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Plant Physiol. (1999) 120: 453-462 Ascorbic Acid Metabolism in Pea Seedlings. A Comparison of D-Glucosone, L-Sorbosone, and L-Galactono-1,4-Lactone as Ascorbate Precursors1
School of Biological Sciences, University of Exeter, Hatherly Laboratories, Prince of Wales Road, Exeter EX4 4PS, United Kingdom
L-Ascorbic acid (AsA) accumulates in pea (Pisum sativum L.) seedlings during germination, with the most rapid phase of accumulation coinciding with radicle emergence. Monodehydroascorbate reductase and dehydroascorbic acid reductase were active in the embryonic axes before AsA accumulation started, whereas AsA oxidase and AsA peroxidase activities increased in parallel with AsA. Excised embryonic axes were used to investigate the osone pathway of AsA biosynthesis, in which D-glucosone and L-sorbosone are the proposed intermediates. [U-14C]Glucosone was incorporated into AsA and inhibited the incorporation of [U-14C]glucose (Glc) into AsA. A higher D-glucosone concentration (5 mM) inhibited AsA accumulation. L-Sorbosone did not affect AsA pool size but caused a small inhibition in the incorporation of [U-14C]Glc into AsA. Oxidase and dehydrogenase activities capable of converting Glc or Glc-6-phosphate to glucosone were not detected in embryonic axis extracts. The osones are therefore unlikely to be physiological intermediates of AsA biosynthesis. L-Galactono-1,4-lactone, recently proposed as the AsA precursor (G.L. Wheeler, M.A. Jones, N. Smirnoff [1998] Nature 393: 365-369), was readily converted to AsA by pea embryonic axes. Although L-galactono-1,4-lactone did not inhibit [14C]Glc incorporation into AsA, this does not mean that it is not a precursor, because competition between endogenous and exogenous pools was minimized by its very small pool size and rapid metabolism.
AsA has an important role in the plant antioxidant system (Foyer,
1993
More recently, a pathway was proposed (Fig. 1) that involves the osones
D-glucosone and L-sorbosone and fits the
labeling data. The pathway predicts no inversion of the hexose
precursor, epimerization at C5, or conservation of the C6 hydroxymethyl
group. C-labeled glucosone and sorbosone are
incorporated into AsA by a number of plant tissues (Saito et al.,
1990 The pathways and enzymes involved in oxidizing AsA and reducing the
unstable oxidation products MDHA and DHA back to AsA are better
characterized than the biosynthetic pathway. AsA is readily oxidized to
the MDHA free radical as part of its antioxidant function. Oxidation is
catalyzed by APX and AOX. MDHA disproportionates to DHA and AsA
if it is not immediately reduced. DHA is unstable above pH 7.0 and
irreversibly delactonizes to 2,3-diketogulonate (Loewus, 1988 We used germinating pea (Pisum sativum L.) seedlings in the
present study, which are a good system with which to study AsA synthesis and turnover, because the embryonic axes accumulate AsA
rapidly and exhibit large increases in APX and AOX activity and lesser
increases in MDHAR and DHAR activity. Therefore, we used these axes to
investigate the incorporation of putative precursors into AsA.
Investigation of the metabolism of glucosone and sorbosone provided
evidence against a physiological role for the osone pathway. This
system has also provided evidence supporting the newly proposed AsA
biosynthetic pathway via GDP-Man, GDP-L-Gal,
L-Gal, and L-GAL (Wheeler
et al., 1998 Plant Culture
Enzyme Assays Embryonic axes were homogenized in the extraction medium described by Smirnoff and Colombé (1988) to extract MDHAR and DHAR. APX and
AOX were extracted as described by Conklin et al. (1997). The assays
were performed according to the methods of the following investigators:
MDHAR and APX, Smirnoff and Colombé (1988); DHAR, Nakano and
Asada (1980); and soluble and cell wall-bound AOX, Conklin et al.
(1997). Protein was measured by the method of Bradford (1976).
AsA Assay Tissue (0.1 g) was frozen in liquid nitrogen, ground to a powder, and extracted in 1 mL of 5% perchloric acid. The homogenate was centrifuged at 12,000g for 2 min, and the supernatant was neutralized with 5 M potassium carbonate using methyl orange indicator. The neutralized supernatant was centrifuged again and used for the AsA assay with AOX (Hewitt and Dickes, 1961).
Total AsA was assayed after first reducing the sample with homocysteine
(Okamura, 1980). The DHA concentration was calculated from the
difference between total and reduced AsA.
Feeding of Potential AsA Precursors to Embryonic Axes After dissection from seedlings, embryonic axes were incubated in glass vials on two layers of 1-cm-diameter filter-paper discs moistened with 0.4 mL of sterile nutrient medium plus the desired additions. To aid in gas exchange, the vials were enclosed in boxes with loosely fitting lids (with wet paper towels to minimize evaporation) and incubated in the dark at 20°C. When radioactive substrates were applied, the boxes were sealed to prevent the loss of 14CO2.Metabolism of 14C-Labeled Compounds by Embryonic Axes Radiolabeled compounds were fed to embryonic axes as described above. The compounds used were D-[U-14C]Glc (3 µCi; specific activity, 295 mCi mmol 1; Amersham) and
D-[U-14C]glucosone (3 µCi;
specific activity, 295 mCi mmol1; prepared as
described below). After incubation the samples were extracted in
perchloric acid, and the supernatant was separated into neutral,
acidic, and basic fractions by ion exchange. AsA was isolated in a
fraction eluted from a strong anion-exchange resin by 60 mM
formic acid, purified by HPLC, and its 14C
content was determined with a liquid scintillation counter. Full
details were given by Conklin et al. (1997).
Synthesis of D-Glucosone D-Glucosone (2-keto-Glc) was prepared from D-Glc by specific oxidation at C2 using pyranose-2-oxidase purified from the basidiomycete fungus Phanerochaete chrysosporium. The fungus was inoculated into a medium containing 2.4% (w/v) Glc, 1.8% (w/v) cornsteep powder (Merck, Rahway, NJ), and 0.18% (w/v) MgSO4·7H2O, pH 5.5, and grown in a shaking culture at 30°C for 11 d until reaching the stationary phase. The mycelium was harvested and homogenized in 50 mM potassium phosphate buffer, pH 7.0. Pyranose-2-oxidase activity was purified by ammonium sulfate precipitation (25% saturation), hydrophobic interaction chromatography (Phenyl-Sepharose, Pharmacia), and anion-exchange chromatography (DEAE-Sephacel, Pharmacia). The enzyme preparation was dialyzed against 20 mM Tris-HCl, pH 6.5, before use and assayed as described by Liu et al. (1983). The purified enzyme (5 units) was
incubated at 28°C with 100 mg of Glc in 4 mL of sterile, distilled
water with 12,500 units of catalase and 10 µL of an
antibiotic/antimycotic mixture (Sigma). After 24 h of
incubation, the reaction mixture was freeze dried.
Assay of Glucosone Formation by Embryonic Axis Extracts Embryonic axes were isolated 22 h after imbibition and homogenized in ice-cold 50 mM Hepes-KOH buffer, pH 7.0, containing 5 mM MgCl2, 0.15% Triton X-100, and 1 mM DTT. The homogenate was centrifuged at 12,000g for 2 min in a microcentrifuge. The supernatant was desalted on Sephadex G-25 (Pharmacia PD-10 columns) equilibrated with the homogenizing medium without Triton X-100. The eluent was used for assays. The reaction mixture contained, in a total volume of 100 µL, 50 µL of 50 mM Hepes-KOH, pH 6.0 or 7.0, 40 µL of desalted extract, 0.2 µCi of D-[U-14C]Glc, and 5 µL of an antibiotic/antimycotic mixture (Sigma). NAD(P) (0.1 mM) was added to determine the cofactor requirement. D-[U-14C]Glc-6-P was generated by adding 10 mM ATP and 6 units of hexokinase (Sigma) to the reaction mixture. After incubation for 2 to 4 h at 25°C, the reactions were stopped by the addition of 0.1 mL of isopropanol. After the sample was centrifuged, 10-µL aliquots were spotted onto a silica-gel TLC plate. The plates were developed with acetonitrile:0.1 M NH4Cl (7:3, v/v). The plate was scanned to detect 14C with a linear analyzer (Berthold Analytical, Gaithersburg, MD). Glc, Glc-6-P, and glucosone were detected on the plates with aniline/diphenylamine stain (Dawson et al., 1969). The reaction mixtures containing ATP and hexokinase were treated with 0.16 unit of alkaline phosphatase before precipitation to dephosphorylate the reaction products.
AsA Accumulation during Pea Seed Germination The AsA concentration in the embryonic axes and cotyledons of pea seeds was measured during the first week of germination in the dark. In all cases DHA constituted 10% to 20% of the total AsA pool; therefore, all results refer to the total. The dry seeds contained very little AsA. The AsA pool of the embryonic axes started to increase 10 h after imbibition, and between 24 and 50 h the concentration increased rapidly (Fig. 2B). The rapid increase corresponded to the emergence of the radicle (Fig. 2A). AsA concentration decreased 50 h after imbibition (Fig. 2B), corresponding to the emergence of the plumule and a phase of rapid growth in fresh and dry weight of the embryonic axis (Fig. 2A), suggesting that the rate of AsA synthesis does not increase to keep pace with growth at this point. The AsA concentration in the cotyledons also increased between 24 and 50 h after imbibition (Fig. 2B); however, the cotyledons always contained considerably less AsA than the embryonic axes (Fig. 2B).
Activity of AsA-Metabolizing Enzymes in Embryonic Axes during Germination APX and AOX activities were both undetectable 10 h after imbibition and then increased rapidly until 40 h after imbibition (Fig. 3). APX and AOX activity followed a time course similar to that of the increase in AsA concentration during germination (Figs. 2B and 3, A and B). The pattern of MDHAR and DHAR activity during germination differed from that of APX and AOX in that appreciable activity was detected early in germination (Fig. 3, C and D). The contrasting patterns of APX/AOX and MDHAR/DHAR activity suggest that the embryonic axis has a relatively high capacity to regenerate oxidized AsA from a very early stage of germination but that the AsA-oxidizing enzymes have very low activity until AsA accumulation begins.
Characteristics of AsA Accumulation by Isolated Embryonic Axes Embryonic axes retained their ability to synthesize and accumulate AsA after the removal of their cotyledons. The total AsA content doubled during the 6 h after embryonic axes were excised from seedlings 24 h after imbibition (Table I), even though 50% of the soluble carbohydrate pool was lost during this time (data not shown). The axes also grew by water uptake during this period (Table I). A range of sugars was supplied to isolated embryonic axes to assess the role of carbohydrate supply in AsA accumulation. Suc, D-Glc, D-Fru, D-Man, L-Man, D-Gal, and L-rhamnose (5-25 mM for periods of 6-24 h) had no effect on the AsA pool size or on the expansion growth of the axes (data not shown). Isolated embryonic axes, therefore, are a suitable system with which to investigate AsA metabolism, because they can accumulate AsA at a rate (0.1-0.2 µmol h1 g1 fresh weight) comparable to that
of intact seedlings in the absence of an exogenous carbon source.
The Effect of Putative AsA Precursors on AsA Pool Size In the absence of an influence of sugars, a range of more specific AsA precursors was fed to the embryonic axes (Table I). L-GAL increased the AsA pool. L-GUL, the AsA precursor in animals (Burns, 1967), caused a small and statistically
insignificant increase in AsA pool size; this effect was consistently
noted in several experiments. D-Glucosone and
L-sorbosone have been suggested as AsA precursors in plants
(Saito et al., 1990). When fed to embryonic axes, up to 5 mM L-sorbosone had no effect on AsA pool size
or expansion growth (Table I). In contrast, 5 mM L-glucosone prevented AsA accumulation (Table I). At 25 mM, glucosone also inhibited embryonic axis expansion
growth, suggesting that it is toxic at high concentrations (Table I).
The effect of L-GAL and glucosone together was also
investigated (Table I). The conversion of L-GAL was
apparently inhibited but not prevented by glucosone. Because the
increase after the addition of both compounds (1.17 µmol
g1 compared with no addition) was similar to
the difference between the calculated net change caused by either
compound alone (1.08 µmol g1), it appears
that glucosone does not interfere directly with the oxidation of
L-GAL to AsA.
Comparison of D-[U-14C]Glc and D-[U-14C]Glucosone Incorporation into AsA Glucosone metabolism was further investigated by following the metabolism of D-[U-14C]Glc and D-[U-14C]glucosone for 2, 4, and 6 h. The labeled glucosone contained no carrier, and its concentration was 25 µM. At this level the glucosone did not inhibit AsA accumulation in the embryonic axes (Table II) compared with unlabeled glucosone at 5 mM (Table I). The uptake of labeled glucosone was 20% to 35% slower than that of Glc (Table II). The tissue was extracted and fractionated by ion-exchange chromatography into major classes of compounds. The distribution of label between these fractions is compared in Table III. Compared with [14C]Glc, more label from glucosone remained in the soluble fraction and less label was incorporated into insoluble material (cell walls and protein) and respired as CO2. Within the soluble fraction the same proportion was incorporated into acidic compounds after metabolism of both compounds; however, a much smaller proportion of label from glucosone was found in the basic fraction (amino acids), whereas a much higher proportion was found in neutral compounds. A detailed study of the products of glucosone metabolism is not presented here, but HPLC analysis of the acidic fraction showed label in compounds with the same retention time as malic and citric acids.
The Effect of Putative Precursors on the Synthesis of AsA from [U-14C]Glc Saito et al. (1990) carried out competition experiments in which
unlabeled osones were allowed to compete with
[14C]Glc for incorporation into AsA over
24 h. Both compounds competed, suggesting that they could be
intermediates in AsA biosynthesis. Similar experiments were carried out
with embryonic axes but with much shorter labeling times (2 h).
D-Glucosone, L-sorbosone, and L-GAL
were fed to embryonic axes, and their effect on the incorporation of
[U-14C]Glc into AsA was determined (Table
IV). None of the precursors affected
[14C]Glc uptake or its incorporation into the
major fractions (data not shown). D-Glucosone inhibited
synthesis of AsA from Glc and decreased the pool size of AsA in the
embryonic axes. L-Sorbosone decreased the proportion of
[14C]Glc incorporated into AsA from 1.76% to
1.54%. Although statistically significant, this effect was very small.
L-GAL increased the AsA pool size but did not decrease
[14C]Glc incorporation into AsA. It was not
possible to detect a peak corresponding to L-GAL after HPLC
of the neutral fraction from the embryonic axes that had been fed with
L-GAL.
D-Glucosone Formation by Embryonic Axis Extracts A predicted requirement for the osone pathway is oxidation of a hexose at C2 to form glucosone. Basidiomycete fungi have a pyranose-2-oxidase that forms glucosone from Glc (Daniel et al., 1994),
and activity has also been reported in a red seaweed (Bean and Hassid,
1956). The possibility that Glc or Glc-6-P could be oxidized to
glucosone was investigated by incubating
14C-labeled Glc with desalted pea embryonic axis
extracts and a range of cofactors. TLC separation of the reaction
products revealed an extremely small radioactive peak with an
RF similar to that of glucosone (Fig.
4A). It did not increase with increased
reaction time (Fig. 4B) and was not affected by the presence of
NAD+ or NADP+ (Fig. 4C).
Because the peak was so small and did not decrease in a time-dependent
manner, there was no strong evidence that it was glucosone. Inclusion
of pyranose-2-oxidase from P. chrysosporium in the reaction
converted all of the [14C]Glc to glucosone
(Fig. 4D), indicating that glucosone is stable under the assay
conditions used. [14C]Glc-6-P was generated by
inclusion of ATP and hexokinase. The reaction products were treated
with alkaline phosphatase to dephosphorylate them and then separated by
TLC. No radioactive peaks corresponding to glucosone were found (data
not shown). Therefore, the embryonic axis extracts assayed under these
conditions did not exhibit oxidase or dehydrogenase activity toward C2
of Glc or Glc-6-P.
Development of the AsA System during Pea Seed Germination Pea seeds contain low concentrations of AsA and DHA. Similarly, low concentrations have been found in bean seeds at maturity (Arrigoni et al., 1992) and in a wide range of other species (N. Smirnoff,
unpublished results). AsA is oxidized and DHA is destroyed during the
desiccation phase of seed development (Arrigoni et al., 1992), which
would explain the lack of AsA in seeds. There is a lag of 20 h
after imbibition before the AsA content of the embryonic axis increases
rapidly. This increase just precedes radicle emergence. The embryonic
axes have very low APX and AOX activity before 20 h. This
corresponds with the lack of AOX activity in developing bean seeds
and the loss of APX activity during desiccation (Arrigoni et al.,
1992). These enzymes have a coordinated increase in their activity in
parallel with AsA content, which suggests that the expression of the
AsA system during germination is highly controlled. The correlation
between growth of the radicle and AOX activity supports the suggestion
that wall AOX activity is related to cell wall expansion (Smirnoff,
1996).
The Osones as Intermediates of AsA Biosynthesis in Pea Embryonic Axes The osone pathway was proposed to explain the lack of inversion of the Glc carbon skeleton during AsA synthesis (Loewus et al., 1990;
Saito et al., 1990). Neither glucosone nor sorbosone has been detected
in plant extracts, and therefore, if they are present their
concentration is very low. Although L-GAL was proposed as a
precursor many years ago, it has been assumed that it could be
synthesized from Glc only by inversion of the carbon skeleton. However,
the recently proposed biosynthetic pathway of AsA in plants suggests
that the immediate precursor is L-GAL, which can be
synthesized from Glc without inversion (Fig. 1; Wheeler et al., 1998).
Therefore, it remains to be explained how L-glucosone and
L-sorbosone can act as AsA precursors. The alternative
possibilities are that the osones provide a second minor
AsA-biosynthesis pathway or that they are analogs of intermediates in
the L-GAL pathway.
L-GAL as an AsA Precursor In the present study, pea embryonic axes converted L-GAL to AsA, as has been found in all higher plant tissues examined thus far. This reaction is catalyzed by a mitochondrial L-GAL dehydrogenase (Mapson and Breslow, 1958; Oba et al.,
1994, 1995; Matsuda et al., 1995; Østergaard et al., 1997).
L-GUL, the AsA precursor in animals, had a small effect on
the AsA pool in pea. Purified cauliflower and spinach L-GAL
dehydrogenase has little or no activity toward L-GUL
(Matsuda et al., 1995; Østergaard et al., 1997). However, partially
purified potato tuber L-GAL dehydrogenase supported L-GUL oxidation at a rate that was 20% of that of
L-GAL (Oba et al., 1994), suggesting that there is
interspecific variability in enzyme specificity.
* Corresponding author; e-mail n.smirnoff{at}exeter.ac.uk; fax 44-1392-263-700. Received January 26, 1999;
accepted February 22, 1999.
Abbreviations: AOX, ascorbate oxidase. APX, ascorbate peroxidase. AsA, L-ascorbic acid. DHA, dehydroascorbic acid. DHAR, dehydroascorbic acid reductase. L-GAL, L-galactono-1,4-lactone. L-GUL, L-gulono-1,4-lactone. MDHA, monodehydroascorbate. MDHAR, monodehydroascorbate reductase.
We are grateful to Dr. Jack Fisher for providing the P. chrysosporium isolate and for advice concerning its culture. The L-sorbosone was a gift from Nippon-Roche. Marjorie Raymond provided technical support.
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  H. Gautier, C. Massot, R. Stevens, S. Serino, and M. Genard Regulation of tomato fruit ascorbate content is more highly dependent on fruit irradiance than leaf irradiance Ann. Bot., February 1, 2009; 103(3): 495 - 504. [Abstract] [Full Text] [PDF]
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I. Pateraki, M. Sanmartin, M. S. Kalamaki, D. Gerasopoulos, and A. K. Kanellis Molecular characterization and expression studies during melon fruit development and ripening of L-galactono-1,4-lactone dehydrogenase J. Exp. Bot., August 1, 2004; 55(403): 1623 - 1633. [Abstract] [Full Text] [PDF]
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J. A. Running, R. P. Burlingame, and A. Berry The pathway of L-ascorbic acid biosynthesis in the colourless microalga Prototheca moriformis J. Exp. Bot., August 1, 2003; 54(389): 1841 - 1849. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
) and embryonic axes (
) during
germination are shown. First arrow in A indicates radicle emergence;
second arrow indicates plumule emergence. Values are means ± SD (n = 3).
-lactone dehydrogenase.
Biochem J
68:
395-406