Ascorbic acid in plants: biosynthesis and function

Ascorbic acid (vitamin C) is an abundant component of plants. It reaches a concentration of over 20 mM in chloroplasts and occurs in all cell compartments, including the cell wall. It has proposed functions in photosynthesis as an enzyme cofactor (including synthesis of ethylene, gibberellins and anthocyanins) and in control of cell growth. A biosynthetic pathway via GDP-mannose, GDP-L-galactose, L-galactose, and L-galactono-1,4-lactone has been proposed only recently and is supported by molecular genetic evidence from the ascorbate-deficient vtc 1 mutant of Arabidopsis thaliana. Other pathways via uronic acids could provide minor sources of ascorbate. Ascorbate, at least in some species, is a precursor of tartrate and oxalate. It has a major role in photosynthesis, acting in the Mehler peroxidase reaction with ascorbate peroxidase to regulate the redox state of photosynthetic electron carriers and as a cofactor for violaxanthin de-epoxidase, an enzyme involved in xanthophyll cycle-mediated photoprotection. The hypersensitivity of some of the vtc mutants to ozone and UV-B radiation, the rapid response of ascorbate peroxidase expression to (photo)-oxidative stress, and the properties of transgenic plants with altered ascorbate peroxidase activity all support an important antioxidative role for ascorbate. In relation to cell growth, ascorbate is a cofactor for prolyl hydroxylase that posttranslationally hydroxylates proline residues in cell wall hydroxyproline-rich glycoproteins required for cell division and expansion. Additionally, high ascorbate oxidase activity in the cell wall is correlated with areas of rapid cell expansion. It remains to be determined if this is a causal relationship and, if so, what is the mechanism. Identification of the biosynthetic pathway now opens the way to manipulating ascorbate biosynthesis in plants, and, along with the vtc mutants, this should contribute to a deeper understanding of the proposed functions of this multifaceted molecule.     

Ascorbic Acid in Plants: Biosynthesis and Function

ABSTRACT

Ascorbic acid (vitamin C) is an abundant component of plants. It reaches a concentration of over 20 mM in chloroplasts and occurs in all cell compartments, including the cell wall. It has proposed functions in photosynthesis as an enzyme cofactor (including synthesis of ethylene, gibberellins and anthocyanins) and in control of cell growth. A biosynthetic pathway via GDP-mannose, GDP-L-galactose, L-galactose, and L-galactono-1,4-lactone has been proposed only recently and is supported by molecular genetic evidence from the ascorbate-deficient vtcl mutant of Arabidopsis thaliana. Other pathways via uronic acids could provide minor sources of ascorbate. Ascorbate, at least in some species, is a precursor of tartrate and oxalate. It has a major role in photosynthesis, acting in the Mehler peroxidase reaction with ascorbate peroxidase to regulate the redox state of photosynthetic electron carriers and as a cofactor for violaxanthin de-epoxidase, an enzyme involved in xanthophyll cycle-mediated photoprotection. The hypersensitivity of some of the vtc mutants to ozone and UV-B radiation, the rapid response of ascorbate peroxidase expression to (photo)-oxidative stress, and the properties of transgenic plants with altered ascorbate peroxidase activity all support an important antioxidative role for ascorbate. In relation to cell growth, ascorbate is a cofactor for prolyl hydroxylase that posttranslationally hydroxylates proline residues in cell wall hydroxyproline-rich glycoproteins required for cell division and expansion. Additionally, high ascorbate oxidase activity in the cell wall is correlated with areas of rapid cell expansion. It remains to be determined if this is a causal relationship and, if so, what is the mechanism. Identification of the biosynthetic pathway now opens the way to manipulating ascorbate biosynthesis in plants, and, along with the vtc mutants, this should contribute to a deeper understanding of the proposed functions of this multifacetted molecule.     

Ascorbate deficiency can limit violaxanthin de-epoxidase activity in vivo

As a response to high light, plants have evolved non-photochemical quenching (NPQ), mechanisms that lead to the dissipation of excess absorbed light energy as heat, thereby minimizing the formation of dangerous oxygen radicals. One component of NPQ is pH dependent and involves the formation of zeaxanthin from violaxanthin. The enzyme responsible for the conversion of violaxanthin to zeaxanthin is violaxanthin de-epoxidase, which is located in the thylakoid lumen, is activated by low pH, and has been shown to use ascorbate (vitamin C) as its reductant in vitro. To investigate the effect of low ascorbate levels on NPQ in vivo, we measured the induction of NPQ in a vitamin C-deficient mutant of Arabidopsis, vtc2-2. During exposure to high light (1,500 micromol photons m(-2) s(-1)), vtc2-2 plants initially grown in low light (150 micromol photons m(-2) s(-1)) showed lower NPQ than the wild type, but the same quantum efficiency of photosystem II. Crosses between vtc2-2 and Arabidopsis ecotype Columbia established that the ascorbate deficiency cosegregated with the NPQ phenotype. The conversion of violaxanthin to zeaxanthin induced by high light was slower in vtc2-2, and this conversion showed saturation below the wild-type level. Both the NPQ and the pigment phenotype of the mutant could be rescued by feeding ascorbate to leaves, establishing a direct link between ascorbate, zeaxanthin, and NPQ. These experiments suggest that ascorbate availability can limit violaxanthin de-epoxidase activity in vivo, leading to a lower NPQ. The results also demonstrate the interconnectedness of NPQ and antioxidants, both important protection mechanisms in plants.     

The physiological roles and metabolism of ascorbate in chloroplasts

Ascorbate is a multifunctional metabolite in plants. It is essential for growth control, involving cell division and cell wall synthesis and also involved in redox signaling, in the modulation of gene expression and regulation of enzymatic activities. Ascorbate also fulfills crucial roles in scavenging reactive oxygen species, both enzymatically and nonenzymatically, a well‐established phenomenon in the chloroplasts stroma. We give an overview on these important physiological functions and would like to give emphasis to less well‐known roles of ascorbate, in the thylakoid lumen, where it also plays multiple roles. It is essential for photoprotection as a cofactor for violaxanthin de‐epoxidase, a key enzyme in the formation of nonphotochemical quenching. Lumenal ascorbate has recently also been shown to act as an alternative electron donor of photosystem II once the oxygen‐evolving complex is inactivated and to protect the photosynthetic machinery by slowing down donor‐side induced photoinactivation; it is yet to be established if ascorbate has a similar role in the case of other stress effects, such as high light and UV‐B stress. In bundle sheath cells, deficient in oxygen evolution, ascorbate provides electrons to photosystem II, thereby poising cyclic electron transport around photosystem I. It has also been shown that, by supporting linear electron transport through photosystem II in sulfur‐deprived Chlamydomonas reinhardtii cells, in which oxygen evolution is largely inhibited, externally added ascorbate enhances hydrogen production. For fulfilling its multiple roles, Asc has to be transported into the thylakoid lumen and efficiently regenerated; however, very little is known yet about these processes.     

Membrane barriers and Mehler-peroxidase reaction limit the ascorbate available for violaxanthin de-epoxidase activity in intact chloroplasts

The presence of an acidic lumen and the xanthophylls, zeaxanthin and antheraxanthin, are minimal requirements for induction of non-radiative dissipation of energy in the pigment bed of Photosystem II. We recently reported that ascorbate, which is required for formation for these xanthophylls, also can mediate the needed lumen acidity through the Mehler-peroxidase reaction [Neubauer and Yamamoto (1992) Plant Physiol 99: 1354–1361]. It is demonstrated that in non-CO₂-fixing intact chloroplasts and thylakoids of Lactuca sativa, L. c.v. Romaine, the ascorbate available to support de-epoxidase activity is influenced by membrane barriers and the ascorbate-consuming Mehler-peroxidase reaction. In intact chloroplasts, this results in biphasic kinetic behavior for light-induced de-epoxidation. The initial relatively high activity is due to ascorbate preloaded into the thylakoid before light-induction and the terminal low activity due to limiting ascorbate from the effects of chloroplast membranes barriers and a light-dependent process. A five-fold difference between the initial and final activities was observed for light-induced de-epoxidation in chloroplasts pre-incubated with 120 mM ascorbate for 40 min. The light-dependent activity is ascribed to the competitive use of ascorbic acid by ascorbate peroxidase in the Mehler-peroxidase reaction. Thus, stimulating ascorbic peroxidase with H₂O₂ transiently inhibited de-epoxidase activity and concomitantly increased photochemical quenching. Also, the effects inhibiting ascorbate peroxidase with KCN, and the K_M values for ascorbate peroxidase and violaxanthin de-epoxidase of 0.36 and 3.1 mM, respectively, support this conclusion. These results indicate that regulation of xanthophyll-dependent non-radiative energy dissipation in the pigment bed of Photosystem II is modulated not only by lumen acidification but also by ascorbate availability.Abbreviations APO ascorbate peroxidase - MP Mehler ascorbate-peroxidase - NIG nigericin - NPQ non-photochemical quenching - F_o dark fluorescence - F fluorescence at any time - F_M maximal fluorescence of the (dark) non-energized state - F_M maximal fluorescence of the energized state - q_P coefficient for photochemical fluorescence quenching - VDE violaxanthin de-epoxidase - k first-order rate constant for violaxanthin de-epoxidase activity     

L-ascorbic acid metabolism in the ascorbate-deficient arabidopsis mutant vtc1.

The biosynthesis of L-ascorbic acid (vitamin C) is not well understood in plants. The ozone-sensitive Arabidopsis thaliana mutant vitamin c-1 (vtc1; formerly known as soz1) is deficient in ascorbic acid, accumulating approximately 30% of wild-type levels. This deficiency could result from elevated catabolism or decreased biosynthesis. No differences that could account for the deficiency were found in the activities of enzymes that catalyze the oxidation or reduction of ascorbic acid. The absolute rate of ascorbic acid turnover is actually less in vtc1 than in wild type; however, the turnover rate relative to the pool of ascorbic acid is not significantly different. The results from [U-14C]Glc labeling experiments suggest that the deficiency is the result of a biosynthetic defect: less L-[14C]ascorbic acid as a percentage of total soluble 14C accumulates in vtc1 than in wild type. The feeding of two putative biosynthetic intermediates, D-glucosone and L-sorbosone, had no positive effect on ascorbic acid levels in either genotype. The vtc1 defect does not appear to be the result of a deficiency in L-galactono-1,4-lactone dehydrogenase, an enzyme able to convert L-galactono-1,4-lactone to ascorbic acid.     

Mehler-peroxidase reaction mediates zeaxanthin formation and zeaxanthin-related fluorescence quenching in intact chloroplasts

Induction of zeaxanthin formation and the associated nonphotochemical quenching in iodoacetamide-treated, non-CO₂-fixing intact chloroplasts of Lactuca sativa L. cv Romaine is reported. The electron transport needed to generate the required ΔpH for zeaxanthin formation and nonphotochemical quenching are ascribed to the Mehler-ascorbate peroxidase reaction. KCN, an inhibitor of ascorbate peroxidase, significantly affected these activities without affecting linear electron transport to methyl viologen or violaxanthin deepoxidase activity. At 1 millimolar KCN, zeaxanthin formation and ΔpH were inhibited 60 and 55%, respectively, whereas ascorbate peroxidase activity was inhibited almost totally. The KCN-resistant activity, which apparently was due to electron transport mediated by the Mehler reaction alone, however, was insufficient to support a high level of nonphotochemical quenching. We suggest that in vivo, as CO₂ fixation becomes limiting, the Mehler-peroxidase reaction protects photosystem II against the excess light by supporting the electron transport needed for zeaxanthin-dependent nonphotochemical quenching and concomitantly scavenging H₂O₂. Ascorbate is essential for this process to occur.     

The biosynthesis of erythroascorbate in Saccharomyces cerevisiae and its role as an antioxidant

This study investigated the ability of the yeast Saccharomyces cerevisiae to synthesize ascorbate and its 5-carbon analogue erythroascorbate from a variety of precursors, and their importance as antioxidants in this organism. Studies of ascorbate and analogues in micro-organisms have been reported previously, but their function as antioxidants have been largely ignored. Ascorbate and erythroascorbate concentrations in yeast extracts were measured spectrophotometrically, and their levels and identity were checked using liquid chromatography-electrospray mass spectrometry. The yeast was readily able to synthesize ascorbate from L-galactono-1,4-lactone or erythroascorbate from D-arabinose and D-arabino-1,4-lactone, whereas L-gulono-1,4-lactone was a much poorer substrate for ascorbate biosynthesis. In untreated cells, the concentration of ascorbate-like compounds was below the level of detection of the methods of analysis used in this study (approximately 0.1 mM). Intracellular ascorbate and erythroascorbate were oxidized at high concentrations of tert-butylhydroperoxide, but not hydrogen peroxide. Their synthesis was not increased in response to low levels of stress, however, and preloading with erythroascorbate did not protect glutathione levels during oxidative stress. This study provides new information on the metabolism of ascorbate and erythroascorbate in S. cerevisiae, and suggests that erythroascorbate is of limited importance as an antioxidant in S. cerevisiae.     

BOTANICAL BRIEFING: The Function and Metabolism of Ascorbic Acid in Plants

Ascorbate is a major metabolite in plants. It is an antioxidantand, in association with other components of the antioxidantsystem, protects plants against oxidative damage resulting fromaerobic metabolism, photosynthesis and a range of pollutants.Recent approaches, using mutants and transgenic plants, areproviding evidence for a key role for the ascorbate–glutathionecycle in protecting plants against oxidative stress. Ascorbateis also a cofactor for some hydroxylase enzymes (e.g. prolylhydroxylase) and violaxanthin de-epoxidase. The latter enzymelinks ascorbate to the photoprotective xanthophyll cycle. Arole in regulating photosynthetic electron transport has beenproposed. The biosynthetic pathway of ascorbate in plants hasnot been identified and evidence for the proposed pathways isreviewed. Ascorbate occurs in the cell wall where it is a firstline of defence against ozone. Cell wall ascorbate and cellwall-localized ascorbate oxidase (AO) have been implicated incontrol of growth. High AO activity is associated with rapidlyexpanding cells and a model which links wall ascorbate and ascorbateoxidase to cell wall extensibility is presented. Ascorbate hasalso been implicated in regulation of cell division by influencingprogression from G1 to S phase of the cell cycle. There is aneed to increase our understanding of this enigmatic moleculesince it could be involved in a wide range of important functionsfrom antioxidant defence and photosynthesis to growth regulation. Ascorbic acid; ascorbate oxidase; cell division; cell wall; growth; oxidative stress; photosynthesis; ozone; vitamin C     

Ultraviolet-B-induced oxidative stress and antioxidant defense system responses in ascorbate-deficient vtc1 mutants of Arabidopsis thaliana

Ultraviolet-B (UV-B) radiation has a negative impact on plant cells, and results in the generation of reactive oxygen species (ROS). In order to increase our understanding of the effects of UV-B on antioxidant processes, we investigated the response of an ascorbate-deficient Arabidopsis thaliana mutant vtc1 to short-term increased UV-B exposure. After UV-B supplementation, vtc1 mutants exhibited oxidative damage. Evidence for damage included an increase in H(2)O(2) content and the production of thiobarbituric acid reactive substances (TBARS); a decrease in chlorophyll content and chlorophyll fluorescence parameters were also reported. The vtc1 mutants had higher total glutathione than the wild type (WT) during the first day of UV-B treatment. We found reduced ratio of glutathione/total glutathione and increased ratio of dehydroascorbate/total ascorbate in the vtc1 mutants, compared to the WT plants. In addition, the enzymes responsible for ROS scavenging, including superoxide dismutase, catalase, and ascorbate peroxidase, had insufficient activity in the vtc1 mutants, compared to the WT plants. The same reduced activity in the vtc1 mutants was reported for the enzymes responsible for the regeneration of ascorbate and glutathione (including monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase). These results suggest that the ascorbate-deficient mutant vtc1 is more sensitive to supplementary UV-B treatment than WT plants and ascorbate can be considered an important antioxidant for UV-B radiation.     

Ascorbate-deficient mutants of Arabidopsis grow in high light despite chronic photooxidative stress

Acclimation to changing environments, such as increases in light intensity, is necessary, especially for the survival of sedentary organisms like plants. To learn more about the importance of ascorbate in the acclimation of plants to high light (HL), vtc2, an ascorbate-deficient mutant of Arabidopsis, and the double mutants vtc2npq4 and vtc2npq1 were tested for growth in low light and HL and compared with the wild type. The vtc2 mutant has only 10% to 30% of wild-type levels of ascorbate, vtc2npq4 has lower ascorbate levels and lacks non-photochemical quenching of chlorophyll fluorescence (NPQ) because of the absence of the photosystem II protein PsbS, and vtc2npq1 is NPQ deficient and also lacks zeaxanthin in HL but has PsbS. All three genotypes were able to grow in HL and had wild-type levels of Lhcb1, cytochrome f, PsaF, and 2-cysteine peroxiredoxin. However, the mutants had lower electron transport and oxygen evolution rates and lower quantum efficiency of PSII compared with the wild type, implying that they experienced chronic photooxidative stress. The mutants lacking NPQ in addition to ascorbate were only slightly more affected than vtc2. All three mutants had higher glutathione levels than the wild type in HL, suggesting a possible compensation for the lower ascorbate content. These results demonstrate the importance of ascorbate for the long-term acclimation of plants to HL.     

Conversion of L-galactono-1,4-lactone to L-ascorbate is regulated by the photosynthetic electron transport chain in Arabidopsis

In this study we focused on the effects of light irradiation and the addition of L-galactono-1,4-lactone (L-GalL) on the conversion of exogenous L-GalL to L-ascorbate (AsA) and the total AsA pool size in detached leaves of Arabidopsis plants and transgenic plants expressing the rat L-gulono-1,4-lactone oxidase gene. Increases in the total AsA level in L-GalL-treated leaves depended entirely on light irradiation. Treatment with an inhibitor of photosynthetic electron transport together with L-GalL reduced the increase in total AsA under light. Light, particularly the redox state of photosynthetic electron transport, appeared to play an important role in the regulation of the conversion of L-GalL to AsA in the mitochondria, reflecting the cellular level of AsA in plants.     

Antisense suppression of l-galactose dehydrogenase in Arabidopsis thaliana provides evidence for its role in ascorbate synthesis and reveals light modulated l-galactose synthesis

l-Galactose dehydrogenase (l-GalDH), a novel enzyme that oxidizes l-Gal to l-galactono-1,4-lactone (l-GalL), has been purified from pea seedlings and cloned from Arabidopsis thaliana. l-GalL is a proposed substrate for ascorbate biosynthesis in plants, therefore the function of l-GalDH in ascorbate biosynthesis was investigated by overexpression in tobacco and antisense suppression in A. thaliana. In tobacco the highest expressing lines had a 3.5-fold increase in extractable activity, but this did not increase leaf ascorbate concentration. Arabidopsis thaliana, transformed with an antisense l-GalDH construct, produced lines with 30% of wild-type activity. These had lower leaf ascorbate concentration when grown under high light conditions. l-Gal pool size increased in antisense transformants with low l-GalDH activity, and l-Gal concentration was negatively correlated with ascorbate. The results provide direct evidence for a role of l-GalDH in ascorbate biosynthesis. Ascorbate pool size in A. thaliana is increased by acclimation to high light, but l-GalDH expression was not affected. l-Gal accumulation was higher in antisense plants acclimated to high light, indicating that the capacity to synthesize l-Gal from GDP-mannose is increased. Because the only known function of l-GalL is ascorbate synthesis, these antisense plants provide an opportunity to investigate ascorbate function with minimal effects on carbohydrate metabolism. Measurements of other antioxidants revealed an increase in ascorbate- and pyrogallol-dependent peroxidase activity in low-ascorbate lines. As ascorbate is the major hydrogen peroxide-scavenging antioxidant in plants, this could indicate a compensatory mechanism for controlling hydrogen peroxide concentration.     

Immunocytochemical determination of the subcellular distribution of ascorbate in plants

Ascorbate is an important antioxidant in plants and fulfills many functions related to plant defense, redox signaling and modulation of gene expression. We have analyzed the subcellular distribution of reduced and oxidized ascorbate in leaf cells of Arabidopsis thaliana and Nicotiana tabacum by high-resolution immuno electron microscopy. The accuracy and specificity of the applied method is supported by several observations. First, preadsorption of the ascorbate antisera with ascorbic acid or dehydroascorbic acid resulted in the reduction of the labeling to background levels. Second, the overall labeling density was reduced between 50 and 61% in the ascorbate-deficient Arabidopsis mutants vtc1-2 and vtc2-1, which correlated well with biochemical measurements. The highest ascorbate-specific labeling was detected in nuclei and the cytosol whereas the lowest levels were found in vacuoles. Intermediate labeling was observed in chloroplasts, mitochondria and peroxisomes. This method was used to determine the subcellular ascorbate distribution in leaf cells of plants exposed to high light intensity, a stress factor that is well known to cause an increase in cellular ascorbate concentration. High light intensities resulted in a strong increase in overall labeling density. Interestingly, the strongest compartment-specific increase was found in vacuoles (fourfold) and in plastids (twofold). Ascorbate-specific labeling was restricted to the matrix of mitochondria and to the stroma of chloroplasts in control plants but was also detected in the lumen of thylakoids after high light exposure. In summary, this study reveals an improved insight into the subcellular distribution of ascorbate in plants and the method can now be applied to determine compartment-specific changes in ascorbate in response to various stress situations.     

Effect of nitrogen deficiency on antioxidant status and Cd toxicity in rice seedlings

Effect of nitrogen (N) deficiency on antioxidant status and Cd toxicity in rice seedlings was investigated. N deficiency resulted in a reduction of shoot growth but not root growth. The contents of N-containing compounds such as nitrate, chlorophyll, and protein decreased in leaves of rice seedlings grown under N deficiency. Accumulation of abscisic acid and H₂O₂ in leaves was induced by N deficiency. The content of ascorbate and the activities of ascorbate peroxidase, glutathione reductase, and catalase in N-deficient leaves were lower than their respective control leaves. However, glutathione content was not affected and superoxide dismutase activity was increased by N deficiency. Cd toxicity in N-deficient seedlings was more pronounced than that in N-sufficient ones. Pretreatment with ascorbate or L-galactono-1,4-lactone, a biosynthetic precursor of ascorbate resulted in a reduction of Cd toxicity enhanced by N deficiency. N deficiency also resulted in an enhancement of Cd uptake in rice seedlings. The possible mechanism of Cd toxicity enhanced by N deficiency is discussed.