Arsenic–iron interaction: Effect of additional iron on arsenic-induced chlorosis in barley grown in water culture

ORIGINAL ARTICLE

Arsenic–iron interaction: Effect of additional iron onarsenic-induced chlorosis in barley grown in water culture

Molla R. SHAIBUR1, Nobuyuki KITAJIMA2, S. M. IMAMUL HUQ3

and Shigenao KAWAI4

1The United Graduate School of Agricultural Sciences, Iwate University, Morioka 020-8550, 2Fujita Corporation, Atsugi City,

Kanagawa 243-0125, 4Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan, and 3Bangladesh–Australia Centre for

Environmental Research, Department of Soil, Water and Environment, University of Dhaka, Dhaka 1000, Bangladesh

Abstract

The effect of additional iron (Fe) on arsenic (As) induced chlorosis in barley (Hordeum vulgare L. cv. Minori-

mugi) was investigated. The treatments were: (1) 0 lmol L)1 As + 10 lmol L)1 Fe3+ (control), (2)

33.5 lmol L)1 As + 10 lmol L)1 Fe3+ (As-treated) and (3) 33.5 lmol L)1 As + 50 lmol L)1 Fe3+ (additional-

Fe3+) for 14 days. Arsenic and Fe3+ were added as sodium-meta arsenite (NaAsO2) and ethylenediaminetetra-

acetic acid-Fe3+, respectively. Chlorosis in fully developed young leaves was observed in the As-treated plants.

The chlorophyll index and the Fe concentration decreased in shoots of the As-treated plants compared with the

control plants. Arsenic reduced the concentration of phosphorus, potassium, calcium, magnesium, manganese,

zinc and copper. The additional-Fe3+ treatment increased the chlorophyll index in plants compared with the As-

treated plants. Among the elements, Fe concentration and accumulation specifically increased in the shoots of

additional-Fe3+ plants compared with As-treated plants, indicating that As-induced chlorosis was Fe-chlorosis.

Arsenic and Fe were mostly concentrated in the roots of the As-treated plants. Despite inducing chlorosis in the

As-treated plants, phytosiderophores (PS) accumulation in the roots and release from the roots did not increase,

rather PS accumulation decreased, indicating that As toxicity hindered PS production in the roots. The PS accu-

mulation in the roots was further reduced in the additional-Fe3+ treatment.

Key words: arsenic, chlorophyll index, chlorosis, iron, phytosiderophores.

INTRODUCTION

Arsenic (As) is widely distributed in nature and occurs in

soil, water, air, plants and animals (Mandal and Suzuki

2002). Arsenic is the 20th most abundant element in the

earth’s crust (Shemirani et al. 2005) and the second most

common inorganic constituent after lead (Pb) in the Uni-

ted States Environmental Protection Agency (USEPA)

National Priority List (United States Environmental Pro-

tection Agency 2001), which includes in excess of 2000

contaminated sites that pose environmental health risks

(Davis et al. 2001). Severe As problems in groundwater

have been found in Bangladesh, West Bengal (Indian),

China and Taiwan (World Health Organization 2001). In

Bangladesh, As-contaminated underground water is being

used to irrigate crops. Arsenic-contaminated water causes

toxic effects to plants (Huq et al. 2003), for example,

whitish chlorosis in barley leaves (Shaibur et al. 2008b).

Studies are required to ascertain the reason for the chloro-

sis induced by As in barley.

Iron oxides and hydroxides could reduce the lability of

As and could effectively be used to attenuate As in As-con-

taminated soil (Hartley et al. 2004). It has been reported

that Fe oxide can decrease approximately 50% of water-

extractable As in garden soil (Mench et al. 1998). The

first goethite has been shown to reduce As toxicity in con-

taminated soil (Sun and Doner 1998). Carbonell-Barra-

china et al. (2000) demonstrated that water-soluble

Fe-hydrous oxides controlled the As adsorption–desorption

reaction in sludge. Ferrous sulfate (Artiola et al. 1990)

and amorphous Fe hydroxide (am-Fe(OH)3) also have a

high adsorptive capacity for As (Vangronsveld et al.

1994).

We have shown that As can induce whitish chlorosis in

fully developed young leaves of barley at 33.5 lmol L)1

Correspondence: S. KAWAI, Faculty of Agriculture, Iwate Uni-versity, Morioka 020-8550, Japan. Email: [email protected]

Received 11 February 2009.Accepted for publication 19 August 2009.

� 2009 Japanese Society of Soil Science and Plant Nutrition

Soil Science and Plant Nutrition (2009) 55, 739–746 doi: 10.1111/j.1747-0765.2009.00414.x

(Shaibur et al. 2008b). Arsenic may induce Fe-deficiency

if the Fe movement from the root to shoots is reduced by

high As content in the growth medium. In 1843, Griss first

observed that the young leaves of plants show chlorosis at

deficient levels of Fe (Wallace and Lunt 1960). The defini-

tion of Fe-chlorosis is that if the chlorosis is alleviated

with additional Fe, it is Fe-chlorosis (Shenker and Chen

2005). It is known that graminaceous plants release non-

proteinogenic amino acid phytosiderophores (PS) under

Fe-deficient conditions (Takagi et al. 1984). Further stud-

ies investigating PS are necessary in relation to Fe physiol-

ogy in Gramineae grown under As-contaminated

conditions. Furthermore, we have shown that the concen-

trations of manganese (Mn), zinc (Zn) and copper (Cu) in

the shoots of barley were decreased by As at a level of

33.5 lmol L)1 and we suggested that As-induced chloro-

sis was not the result of heavy metal induced Fe-deficiency

(Shaibur et al. 2008b).

In the present experiment we added an additional

40 lmol L)1 Fe3+ with the 33.5 lmol L)1 As treatment to

obtain data demonstrating that As-induced chlorosis was

Fe-chlorosis. We measured the PS, growth and chloro-

phyll content to investigate physiological changes in

As-induced chlorosis in barley.

MATERIALS AND METHODS

Seedling preparation

Seedlings of barley (Hordeum vulgare L. cv. Minorimugi)

were grown as previously described and a 33.5 lmol L)1

As concentration was chosen because the leaves were most

chlorotic at this concentration (Shaibur et al. 2008b). The

plants were grown for up to 14 days after treatment

(DAT) and the applied treatments were: (1) 0 lmol L)1

As + 10 lmol L)1 Fe3+ (control), (2) 33.5 lmol L)1 As +

10 lmol L)1 Fe3+ (As-treated) and (3) 33.5 lmol L)1

As + 50 lmol L)1 Fe3+ (additional-Fe3+). Arsenic and

Fe3+ were added as sodium-meta arsenite (NaAsO2) and

ethylenediaminetetraacetic acid-Fe3+, respectively. The pH

was adjusted to 6.5 using 1 mol L)1 NaOH or 1 mol L)1

HCl after testing with a digital pH meter (Horiba Korea,

Seoul, Korea) every 24 h.

Chlorophyll index (SPAD value)

The chlorophyll index of the fully developed third leaf

showing whitish chlorosis on 14 DAT was measured

using a SPAD-502 chlorophyll meter (Minolta Camera

Company, Tokyo, Japan).

Collection and measurement of the phytosidero-phores released or accumulated in the roots

The collection and measurement of PS were carried out

using the methods of Takagi (1976). Roots of a bunch of

plants were soaked in beakers containing 500 mL deion-

ized water for 3 h starting at 08.00 AM on 14 DAT. The

concentration of PS in the lyophilized roots was measured

as previously described (Kawai et al. 1993).

Other parameters

The analysis of the plant samples, the As determination

and experimental set up were described in our previous

studies (Shaibur et al. 2008b, 2009).

Calculations for the parameters

The PS accumulation is expressed in lg PS g)1 root dry

weight (DW). The concentration of an element is defined

as the amount of the element g)1 dry weight (mg or lg ele-

ment g)1 DW), and accumulation refers to the total

amount of element plant)1 shoot or plant)1 root (mg or

lg of element plant)1).

Statistical analyses

The data were subjected to ANOVA. Differences between

means were evaluated using a Ryan–Einot–Gabriel–Wel-

sch multiple range test (P < 0.05) (SAS 1988) using com-

puter origin 5 of Iwate University, Japan.

RESULTS AND DISCUSSION

Visible symptoms in the shoots and roots ofAs-treated barley

Recently, we reported the visible symptoms of As-treated

hydroponic barley (Shaibur et al. 2008b) and rice (Shai-

bur et al. 2006). In the present experiment, chlorosis

induced by As was partially reduced in the additional-Fe3+

treatment, indicating that As-induced chlorosis was

Fe-chlorosis. Other possible reasons for the chlorosis may

be Mn, Zn or Cu deficiency or other heavy metal induced

chlorosis (Marschner 1998). In the roots, a reddish color

appeared in the As-treated condition, most probably

because of the formation of Fe plaque (Chen et al. 2005;

Liu et al. 2005).

Dry matter yield

The highest shoot dry weight (DW) was recorded in the

control and the lowest was recorded in the As-treated

plants (Fig. 1a). In the presence of additional Fe3+, the

shoot DW did not increase (Fig. 1a), indicating that As

toxicity in barley shoots at 33.5 lmol L)1 level could

not be recovered by additional Fe3+. The As in the nutri-

ent solution was accountable for an almost 44% shoot

DW reduction, but the value for the roots was almost

12%, indicating that the shoots were more sensitive to

As toxicity than the roots in barley (Shaibur et al.

2008b). The reduction in the shoot DW most probably

resulted from a reduction in shoot height, tiller number,


740 M. R. Shaibur et al.

leaf number and width of the leaf blade caused by As

toxicity (Figs 1b,2). The dry weight reduction in the As-

treated shoot also probably resulted from a reduction in

net photosynthesis and photosynthetic capacity in the

shoots (Rahman et al. 2007) and not in the roots. Marin

et al. (1993) found that As at 0.8 and 1.6 mg L)1 levels

(dimethyl arsenic acid [DMAA]) decreased net photosyn-

thesis and photosynthetic capacity, thereby decreasing

growth. It has been reported that As inhibits respiration

by blocking the electron transport chain of mitochondria

or uncoupling oxidative phosphorylation (Siegel and Sis-

ler 1977). Abedin et al. (2002) found a considerable

reduction in straw and root biomass with 4 and

8 mg As L)1 in rice.

Shoot height, root length, tiller number, leafnumber and width of the leaf blade

Shoot height and root length (Fig. 1b) decreased in

As-treated plants compared with the control plants. In

addition, tiller number (Fig. 2a), leaf number (Fig. 2b)

and the width of leaf blade (Fig. 2c) decreased with As

compared with the control plants. Additional Fe3+ did not

increase shoot height (Fig. 1b), tiller number (Fig. 2a),

leaf number (Fig. 2b) or the width of the leaf blade

(Fig. 2c) compared with the As-treated plants, indicating

that the additional-Fe3+ treatment did not reduce the

effect of As toxicity in the shoots. The dry weight of the

roots (Fig. 1a) was not really affected by the treatments.

However, the decrease in root length observed in the As

treatment was recovered in the additional-Fe3+ treatment

(Fig. 1b). The reason for this alleviation in root length in

the additional-Fe3+ treatment is not known.

Chlorophyll index (SPAD value)

The chlorophyll index decreased significantly in the

As-treated plants compared with the control plants

(Fig. 3a). Similar results have been obtained in hydro-

ponic barley, rice and sorghum (Shaibur et al. 2006; Shai-

bur et al. 2008a,b). The chlorophyll index increased in

the additional-Fe3+ treatment plants compared with the

As-treated plants, but was still lower than the index

recorded in the control plants (Fig. 3a). A partial reduc-

tion in chlorosis in the additional-Fe3+ treatment was

observed. This result suggested that As hindered chloro-

phyll formation by decreasing the Fe concentration

(Table 1) in shoots, and this might result from problems

in Fe translocation (Table 2).

Phytosiderophore accumulation and release

The control plants accumulated the highest amount of PS

in the roots (Fig. 3b). In As-treated plants, despite the

occurrence of Fe-chlorosis in young leaves, the accumula-

tion and release of PS did not increase; rather PS accumula-

tion decreased compared with the control plants (Fig. 3b),

indicating a toxic effect of As on PS accumulation in roots.

In the presence of additional Fe3+, PS accumulation further

Figure 1 (a) Dry matter yield and (b) shoot height and rootlength of barley seedlings with different treatments of arsenic(As) and ethylenediaminetetraacetic acid-Fe3+. Bars with differentletters are significantly different (P < 0.05) according to a Ryan–Einot–Gabriel–Welsch multiple range test.

Figure 2 (a) Tiller number bunch)1, (b) leaf number bunch)1

and (c) leaf blade width of barley seedlings with different treat-ments of arsenic (As) and ethylenediaminetetraacetic acid-Fe3+.Bars with different letters are significantly different (P < 0.05)according to a Ryan–Einot–Gabriel–Welsch multiple range test.


Arsenic–iron interaction 741

decreased compared with the As-treated plants (Fig. 3b).

This may result from a combined effect of As toxicity

(Shaibur et al. 2008b, 2009) and additional Fe3+(Takagi

et al. 1984). We have previously shown that As at a level

of 33.5 lmol L)1 induced chlorosis in barley, but did not

enhance PS accumulation or release in barley grown in a

medium with Fe (Shaibur et al. 2008b). Arsenic at a level

of 67 lmol L)1 decreased PS production and release in

barley (Shaibur et al. 2009). In the current experiment, the

release of PS was not detected, probably because the plants

were grown under Fe3+ conditions. It is possible that As at

a level of 33.5 lmol L)1 reduced the activity of the apical

root (Orwick et al. 1976) and decreased PS accumulation.

The shoot Fe content of barley regulates PS release rates

(Gries et al. 1995) and this release is highly dependent on

metabolic energy (Takagi 1990).

Iron concentration, accumulation andtranslocation

The Fe concentration in the shoots decreased in the

As-treated plants to 44.4 lg g)1 DW compared with the

control plants (80.7 lg g)1 DW) (Table 1), resulting in

chlorosis in the fully expanded young leaves. The critical

deficient level (CDL) of Fe is reported to be 30–50 lg g)1

DW (Bergmann 1988). The Fe concentration increased in

the shoots of the additional-Fe3+ treatment plants com-

pared with the As-treated plants (Table 1). The Fe con-

centration in the shoots of the additional-Fe3+ plants was

69.3 lg g)1 DW and the chlorosis partially disappeared,

indicating that chlorosis was caused by Fe-deficiency

induced by As toxicity.

The Fe concentration in the roots increased in the

As-treated plants to 440 lg g)1 DW compared with

the control plants (281 lg g)1 DW) (Table 1). The Fe

Table 1 Concentration and accumulation of nutrients in the shoots and roots of barley seedlings grown in nutrient solution withdifferent treatments of arsenic and ethylenediaminetetraacetic acid-Fe3+

Treatments (lmol L–1) P K Ca Mg Fe Mn Zn Cu

As EDTA-Fe3+ mg g)1 DW lg g)1 DW

Concentration in shoot

0 10 5.02a 88.3a 5.15a 1.51a 80.7a 23.7a 26.1a 6.66a

33.5 10 1.20b 60.9b 3.60b 1.11b 44.4c 12.7b 15.0b 3.34b

33.5 50 0.32c 58.5b 3.90b 1.08b 69.3b 11.1b 16.1b 1.89c

Concentration in root

0 10 9.84a 80.0a 1.90a 1.67a 281c 44.7a 33.8a 10.8a

33.5 10 5.64b 43.0b 1.26b 1.08b 440b 13.6b 29.8b 5.83b

33.5 50 4.97b 41.0b 1.34b 0.97b 675a 13.4b 22.5c 2.50c

mg plant)1 lg plant)1

Accumulation in shoot

0 10 0.593a 10.4a 0.62a 0.181a 9.53a 2.77a 3.08a 0.79a

33.5 10 0.082b 4.12b 0.24c 0.075b 3.01c 0.86b 1.01b 0.22b

33.5 50 0.025c 4.48b 0.30b 0.084b 5.30b 0.85b 1.24b 0.14c

Accumulation in root

0 10 0.304a 2.48a 0.059a 0.052a 8.73c 1.40a 1.05a 0.34a

33.5 10 0.156b 1.19b 0.035b 0.030b 12.2b 0.38c 0.82b 0.16b

33.5 50 0.161b 1.37b 0.045ab 0.033b 22.8a 0.45b 0.76b 0.10c

Means followed by different letters in each column are significantly different (P = 0.05) according to a Ryan–Einot–Gabriel–Welsch multiple range test.Concentration is defined as mg or lg of element g)1 dry weight (DW); accumulation is defined as mg or lg of element plant)1 shoot or root. Accumulationwas calculated by multiplying the concentration value by the DW of the plant samples. EDTA, ethylenediaminetetraacetic acid.

Figure 3 (a) Chlorophyll index (SPAD value) in fully developedyoung leaves and (b) the phytosiderophores (PS) concentration inthe roots of barley seedlings with different treatments of arsenic(As) and ethylenediaminetetraacetic acid-Fe3+. Bars with differentletters are significantly different (P < 0.05) according to a Ryan–Einot–Gabriel–Welsch multiple range test. The roots were col-lected just before the PS release time and the PS accumulation inthe roots was measured on a root dry weight (DW) basis.



concentration further increased in the roots in the addi-

tional-Fe3+ plants to 675 lg g)1 DW compared with the

roots of the As-treated plants. Because As has a high affin-

ity to the sulfhydryl group of root proteins (Speer 1973),

As may be bound with the protein and repress the func-

tion of the root membrane. In addition, Fe3+ has high

affinity to adsorb As (Hartley et al. 2004). Therefore, a

Fe–As complex may be formed and adsorbed to the cell

wall or the membrane of the roots. The increase in the Fe

concentration in the roots of the As-treated plants most

likely resulted from the formation of Fe plaque (Yamane

1989). Reddish-colored Fe plaque is formed on the root

surface of aquatic plants by the oxidation of Fe on the

root surface (Armstrong 1967; Chen et al. 1980). Forma-

tion of reddish-colored Fe plaque (Chen et al. 2005; Liu

et al. 2005) and an increase in Fe and As concentrations

in the roots of As-treated plants has been described previ-

ously (Shaibur et al. 2006, 2008a,b, 2009). It is also possi-

ble that the root might absorb more Fe3+ under higher

Fe3+ conditions, resulting in a high Fe concentration and

accumulation in roots compared with the As-treated

plants (Table 1). However, for the purpose of discussing

Fe absorption, the amount of absorbed Fe in the roots

needs to be measured after the removal of the apoplastic

Fe in the roots. In future studies, we will measure the con-

tent of absorbed Fe in roots without apoplastic Fe.

Arsenic reduced the translocation of Fe from the roots

to the shoots in As-treated plants compared with control

plants, resulting in a low concentration and accumulation

of Fe in the shoots and a high concentration and accumu-

lation of Fe in the roots. Iron translocation was the most

affected (>50%; almost 2.5-fold lower than the control)

among the elements in the As-treated plants (Table 2).

Concentration, accumulation and translocationof other elements

Phosphorus

The concentration and accumulation (Table 1) of P

decreased significantly in the shoots and roots of As-trea-

ted plants compared with the control plants. The concen-

tration of P in the shoots of the As-treated plants

(1.20 mg g)1 DW) was within the range of CDL of P in

shoots (1–2 mg g)1 DW; Mengel and Kirkby 2001),

whereas the concentration in the control plants was

5.02 mg g)1 DW. It is known that arsenite has antagonis-

tic interactions with P in nutrient ⁄ soil solution (Woolson

et al. 1973) and within the plant (Wallace et al. 1980).

Some arsenite in the medium might be converted to arse-

nate under experimental conditions with aeration. Arse-

nate and phosphate may also compete with each other

during uptake by the roots because arsenate is taken up

by the phosphate transport system (Rahman et al. 2008).

Arsenic partially decreased the concentration, accumula-

tion and translocation of P (Tables 1,2).

The concentration and accumulation of P (Table 1) fur-

ther decreased in the shoots of additional-Fe3+ plants

(0.32 mg g)1 DW) compared with As-treated plants,

which is the effect of Fe on P. It is well known that Fe has

an antagonistic relationship with P. The lower concentra-

tion of P in the additional-Fe3+ treatment plants may acti-

vate Fe in the shoots (Pushnik et al. 1984) and the

formation of chlorophyll may be elevated in additional-

Fe3+ plants compared with As-treated plants. Transloca-

tion of P was negatively affected in the As-treated plants

(34%) and in the additional-Fe3+ treated plants (13%)

compared with the control plants (Table 2). A relation-

ship between As, P and Fe may be involved in the appear-

ance of chlorosis.

Potassium

In the As-treated and additional-Fe3+ treated plants, the

concentration of K significantly decreased in the shoots to

60.9 and 58.5 mg g)1 DW compared with the control

(88.3 mg g)1 DW), respectively (Table 1). Control plants

contained the highest content of K and As-treated plants

contained the lowest (Table 1). The concentration of K in

the shoots was higher than the CDL of the leaves

(23 mg g)1 DW in sweet potato; O’Sullivan et al. 1993).

Potassium-deficiency may not be induced by As. Addi-

tional Fe3+ did not affect the K concentration in the shoots

and roots. Competition between K and As has not been

observed. It is well known that As can block key enzyme

activity by reacting with sulfhydryl groups of protein

Table 2 Translocation (%) of elements from roots to shoots in barley seedlings grown in nutrient solution with different treatments ofAs and EDTA-Fe3+

Treatments (lmol L)1)

As P K Ca Mg Fe Mn Zn CuAs EDTA-Fe3+

0 10 0 66a 81a 91a 78a 52a 67a 75a 70a

33.5 10 1.11c 34b 78a 87a 71b 20b 69a 55c 58b

33.5 50 2.61b 13c 77a 87a 72b 19b 65a 62b 56b

aMeans followed by the different letters in each column are significantly different (P = 0.05) according to Ryan–Einot–Gabriel–Welsch multiple range test.Translocation is expressed in percent (%) of element accumulated in shoot to the total accumulation (accumulation in shoot + root).



(Speer 1973), repressing root function (Orwick et al.

1976). Arsenic might block the K absorption site in the

roots. Translocation of K was not affected by additional

Fe3+.

Calcium

Higher plants often contain 5–30 mg Ca g)1 DW (Men-

gel and Kirkby 2001) or 1–50 mg Ca g)1 DW (Marsch-

ner 1998). Dell and Robinson (1993) suggested that the

CDL of Ca is 1.5–2.0 mg g)1 DW in the youngest leaves

of Eucalyptus maculata Hook. Shoots of the control

plants contained 5.15 mg Ca g)1 DW, but As-treated and

additional-Fe3+ plants contained smaller Ca concentra-

tions, 3.60 and 3.90 mg Ca g)1 DW, respectively

(Table 1). The level of Ca in the As-treated and addi-

tional-Fe3+ plants appeared to be in the normal range.

Additional Fe3+ did not recover the concentrations of Ca

in the shoots and roots. Calcium2+ can be absorbed only

by young root tips (Clarkson and Sanderson 1978) and is

absorbed to the negative charge of the phosphate groups

in the membrane lipids (Caldwell and Haug 1982).

Because of an antagonistic relationship between As and P,

P absorption was reduced by As (Rahman et al. 2008).

Therefore, it was inferred that Ca absorption could be

decreased by decreasing phosphate absorption in the roots

and accumulation to the shoots. Translocation of Ca,

however, was not affected by additional Fe3+.

Magnesium

Similarly to Ca, the Mg concentration in the shoots

(1.11 mg g)1 DW) was significantly decreased by As com-

pared with the control plants (1.51 mg g)1 DW)

(Table 1). The Mg concentration in the shoots of As-trea-

ted and additional-Fe3+ treated plants was lower than the

CDL (1.5–3.5 mg g)1 DW) (Marschner 1998). It was

suspected that the chlorosis induced by As was

Mg-deficiency. However, the chlorosis induced by As

appeared in the new leaves. Furthermore, additional Fe3+

could not increase the Mg concentration in the shoots and

roots. These results do not support Mg-deficiency in

As-treated plants. Translocation of Mg was not affected

by additional Fe3+(Table 2).

Manganese

The Mn concentration decreased in the shoots and roots

of As-treated and additional-Fe3+ treated plants compared

with the control plants (Table 1). The CDL of Mn (Ohki

1981) for most plant species is in the range 10–20 lg g)1

DW of mature leaves (Mengel and Kirkby 2001).

The concentrations of Mn in the shoots in As-treated and

additional-Fe3+ treated plants were 12.7 and 11.1 lg g)1

DW, respectively, and in the CDL. The concentration of

Mn was similar in the As-treated and additional Fe3+

plants (Table 1); however, the chlorosis partially

disappeared in the additional-Fe3+ treatment. Therefore,

Mn might not be responsible for the chlorosis.

Yamane (1989) found that the Mn concentration in the

roots of rice increased with the application of As (III) and

As (V) at rates of 33.5, 67 and 134 mg kg)1 dry soil. It is

known that divalent Mn is absorbed by facilitated diffu-

sion across the plasmalemma (Fox and Guerinot 1998). It

is possible that As toxicity may hamper the activity of the

root plasmalemma and reduce Mn2+ absorption. Translo-

cation of Mn was not affected by additional Fe3+.

Zinc

The concentration of Zn decreased significantly in the

shoots and roots of the As-treated and additional-Fe3+

treated plants compared with the control plants (Table 1).

The concentration of Zn in the shoots of the control

plants was 26.1 lg g)1 DW and this was a sufficient

amount of Zn (20–100 lg g)1 DW; Boehle and Lindsay

1969), but the concentration of Zn in the shoots of the

As-treated plants was 15.0 lg g)1 DW and this was defi-

cient (10–15 lg g)1 DW; Boehle and Lindsay 1969). The

Zn concentration in the shoots of the additional-Fe3+

plants was 16.1 lg g)1 DW. Additional Fe3+ did not

increase Zn concentration or translocation. Therefore, Zn

may not be responsible for the chlorosis.

Copper

The Cu concentration decreased in the shoots and roots of

the As-treated plants compared with the control plants

(Table 1). The concentration of Cu in the shoots of the

As-treated plants was within the range of the CDL of

Cu (1–5 lg g)1 DW) (Marschner 1998), suggesting

Cu-deficiency. Additional-Fe3+ treated plants showed a

further reduction in Cu concentration and accumulation

(Table 1). The concentration of Cu was within the CDL

in the shoots of the As-treated plants and the Cu concen-

tration further decreased in the additional-Fe3+ treated

plants. Thus, the chlorosis may not be Fe-chlorosis

induced by Cu toxicity. The translocation of Cu was not

affected by additional Fe3+.

Arsenic concentration, accumulation andtranslocation

In the As-treated plants, the As concentration was almost

10 lg g)1 DW in the shoots and 900 lg g)1 DW in the

roots, indicating that the roots contained an almost 90-

fold higher As concentration than the shoots (Fig. 4a).

This result suggested that the roots did not easily permit

translocation of As and, therefore, that As was concen-

trated and accumulated in the roots (Fig. 4a,b). The

arsenic concentrations were similar in the shoots of the

As-treated and additional-Fe3+ treated plants; however,

the As concentration was lower in the roots of the addi-

tional-Fe3+ treated plants compared with the As-treated



plants (Fig. 4a). This result indicated that additional Fe3+

decreased only marginally the concentration of As in the

roots. The translocation of As was increased by additional

Fe3+ (Table 2). Recently, in a separate experiment

(Shaibur et al. 2009), we found that Fe translocation was

increased by increasing the As concentration in the

medium when the plants were treated under Fe-deficient

conditions. This result requires further investigation.

Conclusion

Arsenic induced chlorosis in the fully developed young

leaves of hydroponic barley. The chlorophyll index and

the Fe concentration decreased in the As treatment. Chlo-

rosis and Fe concentration were partially recovered with

additional Fe3+. Arsenic toxicity reduced the concentra-

tions of elements such as P, K, Ca, Mg, Fe, Mn, Zn and

Cu in the shoots. Additional Fe3+ did not change the con-

centrations of K, Ca, Mg, Mn and Zn. Moreover, addi-

tional Fe3+ decreased the concentration of P and Cu in the

shoots. Considering the effect of the additional-Fe3+ treat-

ment on the concentration of the elements and the defini-

tion of Shenker and Chen (2005), As-induced chlorosis

was Fe-chlorosis caused by As toxicity and was not heavy

metal induced Fe-deficiency (Mengel and Kirkby 2001).

Translocation of P was uniquely reduced in the addi-

tional-Fe3+ treatment. Phosphorus may also be involved in

the partial greening of shoots in the additional-Fe3+ trea-

ted plants. The production of PS, which functions in Fe

translocation, was repressed by As and further repressed

by the elevation of the Fe concentration in the medium.

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Arsenic–iron interaction: Effect of additional iron on arsenic-induced chlorosis in barley grown in water culture