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Keywords:

  • aluminum toxicity;
  • plasma membrane H+-ATPase;
  • root apex;
  • soybean;
  • tolerant

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Seventeen soybean cultivars were screened to discern differences in aluminum (Al) sensitivity. The Sowon (Al-tolerant) and Poongsan (Al-sensitive) cultivars were selected for further study by simple growth measurement. Aluminum-induced root growth inhibition was significantly higher in the Poongsan cultivar than in the Sowon cultivar, although the differences depended on the Al concentration (0, 25, 50, 75 or 100 μmol L–1) and the amount of exposure (0, 3, 6, 12 or 24 h). Damage occurred preferentially in the root apex. High-sensitivity growth measurements using India ink implicated the central elongation zone located 2–3 mm from the root apex. The Al content was lower 0–5 mm from the root apices in the Sowon cultivar than in the apices of the Poongsan cultivar when exposed to 50 μmol L–1 Al for 12 h. Furthermore, the citric acid exudation rate was more than twofold higher in the Sowon cultivar. Protein production of plasma membrane (PM) H+-ATPase from the root apices (0–5 mm) was upregulated in the presence of Al for 24 h in both cultivars. This activity, however, decreased in both cultivars treated with Al and the Poongsan cultivar was more severely affected. We propose that Al-induced growth inhibition is correlated with changes in PM H+-ATPase activity, which is linked to the exudation of citric acid in the root apex.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Poor crop growth in acid soils can be directly correlated with the degree of aluminum (Al) saturation in the soil solution (Matsumoto 2000). Aluminum is the most abundant metal within the earth’s crust. It was implicated as a cause of root-growth reduction in barley and rye plants growing in acid soils as early as 1918 (Foy 1988; Kochian 1995). Aluminum solubility in the soil solution increases as the soil pH decreases. At low pH values (pH <5), the primary species is (Al(H2O)6)3+, the hydrated form of the metal. Al3+ is likely to be the principal biologically reactive forms.

The root apex is a primary target and plays a central role in Al tolerance and toxicity (Ahn et al. 2001). In particular, inhibition of root growth as a result of Al-induced impairment of cell division and elongation is a well-known early and dramatic symptom of Al phytotoxicity in acid soils (Horst 1995; Kochian 1995; Matsumoto 2000; Taylor 1995). In the case of Al-tolerant plants, organic acids are exudated to resist Al toxicity. Many plants secrete organic acid in response to Al stress. For example, citrate is released from the roots of soybean (Glycine max; Yang et al. 2000) and maize (Zea mays; Pineros et al. 2002), oxalate is released from buckwheat (Fagopyrum esculentum; Ma et al. 1997) and malate is released from the roots of Al-tolerant genotypes of wheat (Triticum aestivum; Delhaize et al. 1993; Ryan et al. 1995). Very little research examining Al-induced inhibition of root growth on Al toxicity has focused on dicot plants. Soybean is an important dicot crop for human nutrition and industrially as a raw material because of its stores of vitamin B1, B2, protein and suitable iron, and because it is amenable to cultivation on acidic soils (von Uexküll and Mutert 1995).

Recent studies have indicated that plasma membrane (PM) H+-ATPase, which is the most abundant protein in this membrane, is involved in the regulation of responses to a variety of environmental stimuli, including stress resulting from Al (Ahn et al. 2004), NaCl (Niu et al. 1993, 1996), phosphorus deficiency (Yan et al. 2002), ammonium (Jernejc and Legisa 2001) and sucrose (Requena et al. 2003). Transcript levels of PM H+-ATPase increase more in the roots of halophytes treated with NaCl than in glycophytes (Niu et al. 1993), suggesting that the capacity of the PM H+-ATPase gene to respond to NaCl may contribute to the greater salt tolerance of halophytes. In addition, our previous study showed that upregulation of PM H+-ATPase activity coupled with citric acid is associated with Al resistance of a soybean cultivar under Al stress (Shen et al. 2005).

In the present paper, we report on the differential growth inhibition observed with screened Al-tolerant (Sowon) and Al-sensitive (Poongsan) soybean cultivars after treatment with Al in vivo. Moreover, Al-induced exudation of organic acids is linked to the expression of PM H+-ATPase at the transcriptional and translational levels.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Plant materials and growth conditions

Seventeen soybean cultivars (Jinmi, Chungdoo, Dawon, Enha, Danmi, Seonhk, Jangwon, Saeol, Gumjung, Seonnock, Dawol, Poongsan, Chungja, Nockchae, Daemang, Sinki and Sowon) were screened to assess differences in Al sensitivity. Seeds were surface-sterilized with 1.5% (v/v) NaOCl + 1.0% sodium dodecyl sulfate (SDS) for 10 min, rinsed thoroughly with distilled water (DW) and germinated on filter paper moistened with DW at 25°C in the dark for 3 days. Germinated seedlings were transferred to modified Hoagland solution (HS) adjusted to pH 4.5 with 0.1 mol L–1 HCl in polyethylene pots and grown in a controlled-environment chamber (light/dark temperature of 22 ± 1°C and relative humidity of 65%) with a 16 h light/8 h dark cycle. The modified HS contained 1.25 mmol L–1 KNO3, 1.5 mmol L–1 Ca(NO3)2, 0.75 mmol L–1 MgSO4, 0.5 mmol L–1 KH2PO4, 75 μmol L–1 Fe-ethylenediaminetetaacetic acid (EDTA), 50 μmol L–1 H3BO3 10 μmol L–1 MnCl, 2 μmol L–1 ZnSO4, 1.5 μmol L–1 CuSO4 and 0.075 μmol L–1 (NH4)6Mo7O24. After germination, uniform seedlings were grown for 24 h in polyethylene pots containing modified HS adjusted to pH 4.5. Four-day-old seedlings were transferred to phosphate-free HS at least 12 h prior to treatment. For further analysis of Al stress, these cultivars were subjected to Al concentrations of 0, 25, 50, 75 or 100 μmol L–1 and Al exposure times of 0, 3, 6, 12 or 24 h.

Segmental root growth measurements

Four-day-old seedlings were transferred to phosphate-free HS at least 12 h before the root growth experiments began. The segmental (2 mm and 5 mm) elongation rate was measured after marking the 2 mm and 5 mm positions up to 10 mm from the apex using a fine brush with India ink. Plants with marked roots were transferred to modified HS containing 0 or 50 μmol L–1 Al (pH 4.5), and movement of the markings on the growing roots was photographed at designated times (0, 3, 6, 12 and 24 h) using a digital camera (Nikon, coolpix5400).

Hematoxylin staining of Al-treated roots

Ten five-day-old intact seedlings were grown in modified HS containing 50 μmol L–1 Al. After 24 h exposure to Al, the seedlings were removed from the modified HS and stained for visual detection of Al as previously described (Polle et al. 1978). The seedlings were washed with DW for approximately 5 min and stained with 0.2% hematoxylin solution (Sigma-Aldrich, St Louis, MO, USA) for 10 min. The seedlings were washed again with DW for 15 min to remove any excess stain and were photographed.

Determination of Al in the root apices

After treatment with 50 μmol L–1 Al for 12 h, the roots were washed with DW and the root apices (0–5 mm as measured from the root apex including the root cap) were excised using a blade. Ten excised segments (representing one replicate) were transferred to 1.5 mL Eppendorf tubes for 24 h; each tube contained 6 mL of 2 mol L−1 HCl. The Al content in the HCl digest of the root tissue was determined by inductively coupled plasma mass spectroscopy (IRIS-AP; Thermo Scientific, Pittsburgh, PA, USA) after dilution.

Analysis of citric acid

Seedlings were treated with 0.5 mmol L–1 CaCl2 (pH 4.5) containing 50 μmol L–1 Al for 24 h. After treatment with Al, the collected solutions containing the root exudates were passed through a cation-exchange column (16 mm × 14 cm) filled with 5 g Amberlite IR-120 B resin (H+ form; Muromachi Chemical, Tokyo, Japan) and then through an anion-exchange column (16 mm × 14 cm) filled with 2.5 g Bio-Rad AG 1-X8 resin. These procedures were carried out in a cold room and the organic acid anions retained in the anion exchange resin were eluted with 2 mol L−1 HCl and concentrated to dryness using a rotary evaporator (N-N; Rikakikai, Tokyo, Japan) at 40°C. The residue was re-dissolved in 1 mL Milli-Q (18.2 MΩ) water adjusted to pH 2.1 with perchloric acid. The citric acids were detected by High Performance Liquid Chromatography (LC-10A equipped with the ion-exclusion column Shim-pack SCR-102 H, 0.8 × 30 cm; Shimadzu, Kyoto, Japan) as described in detail by Yang et al. (2000).

Isolation of the plasma membrane vesicles

Plasma membrane vesicles were prepared at 4°C as previously described (Palmgren et al. 1990). In brief, collected samples were ground in ice-cold homogenization buffer containing 50 mmol L–1 morpholino-ethane-sulfonic-bis-tris-propane (MOPS-BTP; pH 7.5), 330 mmol L–1 sucrose, 5 mmol L–1 EDTA, 5 mmol L–1 dithiothreitol (DTT), 0.5 mmol L–1 phenylmethanesulphonylfluoride, 0.2% (w/v) casein, 0.2% (w/v) bovine serum albumin and 0.5% polyvinylpyrrolidone. The homogenate was filtered through three layers of cheesecloth and centrifuged at 10,000 g for 15 min at 4°C. The supernatant was centrifuged at 30,000 g for 120 min, and the resulting precipitate was resuspended in suspension buffer consisting of 330 mmol L–1 sucrose, 5 mmol L–1 potassium phosphate (KH2PO4; pH 7.8), 5 mmol L–1 KCl, 0.1 mmol L–1 EDTA and 1 mmol L–1 DTT. The homogenate was loaded on a 12 g L−1 two-phase system containing 6.5% (w/w) dextran T-500 (Sigma-Aldrich), 6.5% (w/w) polyethylene glycol PEG-3350 (Sigma-Aldrich), 250 mmol L–1 sucrose, 5 mmol L–1 KH2PO4 (pH 7.8), 4 mmol L–1 KCl and sterile DW. After the batch procedure, the resulting upper phase was mixed with a dilution buffer that consisted of 5 mmol L–1 MOPS-BTP (pH 7.5), 330 mmol L–1 sucrose and 5 mmol L–1 KCl, and centrifuged at 100,000 g for 60 min. The carefully obtained PM vesicles were either used immediately or stored at −80°C until further analysis. The protein concentration was determined as previously described (Bradford 1976).

Electrophoresis and western blotting of plasma membrane H+-ATPase

Determined PM proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as previously described (LaemmLi 1970). Membrane vesicles were solubilized in SDS loading buffer. The samples were loaded onto a SDS-PAGE system consisting of a 5.6% (w/v) stacking gel and a 12% (w/v) separating gel. For western-blot analysis, the SDS-PAGE resolved proteins were transferred to polyvinylidene fluoride filters. To identify and quantify PM H+-ATPase, we incubated each blot with a 1:1,000 dilution in phosphate buffered saline–Tween 20 (PBST) of antiserum containing polyclonal antibody against goat PM H+-ATPase (obtained from Dr Hideo Sasagawa, Okayama University, Japan). After incubation at room temperature for 1 h, each membrane was rinsed with PBST prior to incubation at room temperature for 1 h with a 1:2,000 dilution of secondary antibody. Bound antibody was detected with alkaline phosphatase-conjugated anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, California, USA) using a 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium liquid substrate system (catalog no. B-1911; Sigma-Aldrich).

Assay of plasma membrane H+-ATPase activity

Plasma membrane H+-ATPase was measured in 0.5 mL of reaction solution that contained 300 mmol L–1 MOPS-BTP (pH 6.0), 60 mmol L–1 MgSO4, 1 mol L–1 KCl, 30 mmol L–1 ATP and 1% Triton-X100. After 30 min at 37°C, the reaction was stopped by adding 500 μL of 5% trichloroacetic acid, 2 mL of 100 mmol L–1 sodium acetate, 300 μL of 1% ascorbic acid, 60 μL of 10 mmol L–1 CuSO4 and 300 μL of 1% ammonium molybdate in 0.025 mol L–1 H2SO4. After 10 min at 30°C, the absorbance at 720 nm was measured with a spectrophotometer (Optizen 2120 UV Mecasys Co. Ltd, Daejeon, South Korea). The difference between the samples with and without 0.1 mmol L–1 vanadate was expressed as the PM H+-ATPase activity. A standard curve of phosphate in the reaction mixture was included in each assay.

Data analysis

All experiments were arranged in a randomized complete design and the data were statistically evaluated using standard deviation and Student’s t-tests.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Screening for Al resistance

Plants were grown in HS adjusted to pH 4.5 for 24 h after germination. The Al treatments were carried out in modified (phosphate-free) HS, and the plants were cultured in the absence of phosphate for at least 12 h prior to treatment. Three parameters were used to measure Al toxicity in soybean seedlings: root growth rate (Fig. 1), hematoxylin staining (Fig. 2) and the segmental elongation rate of the roots (Fig. 3). As a first approach, the root length of 17 soybean cultivars in routine use in South Korea was measured before and after treatment with Al. Among these cultivars, the Sowon cultivar showed the highest resistance to 50 μmol L–1 Al for 24 h (90% root growth of the control) and the Poongsan cultivar displayed the lowest resistance (43% root growth of the control) (Fig. 1). Main and branch Al-treated roots were severely affected in the latter cultivar, and only a few roots were evident. These two cultivars were selected for further study.

image

Figure 1.  Screening of 17 soybean cultivars for Al resistance. Root growth rate in the presence of 50 μmol L–1 Al was measured after 24 h. Data are means ± standard deviations (= 10) of three separate experiments. *< 0.05; **< 0.025 (Student’s t-test).

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image

Figure 2.  Effect of Al (0, 25, 50, 75 and 100 μmol L–1) supply on (a) the root growth rate and (b) hematoxylin-stained roots of soybean after 24-h treatment periods. The Al treatments were carried out in phosphate-free Hoagland solution (HS). Seedlings of the Sowon and Poongsan cultivars were grown in HS at 0, 25, 50, 75 and 100 μmol L–1 Al for 24 h and stained with 0.2% hematoxylin. Data are means ± deviations (= 10). *< 0.05; **< 0.025 (Student’s t-test).

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image

Figure 3.  Effects of Al (50 μmol L–1) supply on (a) the root growth rate after 0, 3, 6, 12 and 24 h treatment periods and (b) the specific segmental pattern of root growth inhibition after 12 h of Al treatment in soybean. The Al treatments were carried out in Hoagland solution without phosphate. Figure part (b) shows the main roots of the Sowon (left) and Poonsan (right) cultivars marked with India ink at 1-mm intervals at 0 h (small panel). The arrows indicate subsequent alteration to their position; the Sowon cultivar displayed only the disappearance of the 2 and 3 mm marks after 12 h. Data are means ± standard deviations (= 10). *< 0.05; **< 0.025 (Student’s t-test).

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Aluminum-induced growth inhibition in the root apex

The selected cultivars were exposed to 0, 25, 50, 75 and 100 μmol L–1 Al for 24 h (Fig. 2) and for 0, 3, 6, 12 and 24 h (Fig. 3). Treatment in the absence of Al or with 25 μmol L–1 Al did not inhibit the roots of either cultivar. Treatment with 50, 75 and 100 μmol L–1 Al inhibited root growth in the Sowon cultivar by 5, 21 and 38%, respectively, and in the Poonsan cultivar by 22, 43 and 55%, respectively, compared with the control (Fig. 2a). To compare the Al-induced root growth inhibition and staining pattern, hematoxylin was used to stain the soybean roots. Interestingly, hematoxylin staining was markedly observed only in the Poongsan cultivar; roots treated with high Al concentrations (50, 75 and 100 μmol L–1) for 24 h were severely affected, becoming thick, stubby and dark in the apex region, compared with the Sowon cultivar (Fig. 2b). For further analysis, the segmental elongation rate using India ink at 5-mm intervals after the 24 h Al treatment was determined (data not shown). Untreated roots and those treated with 25 and 50 μmol L–1 Al were not significantly inhibited in the Sowon cultivar, whereas significant inhibition was evident in the root growth of the Poongsan cultivar following 24 h exposure to 50, 75 and 100 μmol L–1 Al. The latter inhibition occurred mostly in the 0–5 mm root apex portion. Subsequent experiments were carried out with roots grown in the absence of Al or in 50 μmol L–1 Al.

Figure 3 summarizes the root growth results following the application of 50 μmol L–1 Al for varying times (0, 3, 6, 12 and 24 h). Significant inhibition of root growth was observed in the Poongsan cultivar beginning at 6 h; by 24 h, root growth was approximately 20% reduced compared with the Sowon cultivar (Fig. 3a). With regard to segmental elongation, almost no inhibition of growth was noted in the control and 50 μmol L–1 Al-treated roots of the Sowon cultivar, whereas root growth of the Poongsan cultivar was inhibited significantly within 6 h. Roots of the Sowon cultivar displayed only the disappearance of the 2 mm and 3 mm marks after 12 h. These marks faded after 12 h from the apex of the root extending approximately 5 mm in the Sowon cultivar and 3 mm in the Poongsan cultivar from their initial position at 0 h, which was evidence of Al-induced growth inhibition in the Poongsan cultivar (Fig. 3b). The Al contents in the 0–5 mm region from the root apices of the soybean plants were quantified after 12 h with 50 μmol L–1 Al. Aluminum was almost undetectable in control root apices in both cultivars. However, Al treatment enhanced Al accumulation by 25% in the Poongsan cultivar (Fig. 4). The contents of potassium, calcium and magnesium were inhibited after Al treatment in both cultivars (data not shown).

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Figure 4.  Effect of Al (50 μmol L–1) on Al accumulation at 5-mm specific segments of soybean roots after 12 h of Al treatment. The Al contents were determined in three replicates, each comprising 10 root apexes of the same distance from the 5-mm root tip. Data are means ± standard deviations (= 5). *< 0.05; **< 0.025 (Student’s t-test).

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It is well known that Al-resistant soybean cultivars exhibit Al-induced exudation of citric acid (Yang et al. 2000). We collected root exudates from the two soybean cultivars after 24 h and analyzed them for citric acids. Efflux of citric acid was significantly enhanced in the Sowon cultivar compared with the Poongsan cultivar in the 50 μmol L–1 Al treatments. No citric acid efflux was detected in the absence of Al in either cultivar (Fig. 5).

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Figure 5.  Effect of Al (50 μmol L–1) on Al-induced citric acid efflux from soybean roots. Seedlings were subjected to 0.5 mmol L–1 CaCl2 solution (pH 4.5) containing Al. Root exudates were collected after 24 h exposure and citric acid were analyzed by High Performance Liquid Chromatography. Data are means ± standard deviations (= 5). *< 0.05; **< 0.025 (Student’s t-test). FW, fresh weight.

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Translational regulation of plasma membrane H+-ATPase by Al

To investigate whether translational regulation was involved in the response to Al, SDS-PAGE of PM proteins isolated from the roots of the two cultivars 0, 1, 3 and 6 days after 50 μmol L–1 Al treatment was conducted. The electrophoretic analyses revealed that several protein bands appeared or disappeared under Al stress. Plasma membrane H+-ATPase protein was detected using western blotting. Using a polyclonal antibody against maize PM H+-ATPase, we observed a signal band at 100 kDa corresponding to PM H+-ATPase. When plants were treated with 50 μmol L–1 Al for 0, 1, 3 and 6 days, the amount of PM H+-ATPase was observed to decrease in whole root fractions after 6 days in the Poongsan cultivar and no change was observed until 6 days in the Sowon cultivar (Fig. 6a). However, Al stress increased the protein amount of PM H+-ATPase in the root apices in both the Al-tolerant (Sowon) and Al-sensitive (Poongsan) cultivars (Fig. 6b). Furthermore, the rate of increase was higher in the Al-tolerant cultivar than in the Al-sensitive cultivar, suggesting that PM H+-ATPase synthesis was stimulated by this treatment. In addition, the amount of PM H+-ATPase protein decreased in the 50 μmol L–1 Al treatment plus citric acid for 24 h in vivo (Fig. 6b), perhaps reflecting Al-induced chelation of citric acid.

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Figure 6.  Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot of plasma membrane (PM) H+-ATPase from (a) whole roots and (b) root apices. (a) The Al-tolerant (Sowon) and Al-sensitive (Poongsan) cultivars were treated with 50 μmol L–1 Al for 0, 1, 3 and 6 days in Hoagland solution without phosphate (pH 4.5) and then the PM was isolated from the whole roots. (b) The two cultivars were treated in the same way with 50 μmol L–1 Al or Al + 50 μmol L–1 citric acid for 24 h and then the PM was isolated from the root apices. Ten micrograms of the protein sample was separated on 12% SDS-PAGE and analyzed by western-blot using a PM H+-ATPase antibody. M; approximate Mr in kDa.

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Impact of Al on plasma membrane H+-ATPase activity

Using the PM fraction obtained from the upper phase of the two-phase partitioning (Palmgren et al. 1990), the effect of various inhibitors on H+-ATPase activity was measured to confirm the purity of the PM vesicles. The H+-ATPase activity in the presence of nitrate or azide, which are specific inhibitors of tonoplast and mitochondrial ATPase (Widell and Larsson 1990), was slightly inhibited, whereas vanadate, which is a specific inhibitor of PM H+-ATPase activity, inhibited approximately 80% of the activity (data not shown). There was no difference in response to the inhibitors between the cultivars. In addition, when ATP was provided as the substrate, PM H+-ATPase activity increased by approximately 90%, whereas activity increased by approximately 10% in the absence of ATPase. The pH dependence of ATPase activity revealed a peak at approximately pH 6.0 (data not shown), suggesting that the soybean preparations were rich in PM (approximately 85% purity). To investigate the impact of Al on PM H+-ATPase activity isolated from whole roots and root apices, soybean plants were exposed to 50 μmol L–1 Al for 24 h. There was an increase in the activity of whole root PM in the Al-tolerant Sowon cultivar, whereas a marginal decrease was observed in the Al-sensitive Poongsan cultivar. Interestingly, however, the H+-ATPase activity of the PM vesicles isolated from the 0–5 mm root apices treated with 50 μmol L–1 Al for 24 h decreased by 28 and 54% in the Sowon and Poongsan cultivars, respectively (Fig. 7). As the 2–3 mm region represents a sensitive target site for Al toxicity (Fig. 3b), these results support the suggestion that the root apex is more sensitive to Al in terms of PM H+-ATPase activity in the Al-sensitive cultivar.

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Figure 7.  Effects of Al (50 μmol L–1) treatment for 24 h on the H+-ATPase activity of plasma membrane (PM) vesicles isolated from whole roots and from the 5-mm root apices of soybean. Soybean seedlings were grown in Hoagland solution without phosphate for at least 12 h prior to the Al treatment. The 5-mm segments were constructed from approximately 300 individual plants and the isolated PM vesicles were pooled to increase the precision of the measurements. Data are means ± standard deviations (= 3). *< 0.05; **< 0.025 (Student’s t-test).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

A number of decades ago, two pioneering works postulated that decreased root growth is a consequence of the inhibition of cell division (Clarkson 1965) and cell elongation (Klimashevski and Dedov 1975). The root apex has been recognized as a primary site of Al-induced injury in plants (Ryan et al. 1993). To survive in the elevated Al of acidic soils, proper root growth is essential. Therefore, we have investigated a mechanism associated with Al tolerance by comparing it with Al sensitivity in soybeans. Short-term experiments using hydroponic culture have been conducted because this approach is rapid and highly reproducible (Campbell and Carter 1990) and, hence, has been used widely to screen soybean cultivars for Al resistance (Horst et al. 1992, 1997; Sartain and Kamprath 1978). In the present study, root growth rate was used as an index for Al-tolerance variation. The results show that the Sowon cultivar was the most Al tolerant and the Poongsan cultivar was the most Al sensitive among the 17 tested cultivars (Fig. 1). To better understand the Al-tolerant and Al-sensitive mechanisms, it was necessary to elucidate whether the concentration of Al in the tissue was responsible for the onset of root growth inhibition. Visual evaluation of stained roots can be used to detect Al accumulation in root tissue (Polle et al. 1978). In the present study, hematoxylin staining showed that Al accumulated mainly in the apical root region of both the Al-tolerant Sowon and the Al-sensitive Poongsan cultivars. The staining intensity in the Al-sensitive root apices increased markedly during a 24 h exposure to 50–100 μmol L–1 Al. These results suggest that root apices acquire more significant injury under higher concentrations of Al stress in sensitive cultivars (Fig. 2). Aluminum (50 μmol L–1) inhibited root apical growth within 6 h and the inhibition was significant at 12 h in the Al-sensitive cultivar (Fig. 3). The pattern of Al inhibition observed was similar to the time-dependent and concentration-dependent inhibitory effect of Al reported for squash roots (Ahn et al. 2002). The results of our spatial analysis of root growth suggest that the 2–3 mm region is the central elongation zone for soybean. Aluminum inhibited root growth preferentially in this region after 12 h. This pattern of Al inhibition is similar to the inhibitory effect of Al described for maize roots (Blancaflor et al. 1998) and in squash (Ahn et al. 2001). Therefore, the inhibition of total root elongation is derived from the effect on the central elongation zone. In accordance with our results, Al-induced root growth inhibition occurs only in the few most apical millimeters of the root in maize plants (Ryan et al. 1993; Sivaguru and Horst 1998). Thus, the root apex is the primary target and plays a central role in Al tolerance and toxicity. Numerous reports in the literature describe Al-induced alterations occurring particularly in the apical regions of roots, leading to, for example, changes in root cell patterning (Doncheva et al. 2005), irregular cell division and alterations in cell shape (Čiamporová 2000; Vázquez et al. 1999), disintegration of the cytoskeleton (Sivaguru et al. 1999, 2003), disturbance of PM properties (Ahn and Matsumoto 2006; Ahn et al. 2001, 2002, 2004; Miyasaka et al. 1989; Olivetti et al. 1995Pavlovkin and Mistrík 1999; Sivaguru et al. 1999, 2003), as well as the production of reactive oxygen species (Darkóet al. 2004; Jones et al. 2006).

Akeson et al. (1989) reported that Al3+ has a very strong affinity for the PM surface (e.g. 56-fold higher than Ca2+). In the present study, root growth of the Al-tolerant Sowon cultivar was affected less than the Al-sensitive Poongsan cultivar based on Al accumulation in the root apices (Fig. 4). These results are consistent with the Al tolerance of the Sowon cultivar being closely associated with an Al exclusion mechanism induced by citric acid, as demonstrated in our present study (Fig. 5). Similar observations of organic exclusion have been made in comparisons of Al-tolerant and Al-sensitive cultivars of malate for wheat (Delhaize et al. 1993), citrate for soybean (Lazof et al. 1994; Yang et al. 2000) and malate/citrate for maize (Llugany et al. 1994). We also observed that K+, Ca2+ and Mg2+ ions were more inhibited after Al treatment in root apices of Al-sensitive cultivars compared with Al-tolerant cultivar (data not shown). Nichol et al. (1993) reported that 100 μmol L–1 Al inhibits the influx of Ca2+ by 69%, NH4+ by 40% and K+ by 13%, whereas it inhibits the influx of NO3 by 44% and phosphate by 17% in an Al-sensitive cultivar of barley. It has been suggested that Al binds to the PM phospholipids, forming a positively charged layer that influences ion movement to the binding sites of the transport proteins, such as PM H+-ATPases, which has been previously discussed (Ahn et al. 2001, 2004).

In soybeans, PM H+-ATPase is encoded by gene accession number AF091303. Until now, this gene was well known only as a PM H+-ATPase among the P-type ATPase family in soybeans. Aluminum induced gene expression of PM H+-ATPase in the Al-tolerant cultivar of soybean in a time-dependent manner, but gene expression of PM H+-ATPase in the Al-sensitive cultivar was decreased by Al treatment after 4 h via RT-PCR (see Data. S1). These results suggest that the PM H+-ATPase gene is positively related to Al stress at the level of transcription. The amount of PM H+-ATPase protein increased more with Al treatment in the Al-tolerant cultivar, suggesting that PM H+-ATPase synthesis is stimulated by Al. Treatment with Al inhibited root growth in the Poongsan cultivar, but not in the Sowon cultivar because the Al-tolerant cultivar rapidly exudes large amounts of citric acid under Al stress; the citric acid chelates the Al into a non-toxic form (Fig. 5). Consistent with this, when citric acid was added to the Al-sensitive Poongsan cultivar in vivo, the Al-induced expression level on PM H+-ATPase protein was ameliorated. The increased amount of PM H+-ATPase protein observed in the present study is similar to the increased effect of Al reported for soybean roots under Al stress (Shen et al. 2005). However, when the Poongsan cultivar was treated with Al and citric acid for 24 h in vivo, the Al-induced deleterious effect of PM H+-ATPase was abrogated, indicating that the increased PM H+-ATPase in Al-tolerant root apices under Al stress is caused by enhancement at translational levels (Fig. 6).

Plasma membrane H+-ATPase activity plays a central role and is a crucial factor for plant survival under various environmental stresses, including Al toxicity. Nevertheless, the relationship between Al toxicity and H+-ATPase activity remains controversial (Kinraide 1988; Ahn et al. 2001, 2004). In the present study, Al faintly altered the activity of PM H+-ATPase isolated from whole roots in the two cultivars and markedly diminished the activity of PM H+-ATPase isolated from the Al-sensitive cultivar, but the capacity for H+-efflux from the root apices in the Al-tolerant cultivar was reduced (Fig. 7). In addition, when we analyzed the difference between the two cultivars statistically, we found significantly inhibited activity in the Al-sensitive cultivar compared with the Al-tolerant cultivar (P < 0.05; Student’s t-test). Previously, Yang et al. (2000) demonstrated that genetic Al tolerance in soybean is related to enhanced root exudation of citric acid induced specifically by Al. In the present study, citric acid exudation by Al was elevated twofold in the Al-tolerant cultivar compared with the Al-sensitive cultivar. These observations may indicate that the relatively higher PM H+-ATPase activity in the Al-tolerant cultivar reflects less disorder in PM function. And this is accompanied by Al-induced exudation of citric acid as a basis for the mechanism of Al tolerance. Furthermore, using bromocresol purple in agarose gel, a more vigorous H+-influx into root apex was found in the Al-tolerant cultivar than in the Al-sensitive cultivar after Al treatment of the roots (data not shown). The results of the visualizing surface pH and enzyme activity suggest that H+-flux across the PM of root apices can be impaired by Al stress in the Al-sensitive cultivar only.

In good agreement with our hypothesis, Yang et al. (2007) proposed that the addition of Mg to the toxic Al solution helps to restore the activity of PM H+-ATPase and that these changes enhanced the Al-dependent exudation of citric acid. Under P-deficient conditions, Tomasi et al. (2009) demonstrated a positive relationship between root exudation of citric acids and PM H+-ATPase-catalyzed H+ efflux in active proteoid roots of white lupin. In contrast, Zhu et al. (2005) showed that export of citric acid across the PM of proteoid root cells is not strictly related to H+ efflux. Therefore, we cannot exclude the possibility that the changes in PM H+-ATPase activity and in the exudation of citric acid under Al stress occur independently.

In conclusion, our results demonstrate that PM H+-flux may play a central role not only in the physiological effect, but also in a translational aspect under Al stress. Further research into the proteomics of the PM isolated from whole root and root apices in Al-tolerant and Al-sensitive cultivars will help confirm the primary role of the PM H+-ATPase in Al toxicity and tolerance.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

This work was supported in part by the Korea Science and Engineering Foundation through the Agricultural Plant Stress Research Center (R 11-2001-092-01004-0) of Chonnam National University and by the Friendly Environment Bio-Energry Research Center (project 20070201036014) and the World Class University project of the Ministry of Science and Technology of Korea (R31-2009-000-20025-0). We also acknowledge the Bioenergy Research Institute, Chonnam National University, for its support of this research.

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  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article:

Data S1. Semiquantitative RT-PCR analysis of the transcriptional level (A) and relative induction of gene expression (B) of the PM H+-ATPase in soybean roots. Roots of Al-tolerant (Sowon) and Al-sensitive (Poongsan) cultivars were treated with 50 μmol L−1 Al during 0, 2 and 4 h in HS without phosphate (pH 4.5).

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