SEARCH

SEARCH BY CITATION

Keywords:

  • aluminum accumulator;
  • cation adsorption;
  • Melastoma malabathricum;
  • polysaccharides;
  • root mucilage;
  • uronic acids;
  • Zea mays;
  • 27Al NMR

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    Plant roots exude viscous polysaccharides, called mucilage. One of the suggested roles of mucilage is immobilization of toxic metal cations, including aluminum (Al), in the rhizosphere.
  • • 
    Mucilage exuded from roots of Melastoma malabathricum (Al accumulator) was characterized in comparison with that of Zea mays (maize; Al nonaccumulator).
  • • 
    Removal of mucilage significantly reduced Al accumulation in M. malabathricum. The cation adsorption affinity of M. malabathricum mucilage was higher for Al and lanthanum (La) than for barium (Ba), whereas that of maize mucilage was in the order Ba > La > Al. A 27Al nuclear magnetic resonance (NMR) spectrum of the Al-adsorbed mucilage and bioassay with alfalfa seedlings indicated that the concentrated Al in the mucilage of M. malabathricum, unlike that of maize, bound very weakly to cation exchange sites of mucilage.
  • • 
    The higher charge density in M. malabathricum mucilage, derived from unmethylated uronic acid, is inferred to be related to preferential adsorption of trivalent cation. Not only a higher degree of methylation in the uronic acid (glucuronic acid) but also H+ release from roots to the mucilage appears to be responsible for the loose binding of Al in M. malabathricum mucilage. These characteristics of mucilage may help Al hyperaccumulation in M. malabathricum.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plants exude various chemical compounds from their roots. Mucilage is the gelatinous material composed of high-molecular-weight compounds that is exuded from the outer layers of the root cap (Sievers et al., 2002). Mucilage consists mainly of polysaccharides, and is transported to the plasma membrane by vesicles of the Golgi apparatus. Several roles of mucilage for plant growth have been suggested, including as a lubricant in root elongation (Iijima et al., 2003) and as a carbon source for rhizosphere microbes (Knee et al., 2001). Detoxification (fixation) of toxic metal cations is also considered to be an important role of mucilage in the rhizosphere (Morel et al., 1986), because sugars in mucilage contain uronic acids (Moody et al., 1988), the carboxyl groups of which may adsorb metal cations as a nonbioavailable form (Morel et al., 1986).

Acidic soils often contain a high concentration of soluble Al that restricts plant growth (Kochian et al., 2004). In general, the mechanism of Al tolerance is categorized into external and internal detoxification mechanisms (Kochian, 1995). One of the external detoxification mechanisms is the exudation of organic acids from roots into the rhizosphere, where they make stable complexes with Al (Ma et al., 2001). Since mucilage can form complexes with Al similar to the organic acids, the mucilage may protect roots from Al toxicity. Horst et al. (1982) reported that 50% of the total Al in 5 mm root tips of cowpea (Vigna unguiculata (L.) Walpers) was bound to mucilage. Also, removal of mucilage from the root tips enhanced Al rhizotoxicity and increased Al accumulation in root tips (Horst et al., 1982). Mucilage strongly binds Al; in wheat roots, mucilage-bound Al accounted for approx. 25–35% of the Al remaining after desorption with 0.5 mm citric acid solution (pH 4.5, 30 min) (Archambault et al., 1996). Li et al. (2000) also reported that maize mucilage strongly binds Al, and Al bound to mucilage is not phytotoxic.

However, it is likely that the contribution of mucilage to Al tolerance (exclusion) is limited in several plant species. Li et al. (2000) reported that inhibition of maize root elongation by 5 µm Al was independent of the presence or absence of mucilage before the Al treatment. A small binding capacity of mucilage to Al was considered to be one reason why the mucilage failed to protect maize roots from Al toxicity. These investigators also suggested that the excretion site of mucilage, localized at the root tip (the apical 1 mm), was induced by another factor because the distal part (2–3 mm from the root tip) is the most Al-sensitive site of the maize root apex (Sivaguru & Horst, 1998). Wagatsuma et al. (2001) reported no detectable differences in the amount of root mucilage between Al-tolerant and Al-sensitive cultivars of maize, wheat, rice (Oryza sativa L.), pea (Pisum sativum L.), and sorghum (Sorghum bicolor (L.) Moench).

Melastoma malabathricum (Melastomataceae) is an Al accumulator growing in tropical acidic soils and is one of the dominant woody species in acid sulfate soils with very high acidity and very poor concentrations of nutrients (e.g. N, P, and basic cations) in tropical Asia, Australia and Polynesia (Osaki et al., 1998). This species accumulates more than 10 mg Al g−1 DW in leaves and roots, and Al seems to be a beneficial element for this species (Watanabe et al., 2005, 2006). The internal mechanisms of Al hyperaccumulation in M. malabathricum have been well investigated so far (Watanabe et al., 1998, 2001, 2005). M. malabathricum often accumulates high concentrations of Al in the leaves, even in peat soils with low exchangeable Al (Osaki et al., 1998), predicting that M. malabathricum has some specific mechanisms to enhance Al absorption. In hydroponic culture, we found that roots of M. malabathricum exuded large amounts of mucilage (Fig. 1a). If the mucilage adsorbs Al as a nonbioavailable form, as reported in maize, the presence of large quantities of root mucilage seems contradictory for the plant with high Al concentrations in the leaves. In the present study, therefore, we characterized root mucilage of M. malabathricum and examined its role in Al hyperaccumulation. Maize, of which mucilage strongly binds to Al, was used as a control plant.

image

Figure 1. Mucilage exuded from roots in a hydroponic culture. (a) Photographs of mucilage of Melastoma malabathricum (main photograph) and maize (Zea mays, inset). (b) Effect of aluminum (Al) on mucilage exudation in Melastoma malabathricum. Plants were grown with or without 0.5 mm AlCl3 (pH 4.0) for 4 d to determine the amount of sugar (glucose equivalents) released from roots (bar graph) or 1 wk to observe the mucilage (photograph). Data are means of three replicates ± SE. Different letters indicate significant differences at P < 0.05 (n = 3).

Download figure to PowerPoint

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Effect of aluminum on mucilage exudation from roots

Uniform cuttings from 1-yr-old M. malabathricum L. plants were rooted in a 40 l container containing an Al-free standard nutrient solution that was aerated constantly for 1 month in a glasshouse at Hokkaido University under natural conditions (13–15 h photoperiod and a day : night temperature of 25–28 : 18–22°C). The standard nutrient solution contained 0.54 mm N (NH4NO3), 64 µm P (NaH2PO4·2H2O), 0.15 mm K (K2SO4 : KCl = 1 : 1), 0.25 mm Ca (CaCl2·2H2O), 0.16 mm Mg (MgSO4·7H2O), 7.2 µm Fe (FeSO4·7H2O), 1.8 µm Mn (MnSO4·4H2O), 9.26 µm B (H3BO3), 0.62 µm Zn (ZnSO4·7H2O), 0.036 µm Cu (CuSO4·5H2O), and 0.01 µm Mo ((NH4)6Mo7O24·4H2O); total SO4 = 0.21 mm. Well-rooted cuttings were used for each experiment. Before the treatment, the mucilage was gently removed from roots with fingers. Plants were transferred to 12 l pots filled with the standard nutrient solution with or without 0.5 mm AlCl3 ([Al3+] = 0.42 mm, calculated by a computer program developed by Wada & Seki (1994)) at pH 4.0 under aeration and grown for 1 wk. After the treatment, the mucilage adhering to the roots was visualized by 0.5% (v/v) India ink. In a different experiment, plants were cultured in a nutrient solution (100 ml), with or without 0.5 mm AlCl3 (pH 4.0), under aeration for 4 d to determine the amount of sugars excreted from roots. After the treatment, the mucilage adhering to the roots was gently removed, and the nutrient solution was dried with a rotary evaporator. Total sugar concentrations (monosaccharides and polysaccharides) in the mucilage and in the dried nutrient solution were determined by the phenol sulfuric acid method using glucose as a standard (Dubois et al., 1956).

Effect of removing root mucilage on cation uptake

Melastoma malabathricum plants from which mucilage was removed were transferred to 40 l containers containing the standard nutrient solution with 0.5 mm AlCl3 at pH 4.2 (−muci) under aeration. As a control, the plants with the mucilage were also treated (+muci). Mucilage in the −muci treatment was removed every day. At the end of the 10 d treatment period, the plants were sampled to determine the mineral concentrations in shoots. Shoots were dried in a forced-air oven at 80°C for 72 h and ground with a vibrating sample mill (TI-100, CMT, Saitama, Japan). Dry samples, 100 mg, were digested by H2SO4-H2O2, and the mineral concentrations were determined by inductively coupled plasma atomic emission spectrophotometry (ICPAES; ICPS-7000, Shimadzu, Kyoto, Japan).

Selectivity for the adsorption of cations in root mucilage

The seeds of maize (Zea mays L. cv. Freya) were sterilized with sodium hypochlorite for 10 min, washed with deionized water, and sown on a moderately moist perlite. Uniform plants (shoot height = 15 cm) were transferred to hydroponic culture. Plants of M. malabathricum and maize were grown in 40 l containers containing an Al-free standard nutrient solution (pH 4.0) with aeration, and the mucilage was collected as described earlier. A mixed cation solution (1200 µl) was added to the mucilage (300 µl), derived from M. malabathricum or maize plants, and shaken for 2 h. The mixed cation solution contained 0.1 mm AlCl3, 0.1 mm BaCl2, and 0.1 mm LaCl3, at pH 4.2. The mucilage in the solution was removed by Microcon-3 Centrifugal Filters (cutoff 3 kDa, Millipore Corporation, Bedford, MA, USA), and concentrations of each cation were determined by ICPAES. For comparison, selectivity for the adsorption of cations in two cation exchange resins, DOWEX 50W-X8 (sulfonic acid resin, Na+-form, 100–200 mesh, cation exchange capacity (CEC) = 1.7 meq ml−1 resin; Muromachi Chemicals, Tokyo, Japan) and Muromac A-1 (chelating resin, Na+-form, 100–200 mesh, CEC = 2.8 meq ml−1 resin; Muromachi Chemicals), were also examined using the mixed cation solution. One milliliter of the suspension of the cation exchange resin (2.09 µl resin (3.55 µeq CEC) in DOWEX, 1.04 µl resin (2.91 µeq CEC) in Muromac) was added to 4 ml of the mixed cation solution, shaken gently for 2 h and filtered through a membrane filter (nitrocellulose, pore size = 0.45 µm). Cation concentrations in the filtrate were determined by ICPAES.

Speciation of the aluminum form in root mucilage

One milliliter of mucilage exuded from roots of M. malabathricum grown in the standard nutrient solution with aeration was collected as described earlier, added to 4 ml of 0.1 mm AlCl3 (pH 4.4), shaken for 2 h, and centrifuged at 12 000 g to obtain a dense mucilage bound to Al (mucilage-Al). Liquid-state 27Al nuclear magnetic resonance (NMR) was used to determine the Al form in the mucilage. The 27Al NMR spectrum of the M. malabathricum mucilage was recorded at 156.3 MHz (JNM-α600 spectrometer; JEOL, Tokyo, Japan). The parameters used were as follows: frequency range, 62.5 kHz; data point, 33 k; acquisition time, 0.52 s. An aluminum chloride solution (1 mm AlCl3 in 0.1 M HCl) was used as an external reference for calibration of the chemical shift (0 ppm). The total Al concentration in the Al-treated mucilage was determined by ICPAES after digestion with H2SO4-H2O2.

Determination of bioavailability of Al in root mucilage

To estimate Al bioavailability in the M. malabathricum mucilage, Al phytotoxicity in the Al solution containing the mucilage was compared with that containing citrate or that not containing any chelator, by evaluating root elongation of alfalfa (Medicago sativa L. cv. Hisawakaba). Alfalfa is known to be highly sensitive to Al toxicity (Rechcigl et al., 1988). The mucilage was collected from roots of M. malabathricum plants grown in 40 l containers containing the standard nutrient solution (pH 4.0, Al-free). The seeds of alfalfa were surface-sterilized with sodium hypochlorite for 10 min, washed with deionized water, and germinated on filter paper dipped in a 200 µm CaCl2 solution (pH 5.5), in darkness, at 25°C for 24 h. A seedling was transferred to a 2 ml plastic cuvette containing 1.8 ml of treatment solution. The cuvette was covered with nylon net and the seedling was placed on the net. This experiment was a 3 (+mucilage, +citrate, control) × 2 (low Al, high Al) factorial treatment design. The treatment solution in the +mucilage, +citrate, and control treatments contained mucilage as 0.436 mm total uronic acids (determined by the m-phenyl phenol method; Blumenkrantz & Asboe-Hansen, 1973), 0.145 mm citrate (= 0.436 mm carboxyl group), and none of any chelators, respectively. Since the +mucilage treatment solution contained 0.05 mm K, 0.11 mm Ca, 0.05 mm Mg, and 0.01 mm Pi, derived from the mucilage (0.6 ml), these elements were added as KCl, CaCl2, MgCl2, and NaH2PO4 to the other treatment solutions at the same concentration for each element. Although Al was not added in a nutrient solution during the cultivation of M. malabathricum, the collected mucilage contained 0.02 mm Al, possibly because of contamination. Therefore, we applied the Al treatment at two different Al concentrations, 0.02 mm (low Al) and 0.10 mm (high Al). Al was applied as AlCl3. The pH in each treatment solution was adjusted to 4.1. At the end of the 40 h treatment period, the root length of the alfalfa seedlings was measured. The alleviation effects of mucilage and citrate were individually assessed by comparing the root length between the low-Al and high-Al treatments. There was no significant pH change in the medium during the treatment.

Chemical properties of root mucilage

Mucilage was collected from roots of M. malabathricum and maize grown in a standard nutrient solution (pH 4.0, Al-free). In order to determine the component sugars, hydrolysis of the mucilage was carried out in 4 m TFA at 100°C for 3 h. An anion exchange column (CarboPac PA1, Dionex Corp, Sunnyvale, CA, USA) was used for the determination of monosaccharides. The column was eluted isocratically using the pump (L-7100, Hitachi, Tokyo, Japan) with a flow rate of 1.0 ml min−1 at 25°C. The separated sugars were detected by a pulsed amperometric detector (ESA Coulochem II electrochemical detector with ESA 5040 analytical cell; ESA, Bedford, USA). The pulsed amperometric detector settings were as follows: E1 (detection potential) = +200 mV, T1 = 500 ms (300 ms acquisition delay), E2 (oxidative cleaning) = +700 mV, T2 = 100 ms, E3 (regeneration) = −900 mV, T3 = 100 ms. For the determination of neutral sugars and uronic acids, 18 mm NaOH and 100 mm NaOH/150 mm AcONa were used as the eluents, respectively. Total sugar concentration was determined by the phenol-sulfuric acid method (Dubois et al., 1956).

The degree of methylation of uronic acids in the mucilage was determined by the method of Wojciechowski & Fall (1996). Briefly, 1 ml of the mucilage and 600 µl of 0.25 M NaOH were placed into a 2 ml tube. The tube was shaken for 10 min and stored for 4 h in a refrigerator at 4°C. After centrifuging the samples at 12 000 g for 10 min, methanol concentration in the supernatant was determined fluorometrically. Total uronic acid concentration was determined by the method of Blumenkrantz & Asboe-Hansen (1973). The pH of the mucilage was measured by a pH meter directly after the homogenization.

Furthermore, organic acid concentration and 13C NMR spectra were determined for the mucilage of M. malabathricum. For the determination of organic acids in the mucilage, 10 mg of the lyophilized mucilage was extracted with 1 ml of 0.05 m HCl. The extract was filtered through a membrane filter (pore size = 0.45 µm) and organic acid concentrations were determined by capillary electrophoresis (Quanta 4000CE, Waters, Milford, MA, USA), as described by Watanabe et al. (1998). Solid-state 13C NMR spectrum of mucilage was obtained with a FT NMR system (JNM-α300 spectrometer; JEOL) employing the cross-polarization magic angle spinning (CPMAS) technique. Lyophilized mucilage was transferred into a high-speed spinning tube (rotor = zirconia, cap = Kel-F, 6 mm of inner diameter; JEOL). Signals of 13C were recorded at 75.45 MHz. The parameters used were as follows: contact time, 1 ms; frequency range, 35.1 kHz; data point, 4096; acquisition time, 0.12 s; magic angle spinning, 6 kHz. Chemical shifts were quoted with respect to tetramethylsilane but were determined by referring to an external sample of adamantine (29.50 ppm).

Statistics

The results were analyzed with analysis of variance and Tukey's multiple comparison test, or with Student's t-test (P < 0.05).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Relationship between mucilage exudation and aluminum in M. Malabathricum

Mucilage exudation in M. malabathricum was observed when plants were grown in a nutrient solution without Al, whereas the mucilage was hardly visible in roots of plants grown in 0.5 mm Al (photograph in Fig. 1b). The amount of sugar exuded from M. malabathricum roots without the Al application was more than five times larger than that with 0.5 mm Al application (graph in Fig. 1b). Approximately 70% of the total sugars exuded from the roots without 0.5 mm Al application constituted the mucilage adhering to the roots.

The effect of removing root mucilage on cation uptake of M. malabathricum grown in nutrient solutions containing K, Ca, Mg, and Al was examined. The removal of root mucilage did not affect K, Ca, or Mg concentrations in shoots, but significantly reduced Al concentrations (Fig. 2).

image

Figure 2. Concentrations of aluminum (Al), potassium (K), calcium (Ca) and magnesium (Mg) in shoots of Melastoma malabathricum grown in a nutrient solution with Al. In the –muci treatment, the mucilage was removed from the roots throughout the Al exposure (10 d). Closed bars, + muci; open bars, –muci. Data are means of three replicates ± SE. Different letters indicate significant differences at P < 0.05.

Download figure to PowerPoint

Selectivity for the adsorption of cations in root mucilage

Selectivity for the adsorption of Al, lanthanum (La), and barium (Ba) in the isolated mucilage was determined to know the characteristics of mucilage as a cation exchanger. Mucilage of M. malabathricum adsorbed more Al and La (trivalent cations) than Ba (divalent cation) (Fig. 3). On the other hand, the cation adsorption affinity of maize mucilage was Ba > Al > La. In the case of artificial cation exchange resins, DOWEX 50W-X8 (a sulfonic acid resin, ion exchange group = sulfo group) did not show selectivity for the adsorption of cations used in this experiment, and Muromac A-1 (a chelating resin, ion exchange group = carboxyl group) adsorbed more Al and La than Ba, a result similar to the mucilage of M. malabathricum.

image

Figure 3. Concentrations of cations (aluminum (Al), lanthanum (La) and barium (Ba)) adsorbed on mucilages (Melastoma malabathricum and maize (Zea mays)) or cation exchange resins (DOWEX and Muromac) equilibrated with a mixed cation solution. Data are means of five replicates ± SE. Different letters indicate significant differences at P < 0.05.

Download figure to PowerPoint

Form and activity of aluminum in root mucilage

The 27Al NMR spectrum of the Al-adsorbed mucilage showed a single broad resonance peak at 0.354 ppm, shifted slightly downfield compared with 1 mm AlCl3 in 0.1 M HCl (Fig. 4). A relative peak area of 191 for the mucilage spectrum was calculated in comparison with the reference signal of 1 mm Al in 0.1 m HCl (1000).

image

Figure 4. 27Al nuclear magnetic resonance (NMR) spectra of mucilage (lower) and 1 mm AlCl3 solution in 0.1 M HCl (upper). Total aluminum (Al) concentration in the mucilage was 0.24 mm. The AlCl3 solution (1 mm Al in 0.1 M HCl) was also used as an external reference for calibration of chemical shift (0 ppm).

Download figure to PowerPoint

The effect of M. malabathricum mucilage application on Al-induced inhibition of alfalfa root elongation was examined to assess bioavailability of Al in the mucilage, in comparison with citrate as a control chelator. The citrate application completely alleviated the Al-induced elongation inhibition, whereas the M. malabathricum mucilage application, which contains the same amount of carboxyl groups as in the citrate application, showed no significant alleviation effect (Fig. 5).

image

Figure 5. Effect of mucilage or citrate application on aluminum (Al) toxicity in elongation of alfalfa (Medicago sativa) roots. Low Al, 0.02 mm Al; high Al, 0.10 mm Al; + mucilage, 0.436 mm uronic acid equivalents of Melastoma malabathricum mucilage; citrate, 0.145 mm citrate. Data are means of four replicates ± SE. Different letters indicate significant differences at P < 0.05.

Download figure to PowerPoint

Chemical properties of root mucilage

The composition of monosaccharides in mucilage of M. malabathricum was compared with that of maize. Mucilage of M. malabathricum contained a lower proportion of fucose and arabinose and higher proportion of xylose and glucuronic acid than that of maize (Table 1). Glucuronic acid was the primary monosaccharide component of M. malabathricum mucilage. Total sugar concentrations of mucilage determined by a colorimetric method are significantly higher in M. malabathricum. The degree of methylation of uronic acid carboxyl groups in M. malabathricum mucilage was more than three times higher than that in maize (Table 1). The amounts and viscoelasticity of mucilage exuded from roots were obviously higher in M. malabathricum than in maize (data not shown). Organic acids were not detected in M. malabathricum mucilage (Table 1). The pH of M. malabathricum mucilage (pH 3.86) was significantly lower than that of a nutrient solution (pH 4.00) and of maize mucilage (pH 3.99), indicating accumulation of protons in the mucilage.

Table 1.  Chemical properties of root mucilage of Melastoma malabathricum and maize (Zea mays)
 M. malabathricumMaize
  1. ND, not detected; –, not determined.Values are the means of three replicates ± SE. Different letters in each parameter indicate significant differences at P < 0.05.

Sugar (molar ratio, %)
 Fucose3.2 ± 0.1 b14.9 ± 0.3 a
 Rhamnose0.8 ± 0.6 a1.5 ± 0.4 a
 Arabinose6.3 ± 0.7 b8.9 ± 0.5 a
 Galactose35.5 ± 1.4 a35.4 ± 0.3 a
 Glucose1.0 ± 0.2 a2.1 ± 0.6 a
 Xylose11.3 ± 0.6 a8.6 ± 0.4 b
 Glucuronic acid42.0 ± 2.1 a28.6 ± 1.6 b
 Galacuturonic acidNDND
Degree of methylation (%)7.3 ± 0.1 a1.9 ± 1.1 b
Total sugar (mmol l−1 mucilage)4.8 ± 0.0 a3.6 ± 0.1 b
Organic acidND
pH3.86 ± 0.01 b3.99 ± 0.03 a

A typical solid-state 13C NMR spectrum of M. malabathricum mucilage is shown in Fig. 6. The spectrum indicated that the major organic components of the M. malabathricum mucilage were carbohydrates (polysaccharides and monosaccharides, resonated at 50–110 ppm) (Wooten, 1995). The spectrum also showed the presence of significant amounts of carboxylic C (160–185 ppm), and trace amounts of aromatic (110–160 ppm) and aliphatic C (10–50 ppm) in M. malabathricum mucilage.

image

Figure 6. A solid-state 13C nuclear magnetic resonance (NMR) spectrum of the mucilage of Melastoma malabathricum.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

As described in the introduction, it has been reported in maize that root mucilage very strongly binds Al, that the bound Al is less toxic, and that root mucilage does not enhance Al uptake (Li et al., 2000). M. malabathricum, an Al accumulator, exudes large amounts of mucilage from its roots (Fig. 1a), yet its role in Al absorption has not been examined. The amount of exuded mucilage in a nutrient solution with 0.5 mm Al was < 20% of that in an Al-free nutrient solution (Fig. 1b). Such a decline in the exuded mucilage is often observed and is considered to be the result of Al-induced disorder of root cell function (Puthota et al., 1991). However, the metabolic activity in root tips of M. malabathricum was not inhibited, but rather enhanced by the application of 0.5 mm Al (Watanabe et al., 2005), indicating that the decline in the exuded mucilage is not caused by Al injury. Consequently, we hypothesize that root exudation of mucilage is induced under ‘Al deficient’ conditions. Horst et al. (1982) reported that the remova1 of mucilage before the Al treatment enhanced the entry of A1 into root tissue and rendered roots more sensitive to Al. In the present study, the removal of root mucilage reduced the Al concentration in M. malabathricum shoots significantly, whereas the K, Ca, and Mg concentrations were not affected (Fig. 2). This result indicates that root mucilage enhances Al absorption in M. malabathricum.

One of the most important characteristics of mucilage is the cation adsorption ability caused by uronic acids in its structural polysaccharides. Pectin is also a polysaccharide containing uronic acid as a component sugar and is responsible for the CEC of cell walls. Purified walls of carrot cell suspensions have the ionic selectivity sequence of Ca2+ > Mg2+ ≈ K+ (Messiaen et al., 1997), and the cell walls of clover and rye-grass have the ionic selectivity sequence of Ca2+ > Mg2+ >> K+ (Amory & Dufey, 1984). By contrast, there are no reports on the cation adsorption selectivity of mucilage and, of course, differences between plant species have not been examined. The present study elucidated that the characteristics of mucilage in adsorption of cations, including Al ion, differ completely between M. malabathricum and maize; namely, M. malabathricum mucilage has higher affinities for trivalent cations (Al and La), whereas maize mucilage has a higher affinity for the divalent cation (Ba) (Fig. 3).

In the Donnan free space of roots, the preferential binding of polyvalent cations increases the concentration of these cations in the apoplasm, thereby inducing cation uptake (Rengel, 1990; Marschner, 1995). Although this observation may suggest that the preferential binding of Al to mucilage increases Al absorption by roots, Al cannot be absorbed directly if Al binds very tightly to mucilage, as reported in maize (Li et al., 2000). Cu ion complexed with polyuronates can be liberated by organic acids, and the Cu-organic acid complexes can be absorbed by roots (Deiana et al., 2003). Therefore, we expected that M. malabathricum roots would release organic acids into mucilage, making the adsorbed Al available for absorption by the roots; however, organic acids were not detected in M. malabathricum mucilage (Table 1). Phenolics can also make complex with Al (Barceló & Poschenrieder, 2002). Although we did not determine phenolics concentration in the mucilage, results of 13C NMR indicated the presence of a trace amount of aromatic C in M. malabathricum mucilage (Fig. 6).

To identify the form of Al in M. malabathricum mucilage directly, 27Al NMR was applied. 27Al NMR is a very efficient tool for the speciation of Al in solution (Kerven et al., 1995), although Al tightly bound to the solid phase cannot be detected by solution-state NMR. Li et al. (2000) measured the 27Al NMR spectra of maize mucilage, but no peaks were detectable, indicating that Al binds to the mucilage very tightly. By contrast, the Al adsorbed on the mucilage of M. malabathricum was visible in 27Al NMR and the major chemical form of the detected Al was inorganic monomeric ion (Fig. 4). Comparing the relative peak area of the 27Al NMR spectrum (equivalent to 0.191 mm Al) with the total Al concentration determined by ICPAES (0.24 mm Al) in the M. malabathricum mucilage indicates that the major part of Al quickly drifts between the liquid phase and negatively charged mucilage with very weak interactions. Taken together, the majority of Al in the mucilage is considered to be in a form that can be absorbed easily by the roots of M. malabathricum. The fact that Al in the M. malabathricum mucilage has a similar toxicity to that of AlCl3 solution for alfalfa seedlings (Al-sensitive) also demonstrates the higher bioavailability of Al in the M. malabathricum mucilage (Fig. 5). By contrast, it has been reported that the bioavailability of Al bound to the maize mucilage is less toxic (Li et al., 2000).

So what is the primary factor behind such interesting characteristics of Al adsorption in the mucilage of M. malabathricum? The solid-state 13C NMR spectrum of M. malabathricum mucilage indicated that carbohydrates are the main component (Fig. 6). As described earlier, the component sugars of mucilage are expected to affect ion adsorption by mucilage. Fucose is a specific constituent of mucilage (Roy et al., 2002), and is the second major component of neutral sugar in maize mucilage (Table 1). The proportion of fucose to total sugar in maize is more than four times higher than that in M. malabathricum (Table 1). Although the composition of neutral sugars may affect mucilage structure, it is not clear to what extent neutral sugars affect cation adsorption. By contrast, uronic acid, which has a carboxyl group, must be responsible for the CEC of mucilage. Glucuronic acid was detected in the mucilage of both M. malabathricum and maize, and glucuronic acid was the most abundant sugar in the mucilage of M. malabathricum (Table 1). Since organic acids could not be detected (Table 1), the majority of carboxylic C in the mucilage of M. malabathricum (Fig. 6, 160–185 ppm) would be derived from glucuronic acid. In the case of artificial cation exchange resins, Muromac A-1 preferentially adsorbed trivalent cations, whereas DOWEX 50W-X8 did not show selectivity in cation adsorption (Fig. 3). These different characteristics may result partly from the difference in CEC per unit volume between Muromac A-1 (2.8 meq ml−1 resin) and DOWEX 50W-X8 (1.7 meq ml−1 resin), because a cation exchanger with a high charge density exhibits preferences for trivalent cations (Falke et al., 1991; Reusch, 2000). If all the cation exchange sites in mucilage are derived from the unmethylated carboxyl group of glucuronic acid, the cation exchange capacities per unit volume are 1.9 and 1.0 meq l−1 mucilage in M. malabathricum and maize, respectively. These results suggest that the charge density in the mucilage of M. malabathricum is nearly twice as high as that of maize. This high charge density in the mucilage of M. malabathricum is probably the primary factor for the preferential adsorption of trivalent cations (Fig. 3).

One of the possible factors responsible for the binding strength between mucilage and Al is the degree of methylation of uronic acids. The polyuronic acid region of mucilage may form ‘egg box’ junctions with cations, including Al ion, bridging more than one sugar chain (Grant et al., 1973). Methylated polyuronic acid can also make the junctions, but the binding strength is less firm (Carpita & McCann, 2000). In fact, the degree of methylation in the mucilage of M. malabathricum is four times higher than that of maize (Table 1), suggesting that a higher degree of methylation in M. malabathricum mucilage causes the loose binding of Al. However, because a major part of uronic acid is not methylated in both M. malabathricum and maize mucilages, the effect of the degree of methylation may be limited. Another highly possible factor is H+ release from roots to the mucilage. The difference in H+ activity between the M. malabathricum mucilage and the external nutrient solution was 0.04 mm ([H+] = 0.1 and 0.14 mm in the nutrient solution and in the M. malabathricum mucilage, respectively), whereas there was no significant difference in maize (Table 1). Since the mucilage was homogenized before the pH measurement, pH in the vicinity of the root surface was probably much lower than in the nutrient solution, increasing availability of Al in mucilage. For further understanding of their characteristics in Al adsorption, the linkage structure and the sequence of sugars in the mucilages need to be determined as well.

Melastoma malabathricum accumulates thousands of mg Al kg−1 DW in leaves growing even in tropical peat soils that scarcely contain available Al ions (Osaki et al., 1998). So far, the mechanisms to explain this phenomenon have not been elucidated. The present study showed a possible mechanism: M. malabathricum exudes mucilage from its roots under ‘Al deficient’ conditions and the mucilage traps and concentrates Al from a soil solution with low Al concentration. By concentrating Al in the mucilage as a bioavailable form, M. malabathricum may absorb Al efficiently even under such conditions.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We would like to thank Dr O. Nakahara (Hokkaido University) for useful discussions. This study was supported financially by grants-in-aid for scientific research (no. 16208008) from the Japanese Society for the Promotion of Science and by a grant-in-aid for scientific research (No. 16780043) from the Ministry of Education, Culture, Sports, Science, and Technology.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Amory DE, Dufey JE. 1984. Adsorption and exchange of Ca, Mg and K-ions on the root cell walls of clover and rye-grass. Plant and Soil 80: 181190.
  • Archambault DJ, Zhang G, Taylor GJ. 1996. Accumulation of Al in root mucilage of an Al-resistant and an Al-sensitive cultivar of wheat. Plant Physiology 112: 14711478.
  • Barceló J, Poschenrieder C. 2002. Fast root growth responses, root exudates, and internal detoxification as clues to the mechanisms of aluminium toxicity and resistance: a review. Environmental and Experimental Botany 48: 7592.
  • Blumenkrantz N, Asboe-Hansen G. 1973. New method for quantitative determination of uronic acids. Analytical Biochemistry 54: 484498.
  • Carpita NC, McCann MC. 2000. Chapter 2. The cell wall. In: BuchananBB, GruissemW, JonesRL, eds. Biochemistry & molecular biology of plants. Rockville, MD, USA: American Society of Plant Physiologists, 52108.
  • Deiana S, Gessa C, Palma A, Premoli A, Senette C. 2003. Influence of organic acids exuded by plants on the interaction of copper with the polysaccharidic components of the root mucilages. Organic Geochemistry 34: 651660.
  • Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. 1956. Colorimetric method for determination of sugars and related substances. Analytical Chemistry 28: 350356.
  • Falke JJ, Snyder EE, Thatcher KC, Voertler CS. 1991. Quantitating and engineering the ion specificity of an EF-hand-like Ca2+ binding. Biochemistry 30: 86908697.
  • Grant GT, Morris ER, Rees DA. 1973. Biological interactions between polysaccharides and divalent cations: the egg-box model. FEBS Letter 32: 195198.
  • Horst WJ, Wagner A, Marschner H. 1982. Mucilage protects root meristems from aluminium injury. Zeitschrift für Pflanzenphysiologie 109: 95103.
  • Iijima M, Higuchi T, Barlow PW, Bengough AG. 2003. Root cap removal increases root penetration resistance in maize (Zea mays L.). Journal of Experimental Botany 54: 21052109.
  • Kerven GL, Larsen PL, Bell LC, Edwards DG. 1995. Quantitative 27Al NMR spectroscopic studies of Al(III) complexes with organic acid ligand and their comparison with GEOCHEM predicted values. Plant and Soil 171: 3539.
  • Knee EM, Gong FC, Gao M, Teplitski M, Jones AR, Foxworthy A, Mort AJ, Bauer WD. 2001. Root mucilage from pea and its utilization by rhizosphere bacteria as a sole carbon source. Molecular Plant–Microbe Interactions 14: 775784.
  • Kochian LV. 1995. Cellular mechanisms of aluminum toxicity and resistance in plants. Annual Review of Plant Physiology and Plant Molecular Biology 46: 237260.
  • Kochian L, Hoekenga OA, Piñeros MA. 2004. How do crop plants tolerate acid soils? Mechanisms of aluminum tolerance and phosphorous efficiency. Annual Review of Plant Biology 55: 459493.
  • Li XF, Ma JF, Hiradate S, Matsumoto H. 2000. Mucilage strongly binds aluminum but does not prevent roots from aluminum injury in Zea mays. Physiol Plantarum 108: 152160.
  • Ma JF, Ryan PR, Delhaize E. 2001. Aluminium tolerance in plants and the complexing role of organic acids. Trends in Plant Science 6: 273278.
  • Marschner H. 1995. Mineral nutrition of higher plants, 2nd edn . New York, NY, USA: Academic Press.
  • Messiaen J, Cambier P, Cutsem PV. 1997. Polyamines and pectins. Plant Physiology 113: 387395.
  • Moody SF, Clarke AE, Bacic A. 1988. Structural analysis of secreted slime from wheat and cowpea roots. Phytochemistry 27: 28572861.
  • Morel JL, Mench M, Guckert A. 1986. Measurement of Pb2+, Cu2+ and Cd2+ binding with mucilage exudates from maize (Zea mays L.) roots. Biology and Fertility of Soils 2: 2934.
  • Osaki M, Matsumoto M, Watanabe T, Kawamukai T, Shinano T, Nuyim T, Nilnond C, Tadano T. 1998. Strategies for adaptation of plants grown in adverse soils. In: BassamNE, BehlRK, ProchnowB, eds. Sustainable agriculture for food, energy, and industry. London, UK: James & James Ltd, 537546.
  • Puthota V, Cruz-Ortega R, Johnson J, Ownby J. 1991. An ultrastructural study of the inhibition of mucilage secretion in the wheat root cap by aluminium. In: WrightRJ, BaligarVC, MurrmannRP, eds. Plant–soil interactions at low pH. Dordrecht, the Netherlands: Kluwer Academic Publishers, 779787.
  • Rechcigl JE, Reneau RB, Zelazny LW. 1988. Soil solution Al as a measure of Al toxicity to alfalfa in acid soils. Communications in Soil Science and Plant Analysis 19: 9891001.
  • Rengel Z. 1990. Net Mg2+ uptake in relation to the amount of exchangeable Mg2+ in the Donnan free space of ryegrass roots. Plant and Soil 128: 185189.
  • Reusch RN. 2000. Transmembrane ion transport by polyphosphate/poly-(R)-3-hydroxybutyrate complexes. Biochemistry (Moscow) 65: 280295.
  • Roy SS, Mittra B, Sharma S, Das TK, Babu CR. 2002. Detection of root mucilage using an anti-fucose antibody. Annals of Botany 89: 293299.
  • Sievers A, Braun M, Monshausen GB. 2002. Chapter 3. The root cap: structure and function. In: WaiselY, EshelA, KafkafiU, eds. Plant roots. New York, NY, USA: Marcel Dekker, 3347.
  • Sivaguru M, Horst WJ. 1998. The distal part of the transition zones the most aluminum-sensitive apical root zone of maize. Plant Physiology 116: 155163.
  • Wada S-I, Seki H. 1994. A compact computer cod for ion speciation in aqueous solutions based on a robust algorithm. Soil Science and Plant Nutrition 40: 165172.
  • Wagatsuma T, Ishikawa S, Ofei-Manu P. 2001. The role of the outer surface of the plasma membrane in aluminum tolerance. In: AeN, AriharaJ, OkadaK, SrinivasanA, eds. Plant nutrient acquisition. Tokyo, Japan: Springer-Verlag, 159184.
  • Watanabe T, Jansen S, Osaki M. 2005. The beneficial effect of aluminium and the role of citrate in Al accumulation in Melastoma malabathricum. New Phytologist 165: 773780.
  • Watanabe T, Jansen S, Osaki M. 2006. Al-Fe interactions and growth enhancement in Melastoma malabathricum and Miscanthus sinensis dominating acid sulphate soils. Plant, Cell & Environment 29: 21242132.
  • Watanabe T, Osaki M, Tadano T. 2001. Al uptake kinetics in roots of Melastoma malabathricum L.-an Al accumulator plant. Plant and Soil 231: 283291.
  • Watanabe T, Osaki M, Yoshihara T, Tadano T. 1998. Distribution and chemical speciation of aluminum in the Al accumulator plant, Melastoma malabathricum L. Plant and Soil 201: 165173.
  • Wojciechowski CL, Fall F. 1996. A continuous fluorometric assay for pectin methylesterase. Analytical Biochemistry 137: 103108.
  • Wooten JB. 1995. 13C CPMAS NMR of bright and burley tobaccos. Journal of Agricultural and Food Chemistry 43: 28452868.