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. 2006 Sep;98(3):631–636. doi: 10.1093/aob/mcl139

Phosphate (Pi) and Arsenate Uptake by Two Wheat (Triticum aestivum) Cultivars and Their Doubled Haploid Lines

YONG-GUAN ZHU 1,*, CHUN-NU GENG 2, YI-PING TONG 3, SALLY E SMITH 4, F ANDREW SMITH 4
PMCID: PMC2803564  PMID: 16803848

Abstract

• Background and Aims Arsenic accumulation in cereal crops represents an important pathway for human exposure to arsenic from the environment. The objectives of the present work were to find whether the relationship between arsenate and phosphate (Pi) uptake rate differs among genotypes and to select genotypes with a low arsenate uptake rate with the aim of improving food safety and human health.

• Methods A hydroponic experiment was conducted using two wheat (Triticum aestivum) cultivars (Hanxuan 10 and Lumai 14) and ten doubled haploid (DH) lines derived from them to investigate Pi and arsenate uptake over 48 h. Ten plants were transferred to bottles containing 50 mL of pre-treatment solution containing 0·5 mm CaCl2 and 5 mm MES set at pH 6.0 with 330 µm Pi as KH2PO4 and 7·33 µm arsenate. The solutions were aerated continuously. At 8, 24 and 48 h after uptake, 1 mL of test solution was sampled for determination of Pi and arsenate concentrations.

• Key Results and Conclusions For each wheat line, Pi and arsenate concentrations in the test solution decreased with uptake time. Exponential (for Pi) or polynomial (for arsenate) regression plots fitted the data closely. For all genotypes, net Pi uptake rates decreased with time (from 0 to 48 h). However, net arsenate uptake rates decreased with time for D5, changed little with time for the male parent, D4 and D6, and increased with time for the others. An inflexion of about 25 µm Pi was observed for the relationship between arsenate and Pi concentrations in the test solution, indicating that 25 µm could be the point where the high-affinity uptake system ‘switches on’, or dominates over low-affinity uptake. In addition, the male parent, D1, D6 and D10 were considered ideal genotypes because they possess Pi transporters that discriminate strongly against arsenate and are expected to accumulate less arsenate in the field.

Keywords: Arsenic, phosphate transporters, ion selectivity, plant membrane transport

INTRODUCTION

Arsenate, the dominant form of arsenic (As) in aerobic conditions, is taken up by plants via the phosphate (Pi) transport systems because of the chemical similarity between arsenate and Pi (Dixon, 1997). It has been demonstrated that arsenate inhibits Pi uptake by yeast (Rothstein and Donovan, 1963), phytoplankton (Blum, 1966), Arabidopsis thaliana (Clark et al., 2003), wheat (Geng et al., 2006) and the As hyperaccumulator, Chinese brake fern Pteris vittata (Wang et al., 2002). Similarly, Pi suppresses arsenate uptake by phytoplankton (Planas and Healey, 1978), rice (Abedin et al., 2002), Lupinus albus (Esteban et al., 2003) and the As-tolerant plants Holcus lanatus, Cytisus striatus (Meharg and MacNair, 1992; Bleeker et al., 2003) and P. vittata (Wang et al., 2002; Tu and Ma, 2003). Arsenate in the plant is converted to arsenite and then methylated in the leaves, accompanied by the induction of arsenic methyltransferase activities (Wu et al., 2002; Duan et al., 2005).

One way of reducing As accumulation in food and toxicity to humans is to select species or genotypes with low As uptake rates. Arsenate resistance has been identified in a number of species occurring on As-contaminated soils, including Andropogon scoparius, Agrostis castellana, A. delicatula, A. capillaris, Deschampsia cespitosa, H. lanatus, Silene vulgaris, Plantago lanceolata and Calluna vulgaris (Sharples et al., 2000; Meharg and Hartley-Whitaker, 2002). However, in populations of H. lanatus from uncontaminated soils across the UK, approx. 45 % of seeds gave rise to As-resistant plants (Meharg et al., 1993), whilst individuals of S. vulgaris (Paliouris and Hutchinson, 1991) and P. lanceolata (Pollard, 1980) from uncontaminated soils also exhibited As resistance. This polymorphism implies that the cost of As resistance is low (Meharg et al., 1993; Meharg and Hartley-Whitaker, 2002), and thus As resistance is common. Accordingly, it should be possible to select As-resistant genotypes from crops such as rice and wheat.

One hundred and twenty doubled haploid (DH) lines (derived from parents Hanxuan 10 and Lumai 14) have been demonstrated to differ in their rate of phosphorus (P) uptake in a pot trial at seedling stage (Yiping Tong, unpubl. res.). Parents and ten DH lines have been selected in the present work based on these large differences in P uptake in the field (Table 1). The objectives of the present work were to find whether the arsenate uptake rate had a relationship to Pi uptake rate among genotypes and to select some genotypes with low arsenate uptake rate for the purpose of food safety and human health.

Table 1.

The parents and ten doubled haploid lines used in the present work

Genotypes P efficiency in field soil Abbreviations in the text
Male Lumai 14 M
Female Hanxuan 10 F
DH lines DH04-08 High D1
DH04-14 High D2
DH04-24 High D3
DH04-38 High D4
DH04-65 High D5
DH04-03 Low D6
DH04-31 Low D7
DH04-99 Low D8
DH04-109 Low D9
DH04-120 Low D10
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MATERIALS AND METHODS

Preparation of seedlings

Seeds of the two parental genotypes of Triticum aestivum L. and ten DH lines were sterilized in 10 % H2O2 (v/v) for 10 min followed by thorough washing in de-ionized water, and then germinated on moist filter paper for 2 d Germinated seeds were transferred to moist perlite and cultivated for 9 d. The seedlings were removed from the perlite and washed carefully under tap water to remove adhering particles. They were then transferred to PVC pots containing 1100 mL of modified Hoagland's solution containing: (in mm), KNO3, 2·0; Ca(NO3)2, 2·0; MgSO4, 0·7; KH2PO4, 0·7; and (in µm), FeEDTA, 50; ZnSO4, 0·5; CuSO4, 0·5; MnSO4, 2·5; H3BO3, 5; Na2MoO4, 0·25; CoSO4, 0·09; and NaCl, 50. Each genotype was planted into one pot with three holes, and with ten plants in each hole. The nutrient solution was renewed twice a week and aerated continuously. Pots were randomly arranged every day during the growth period. The seedlings were grown in a growth chamber with 14/10 h light/dark cycles. Light intensity was approx, 280 µmol m−2 s−1.

Treatments

After 6 d of pre-culture in hydroponics, roots of intact plants were rinsed with de-ionized water and plants were transferred to bottles containing 50 mL of pre-treatment solution containing 0·5 mm CaCl2 and 5 mm MES set at pH 6·0. Altogether there were 12 genotypes (lines) with three replicates for each, giving 36 bottles in total. Each bottle had ten plants for each genotype. Twelve hours later (also 2 h after the light period started), the pre-treatment solution was replaced with 50 mL of test solutions. Test solutions contained 330 µm Pi as KH2PO4 and 7·33 µm arsenate as Na3AsO4. Otherwise, micro- and macronutrients were the same as in the pre-culture solutions. The test solutions were aerated continuously. At 8, 24 and 48 h after uptake, 1 mL of test solution was sampled for determination of Pi and arsenate concentrations, and 1 mL of de-ionized water was added to each bottle to replenish the volume of sampling solution. Water losses through transpiration were also compensated by the addition of de-ionized water at hourly intervals. The temperature was maintained at 25 ± 0·5°C.

Pi and arsenate analysis

The Pi and arsenate concentrations in test solutions were determined by ICP-OES (Inductively Coupled Plasma Optical Emission Spectrometer, Optima 2000 DV, Perkin Elmer, USA). At 48 h after uptake, arsenate concentrations in test solutions were determined by an AF-610A atomic fluorescence spectrometer (Beijing Ruili Analytical Instrument Co., Beijing, China). At 48 h, plants were harvested and divided into roots and shoots. Plant materials were then oven dried at 70°C for 48 h, and the dry weights of shoots and roots were recorded.

Data analysis

There are five steps of data analysis as follows: (1) for each wheat line, the Pi or arsenate concentrations were plotted against four time points (0, 8, 24 and 48 h); (2) each line was fitted by an exponential (for Pi) or polynomial (for arsenate) equation; (3) differentiation of this equation gives the instantaneous uptake rate, which can be converted into sensible units from the solution volume and plant weights; (4) the uptake rate for each time point was calculated by inserting 0, 8, 24 and 48 h into the rate equation and these rates were then plotted against the solution concentrations measured at that time point; and (5) average arsenate and Pi uptake rates were also calculated during the 48 h uptake period for all 12 lines.

All data were subjected to an analysis of variance (ANOVA) performed on a Windows-based Genstat (6th edn., NAG Ltd, UK).

RESULTS

Biomass

Parental genotypes had similar root biomass (Fig. 1). DH lines D2, D4, D5, D7 and D8 had similar root biomass to their parents, and D1, D3, D6, D9 and D10 had lower root biomass than their parents. The female parent had higher shoot biomass than the male parent. D3 had lower shoot biomass than the male parent, and D4, D5 and D8 had higher shoot biomass than the male parent, and similar shoot biomass to the female. D1, D2, D3, D6, D7, D9 and D10 had lower shoot biomass than the female parent and no DH lines had higher shoot biomass than the female parent.

Fig. 1.

Fig. 1.

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Root (A) and shoot (B) biomass of parents and the ten doubled haploid (DH) lines 17 d after sowing. Error bars represent the s.e.m. from three replicates with ten plants for each replicate.

Pi and arsenate concentrations in the test solution

For each wheat line, Pi and arsenate concentrations in the test solutions decreased with uptake time, which was fitted well by exponential (for Pi) or polynomial (for arsenate) equations (Table 2).

Table 2.

The relationship between Pi or arsenate concentrations in the test solution and time (T, h) fitted by exponential (for Pi) or polynomial (for arsenate) regression for parents and the ten doubled haploid lines

R2
Pi (μm)
M Pi = 325·39 × e (−0·038 × T) 0·9995
F Pi = 340·45 × e (−0·0775 × T) 0·9956
D1 Pi = 351·09 × e (−0·0525 × T) 0·9949
D2 Pi = 473·17 × e (−0·1127 × T) 0·9539
D3 Pi = 331·54 × e (−0·0585 × T) 0·9958
D4 Pi = 340·45 × e (−0·0775 × T) 0·9956
D5 Pi = 369·99 × e (−0·0968 × T) 0·9973
D6 Pi = 311·73 × e (−0·0488 × T) 0·9957
D7 Pi = 324·85 × e (−0·0629 × T) 0·9959
D8 Pi = 421·17 × e (−0·0953 × T) 0·9645
D9 Pi = 451·88 × e (−0·1068 × T) 0·9595
D10 Pi = 351·36 × e (−0·0783 × T) 0·9969
Arsenate (μm)
M Arsenate = −4E-05 × T2 − 0·0603 × T + 7·4106 0·9935
F Arsenate = −0·0013 × T2 − 0·0418 × T + 7·3675 0·9995
D1 Arsenate = −0·0007 × T2 − 0·0414 × T + 7·429 0·9930
D2 Arsenate = −0·0014 × T2 − 0·0334 × T + 7·2674 0·9986
D3 Arsenate = −0·0005 × T2 − 0·0607 × T + 7·3602 0·9995
D4 Arsenate = −0·0002 × T2 − 0·099 × T + 7·4682 0·9937
D5 Arsenate = 0·0004 × T2 − 0·1368 × T + 7·5407 0·9878
D6 Arsenate = 0·0001 × T2 − 0·0733 × T + 7·2554 0·9951
D7 Arsenate = −0·0006 × T2 − 0·0635 × T + 7·3302 1·0000
D8 Arsenate = −0·0003 × T2 − 0·091 × T + 7·2065 0·9949
D9 Arsenate = −0·0008 × T2 − 0·0569 × T + 7·1721 0·9902
D10 Arsenate = −0·0003 × T2 − 0·0695 × T + 7·2803 0·9987
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For individual replicates of all wheat lines, the relationship between arsenate and Pi concentrations in the test solutions at 8, 24 and 48 h was also fitted well by a regression equation: arsenate = 4·25Pi/(2·19 + Pi) + 0·01Pi (r = 0·96, n = 106, P < 0·001). An inflexion of about 25 µm Pi can be derived from Fig. 2. When Pi concentrations in the test solutions were higher than approx. 25 µm, arsenate concentrations decreased linearly with Pi concentrations and the slope of the line was 0·01. However, when Pi concentrations were lower than approx. 25 µm, arsenate concentrations decreased sharply compared with Pi concentrations.

Fig. 2.

Fig. 2.

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The relationship between arsenate and Pi concentrations in the test solutions during the 48 h uptake period for parents and the ten doubled haploid lines. Values are from individual replicates of 12 genotypes.

Net Pi and arsenate uptake rates

For all wheat lines, net Pi uptake rate decreased with time (Fig. 3A). However, the changes in net arsenate uptake rate with time were variable for different DH lines. For D5, the net arsenate uptake rate decreased with time; for the male parent, D4 and D6, net arsenate uptake rate changed little with time; and for the rest of the lines, net arsenate uptake rate increased with time (Fig. 3B).

Fig. 3.

Fig. 3.

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The relationship of net Pi (A) or arsenate (B) uptake rates to time for parents and the ten doubled haploid lines at 0, 8, 24 and 48 h.

The relationship of average arsenate and Pi uptake rates during the 48 h uptake period for the male parent, D1, D6 and D10 was fitted well by a linear regression of y = 19·558x—5·7715 (R2 =yy 0·9971, P < 0·001, Fig. 4). The other wheat lines were all above the regression line.

Fig. 4.

Fig. 4.

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Average Pi and arsenate uptake rates during the 48 h uptake period for parents and the ten doubled haploid lines. Each value was the mean from three replicates with ten plants for each replicate.

DISCUSSION

Nutrient uptake is frequently described by two additive Michaelis–Menten functions representing two carrier sites (transporters), one dominant at substrate concentrations below 100 µm, termed the high-affinity uptake system, and the other dominant at high substrate concentrations, termed the low-affinity uptake system; the latter is constitutively expressed (Epstein, 1976; Meharg and Macnair, 1992; Schachtman et al., 1998; Raghothama, 2000). Pi concentrations in soil solution range from 0·5 to 2 µm, depending on the soil composition (Bieleski and Ferguson, 1983). Therefore, a high-affinity Pi uptake system in the plant root should operate with a Km value for Pi uptake that covers this concentration range. PHT1, a high-affinity Pi transporter gene, isolated from Arabidopsis thaliana showed a Km of 3·1 µm when expressed in cultured cells of tobacco plants (Mitsukawa et al., 1997). From these results, the inflexion of 25 µm shown in Fig. 2 could be the point where the high-affinity uptake system ‘switches on’, or dominates over the low-affinity uptake system.

In the low-affinity stage (when the Pi concentration was >25 µm), the slope of the line was 0·01, but the ratio of the original arsenate concentration (7·33 µm) to the original Pi concentration (330 µm) was 0·022, showing that relatively more Pi was taken up than arsenate. This result is in accordance with previous findings that the transporters are more selective for Pi than for arsenate (e.g. Meharg et al., 1994). This selectivity is adopted by plants to alleviate arsenate stress through reducing arsenate absorption. In the high-affinity stage (when Pi was lower than 25 µm), however, the slope of the line was much higher than 0·022, indicating that more arsenate was taken up than Pi, which was due to Pi exhaustion in the nutrient solution. Because of Pi exhaustion in the solution near the end of the 48 h uptake (Table 2), high-affinity Pi transporters should be highly expressed, and thus the arsenate uptake was expected to increase. Such an effect has been reported in barley (Hordeum vulgare; Lee, 1982), the As-sensitive population of H. lanatus (Meharg and Macnair, 1992), Brassica napus (Quaghebeur and Rengel, 2004) and P. vittata (Wang et al., 2002).

The ultimate purpose of this study was to identify genotypes that take up more Pi and less arsenate. The linear relationship of average arsenate and Pi uptake rates for the male parent, D1, D6 and D10 (Fig. 4) indicated that in these four wheat lines the Pi transporters were absorbing arsenate and Pi in a similar ratio. However, in the remaining lines positioned above the regression line, the Pi transporters appear to absorb more arsenate than the male parent, D1, D6 or D10. These lines are thus considered to be desirable candidate genotypes since they appear to possess Pi transporters that discriminate better against arsenate and are likely to accumulate less arsenate in the field. The molecular basis of this difference in Pi transporter properties remains to be explored. The genetics of arsenate tolerance and uptake are not fully understood. Dasgupta et al. (2004) have revealed the presence of an arsenate resistance gene in rice and have mapped its location using a quantitative trait locus (QTL) approach. This As tolerance QTL was found to be closely linked to that of P efficiency (Wissuwa and Ae, 2001), indicating pleiotropic actions for the genes responsible for As tolerance and P uptake/use efficiency. Further studies are under way to dissect further the mechanism of arsenate uptake and tolerance in this wheat DH population.

Acknowledgments

The work was financially supported by the Natural Science Foundation of China (40321101 and 40225002), Ministry of Science and Technology of China (2002CB410808).

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