JXB Advance Access originally published online on April 4, 2006
Journal of Experimental Botany 2006 57(9):1899-1908; doi:10.1093/jxb/erj131
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RESEARCH PAPER |
Oxalate accumulation and regulation is independent of glycolate oxidase in rice leaves
1Laboratory of Molecular Plant Physiology, College of Life Sciences, South China Agricultural University, Guangzhou 510642, PR China
2Department of Biotechnology, Zhengzhou University, Zhengzhou 450052, PR China
3Department of Biology, San Francisco State University, 1600 Holloway Avenue, San Francisco, CA 94132, USA
To whom correspondence should be addressed. E-mail: xpeng{at}scau.edu.cn
Received 23 October 2005; Accepted 23 January 2006
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Abstract |
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Cellular oxalate, widely distributed in many plants, is implicated to play important roles in various functions and is also known to affect food qualities adversely in fruits and vegetables. How oxalate is regulated in plants is currently not well understood. Glycolate oxidase (GLO) has long been considered as an important player in oxalate accumulation in plants. To gain further insight into the biochemical and molecular mechanisms, the possible roles of GLO in the process were studied. Drastically different levels of oxalate could be achieved by treating rice with various nitrogen forms (nitrate versus ammonium). While nitrate stimulated oxalate accumulation, ammonium reduced its level. Such treatments resulted in similar pattern changes for some other related organic acids, such as glycolate, oxaloacetate, and malate. By feeding plants with exogenous glycolate it was possible almost completely to restore the ammonium-decreased oxalate level. Under the two treatments few differences were observed for GLO mRNA levels, protein levels, and in vitro activities. Both Km for glycolate/glyoxylate and Ki for oxalate remained almost the same for GLO purified from either nitrate- or ammonium-fed leaves. A further in vivo study, with transgenic plants carrying an estradiol-inducible GLO antisense gene, showed that, while the estradiol-induced antisense expression remarkably reduced both GLO protein levels and activities, oxalate levels were not significantly altered in the estradiol-treated transgenic plants. Taken together, it is suggested that oxalate accumulation and regulation is independent of GLO in rice leaves.
Key words: Glycolate oxidase, oxalate accumulation, rice
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Oxalate is widely distributed in the plant kingdom, and many plant species accumulate oxalate in a range of 3–15% (w/w) of their dry weight (Zindler-Frank, 1976; Libert and Franceschi, 1987; Nakata, 2003; Franceschi and Nakata, 2005). Studies have shown that oxalate may play various roles in plants including calcium regulation, ion balance (e.g. Na and K), plant protection, tissue support, and heavy metal detoxification (Libert and Franceschi, 1987; Franceschi and Nakata, 2005). Some plants, such as buckwheat, taro, and rice, exude and/or in vivo accumulate oxalate to detoxify aluminium and lead (Ma et al., 1997; Ma and Miyasaka, 1998; Yang et al., 2000). Oxalate may also be involved in the detoxification of other hazardous metals such as strontium (Franceschi and Schueren, 1986), cadmium (Choi et al., 2001), and copper (Mazen and Maghraby, 1997). Inoculation of tobacco leaves with an oxalate-deficient non-pathogenic mutant of Sclerotinia sclerotiorum induced measurable oxidant biosynthesis, but inoculation with an oxalate-secreting strain did not, indicating that oxalate was able to quench the oxidative burst in plants in responding to pathogen attack (Cessna et al., 2000). Despite the broadly suggested oxalate functional roles in plants, high oxalate content can also be a concern to human nutrition and health (Franceschi and Nakata, 2005). Excess levels of oxalate in any edible parts of plants significantly lower the nutritional quality, as oxalate decreases calcium bioavailability and may cause kidney stones (Libert and Franceschi, 1987; Horner and Wagner, 1995; Massey, 2003; Franceschi and Nakata, 2005). Therefore, elucidating oxalate metabolic and regulatory mechanisms becomes increasingly important in both scientific and applied aspects.
Efforts have been made to elucidate the metabolic pathways of oxalate biosynthesis and to reduce the oxalate levels in some crop plants (Libert and Franceschi, 1987). Several pathways were hypothesized, including photorespiratory glycolate/glyoxylate oxidation, cleavage of ascorbate, hydrolysis of oxaloacetate (Horner and Wagner, 1995; Nakata, 2003; Franceschi and Nakata, 2005). Glycolate/glyoxylate oxidation has long been proposed as an important pathway for oxalate biosynthesis in plants (Libert and Franceschi, 1987; Fujii et al., 1993; Nakata, 2003; Franceschi and Nakata, 2005). This proposal was ultimately based on the enzymatic evidence, in which glycolate oxidase (GLO) was shown to be able to catalyse glyoxylate oxidation to oxalate, although its ability to convert glycolate to glyoxylate during photorespiration was well known (Richardson and Tolbert, 1961; Tolbert, 1981). So far this is the sole enzyme in plants convincingly justified to catalyse oxalate formation. However, there are several lines of evidence challenging this pathway. Raven et al. (1982) showed that 18O2 incorporation into glycolate did not extend to oxalate. Some C4 plants such as amaranth, with minor photorespiration, still accumulated high concentrations of oxalate (Libert and Franceschi, 1987; Franceschi, 1987; Li et al., 2000). Oxalate accumulates even in the dark or in callus where there is supposedly no photorespiration (Franceschi and Horner, 1979; Franceschi, 1987). The calcium oxalate crystal idioblasts even lack GLO and/or photorespiration (Kausch and Horner, 1985; Li and Franceschi, 1990; Franceschi and Nakata, 2005). More recent work points towards an ascorbate cleavage as a major pathway for oxalate production in some plant species (Horner et al., 2000; Keates et al., 2000; Kostman et al., 2001). Green and Fry (2005) found that this pathway could even operate extracellularly. Nevertheless, the biochemical data for this proposed pathway are lacking so far. Interestingly, Franceschi (1987) showed that glycolate/glyoxylate may have served as the intermediates in catabolism of ascorbate to oxalate. Oxaloacetase, commonly found in microbes, can cleave oxaloacetate into oxalate and acetate (Gadd, 1999). Although oxaloacetase was once reported to exist in crude preparations of beetroot and spinach (Chang and Beevers, 1968), its existence in plants remains to be confirmed. Overall, an oxalate biosynthetic pathway in plants remains controversial, but more researchers tend to the view that the predominant biosynthetic pathway of oxalate may vary from plant species to species.
GLO has long been considered as a key component mediating oxalate biosynthesis in some plants (Millerd et al., 1963a, b; Piquemal et al., 1980; Chang and Huang, 1981; Franceschi, 1987; Li and Franceschi, 1990), yet its role in regulating oxalate accumulation is not defined. It has been observed that GLO activity and its kinetic property did not differ between oxalate-accumulating and oxalate non-accumulating plant species (Watanabe et al., 1995; Li et al., 2000) and that the Km of GLO for glyoxylate was determined to be much higher than the measured physiological glyoxylate concentration (Davies and Asker, 1983; Libert and Franceschi, 1987). In this study, the role of GLO in oxalate accumulation and regulation was investigated in rice plants. Biochemical and transgenic analyses have suggested that oxalate accumulation and regulation is independent of GLO in rice leaves, although the metabolites glycolate/glyoxylate still appear to be involved in the process.
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Materials and methods |
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Plant materials
Two varieties of rice (Oryza sativa L.) were used in the experiment: Xiangzhongxian 2 for the biochemical study and Shishoubaimao for antisense transformation.
Growth conditions and treatments
Pre-germinated seeds were grown in Kimura B complete nutrient solution (Yoshida et al., 1976) under greenhouse conditions [average temperature of 30/25 °C (day/night), relative humidity 60–80%, average photosynthetically active radiation 600–1000 µmol m–2 s–1 and photoperiod of 14 h day/10 h night]. Until the seedlings had four leaves they were treated with the two forms of nitrogen by replacing the nitrogen element in the complete solution with sole nitrate or sole ammonium (2.86 mM). The solution was renewed every 3 d. For the other treatments (i.e. feeding of glycolate, glyoxylate, ascorbate, oxaloacetate, isocitrate), the chemicals were added to the ammonium nutrient solution without altering the other elements. Analysis was made at regular time intervals after the treatments (0, 3, 6, 9, and 12 d or as specified elsewhere).
Growth of the transgenic plants and the estradiol-induced treatment were conducted in a growth chamber. The condition was controlled as follows: temperature 30/25 °C (day/night), relative humidity 
70%, photosynthetically active radiation about 600 µmol m–2 s–1 and photoperiod of 14 h day/10 h night.
Determination of oxalate and other organic acids
Extraction of organic acids:
Aliquots of 0.2–0.5 g FW, depending on sample availability, were homogenized in 1–4 ml of 0.5 N HCl. The homogenate was heated at 80 °C for 10 min with intermittent shaking. To the homogenate was added distilled water up to a volume of 5–25 ml. About 2–3 ml of the solution was withdrawn and centrifuged at 12 000 g for 10 min. One millilitre of supernatant was passed through a filter (0.45 µm) before HPLC analysis.
Analysis of oxalate, citrate, and malate:
The analysis was made according to Libert (1981) with modifications (Yu et al., 2002). Hypsil C18 column (5 µM, 4.6 mmx250 mm) equipped Waters 550 (Waters, MA, USA) was used as the static phase and the mobile phase was a solution containing 0.5% KH2PO4 and 0.5 mM TBA (tetrabutylammonium hydrogen sulphate) buffered at pH 2.0 with orthophosphoric acid. Flow rate was 1 ml min–1 and detection wavelength was at 220 nm.
Analysis of glycolate, glyoxylate, and oxaloacetate:
The analysis was made according to Petrarulo et al. (1989, 1990) with modifications (Ji et al., 2005). Glyoxylate and oxaloacetate were first derivatized by phenylhydrazine to form phenylhydrazone, then the derivatives were separated and quantified by reversed-phase HPLC. Hypersil C18 column (5 µM, 4.6x250 mm), a mobile phase containing 5% methanol and 95% phosphate buffer (13 mM potassium biphosphate; 1 mM potassium phosphate dibasic pH 6.0) and detection at 324 nm were applied in the system. Glycolic acid was determined by oxidizing glycolate into glyoxylic acid with purified GLO, then followed by the same procedure as above for glyoxylate determination.
For determination of ascorbic acid, 0.5 g of fresh leaves was homogenized in 3.5 ml 5% TCA solution and the homogenate was centrifuged at 20 000 g for 10 min. The supernatant was used for further analysis according to Okamura (1980).
Purification of GLO
Twenty grams of fresh leaves were homogenized in 80 ml of 100 mM phosphate buffer (pH 8.0) with a homogenizer. The homogenate was filtered through two layers of cheesecloth to remove fibrous materials. The filtrate was centrifuged at 15 000 g for 10 min and the precipitate was discarded. The pH of the supernatant was carefully adjusted to 5.5 with diluted HCl and then centrifuged at 15 000 g for 10 min. The supernatant was then fractionated between 20% and 40% of ammonium sulphate and the final precipitate was resuspended in 15 ml 5 mM TRIS-HCl (pH 7.5) and centrifuged again. The resultant supernatant was desalted through a Sephadex G-25 column (30 mmx200 mm) and the desalted solution was then loaded onto a DEAE-Sepharose Fast Flow column (20 mmx150 mm). The column was first flushed with 5 mM TRIS-HCl buffer (pH 7.5) and the enzyme was subsequently eluted with 100 mM TRIS-HCl buffer (pH 7.5).
RNA, protein blot, and activity assay of GLO
RNA blot:
Total RNA was extracted from rice leaves according to Logemann et al. (1987). Northern hybridization was after Sambrook et al. (1989). Thirty micrograms of total RNA were size-fractionated on a formaldehyde agarose gel and transferred to Hybond-N+ nylon membrane (Amersham). The blot was hybridized with a random prime [
-32P]-labelled cDNA probe: the complete GLO cDNA (1.3 kb) synthesized on RT-PCR product template with the upstream primer 5'-GAGAGAACTAGTGCAGGGTTCACAAGGCAGGAGAAAA-3' and the downstream primer 5'-GTGTCTCTCGAGCATGAACGACCCAGTTACGA-3'.
Protein blot:
Proteins were extracted by homogenizing 0.5 g of fresh leaves in 4 ml 20 mM phosphate buffer (pH 8.0). The homogenate was centrifuged at 15 000 g for 15 min. Equally loaded samples (15 µg protein) were fractionated on a 4–20% gradient SDS-PAGE, and then electroblotted onto a nitrocellulose membrane using a Mini Trans-Blot cell (Bio-Rad). GLO protein was detected using a rabbit polyclonal GLO antibody (1:1000). The antibody was prepared by expressing the complete GLO cDNA (inserted to a pET-23d vector; Novagen) in Escherichia coli (BL21) after Dumbroff and Gepstein (1993), and the expressed GLO protein induced by IPTG was purified on gradient SDS-PAGE (4–20%) and then injected into a rabbit. The serum was withdrawn as the antibody.
Activity assay:
GLO activity was assayed according to Hall et al. (1985) with some modifications. Samples (0.1–0.5 g) of fresh leaves were homogenized in 1–5 ml extraction buffer (100 mM phosphate, pH 8.0) at 4 °C. The homogenate was then centrifuged at 15 000 g for 15 min. The supernatant was used for the enzyme activity assay. A 1.5 ml sample of the reaction mixture contained 66 mM phosphate buffer (pH 8.0), 1 mM 4-amino-antipyrine, 5 units of horseradish peroxidase, 2 mM phenol, 0.1 mM FMN, 5 mM glycolate/glyoxylate, and 0.05 ml of enzyme extract. The enzyme was added last to start the reaction and distilled water substituted the substrate as the blank. H2O2 produced in the reaction mixture was determined spectrophotometrically at 520 nm and under 30 °C.
Construction of transgenics and induced down-regulation of GLO expression
The estradiol-inducible transformation vector pER 8 was kindly provided by Dr Nam-Hai Chua (Rockefeller University, New York) (Zuo et al., 2000). To generate the pER 8-GLO antisense construct, the complete cDNA of GLO was cloned by RT-PCR as described above, then inserted into pER8 between SpeI and XhoI restriction sites. First, PCR with specific primers and cutting with restriction enzymes showed that the target fragment had been correctly ligated. DNA sequencing further confirmed the correct orientation and cDNA identity [100% identical to that reported in the gene bank (AK098878)]. The pER8-GLO was transformed into rice by Agrobacterium-mediated infection (strain EHA105). The first two cycles of selection by hygromycin (50 mg l–1) was followed by HPT marker PCR amplification to select the positive transformants (since it is known that nos promoter governing the hygromycin resistance gene works less efficiently in monocotyledonous plants, more cycles of hygromycin selection were avoided). For further estradiol-inducibility tests, the seeds harvested from the positive T0 lines were germinated and grown in Kimura B complete nutrient solution. Until the T1 plants had three leaves they were treated with estradiol (5 µmol l–1) by adding it into the nutrient solution which was renewed every 3 d. GLO activity was determined 8 d after the treatment. The plants with markedly decreased GLO activity were selected as inducibility positive, and were transferred to normal soil conditions to grow until the seeds were harvested.
The seeds harvested from the positive T1 lines (26-18, 26-19, 26-23), originated from T0 line no. 26, were germinated and grown in Kimura B complete nutrient solution. Until the seedlings of T2 heterozygous plants had four leaves they were treated with estradiol (5 µmol l–1) and, at the same time, the nitrogen element in the nutrient solution was replaced with sole nitrate. For the controls, wild-type plants were grown and treated in the same manner. After treatment for 6 or 12 d, the first and second leaves from the top were detached for determining oxalate content, GLO activity, and enzyme protein.
Protein determination
Protein content was determined according to Bradford (1976).
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Results |
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Effects of nitrate/ammonium on the metabolism of organic acids
Nitrogen nutrition is known to cause changes in oxalate accumulation in certain plants (Libert and Franceschi, 1987). To identify the potential regulators for oxalate metabolism, an attempt was made to utilize different nitrogen forms to achieve various levels of oxalate accumulation. Rice seedlings treated for different periods with either nitrate or ammonium were harvested and assayed for oxalate content. As previously reported, nitrogen forms dramatically affected oxalate accumulation in rice. As shown in Fig. 1, while nitrate treatment significantly stimulated the foliar oxalate accumulation, ammonium had a suppressive effect. Three days after treatment, the oxalate content had increased by nearly 100% in nitrate-treated leaves but had decreased by >65% in ammonium-treated ones. As a result of the opposite effects, oxalate level in nitrate-treated leaves was five times higher than that in ammonium-treated ones at 3 d after treatment. Oxalate levels remained low in ammonium-treated leaves thereafter. By contrast, oxalate levels in nitrate-treated leaves continued increasing. At 12 d after treatment, the oxalate level in nitrate-treated leaves was nine times higher than that in ammonium-treated ones (Fig. 1).
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Metabolism of organic acids is regarded as well co-ordinated (Ryan et al., 2001). In order to understand how oxalate accumulation is associated in the metabolic network, the rice seedlings treated with either nitrate or ammonium were measured for levels of glycolate, glyoxylate, ascorbate, oxaloacetate, citrate, and malate. As shown in Fig. 2, while some of the organic acids demonstrated a similar time-course, others showed delayed responses. Glycolate, oxaloacetate, and malate showed a similar time-course to that of oxalate (Fig. 2A–C versus Fig. 1), whereas ascorbate and citrate responded noticeably later than oxalate to the treatments (Fig. 2D, E versus Fig. 1). By contrast, glyoxylate accumulated in ammonium-fed leaves throughout the whole treatment course (Fig. 2F) and changes in oxalate and glyoxylate were in a negative parallel.
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Effects of oxalate precursors on ammonium-suppressed oxalate accumulation
Glycolate, glyoxylate, ascorbate, oxaloacetate, and isocitrate have been suggested as the metabolic precursors for oxalate formation (Nakata, 2003). To find out how these precursors would affect oxalate accumulation they were fed exogenously to rice seedlings. As shown in Table 1 and Fig. 3, when glycolate was fed to the ammonium-treated rice plants it could reverse the decreased oxalate up to the level in the nitrate-fed leaves. Glyoxylate and oxaloacetate restored it to
50% of the nitrate-fed leaves. Ascorbate was less effective, with only about 30% of the nitrate-fed leaves. Isocitrate had no significant effect on the ammonium-suppressed oxalate level (Table 1). These results suggest that glycolate may be the predominant precursor and/or regulator for oxalate formation in rice leaves.
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GLO expression, activity, and catalytic kinetics as affected by nitrate/ammonium
The experiments mentioned above showed that glyoxylate and oxalate levels were adversely correlated (Fig. 1 versus Fig. 2F); feeding plants with either glycolate or glyoxylate could significantly increase the ammonium-suppressed oxalate level (Table 1; Fig. 3). These results hint at a possible involvement of glycolate metabolism in oxalate accumulation and regulation in rice. GLO, the key enzyme of glycolate metabolism (Tolbert, 1981), has long been suggested as an important player in oxalate accumulation in various plant species (Franceschi, 1987; Franceschi and Nakata, 2005). To define the potential role of GLO during oxalate accumulation in rice, GLO expressions were examined in both nitrate- and ammonium-fed rice leaves. Total RNA was extracted from the treated leaves and used for northern blotting. As shown in Fig. 4A, the two nitrogen forms had little effect on GLO transcript levels at the time intervals tested. Total proteins were prepared from the treated samples and analysed by western blotting. GLO protein levels remained unchanged between the two treatments during the time course (Fig. 4B). GLO activities in the various treated samples were also determined using either glycolate (Fig. 4C) or glyoxylate (Fig. 4D) as the substrate. There was no significant difference for GLO catalytic activities between the two treatments. Taken together, it is suggested that nitrate/ammonium treatments had little effect on both GLO expressions and activities, although, in this case, a dramatic difference in oxalate accumulation occurred (Fig. 1).
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To obtain detailed kinetic data, GLO was purified from both nitrate-fed and ammonium-fed leaves. The purification efficiency was monitored by measuring the specific activity at various fractionation steps. The final GLO obtained carried a purification factor of 58-fold. Kinetic analysis revealed that the Km of GLO for glycolate and glyoxylate was about 0.4 mM and 4 mM, respectively. Little difference was observed for the Km of GLO between the treatments with the two forms of nitrogen (Table 2). Oxalate, the product of glyoxylate oxidation, was able competitively to inhibit the activity, with a Ki of 1.5 mM. The Ki values remained the same between the two treatments (Table 2).
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Antisense suppression of GLO and oxalate accumulation
To verify the role of GLO in oxalate accumulation and regulation further, an estradiol-inducible system was used to express the GLO antisense gene. Transgenic GLO antisense rice plants were analysed for GLO proteins, activities, and oxalate levels (Fig. 5). Application of the inducer estradiol effectively suppressed GLO expression. Six days after estradiol treatment, both GLO enzymatic activities and protein levels in the antisense plants were significantly reduced, as compared with the wild-type controls (Fig. 5A, C). Twelve days after estradiol treatment, the enzymatic activities were minimal (>90% down-regulated) and GLO proteins became undetectable in the transgenic antisense plants (Fig. 5A, C). It can also be noted that there was a good correlation between the amount of GLO protein and GLO activity for these plants (Fig. 5A, C). Oxalate contents in the transgenic antisense plants remained almost the same throughout the estradiol treatment (Fig. 5B). These results further confirmed that oxalate accumulation is GLO-independent.
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Discussion |
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Plants may accumulate oxalate in a range of 3–15% (w/w) of their dry weight (Zindler-Frank, 1976; Libert and Franceschi, 1987; Nakata, 2003). Rice, as a model monocotyledonous plant, generally contains 3–6% of oxalate depending on growth stages and culture conditions (Libert and Franceschi, 1987; Ji and Peng, 2005; Fig. 1), with 40–50% of the soluble form and without any noticeable crystals during the growth stage being investigated in the present study (data not shown). Various forms of evidence have supported the idea that oxalate does not originate from the same source in different plant species. Through a number of isotope-labelling experiments, it was considered to be a strong possibility that the photorespiratory glycolate pathway is responsible for oxalate synthesis in various plants (Millerd et al., 1963b; Chang and Beevers, 1968; Osmond and Avadhani, 1968; Seal and Sen, 1970; Piquemal et al., 1980; Fujii et al., 1993; Franceschi and Nakata, 2005), although C2/C3 cleavage of ascorbate was recently suggested as a major oxalate source in Lemna minor L. and Pistia stratiotes (Keates et al., 2000; Kostman et al., 2001). It was initially detected that nitrate/ammonium was able to alter markedly oxalate levels in rice leaves (Ji and Peng, 2005; Fig. 1; Table 1) and provided further evidence that such an alteration was due to the efficiently regulated oxalate metabolism within rice leaves rather than due to its different downwards transportation and exudation (Ji and Peng, 2005). The mechanistic basis underlying this regulation may bring to light the general mechanism of oxalate accumulation and regulation in rice and, perhaps, in other plants.
Apart from oxalate, some other organic acids were also suppressed by ammonium treatment (Fig. 2). Sole ammonium source is known to cause toxicity to many plant species (Britto et al., 2001), so that one may consider that the ammonium-suppression of oxalate and some other organic acids could simply result from the toxicity-induced whole metabolism breakdown in plants. It should be noted, however, that rice is a typical ammonium-preferring plant, which usually does not suffer from ammonium toxicity (Magalhaes et al., 1995; Britto et al., 2001). By contrast, sole nitrate nutrition stimulated not only the accumulation of oxalate but also some other organic acids, such as glycolate, oxaloacetate, malate, ascorbate, and citrate (Fig. 2). This observation is consistent with a previous report that nitrate itself acted as a direct signal to induce organic acid accumulation (Scheible et al., 1997). It is yet to be defined how oxalate is metabolically associated with the other organic acids. Ascorbate and citrate accumulated noticeably later than oxalate during nitrate treatment (Fig. 2D, E versus Fig. 1). By contrast to glycolate, neither ascorbate nor isocitrate was able to greatly enhance the ammonium-decreased oxalate (Table 1). This result suggested that the level of either ascorbate or isocitrate may not have played the predominant role in mediating oxalate accumulation in rice plants. Interestingly, glyoxylate accumulated in ammonium-fed leaves and in a negative correlation with oxalate (Figs 1, 2F), suggesting that the downstream of glyoxylate metabolism including glyoxylate oxidation to oxalate could be interrupted under ammonium treatment. Considered together with the fact that glycolate/glyoxylate significantly restored the ammonium-suppressed oxalate level (Table 1; Fig. 3) it is possible that glycolate/glyoxylate is involved in oxalate accumulation in rice leaves. In this case, glycolate/glyoxylate may have acted as a metabolic precursor to activate and/or protect from the inhibition of an oxalate-forming enzyme. However, it is unclear why glycolate was even more effective than glyoxylate in the effect (Table 1; Fig. 3), since glyoxylate had been frequently shown to be a more direct and efficient precursor for oxalate formation in oxalate-accumulating plants (Millerd et al., 1963a, b; Chang and Beevers, 1968; Osmond and Avadhani, 1968; Seal and Sen, 1970; Fujii et al., 1993). The possible involvement of glycolate/glyoxylate in rice oxalate accumulation re-prompted interest as to the role of GLO in the process.
So far, GLO is the only enzyme convincingly proved to be capable of catalysing oxalate synthesis in plants. GLO from various plant species was found to be able to catalyse not only glycolate oxidation to glyoxylate but also glyoxylate oxidation to oxalate (Richardson and Tolbert, 1961; Havir, 1983). Evidence was further presented that GLO mediated in vivo oxalate accumulation and regulation in plants (Millerd et al., 1963a, b; Piquemal et al., 1980; Chang and Huang, 1981; Franceschi, 1987). The present results, however, suggest that GLO may not be involved in oxalate accumulation and regulation in rice leaves based on the following evidence. There was no difference between the leaves with contrasting oxalate content regulated by nitrate/ammonium in terms of GLO expressions, activities, and kinetic properties (Fig. 4; Table 2). Both Km for glycolate/glyoxylate and Ki for oxalate remained the same for GLO purified from either nitrate- or ammonium-fed leaves. Further in vivo study with antisense transgenic plants showed that, while GLO was >90% down-regulated, foliar oxalate levels were not altered, (Fig. 5) and correlation analysis assured that oxalate content and GLO activities were not significantly correlated (data not shown). Previous reports have also indicated that GLO activity and kinetic property were not different between the oxalate-accumulating and oxalate-non-accumulating plant species (Watanabe et al., 1995; Li et al., 2000), and the Km of GLO for glyoxylate was determined to be much higher than the measured physiological glyoxylate concentration (Davies and Asker, 1983; Libert and Franceschi, 1987). Here, it was also shown that the Km of rice GLO for glyoxylate was 4 mM while glyoxylate concentration in cells could be much lower than this value (Heupel and Heldt, 1994; Fig. 2B). GLO-catalysed glyoxylate oxidation was competitively inhibited by the product oxalate, with a Ki about 1.5 mM, whereas cellular oxalate may be much higher than this value (Fig. 1; Table 2). The value could be even higher at the subcellular level if oxalate is truly produced in peroxisomes by GLO catalysis. As far as is known, the present results have provided the strongest evidence so far, arguing against GLO's role in oxalate accumulation and regulation in plants, although glycolate/glyoxylate's involvement in this process is still not excluded.
How exactly oxalate accumulation is regulated in plants is currently not clear. It is proposed that there is a novel and undetermined enzyme(s) in rice responsible for oxalate synthesis from glycolate/glyoxylate. Millerd et al. (1963b) mentioned that an enzyme oxidizing glycolate/glyoxylate in Oxalis pes-caprae differed from the defined GLO since it catalysed glyoxylate turnover more efficiently than glycolate. While evidence for multiple enzymes with glycolate and glyoxylate oxidase activity was also demonstrated in tobacco leaves by Havir (1983), Nishimura et al. (1983) claimed existence of only one glycolate oxidase in spinach leaves. Glyoxylate dehydrogenase for oxalate production was detected in animals and microbes (Tokimatsu et al., 1998) but has not yet been identified in plants. A glycolate dehydrogenase, catalysing direct oxidation of glycolate to oxalate without forming glyoxylate as an intermediate, was only reported in animals (Fry and Richardson, 1979). Another glycolate dehydrogenase, recently described in Arabidopsis mitochondria, catalyses glycolate oxidation to glyoxylate (Bari et al., 2004), and, similarly, Goyal and Tolbert (1996) observed a glycolate-quinone oxidoreductase system, which is associated with the chloroplast photosynthetic electron transport chain catalyses the oxidation of glycolate to glyoxylate. Whether such glycolate dehydrogenases could also catalyse the oxidation of glyoxylate to oxalate in plants is unknown. Lactate dehydrogenase might also act on glyoxylate to produce oxalate in plants (Davies and Asker, 1983). Further biochemical, molecular, and genetic analysis will help to identify the potential novel enzyme(s) and finally define the metabolic mechanism of oxalate accumulation and regulation in plants.
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Acknowledgements |
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We thank Dr Li-Zhen Tao (Department of Biochemistry and Molecular Biology, University of Massachusetts) for critically reading the manuscript. This work was supported by The National Science Foundation of China (No. 30270799, 30470152).
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Footnotes |
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* The first two authors contributed equally to this work.
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References |
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