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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.
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.
MATERIALS AND METHODS
Cuttings were prepared from M. malabathricum plants grown in an Al-free standard nutrient solution that was aerated constantly for more than 1 year. The cuttings were rooted in a one-fifth strength standard nutrient solution (Watanabe, Misawa & Osaki 2005b) for 30 d in a glasshouse at Hokkaido University. Seeds of M. sinensis were surface sterilized with 1% sodium hypochlorite for 10 min, washed with deionized water and sown in dried Sphagnum moss with moderate moisture. After germination, the seedlings of M. sinensis were cultivated in a medium during 30 d. They were then pre-cultivated in 40 L containers containing the one-fifth strength standard nutrient solution for 30 d. The one-fifth strength standard nutrient solution contained 0.54 mm N (NH4NO3), 32 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. In this study, a poor nutrient solution was used to simulate natural growth conditions for M. malabathricum and M. sinensis. Then, the seedlings were transferred to 12 L pots filled with four different treatment solutions, consisting of the one-fifth strength standard nutrient solution (Fe free) with defined concentrations of Al and Fe. The treatments were: 10Fe0Al (10 µm FeCl2, 0 µm AlCl3), 10Fe500Al (10 µm FeCl2, 500 µm AlCl3), 100Fe0Al (100 µm FeCl2, 0 µm AlCl3) and 100Fe500Al (100 µm FeCl2, 500 µm AlCl3). The pH of the solutions was adjusted to 4.0 with NaOH or HCl every day, and the solutions were renewed every week. The seedlings were sampled 30 d after the treatment started. The roots of the seedlings were washed with deionized water. The plants were then separated into roots and shoot, lyophilized, weighed and ground. The basal part of shoots was not used for mineral analysis to avoid contamination.
Analysis of mineral and organic acid concentrations
After the lyophilized samples were digested by H2SO4—H2O2 for mineral analysis, Al and Fe concentrations were determined by inductively coupled plasma emission spectrophotometry (ICPS-7000; Shimadzu, Kyoto, Japan). Organic acids in lyophilized samples of M. malabathricum were extracted with 0.05 m HCl and analysed by capillary electrophoresis (Quanta 4000CE; Waters, Milford, MA, USA) (Watanabe et al. 1998).
Determination of lignin
Lignin concentrations in the roots of M. malabathricum were determined by the detergent method (Van Soest 1963a,b; Watanabe et al. 1998). Dried and ground root samples (100 mg) were digested with a 10 mL acid detergent solution (2% cetyl trimethylammonium bromide, 0.5 m H2SO4) at 100 °C for 1 h. After digestion, the solution was filtered on a glass filter (GA200; Advantec Toyo, Tokyo, Japan) under vacuum. The residue on the filter was washed with hot water and acetone, then dried at 80 °C and covered with 72% H2SO4. After 3 h, the residue was washed with hot water until it was acid free. The residue (lignin + crude silica) was dried at 80 °C, weighed, then ignited at 550 °C for 10 h in a muffle furnace and the ash (crude silica) was weighed to calculate the lignin content.
The seedlings were prepared as described in experiment 1. The seedlings of M. malabathricum were transferred to 12 L pots filled with four treatment solutions, consisting of the one-fifth strength standard nutrient solution (Fe free) and defined concentrations of Al and Fe. The treatments were: 10Fe0Al (10 µm FeCl2, 0 µm AlCl3), 10Fe500Al (10 µm FeCl2, 500 µm AlCl3), 40Fe0Al (40 µm FeCl2, 0 µm AlCl3) and 40Fe500Al (40 µm FeCl2, 500 µm AlCl3). We applied 40 µm Fe to the treatments because a similar Fe concentration was used in previous studies in which the beneficial effects of Al were observed (Osaki et al. 1997; Watanabe, Osaki & Tadano 1997; Watanabe & Osaki 2001; Watanabe et al. 2005a). The pH of the solutions was adjusted to 4.0. The seedlings were grown in the treatment solution for 1 week.
Determination of lipid peroxidation
Lipid peroxidation in roots was estimated by measuring the content of MDA as described by Munné-Bosch et al. (2004). Briefly, 70 mg of fresh root tips (< 2.0 mm) was repeatedly extracted (four times) with 80:20 (v/v) ethanol/water containing 1 µg mL−1 BHT using a homogenizer and ultrasonication. After centrifugation, the supernatants were pooled and 1.2 mL of the sample was added to a test tube with 1.2 mL of either: (1) –TBA solution containing 20% (w/v) trichloroacetic acid and 0.01% (w/v) BHT; or (2) +TBA solution containing the above plus 0.65% (w/v) TBA. The samples were heated at 95 °C for 25 min and, after cooling, the absorbance was read at 440, 532 and 600 nm. MDA equivalents (nmol mL–1) were calculated as 106 × [(A − B)/157 000], where A=[(Abs532+TBA− Abs600+TBA) − (Abs532–TBA− Abs600–TBA)], and B = [(Abs440+TBA − Abs600+TBA) × 0.0571].
Characterization of root pigments
In M. malabathricum grown without Al in the standard nutrient solution, young roots show a blackish colour (Watanabe et al. 2005a). Therefore, the effect of the treatments on anthocyanin concentration in roots was determined as described by Hodges et al. (1999). Approximately 0.1 g of fresh roots was homogenized in 5 mL methanol–1% HCl. The total amount of anthocyanins was determined as the difference between the absorbance at 536 and 600 nm. The results were expressed as cyanidin-3-glucoside equivalents. The effect of the chelate reagent on the root colour was also examined. Fresh excised roots (0.25 g) in the 40Fe0Al treatment were washed with deionized water, put into a Petri dish and 15 mL of 1 mm CaCl2 with or without 10 mm citrate (pH 4.0) was added. For comparison, excised roots in the 10Fe500Al treatment were also incubated with 1 mm CaCl2 without citrate. After 15 min, changes in root colour were observed. The Fe concentration in the solution was also determined by inductively coupled plasma emission spectrophotometry.
The results were analysed with analysis of variance and Tukey’s multiple comparison test (P < 0.05). All experiments were carried out with three replicates.
The growth of M. malabathricum seedlings decreased by the increase of Fe concentration in the medium in the absence of Al, but was not affected in the presence of Al (Fig. 1). The Al-induced growth enhancement was observed in the presence of 100 µm Fe, but not in the presence of 10 µm Fe. The roots showed a blackish colour in the 100Fe0Al treatment, whereas they were white in the other treatments, especially in the 10Fe500Al treatment (Fig. 2). The symptom of rolling the leaves, which is often found in M. malabathricum grown in the standard nutrient solution without Al for long periods, was also observed in the 100Fe0Al treatment (Fig. 2). By contrast, the growth of M. sinensis was not significantly affected by the treatments (Fig. 1), and there was no visual toxicity symptom in the seedlings growing in any treatment (Fig. 2).
In the absence of Al, the Fe concentrations in both shoots and roots in M. malabathricum significantly increased by the Fe application, whereas they remained at the control level (10Fe treatment) in the presence of Al (Fig. 3). The Fe concentrations of shoots and roots of M. malabathricum in the 100Fe0Al treatment were ca. 1 and 10 mg g−1, respectively. By contrast, the Al concentrations in both shoots and roots of M. malabathricum after Al application were not significantly affected by the Fe application, resulting in relatively high concentrations (ca. 7 and 6 mg g−1 in shoots and roots, respectively) (Fig. 4). In shoots of M. sinensis as well, the Fe concentration increased by the Fe application, whereas the Al application did not affect it (Fig. 3). A significantly high concentration of Fe was found in the roots of M. sinensis with the 10Fe0Al treatment compared to those with the other treatments (Fig. 3). In M. sinensis, the Al application increased the Al concentration in roots, but not in shoots (Fig. 4). The Al concentration in roots of M. sinensis with the 100Fe500Al treatment decreased by 50% compared to the 10Fe500Al treatment (Fig. 4).
The lignin concentrations in roots of M. malabathricum increased with the Fe application and decreased with the Al application (Fig. 5a). Both oxalate and citrate concentrations in shoots of M. malabathricum significantly increased in the presence of Al (Table 1). Whereas citrate concentrations in roots also increased by the Al application, oxalate concentrations were consistently high regardless of the treatments. A difference in Fe concentration (10 and 100 µm) in the medium did not affect these organic acid concentrations.
Table 1. Concentrations of oxalate and citrate [µmol g−1 dry weight (DW)] in shoots and roots of Melastoma malabathricum grown in four different concentrations of Al and Fe for 30 d (experiment 1)
Values are the means of three replicates ± SE. Different letters indicate significant differences at P < 0.05.
28.5 ± 2.0 b
57.7 ± 15.7 a
121.6 ± 9.4 a
105.7 ± 20.5 a
10.6 ± 3.6 b
54.6 ± 7.9 a
131.9 ± 16.2 a
73.7 ± 9.3 a
2.4 ± 0.5 b
6.2 ± 0.5 b
13.0 ± 1.4 a
24.4 ± 3.3 a
1.3 ± 0.4 b
2.3 ± 0.4 b
16.4 ± 1.1 a
18.6 ± 1.4 a
TBA-reactive products (MDA) in the roots of M. malabathricum were determined to estimate lipid peroxidation. The accumulation of MDA in roots was significantly higher in the 40Fe0Al treatment (Fig. 5b). There was no significant difference in MDA contents in roots among the other treatments (Fig. 5b).
Although the Fe application made roots blackish in colour as shown in Fig. 1, the anthocyanin concentrations in roots were very low and did not differ among treatments [< 0.02 µmol g−1 fresh weight (FW)]. Interestingly, the blackish colour in roots with the 40Fe0Al treatment disappeared when extracted with methanol–HCl. To find out whether the disappearance of the blackish colour in roots was caused by a decrease of pH or the liberation of Fe adsorbed by the roots, the root colour was examined after washing with a citrate solution (pH 4.0). The citrate application rapidly bleached the roots with the 40Fe0Al treatment (Fig. 6). The amounts of Fe eluted in the washing solution with or without citrate were 59.6 ± 3.7 and 1.0 ± 0.1 µg, respectively, in roots treated with 40Fe0Al (± SE, n = 3). There was no difference in colour between the citrate-washed roots with the 40Fe0Al treatment and roots with the 10Fe500Al treatment (Fig. 6).
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
As this was a preliminary experiment, no replication was applied. These data were not shown in Osaki et al. (1997).
Acacia mangium Willd.
Brachiaria ruziziensis Germain & Evrard
Fagopyrum esculentum Moench
Hordeum vulgare L.
Hydrangea macrophylla Ser.
Ischaemum barbatum Retz.
Leucaena leucocephala (Lam.) de Wit
Melaleuca cajuputi Roxb.
Melastoma malabathricum L.
Oryza sativa L.
Polygonum sachalinense Schmidt ex Maxim.
Stylosanthes guianensis Swatz
Vaccinium macrocarpon Ait.
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.
This study was supported financially by two Grants-in-Aid for Scientific Research (nos. 15208008 and 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.