JXB Advance Access originally published online on May 28, 2003
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Journal of Experimental Botany, Vol. 54, No. 388, pp. 1753-1759,
July 1, 2003
© 2003 Oxford University Press
Al-induced efflux of organic acid anions is poorly associated with internal organic acid metabolism in triticale roots
Received 4 December 2002; Accepted 4 April 2003
,
Faculty of Agriculture, Kagawa University, Ikenobe 2393, Miki-cho, Kita-gun, Kagawa 761-0795, Japan
* Present address: Environmental Biology, School of Earth and Environmental Sciences, Adelaide University, SA 5005, Australia. To whom correspondence should be addressed. Fax: +81 87 891 3137. E-mail: maj{at}ag.kagawa-u.ac.jp
Abbreviations: Al, aluminium; CS, citrate synthase; MDH, malate dehydrogenase; NADP-ICDH, NADP-isocitrate dehydrogenase; PEPC, phosphoenolpyruvate carboxylase.
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Abstract |
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The secretion of organic acid anions from roots has been identified as a mechanism of resistance to Al. However, the process leading to the secretion of organic acid anions is poorly understood. The effect of Al on organic acid metabolism was investigated in two lines of triticale (xTriticosecale Wittmark) differing in Al-induced secretion of malate and citrate and in Al resistance. The site of Al-induced secretion of citrate and malate from a resistant line was localized to the root apices (terminal 5 mm). The levels of citrate (root apices and mature root segments) and malate (mature segments only) in roots increased during exposure to Al, but similar changes were observed in both triticale genotypes. The in vitro activities of four enzymes involved in malate and citrate metabolism (citrate synthase, phosphoenolpyruvate carboxylase, malate dehydrogenase, and NADP-isocitrate dehydrogenase) were similar for sensitive and resistant lines in both root apices and mature root segments. The response of these enzymes to pH did not differ between tolerant and sensitive lines or in the presence and absence of Al. Moreover, cytoplasmic and vacuolar pH were not affected by exposure to Al in either line. Together, these results indicate that the Al-dependent efflux of organic acid anions from the roots of triticale is not regulated by their internal levels in the roots or by the capacity of the root cells to synthesize malate and citrate.
Key words: Aluminium resistance, citrate, malate, organic acid anion efflux, organic acid metabolism, triticale.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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There is wide genetic variation, both within and between species, in the resistance of plants to aluminium (Al). Such variation provides breeders with a strategy for improving the ability of agricultural systems to cope with Al toxicity, a major problem in many of the world’s arable soils.
A well-characterized mechanism of resistance to Al is the specific release of organic acid anions, such as malate, citrate and oxalate, from the roots of Al-resistant plants (Delhaize et al., 1993; Pellet et al., 1995; Ma et al., 1997). Two patterns of organic acid release have been classified, on the basis of the timing of secretion (Ma, 2000). In Pattern I, no discernible delay is observed between the addition of Al and the onset of secretion, while in Pattern II, organic acid anion secretion is delayed for several hours after exposure to Al. Different mechanisms have been suggested to be involved in the two secretion patterns. The rapidity of the Pattern I response suggests that Al activates a pre-existing anion channel on the plasma membrane and that the induction of proteins is not required (Ma, 2000; Ma et al., 2001b). For example, Al rapidly triggers the opening of malate-permeable channels in the plasma membrane of wheat root cells, which facilitates malate efflux (Ryan et al., 1997; Zhang et al., 2001). There is a rapid release of malate from wheat roots in response to Al exposure (Delhaize et al., 1993; Ryan et al., 1995). By contrast, the delay observed in Pattern II-type secretion might indicate that protein induction is required. These induced proteins could be involved in organic acid metabolism or in the transport of organic acid anions, but the exact mechanisms are not understood.
Triticale (Triticosecalexspp.) is a synthetic hybrid species formed between wheat and rye. Ma et al. (2000) recently developed a set of chromosome substitution lines of triticale and screened them for resistance to Al. ST2, a non-substitution line, is relatively resistant. This line also releases substantial quantities of two organic acid anions, malate and citrate, from the roots following exposure to Al. By contrast, ST22 triticale is sensitive to Al and does not show the same induction of organic acid anion release. ST22 triticale was created by substitution of the short arm of chromosome 3R with genetic material from wheat (3D) (Ma et al., 2000). It was concluded that genes responsible for Al resistance in triticale were located on the short arm of chromosome 3R.
In contrast to the pattern of efflux observed in wheat, a delay of 6–12 h was observed between the initiation of Al treatment and the increase in organic anion efflux from ST2 triticale (Ma et al., 2000). Such a delay indicates Pattern II-type secretion and that gene transcription and new or increased synthesis of specific proteins is required (Ma et al., 2001b). On the basis of correlations between efflux and metabolism, several authors have speculated that internal organic acid anion contents and enzyme activities of roots are directly responsible for the Al-stimulated efflux of organic anions (Li et al., 2000; Gaume et al., 2001). Recent work has also suggested that genetic transformation of plants to over-express organic acid metabolic enzymes sometimes leads to increased efflux of organic acid anions and Al resistance (de la Fuente et al., 1997; Tesfaye et al., 2001). However, other studies using transgenic plants indicate that organic acid anion levels in cells, and their efflux, are not regulated at the level of gene transcription or protein synthesis (Kruse et al., 1998; Delhaize et al., 2001). In this study, the effect of Al on organic acid metabolism in roots of triticale was investigated in order to determine whether changes in enzymes involved in organic acid biosynthesis or in root organic acid anion concentrations are associated with the onset of Al-stimulated citrate and malate efflux.
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Materials and methods |
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Seedling culture and sample collection
Two triticale lines (ST2=Al-resistant; ST22=Al-sensitive) from a set of previously characterized chromosome substitution lines were used (Ma et al., 2000). Seeds were surface-sterilized for 30 min in 0.5% (v/v) sodium hypochlorite solution containing 0.05% (w/v) sodium dodecyl sulphate, and were germinated for 2 d on damp filter paper in Petri dishes. Germinated seedlings were transferred to a plastic net that was floated on 1.0 l of 0.5 mM CaCl2. The solutions were adjusted to pH 4.5 using dilute HCl and were replaced daily over 3 d of seedling growth. The seedlings were maintained in a growth cabinet with a day/night temperature regime of 25/20 °C and 14/10 h.
Seedlings (80–100 per box) were transferred to boxes containing 1.0 l of 0.5 mM CaCl2 solution (pH 4.5), either Al-free or amended with 50 µM AlCl3. At each collection time, the root apices (terminal 5 mm) were excised with a razor blade in ice-cold treatment solution. For each sample, 50 apices were collected in an Eppendorf tube, snap-frozen and stored at –80 °C for later analysis of organic acids or enzyme activities. Freezing and storage did not affect the determination of malate and citrate or of enzyme activities in the samples. Mature segments of root (30 segments sample–1) were also collected, by excision of portions of root located at between 1 and 2 cm behind the apex.
Root elongation was routinely measured during sampling, as described previously (Ma et al., 2000), to verify the differential response of ST2 and ST22 to Al treatment.
Location of organic acid anion efflux
Al-stimulated efflux of malate and citrate has been described previously (Ma et al., 2000), but was repeated in this study to verify that ST2 and ST22 respond differently to Al. In order to determine the location of Al-stimulated organic acid anion release in ST2 triticale, plastic trays were used that could be divided into compartments of 1 cm length with removable dividers (Ma et al., 2001a). All but the longest roots of selected, uniform seedlings were removed. A single remaining root of each of 20 seedlings was lain in a tray and the dividers placed carefully on top, using vacuum grease to form leak-proof seals. Treatment solution (4 ml) was added to each compartment in the tray and collected after incubation, with the roots shaded from light, for 6 h. Similar experiments using ST22 triticale could not be undertaken because insufficient malate and citrate was released from this line.
Determination of organic acid anions
Malate and citrate were determined in exudate and root samples according to enzymic procedures described by Delhaize et al. (1993). Root samples were extracted in 1 ml ice-cold 0.6 N perchloric acid, and the supernatants neutralized with 5 M K2CO3 (90 µl ml–1) prior to analysis. Volumes of 0.3 ml and 0.4 ml of extract solution, and 0.1 ml and 0.2 ml of exudate solution, were assayed for L-malate and citrate contents, respectively. Formation or oxidation of NADH was recorded in the assay samples by spectrophotometric analysis at 340 nm.
Measurement of enzyme activities
For the determination of phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH) and NADP-isocitrate dehydrogenase (NADP-ICDH) activities, root apices (0–5 mm) or mature root segments (1–2 cm) were extracted in a buffer containing 0.1 M TRIS-HCl (pH 8.0), 5 mM MgCl2, 5 mM EDTA, 10% glycerol, 0.1% Triton X-100, 0.5 mM phenylmethylsulphonyl fluoride (PMSF), and 5 mM 1,4-dithio-DL-threitol (DTT). Samples were centrifuged for 15 min (15 000 rpm at 4 °C) and the supernatants retained on ice for determination of enzyme activities. Minimal activity loss was observed when the extract solutions were stored on ice for up to 3 h. No more than six samples were processed at any one time, to ensure that enzyme activities were determined soon after sample preparation.
PEPC activity was measured by incubation of 0.2 ml extract in 1 ml of solution containing 50 mM TRIS-HCl (pH 8.0), 0.2 mM NADH, 5 mM MgCl2, 1 mM glucose-6-phosphate, 2.5 mM phosphoenolpyruvate (PEP), and 2 U malate dehydrogenase. The reaction was initiated by the addition of PEP, and NADH oxidation was followed by monitoring the rate of absorbance change at 340 nm for 2 min. MDH activities were also measured by monitoring the oxidation of NADH. Fifty-fold dilutions of extract (40 µl) were incubated in 1 ml assay solution containing 50 mM TRIS-HCl (pH 8.0), 0.5 mM EDTA, 0.2 mM NADH, and 1 mM oxaloacetic acid. The reaction was initiated by the addition of oxaloacetic acid.
For the determination of NADP-ICDH activity, extract (0.1 ml) was incubated in 1 ml of assay solution, composed of 50 mM TRIS-HCl (pH 8.0), 5 mM MgCl2, and 0.1 mM NADP and with 2 mM DL-isocitrate added to start the reaction. Activity was recorded as the rate of reduction of NADP, monitored at 340 nm.
For CS activity measurement, root samples were extracted in the extraction buffer described above, without added DTT. Extracts were first desalted on Sephadex PD10 columns (Sephadex G-25 M; Pharmacia), to obtain more consistent activity determinations. Prepared solution (0.4 ml) was incubated in a reaction buffer containing 50 mM TRIS-HCl (pH 8.0), 5 mM MgCl2, 100 µM 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB), 0.2 mM acetyl CoA, and 0.5 mM oxaloacetate. The CS reaction was begun by the addition of oxaloacetate, and measured by following reduction of acetyl CoA in the presence of DTNB at 412 nm (Srere, 1967).
Enzyme activities were expressed in units (U), where one unit is the activity required to produce 1 µmol of the monitored reaction product min–1.
Effect of pH on enzyme activity
To examine the effect of pH on the activity of CS, PEPC, MDH, and NADP-ICDH, root apices (0–1 cm) were excised from ST2 and ST22 exposed to 0.5 mM CaCl2 solution (pH 4.5) with or without 50 µM Al for 24 h. The extraction and measurement of enzyme activity were as described above, using MES and TRIS buffers adjusted to different pH values as indicated in Fig. 4. The actual pH of each sample was measured after enzyme activities were determined.
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In vivo pH measurement by 31P-NMR
Root apices (0–5 mm) were excised from ST2 and ST22 triticale seedlings exposed to 0.5 mM CaCl2 solution (pH 4.5) with or without 50 µM Al for 18 h and subjected to 31P-NMR measurement. The NMR spectra were recorded at 161.92 MHz (GSX-400). For a single sample, 250–300 root apices were used and the measurement was conducted three times independently. Values for cytoplasmic and vacuolar pH were estimated based on a standard curve between chemical shift and pH (Roberts et al., 1980)
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Results |
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Effects of Al on organic acid anion efflux
The effects of Al on root elongation and organic acid anion efflux in ST2 and ST22 triticale have been published in detail in a previous manuscript (Ma et al., 2000). On the basis of root growth measurements, ST2 is more resistant than ST22 triticale to 50 µM AlCl3 (46% and 83% inhibition of elongation of ST2 and ST22 roots, respectively, following treatment in a simple Ca solution for 24 h). Large amounts of malate and citrate are released from the roots of ST2 seedlings with exposure to Al. For example, over 20 h, malate and citrate efflux from ST2 roots were 19.8±1.88 (n=3) and 9.6±0.79 nmol seedling–1, respectively, while 3.8±0.58 nmol malate and 3.1±0.45 nmol citrate seedling–1 were released from ST22 roots. Negligible amounts of malate and citrate were released from either triticale line in the absence of Al.
Al-stimulated citrate and malate efflux in ST2 triticale were localized to the root apex (Table 1). On a root length basis, 16- and 20-fold more citrate and malate, respectively, was released from 5 mm root apices compared to the root zone behind the apex. This result is consistent with observations for other species that show Al-stimulated organic acid anion release, including wheat (Ryan et al., 1995), maize (Pellet et al., 1995) and buckwheat (Zheng et al., 1998).
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Changes in malate and citrate content of roots
Malate content within the root apices (5 mm) of Al-resistant and Al-sensitive triticale seedlings remained constant during exposure to 50 µM AlCl3 for 24 h (Fig. 1A). However, levels of citrate were increased by around 2-fold after 12 h Al treatment (Fig. 1B). In a separate experiment higher levels of citrate were still observed in Al-treated root apices after 72 h treatment (data not shown). Changes with Al treatment were the same for the Al-resistant (ST2) and Al-sensitive (ST22) triticale lines (Fig. 1).
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In more mature root segments, sampled from between 1 and 2 cm behind the root apex, the contents of both malate and citrate increased with Al treatment (Fig. 2). After 24 h exposure to 50 µM AlCl3, citrate levels had increased by around 2- and 3-fold in ST22 and ST2 root segments, respectively, with significant differences between treatments (P <0.05) first observed following 12 h of Al exposure. Malate contents of mature root segments were increased in both triticale lines by around 2-fold after 24 h treatment with 50 µM AlCl3. Changes in levels of malate were observed as early as 3 h after the start of Al exposure.
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Smaller increases in both citrate (apices and mature segments) and malate (mature segments only) were observed during exposure to an intermediate level of Al (20 µM AlCl3; data not shown).
Enzyme activities
Activities of some key enzymes responsible for malate and citrate synthesis and degradation were measured in extracts prepared from Al-treated and untreated root apices of triticale (Fig. 3). No significant changes were observed with Al exposure, for either ST2 or ST22, in the activities of CS, PEPC or MDH. NADP-ICDH activities were increased, by around 20–30%, with Al stress in both triticale lines. There were similar observations when enzyme activities were expressed relative to root apex.
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The effect of Al on the activities of CS, PEPC, MDH, and NAPD-ICDH was also investigated in extracts prepared from mature root segments (1–2 cm) of ST2. No differences were observed for any enzyme activities, between Al-treated and untreated root segments (Table 2).
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Effect of pH on enzyme activity
Activities of the organic acid metabolic enzymes were dependent on pH (Fig. 4). However, sensitivity to pH did not differ between enzyme preparations obtained from ST2 and ST22 triticale roots treated with or without Al.
Cytoplasmic and vacuolar pH
The pH sensitivities of the enzymes suggested that changes in cellular pH could alter enzyme activities, which would not be evident in measurements made in the buffered extracts. However, Al treatment did not affect cytoplasmic or vacuolar pH in either ST2 or ST22 (Table 3). Cytoplasmic and vacuolar pH also did not differ between the two triticale lines (Table 3).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Many Al-resistant species and cultivars respond to Al stress by the secretion of specific organic acid anions (e.g. oxalate, malate, citrate) from the roots (for reviews see Ma, 2000; Ma et al., 2001b). However, the process leading to the secretion of organic acid anions is poorly understood. In the present study, the effect of Al on the metabolism of organic acid anions was investigated in relation to the secretion of malate and citrate secretion in two lines of triticale differing in Al resistance.
The internal contents of malate and citrate in root apices were examined. Citrate content increased with Al exposure, while malate content did not change (Fig. 1). However, there was no difference in the citrate content between the Al-tolerant (ST2) and Al-sensitive (ST22) triticale lines (Fig. 1), suggesting that the secretion of citrate and malate is not associated with the accumulation of citrate and malate in the root apices.
To investigate the Al-induced increase in citrate content of the root apices further, the effect of Al on the activities of citrate synthase (CS), phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), and NADP-isocitrate dehydrogenase (NADP-ICDH) was investigated. These four enzymes are closely associated with the synthesis and degradation of malic and citric acids. However, no major difference in the activities of these four enzymes was detected between ST2 and ST22 or between roots with and without Al exposure (Fig. 3). These results suggest that the secretion of organic acid anions is poorly associated with their metabolism.
The increase in citrate in the root apices may be due to the transport of citrate from the mature zone of the roots, and Al may stimulate synthesis of citrate in the mature root zone. To examine this possibility, the internal content of malate and citrate in older root segments (1–2 cm) was compared between ST2 and ST22 in the presence and absence of Al. Although the secretion of malate and citrate is restricted to the root apex, the internal contents of malate and citrate in the mature root zone were increased by Al exposure (Fig. 2). However, no difference in malate and citrate contents was observed between ST2 and ST22 or between Al-treated and untreated roots. The activity of the four tested organic acid metabolic enzymes was also not affected by Al in root segments of ST2 roots (Table 2).
In vitro measurements of enzyme activity are usually conducted under optimum conditions. No detectable differences in the measured enzyme activities between Al-treated and untreated roots suggests that the levels of enzyme protein were not increased by Al. In support of this, no change was detected in protein levels of CS with exposure to Al, using an antibody for the enzyme (data not shown). However, it is possible that Al alters the enzyme activity by changing cellular pH or other factors, which would not be detected in vitro. The enzyme preparations were buffered to pH 8.0 for activity determinations, and this may well have masked cellular pH differences between Al treatments and triticale lines. Therefore, the effect of pH on enzyme activity was investigated. Although the activities of four enzymes were sensitive to pH, sensitivities were similar for samples prepared from ST2 and ST22 in the presence and absence of Al (Fig. 4). Moreover, Al exposure did not lead to changes in cytoplasmic or vacuolar pH in triticale root tips (Table 3). Citric and malic acids are primarily synthesized in mitochondria. Although the pH of mitochondrial compartments could not be measured, it seems unlikely that Al causes a pH change in this organelle because there were no measurable changes in cytoplasmic or vacuolar pH (Table 3).
The Al-induced increase in citrate and malate contents may be a stress response to Al toxicity. In wheat, which responds to Al by the rapid secretion of malate, similar changes in the internal levels of both citrate and malate were observed with exposure to Al, for both an Al-tolerant and Al-sensitive line (data not shown). Other studies have shown that while only one or two organic acid anions are released by roots, the levels of a range of organic anion species within roots change with Al exposure (Cambraia et al., 1983; Scott et al., 1991; Pellet et al., 1995; Gaume et al., 2001). The mechanism for the Al-induced increase in the content of organic acid anions in roots remains to be examined.
The over-expression of organic acid enzymes can sometimes lead to changes in internal organic acid anions and in efflux, as was found for tobacco transformed with a gene for CS (de la Fuente et al., 1997), and demonstrated more recently in lucerne that was engineered to over-express MDH (Tesfaye et al., 2001). However, other studies using transgenic plants indicate that organic acid anion levels in cells, and their efflux, are not regulated at the level of gene transcription or protein synthesis (Kruse et al., 1998; Delhaize et al., 2001). The authors’ results have shown that the Al-dependent efflux of malate and citrate from roots of an Al-resistant line of triticale is poorly correlated with internal organic acid anion contents and with the in vitro activities of CS, MDH, PEPC, and NADP-ICDH. The release of organic acid anions does not appear to be regulated by their levels in the roots or by the capacity of root tissues to synthesize them. Rather, it is suggested that the delayed increase in efflux from ST2 with Al stress may be related to the up-regulation of a gene or genes involved in the transport of organic acid anions out of the cell.
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Acknowledgements |
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JEH was a recipient of a Postdoctoral Fellowship from the Japan Society for the Promotion of Science (JSPS). This study was supported by a Grant-in-Aid for General Scientific Research (Grant no. 13660067 to JF Ma) from the Ministry of Education, Science, Sports and Culture and by NSFC (no. 30228023 to JF Ma). We are very grateful for the helpful comments and advice provided by Drs Manny Delhaize and Peter Ryan (CSIRO Plant Industry, Australia), and thank Dr Shin Taketa for the supply of triticale seeds. Thanks are also given to Hideko Yomo and Sakiko Nagao for their assistance in NMR measurements.
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