Annals of Botany 111: 1189– 1195, 2013
doi:10.1093/aob/mct072, available online at www.aob.oxfordjournals.org
Root iron plaque formation and characteristics under N2 flushing and its effects
on translocation of Zn and Cd in paddy rice seedlings (Oryza sativa)
Bo Xu1,2 and Shen Yu1,*
1
Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences,
Xiamen 361021, China and 2University of Chinese Academy of Sciences, Beijing 100049, China
* For correspondence. E-mail [email protected]
Received: 14 November 2012 Revision requested: 4 January 2013 Accepted: 14 February 2013 Published electronically: 9 April 2013
† Background and Aims Anoxic conditions are seldom considered in root iron plaque induction of wetland plants
in hydroponic experiments, but such conditions are essential for root iron plaque formation in the field. Although
ferrous ion availability and root radial oxygen loss capacity are generally taken into account, neglect of anoxic
conditions in root iron plaque formation might lead to an under- or over-estimate of their functional effects, such
as blocking toxic metal uptake. This study hypothesized that anoxic conditions would influence root iron plaque
formation characteristics and translocation of Zn and Cd by rice seedlings.
† Methods A hydroponic culture was used to grow rice seedlings and a non-disruptive approach for blocking air
exchange between the atmosphere and the induction solution matrix was applied for root iron plaque formation,
namely flushing the headspace of the induction solution with N2 during root iron plaque induction. Zn and Cd
were spiked into the solution after root iron plaque formation, and translocation of both metals was determined.
† Key Results Blocking air exchange between the atmosphere and the nutrient solution by N2 flushing increased
root plaque Fe content by between 11 and 77 % (average 31 %). The N2 flushing treatment generated root iron
plaques with a smoother surface than the non-N2 flushing treatment, as observed by scanning electron microscopy, but Fe oxyhydroxides coating the rice seedling roots were amorphous. The root iron plaques sequestrated
Zn and Cd and the N2 flushing enhanced this effect by approx. 17 % for Zn and 71 % for Cd, calculated by both
single and combined additions of Zn and Cd.
† Conclusions Blocking of oxygen intrusion into the nutrient solution via N2 flushing enhanced root iron plaque
formation and increased Cd and Zn sequestration in the iron plaques of rice seedlings. This study suggests that
hydroponic studies that do not consider redox potential in the induction matrices might lead to an under-estimate
of metal sequestration by root iron plaques of wetland plants.
Key words: Anoxic conditions, root iron plaque, zinc, cadmium, translocation, rice seedlings, Oryza sativa.
IN T RO DU C T IO N
Wetland plant roots are ubiquitously coated with iron oxides,
namely root iron plaques (Emerson et al., 1999). Formation of
root iron plaque has been considered as a survival strategy for
wetland plants in anoxic and flooded environments (Smolders
and Roelofs, 1996; Chabbi, 1999). Root iron plaques provide a
barrier for metals toxic to wetland plants, such as paddy rice, a
main food source in south-east Asia. Root iron plaque has
been shown to reduce uptake of Al, As, Sb, Pb and Zn in rice
plants (Liu et al, 2004a, b; Chen et al., 2006; Deng et al.,
2010; Lei et al., 2011; Li et al., 2011; Huang et al., 2012). As
Mendelssohn et al. (1995) note, the presence and degree of
root iron plaque formation are controlled by multiple abiotic
and biotic factors. The oxidizing capacity, characterized as
radial oxygen loss (ROL), of plant roots and ferrous ion availability are the most important biotic and abiotic factors, respectively. Wetland plant species, including rice cultivars, show a
significant species-specificity in their ROL capability (Deng
et al., 2009; Yao et al., 2011; Wu et al., 2011). Numerous
wetland plant studies have induced root iron plaque in a
ferrous ion-enriched nutrient solution over a short period of
time, typically 12– 72 h. Mendelssohn et al. (1995) assumed
that low redox potential might promote iron plaque formation
on wetland plant root surfaces, although suspecting that high
or low redox potential might prevent iron plaque formation.
However, to our knowledge, no studies have testifies to the
low redox potential effect on root iron plaque formation by
hydroponic cultures, although numerous studies of the effects
of root iron plaques on metal uptake or translocation in
wetland plants have been conducted with hydroponic cultures.
Iron oxyhydroxide minerals constitution in iron plaque has
been shown to be different in rice roots grown in the field (goethite and lepidocrocite, Chen et al., 1980) versus sand hydroponics
(lepidocrocite only, Bacha and Hossner, 1977). The differences
might be attributed partially to differing anoxic conditions
between field and sand hydroponic cultures rather than to
mineral ageing, high CO2-favouring conditions or Al suppression (Chen et al., 1980). Chabbi (1999) found crystalline goethite coating Juncus bulbosus roots in wetland soils. Wetlands in
Virginia and Maryland, USA, were found in which the plant
rhizsophere had a higher percentage of poorly crystalline
Fe(III) and a lower percentage of crystalline Fe(III) than nonrhizosphere, possibly also explained by redox potential differences,
as a result of, among other things, more active microbial Fe cycling
in the rhizosphere (Weiss et al., 2004). Neubauer et al. (2007)
# The Author 2013. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
For Permissions, please email: [email protected]
1190
Xu & Yu — Root iron plaque induction and function under anoxic conditions
confirmed that iron-oxidizing bacteria could enhance plaque
formation on Juncus effusus roots. Normal root iron plaque
induction in hydroponic cultures allows air exchange between
air and nutrient solution, except Deng et al. (2010) who
150
ab
a
a
b
Leaf
Biomass (mg per plant)
120
90
60
ab
a
a
M AT E R I A L S A N D M E T H O D S
b
Root
Preparation of rice seedlings
30
0
flushed the nutrient solution with N2. However, flushing the nutrient solution with N2 might disturb the oxygen gradient around
plant roots and influence iron plaque formation. We hypothesized greater root iron plaque formation under more reduced
conditions in the hydroponic matrix. Reduction of air intrusion
into hydroponic cultures might change the density or other characteristics of iron plaques on rice seedling roots in comparison
with normal hydroponic cultures. This study implemented an
oxygen intrusion reduction by flushing the headspace with N2
rather than flushing throughout the nutrient solution to test this
hypothesis. The effects of N2 flushing on root iron plaque formation and on translocation of Zn and Cd in rice seedlings were
tested in the study.
+Fe+N2
–Fe+N2
+Fe–N2
–Fe–N2
Treatment
F I G . 1. Effects of FeSO4 addition and N2 flushing on leaf and root biomass of
rice seedlings in hydroponic culture. Error bars represent standard deviation.
Different letters above the bars indicate that differences are significant
among treatments at P , 0.05 by a post-hoc test of Tukey’s HSD (n ¼ 36,
nine replicates). The dashed line represents the mean and the solid line represents the median. 2Fe2N2, control without Fe spiking and N2 flushing;
2Fe+N2, control without Fe spiking but with N2 flushing; +Fe2N2, treatment
with Fe spiking but without N2 flushing; +Fe+N2, treatment with both Fe
spiking and N2 flushing.
Rice seeds (Oryza sativa, ‘Teyou180’, a local cultivar in Fujian
Province, China) were surface-sterilized in 30 % (v/v) H2O2 solution for 15 min, washed thoroughly with deionized water and
then geminated in acid-washed quartz sand. The 2-week-old
seedlings were grown for 3 weeks in a full-strength Hoagland nutrient solution ( pH 5.5), consisting of (mol L21): KNO3, 5 ×
1023; KH2PO4, 1 × 1023; MgSO4.7H2O, 2 × 1023; Ca(NO3)2.
4H2O, 5 × 1023; H3BO3, 4.63 × 1025; MnCl2.4H2O, 9.15 ×
1026; CuSO4.5H2O, 3.2 × 1027; ZnSO4.7H2O, 7.65 × 1027;
H2MoO4.4H2O, 3.85 × 1027; Fe(II)-ethylenediaminetetraacetic
acid (EDTA), 2 × 1025, which was changed every 3 days. The
experiment was carried out in a greenhouse with light exposure
of 12– 14 h d21.
TA B L E 1. Analysis of variance for biomass weight of shoots and roots and concentrations of Fe, Cd and Zn in leaf, root and root
plaque of rice seedling after treatment with FeSO4 addition, N2 flushing and Zn/Cd addition (n ¼ 36; significant effects highlighted
in bold)
Biomass weight
Total Fe
Total Cd
Total Zn
Part
Effect†
d.f.
F value
P,F
F value
P,F
F value
P,F
F value
P,F
Leaf
FeSO4 addition (A)
N2 flushing (B)
Zn/Cd addition (C)
A*B
A*C
B*C
A*B*C
FeSO4 addition (A)
N2 flushing (B)
Zn/Cd addition (C)
A*B
A*C
B*C
A*B*C
FeSO4 addition (A)
N2 flushing (B)
Zn/Cd addition (C)
A*B
A*C
B*C
A*B*C
1
1
2
1
2
2
2
1
1
2
1
2
2
2
1
1
2
1
2
2
2
2.7
6.2
0.5
2.6
1.2
0.8
0.1
7.1
14.2
2.4
0.1
1.7
0.8
0.5
0.113
0.020
0.632
0.119
0.321
0.451
0.937
0.013
0.001
0.116
0.805
0.206
0.470
0.625
69.3
0.0
0.6
0.0
0.2
1.1
0.5
147.0
11.6
0.8
3.6
0.8
1.0
0.6
1240.6
27.7
0.1
22.8
0.1
1.7
1.2
< 0.0001
0.895
0.586
0.939
0.811
0.339
0.630
< 0.0001
0.002
0.465
0.071
0.472
0.370
0.549
< 0.0001
< 0.0001
0.868
< 0.0001
0.930
0.199
0.317
0.4
3.5
92.5
2.1
3.3
1.2
1.2
25.8
28.4
263.4
37.2
10.2
7.4
14.4
132.3
42.7
266.1
9.3
57.2
13.9
2.9
0.519
0.075
< 0.0001
0.157
0.054
0.321
0.313
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
0.003
< 0.001
< 0.0001
< 0.0001
< 0.0001
0.006
< 0.0001
< 0.0001
0.072
6.3
3.8
159.7
0.1
1.5
0.5
0.1
10.2
1.0
134.9
24.5
2.8
0.1
7.6
276.5
7.4
499.9
1.0
62.7
3.4
0.6
0.019
0.062
< 0.0001
0.775
0.250
0.611
0.888
0.004
0.335
< 0.001
< 0.001
0.080
0.906
0.003
< 0.0001
0.012
< 0.0001
0.321
< 0.0001
0.049
0.542
Root
Plaque
†
FeSO4 addition means that the rice seedlings were treated with and without 50 mg Fe2+ L21; N2 flushing means that the flasks with rice seedlings were
flushed with and without N2 to prevent O2 intrusion; metal addition means the rice seedlings were treated with Zn and Cd individually and interactively.
Xu & Yu — Root iron plaque induction and function under anoxic conditions
Iron plaque formation and metal treatment
After 3 weeks in the Hoagland nutrient solution rice seedlings were transferred to deionized water for 1 d to minimize
interference of elements with iron. Seedlings were then
grown in four pots (nine seedlings per pot) with 10 litres fullstrength Hoagland nutrient solution for a 2-d adaption. Four
pots were then treated to form rice root iron plaques for 3 d
as (1) Control: full-strength Hoagland nutrient solution; (2)
FeSO4 addition: full-strength Hoagland nutrient solution with
FeSO4.7H2O (50 mg Fe2+ L21) substituting for Fe-EDTA;
(3) Control with N2 flushing: full-strength Hoagland nutrient
solution flushed with N2 gas at a flow rate of 40 mL min21;
and (4) FeSO4 addition with N2 flushing: full-strength
Hoagland solution with FeSO4.7H2O (50 mg Fe2+ L21) substituting for Fe-EDTA continuously flushed with N2 gas over the
whole period of iron plaque induction at a flow rate of 40 mL
A
IUE 5·0kV 8·2mm ×2·00k SE(M)
min21. The 50 mg Fe2+ L21 as FeSO4.7H2O was determined
when considering ferrous toxicity. N2 flushing was used to
block oxygen intrusion at the air – solution interface. The
pure N2 gas was controlled by a manifold with a gas flowmeter
and microvalves. A preliminary experiment indicated that dissolved oxygen in the N2 bubbled nutrient solution without rice
seedlings remained at 2.6 mg L21 by flushing the pot headspace continuously at 40 mL min21 over 1 h.
The iron-coated roots were imaged using an Hitachi S-4800
field emission scanning electron microscope (Tokyo, Japan) and
their Fe oxyhydroxide minerals were examined using X-ray diffractometry (XRD; X’Pert PRO, Almelo, Netherlands).
In each pot the nine root-iron-coated seedlings were divided
into three groups to grow in small pots with 1 litre full-strength
Hoagland nutrient solution after a thorough rinse in deionized
water. One small pot hosted one rice seedling for a 2-d
B
20·0µm
C
IUE 5·0kV 8·6mm ×2·00k SE(M)
1191
IUE 5·0kV 8·1mm ×2·00k SE(M)
20·0µm
D
20·0µm
IUE 5·0kV 8·4mm ×2·00k SE(M)
20·0µm
F I G . 2. Scanning electron micrographs of iron plaques on rice roots in nutrient solution with or without FeSO4 addition and N2 flushing. (A) FeSO4 addition:
full-strength Hoagland nutrient solution with FeSO4.7H2O (50 mg Fe2+ L21) substituting for Fe-EDTA; (B) FeSO4 addition with N2 flushing: full-strength
Hoagland solution with FeSO4.7H2O (50 mg Fe2+ L21) substituting for Fe-EDTA flushed with N2 gas at a flow rate of 40 mL min21; (C) Control: full-strength
Hoagland nutrient solution; (D) Control with N2 flushing: full-strength Hoagland nutrient solution flushed with N2 gas at a flow rate of 40 mL min21. The N2
flushing was designed to block oxygen intrusion at the air –solution interface. The treatments were carried out for 3 d.
Xu & Yu — Root iron plaque induction and function under anoxic conditions
The fresh roots were extracted for iron plaque using the
dithionite– citrate – bicarbonate method (DCB) (Taylor and
Crowder, 1983). The iron-coated roots of each seedling were
extracted for 60 min at 25 8C in a 30-mL mixture of 0.03 M
sodium citrate (Na3C6H5O7.2H2O), 0.125 M sodium bicarbonate (NaHCO3) and 0.8 g sodium dithionite (Na2S2O4). The
roots were then rinsed three times with deionized water
which was added to the DCB extracts. The extracts were
then transferred to a 100-mL flask and the volume of liquid
brought to 100 mL using deionized water. The well-shaken
extracts were filtered through a Whatman 42 filter prior to
analyses of Fe, Zn and Cd.
After the DCB extraction, roots and leaves were oven-dried at
70 8C to a constant weight. Oven-dried root and leaf samples
were ground prior to digestion with concentrated HNO3HClO4 at 4 : 1 volumetric ratio (Liu et al., 2007a). Plant
samples were placed in digestion tubes and reacted overnight
with a 5 mL mixture of HNO3 and HClO4 (4 : 1, v/v). On the following day, the tubes were heated on a digestion block at 90 8C
for 3 h, then at 140 8C for 5 h and at 180 8C for a further 2 h.
After cooling the digests were transferred into 25-mL flasks
and the volume of liquid brought to 25 mL with deionized
water. Samples were then filtered through a Whatman 42 filter
and collected into polyethylene bottles. A reagent blank and a
standard reference material (tomato, GSBZ 51001-94, Chinese
National Certified Reference Material) were included as a
quality control for the digestion procedure and analysis.
Concentrations of Fe, Zn and Cd in the DCB extracts and plant
tissue digests were determined using a flame atomic absorption
spectrophotometer (FAAS Solaar M6; Thermo Electron Corp.,
Waltham, MA, USA).
Statistical analysis
Factorial analyses of variance (factorial ANOVAs) were
used to test differences in metal concentrations in iron
plaque, root and leaf tissues, under the various treatment conditions. A post-hoc test for means was carried out using
Tukey’s HSD method at a significant level of ,5 %. The statistical analyses were conducted using SAS software (ver. 9.1.3
for Windows, SAS Institute Inc., Cary, NC, USA).
RES ULT S
Responses of rice seedlings and root iron plaque to N2 flushing
in the headspace
N2 flushing slightly reduced both root and leaf biomass of rice
seedlings, but only significantly without FeSO4 addition (P ,
0.05, n ¼ 36) (Fig. 1 and Table 1). Regardless of the addition
Intensity (counts)
800
600
400
200
0
20
40
60
Degrees 2q (Cu, Ka)
80
F I G . 3. Amorphous Fe oxyhydroxides in root iron plaques of rice seedlings
detected by XRD. The root iron plaques were induced in a ferrous-enriched
Hoagland solution (50 mg Fe2SO4 L21) with N2 flushing in the headspace for 72 h.
2·5
Leaf Fe content
(g kg–1 d. wt)
Analysis of Fe, Zn, and Cd in root, leaf, and iron plaque
1000
2·0
1·5
1·0
a
a
0·5
0
Root Fe content
(g kg–1 d. wt)
adaptation in deionized water. Each group of three replicates
was then treated with metal addition for 3 d. Treatments
included Zn at 200 mg L21, Cd at 1 mg L21, or a combination
of the two (Zn + Cd) in full-strength Hoagland nutrient solution. The treated rice seedlings were harvested, rinsed with
deionized water, and divided into leaf and root portions prior
to analyses of Fe, Zn and Cd.
b
b
a
2·0
b
1·5
1·0
c
0·5
c
0
Plaque Fe content
(g kg–1 root d. wt)
1192
200
a
150
b
100
50
c
0
+Fe+N2
c
–Fe+N2
+Fe–N2
–Fe–N2
Treatment
F I G . 4. Effects of FeSO4 addition and N2 flushing on Fe loadings in (A) leaf,
(B) root and (C) root plaque of rice seedlings in hydroponic culture. Error bars
represent standard deviation. Total Fe contents in root plaque were presented
on the basis of dry root weight. Different letters above the bars indicate that
differences are significant among treatments at P , 0.05 by a post-hoc test
of Tukey’s HSD (n ¼ 36, nine replicates). The dashed line represents the
mean and the solid line represents the median. 2Fe2N2, control without Fe
spiking and N2 flushing; 2Fe+N2, control without Fe spiking but with N2
flushing; +Fe2N2, treatment with Fe spiking but without N2 flushing;
+Fe+N2, treatment with both Fe spiking and N2 flushing.
Xu & Yu — Root iron plaque induction and function under anoxic conditions
or not of FeSO4, smooth iron plaques were observed on the
root surface with the N2 flushing and a relatively coarse
surface of iron plaques was observed without N2 flushing
(Fig. 2). However, these Fe oxyhydroxides on the rice root
surface were amorphous, even with N2 flushing (Fig. 3). N2
flushing significantly enhanced Fe density in root iron plaque
(P , 0.0001, n ¼ 36, Table 1), on average by 31 % (range
11– 77 %, n ¼ 36) with FeSO4 addition (Fig. 4). Meanwhile,
mean root Fe content increased over 50 % with N2 flushing
(P , 0.05) while leaf Fe content did not show an significant
changes (Fig. 4). On the other hand, FeSO4 addition significantly increased Fe contents in leaf, root and plaque of rice
seedlings (Fig. 4). However, Zn and Cd additions did not
show significant effects on Fe contents in rice seedlings and
root plaques (Table 1).
Zn and Cd in root iron plaques and transportation
in rice seedlings
Although single and combination additions of Zn and Cd
did not influence rice seedling growth and iron plaque formation, Zn was doubly entrapped by root iron plaques versus
non-iron plaques regardless of whether it was added singly
or in combination with Cd (Fig. 5). Cd in root iron plaques
was dramatically increased with FeSO4 addition, by approx.
1·2
Zn
Zn+Cd
ns
ns
101– 519 % (P , 0.05, n ¼ 12) when Cd was added alone
and by 54– 224 % when Cd was added with Zn (Fig. 5). N2
flushing significantly increased Cd in root iron plaques, but
not Zn (P , 0.05, Fig. 5).
Both Zn and Cd contents in leaves of rice seedlings treated
by the metal singly showed only slight effects of root iron
plaque formation; i.e. with iron plaques Zn and Cd contents
in leaves were slightly lower than without iron plaques, but
not significant so, and N2 flushing did not enhance the
effects. The combination addition of Zn and Cd was an exception, which did not lower Cd content in leaves (Fig. 5).
However, Cd contents in roots showed significant iron
plaque effects, i.e. the control treatment (without FeSO4 addition or N2 flushing) had significantly greater Cd contents in
roots than other treatments (Fig. 5). This was the case for Zn
only when it was added with Cd together (Fig. 5). Similarly,
N2 flushing did not enhance the iron plaque effects on Cd
and Zn contents in rice seedling roots.
DISCUSSION
We hypothesized that more iron oxides would be deposited on
root surfaces in the more reduced hydroponic matrix. N2 flushing, a simple non-disturbance treatment blocking air exchange
between the atmosphere and nutrient solution, significantly
0·9
0·6
0·3
0·9
a
ab
0·6
b
c
0·3
Root Cd content
(mg kg–1 d. wt)
ns
1·2
0
ns
20
15
10
5
b
4·0
b
b
a
b
b
100
b
b
50
b
b
b
b
a
a
a
6·0
a
150
0
a
a
Plaque Zn content
(g kg–1 root d. wt)
ns
25
0
b
2·0
0
Plaque Cd content
(mg kg–1 root d. wt)
Root Zn content
(g kg–1 d. wt)
0
Zn+Cd
Cd
30
Leaf Cd content
(mg kg–1 d. wt)
Leaf Zn content
(g kg–1 d. wt)
1·5
1193
600
b
400
a
c
200
c
b
0
N2
+
Fe
+
N2
N2
N 2 –N 2
N 2 –N 2
N2
e–
e–
e
e
e+
e+
–F
–F
+F
+F
–F
+F
Treatment
–
+
Fe
N2
e+
+F
N2
N2
N 2 –N 2
N 2 –N 2
N2
e–
e–
e
e
e+
e+
–F
–F
+F
–F
+F
+F
Treatment
e+
–F
F I G . 5. Effects of FeSO4 addition and N2 flushing on Zn and Cd loadings in leaf, root and root plaque of rice seedlings in hydroponic culture. Error bars represent
standard deviation. Total contents of Zn and Cd in root plaque were presented on the basis of dry root weight. Different letters above the bars indicate that differences
are significant among treatments at P , 0.05 and ‘ns’ represents ‘not significant at P , 0.05’ by a post-hoc test of Tukey’s HSD (n ¼ 12, three replicates). 2Fe2N2,
control without Fe spiking and N2 flushing; 2Fe+N2, control without Fe spiking but with N2 flushing; +Fe2N2, treatment with Fe spiking but without N2 flushing;
+Fe+N2, treatment with both Fe spiking and N2 flushing.
1194
Xu & Yu — Root iron plaque induction and function under anoxic conditions
facilitated more Fe deposition on the root surface of rice seedlings in this study. This is also the case as compared with other
studies with comparable initial ferrous ion concentration in
hydroponic culture without considering redox potential but
short induction periods (12 or 24 h) (Liu et al., 2004a;
Huang et al., 2012). The smooth iron plaque surface with N2
flushing treatments observed by SEM (Fig. 2) leads to a hypothesis that lowering redox potential by N2 flushing might
result in a mineralogical change of Fe oxyhydroxides in root
iron plaques or its degree of crystallinity. Field studies indicated that crystalline Fe oxides occur in iron plaques, such
as ferrihydrite (63 %), goethite (32 %) and siderite (5 %) in
root iron plaques of Phalaris arundinacea (Hansel et al.,
2001) and crystalline goethite dominating root iron plaques
of Juncus bulbosus (Chabbi, 1999). However, XRD indicated
that Fe oxyhydroxides in root iron plaques with N2 flushing in
this study were amorphous (Fig. 3). Improving the crystallinity
of oxyhydroxides in root iron plaques requires a relatively long
ageing time (Chen et al., 1980). Root iron plaque formation in
this study occurred in a relatively short period of 72 h, which is
not long enough to test for N2 flushing changing the crystallinity of oxyhydroxides in root iron plaques. As Weiss et al.
(2004) have shown, oxyhydroxides in root iron plaques of
Typha were poorly crystallized even in the field, comprising
18 % crystalline Fe and 66 % poorly crystalline Fe.
Although oxyhydroxides in root iron plaques were amorphous with N2 flushing, sequestrations of Zn and Cd by root iron
plaques were enhanced by N2 flushing (Fig. 5). These results
matched positively with increased Fe density in root iron
plaques with N2 flushing. This suggests that N2 flushing blocking air exchange between the atmosphere and solution matrix
lowered air oxygen diffusion in the nutrient solution matrix,
promoted root iron plaque formation by root radial oxygen gradient and altered its metal sequestration capacity. The reduced
difference between Cd concentration in rice shoots of hydroponic and soil pot culture by Liu et al. (2007b, 2008) strongly
supports our findings. The Cd concentration in shoots of rice
seedlings was not significantly different with and without
iron plaque induction in the hydroponic culture (Liu et al.,
2007b) but was significantly reduced by iron plaques in the
soil pot culture (Liu et al., 2008). Therefore, previous studies
that induced root iron plaques in ferrous ion-enriched solution
without considering oxygen intrusion effect from the headspace to the nutrient solution might have underestimated the
capacity of iron plaques to sequester toxic metals. However,
in a greenhouse study, 30-d-old rice seedlings grown in contaminated paddy soils had 30– 88 % As as well as 11– 43 %
Cd and 14– 40 % Pb sequestrated in their root iron plaques
(Lei et al., 2011), but Liu et al. (2004a) indicated that 75–
89 % As(V) (initially treated at 0.5 mg L21) was sequestrated
by root iron plaque (12-h induction) of rice seedlings in hydroponic culture. Although As(V) retention in root iron plaque of
paddy rice could be regulated by phosphorus level (Liu et al.,
2004b), the comparable sequestration rates of As(V) in root
iron plaques between rice seedlings grown in the contaminated
paddy soil and induced in ferrous ion-enriched nutrient solution might suggest factors other than redox potential in the
matrix regulating root iron plaque functions, such as exposure
levels or availability of toxic metals. Deng et al. (2010) found
that As(V) uptake in rice seedlings was reduced only by iron
plaque (24-h induction) at low As(V) concentrations
(,2 mg L21) but was increased at high As(V) concentrations
(.6 mg L21). These studies suggest that high exposure levels
of toxic metals might seriously impact plant growth and root
cell permeability, and further influence formation of root
iron plaque and reduce its sequestration rate of toxic metals.
In this study, both Zn and Cd treatments and addition of
ferrous ion did not cause any significant impacts on rice seedling growth (Fig. 1 and Table 1).
Additionally, matrix pH might influence metal sequestration
in root iron plaques; for example, Cu and Mn sequestrated by
root iron plaque of Phragmites australis were higher at pH 6.0
than at pH 3.5 in the matrix (Batty et al., 2000). The effect of
different genotypes on on toxic metal sequestration and translocation by iron plaques within rice seedlings should also be
considered (Liu et al., 2004a).
In short, this study indicates that redox potential in the
matrix is an important factor influencing root iron plaque formation and its characteristics, further shifting root iron plaque
functions in regulating metal translocation in plants. Previous
studies of metal sequestration by root iron plaque induced
without consideration of anoxic conditions of the matrix
might underestimate root iron plaque functions on metal sequestration in the field. This is a preliminary study on redox
potential effects on root iron plaque formation and its metal sequestration. To what extent the artificially induced root iron
plaques can explain the ‘real story’ of metal retention in the
field requires further studies.
ACK N OW L E DG E M E N T S
We thank Dr Beth Ravit from Rutgers, The State University of
New Jersey, for comments. This study was financed by Chinese
Academy of Sciences ‘Knowledge Innovation Programs’
(KZCX2-YW-JC402) and the National Science Foundation of
China (40871244 and 31070463).
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