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Plants growing in acid sulphate soils are subject to high levels of Al availability, which may have effects on the growth and distribution of these species. Although Fe availability is also high in acid sulphate soils, little is known about the effect of Fe on the growth of native plants in these soils. Two species dominating this soil type in Asia, viz. Melastoma malabathricum and Miscanthus sinensis were grown hydroponically in a nutrient solution with different concentrations of Al and Fe. Melastoma malabathricum is found to be sensitive to Fe (40 and 100 µm). Application of 500 µm Al, however, completely ameliorates Fe toxicity and is associated with a decrease of Fe concentration in shoots and roots. The primary reason for the Al-induced growth enhancement of M. malabathricum is considered to be the Al-induced reduction of toxic Fe accumulation in roots and shoots. Therefore, Al is nearly essential for M. malabathricum when growing in acid sulphate soils. In contrast, application of both Fe and Al does not reduce the growth of M. sinensis, and Al application does not result in lower shoot concentrations of Fe, suggesting that this grass species has developed different mechanisms for adaptation to acid sulphate soils.
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In acid soils, the excess of Al ions may restrict the growth of some plant species, including various crop plants such as wheat, corn and soybean (Kochian, Hoekenga & Piñeros 2004). It has been reported that Al inhibits root growth and disorders root functions, such as plasma membrane permeability (Ishikawa & Wagatsuma 1998), lipid peroxidation (Ikegawa, Yamamoto & Matsumoto 2000) and cell wall rigidity (Wehr, Menzies & Blamey 2004). However, many native plant species grow well in most acid soils, even in acid sulphate soils with an extremely low soil pH (< 3.5) (Osaki et al. 1998a,b). These species are well adapted to high levels of Al in the soils. Although Al exhibits toxic effects, it has often been reported that Al application enhanced the growth of native plant species in acid soils, including Arnica montana L., Deschampsia flexuosa Trin., Hydrangea paniculata Siebold, Melaleuca cajuputi Roxb. and Polygonum sachalinense Schmidt ex Maxim. (Yoshii 1937; Pegtel 1987; Osaki, Watanabe & Tadano 1997). Two major hypotheses have been suggested to explain the mechanisms of Al-induced growth enhancement: (1) Al alleviates H+ toxicity at low pH (Kinraide 1993; Llugany, Poschenrieder & Barceló 1995); and (2) Al alleviates excess P toxicity (Clark 1977). Nevertheless, these hypotheses may only be adopted in a restricted number of species because the beneficial effect of Al was often observed in low pH-tolerant species under low P conditions (Osaki et al. 1997). This means that the Al-induced growth stimulation can be observed even in the absence of the excess H+ and P toxicities.
Some plant species growing in acid soils accumulate huge amounts of Al in both roots and shoots (Watanabe & Osaki 2002). These plants are called ‘Al accumulators’. Haridasan & Araújo (1988) determined the Al concentrations of 39 plant species growing in the cerrado forest region of central Brazil. They reported that Al accumulators accounted for 17.3% of the total importance value in strongly acidic soils [pH (KCl) = 3.7, surface soils] and 11.7% in slightly acidic soils [pH (KCl) = 4.3]. This may indicate that Al accumulators have advantages in terms of their ability to survive in soils with high levels of soluble Al. Al accumulators have developed mechanisms to detoxify Al in their tissues by making stable complexes with organic and inorganic ligands (Watanabe & Osaki 2002). For example, an Al–citrate complex is formed in leaves of Hydrangea macrophylla, an Al accumulator growing in temperate regions (Ma et al. 1997).
Melastoma malabathricum L. (Melastomataceae) is an Al accumulator growing in tropical acid soils and one of the dominant woody species in acid sulphate soils with very high acidity and very poor mineral levels in tropical Asia, Australia and Polynesia (Osaki et al. 1998a,b). This species accumulates more than 10 mg Al g−1 in leaves and roots in the form of monomeric Al. The growth of M. malabathricum is remarkably enhanced by Al application in hydroponics (Osaki et al. 1997), but the detailed process behind this mechanism has not been well elucidated. So far, we reported that Al application in M. malabathricum induces an increase in dry weight; nutrient uptake; number of white, fine roots; and root metabolic activity, but a decrease in root lignin contents (Osaki et al. 1997; Watanabe, Jansen & Osaki 2005a). Moreover, when M. malabathricum is cultivated without Al for long periods, abnormal visual symptoms, such as rolling of the leaves, are often observed.
Miscanthus sinensis Andersson (Poaceae) is one of the dominant grass species in acid sulphate soils in temperate climates and has many widespread cultivars. Although it has been reported that the growth of M. sinensis is enhanced by Al application (Yoshii 1937), this species does not seem to be an Al accumulator (Kayama 2001). Moreover, the Al-induced growth enhancement in M. sinensis has not been examined in detail and it is unknown how this species deals with high levels of Al.
The low pH in acid sulphate soils is due to high concentration of sulphuric acid derived from the oxidation of pyrite (FeS2). Oxidation of pyrite also produces large amounts of Fe ions (Shamshuddin et al. 2004). Although most Fe ions in soils consist of Fe3+, which easily makes insoluble precipitations (non-toxic for plants) under dry conditions, parts of Fe3+ change to soluble Fe2+ under wet conditions (e.g. rainfall), inducing the increase of soluble Fe concentration in soils. In addition, Fe3+ itself can be solubilized in acid sulphate soils with a low pH (Mengel & Kirkby 2001). Therefore, plants growing in acid sulphate soils are exposed to the excess of two metals, Al and Fe. Although Fe is an essential element for plants, a high concentration of Fe is toxic because it induces an excess of reactive oxygen species. This may lead to the disorder of various cell functions, including the inhibition of plasma membrane H+–ATPase and lipid peroxidation (Fang et al. 2001; Yang et al. 2003). Whereas it was reported that the coexistence of Al ions with Fe2+ ions stimulated lipid peroxidation in cultured tobacco cells and pea roots (Ikegawa et al. 2000; Yamamoto, Kobayashi & Matsumoto 2001), the interactive effects of Al and Fe on the growth of plants growing in acid sulphate soils have not been elucidated.
In the present study, the effect of different concentrations of Al and Fe on growth, mineral uptake, lipid peroxidation and lignin accumulation in M. malabathricum was examined and compared to similar experiments on M. sinensis.
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Although previous studies reported the enhanced growth of M. malabathricum and M. sinensis when growing in the presence of Al (Yoshii 1937; Osaki et al. 1997), the exact mechanisms behind this process remained unclear. This study clearly shows that there is a significant interaction between Al and Fe in M. malabathricum, namely that the primary reason for the Al-induced growth enhancement in this species is the alleviation of Fe toxicity by Al. As far as we know, this mechanism may be considered as a specialized adaptation to its distribution in acid sulphate soils and has not been reported in any other plants. By contrast, 100 µm Fe did not reduce the growth and Al had no beneficial effect on M. sinensis (Fig. 1). This indicates that M. sinensis is highly tolerant to the excess of Fe in comparison with M. malabathricum and that the Al-induced growth enhancement in this species reported in a previous study (Yoshii 1937) was probably caused by another mechanism, such as amelioration of the excess of H+ and/or P toxicities (Clark 1977; Kinraide 1993; Llugany et al. 1995).
As described in the introduction, Fe excess toxicity in plants is essentially due to oxidative stress. In M. malabathricum roots as well, symptoms concerning oxidative stress, including lipid peroxidation and lignin deposition, were induced by the Fe application, in the absence of Al (Fig. 5). Membrane lipid peroxidation would induce disorders of membrane functions, resulting in the decrease of nutrient uptake and translocation. Although lignin precursors work as reactive oxygen species-scavenging compounds (Blokhina, Virolainen & Fagerstedt 2003), lignin deposition in root cell walls may inhibit the elongation of the cells (Sasaki, Yamamoto & Matsumoto 1996), followed by an inhibition of the whole root development. It is well known that rice leaves affected by excess Fe stress reveal brown spots on their tips and finally turn entirely brown (Tanaka, Loe & Navasero 1966). This symptom is called ‘bronzing’. Although the abnormal symptom in leaves of M. malabathricum appeared to differ from typical bronzing (Fig. 2), it is obvious that the excess of Fe directly or indirectly inhibited the functions of leaves, possibly including the decrease of the photosynthetic rate (Kampfenkel, Montagu & Inzé 1995).
The Al application ameliorated all these Fe-induced disorders in M. malabathricum (Figs 1, 2 & 5). This indicates that Al is required for a healthy growth of M. malabathricum in soils rich in Fe. Most studies of Fe toxicity in plants were carried out using lowland rice because Fe toxicity in uplands is very rare due to the oxidative conditions (Fageria & Rabelo 1987; Luo et al. 1997; Audebert & Sahrawat 2000). In these studies, the toxic levels of Fe in the treatment solutions were relatively high (ca. 1–10 mm). By contrast, in case of M. malabathricum, 40 µm Fe induced lipid peroxidation in roots and formation of blackish roots (Figs 5b & 6), 100 µm Fe inhibited the growth and increased root lignin contents (Figs 1 & 5a) and 500 µm Fe induced necrosis of root tips (data not shown). These results show that M. malabathricum is very sensitive to Fe when Al is not applied to the medium. The Fe levels in the standard nutrient solution used for general crops seem to be sufficient to induce disorders of M. malabathricum (Osaki et al. 1997).
Then, how does Al ameliorate Fe toxicity in M. malabathricum? As the Al application significantly decreased the Fe concentration in shoots and roots of M. malabathricum (Fig. 3), the antagonistic suppression of Fe uptake by Al is strongly suggested as a mechanism of the Fe toxicity amelioration. However, because Al did not decrease the Fe concentration in shoots of M. malabathricum in the presence of 10 µm Fe (Fig. 3), there may be at least two different Fe uptake systems: an Al sensitive mechanism and one that is uncoupled from Al uptake. Furthermore, Al may reduce the cell surface negativity in the roots, inducing the reduction of the toxic Fe2+ (and/or Fe3+) concentrations on cell surface that in turn results in reduced Fe uptake (Kinraide 1998). Although Al and Fe may share the same absorption system in M. malabathricum, the Al concentration in both shoots and roots of M. malabathricum was not significantly affected by the medium Fe levels (Fig. 4), indicating that this uptake and/or translocation in M. malabathricum is highly specific for Al in comparison with Fe. Al-specific induction of syntheses of organic acids, which are the ligands for accumulation and translocation of Al (Watanabe et al. 1998, 2005b; Watanabe & Osaki 2001), may be related to the specific mechanisms (Table 1). In case of the non-Al-accumulating M. sinensis, the Al application did not decrease the Fe concentration in shoots, whereas the Fe application significantly decreased the Al concentration in roots (Figs 3 & 4). Contrary to M. malabathricum, the uptake and/or translocation in M. sinensis seems to be highly specific for Fe. Roots of M. sinensis with the 10Fe0Al treatment showed a higher Fe concentration. This may be due to changes of the root apoplast structure (e.g. decrease of cation exchange capacity). Indeed, the concentration of other polyvalent cations, such as Ca and Mg, was also high in this treatment (data not shown).
Previously, we examined in a hydroponic culture the effect of Al on the growth of 13 plant species (Osaki et al. 1997). Although some of these can grow well in acid sulphate soils, the Fe concentration in leaves of all these species except for M. malabathricum did not decrease with Al application (Table 2), suggesting that the amelioration of Fe toxicity by Al is restricted to few plant groups, including M. malabathricum. Although Al accumulation is relatively common within the Melastomataceae family and related families of the order Myrtales (Jansen, Watanabe & Smets 2002), it is unclear whether these Al accumulators show similar Al–Fe interactions as M. malabathricum. Moreover, the beneficial effect of Al on the growth of Al accumulators may not be explained by a single factor only, because Al may well have other physiological effects on plant tissues, such as a possible impact on photosynthesis (von Faber 1925). Recently, Ghanati, Morita & Yokota (2005) reported that Al activates antioxidant enzymes and ameliorates oxidative stress in tea plants. In M. malabathricum as well, Al-induced activation of these enzymes may contribute to the growth enhancement, synergistically with the decrease of Fe accumulation.
Table 2. Fe concentration (mg kg−1) in mature leaves of 13 plant species grown for 2–4 weeks in the standard nutrient solution with or without 110 µm Al [for more details on methods, see Osaki et al. (1997)]
|0 µm Al||110 µm Al|
|Acacia mangium Willd.||152||301|
|Brachiaria ruziziensis Germain & Evrard||160||230|
|Fagopyrum esculentum Moench||429||299|
|Hordeum vulgare L.|| 67||329|
|Hydrangea macrophylla Ser.||264||233|
|Ischaemum barbatum Retz.||181||207|
|Leucaena leucocephala (Lam.) de Wit||220||315|
|Melaleuca cajuputi Roxb.||179||182|
|Melastoma malabathricum L.||1124||128|
|Oryza sativa L.||163||114|
|Polygonum sachalinense Schmidt ex Maxim.||442||427|
|Stylosanthes guianensis Swatz||173||217|
|Vaccinium macrocarpon Ait.||294||474|
Typical visual symptoms in M. malabathricum roots without Al application are a decrease of fine roots and an increase of blackish or blackish brown roots in solution culture (Watanabe et al. 2005a). In the present study as well, roots turned blackish in the 100Fe0Al and 40Fe0Al treatments (Figs 2 & 6). Oxidative stress-induced disorders including lipid peroxidation (Fig. 5b) or enhancement of lignin deposition (Fig. 5a) should be responsible for these symptoms. Furthermore, as it is known that anthocyanin synthesis is induced by the generation of active oxygen species (Nagata et al. 2003), this blackish colour was suspected to be derived from its accumulation in roots. However, the anthocyanin concentration in roots was very low and not affected by the treatments. An additional experiment revealed that roots turned blackish directly by adsorption of large amounts of Fe in the root apoplast (Fig. 6). This adsorption is reversible and roots become white in colour when Fe in the root apoplast is removed by the chelater (citrate) (Fig. 6). The effect of the formation of Fe-bound pigments on the growth of M. malabathricum will be examined in a future study.
In conclusion, it is likely that M. malabathricum has evolved mechanisms of internal Al tolerance and uses Al to avoid Fe toxicity. These characteristics have allowed M. malabathricum to distribute as a dominant plant species in acid sulphate soils. For that reason, Al is almost an essential element for M. malabathricum when growing in acid sulphate soils. Due to its sensitivity to Fe tolerance, M. malabathricum may not be able to grow well even in a suitable growth medium for general plant species, such as the standard nutrient solution, without Al. By contrast, M. sinensis has acquired mechanisms of tolerance to excessive levels of both Al and Fe.