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Aluminium (Al) toxicity is a very serious problem for the cultivation of many crop plants in acid soils all over the world (Foy et al., 1978). Excess of soluble and bioavailable Al especially inhibits the root growth of Al-sensitive plants such as wheat (Triticum aestivum), maize (Zea mays) and soybean (Glycine max). Because the normal metabolic activities of root cells can be hindered by apoplastic as well as symplastic Al effects, the growth of the whole plant can be seriously reduced. Several mechanisms dealing with the toxic properties of Al have been proposed (Kochian, 1995; Matsumoto, 2000; Barceló & Poschenrieder, 2002).
The majority of plant species avoids Al stress by excluding Al from the roots and can therefore be termed Al excluders. At present, exudation of organic acids from the roots is considered as the most common Al exclusion mechanism (Ma et al., 2001a). Some organic acids (e.g. citrate) have a high chelating affinity to Al and this may substantially reduce the uptake and toxicity of Al. Not only organic acids, but also some inorganic anions, such as fluorine (F), silicon (Si) or sulfate (SO4) can make stable complexes with Al and reduce the harmful Al effects (Tanaka et al., 1987; Hodson & Evans, 1995). Among these anions, the effect of Si application on Al toxicity has been well studied. For example, in the case of maize, the concentration of Al3+ in culture solution is strongly reduced when Si is applied (Ma et al., 1997a). Silicon has also been demonstrated to inhibit Al penetration into the root cortex of Sorghum bicolor, indicating that Si makes a complex with Al in the medium and/or roots and contributes to the detoxification of Al (Hodson & Sangster, 1993).
While many plants exclude Al from the roots, some plant species accumulate huge amounts of Al in both roots and shoots (Haridasan, 1982; Geoghegan & Sprent, 1996; Osaki et al., 1998; Jansen et al., 2002a,b; Watanabe & Osaki, 2002). Generally, plant species are classified as Al accumulators if they accumulate at least 1000 mg kg−1 in their leaves (Chenery, 1948). The Al accumulators often exhibit a high Al tolerance despite the high Al concentration in their above-ground tissues (Haridasan, 1988; Osaki et al., 1997; Watanabe et al., 1997). Several researchers considered that Al-accumulating species are able to detoxify Al, for example, by binding to the cell walls, compartmentalization in the vacuole or formation of Al chelates. Examples that have demonstrated the occurrence of chelated Al forms in plant leaves are Al-catechin in the tea bush (Camellia sinensis), Al-citrate in Hydrangea macrophylla, and Al-oxalate in buckwheat (Fagopyrum esculentum) and Melastoma malabathricum (Nagata et al., 1992, 1993; Ma et al., 1997b,c; Watanabe et al., 1998a). Chelation of Al by inorganic ligands has not yet been reported in Al accumulators, although Al–F was considered as the translocation form in the tea bush (Nagata et al., 1993; Liang et al., 1996). Moreover, Masunaga et al. (1998) found a positive correlation between Al and Si levels in leaves of various Al accumulators (3000 mg Al kg−1) growing in a tropical rain forest in Indonesia, which may contribute to the alleviation of Al toxicity in several Al accumulators.
Al accumulators are not randomly distributed among the flowering plants, but frequently characterize families such as Rubiaceae, Melastomaceae or Vochysiaceae (Chenery, 1948; Haridasan, 1982; Jansen et al., 2002a,b). Especially in Rubioideae, one of the three subfamilies within Rubiaceae, strong Al accumulators have been reported (Jansen et al., 2000a,b). We have previously reported on the chemical characterization of plant species from the swamp forest in the Ilha do Mel, Brazil (Britez et al., 1997). These preliminary results indicated that Faramea marginata, a woody member of the Rubiaceae family that grows on mainly acid soils, is not only an Al accumulator, but also appears to accumulate Si in its leaves. Since Si can form stable complexes with Al, we hypothesize that an Al–Si complex is formed in F. marginata and that this may contribute to an increased Al tolerance in its above-ground tissues. Therefore, this study investigates in detail the relationship between Al and Si accumulation in the leaves of this species.
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Since the mean Al concentration in all 30 leaf samples of F. marginata analysed is 18 100 mg kg−1, this species is indisputably an Al accumulator. Even the lowest Al level obtained is much higher than the criterion of 1000 mg kg−1 used to define Al accumulators. Moreover, the Al levels found in F. marginata are in agreement with previous studies that reported Al concentrations ranging from 16 000 to 36 900 mg kg−1 in several Faramea species (Chenery, 1946). We suggest that probably all Faramea species are strong Al accumulators, since there are no records of nonaccumulating specimens in this genus (Chenery, 1946, 1948; Jansen et al., 2000a). Aluminium accumulation was also reported in all wood samples of Faramea that were tested using the chrome azurol-S test (Kukachka & Miller, 1980; Jansen et al., 2000b).
It is well known that chelation is one of the most important mechanisms to detoxify Al in the shoot of Al accumulators (Ma et al., 2001b; Watanabe & Osaki, 2002). The Al forms in shoots of several Al accumulators have been identified to be Al complexes with organic ligands (Nagata et al., 1992, 1993; Ma et al., 1997b,c; Watanabe et al., 1998a). In the present study, however, the results obtained from the extraction with ethanol and the analysis of organic acids in the water extract indicate that neither Al–organic acid complexes nor monomeric Al occur in the leaves of F. marginata.
Silicon is generally considered to interact with Al at the root level, and the formation of an Al–Si complex in above-ground tissues has not yet been well demonstrated. Si accumulation is common in the shoot of several monocots, pteridophytes and bryophytes, but this feature is relatively rare in dicots (Ma & Takahashi, 2002). Silicon accumulators are defined as plants with Si levels above 10 000 mg kg−1 and a Si : Ca ratio higher than 1, because plants that accumulate Si tend to have a low Ca concentration (Ma et al., 2001b; Ma & Takahashi, 2002). The Si : Ca mole ratio in the leaves of F. marginata is higher than 1, but Si levels are above 10 000 mg kg−1 in only five out of the 30 samples analysed . This suggests that F. marginata can be no more than an intermediate or weak Si accumulator (Fig. 1, Table 1).
The most important conclusion from this study is that there is strong evidence to suggest that an Al–Si complex may be formed in shoots of F. marginata. This hypothesis is based on the following results: (1) the striking positive correlation between Al and Si in various aged leaves (Fig. 1); (2) the extraction patterns of leaves are similar to those of aluminium silicate, not to Si in rice or SiO2 (Figs 2,3), and (3) the two elements show a similar localization in both leaves and stems (Figs 4 and 5). It has been documented that Al and Si(OH)4 form hydroxyaluminosilicate (HAS) with a Si : Al ratio of approx. 0.5 in acidic solutions (Exley et al., 2002). Since the mole ratio of Si : Al in the leaves of F. marginata estimated from the regression line in Fig. 1a is approximately 0.5, the formation of HAS may occur in the shoot of this species. However, the variations in the Si : Al ratio are not negligible (0.34–0.60, see Fig. 1b,c), which may indicate that more than one Al–Si compound occurs. This can be supported by the slight differences found in the extraction patterns of Al and Si between F. marginata and aluminium silicate. However, these differences may be affected by the chemical composition of the aluminium silicate used, because it is not known whether this reagent contains only a single or more than one Al–Si compound. Interestingly, the mole ratio of Si : Al shows a significant positive correlation as a function of Si concentrations (Fig. 1c). Therefore, Si seems to regulate the Al accumulation in leaves of F. marginata. Moreover, data on the Si : Al ratio in other Al-accumulating Rubiaceae are found to differ widely (from 0.23 in Alberta minor Baillon to 17.4 in Emmeorhiza umbellata K. Schum.), and there appear to be different possibilities for Al–Si associations (S. Jansen et al., unpublished). An alternative possibility may be that Al–Si compounds occur as solid compounds such as aluminosilicate (AS) or phytoliths (plant opal) that mainly consist of hydrated silica. It has been reported that 60–95% of the total Al content in leaf tissues of plants growing on acid soils is incorporated into phytolith structures (Bartoli & Wilding, 1980). Klinowski et al. (1998) found that Al in bamboo species (Melocanna bambusoides Trin.) was incorporated into the silicate network. However, this hypothesis is unlikely in F. marginata because phytoliths are absent in this species (Fig. 3), as well as in all other Al-accumulating Rubiaceae. Further research is needed to identify the actual chemical forms of the Al–Si compounds. Since HAS and solid Al–Si compounds are considered to be nontoxic or less toxic than monomeric Al, F. marginata may be able to grow successfully in acid soils despite high concentrations of Al in its shoots.
Compartmentalization, especially localization of Al in the cell wall or vacuole so that Al3+ cannot interfere with the cytoplasmic activities, has been suggested by several authors (Ma et al., 2001a; Watanabe & Osaki, 2002). Observations on the localization of Al in the shoot of Rubiaceae have, as far as we know, only been investigated in three species of Palicourea (Haridasan et al., 1986; Cuenca & Herrera, 1987). As suggested for several other Al accumulators, Al occurs mainly in the epidermis of the leaves in Palicourea and F. marginata (Matsumoto et al., 1976; Haridasan et al., 1986; Cuenca et al., 1991; Watanabe et al., 1998a). This may suggest that Al accumulators avoid Al toxicity in their leaves by localizing Al in the epidermal cells, which do not directly participate in photosynthesis in general. Accumulation in the epidermis of leaves has also been reported for nickel (Ni) and zinc (Zn) in heavy metal hyperaccumulators (Krämer et al., 1997; Küpper et al., 1999; Frey et al., 2000) and with respect to Ca and chlorine (Cl) in Hordeum vulgare (Leigh & Storey, 1993). Karley et al. (2000) suggested that ions that are not preferentially taken up at the bundle sheath plasma membrane move from the leaf vascular tissue to the epidermis via vein extensions. Localization of Al and other elements in the epidermis of the leaves may be explained by this hypothesis.
In addition to the epidermis, Al occurs in the mesophyll of the leaves in F. marginata (Fig. 3a), as was also reported for the Al accumulator M. malabathricum (Watanabe et al., 1998a). Since fluorescent induction of the leaves of M. malabathricum changed with increasing Al concentration in the leaves, this may indicate that Al affects the photosynthetic reaction in the mesophyll cells (Watanabe et al., 1998b). By contrast, the photosynthetic performance of two mistletoes species (Phthirusa ovata Eichler and Phoradendron crassifolium Nutt.) was found to be independent of whether they parasitized Al-accumulating or nonaccumulating host species (Lüttge et al., 1998). It may be worth studying in more detail the effect of Al on the photosynthesis in Al accumulators.
In general, the concentration of an element with low mobility in the plant body is higher in mature leaves than in young leaves. The mature : young leaf ratios found for Al, Si and Ca in F. marginata are 1.24, 1.36 and 1.56, respectively. This may indicate that the mobility is slightly higher for Al and Si than for Ca. With respect to Al, a significant difference between mature and young leaves has been observed in Al nonaccumulators and in at least few Al accumulators (e.g. the tea bush, F. esculentum) (Matsumoto et al., 1976; Liang et al., 1996; Osaki et al., 1997; Shen & Ma, 2001). Therefore, Al can be suggested to have a rather low mobility in these species. In most Al accumulators, however, Al concentrations do not appear to show considerable differences between young and mature leaves and this is also observed in F. marginata (de Medeiros & Haridasan, 1985; Osaki et al., 1997; Watanabe et al., 1997, 1998a,b; Masunaga et al., 1998). Hence, Al seems to behave as an element with high mobility in the majority of Al accumulators including F. marginata. Although Al localization is not observed in phloem in the present study (Fig. 4b,c), further research is required because Al has been suggested to be transported through the phloem elements in several Al accumulators (Haridasan et al., 1986).
In conclusion, this study strongly suggests that Si may not only alleviate Al toxicity near roots or in the rhizosphere, but also in shoots of some Al accumulators, such as F. marginata. We believe this is the first study providing evidence for the formation of an Al complex with an inorganic ligand in the shoot of an Al accumulator. Not only F. marginata, but also many species in Rubiaceae tend to accumulate Al and Si in their shoots (Golley et al., 1980; S. Jansen et al. unpublished). Further research is required to determine whether all Al-accumulating Rubiaceae form Al–Si complexes in their shoot tissues.