in soils may be an important factor in the distribution of plant species, subspecies and varieties. Therefore, we
can infer that the japonica group may have grown originally in
an environment with more, while indica may have grown in an
environment with less, potential for Al toxicity.
Although NH+
4 is generally accepted to be the preferred N
source of rice, different conclusions have often been obtained
depending on experimental conditions and rice varieties. The
indica group utilizes NO2
3 as an N source more efficiently
than japonica (Ta et al., 1981; Fan et al., 2005), and this superiority can be influenced by various environmental and
medium conditions (Ta and Ohira, 1981, 1982a, b). The
present study confirms the superiority of the indica group in
utilizing NO2
3 -N compared with japonica (Fig. 2A, B).
However, in previous reports (Ta et al., 1981; Ta and Ohira,
1981, 1982a, b), the japonica and indica groups responded
similarly to NH+
4 -N supply, which is inconsistent with the
present study, where the japonica group grew better under a
sole NH+
4 -N supply than did the indica group (Fig. 2C, D).
This discrepancy may be attributed to the pH medium used.
Ta et al. (1981) used a medium of pH 4.5, 4.5 and 6.0, respect2
+
+
ively, in the NO2
3 , NO3 + NH4 and NH4 treatments. In

80
60

40
20
0

japonica

indica

F I G . 4. The effect of Al on the growth of japonica and indica rice plants

2
grown with NH+
4 or NO3 alone. Six-day-old rice seedlings were grown in nu2
trient solutions with or without 50 mM Al at NH+
4 /NO3 ratios of 100 : 0 or 0 :
100 at an N level of 2 mM for 24 d. The calculation method of relative root dry
weight increment and relative shoot dry weight increment is described in the
Materials and Methods. (A) Relative root dry weight increment; (B) relative
shoot dry weight increment; (C) root Al concentrations; (D) shoot Al concentrations. Data are means + s.d. (n 5) for five japonica varieties or five indica
varieties as described in the Materials and Methods. The data of Al concentrations of roots and shoots for the absence of Al are not shown due to very small
values. Different letters above the column indicate statistically significant differences among different columns (P , 0.05 by Tukeys test).

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Relative shoot dry weight increment (%)

80

Zhao et al. Relationship of aluminium and nitrogen in rice

Interestingly, in this study, Al increased the dry weights of

rice supplied with NH+
4 -N, which is consistent with previous
reports on grasses (Rorison, 1985) and tropical trees
(Watanabe et al., 1998). The synergism between Al and
NH+
4 can be understood in two ways: one is that Al increases
the NH+
4 -N utilization of rice, and the other is that Al
decreases possible NH+
4 toxicity in the rice, as higher concentrations of NH+
-N
are
often toxic to plants (Britto and
4
Kronzucker, 2002). The mechanisms responsible for the
improved rice growth in the presence of Al and an NH+
4 -N
supply cannot be elucidated from the present data. One
reason for this may be that Al ions reduced the toxicity of
protons secreted from rice roots after NH+
4 -N was taken up,
as the Al3+ and H+ ions are mutually antagonistic (Kinraide,
2003). In the present study, the pH of the NH+
4 -N and
.
NO2
-N
media
changed
from
an
initial
4
5
to
final
values
of
3
3.5 3.8 and 4.5 5.5, respectively, after 24 h of culture, although the nutrient solutions were renewed every day.
Another possible reason is that Al can increase the activity
of NH+
4 -N assimilatory enzymes. Some reports indicate that
Al activated chloroplastic glutamine synthetase in wheat
(Pecsvaradi et al., 2009) and enhanced the expression of the
gene encoding glutamine synthetase in rice (Zhang et al.,
2007). The activation of glutamine synthetase can transform
+
NH+
4 -N into glutamine, so NH4 toxicity was alleviated in
the presence of Al compared with in the absence of Al.
Thus, rice growth was increased under the combination of
+
Al and NH+
4 -N relative to the only NH4 -N supply.
It is generally accepted that wheat and barley are both
NO2
3 -N-preferring and Al-sensitive, while tea trees are
NH+
4 -N- and Al-preferring. On the basis of this and our
results, we summarized the interactive behaviour of Al and N
in soil plant ecosystems by a schematic diagram (Fig. 5).
From this diagram, we observe that neutral and calcareous
soils are chemically dominated by lower Al3+ and higher
NO2
3 concentrations, while acidic soils are characterized by
more Al3+ and more NH+
4 . Correspondingly, plants originally
growing in neutral and calcareous soils might be both
Al-sensitive and NO2
3 -N-preferring while plants originally
growing in acidic soils might be both Al-tolerant and
2
+
NH+
4 -N-preferring. Moreover, NO3 decreases, while NH4
2
increases, toxic Al concentration in soils as NO3 increases
while NH+
4 decreases soil pH due to their uptake and N transformation. However, within plants, Al and NO2
3 are antagonistic
while Al and NH+
4 are synergistic. Thus, plants are able to make
full use of various inherent mechanisms to resolve the different
situations in soils. However, why the NO2
3 -preferring and
Al-sensitive indica rice can extensively distribute in acid soils
remains unclear. Therefore, there are other important factors
such as temperature and water to drive the geographical distribution of plants than Al and N.
It should be emphasized that the schematic diagram shown
in Fig. 5 is suitable for most plant species and soil types, but
some particular plants and soils may be excluded from the predicted patterns. More experimental results are needed to test
and develop this. It was mainly developed based the results
of our research described here. To test it further, the relationships between Al tolerance and N source preference need to be
investigated, both with different plant species and with different varieties of the same plant species. In addition, the effects

Downloaded from http://aob.oxfordjournals.org/ by guest on April 9, 2015

contrast, the initial pH of the medium we used was 4.5 in all N

treatments. Improved rice growth at higher pH may reduce or
mask differences in NH+
4 -N utilization between japonica and
indica varietal groups previously reported. Results in this
study demonstrate that indica group plants grew better in the
+
2
medium containing only NO2
3 or NH4 NO3 mixtures than
did the japonica group plants while the latter did better only
in conditions of NH+
4 -N supply. Thus, as with Al, it is inferred
that the japonica varietal group originally grew in environments with more NH+
4 -N while the indica group occupied
environments with more NO2
3 -N.
Plant species adapted to acidic soils have a preference for
NH+
4 -N while plants with a preference for neutral to calcareous
soils preferentially utilize NO2
3 -N (Rorison, 1985; FalkengrenGrerup, 1995; Marschner, 1995). Accordingly, the preference
2
for NH+
4 -N or NO3 -N in plants should be closely related to
soil pH. Thus, acid-sensitive plants should prefer a NO2
3 -N
source, but acid-tolerant plants should prefer an NH+
4 -N
source. Because toxic Al levels in soils are mainly affected
by pH and a decrease in pH lower than 5.0 can rapidly increase
Al to toxic concentrations, it is reasonable to guess that the
2
preference of plants for NH+
4 -N and NO3 -N should have
some relationship with Al tolerance. This suggestion is supported by the data shown here, in which Al tolerance in the
rice varieties was significantly negatively with a preference
for NO2
3 -N (Fig. 3). This information can be used to screen
for rice varieties that are both Al-tolerant and NH+
4 -efficient.
If such rice varieties can be grown in acidic soils, Al tolerance
and N utilization might be improved simultaneously. This may
be useful for enabling integrated management of acid soils and
increasing plant and grain production in the future.
The indica and japonica subspecies, the two major varieties
of cultivated rice, have distinct eco-geographical distribution
ranges (Zhang et al., 1992). Rice subspecies differ in many
trait performances with respect to biotic and abiotic stress
(Famoso et al., 2010); indica rice is predominant in tropical
and subtropical regions that comprise the major part of the
global rice-growing areas, while japonica rice is more
adapted to temperate regions (Zhang et al., 1992). However,
acidic soils are widely distributed in humid zones of tropical,
subtropical and temperate regions (von Uexkull and Mutert,
1995). This indicates that japonica and indica are not distributed entirely according to soil pH. Therefore, there must be
other factors other than N and Al driving the differentiation
of japonica and indica rice.
The data presented here demonstrate that Al is beneficial to
rice growth in a medium containing NH+
4 -N, but is harmful in
an NO2
3 -N medium (Fig. 4). This suggests a synergistic effect
2
between NH+
4 and Al, but an antagonistic effect between NO3
and Al. There is extensive literature on the alleviating effect of
NH+
4 on Al toxicity in other plant species (e.g. McCain and
Davies, 1983; Rorison, 1985; Klotz and Horst, 1988;
Cumming, 1990; Cumming and Weinstein, 1990; Grauer and
Horst, 1990; Schier and McQuattie, 1999). We have previously
demonstrated that NH+
4 -N can alleviate Al toxicity in rice and
Lespedeza compared with NO2
3 -N. The mechanism is that
NH+
4 ions decrease the cation-binding sites of the cell wall
through direct NH+
4 and indirect proton competition with Al
ions (Zhao et al., 2009; Chen et al., 2010).

75

76

Zhao et al. Relationship of aluminium and nitrogen in rice

PLANTS

Al and NO3 are antagonistic within plants

Al-sensitive

Prefer NO3

Plants

Al and NH4+ are synergistic within plants

Prefer NH4+

Al-tolerant

Neutral to
calcareous soils

More NO3

More NH4+

Acidic soils

More Al3+

AlN interaction in soils: NO3 may reduce

toxic Al concentration of soils since
increase soil pH due to

NO3

NO3

can

uptake by plants.

AlN interaction in soils: NH4+ may increase

toxic Al concentration of soils since NH4+ can
reduce soil pH due to NH4+ uptake by plants.

F I G . 5. Schematic diagram of the relationship between N and Al in soil plant ecosystems with regard to soil pH. The top half above the line indicates N and Al
behaviours within plants. The bottom half below the line indicates N and Al behaviours within soils. The left side represents neutral to calcareous soils; the right
side represents acid soils. The circles indicate the characteristics of the plants and soils, whilst the rectangles indicate the N Al interactions within them.
2
of Al on the chemical processes of NH+
4 and NO3 in soils, especially nitrification, remain poorly known. This information
is essential if we are to identify the role of Al in the global
N cycle.
Finally, we conclude that Al and NH+
4 show synergistic behaviour while Al and NO2
3 are antagonistic in rice under acid
conditions. This close N Al relation in plants will provide
integrated knowledge enabling increases in plant production
by increasing the coordinated adaptation of plants to NH+
4
and Al coexisting in acid soils.

AC KN OW LED GEMEN T S
This work was supported by the National Natural Science
Foundation of China (No. 41025005, 31000933), and
National Natural Science Foundation of China (NSFC)
Japan Science and Technology Agency (JST) Cooperative
Research Project (No. 30821140538). We thank Philip
C. Brookes (Rothamsted Research) for his kind assistance in
revising this paper, Yongchun Zhang (Jiangsu Academy of
Agricultural Science) and Hongsheng Zhang (Nanjing
Agricultural University) for their generous supply of rice
seeds, and Ge Song (Institute of Soil Science, Chinese
Academy of Sciences) for her assistance in determining N concentrations in rice.

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