Biosynthesis and metabolism of ascorbic acid in plants

The biosynthesis of L‐ascorbic acid in plants differs from that encountered in ascorbic acid‐synthesizing animals. Enzymic details are sparse, but in vivo studies with tracers clearly establish the stereochemical detail of both processes. Examples of each process are found in separate classes of algae. Plants utilize L‐ascorbic acid as the carbon source for the biosynthesis of two important plant acids, oxalic acid and L‐tartaric acid. Here, cleavage of L‐ascorbic acid between carbons 2 and 3 releases the 2 and 4 carbon intermediates. A second L‐tartaric acid‐synthesizing process peculiar to vitaceous plants, i.e., grape, cleaves ascorbic acid between carbons 4 and 5. The physiological significance of these metabolic interconversions is discussed. Other metabolic processes such as the oxidation/reduction properties of L‐ascorbic acid are also considered.     

On the brain ascorbic acid and its importance in metabolism of biogenic amines.

Practically no work has been done on the role of ascorbic acid in brain, presumably assuming the homogeneity of the vitamin in the brain. On the contrary, the regional distribution of ascorbic acid differs very much and could be altered by conditions enhancing the neurotransmitter concentration in the different regions. The importance of ascorbic acid in the metabolism of the putative neurotransmitters has been surveyed and a hitherto unexplored area of the role of vitamin C has been given importance in this article.     

Concerning the presence and formation of ascorbic acid in yeasts

The selective, sensitive method of analysis of ascorbic acid by high performance liquid chromatography with electrochemical detection (HPLC/EC) has been used to determine the ascorbic acid content of cell extracts from yeasts grown in glucose-free medium, 0.3 M D-glucose, and 0.112 M L-galactono-1,4-lactone. Saccharomyces cerevisiae (strain G-25 and its tetraploid) and a commercial baker's yeast contained less than 2 μg ascorbic acid g^?1 wet wt. of cells when grown for 22 h in glucose-free medium. In 0.3 M D-glucose, only the commercial baker's yeast gave a slight increase (2–50 μg g^?1 wet wt. in 22 h). In 0.112 M L-galactono-1,4-lactone, all three strains produced ascorbic acid (372–587 μg g^?1 wet wt. in 22 h). Lypomyces starkeyi, a species previously reported to contain a significant amount of ascorbic acid (Heick et al., Can. J. Biochem., 47 (1972) 752), was essentially devoid of ascorbic acid under all three conditions of incubation although it did contain an HPLC/EC reactive peak (R_T = 0.87 relative to ascorbic acid) that was readily oxidized by charcoal in the presence of oxygen. The identity of this new compound remains to be determined.     

Recent advances in the role and biosynthesis of ascorbic acid in plants

The past few years have provided many advances in the role and biosynthesis of L -ascorbic acid (AsA) in plants. There is an increasing body of evidence confirming that AsA plays an important role in the detoxification of reactive oxygen species. The role of AsA in photoprotection has been confirmed in vivo with the use of Arabidopsis mutants. A player in the defence against reactive oxygen species, AsA peroxidase, has been extensively studied at the molecular level, and regulation of this key enzymatic activity appears to occur at several levels. As a cofactor in the hydroxylation of prolyl and lysl-residues by peptidyl-prolyl and -lysyl hydroxylases, AsA plays a part in cell wall synthesis, defence, and possibly cell division. The maintenance of reduced levels of AsA appears to be highly regulated, involving the interplay of both monodehydroascorbate and dehydroascorbate reductases and possibly auxin. A major breakthrough in plant AsA biosynthesis has been made recently, and strong biochemical and genetic evidence suggest that GDP-mannose and L -galactose are key substrates. In addition, evidence for an alternative AsA biosynthetic pathway(s) exists and awaits additional scrutiny. Finally, newly described Arabidopsis mutants deficient in AsA will further increase our understanding of AsA biosynthesis     

Progress in manipulating ascorbic acid biosynthesis and accumulation in plants

l -Ascorbic acid (vitamin C) is synthesized from hexose sugars. It is an antioxidant and redox buffer, as well as an enzyme cofactor, so it has multiple roles in metabolism and in plant responses to abiotic stresses and pathogens. Plant-derived ascorbate also provides the major source of vitamin C in the human diet. An understanding of how ascorbate metabolism is controlled should provide a basis for engineering or otherwise manipulating its accumulation. Biochemical and molecular genetic evidence supports synthesis from GDP- d -mannose via l -galactose ( d -Man/ l -Gal pathway) as a significant source of ascorbate. More recently, evidence for pathways via uronic acids has been obtained: overexpression of myo -inositol oxygenase, d -galacturonate reductase and l -gulono-1,4-lactone oxidase all increase leaf ascorbate concentration. Interestingly, this has proved more effective in pathway engineering than overexpressing various d -Man/ l -Gal pathway genes. Ascorbate oxidation generates the potentially unstable dehydroascorbate, and the overexpression of glutathione-dependent dehydroascorbate reductase has resulted in increased ascorbate. Ascorbate is catabolized to products such as oxalate, l -threonate and l -tartrate. The enzymes involved have not been identified, so catabolism is not yet amenable to manipulation. In the examples of pathway engineering so far, the increase in ascorbate has been modest on an absolute or proportional basis. Therefore, a deeper understanding of ascorbate metabolism is needed to achieve larger increases. Identifying genes that control ascorbate accumulation by techniques such as analysis of quantitative trait loci (QTL) or activation tagging may hold promise, particularly if regulatory genes can be identified.     

Alicyclic acid metabolism in plants

When the epicotyls of etiolated pea seedlings were fed with 40 mM potassium quinate solution in the dark for 24 hr, a marked accumulation of shikimic acid occurred in the tissue. This effect was much more pronounced in epicotyls preliminarily starved in a phosphate solution for 24 hr. On the other hand, supplying shikimate to the epicotyls brought about no significant accumulation of quinic acid. Tracer studies with¹⁴C-shikimate have shown that, in the epicotyls, shikimic acid was rapidly metabolized and fairly high radioactivities were observed in the amino acid fraction. However, feeding of¹⁴C-shikimate together with unlabeled alicyclic acids resulted in a reduction of shikimate utilization. When³H-quinate was fed to the epicotyls, radioactivities were retained mostly in an acidic fraction, indicating the sluggish conversion of quinic acid. In starved epicotyls, however, nearly half of the absorbed radioactivity was consumed. In tracer experiments the conversion of quinate to shikimate was clearly observed, whereas the reverse reaction was not. From these findings the metabolic role of quinic acid in quinate-less pea seedlings is discussed.     

Cytochrome oxidase and ascorbic acid oxidase activities in cereal plants

Cytochrome oxidase and ascorbic acid oxidase activities were investigated in rye, wheat, barley and oat plants. The variations in the activity of both enzymes was followed in the course of the initial 28 days of growth, as well as at the phase of milk ripeness, namely in the cytoplasmic and mitochondrial cell fractions of roots, leaves and spikes. Both enzymes were active in all measurements. Cytochrome oxidase mostly exhibited a higher activity than ascorbio acid oxidase. The activity of the former enzyme was substantially higher in the mitochondrial fraction of leaves, roots and spikes of the four experimental plants in comparison with the cytoplasmic fraction. On the contrary, the ascorbic acid oxidase activity varied in both cell fractions according to the plant species, organ and growth phase. The variations in the activity of both enzymes exhibited on the whole a course similar to that of the respiration rate. During the first 14 to 21 days of growth the enzyme activities increased up to the maximum. This was thon followed at first by a rapid, later on by a slow decrease. The course of variations in the enzyme activities was, with certain exceptions, alike in all the four plant species investigated.     

Impaired ascorbic acid metabolism in streptozotocin-induced diabetic rats

Ascorbic acid (AA) metabolism in streptozotocin (STZ)-induced diabetic rats was determined by examining urinary excretion, renal reabsorption, reductive regeneration, and biosynthesis of AA at 3 and 14 days after STZ administration. AA concentrations in the plasma, liver, and kidney of the diabetic rats were significantly lower than those of controls on d 3, and decreased further as the diabetic state continued. Hepatic AA regeneration significantly decreased in the diabetic rats on d 3 in spite of increased gene expressions of AA regenerating enzymes and was further reduced on d 14. Hepatic activity of L-gulono-gamma-lactone oxidase, a terminal enzyme of hepatic AA biosynthesis, also decreased significantly on d 3 and decreased further on d 14. Urinary excretion of AA was significantly increased on d 3, with an increase in urine volume but no change in gene expressions of renal AA transporters (SVCT1 and SVCT2). Urinary excretion of AA was normalized on d 14. The results suggest that impaired hepatic and renal regeneration, as well as increased urinary excretion and impaired hepatic biosynthesis of AA, contributed to the decrease in AA in plasma and tissues of STZ-induced diabetic rats.     

Changes in catecholamine metabolism by ascorbic acid deficiency in spontaneously hypertensive rats unable to synthesize ascorbic acid

We have previously reported the establishment of a novel rat strain, SHR-od, with both spontaneous hypertension and a defect of ascorbic acid biosynthesis. Blood pressure in mature SHR-od fed an ascorbic acid-supplemented diet is over 190-200 mmHg, while it decreased to around 120 mmHg at 4-5 weeks after the cessation of ascorbic acid supplementation. With regard to possible mechanisms of blood pressure lowering, we focused on catecholamine synthesis in adrenal glands, since catecholamine is a major factor for blood pressure regulation and ascorbic acid is a co-factor of dopamine beta-hydroxylase (DBH) in catecholamine biosynthesis. Male SHR-od (25-week-old) and normotensive ODS rats with a defect in ascorbic acid biosynthesis (25-week-old) were fed a Funabashi-SP diet with or without ascorbic acid (300 mg/kg diet) for 28 days or 35 days. In SHR-od, systolic blood pressure (191 +/- 6 mmHg) began to decrease from day 21 in the ascorbic acid-deficient group, whereas no significant difference was found in ODS rats. In spite of significant lowering of blood pressure, no significant differences were found in catecholamine levels in serum, adrenal glands and brain on day 28. On day 35, however, urinary excretion of norepinephrine and epinephrine in the ascorbic acid-deficient SHR-od were higher at 490% (P < 0.05) and 460% (P < 0.05) of the respective control. Serum catecholamine concentrations and the adrenal catecholamine content tended to be higher in the ascorbic acid-deficient SHR-od than the control of SHR-od and reached to similar level in ODS rats. The administration of ascorbic acid (intraperitoneal injection, 60 mg ascorbic acid/kg body weight, once a day) to the ascorbic acid-deficient SHR-od restored blood pressure to the range 180-190 mmHg within two days. These findings indicate that ascorbic acid deficiency affects catecholamine metabolism in the adrenal glands of SHR-od in response to blood pressure lowering, suggesting catecholamines are not involved in the mechanism for the remarkable reduction in blood pressure in response to ascorbic acid deficiency.     

Peroxisomal metabolism of propionic acid and isobutyric acid in plants

The subcellular sites of branched-chain amino acid metabolism in plants have been controversial, particularly with respect to valine catabolism. Potential enzymes for some steps in the valine catabolic pathway are clearly present in both mitochondria and peroxisomes, but the metabolic functions of these isoforms are not clear. The present study examined the possible function of these enzymes in metabolism of isobutyryl-CoA and propionyl-CoA, intermediates in the metabolism of valine and of odd-chain and branched-chain fatty acids. Using (13)C NMR, accumulation of beta-hydroxypropionate from [2-(13)C]propionate was observed in seedlings of Arabidopsis thaliana and a range of other plants, including both monocots and dicots. Examination of coding sequences and subcellular targeting elements indicated that the completed genome of A. thaliana likely codes for all the enzymes necessary to convert valine to propionyl-CoA in mitochondria. However, Arabidopsis mitochondria may lack some of the key enzymes for metabolism of propionyl-CoA. Known peroxisomal enzymes may convert propionyl-CoA to beta-hydroxypropionate by a modified beta-oxidation pathway. The chy1-3 mutation, creating a defect in a peroxisomal hydroxyacyl-CoA hydrolase, abolished the accumulation of beta-hydroxyisobutyrate from exogenous isobutyrate, but not the accumulation of beta-hydroxypropionate from exogenous propionate. The chy1-3 mutant also displayed a dramatically increased sensitivity to the toxic effects of excess propionate and isobutyrate but not of valine. (13)C NMR analysis of Arabidopsis seedlings exposed to [U-(13)C]valine did not show an accumulation of beta-hydroxypropionate. No evidence was observed for a modified beta-oxidation of valine. (13)C NMR analysis showed that valine was converted to leucine through the production of alpha-ketoisovalerate and isopropylmalate. These data suggest that peroxisomal enzymes for a modified beta-oxidation of isobutyryl-CoA and propionyl-CoA could function for metabolism of substrates other than valine.