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

Abstract

The effect of additional iron (Fe) on arsenic (As) induced chlorosis in barley (Hordeum vulgare L. cv. Minorimugi) was investigated. The treatments were: (1) 0 μmol L⁻¹ As + 10 μmol L⁻¹ Fe³⁺ (control), (2) 33.5 μmol L⁻¹ As + 10 μmol L⁻¹ Fe³⁺ (As-treated) and (3) 33.5 μmol L⁻¹ As + 50 μmol L⁻¹ Fe³⁺ (additional-Fe³⁺) for 14 days. Arsenic and Fe³⁺ were added as sodium-meta arsenite (NaAsO₂) and ethylenediaminetetraacetic 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 accumulation 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 United States Environmental Protection Agency (USEPA) National Priority List (United States Environmental Protection 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 chlorosis 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-contaminated 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 contaminated soil (Sun and Doner 1998). Carbonell-Barrachina 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 μmol L⁻¹ (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 definition 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 studies investigating PS are necessary in relation to Fe physiology in Gramineae grown under As-contaminated conditions. Furthermore, we have shown that the concentrations of manganese (Mn), zinc (Zn) and copper (Cu) in the shoots of barley were decreased by As at a level of 33.5 μmol L⁻¹ and we suggested that As-induced chlorosis was not the result of heavy metal induced Fe-deficiency (Shaibur et al. 2008b).

In the present experiment we added an additional 40 μmol L⁻¹ Fe³⁺ with the 33.5 μmol L⁻¹ As treatment to obtain data demonstrating that As-induced chlorosis was Fe-chlorosis. We measured the PS, growth and chlorophyll 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 μmol 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 μmol L⁻¹ As + 10 μmol L⁻¹ Fe³⁺ (control), (2) 33.5 μmol L⁻¹ As + 10 μmol L⁻¹ Fe³⁺ (As-treated) and (3) 33.5 μmol L⁻¹ As + 50 μmol 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 phytosiderophores 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 deionized 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 μg 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 μg element g⁻¹ DW), and accumulation refers to the total amount of element plant⁻¹ shoot or plant⁻¹ root (mg or μg of element plant⁻¹).

Statistical analyses

The data were subjected to ANOVA. Differences between means were evaluated using a Ryan–Einot–Gabriel–Welsch multiple range test (P < 0.05) (SAS 1988) using computer origin 5 of Iwate University, Japan.

Results and discussion

Visible symptoms in the shoots and roots of As-treated barley

Recently, we reported the visible symptoms of As-treated hydroponic barley (Shaibur et al. 2008b) and rice (Shaibur 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 μmol L⁻¹ level could not be recovered by additional Fe³⁺. The As in the nutrient 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, 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 photosynthesis 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 Sisler 1977). Abedin et al. (2002) found a considerable reduction in straw and root biomass with 4 and 8 mg As L⁻¹ in rice.

Figure 1 (a) Dry matter yield and (b) shoot height and root length of barley seedlings with different treatments 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.

Shoot height, root length, tiller number, leaf number 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.

Figure 2 (a) Tiller number bunch−1, (b) leaf number bunch−1 and (c) leaf blade width of barley seedlings with different treatments 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.

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 hydroponic barley, rice and sorghum (Shaibur et al. 2006; Shaibur 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 reduction in chlorosis in the additional-Fe³⁺ treatment was observed. This result suggested that As hindered chlorophyll 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 accumulation and release of PS did not increase; rather PS accumulation 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 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 μmol 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 μmol 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 μmol 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).

Figure 3 (a) Chlorophyll index (SPAD value) in fully developed young leaves and (b) the phytosiderophores (PS) concentration in the roots of barley seedlings with different treatments 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. The roots were collected just before the PS release time and the PS accumulation in the roots was measured on a root dry weight (DW) basis.

Iron concentration, accumulation and translocation

The Fe concentration in the shoots decreased in the As-treated plants to 44.4 μg g⁻¹ DW compared with the control plants (80.7 μg 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 μg g⁻¹ DW (Bergmann 1988). The Fe concentration increased in the shoots of the additional-Fe³⁺ treatment plants compared with the As-treated plants (Table 1). The Fe concentration in the shoots of the additional-Fe³⁺ plants was 69.3 μg 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 μg g⁻¹ DW compared with the control plants (281 μg g⁻¹ DW) (Table 1). The Fe concentration further increased in the roots in the additional-Fe³⁺ plants to 675 μg g⁻¹ DW compared with the roots of the As-treated plants. Because As has a high affinity to the sulfhydryl group of root proteins (Speer 1973), As may be bound with the protein and repress the function 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). Formation 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 previously (Shaibur et al. 2006, 2008a,b, 2009). It is also possible 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 content of absorbed Fe in roots without apoplastic Fe.

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

Treatments (μmol L^–1)		P	K	Ca	Mg	Fe	Mn	Zn	Cu
As	EDTA-Fe^3+	mg g^−1 DW				μg 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				μg 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 μg of element g^−1 dry weight (DW); accumulation is defined as mg or μg of element plant^−1 shoot or root. Accumulation was calculated by multiplying the concentration value by the DW of the plant samples. EDTA, ethylenediaminetetraacetic acid.

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

Treatments (μmol L^−1)		As	P	K	Ca	Mg	Fe	Mn	Zn	Cu
As	EDTA-Fe^3+
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).

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 accumulation 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 translocation of other elements

Phosphorus

The concentration and accumulation (Table 1) of P decreased significantly in the shoots and roots of As-treated plants compared with the control plants. The concentration 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 antagonistic 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 arsenate under experimental conditions with aeration. Arsenate 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, accumulation and translocation of P (Tables 1,2).

The concentration and accumulation of P (Table 1) further 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 concentration of P in the additional-Fe³⁺ treatment plants may activate 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. Translocation 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 relationship between As, P and Fe may be involved in the appearance 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. Additional 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 (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 (Mengel and Kirkby 2001) or 1–50 mg Ca g⁻¹ DW (Marschner 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 concentrations, 3.60 and 3.90 mg Ca g⁻¹ DW, respectively (Table 1). The level of Ca in the As-treated and additional-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 compared with the control plants (1.51 mg g⁻¹ DW) (Table 1). The Mg concentration in the shoots of As-treated 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 μg 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 μg 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 diffusion 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. Translocation 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 μg g⁻¹ DW and this was a sufficient amount of Zn (20–100 μg g⁻¹ DW; Boehle and Lindsay 1969), but the concentration of Zn in the shoots of the As-treated plants was 15.0 μg g⁻¹ DW and this was deficient (10–15 μg g⁻¹ DW; Boehle and Lindsay 1969). The Zn concentration in the shoots of the additional-Fe³⁺ plants was 16.1 μg 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 μg 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 concentration 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 and translocation

In the As-treated plants, the As concentration was almost 10 μg g⁻¹ DW in the shoots and 900 μg 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 concentrated 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 additional-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.

Figure 4 Arsenic (a) concentration and (b) accumulation in the shoots and roots of barley seedlings with different treatments 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. Concentration is defined as mg or μg of element g−1 dry weight (DW); accumulation is defined as mg or μg of element plant−1 shoot or root. Accumulation was calculated by multiplying the concentration value by the DW of the plant samples. nd, not detected.

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. Chlorosis and Fe concentration were partially recovered with additional Fe³⁺. Arsenic toxicity reduced the concentrations of elements such as P, K, Ca, Mg, Fe, Mn, Zn and Cu in the shoots. Additional Fe³⁺ did not change the concentrations of K, Ca, Mg, Mn and Zn. Moreover, additional Fe³⁺ decreased the concentration of P and Cu in the shoots. Considering the effect of the additional-Fe³⁺ treatment on the concentration of the elements and the definition 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 additional-Fe³⁺ treatment. Phosphorus may also be involved in the partial greening of shoots in the additional-Fe³⁺ treated 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.