The relationship between aluminium and silicon

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1 Research The relationship between aluminium and silicon Blackwell Science, Ltd accumulation in leaves of Faramea marginata (Rubiaceae) Ricardo M. Britez 1, Toshihiro Watanabe 2,4, Steven Jansen 3,5, Carlos B. Reissmann 1 and Mitsuru Osaki 2 1 Department of Soils, Federal University of Paraná, C. Postal 2959, , Curitiba/PR, Brazil; 2 Graduate School of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kitaku, Sapporo , Japan; 3 Laboratory of Plant Systematics, Institute of Botany and Microbiology, K. U. Leuven, Kasteelpark Arenberg 31, B 3001 Leuven, Belgium; 4 Present address: Japan International Research Center for Agricultural Sciences, 1 1 Ohwashi, Tsukuba , Japan; 5 Present address: Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK Summary Author for correspondence: Mitsuru Osaki Tel: Fax: mosaki@chem.agr.hokudai.ac.jp Received: 26 July 2002 Accepted: 9 September 2002 The relationship between high aluminium (Al) and silicon (Si) levels in Faramea marginata was investigated and the hypothesis tested that the coexisting accumulation of these elements is associated. Mineral concentrations of Al, Si and calcium (Ca) were analysed in 30 samples by atomic absorption spectrophotometry and the spectrophotometric molybdenum blue method. Extraction patterns of Al and Si from leaves were compared with Melastoma malabathricum, rice (Oryza sativa), aluminium silicate, and silicon dioxide. The localization of Al and Si was studied using pyrocatechol violet staining of sections and fluorescent X-ray analytical microscopy. A positive correlation occurred between the Al and Si levels and both elements showed a similar distribution in leaf and stem tissues. The Al and Si elution patterns were similar to those of aluminium silicate. These results suggest the formation of an Al Si complex in the shoot tissues of F. marginata, which may substantially contribute to the internal detoxification of Al. Key words: aluminium, Al accumulation, Al localization, Al Si complex, Faramea marginata, Rubiaceae, silicon. New Phytologist (2002) 156: Introduction 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 (SO 4 ) 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 Al 3+ 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 New Phytologist (2002) 156:

2 438 Research 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. Materials and Methods Sampling and mineral analysis of leaves Leaves of F. marginata Cham. were sampled at the swamp forest in the Ilha do Mel, Brazil (25 30 S; W, soil ph (CaCl 2 ) 3.4, A layer). The soil was classified as a Podzol (Podosol) with sandy texture, developed on the quaternary coastal plains and was considered as relatively homogeneous. A total of 30 samples was collected from 10 plants, including 10 young, 10 intermediate and 10 mature leaves. All 30 leaf samples were dried at 80 C for 48 h. Mineral concentrations in the leaves were analysed after wet digestion with an acid mixture (HNO 3 HClO 4 H 2 SO 4, 5 : 2 : 1). The concentrations of Al and Ca were determined by atomic absorption spectrophotometry (AA-6200; Shimadzu, Kyoto, Japan). The concentrations of Si were determined by atomic absorption spectrophotometry for acid-soluble Si and the spectrophotometric molybdenum blue method for acid-insoluble Si. Therefore, acid-insoluble Si was isolated from the digested solution (0.9 N H 2 SO 4 ) by filtering through a filter paper (No. 5C; Advantec Toyo, Tokyo, Japan) and extracted with 20 ml of 5% NaOH at 80 C for 4 h. The extract was made up to 250 ml. An adequate amount of the extract (< 0.06 mg Si) was diluted with deionized water to approx. 15 ml and neutralized with 0.5 N HCl. Then 0.6 ml of 0.5 N HCl and 1.25 ml of 10% ammonium molybdate solution were added to the sample solution and allowed to stand for 3 min. Finally, 2.5 ml of 17% sodium sulphite solution was added to the sample and made up to 25 ml. The absorbance at 650 nm was measured after 10 min. Extraction patterns of Al and Si from leaves Extraction with ethanol Mature leaves of eight plants of F. marginata were lyophilized. The leaves were well ground, extracted with ethanol (sample ethanol, 1 : 100; ph was not adjusted or adjusted to 1.0), and shaken for 1 h. The extract was filtered with filter paper (No. 5C; Advantec) and the concentration of Al and Si in the extract was determined by atomic absorption spectrophotometry. For comparison, the leaves of M. malabathricum L., which is an Al accumulator but not a Si accumulator, were similarly extracted with ethanol (Watanabe et al., 1997). Each extraction was repeated three times. Extraction with water Lyophilized mature leaves were extracted with deionized water with different ph (sample water, 1 : 100), and shaken for 1 h. The ph of deionized water was adjusted to 1, 4, and 12 with NaOH or HCl, or not adjusted. The extract was filtered with a membrane filter (pore size 0.45 µm), and the concentration of Al and Si was determined by atomic absorption spectrophotometry. The concentration of organic acids in the extract of the leaves was also determined by capillary electrophoresis (Watanabe et al., 1998a). In addition, shoot samples of rice (O. sativa L.), which is a typical Si accumulator, and the chemical reagent of aluminium silicate (Wako Chemicals, Osaka, Japan; approximately Al 2 O 3.3SiO 2 = ) and SiO 2 (Wako Chemicals, Japan) were extracted with deionized water with different ph. The concentration of Al and Si was determined as described above. Samples of rice were obtained from the field at the Japan International Research Center for Agricultural Sciences (Tsukuba, Japan). Each extraction was carried out with three replicates. New Phytologist (2002) 156:

3 Research 439 Fig. 1 The relationship between (a) aluminium (Al) and silicon (Si) concentrations, (b) mole ratio of Si : Al and Al concentration, and (c) mole ratio of Si : Al and Si concentration in various aged leaves of Faramea marginata growing in a Brazilian swamp forest. a, Regression equation in mmol kg 1 ; ***, significant at P < Al and Si localization in the shoot The Al localization in a fresh mature leaf and a young stem was determined by the PCV (pyrocatechol violet) staining method (Watanabe et al., 1998a). Transverse leaf and twig sections were obtained by hand-sectioning with a razor blade. The sections were placed on a slide and stained with 0.02% PCV in 2.5% hexamine NH 4 OH buffer (ph 6.2) for 15 min, washed with 2.5% hexamine NH 4 OH buffer (ph 6.2) and observed under a light microscope. Fresh samples of a whole leaf and stem cross-sections were obtained from seedlings of F. marginata growing on a pot containing 12 l of soil in a greenhouse at the Hokkaido University, Japan. The samples were dried using an ironing press and the Al and Si distribution was determined by a fluorescent X-ray analytical microscope (XGT-2000W; Horiba, Kyoto, Japan). The analytical parameters were as follows: X-ray tube target = Rh, tube voltage = 15 kv, tube current = 1.0 ma. Results Interaction between Al and Si concentrations in leaves The Al and Si concentrations in all leaves of F. marginata analysed ranged from to mg kg 1 and from 6500 to mg kg 1, respectively (Fig. 1). The mean values of Al and Si concentrations were and 8480 mg kg 1, respectively. There was a significant positive correlation between Al and Si concentrations in leaves (P < 0.001, Fig. 1). The mole ratio of Si : Al was approximately 0.5, which was calculated by Table 1 The mean values of aluminium (Al), silicon (Si) and calcium (Ca) concentrations, and mole ratios of Si : Al, Al : Ca and Si : Ca in young, intermediate, and mature leaves of Faramea marginata. Young Intermediate Mature Al (mg kg 1 ) ± ± ± 537 Si (mg kg 1 ) ± ± ± 591 Ca (mg kg 1 ) ± ± ± 159 Si : Al 0.44 ± ± ± 0.02 Al : Ca ± ± ± 0.70 Si : Ca 6.35 ± ± ± 0.44 Values are means of 10 samples ± SE. The data are included in Fig. 1. a slope of the regression line between Al and Si concentrations (Fig. 1a). However, considerable variation was found in the mole ratio of Si : Al. The Si concentration showed a significant positive correlation with the mole ratio of Si : Al, while Al concentrations did not (P < 0.001, Fig. 1b,c). The mean values of Al, Si and Ca concentrations in young, intermediate, and mature leaves were summarized in Table 1. The concentrations of all three elements increased in mature leaves, but the differences between young and mature leaves were not very large. Extraction patterns of Al and Si from leaves Aluminium in the leaves of F. marginata could not be extracted with ethanol, even at ph 1.0 (Table 2). However, approx. 40% of the total Al content in leaves of M. Table 2 Concentrations of Al, Si, oxalate and citrate (mm) in the ethanol- or waterextract of leaves from Melastoma malabathricum and Faramea marginata. Species/extractant Al Si Oxalate Citrate F. marginata Ethanol (ph not adjusted) Trace Trace n.d. n.d. Ethanol (ph 1) Trace Trace n.d. n.d. Water (ph not adjusted) 3.03 ± 0.02 (39) ± ± ± 0.03 M. malabathricum Ethanol (ph not adjusted) 1.53 ± 0.14 (42) 1 n.d ± ± 0.07 Water (ph not adjusted) 2.70 ± 0.06 (74) 1 n.d ± ± 0.03 Values are means of three replicates ± SE. 1 Percentage of total Al in the leaves; n.d., not determined. New Phytologist (2002) 156:

4 440 Research Since the leaves and chemical reagents were well ground and mixed, the standard errors of Al, Si and ph were 7.7, 7.2 and 4.3% at maximum cases (except for the data near zero). Fig. 2 Extraction patterns of aluminium (Al) from leaves of Faramea marginata and aluminium silicate. *, Percentage of total Al in leaves. The lines were fitted by hand. Fig. 3 Extraction patterns of silicon (Si) from leaves of Faramea marginata, aluminium silicate, rice shoots, and SiO 2. *, Percentage of total Si in leaves. The lines were fitted by hand. malabathricum, in which soluble Al mainly consisted of monomeric Al ions and an Al oxalate complex, was extracted with ethanol (ph not adjusted, Table 2). When organic acids in leaves of F. marginata were extracted with water at ph 1.0, the mole amounts of extracted citrate and oxalate, which were the primary organic acids in the extract, were less than 10% of extracted Al (Table 2). The Al and Si in leaves of F. marginata could be extracted with water, especially at low ph (Fig. 2). Near ph 1, 90% of the total Al and 35% of the total Si content was detected in the extract. Extraction patterns of Al and Si in leaves of F. marginata were similar to those of aluminium silicate, although they did not completely correspond with each other (Figs 2 and 3). By contrast, Si in shoots of rice, in which SiO 2 was the main form of Si, was only soluble at high ph and extraction patterns of Si in leaves of rice completely corresponded with those of SiO 2 (Ma et al., 2001b; Fig. 2). Al and Si localization in the shoot The PCV formed a blue colour by making a chelating complex with Al. Although PCV would also chelate with other metal cations, such as Fe, the disturbance by these elements could be ignored because the concentrations of these elements were much lower compared with that of Al (Britez et al., 1997). The PCV staining indicated high Al levels in both the upper and lower epidermis, and also showed Al accumulation in the cell walls of the palisade and the spongy mesophyll (Fig. 4a). The transverse section of the midrib illustrated that Al is localized in the cell walls of the upper and lower epidermis, parenchyma and collenchyma, but absent in sclerenchyma, xylem and phloem (Fig. 4b). The phloem and part of the ground tissue under the upper epidermis showed a dark colour, possibly because these areas included tanniniferous cells or secretory cells with unidentified contents. As this dark colour was also observed in sections that were not stained with PCV, these cells did not appear to react with PCV and therefore provided no evidence for Al localization. The transverse stem section clearly illustrated that the epidermis and cortex cells (collenchyma and parenchyma) were strongly stained with PCV, while the cuticle, sclerenchyma, phloem, xylem, and pith parenchyma did not stain blue (Fig. 4c). The images obtained using the fluorescent X-ray analytical microscope showed a similar distribution regarding Al and Si in a whole leaf (Fig. 5a,b) and stem section (Fig. 5c,d). Both Al and Si were found all over the leaf and mainly occurred in the cortex of the stem sections (Fig. 5c,d) which corresponded with the PCV staining (Fig. 4c). Discussion Since the mean Al concentration in all 30 leaf samples of F. marginata analysed is 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 to 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 New Phytologist (2002) 156:

5 Research 441 Fig. 4 Transverse sections of Faramea marginata after pyrocatechol violet staining, the blue-coloured tissues indicate the localization of aluminium (Al): (a) leaf lamina; (b) leaf lamina with midrib; (c) stem section; e, epidermis; c, cortex; le, lower epidermis; m, mesophyll; p/c, parenchyma/collenchyma; pa, palisade parenchyma; ph, phloem; pi, pith parenchyma; s, sclerenchyma; ue, upper epidermis; x, xylem. 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 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 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 SiO 2 (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 ( , 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 New Phytologist (2002) 156:

6 442 Research Fig. 5 The localization of aluminium (Al) and silicon (Si) in Faramea marginata as determined by fluorescent X-ray analysis: (a,b) whole leaf (c,d) stem section. 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 Alaccumulating 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 Al 3+ 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 New Phytologist (2002) 156:

7 Research 443 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. 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