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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.
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).
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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.