Plant Physiol. (1998) 117: 753-759

High Aluminum Resistance in Buckwheat¹
II. Oxalic Acid Detoxifies Aluminum Internally

Jian Feng Ma^*, Syuntaro Hiradate, and Hideaki Matsumoto

Research Institute for Bioresources, Okayama University, Chuo 2-20-1, Kurashiki 710, Japan (J.F.M., H.M.); Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Tsukuba, Japan (H.M.); and Division of Plant Ecology, National Institute of Agro-Environmental Sciences, Tsukuba Norin Danchi, P.O. Box 2, Tsukuba, Ibaraki 305, Japan (S.H.)

	ABSTRACT

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Buckwheat (Fagopyrum esculentum Moench. cv Jianxi), which shows high Al resistance, accumulates Al in the leaves. The internal detoxification mechanism was studied by purifying and identifying Al complexes in the leaves and roots. About 90% of Al accumulated in the leaves was found in the cell sap, in which the dominant organic acid was oxalic acid. Purification of the Al complex in the cell sap of leaves by molecular-sieve chromatography resulted in a complex with a ratio of Al to oxalic acid of 1:3. A ¹³C-nuclear magnetic resonance study of the purified cell sap revealed only one signal at a chemical shift 164.4 ppm, which was assigned to the Al-chelated carboxylic group of oxalic acid. A ²⁷Al-nuclear magnetic resonance analysis revealed one major signal at the chemical shift of 16.0 to 17.0 ppm, with a minor signal at the chemical shift of 11.0 to 12 ppm in both the intact roots and their cell sap, which is consistent with the Al-oxalate complexes at 1:3 and 1:2 ratios, respectively. The purified cell sap was not phytotoxic to root elongation in corn (Zea mays). All of these results indicate that Al tolerance in the roots and leaves of buckwheat is achieved by the formation of a nonphytotoxic Al-oxalate (1:3) complex.

	INTRODUCTION

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Al³⁺ is toxic to most plant species, but some can accumulate Al without showing any toxicity. Tea and hydrangea are well-known Al accumulators. Old leaves of tea can accumulate Al up to 30,000 mg kg¹ on a dry-weight basis (Matsumoto et al., 1976), and the Al content in the leaves of hydrangea plants with blue-sepaled flowers can become higher than 3,000 mg kg¹ (Ma et al., 1997a). Some trees in tropical cloud forests (such as Richeria grandis) have also been reported to accumulate high levels of Al (more than 1,000 mg kg¹; Cuenca et al., 1990). Recently, several plants (Melastoma malabathricum and Vaccinium macrocarpon; Osaki et al., 1997) adapted to low-pH soils were found to accumulate high Al in either roots or leaves. Although localization of Al in the cells of these plants has not yet been well identified, several reports have suggested the presence of Al in the symplasm in a soluble form (Cuenca et al., 1990). At the pH of symplasmic solution (>7.0), the concentration of free Al³⁺ is decreased to less than 10¹⁰ M due to the formation of insoluble Al(OH)₃ (Martin, 1988), but this does not imply that such low concentrations are biologically inert (Taylor, 1991). Because of the high affinity of Al for O₂-donor compounds such as Pi, nucleotides, RNA, DNA, proteins, carboxylic acids, phospholipids, polygalacturonic acids, heteropolysaccharides, lipopolysaccharides, flavonoids, anthocyanins, etc. (Haug, 1984; Martin, 1986), very low concentrations of free Al in the symplasm are potentially phytotoxic (Taylor, 1991). For example, Al³⁺ binds almost 10⁷ times more strongly to ATP than does Mg²⁺; therefore, less than nanomolar amounts of Al³⁺ can compete with Mg for the P sites (Martin, 1988). These facts suggest that Al-accumulating plants must possess effective mechanisms for detoxifying Al internally. However, these mechanisms have not been well documented.

The buckwheat (Fagopyrum esculentum Moench.) is an important economic crop in Asia. The cv Jianxi, which is cultivated in the acid-soil area of southern China, was found to show high resistance to Al toxicity (Zheng et al., 1998a, 1998b), and one of the mechanisms responsible for the high Al resistance in this cultivar was the secretion of oxalic acid, a strong Al chelator, by the roots (Ma et al., 1997b; Zheng et al., 1998b). This response was very rapid (occurring within 30 min after exposure to Al solution) and was specific to Al stress; neither P deficiency nor other toxic metals such as La could induce the secretion of oxalic acid. Furthermore, we found that cv Jianxi accumulated Al in the leaves.

Following 10 d of intermittent treatment with 50 µM Al, the Al concentration of the buckwheat leaves reached about 450 mg Al kg¹ on a dry-weight basis, in contrast to other species such as wheat, oat, radish, and rape, which contained less than 50 mg Al kg¹ after the same treatment (Ma et al., 1997b). However, the Al concentration in buckwheat roots was less than that of other species. Based on a ²⁷Al-NMR study, the form of Al in the buckwheat leaves has been suggested to be an Al-oxalate complex at a 1:3 ratio (Ma et al., 1997b). In the present study the Al complex in the leaves was purified and identified using ¹³C-NMR, and the form of Al in the roots was also examined using ²⁷Al-NMR. The results indicate that accumulation of Al as Al-oxalate (1:3) complex, a nonphytotoxic form, is also responsible for high Al resistance in buckwheat.

	MATERIALS AND METHODS

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Plant Materials and Al Treatment

2

Extraction and Purification of the Cell Sap

1

¹³C- and ²⁷Al-NMR Measurement

²⁷Al-NMR spectra were obtained at 156.3 MHz (JNM--600 spectrometer, JEOL). The roots were cut with scissors and then placed in NMR tubes 10 mm in diameter. Solution samples were analyzed using NMR tubes 5 mm in diameter. The parameters used were: frequency range, 62.5 kHz; data points, 33,000; acquisition time, 0.52 s. AlCl₃ (0.2 mM in 0.1 M HCl) was used as an external reference for calibration of the chemical shift (0 ppm).

Complexes of Al-oxalate at a molar ratio of 1:1, 1:2, and 1:3 were prepared by mixing an equal volume of 4 mM AlCl₃ and 4, 8, and 12 mM Na₂C₂O₄. After the solution pH was adjusted to 4.6, the complexes were subjected to ²⁷Al-NMR measurement as described above.

Bioassay of Al Toxicity

To examine different binding of Al-oxalate complexes with different molar ratios of Al to oxalic acid, staining patterns of roots with Eriochrome Cyanine R were compared using an Al-sensitive cultivar of wheat (Tritium aestivum L. cv Scout 66). Seeds were soaked in distilled water for 10 h and then germinated on a net tray in the dark at 25°C. After 2 d the tray was put in a plastic container containing 100 µM CaCl₂ solution, pH 4.5. After a further 2 d seedlings selected for uniform size were exposed to 100 µM CaCl₂ solutions, pH 4.5, without Al (Al) or with 20 µM AlCl₃ (+Al³⁺) or 20 µM Al-oxalate complex at a 1:1, 1:2, or 1:3 molar ratio. After 20 h the roots were placed in distilled water for 5 min and then stained with a 0.1% aqueous solution of Eriochrome Cyanine R solution (Sigma) for 10 min. The excess dye was removed by washing with distilled water and then the roots were observed with a light microscope (model B061, Olympus). The root length was measured before and after treatment.

	RESULTS

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Buckwheat leaves accumulated as much as 2.01 mmol Al kg¹ fresh weight following 10 d of intermittent treatment with 50 µM Al (Table I), and the roots contained 3.45 mmol Al kg¹ fresh weight after 20 h of exposure to Al solution. Roots intermittently treated with Al for 10 d contained a similar Al concentration (data not shown). About 90% of the Al in the leaves was found in the cell sap, where the concentration was higher than 2 mM (Table I). About 60% of Al in the roots was extracted in the cell sap. The major organic acid was oxalic acid (Fig. 1), at a concentration of about 50 mM in the cell sap of leaves with or without Al treatment (Table I).

View larger version (10K): [in this window] [in a new window]   Figure 1. HPLC profile of ligands in the Al complex in the crude and purified cell sap of buckwheat leaves on an ion-exclusion column. Buckwheat was intermittently exposed to 50 µM Al in 0.5 mM CaCl2, pH 4.5, for 10 d. The crude cell sap was purified four times by Sephadex G-10 column chromatography. Detection was at 425 nm. OX, Oxalic acid; Cit, citric acid; and Mal, malic acid.

The cell sap from the leaves was purified by molecular-sieve chromatography (Sephadex G-10). After four purification procedures, a pure peak having the same retention time as oxalic acid was obtained (Fig. 1), suggesting the separation of the Al-oxalate complex from other compounds; 67% of Al in the crude cell sap was recovered (Table II). The ratio of oxalic acid to Al decreased from about 25 to nearly 3. At the fourth round of purification, the ratio of oxalic acid to Al in each fraction was approximately 3 (Fig. 2). All of these findings indicate the presence of the Al-oxalate (1:3) complex in the leaves.

View larger version (18K): [in this window] [in a new window]   Figure 2. Molecular-sieve Sephadex G-10 chromatography (fourth) of the cell sap of buckwheat leaves. The crude cell sap was prepared from the leaves of buckwheat intermittently exposed to 50 µM Al in 0.5 mM CaCl2, pH 4.5, for 10 d. Dilute HClO4 solution, pH 4.6, was used as the eluant, and fractions (3 mL each) were collected at a flow rate of 0.72 mL min1. , Al; , oxalic acid.

To further identify the structure of the purified Al complex from the leaves, the purified cell sap was subjected to ¹³C-NMR. The complex of Al-oxalate at 1:25 gave two peaks at a chemical shift of 164.4 and 171.6 ppm at ¹³C-NMR spectra (Fig. 3A). However, only one signal at a chemical shift of 164.4 ppm was observed in purified cell sap (Fig. 3B). The signal at a chemical shift of 171.6 was assigned to the free carboxylic group of oxalic acid, whereas the signal at 164.4 ppm was assigned to the Al-chelated carboxylic group of oxalic acid, confirming that the Al complex in the purified cell sap was Al-oxalate at 1:3. Furthermore, the complex in the purified cell sap was separated into the ligand and Al by passage through a cation-exchange resin column, followed by passage through an anion-exchange resin column. Negative-ion fast-atom-bombardment MS of the anionic fraction exhibited the pseudomolecular ion peak [M-H] at m/z 89 (data not shown), indicating that the ligand chelated with Al in buckwheat leaves is oxalic acid.

View larger version (12K): [in this window] [in a new window]   Figure 3. 13C-NMR spectra of Al-oxalate (1:25) complex (A) and the purified cell sap of buckwheat leaves (B). Buckwheat was intermittently exposed to 50 µM Al in 0.5 mM CaCl2, pH 4.5, for 10 d. The crude cell sap was purified four times by Sephadex G-10 column chromatography. Spectra were measured at 150.8 MHz. See ``Materials and Methods'' for the purification process and measurement conditions.

To investigate the form of Al in the intact roots, the ²⁷Al-NMR spectra were measured. No signal was observed in the roots without Al treatment (Fig. 4A), but one major signal at the chemical shift of 16.0 to 17.0 ppm with a minor signal at the chemical shift of 11.0 to 12.0 ppm was observed in the intact roots exposed to the Al solution for 20 h (Fig. 4B). Crude cell sap of the roots gave a similar chemical shift of ²⁷Al as that in intact tissues (Fig. 4C), suggesting that the Al form in the intact roots and in their cell sap was the same and that the Al was in a hexacoordinated complex (Haraguchi and Fujiwara, 1969). The chemical shift of major signal in the roots was similar to that observed in the leaves (Ma et al., 1997b). Since the chemical shift suggested that the Al may be chelated with organic acids, the organic acids in the cell sap were analyzed by HPLC. The major organic acid was oxalic acid at a concentration of 8.8 mM in the cell sap of the roots (Table I).

View larger version (20K): [in this window] [in a new window]   Figure 4. 27Al-NMR spectra of intact roots exposed to 0 (A) or 50 µM (B) Al for 20 h or the cell sap (C) extracted from the roots exposed to 50 µM Al for 20 h. Spectra were measured at 156.3 MHz. See ``Materials and Methods'' for measurement conditions.

Because oxalic acid can form a complex with Al at an Al:oxalic acid molar ratio of 1:1, 1:2, or 1:3, the chemical shifts for these three Al complexes were compared under the same pH as the cell sap (pH 4.6). Signals were observed at chemical shifts of 6.5, 11.6, and 16.1 ppm (Fig. 5), which are assigned to the 1:1, 1:2, and 1:3 Al-oxalate complex, respectively (Kerven et al., 1995). This suggests that the Al in buckwheat roots is present in the form of Al-oxalate complexes at a molar ratio of 1:3 and 1:2.

View larger version (12K): [in this window] [in a new window]   Figure 5. 27Al-NMR spectra of Al-oxalate complexes with a 1:1 (A), 1:2 (B), or 1:3 (C) molar ratio of Al to oxalic acid. The pH of the solution was 4.6. Spectra were measured at 156.3 MHz.

The primary response of plants to Al toxicity is the inhibition of root elongation, which occurs within several hours after Al treatment (Ryan et al., 1993). The toxicity of the purified cell sap was tested by investigating the effect on the root elongation of corn. A 20-h treatment with 20 µM AlCl₃ in 100 µM CaCl₂ solution inhibited the elongation of corn roots by 50%, whereas the purified cell sap containing the same concentration of Al (20 µM) did not inhibit root elongation (Fig. 6). This suggests that the purified Al complex is not phytotoxic.

View larger version (15K): [in this window] [in a new window]   Figure 6. Effect of the purified cell sap of buckwheat leaves on the root elongation of corn. Roots were exposed to 100 µM CaCl2 solution containing 0 (Al) or 20 µM AlCl3 (+Al) or 20 µM Al complex purified from the cell sap of buckwheat leaves. The treatment period was 20 h. Values are means ± SD of 10 replicates.

The toxicity mechanism of Al is still unknown, but the binding of Al to cellular components such as the cell wall, plasma membrane, DNA, enzymes, ATP, and others is considered to be the initial step in Al toxicity (Kochian, 1995). Binding of Al-oxalate complexes with different ratios of Al to oxalic acid to roots was investigated by staining with Eriochrome Cyanine R using cv Scout 66, an Al-sensitive cultivar of wheat. Heavy staining was observed in the root apex treated with Al³⁺, but no staining was observed in the roots treated with 1:2 and 1:3 Al-oxalate complexes even though roots treated with 1:1 Al-oxalate were also stained (Fig. 7). The 1:3 complex did not inhibit the root elongation at all, but a 20-h exposure to 20 µM AlCl₃ inhibited it by 90% (data not shown). This suggests that oxalic acid prevents binding of Al to the cellular components, thereby detoxifying Al.

View larger version (68K): [in this window] [in a new window]   Figure 7. Staining patterns of wheat cv Scout 66 roots exposed to Al-oxalate complexes with different molar ratios of Al to oxalic acid. Roots were exposed to 100 µM CaCl2 solution containing 0 (Al) or 20 µM AlCl3 (+Al) or 20 µM 1:1, 1:2, or 1:3 Al-oxalate complexes. After 20 h the roots were stained with Eriochome Cyanine R. Pink color shows the binding of Al to the root apex.

	DISCUSSION

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Several potential mechanisms of internal tolerance for Al have been suggested, including chelation in the cytosol, compartmentation in the vacuole, Al-binding proteins, evolution of Al-tolerant enzymes, and elevated enzyme activity (Taylor, 1991; Kochian, 1995). However, there is still little evidence supporting these mechanisms. Because Al³⁺ has a high affinity for cellular components, forms other than Al³⁺ must be present in Al-tolerant plants. However, very little is known about the Al form in plants. In tea leaves most of the Al was proposed to be bound to catechins, based on the observed signal on the ²⁷Al-NMR spectrum (Nagata et al., 1992). However, such a complex has not been isolated or identified. In the vacuoles of the mycorrhizal basidiomycete Laccaria bicolor, Al was present in the form of polyphosphate complexes (Martin et al., 1994). Previously, we found that more than two-thirds of the Al in hydrangea leaves was present in the cell sap in a soluble form, and this Al form has been identified as Al-citrate complex at a 1:1 molar ratio of Al to citric acid (Ma et al., 1997a). We therefore suggest that internal detoxification of Al in hydrangea leaves is achieved by the formation of the Al-citrate complex, a nonphytotoxic form.

Because of the high binding ability of Al³⁺ with cellular components of roots, Al is not usually translocated to the upper parts of plants even when the root growth is severely inhibited. The high Al concentration in buckwheat leaves suggests that this cultivar accumulated Al in the leaves (Table I). It is noteworthy that about 90% of the Al was present in the cell sap in a soluble form and that the Al concentration in the cell sap was as high as 2 mM. An ²⁷Al-NMR study revealed a sharp signal at chemical shift 16.0 to 17.0 ppm in both the intact leaves and extracted cell sap (Ma et al., 1997b), which was apparently different from those observed in hydrangea leaves (broad signal at chemical shift 11-12 ppm) but was consistent with that of Al-oxalate complex at 1:3 ratio. Four purifications by Sephadex G-10 resulted in a complex with a molar ratio of Al to oxalic acid of 1:3 (Table II; Fig. 2). The ligand chelated with Al had the same retention time as oxalic acid on HPLC (Fig. 1). A ¹³C-NMR study revealed that the chemical shift of purified Al complex was consistent with that of a 1:3 Al-oxalate complex (Fig. 3). All of these results suggested that the Al in buckwheat leaves was complexed with oxalic acid at a molar ratio of 1:3.

Oxalic acid was the dominant organic acid in buckwheat leaves; the concentration in the cell sap was 50 mM (Table I). Although it is the simplest dicarboxylic acid, oxalic acid can form a bidentate complex through its two carboxyl functional groups. Formation of a stable five-ring structure with Al results in its strong chelation (Hue et al., 1986). Oxalic acid can form three species of complexes with Al at an Al to oxalic acid molar ratio of 1:1, 1:2, and 1:3, but 1:3 Al-oxalate complex is the most stable, with a stability constant of 12.4 (Nordstrom and May, 1996). This stability constant is much higher than that of Al-citrate (8.1) or Al-ATP (10.9; Martin, 1988), meaning that formation of a 1:3 Al-oxalate complex can prevent binding of Al to cellular components, thereby detoxifying Al.

The detoxifying effect of Al by oxalic acid has been reported (Hue et al., 1986), and we suggest here that the extent of Al detoxification depends on the molar ratio of Al to oxalic acid. Two times more oxalic acid than Al did not inhibit root elongation of cotton, but complexes with less than this ratio inhibited the root elongation (Hue et al., 1986). The Al-oxalate complex at 1:2 did not inhibit root elongation of corn at all (Zheng et al., 1998b). In the present study the purified cell sap containing a 1:3 Al-oxalate complex did not show toxicity affecting elongation in corn roots (Fig. 6). The staining results clearly showed that the 1:1 Al-oxalate complex was bound to the root apex of an Al-sensitive cultivar of wheat (Scout 66), whereas no binding of Al to the roots was observed upon exposure to an Al-oxalate complex with a molar ratio of oxalic acid to Al higher than 2 (Fig. 7). All of these findings indicate that the 1:3 Al-oxalate complex is nontoxic. The difference in Al-oxalate complexes with different ratios in detoxifying Al can be attributable to their different stability constants, which result in different activities of free Al³⁺.

Oxalic acid is a widely occurring natural product of plants, but little is known about its metabolism and function (Libert and Franceschi, 1987). Because there was no big difference in the concentration of oxalic acid in the cell sap between leaves treated with Al and those not treated with Al (Table I), it is unlikely that de novo biosynthesis of oxalic acid was induced by Al. Al was also mainly present in the form of a 1:3 complex in buckwheat roots based on the chemical shift of ²⁷Al (Fig. 4). Thus, the Al complex in the leaves may be translocated from the roots, although the form of translocation remains to be identified.

Buckwheat roots responded to Al stress by rapid secretion of oxalic acid (Ma et al., 1997b). One possibility of Al entering the roots is that secreted oxalic acid chelates with external Al, and then the Al-oxalate is taken up by the roots. However, we found that more Al was accumulated in the leaves when the roots were exposed to the AlCl₃ solution compared with roots exposed to the Al-oxalate complex at 1:3 (data not shown). This result suggests that some of Al³⁺ may be directly transported into the cell, which then forms a complex with internal oxalic acid. Further research is needed to confirm this.

Buckwheat shows a high Al resistance; relatively long-term treatment (10 d) with 50 µM Al in a 0.5 mM CaCl₂ solution, pH 4.5, did not affect the growth of the root or shoot of buckwheat, but the same treatment significantly inhibited the root growth of wheat (cvs Atlas 66 and Scout 66), oat (Avena sativa L. cvs Tochiyutaka and Heoats), and oilseed rape (Brassica rapus L. cvs 94008 and H166; Zheng et al., 1998a). Short-term Al treatment (16 h) also showed that buckwheat had higher resistance to Al compared with an Al-tolerant cultivar of wheat, Atlas 66 (Zheng et al., 1998b). One of the mechanisms responsible for this high resistance in buckwheat is proposed to be rapid and specific secretion of oxalic acid by the roots (Ma et al., 1997b; Zheng et al., 1998b). The secretion of oxalic acid can prevent Al³⁺ from entering the roots. However, recent studies indicate that a fraction of the Al enters the root symplasm fairly rapidly (Lazof et al., 1994) and is thought to interact at many cellular sites (Kochian, 1995). This suggests that internal tolerance mechanisms are also required for high Al resistance. Internal detoxification of Al by the formation of a 1:3 Al-oxalate complex in both the roots and leaves is also responsible for high Al resistance in buckwheat.

In conclusion, buckwheat accumulated Al in the form of a 1:3 Al-oxalate complex, a nonphytotoxic form, in both roots and shoots. Secretion of oxalic acid from the roots and accumulation of Al in a nontoxic form in the leaves indicate that both internal and external Al-detoxification mechanisms contribute to the high Al resistance seen in buckwheat.

	FOOTNOTES

   Received December 5, 1997; accepted April 8, 1998.

	ACKNOWLEDGMENT

We are grateful to Dr. Hideo Naoki (Suntory Institute for Bioorganic Research, Osaka, Japan) for fast-atom bombardment-MS measurement.

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