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Arsenate uptake and translocation in seedlings of two genotypes of rice is affected by external phosphate concentrations
Aquatic Botany 83 (2005) 321–331 www.elsevier.com/locate/aquabot
Arsenate uptake and translocation in seedlings of two genotypes of rice is affected by external phosphate concentrations Chun-Nu Geng a,b, Yong-Guan Zhu a,*, Wen-Ju Liu a,c, Sally E. Smith d a
Research Center for Eco-environmental Sciences, Chinese Academy of Sciences, Beijing, China b Shanghai Academy of Environmental Sciences, Shanghai, China c College of Natural Resources and Environment, Hebei Agricultural University, Baoding, Hebei Province, China d Soil and Land Systems, School of Environmental Sciences, The University of Adelaide, Adelaide, SA 5005, Australia Received 5 September 2004; received in revised form 15 June 2005; accepted 4 July 2005
Abstract Two genotypes of rice (Oryza sativa L.), 94D-54 and 94D-64 were used to investigate the formation of iron plaque controlled by different phosphorus (P) concentrations and the effect of iron plaque on arsenate uptake in a hydroponic experiment. External P concentrations from 10 to 50 mM caused a marked decrease in dithionite-citrate-bicarbonate (DCB)–Fe concentrations for both genotypes, but further increases from 50 to 300 mM only resulted in small decrease. Arsenic (As) concentrations in DCB-extracts were determined by the amounts of iron plaque and the adsorption capacity of As by iron plaque, and both controlled by external P concentrations. At 10 mM external P, genotype 94D-54 had higher Fe, As and P concentrations in DCB-extracts than genotype 94D-64, but the difference disappeared with increasing P concentrations. Increasing P concentrations decreased the percentages of As distributed in iron plaque from around 70 to 10%, and increased the percentages of As in roots and shoots gradually from around 20 to 60% for toots and from 5 to nearly
* Corresponding author. Tel.: +86 10 62396940; fax: +86 10 62396940. E-mail address: [email protected] (Y.-G. Zhu). 0304-3770/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2005.07.003
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35% for shoots, respectively. Moreover, P concentration increased the molar ratio of shoot-to-root As, from 0.05 to nearly 0.2, indicating P concentration may promote As translocation from roots to shoots. # 2005 Elsevier B.V. All rights reserved. Keywords: Arsenate; Iron plaque; Oryza sativa; Phosphate; Uptake
1. Introduction Aquatic plants take up Fe mainly as Fe(II) from soil and high levels of Fe(II) in plant tissues may cause damages by the formation of highly reactive hydroxyl radicals through the reaction of Fe(II) with hydrogen peroxide (Briat and Lobre´aux, 1997). In order to overcome this problem, aquatic plants have evolved a mechanism to oxidize Fe(II) to Fe(III) in the rhizosphere and thus induce the formation of iron plaque on the root surface, which is facilitated by the release of oxygen and oxidants into the rhizosphere (Armstrong, 1967; Chen et al., 1980; Taylor and Crowder, 1983; Taylor et al., 1984). Rice (Oryza sativa L.) is a typically aquatic plant and iron plaque is ubiquitously formed on the roots of paddy rice (Armstrong, 1964; Liu et al., 2004a,b). Because it has higher submergence tolerance and higher ability to form iron plaque than rice, Echinochloa crugalli has been used in breeding rice genotypes with better submergence tolerance. Both derived from rice cv ‘‘Gui 630’’ by means of injecting exogenous DNA of E. crugalli into rice spike-stalk (Zhou et al., 2001), two genotypes (94D-54 and 94D-64) have been demonstrated to have different submergence tolerance under field-flooded conditions, indicating that these two genotypes may have different capacity to form iron plaque on root surface (Damo Li, personal communication). It is also well known that arsenate acts as a phosphate (Pi) analog and is taken up by plants via Pi transporter systems (Asher and Reay, 1979; Macnair and Cumbes, 1987; Meharg and Macnair, 1992; Dixon, 1997; Wang et al., 2002). Phosphorus deficiency or starvation can enhance arsenate uptake and accumulation in barley (Lee, 1982), grasses (e.g. Holcus lanatus L.) (Meharg and Macnair, 1992) and even in the As hyperaccumulator fern Pteris vittata (Wang et al., 2002). However, P deprivation from solution enhanced the formation of iron plaque on the roots of rice plants and P sufficient rice had little iron plaque on the roots (Liu et al., 2004a). Iron plaque altered the phosphate-arsenate interactions, suggesting the possibility of three-way interactions among arsenate, P nutrition and iron plaque development (Liu et al., 2004a). However, little information is available on the quantitative relationship between the amount of iron plaque and P concentrations, and subsequently the sequestration of As in iron plaque in relation to external P supply. The objectives of the present work were, therefore, to investigate the critical P concentration controlling the formation of iron plaque and the interactions between P and As in iron plaque. In addition, we also want to investigate if rice genotypes differ in their ability to form Fe plaque and As uptake and translocation.
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2. Methods and materials 2.1. Preparation of rice seedlings Seeds of two genotypes (94D-54 and 94D-64) of rice were surface sterilized in 10% H2O2 (w/w) solution for 15 min, followed by thorough washing with de-ionized water. The seeds were germinated for 25 days in moist perlite in a plastic pot (size 1.5 L). During the seed germination period, 50 mL modified Hewitt (1966; Liu et al., 2004a) nutrient solution was supplied twice a week. 2.2. Hydroponic experiment After 25 days, uniform seedlings were selected and transplanted into PVC pots (11.5 cm in diameter and 14 cm in height) containing 1100 mL modified Hewitt solution. Rice seedlings were pre-treated in the solution supplemented with four P concentrations (10, 50, 150 and 300 mM P) as KH2PO4 for 10 days. KCl was added compensating for the reduction in K concentrations in the growth due to changes in P concentrations (as KH2PO4). After the pre-treatment, seedlings were exposed to solutions with the corresponding P concentrations and a constant As concentration of 10 mM as Na3AsO4 for another 11 days. All nutrient solutions were changed twice per week, and pH was adjusted to 5.5 using 0.1 M KOH or HCl. Iron in the nutrient solution was supplied as Fe2+ to represent the main iron species in paddy soil solution. The nutrient solution was not aerated during the experimental period to mimic the anaerobic conditions in paddy soils. Each treatment had four replicates. The experiment was carried out in a growth chamber with a 14-h photoperiod (260– 350 mmol m 2 s 1) and temperatures of 28 8C at day and 20 8C at night. The relative humidity was 70%. Pots were arranged haphazardly in the growth chamber and re-arranged every day. 2.3. DCB extraction of iron plaque DCB extraction was conducted in the same manner as described by Liu et al. (2004a). After DCB extraction, roots and shoots were oven dried at 70 8C for 2 days and dry weights of roots and shoots were recorded. 2.4. Chemical analysis of plant tissues Dried plant materials were ground and about 0.25 g was weighed accurately into clean, dry digestion tubes (100 mL for CEM Microwave digestion systems). Five millilitres concentrated HNO3 was added and digested by MARS (Microwave Accelerated Reaction System, MARS 5, CEM Corporation, NC, USA). A reagent blank and a standard reference plant material (tea leaves, GBW07605 from the National Research Center for Standard Materials in China) were included to verify the accuracy and precision of the digestion procedure and subsequent analysis. After digestion the solutions were cooled, diluted to 50 mL with ultra-pure water (Dubugue, IA, USA) and filtered into acid-washed plastic
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bottles. The concentrations of Fe, P and As in the DCB-extracts and in the acid digests were measured by inductively coupled plasma-optical emission spectrometer (ICP-OES, Optima 2000 DV, Perkin-Elmer, USA). For plant tissue samples with low As concentrations, an atomic fluorescence spectrometry (AF-610A, Beijing Haiguang Analytical Instrument Co., Beijing, China) was used instead. 2.5. Statistical analysis Elemental concentrations in DCB-extracts, roots and shoots were calculated on the basis of dry weight. Analysis of variance (ANOVA) on plant biomass and concentrations of P, Fe and As were performed using Windows-based Genstat (6th edition, NAG Ltd., England).
3. Results 3.1. Plant growth For both genotypes, increasing external P concentrations from 10 to 300 mM significantly increased root and shoot biomass (Table 1, P < 0.001). There was little difference in root biomass between genotypes. However, there was significant difference in shoot biomass between genotypes (Table 1, P < 0.001). The leaves of both genotypes with lower external P concentrations (10 mM) became curled and showed toxic symptom, but both genotypes with higher external P concentrations (300 mM) did not show any toxic symptom. 3.2. Fe, P and As in DCB-extracts At harvest, roots of both genotypes with P additions of 10, 50 mM appeared reddish, indicating the formation of iron plaque on root surface, but at P additions of 150, 300 mM, they remained white. This quantitative relationship is clearly shown in Fig. 1a.With Table 1 Shoot and root biomass of rice two genotypes (94D-54 and 94D-64) pre-treated with four P concentrations (10, 50, 150 and 300 mM) for 10 days and then exposed to 10 mM As and the corresponding P concentrations for 11 days P treatment (mM)
10 50 150 300 Analysis of variance Genotypes (G) P treatment (P) GP
Root biomass (mg pot 1)
Shoot biomass (mg pot 1)
94D-64
94D-54
94D-64
94D-54
88 4 104 6 120 6 103 17
61 3 106 5 118 7 137 15
262 7 326 18 400 23 348 47
232 12 412 21 461 28 499 58
NS P < 0.001 P < 0.05
Values are the mean S.E. (n = 4). NS refers to not significant at P = 0.05.
P < 0.01 P < 0.001 NS
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Fig. 1. Fe (a), P (b) and As (c) concentrations in DCB-extracts in the two rice genotypes (94D-54 and 94D-64) pretreated with four P concentrations (10, 50, 150 and 300 mM) for 10 days and then exposed to 10 mM As and the corresponding P concentrations for 11 days. The error bars represent one S.E. of the mean from four replicates.
external P concentrations increasing from 10 to 50 mM DCB–Fe concentrations decreased sharply for both genotypes and further increasing external P concentrations from 50 to 300 mM only resulted in slow decrease in DCB–Fe concentrations (Fig. 1a, Table 2, P < 0.001). Similarly, increasing external P concentrations from 10 to 300 mM decreased DCB-As concentrations significantly (Fig. 1b, Table 2, P < 0.001). However, the pattern of DCB-P concentrations was different (Fig. 1c). With external P concentration of 10 mM, genotype 94D-54 had higher Fe, P and As concentrations in DCB-extracts than genotype 94D-64,
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Table 2 Analysis of variance (two-way ANOVA) on data shown in Figs. 1–6 Indexes
Genotypes (G)
P treatment (P)
GP
Fig. 1
DCB–Fe DCB-As DCB-P
P < 0.001 P < 0.001 P < 0.001
P < 0.001 P < 0.001 NS
P < 0.001 P < 0.001 P = 0.001
Fig. 2
Molar ratios of P/Fe Molar ratios of As/Fe
P = 0.002 P = 0.02
P < 0.001 P < 0.001
NS P = 0.039
Fig. 3
Root As Shoot As
P = 0.002 P = 0.001
P < 0.001 P < 0.001
P < 0.001 P = 0.041
Fig. 4
Molar ratios of shoot-to-root As
P = 0.025
P < 0.001
P = 0.025
Fig. 5
Root P Shoot P
P = 0.001 P < 0.001
P < 0.001 P < 0.001
NS NS
Fig. 6
DCB Root Shoot
P < 0.001 P = 0.001 NS
P < 0.001 P < 0.001 P < 0.001
P < 0.001 P = 0.007 P = 0.045
NS refers to not significant at P = 0.05.
and the difference disappeared with increasing external P concentrations (Fig. 1a–c). For both genotypes, with increasing external P concentrations, the P/Fe ratios increased, but the As/Fe ratios decreased (Fig. 2, Table 2, P < 0.001). 3.3. As and P concentrations in roots and shoots For both genotypes, root As concentrations decreased significantly with increasing external P concentrations from 10 to 300 mM (Fig. 3a, Table 2, P < 0.001), but this is not
Fig. 2. The molar ratios of P/Fe and As/Fe in DCB-extracts in the two rice genotypes (94D-54 and 94D-64) pretreated with four P concentrations (10, 50, 150 and 300 mM) for 10 days and then exposed to 10 mM As and the corresponding P concentrations for 11 days. The error bars represent one S.E. of the mean from four replicates.
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Fig. 3. Arsenic concentrations in roots (a) and shoots (b) of the two rice genotypes (94D-54 and 94D-64) pretreated with four P concentrations (10, 50, 150 and 300 mM) for 10 days and then exposed to 10 mM As and the corresponding P concentrations for 11 days. The error bars represent one S.E. of the mean from four replicates.
the case for As concentrations in shoots. Increasing external P concentrations from 10 to 50 mM decreased shoot As concentrations, but further increasing external P concentrations from 50 to 300 mM increased shoot As concentrations (Fig. 3b, Table 2, P < 0.001). At 10 mM P, genotype 94D-64 had higher As concentrations in the roots than 94D-54 and the difference disappeared with increasing P concentrations. However, at 300 mM P, genotype 94D-64 had higher As concentrations in the shoots than 94D-54 and the difference disappeared with decreasing P concentrations (Fig. 3a and b). For both genotypes, increasing external P concentrations increased the molar ratios of shoot-to-root As linearly (Fig. 4; Table 2, P < 0.001). For both genotypes, root and shoot P concentrations increased significantly with increasing external P concentrations. Genotype 94D-64 had higher tissue P concentrations than genotype 94D-54 (Fig. 5, Table 2, P 0.001). 3.4. As distribution in DCB-extracts, roots and shoots The distribution of As in iron plaques (DCB-extracts), roots and shoots as percentages of total As in plants changed significantly between P concentrations (Fig. 6, Table 2,
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Fig. 4. The relationship between P concentration and molar ratios of shoot-to-root As in two rice genotypes (94D54 and 94D-64) pre-treated with four P concentrations (10, 50, 150 and 300 mM) for 10 days and then exposed to 10 mM As and the corresponding P concentrations for 11 days. The error bars represent one S.E. of the mean from four replicates.
P < 0.001). For both genotypes, increasing P additions from 10 to 300 mM decreased the amounts of As distributed in DCB-extracts, while those in roots and shoots increased gradually with increasing P concentrations.
4. Discussion The toxicity of As may be dependent on As/P ratios within plant tissues, not absolute As concentrations in tissues. Both genotypes had similar shoot As concentrations with external P concentrations increasing from 10 to 300 mM, but plants grown at 10 mM external P concentration did show toxic symptom and those grown at 300 mM P did not show any toxic symptom. This may be due to the detoxification effect of P, since plants with higher P concentration in the solution had higher shoot P concentrations (Fig. 5b). The possible role of P in alleviating As toxicity has also been demonstrated in Arabidopsis recently (Lee et al., 2003). Ars1, an Arabidopsis mutant, could germinate and develop under the As concentrations that completely inhibited the growth of wild type plants, but the mutant accumulated similar As concentration to wild type. Further investigation showed that the mutant plant had higher P concentrations than the wild type. A molar P/As ratio of at least 12 is needed to protect plants against arsenate toxicity (Walsh and Keeney, 1975). Low P in plant tissues might increase the oxygen release from rice roots to the rhizosphere (Kirk and Du, 1997), and rice plants might employ this mechanism as one of the adaptive metabolic reactions to the condition of P deficiency. The increase in oxygen release from rice roots would have had the effect on stimulating the formation of iron plaque. Liu et al. (2004a) found that iron plaque was developed on rice roots 24 h after P
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Fig. 5. P concentrations in roots (a) and shoots (b) of the two rice genotypes (94D-54 and 94D-64) pre-treated with four P concentrations (10, 50, 150 and 300 mM) for 10 days and then exposed to 10 mM As and the corresponding P concentrations for 11 days. The error bars represent one S.E. of the mean from four replicates.
deprivation from the nutrient solution. In the present work, external P concentrations lower than 50 mM stimulated the formation of iron plaque on root surface (Fig. 1a), and different cultivars responded differently to P deficiency. At external P concentration of 10 mM P genotype 94D-54 developed more iron plaque on root surface than genotype 94D-64 (Fig. 1a), which was in agreement with submergence tolerance observed under field conditions (D.M. Li, personal communication). The formation of iron plaque may contribute to the genotypic difference in As uptake and translocation (Liu et al., 2004a; Meharg, 2004). Although it is well known that phosphate inhibits arsenate uptake (Wang et al., 2002), this was not the case for genotype 94D-54 in root As concentrations with external P concentrations between 10 and 50 mM. Furthermore, genotype 94D-54 had lower root As concentrations than 94D-64 only at external P concentration of 10 mM (Fig. 3a), which coincided with the higher As concentrations in DCB-extracts (Fig. 1b), indicating that iron plaque may act as a ‘‘buffer’’ in the rhizosphere for As uptake into rice roots. A number of reports have shown that iron plaque could act as a barrier for the uptake of other toxic metals (Greipsson, 1994, 1995; Otte and Dekkers, 1991; Zhang et al., 1998; Christensen and Sand-Jensen, 1998; Batty and Baker, 2000).
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Fig. 6. Arsenic distribution in DCB-extracts, root and shoot of two rice genotypes (94D-54 and 94D-64) pretreated with four P concentrations (10, 50, 150 and 300 mM) for 10 days and then exposed to 10 mM As and the corresponding P concentrations for 11 days. The error bars represent one S.E. of the mean from four replicates.
Arsenic sequestrated in iron plaque (i.e. in DCB-extracts) could be determined by two factors: one is the amount of iron plaque on root surface regulated by plant P nutrition (Fig. 1a), and the other is the As/Fe ratios also affected by external P concentrations via adsorption–desorption processes (Fig. 2). The opposite pattern of the ratios of As/Fe and P/ Fe with increasing external P concentrations (Fig. 2) suggested the competitive adsorption of arsenate and phosphate in iron plaque. Since the ratios of As/Fe also decreased with increasing external P concentrations, DCB-As concentrations decreased more sharply than DCB–Fe concentrations with increasing external P concentrations, though As and Fe concentrations in the solution remained constant (Fig. 1a and b). For example, at external P concentrations of 10–50 mM, DCB–Fe concentrations decreased by 41 and 59% for genotypes 94D-54 and 94D-64, respectively, but DCB–As concentrations decreased 79 and 86% for the two genotypes, respectively. In conclusion, plant P nutrition controlled the formation of iron plaque and thus had an effect on As sequestration in iron plaque. However, the mechanisms of how P nutrition controls the formation of iron plaque need further investigations.
Acknowledgements We thank Professor DM Li (Institute of Subtropical Regional Agriculture, Chinese Academy of Sciences) for providing rice seeds. The work was supported financially by the Natural Science Foundation of China (40225002), Ministry of Science and Technology of China (2002CB410808) and the Chinese Academy of Sciences (Hundred Talent Program).
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References Armstrong, W., 1964. Oxygen diffusion from the roots of some British bog plants. Nature 204, 801–802. Armstrong, W., 1967. The oxidising activity of roots in waterlogged soils. Physiol. Plant. 20, 920–926. Asher, C.J., Reay, P.F., 1979. Arsenic uptake by barley seedlings. Aust. J. Plant Physiol. 6, 459–466. Batty, L.C., Baker, A.J.M., 2000. The effect of pH and plaque on the uptake of Cu and Mn in Phragmites australis (Cav.) Trin ex.Steudel. Ann. Bot. 86, 647–653. Briat, J.F., Lobre´aux, S., 1997. Iron transport and storage in plants. Trends Plant Sci. 2, 187–193. Chen, C.C., Dixon, J.B., Turner, F.T., 1980. Iron coatings on rice roots: Morpology and models of development. Soil Sci. Soc. Am. J. 44, 1113–1119. Christensen, K.K., Sand-Jensen, K., 1998. Precipitated iron and manganese plaques restrict root uptake of phosphorus in Lobelia dortmanna. Can. J. Bot. 72, 2158–2163. Dixon, H.B.F., 1997. The biochemical action of arsenic acids especially as phosphate analogues. Adv. Inorg. Chem. 44, 191–227. Greipsson, S., 1994. Effect of plaque on roots of rice on growth and metal concentration of seeds and plant tissues when cultivated in excess Cu. Commun. Soil Sci. Plant Anal. 25, 2761–2769. Greipsson, S., 1995. Effect of iron plaque on roots of rice on growth of plants in excess zinc and accumulation of phosphorus in plants in excess copper or nickel. J. Plant Nutr. 18, 1659–1665. Kirk, G.J.D., Du, V.L., 1997. Change in rice root architecture, porosit, and oxygen and proton release under phosphorus deficiency. New Phytol. 135, 191–200. Lee, R.B., 1982. Selectivity and kinetics of ion uptake by barley plants following nutrient deficiency. Ann. Bot. 50, 429–449. Lee, D.A., Chen, A., Schroeder, J.I., 2003. ars1, an Arabidopsis mutant exhibiting increased tolerance to arsenate and increased phosphate uptake. Plant J. 35, 637–646. Liu, W.J., Zhu, Y.G., Smith, F.A., Smith, S.E., 2004a. Do phosphorus nutrition and iron plaque alter arsenate (As) uptake by rice seedlings in hydroponic culture? New Phytol. 162, 481–488. Liu, W.J., Zhu, Y.G., Smith, F.A., Smith, S.E., 2004b. Arsenic uptake by and translocation within rice seedlings (Oryza sativa L.) of three genotypes were influenced by iron plaque on root surface. J. Exp. Bot. 55, 1707– 1713. Macnair, M.R., Cumbes, Q., 1987. Evidence that arsenic tolerance in Holcus lanatus L. is caused by an altered phosphate uptake system. New Phytol. 107, 387–394. Meharg, A.A., 2004. Arsenic in rice–understanding a new disaster for south-east Asia. Trends Plant Sci. 9, 415– 417. Meharg, A.A., Macnair, M.R., 1992. Suppression of the high-affinity phosphate-uptake system-a mechanism of arsenate tolerance in Holcus Lanatus L. J. Exp. Bot. 43, 519–524. Otte, M.L., Dekkers, M.J., 1991. Uptake of arsenic by Aster tripolium in relation to rhizosphere oxidation. Can. J. Bot. 69, 2670–2677. Taylor, G.J., Crowder, A.A., 1983. Use of DCB technique for extraction of hydrous iron oxides from roots of wetland plants. Am. J. Bot. 70, 1254–1257. Taylor, G.J., Crowder, A.A., Rodden, R., 1984. Formation and morphology lf an iron plaque on the roots of Typha Latifolia L. grown in solution culture. Am. J. Bot. 71, 666–675. Wang, J.R., Zhao, F.J., Meharg, A.A., Raab, A., Feldmann, J., McGrath, S.P., 2002. Mechanisms of arsenic hyperaccumulation in Pteris vittata. uptake kinetics, interactions with phosphate, and arsenic speciation. Plant Physiol. 130, 1552–1561. Walsh, L.M., Keeney, D.R., 1975. Behavior and phytotoxicity of inorganic arsenicals in soils. In: Woolson, E.A. (Ed.), .Arsenical Pesticides. ACS Press, Washington, DC, pp. 35–52. Zhang, X., Zhang, F., Mao, D., 1998. Effect of Fe plaque outside roots on nutrient uptake by rice (Oryza sativa L.): zinc uptake. Plant Soil 202, 33–39. Zhou, J.L., Li, Y.S., Li, D.M., 2001. Analysis on RAPD pattern and variations in iron-toxicity tolerance induced by introducing Echinochloa crugalli DNA into rice. Acta Agron. Sin. 27, 529–532.
Arsenate uptake and translocation in seedlings of two genotypes of rice is affected by external phosphate concentrations Chun-Nu Geng a,b, Yong-Guan Zhu a,*, Wen-Ju Liu a,c, Sally E. Smith d a
Research Center for Eco-environmental Sciences, Chinese Academy of Sciences, Beijing, China b Shanghai Academy of Environmental Sciences, Shanghai, China c College of Natural Resources and Environment, Hebei Agricultural University, Baoding, Hebei Province, China d Soil and Land Systems, School of Environmental Sciences, The University of Adelaide, Adelaide, SA 5005, Australia Received 5 September 2004; received in revised form 15 June 2005; accepted 4 July 2005
Abstract Two genotypes of rice (Oryza sativa L.), 94D-54 and 94D-64 were used to investigate the formation of iron plaque controlled by different phosphorus (P) concentrations and the effect of iron plaque on arsenate uptake in a hydroponic experiment. External P concentrations from 10 to 50 mM caused a marked decrease in dithionite-citrate-bicarbonate (DCB)–Fe concentrations for both genotypes, but further increases from 50 to 300 mM only resulted in small decrease. Arsenic (As) concentrations in DCB-extracts were determined by the amounts of iron plaque and the adsorption capacity of As by iron plaque, and both controlled by external P concentrations. At 10 mM external P, genotype 94D-54 had higher Fe, As and P concentrations in DCB-extracts than genotype 94D-64, but the difference disappeared with increasing P concentrations. Increasing P concentrations decreased the percentages of As distributed in iron plaque from around 70 to 10%, and increased the percentages of As in roots and shoots gradually from around 20 to 60% for toots and from 5 to nearly
* Corresponding author. Tel.: +86 10 62396940; fax: +86 10 62396940. E-mail address: [email protected] (Y.-G. Zhu). 0304-3770/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2005.07.003
322
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35% for shoots, respectively. Moreover, P concentration increased the molar ratio of shoot-to-root As, from 0.05 to nearly 0.2, indicating P concentration may promote As translocation from roots to shoots. # 2005 Elsevier B.V. All rights reserved. Keywords: Arsenate; Iron plaque; Oryza sativa; Phosphate; Uptake
1. Introduction Aquatic plants take up Fe mainly as Fe(II) from soil and high levels of Fe(II) in plant tissues may cause damages by the formation of highly reactive hydroxyl radicals through the reaction of Fe(II) with hydrogen peroxide (Briat and Lobre´aux, 1997). In order to overcome this problem, aquatic plants have evolved a mechanism to oxidize Fe(II) to Fe(III) in the rhizosphere and thus induce the formation of iron plaque on the root surface, which is facilitated by the release of oxygen and oxidants into the rhizosphere (Armstrong, 1967; Chen et al., 1980; Taylor and Crowder, 1983; Taylor et al., 1984). Rice (Oryza sativa L.) is a typically aquatic plant and iron plaque is ubiquitously formed on the roots of paddy rice (Armstrong, 1964; Liu et al., 2004a,b). Because it has higher submergence tolerance and higher ability to form iron plaque than rice, Echinochloa crugalli has been used in breeding rice genotypes with better submergence tolerance. Both derived from rice cv ‘‘Gui 630’’ by means of injecting exogenous DNA of E. crugalli into rice spike-stalk (Zhou et al., 2001), two genotypes (94D-54 and 94D-64) have been demonstrated to have different submergence tolerance under field-flooded conditions, indicating that these two genotypes may have different capacity to form iron plaque on root surface (Damo Li, personal communication). It is also well known that arsenate acts as a phosphate (Pi) analog and is taken up by plants via Pi transporter systems (Asher and Reay, 1979; Macnair and Cumbes, 1987; Meharg and Macnair, 1992; Dixon, 1997; Wang et al., 2002). Phosphorus deficiency or starvation can enhance arsenate uptake and accumulation in barley (Lee, 1982), grasses (e.g. Holcus lanatus L.) (Meharg and Macnair, 1992) and even in the As hyperaccumulator fern Pteris vittata (Wang et al., 2002). However, P deprivation from solution enhanced the formation of iron plaque on the roots of rice plants and P sufficient rice had little iron plaque on the roots (Liu et al., 2004a). Iron plaque altered the phosphate-arsenate interactions, suggesting the possibility of three-way interactions among arsenate, P nutrition and iron plaque development (Liu et al., 2004a). However, little information is available on the quantitative relationship between the amount of iron plaque and P concentrations, and subsequently the sequestration of As in iron plaque in relation to external P supply. The objectives of the present work were, therefore, to investigate the critical P concentration controlling the formation of iron plaque and the interactions between P and As in iron plaque. In addition, we also want to investigate if rice genotypes differ in their ability to form Fe plaque and As uptake and translocation.
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2. Methods and materials 2.1. Preparation of rice seedlings Seeds of two genotypes (94D-54 and 94D-64) of rice were surface sterilized in 10% H2O2 (w/w) solution for 15 min, followed by thorough washing with de-ionized water. The seeds were germinated for 25 days in moist perlite in a plastic pot (size 1.5 L). During the seed germination period, 50 mL modified Hewitt (1966; Liu et al., 2004a) nutrient solution was supplied twice a week. 2.2. Hydroponic experiment After 25 days, uniform seedlings were selected and transplanted into PVC pots (11.5 cm in diameter and 14 cm in height) containing 1100 mL modified Hewitt solution. Rice seedlings were pre-treated in the solution supplemented with four P concentrations (10, 50, 150 and 300 mM P) as KH2PO4 for 10 days. KCl was added compensating for the reduction in K concentrations in the growth due to changes in P concentrations (as KH2PO4). After the pre-treatment, seedlings were exposed to solutions with the corresponding P concentrations and a constant As concentration of 10 mM as Na3AsO4 for another 11 days. All nutrient solutions were changed twice per week, and pH was adjusted to 5.5 using 0.1 M KOH or HCl. Iron in the nutrient solution was supplied as Fe2+ to represent the main iron species in paddy soil solution. The nutrient solution was not aerated during the experimental period to mimic the anaerobic conditions in paddy soils. Each treatment had four replicates. The experiment was carried out in a growth chamber with a 14-h photoperiod (260– 350 mmol m 2 s 1) and temperatures of 28 8C at day and 20 8C at night. The relative humidity was 70%. Pots were arranged haphazardly in the growth chamber and re-arranged every day. 2.3. DCB extraction of iron plaque DCB extraction was conducted in the same manner as described by Liu et al. (2004a). After DCB extraction, roots and shoots were oven dried at 70 8C for 2 days and dry weights of roots and shoots were recorded. 2.4. Chemical analysis of plant tissues Dried plant materials were ground and about 0.25 g was weighed accurately into clean, dry digestion tubes (100 mL for CEM Microwave digestion systems). Five millilitres concentrated HNO3 was added and digested by MARS (Microwave Accelerated Reaction System, MARS 5, CEM Corporation, NC, USA). A reagent blank and a standard reference plant material (tea leaves, GBW07605 from the National Research Center for Standard Materials in China) were included to verify the accuracy and precision of the digestion procedure and subsequent analysis. After digestion the solutions were cooled, diluted to 50 mL with ultra-pure water (Dubugue, IA, USA) and filtered into acid-washed plastic
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bottles. The concentrations of Fe, P and As in the DCB-extracts and in the acid digests were measured by inductively coupled plasma-optical emission spectrometer (ICP-OES, Optima 2000 DV, Perkin-Elmer, USA). For plant tissue samples with low As concentrations, an atomic fluorescence spectrometry (AF-610A, Beijing Haiguang Analytical Instrument Co., Beijing, China) was used instead. 2.5. Statistical analysis Elemental concentrations in DCB-extracts, roots and shoots were calculated on the basis of dry weight. Analysis of variance (ANOVA) on plant biomass and concentrations of P, Fe and As were performed using Windows-based Genstat (6th edition, NAG Ltd., England).
3. Results 3.1. Plant growth For both genotypes, increasing external P concentrations from 10 to 300 mM significantly increased root and shoot biomass (Table 1, P < 0.001). There was little difference in root biomass between genotypes. However, there was significant difference in shoot biomass between genotypes (Table 1, P < 0.001). The leaves of both genotypes with lower external P concentrations (10 mM) became curled and showed toxic symptom, but both genotypes with higher external P concentrations (300 mM) did not show any toxic symptom. 3.2. Fe, P and As in DCB-extracts At harvest, roots of both genotypes with P additions of 10, 50 mM appeared reddish, indicating the formation of iron plaque on root surface, but at P additions of 150, 300 mM, they remained white. This quantitative relationship is clearly shown in Fig. 1a.With Table 1 Shoot and root biomass of rice two genotypes (94D-54 and 94D-64) pre-treated with four P concentrations (10, 50, 150 and 300 mM) for 10 days and then exposed to 10 mM As and the corresponding P concentrations for 11 days P treatment (mM)
10 50 150 300 Analysis of variance Genotypes (G) P treatment (P) GP
Root biomass (mg pot 1)
Shoot biomass (mg pot 1)
94D-64
94D-54
94D-64
94D-54
88 4 104 6 120 6 103 17
61 3 106 5 118 7 137 15
262 7 326 18 400 23 348 47
232 12 412 21 461 28 499 58
NS P < 0.001 P < 0.05
Values are the mean S.E. (n = 4). NS refers to not significant at P = 0.05.
P < 0.01 P < 0.001 NS
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Fig. 1. Fe (a), P (b) and As (c) concentrations in DCB-extracts in the two rice genotypes (94D-54 and 94D-64) pretreated with four P concentrations (10, 50, 150 and 300 mM) for 10 days and then exposed to 10 mM As and the corresponding P concentrations for 11 days. The error bars represent one S.E. of the mean from four replicates.
external P concentrations increasing from 10 to 50 mM DCB–Fe concentrations decreased sharply for both genotypes and further increasing external P concentrations from 50 to 300 mM only resulted in slow decrease in DCB–Fe concentrations (Fig. 1a, Table 2, P < 0.001). Similarly, increasing external P concentrations from 10 to 300 mM decreased DCB-As concentrations significantly (Fig. 1b, Table 2, P < 0.001). However, the pattern of DCB-P concentrations was different (Fig. 1c). With external P concentration of 10 mM, genotype 94D-54 had higher Fe, P and As concentrations in DCB-extracts than genotype 94D-64,
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Table 2 Analysis of variance (two-way ANOVA) on data shown in Figs. 1–6 Indexes
Genotypes (G)
P treatment (P)
GP
Fig. 1
DCB–Fe DCB-As DCB-P
P < 0.001 P < 0.001 P < 0.001
P < 0.001 P < 0.001 NS
P < 0.001 P < 0.001 P = 0.001
Fig. 2
Molar ratios of P/Fe Molar ratios of As/Fe
P = 0.002 P = 0.02
P < 0.001 P < 0.001
NS P = 0.039
Fig. 3
Root As Shoot As
P = 0.002 P = 0.001
P < 0.001 P < 0.001
P < 0.001 P = 0.041
Fig. 4
Molar ratios of shoot-to-root As
P = 0.025
P < 0.001
P = 0.025
Fig. 5
Root P Shoot P
P = 0.001 P < 0.001
P < 0.001 P < 0.001
NS NS
Fig. 6
DCB Root Shoot
P < 0.001 P = 0.001 NS
P < 0.001 P < 0.001 P < 0.001
P < 0.001 P = 0.007 P = 0.045
NS refers to not significant at P = 0.05.
and the difference disappeared with increasing external P concentrations (Fig. 1a–c). For both genotypes, with increasing external P concentrations, the P/Fe ratios increased, but the As/Fe ratios decreased (Fig. 2, Table 2, P < 0.001). 3.3. As and P concentrations in roots and shoots For both genotypes, root As concentrations decreased significantly with increasing external P concentrations from 10 to 300 mM (Fig. 3a, Table 2, P < 0.001), but this is not
Fig. 2. The molar ratios of P/Fe and As/Fe in DCB-extracts in the two rice genotypes (94D-54 and 94D-64) pretreated with four P concentrations (10, 50, 150 and 300 mM) for 10 days and then exposed to 10 mM As and the corresponding P concentrations for 11 days. The error bars represent one S.E. of the mean from four replicates.
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Fig. 3. Arsenic concentrations in roots (a) and shoots (b) of the two rice genotypes (94D-54 and 94D-64) pretreated with four P concentrations (10, 50, 150 and 300 mM) for 10 days and then exposed to 10 mM As and the corresponding P concentrations for 11 days. The error bars represent one S.E. of the mean from four replicates.
the case for As concentrations in shoots. Increasing external P concentrations from 10 to 50 mM decreased shoot As concentrations, but further increasing external P concentrations from 50 to 300 mM increased shoot As concentrations (Fig. 3b, Table 2, P < 0.001). At 10 mM P, genotype 94D-64 had higher As concentrations in the roots than 94D-54 and the difference disappeared with increasing P concentrations. However, at 300 mM P, genotype 94D-64 had higher As concentrations in the shoots than 94D-54 and the difference disappeared with decreasing P concentrations (Fig. 3a and b). For both genotypes, increasing external P concentrations increased the molar ratios of shoot-to-root As linearly (Fig. 4; Table 2, P < 0.001). For both genotypes, root and shoot P concentrations increased significantly with increasing external P concentrations. Genotype 94D-64 had higher tissue P concentrations than genotype 94D-54 (Fig. 5, Table 2, P 0.001). 3.4. As distribution in DCB-extracts, roots and shoots The distribution of As in iron plaques (DCB-extracts), roots and shoots as percentages of total As in plants changed significantly between P concentrations (Fig. 6, Table 2,
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Fig. 4. The relationship between P concentration and molar ratios of shoot-to-root As in two rice genotypes (94D54 and 94D-64) pre-treated with four P concentrations (10, 50, 150 and 300 mM) for 10 days and then exposed to 10 mM As and the corresponding P concentrations for 11 days. The error bars represent one S.E. of the mean from four replicates.
P < 0.001). For both genotypes, increasing P additions from 10 to 300 mM decreased the amounts of As distributed in DCB-extracts, while those in roots and shoots increased gradually with increasing P concentrations.
4. Discussion The toxicity of As may be dependent on As/P ratios within plant tissues, not absolute As concentrations in tissues. Both genotypes had similar shoot As concentrations with external P concentrations increasing from 10 to 300 mM, but plants grown at 10 mM external P concentration did show toxic symptom and those grown at 300 mM P did not show any toxic symptom. This may be due to the detoxification effect of P, since plants with higher P concentration in the solution had higher shoot P concentrations (Fig. 5b). The possible role of P in alleviating As toxicity has also been demonstrated in Arabidopsis recently (Lee et al., 2003). Ars1, an Arabidopsis mutant, could germinate and develop under the As concentrations that completely inhibited the growth of wild type plants, but the mutant accumulated similar As concentration to wild type. Further investigation showed that the mutant plant had higher P concentrations than the wild type. A molar P/As ratio of at least 12 is needed to protect plants against arsenate toxicity (Walsh and Keeney, 1975). Low P in plant tissues might increase the oxygen release from rice roots to the rhizosphere (Kirk and Du, 1997), and rice plants might employ this mechanism as one of the adaptive metabolic reactions to the condition of P deficiency. The increase in oxygen release from rice roots would have had the effect on stimulating the formation of iron plaque. Liu et al. (2004a) found that iron plaque was developed on rice roots 24 h after P
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Fig. 5. P concentrations in roots (a) and shoots (b) of the two rice genotypes (94D-54 and 94D-64) pre-treated with four P concentrations (10, 50, 150 and 300 mM) for 10 days and then exposed to 10 mM As and the corresponding P concentrations for 11 days. The error bars represent one S.E. of the mean from four replicates.
deprivation from the nutrient solution. In the present work, external P concentrations lower than 50 mM stimulated the formation of iron plaque on root surface (Fig. 1a), and different cultivars responded differently to P deficiency. At external P concentration of 10 mM P genotype 94D-54 developed more iron plaque on root surface than genotype 94D-64 (Fig. 1a), which was in agreement with submergence tolerance observed under field conditions (D.M. Li, personal communication). The formation of iron plaque may contribute to the genotypic difference in As uptake and translocation (Liu et al., 2004a; Meharg, 2004). Although it is well known that phosphate inhibits arsenate uptake (Wang et al., 2002), this was not the case for genotype 94D-54 in root As concentrations with external P concentrations between 10 and 50 mM. Furthermore, genotype 94D-54 had lower root As concentrations than 94D-64 only at external P concentration of 10 mM (Fig. 3a), which coincided with the higher As concentrations in DCB-extracts (Fig. 1b), indicating that iron plaque may act as a ‘‘buffer’’ in the rhizosphere for As uptake into rice roots. A number of reports have shown that iron plaque could act as a barrier for the uptake of other toxic metals (Greipsson, 1994, 1995; Otte and Dekkers, 1991; Zhang et al., 1998; Christensen and Sand-Jensen, 1998; Batty and Baker, 2000).
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Fig. 6. Arsenic distribution in DCB-extracts, root and shoot of two rice genotypes (94D-54 and 94D-64) pretreated with four P concentrations (10, 50, 150 and 300 mM) for 10 days and then exposed to 10 mM As and the corresponding P concentrations for 11 days. The error bars represent one S.E. of the mean from four replicates.
Arsenic sequestrated in iron plaque (i.e. in DCB-extracts) could be determined by two factors: one is the amount of iron plaque on root surface regulated by plant P nutrition (Fig. 1a), and the other is the As/Fe ratios also affected by external P concentrations via adsorption–desorption processes (Fig. 2). The opposite pattern of the ratios of As/Fe and P/ Fe with increasing external P concentrations (Fig. 2) suggested the competitive adsorption of arsenate and phosphate in iron plaque. Since the ratios of As/Fe also decreased with increasing external P concentrations, DCB-As concentrations decreased more sharply than DCB–Fe concentrations with increasing external P concentrations, though As and Fe concentrations in the solution remained constant (Fig. 1a and b). For example, at external P concentrations of 10–50 mM, DCB–Fe concentrations decreased by 41 and 59% for genotypes 94D-54 and 94D-64, respectively, but DCB–As concentrations decreased 79 and 86% for the two genotypes, respectively. In conclusion, plant P nutrition controlled the formation of iron plaque and thus had an effect on As sequestration in iron plaque. However, the mechanisms of how P nutrition controls the formation of iron plaque need further investigations.
Acknowledgements We thank Professor DM Li (Institute of Subtropical Regional Agriculture, Chinese Academy of Sciences) for providing rice seeds. The work was supported financially by the Natural Science Foundation of China (40225002), Ministry of Science and Technology of China (2002CB410808) and the Chinese Academy of Sciences (Hundred Talent Program).
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