Environ Geochem Health (2007) 29:189–195
DOI 10.1007/s10653-006-9063-z
ORIGINAL PAPER
Variations between rice cultivars in root secretion of
organic acids and the relationship with plant cadmium
uptake
Jianguo Liu Æ Min Qian Æ Guoliang Cai Æ
Qingsen Zhu Æ Ming H. Wong
Received: 21 May 2006 / Accepted: 2 October 2006 / Published online: 8 February 2007

Springer Science+Business Media B.V. 2007
Abstract To attempt to understand certain
mechanisms causing the variations between rice
cultivars with regard to Cd uptake and accumu-
lation, pot soil experiments were conducted with
two rice cultivars at different levels of Cd, i.e., 0
(the control), 10, 50 mg Cd kg–1 soil. The two
rice cultivars differ significantly with regard to
Cd uptake and accumulation. Root secretions of
low-molecular-weight organic acids (LMWOA)
for each treatment were measured with ion
chromatography. The results showed that
LMWOA concentrations in the soil planted with
Shan you 63 (a high soil Cd accumulator) were
all higher than those in the soil planted with Wu
yun jing 7 (low soil Cd accumulator) at different
soil Cd levels, although the magnitudes of the
J. Liu (&)
M. Qian
G. Cai
Department of Environmental and Safety
Engineering, Jiangsu Polytechnic University, Baiyun
Road, Changzhou, Jiangsu 213016, China
e-mail: ljg@jpu.edu.cn
J. Liu
Q. Zhu
Agricultural College, Yangzhou University,
Yangzhou, Jiangsu 225009, China
M. H. Wong
Croucher Institute for Environmental Sciences, Hong
Kong Baptist University, Hong KongKowloon Tong,
China
differences varied for individual LMWOA and
depend on soil Cd concentrations. For all six
LMWOA, there were significant differences at
P < 0.05 or < 0.01 levels for soils treated with 10
and 50 mg kg–1 Cd. The magnitude of the
differences was greater under soil Cd treatments,
especially at relatively low levels (for example,
10 mg Cd kg–1 soil), than in the control. Acetic
acid and formic acid constituted more than 96%
of the total concentration of the six LMWOA,
while citric acid constituted only about 0.1%.
The rice cultivar with higher concentrations of
LMWOA in soil accumulated more Cd in the
plants. The results indicate that LMWOA secre-
tion by rice root, especially in Cd-contaminated
soils, is likely to be one of the mechanisms
determining the plant Cd uptake properties of
rice cultivars.
Keywords Cadmium
Cultivars
Organic acid

Rice
Oryza sativa L.
Secretion
Uptake
Introduction
Cadmium (Cd) is a toxic metal to plants and
animals. Although plants do not require Cd for
growth and reproduction, the bioaccumulation
index of Cd in plants is high and may exceed
many essential elements (Kabata-Pendias & Pen-
dias, 2001). Therefore, Cd is one of the most
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190
important metals to consider in terms of food-
chain contamination.
The availability of metals in soil to plants is one
of the major factors that influence metal uptake
by plants. Phytoavailability of metals is controlled
by the chemical characteristics of the metal
considered, rhizosphere soil properties, and the
specific characteristics of plant species, cultivars,
or populations. Of all the controlling factors,
plants play a decisive role in dominating metal
bioavailability through specific activity causing
metal mobilization such as release of organic
acids and phytosiderophores (Lombi, Wenzel,
Gobran, & Adriano, 2001). It is commonly
observed that the formation of metal chelates or
complexes in soils may result in easier uptake of
soil Cd by plants and more effective transport of
Cd in plants (Tiffin, 1970, 1972; Senden &
Wolterbeek, 1990).
Root exudates released in the rhizosphere are
able to alter the solubility of metals within the
rhizosphere (Cataldo, McFadden, Garland, &
Wildung, 1988). In various root exudates, organic
acids are negative anions under most soil condi-
tions, which allow them to react strongly with
metal ions in soil (Jones & Darrah, 1994; Jones,
Darrah, & Kochian,1996). The interactions of
organic acids with metals in the soil-plant system
may affect solubility and phytoavailability of
metals in soil and their transport in plant. Some
organic acids, such as citric, malic, and oxalic,
have been reported to be potential metal chela-
tors (Naidu & Harter, 1998; Nakayama, 1981).
Our previous studies and studies by others
showed that rice cultivars varied significantly
with regard to Cd uptake and accumulation (Liu
et al., 2003, 2005; Morishita, Fumoto, Yoshizawa,
& Kagawa, 1987; Wu, Chen, & Wang,1999).
However, limited information is available about
the mechanisms of this variation between rice
cultivars.
To attempt to understand these mechanisms,
we studied the relationship between plant Cd
uptake and root secretion of organic acids for
different rice cultivars. Typical rice cultivars with
high or low Cd accumulation abilities were
selected in the present experiment based on the
results of our previous studies (Liu et al., 2003,
2005).
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Environ Geochem Health (2007) 29:189–195
Materials and methods
Soil preparation
The soils for the pot experiments were collected
from uncontaminated fields (0–20 cm). After air-
drying and passing through a 2-mm sieve, the
following soil properties were tested:
1. Particle size using the hydrometer method
2. pH using a pH meter (soil:distilled
water = 1:2)
3. Organic matter using the sequential extrac-
tion method
4. Cation exchange capacity with ammonium
acetate method
5. Total N using the Kjeldahl method (Bremner,
1996)
6. Total Cd using an atomic absorption spectro-
photometer (ASS) following H2O2-HF-
HNO3-HClO4 digestion
Four kilograms of soil were placed in each pot
(18 cm in diameter and 20 cm in height). Cd in the
form of CdCl2 was added to the soil to obtain Cd
levels of 10 and 50 mg Cd kg–1 soil (dry weight).
CdCl2 was dissolved in de-ionized water and
poured into the soil slowly while mixing the soil at
the same time. The thoroughly mixed soils were
stored in pots and submerged in water (2–3 cm
above the soil surface) for a month before rice
seedlings were transplanted. The soils without
added Cd served as controls.
Rice plant materials
Based on our previous studies (Liu et al., 2003,
2005), two cultivars with high (Shan you 63) and
low (Wu yun jing 7) Cd uptake abilities were used
in this experiment. Rice seeds were submerged in
a water bath for about 48 h at room temperature
(20–25C) and germinated under moist conditions
(seeds were covered with two layers of moist
gauze cloth) at 32C for another 30 h and the
germinated seeds were grown in uncontaminated
soils. After 30 days, the seedlings with two tillers
were transplanted into the pots (3 plants per pot).
The pot soil was maintained under flooded
conditions (with 2–3 cm of water above the soil
surface) during the rice growth period.
Environ Geochem Health (2007) 29:189–195
Experimental design
The experiments were carried out in a green-
house, with a 16-h light period and a day/night
temperature of 32–35/23–25C. The pots were
arranged in a randomized complete block design
with five replicates. One gram of urea (containing
46% N) and 1 g of K2HPO4
3H2O were applied
to each pot on two occasions, one on the 3rd day
before seedling transplantation, and the other on
the 20th day after the transplantation.
Determination of Cd concentrations in rice
plants
The whole rice plants were harvested on the 40th
day after seedling transplant. The plant samples
were washed thoroughly with tap water and de-
ionized water, and oven-dried at 70C to a
constant weight. The oven-dried samples were
ground with a stainless steel grinder (FW-100;
Tianjin Taishite Instruments, Tianjin, China) to
pass through a 100-mesh sieve. The Cd concen-
trations of the samples were determined with an
Atomic Absorption Spectrophotometer (Solaar
S4 + Graphite Furnace System 97; Thermo
Elemental, Waltham, MA, USA) following
HNO3-HClO4 (4:1) digestion procedures (Allen,
1989).
Determination of root organic acids secretion
On the 40th day after seedling transplant, soils
were sampled from each pot. Organic acids of the
soil samples were extracted and purified with the
extracting procedure described by Baziramak-
enga Simard and Leroux (1995). Fifteen grams of
soil (fresh weight) were extracted with 100 mM
NaOH for 12 h, filtered through Whatman No.
42, and centrifuged at 15,000 g for 15 min. The
supernatant was acidified to pH 2.5 with 1M HCl
in order to precipitate the humic substances.
After standing for 16 h, the mixture was centri-
fuged at 15,000 g for 15 min. Organic acids were
obtained by extracting the supernatant 3 times
with 10 ml of ethyl acetate for 5 min, evaporating
the solvent to dryness in a rotary evaporator at
60C and redissolving the residue in 3 ml of
distilled water.
191
The concentrations of seven low molecular
weight organic acids (LMWOA), including acetic
acid, propionic acid, formic acid, malic acid,
tartaric acid, oxalic acid, and citric acid, in the
soil extracts were determined using an ion chro-
matograph (Dionex Dx 500; Dionex) equipped
with a U6K injector, a GP40 pump and a CD20
conductivity detector, and a ASRS-ULTRA
anion micromembrane suppressor. The seven
LMWOA are the main LMWOA excreted by
crop roots (Jones et al., 1996; Takijima, 1964).
Columns were purchased from Dionex. The
LMWOA was separated with an IonPac AS-11
(4 · 250 mm) ion-exchange column, an IonPac
AG-11 (4 · 50 mm) guard column, and an IonPac
ATC-3 (9 · 24 mm) anion-trap column. The
organic acid standards were purchased from
Sigma Chemical Company.
Data were analyzed using the statistical pack-
age SPSS 10.0 and Excel 2000 for Windows. Two
significance levels, 0.05 and 0.01, were used in the
presentation of the results.
Results and discussion
The properties of the soil used in this experiment
are shown in Table 1. It was a silty loam with a
low portion of sand and an acidic pH. The soil
contained a moderate level of organic matter,
CEC and nitrogen, and a very low level of Cd.
The elution profile of the seven LMWOA
standard mixtures is shown in Fig. 1. The con-
centrations of the organic acid standards were all
20 mg l–1.
Table 2 shows the contents of six LMWOA
(propionic acid was not detected) in the pot soils
in which two rice cultivars, Shan you 63 and Wu
yun jing 7, were planted at different soil Cd levels.
Shan you 63 is a high Cd accumulator, but Wu
yun jing 7 is not.
The LMWOA concentrations of the soil
planted with Shan you 63 were higher than those
of the soil planted with Wu yun jing 7 at all soil
Cd levels. However, the magnitudes of the
differences between the two cultivars varied with
the kind of LMWOA and depend on soil Cd
concentrations. In the control, the differences
were significant (P < 0.05 or < 0.01) for acetic,
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Table 1 The selected properties of the soil used in this experiment (n = 3)

Soil type Soil texture Particle size (%) pH OMa CECb Total N Total Cd
(g kg–1) (cmol kg–1) (g kg–1) (mg kg–1)
Sand Silt Clay

Paddy soil Silty loam 18.4 42.7 38.9 4.71 26.2 15.9 1.25 NDc
a
Organic matter
b
Cation exchange capacity
c
Not detected

(Shan you 63 was 27.6% higher than Wu yun jing

Fig. 1 Ion chromatogram of organic acid standards
formic, malic, and citric acid, but not significant
(P > 0.05) for tartaric and oxalic acid. Under soil
Cd treatments of 10 mg kg–1 and 50 mg kg–1, the
differences were significant (P < 0.05 or < 0.01)
for all the six LMWOA.
With regard to the cultivar differences in total
concentrations of the six kinds of LMWOA, the
largest was in soil Cd treatment of 10 mg kg–1
(Shan you 63 was 34.5% higher than Wu yun jing
7), followed by 50 mg kg–1 soil Cd treatment
7). The smallest was in the controls (Shan you 63
was 20.6% higher than Wu yun jing 7).
Exudates, especially LMWOA released by
plant root, have the ability to form complexes
with heavy metals, so they are likely to increase
the solubility and phytoavailability of metals in
soil (Jones et al., 1996; Lundstrom, 1993). Yang,
Baligar, Foster, and Martens (1997) reported that
Ni transport and accumulation were closely
related to the transport and accumulation of
organic acids in ryegrass and maize. An increase
in Cr uptake from the Cr3+-treated soils with
increasing supplementation of organic acids may
be ascribed to the interaction of Cr3+ with organic
ligands leading to the formation of mobile
organically-bound Cr3+, and highlights the inter-
actions between Cr3+ and organic acid as a major
contributor for Cr uptake by plants (Srivastava,
Prakash, & Srivastava, 1999).
In our present experiments, the magnitudes of
the cultivar differences in Cd accumulation abil-
ities (Cd concentrations and Cd accumulations)

Table 2 Differences between rice cultivars in organic acid concentrations of pot soil (lg g–1 de-ionized water, n = 3)
Organic acids Control Cd 10a Cd 50b
Shan you 63 Wu yun jing 7 Shan you 63 Wu yun jing 7 Shan you 63 Wu yun jing 7

Acetic acid 57.938 50.016**c 63.067 48.196** 52.202 43.387**

Formic acid 69.570 55.415** 70.501 51.415** 74.000 55.552**
Malic acid 1.649 1.428*d 2.049 1.385** 1.392 1.096**
Tartaric acid 0.747 0.707 NSe 1.316 1.188* 0.883 0.689**
Oxalic acid 1.052 1.032 NS 1.558 0.859** 0.883 0.668**
Citric acid 0.133 0.095** 0.195 0.096** 0.133 0.069**
Total content 131.088 108.693** 138.686 103.138** 129.493 101.462**
a
Soil Cd treatments of 10 mg kg–1
b
Soil Cd treatments of 50 mg kg–1
c
Significant difference between two cultivars at the P = 0.01 level
d
Significant difference between two cultivars at the P = 0.05 level
e
No significant difference between two cultivars

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Environ Geochem Health (2007) 29:189–195
Fig. 2 Relations between organic acid concentrations in
soil and Cd accumulations in rice plants. Different letters
within a Cd treatment indicate significant differences
between two cultivars at the 0.05 level based on the LSD
(least significant difference) test
were in the order: 10 mg kg–1 soil Cd treat-
ment > 50 mg kg–1 soil Cd treatment > than
control. It was consistent with the order of the
differences in total concentrations of the six kinds
of LMWOA in soil. So there were positive
correlations between the LMWOA concentra-
tions in soil and Cd uptake abilities by rice plants
for the two rice cultivars (Fig. 2). The rice cultivar
with higher concentrations of LMWOA in soil
accumulated more Cd in plants (higher Cd
concentration and more Cd accumulation) at all
the soil Cd levels.
193
Cieslinski, Van Rees, Szmigielska, Krishnam-
urti, and Huang (1998) also showed that the total
amount of LMWOA in the rhizosphere soil of the
high Cd-accumulating wheat cultivar was signif-
icantly higher than that of low Cd-accumulating
cultivar, and Cd accumulation by the high and low
accumulating cultivars was proportional to the
levels of LMWOA found in the rhizosphere soil
of each cultivar.
With regard to the composition of the
LMWOA in the present experiments, acetic acid
and formic acid were dominant. They occupied
more than 96% of the total concentration of the
six kinds of LMWOA for the two rice cultivars at
all soil Cd levels. Citric acid only consisted of
about 0.1%. But the relative cultivar differences
were largest for citric acid (Shan you 63 was
40.0%, 103.1%, and 92.8% higher than Wu yun
jing 7 for the control, 10 mg kg–1, and 50 mg kg–1
soil Cd treatments respectively).
It was reported that the root exudates collected
from different plants had different abilities to
mobilize Cd, and the root exudates of Nicotiana
tobacum can mobilize more Cd than those of
Nicotiana rustica and Zea mays (Mench & Mar-
tin, 1991). The affinity of different organic acids
to form complexation with Cd was different
(Nigam, Srivastava, Prakash, & Srivastava, 2001).
Therefore, the role of different kinds of organic
acids secreted by rice roots in Cd uptake requires
further study.
Summary and conclusion
In our present experiment, significant differences
were observed between rice cultivars in the
secretion of LMWOA. LMWOA concentrations
in the soil planted with Shan you 63 (high soil Cd
accumulator) were all higher than those in the soil
planted with Wu yun jing 7 (low soil Cd accumu-
lator) at all the soil Cd levels
The reaction of the rice cultivars in the
secretion of LMWOA to soil Cd treatments also
varied greatly. Under relatively lower soil Cd
treatment (10 mg kg–1, the growth of rice plants
was not affected, and even slightly improved, data
not shown), the secretion of LMWOA was raised
significantly for the high Cd-accumulating
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cultivar, but reduced significantly for the low Cd-
accumulating cultivar. Under relatively higher
soil Cd treatment (50 mg kg–1, the growth of rice
plants was slightly affected, data not shown), the
secretion of LMWOA was hardly affected for the
high Cd-accumulating cultivar, but was signifi-
cantly inhibited for the low Cd-accumulating
cultivar. As a consequence, the magnitudes of
the differences between the two cultivars in
LMWOA secretion were greater under soil Cd
stress, especially in relatively low soil Cd treat-
ment, than the control.
It can be concluded from our present research
that the differences between rice cultivars with
regard to Cd uptake abilities may be related to
their differences in the properties of root
LMWOA secretion, especially in a soil Cd stress
environment. High Cd-accumulating cultivar can
secrete more LMWOA than low Cd-accumulat-
ing cultivar, and therefore move more Cd from
the soil for root uptake.
The magnitudes of the differences between rice
cultivars in LMWOA secretion varied with the
kind of LMWOA. The differences were largest
for citric acid, and least for tartaric acid, but
acetic acid and formic acid played a dominant
role in the composition of LMWOA. Therefore,
the roles of different organic acids in soil Cd
release and root Cd uptake are likely different,
and it is worthwhile carrying on further research.
Acknowledgements The authors would like to thank the
Science and Technology Project of Changzhou
(CS2005003) and the Research Grants Council
(HKBU2181/03M) of Hong Kong for financial support.
References
Allen, S. E. (1989). Analysis of vegetation and other
organic materials. In S. E. Allen (Eds.), Chemical
analysis of ecological materials (pp. 46–61). Oxford:
Blackwell.
Baziramakenga, R., Simard, R. R., & Leroux, G. D.
(1995). Determination of organic acids in soil extracts
by ion chromatography. Soil Biology and Biochemis-
try, 27, 349–356.
Bremner, J. M. (1996). Nitrogen—total. In D. L. Sparks
(Ed.) Methods of soil analysis, part 3. SSSA Book
Series no 5. Madison, WI: SSSA and ASA
Cataldo, D. A., McFadden, K. M., Garland, T. R., &
Wildung, R. E. (1988). Organic constituents and
123
Environ Geochem Health (2007) 29:189–195
complexation of nickel (II), iron (III), cadmium (II)
and plutonium (IV) in soybean xylem exudates. Plant
Physiology, 86, 734–739.
Cieslinski, G., Van Rees, K. C. J., Szmigielska, A. M.,
Krishnamurti, G. S. R., & Huang, P. M. (1998). Low-
molecular-weight organic acids in rhizosphere soils of
durum wheat and their effect on cadmium bioaccu-
mulation. Plant and Soil, 203, 109–117.
Jones, D. L., & Darrah, P. R. (1994). Role of root derived
organic acids in the mobilization of nutrients from the
rhizosphere. Plant and Soil, 166, 247–257.
Jones, D. L., Darrah, P. R., & Kochian, V. L. (1996).
Critical evaluation of organic acid mediated iron
dissolution in the rhizosphere and its potential role in
root iron uptake. Plant and Soil, 180, 57–66.
Kabata-Pendias, A., & Pendias, H. (2001). Trace elements
in soils and plants. Boca Raton: CRC Press.
Liu, J., Li, K., Xu, J., Liang, J., Lu, X., Yang, J., & Zhu,
Q. (2003). Interaction of Cd and five mineral nutri-
ents for uptake and accumulation in different rice
cultivars and genotypes. Field Crops Research, 83,
271–281.
Liu, J., Zhu, Q., Zhang, Z., Xu, J., Yang, J., & Wong, M.
H. (2005). Variations in cadmium accumulation
among rice cultivars and types and the selection of
cultivars for reducing cadmium in the diet. Journal of
the Science of Food and Agriculture, 85, 147–153.
Lombi, E., Wenzel, W. W., Gobran, G. R., & Adriano, D.
C. (2001). Dependency of phytoavailability of metals
on indigenous and induced rhizosphere processes: a
review. In G. R. Gobran, W. W. Wenzel, & E. Lombi
(Eds.), Trace elements in the rhizosphere (pp. 4–23).
Boca Raton: CRC Press.
Lundstrom, U. S. (1993). The role of organic acids in the
soil solution chemistry of a podsolized soil. Soil
Science, 44, 121–133.
Mench, M., & Martin, E. (1991). Mobilization of cadmium
and other metals from two soils by root exudates of
Zea mays L., Nicotiana tobacum L., and Nicotiana
rustica L. Plant and Soil, 132, 187–196.
Morishita, T., Fumoto, N., Yoshizawa, T., & Kagawa, K.
(1987). Varietal differences in cadmium levels of rice
grains of Japonica, Indica, Javanica and hybrid
varieties produced in the same plot of a field. Soil
Science and Plant Nutrition, 33, 629–637.
Naidu, R., & Harter, R. D. (1998). Effect of different
organic ligands on cadmium sorption and extractabil-
ity from soils. Soil Science Society of America Journal,
62, 644–650.
Nakayama, E. (1981). Chemical speciation of chromium
sea water: Effect of naturally occurring organic
materials on the complex formation of chromium
III. Analytica Chimica Acta, 130, 289–294.
Nigam, R., Srivastava, S., Prakash, S., & Srivastava, M. M.
(2001). Cadmium mobilisation and plant availabil-
ity—the impact of organic acids commonly exuded
from roots. Plant and Soil, 230, 107–113.
Senden, M. H. M. N., & Wolterbeek, H. T. (1990). Effect
of citric acid on the transport of cadmium through
xylem vessels of excised tomato stem-leaf systems.
Acta Botanica Neelrlandica, 39, 297–303.
Environ Geochem Health (2007) 29:189–195
Srivastava, S., Prakash, S., & Srivastava, M. M. (1999).
Chromium mobilization and plant availability—the
impact of organic complexing ligands. Plant and Soil,
212, 203–208.
Takijima, Y. (1964). Study on organic acids in paddy field
soils with reference to their effects on the growth of
rice plant. Soil Science and Plant Nutrition, 10, 14–29.
Tiffin, L. O. (1970). Translocation of iron citrate and
phosphorus in xylem exudate of Soybean. Plant
Physiology, 45, 280–283.
Tiffin, L. O. (1972). Translocation of micronutrients in
plants. In J. J. Mortvedt (Ed.), Micronutrients in
195
agriculture (pp. 199–229). Madison: Soil Science
Society of America.
Wu, Q. T., Chen, L., & Wang, G. S. (1999). Differences on
Cd uptake and accumulation among rice cultivars and
its mechanism. Acta Ecologica Sinica, 19, 104–107 (in
Chinese).
Yang, X. E., Baligar, V. C., Foster, J. C., & Martens, D. C.
(1997). Accumulation and transport of nickel in
relation to organic acids in ryegrass and maize grown
with different nickel levels. Plant and Soil, 196, 271–
276.
123