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N Smirnoff

Publications and source records attributed to N Smirnoff.

15 recordsLinked to original sources
PubMed2001

L-ascorbic acid biosynthesis.

Biosynthesis of L-ascorbate (vitamin C) occurs by different pathways in plants and mammals. Yeast contain D-erythroascorbate, a C5 analog of ascorbate. UDP-D-glucuronic acid is the precursor in mammals. Loss of UDP forms glucuronic acid/glucuronolactone. Reduction of these at C-1 then forms L-gulonic acid/L-gulono-1,4-lactone. The lactone is oxidized by a microsomal L-gulono-1,4-lactone oxidase to ascorbate. Only the L-gulono-1,4-lactone oxidase has been purified and cloned, and very little is known about the properties of the other enzymes. Plants form ascorbate from GDP-D-mannose via GDP-L-galactose, L-galactose, and L-galactono-1,4-lactone. The final oxidation of L-galactono-1,4-lactone to ascorbate is catalyzed by a mitochondrial L-galactono-1,4-lactone dehydrogenase located on the inner membrane and using cytochrome c as electron acceptor. GDP-mannose pyrophosphorylase and L-galactono-1,4-lactone dehydrogenase have been cloned. Yeast synthesizes D-erythroascorbate from D-arabinose and D-arabinono-1,4-lactone in a pathway analogous to that in plants. The plant, mammalian, and yeast aldonolactone oxidase/dehydrogenases that catalyze the last step in each pathway have significant sequence homology. L-Gulono-1,4-lactone oxidase is mutated and not expressed in animals, such as primates, that have lost ascorbate biosynthesis capacity. Assessment of the literature reveals that little is known about many of the enzymes involved in ascorbate biosynthesis or about the factors controlling flux through the pathways. There is also a possibility that minor alternative pathways exist in plants and mammals.

Animals↗
PubMed2000

Ascorbate biosynthesis and function in photoprotection.

Ascorbate (vitamin C) can reach very high concentrations in chloroplasts (20-300 mM). The pool size in leaves and chloroplasts increases during acclimation to high light intensity and the highest concentrations recorded are in high alpine plants. Multiple functions for ascorbate in photosynthesis have been proposed, including scavenging of active oxygen species generated by oxygen photoreduction and photorespiration, regeneration of alpha-tocopherol from alpha-tocopheryl radicals, cofactor for violaxanthin de-epoxidase and donation of electrons to photosystem II. Hydrogen peroxide scavenging is catalysed by ascorbate peroxidase (Mehler peroxidase reaction) and the subsequent regeneration of ascorbate by reductant derived from photosystem I allows electron flow in addition to that used for CO2 assimilation. Ascorbate is synthesized from guanosine diphosphate-mannose via L-galactose and L-galactono-1,4-lactone. The last step, catalysed by L-galactono-1,4-lactone dehydrogenase, is located on the inner mitochondrial membrane and uses cytochrome c as electron acceptor. L-galactono-1,4-lactone oxidation to ascorbate by intact leaves is faster in high-light acclimated leaves and is also enhanced by high light, suggesting that this step contributes to the control of pool size by light. Ascorbate-deficient Arabidopsis thaliana vtc mutants are hypersensitive to a number of oxidative stresses including ozone and ultraviolet B radiation. Further investigation of these mutants shows that they have reduced zeaxanthin-dependent non-photochemical quenching, confirming that ascorbate is the cofactor for violaxanthin de-epoxidase and that availability of thylakoid lumen ascorbate could limit this reaction. The vtc mutants are also more sensitive to photo-oxidation imposed by combined high light and salt treatments.

Arabidopsis↗
PubMed2000

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.

Arabinose↗
PubMed2000

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↗
PubMed2000

Rapid recovery of photosystems on rewetting desiccation-tolerant mosses: chlorophyll fluorescence and inhibitor experiments.

In the mosses Racomitrium lanuginosum, Anomodon viticulosus and Rhytidiadelphus loreus, after a few days air dry, F:(v)/F:(m) reached, within the first minute of remoistening in the dark, two-thirds or more of the value attained after 40 min. A fast initial phase of recovery was completed within 10-20 min after which further change was slow. Initial recovery of Phi(PSII) in the light was somewhat slower, but was generally substantially complete within a similar time. Remoistening with 0.3 mM cycloheximide (CHX) or 3 mM dithiothreitol (DTT) made little difference to this short-term (40 min) recovery of either F:(v)/F:(m) or Phi(PSII); 3 mM chloramphenicol (CMP) had little effect on recovery of F:(v)/F:(m), but resulted in substantial (though not total) depression of Phi(PSII) and (14)CO(2) uptake. Effects of the protein-synthesis inhibitors and DTT were much more clearly apparent in longer-term experiments (>20 h) but only in the light. In the dark, the three inhibitors had at most only slight effects over periods of 60-100 h. In the light, CMP-treated samples of all three species showed a progressive decline of dark-adapted F:(v)/F:(m), falling to zero within 1-5 d (possibly due to blocking of the turnover of the D1 protein of PSII) and accelerated by DTT. CHX-treated samples showed a similar but slower decline. In the shade-adapted and relatively desiccation-sensitive Rhytidiadelphus loreus, slow recovery of F:(v)/F:(m) continued in the dark even in the presence of CMP and CHX for much of the 142 h of the experiment. The results indicate that in desiccation-tolerant bryophytes recovery of photosynthesis after periods of a few days air dry requires only limited chloroplast protein synthesis and is substantially independent of protein synthesis in the cytoplasm.

Adaptation, Physiological↗
PubMed2000

Ascorbic acid: metabolism and functions of a multi-facetted molecule.

Ascorbic acid (vitamin C) is the most abundant antioxidant in plants. Its biosynthetic pathway via GDP-D-mannose and L-galactose, which was proposed only recently, is now supported by molecular genetic evidence from Arabidopsis thaliana and transgenic potato plants. Except for the last step (which is located on the inner mitochondrial membrane) the pathway is cytosolic, sharing GDP-sugar intermediates with cell-wall polysaccharide and glycoprotein synthesis. Ascorbate peroxidase is emerging as a key enzyme in the fine control of H(2)O(2) concentration; its expression being controlled by redox signals and H(2)O(2). Convincing evidence of the involvement of ascorbate in cell division and growth is also accumulating. Its role as a cofactor in the synthesis of cell wall hydroxyproline-rich glycoproteins is one mechanism for this function.

Ascorbate Peroxidases↗
PubMed2000

The control of ascorbic acid synthesis and turnover in pea seedlings.

The rate of ascorbate synthesis and turnover in pea seedling embryonic axes was investigated in relation to its pool size. Ascorbate accumulated in embryonic axes of germinating pea seeds which has been supplied with ascorbate. Incorporation of [U-14C]glucose into ascorbate after a 2 h labelling period was reduced by ascorbate loading for 3 h and 20 h, providing evidence that ascorbate biosynthesis is inhibited by endogenous ascorbate. Ascorbate turnover was estimated by following the metabolism of [1-14C]ascorbate over 2 h after ascorbate loading and by the rate of decrease of the ascorbate pool size after ascorbate loading. Ascorbate turnover rate, determined by [1-14C]ascorbate metabolism, increased as a linear function of pool size. The absolute turnover rate was higher in ascorbate-loaded embryonic axes but was always about 13% of the pool per hour. The initial rate of ascorbate turnover, estimated from the net decrease in pool size after ascorbate loading, also showed a similar turnover rate to that estimated from [1-14C]ascorbate metabolism. Ascorbate loading had no effect on ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase or glutathione reductase activity. Ascorbate oxidase activity decreased after ascorbate loading.

Ascorbic Acid↗
PubMed1999

Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis.

Vitamin C (L-ascorbic acid; AsA) acts as a potent antioxidant and cellular reductant in plants and animals. AsA has long been known to have many critical physiological roles in plants, yet its biosynthesis is only currently being defined. A pathway for AsA biosynthesis that features GDP-mannose and L-galactose has recently been proposed for plants. We have isolated a collection of AsA-deficient mutants of Arabidopsis thaliana that are valuable tools for testing of an AsA biosynthetic pathway. The best-characterized of these mutants (vtc1) contains approximately 25% of wild-type AsA and is defective in AsA biosynthesis. By using a combination of biochemical, molecular, and genetic techniques, we have demonstrated that the VTC1 locus encodes a GDP-mannose pyrophosphorylase (mannose-1-P guanyltransferase). This enzyme provides GDP-mannose, which is used for cell wall carbohydrate biosynthesis and protein glycosylation as well as for AsA biosynthesis. In addition to genetically defining the first locus involved in AsA biosynthesis, this work highlights the power of using traditional mutagenesis techniques coupled with the Arabidopsis Genome Initiative to rapidly clone physiologically important genes.

Amino Acid Sequence↗
PubMed1999

Ascorbic acid metabolism in pea seedlings. A comparison of D-glucosone, L-sorbosone, and L-galactono-1,4-lactone as ascorbate precursors

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.

Journal Article↗
PubMed1999

Independent signaling pathways regulate cellular turgor during hyperosmotic stress and appressorium-mediated plant infection by Magnaporthe grisea.

The phytopathogenic fungus Magnaporthe grisea elaborates a specialized infection cell called an appressorium with which it mechanically ruptures the plant cuticle. To generate mechanical force, appressoria produce enormous hydrostatic turgor by accumulating molar concentrations of glycerol. To investigate the genetic control of cellular turgor, we analyzed the response of M. grisea to hyperosmotic stress. During acute and chronic hyperosmotic stress adaptation, M. grisea accumulates arabitol as its major compatible solute in addition to smaller quantities of glycerol. A mitogen-activated protein kinase-encoding gene OSM1 was isolated from M. grisea and shown to encode a functional homolog of HIGH-OSMOLARITY GLYCEROL1 (HOG1), which encodes a mitogen-activated protein kinase that regulates cellular turgor in yeast. A null mutation of OSM1 was generated in M. grisea by targeted gene replacement, and the resulting mutants were sensitive to osmotic stress and showed morphological defects when grown under hyperosmotic conditions. M. grisea deltaosm1 mutants showed a dramatically reduced ability to accumulate arabitol in the mycelium. Surprisingly, glycerol accumulation and turgor generation in appressoria were unaltered by the Deltaosm1 null mutation, and the mutants were fully pathogenic. This result indicates that independent signal transduction pathways regulate cellular turgor during hyperosmotic stress and appressorium-mediated plant infection. Consistent with this, exposure of M. grisea appressoria to external hyperosmotic stress induced OSM1-dependent production of arabitol.

Amino Acid Sequence↗
PubMed1998

The biosynthetic pathway of vitamin C in higher plants.

Vitamin C (L-ascorbic acid) has important antioxidant and metabolic functions in both plants and animals, but humans, and a few other animal species, have lost the capacity to synthesize it. Plant-derived ascorbate is thus the major source of vitamin C in the human diet. Although the biosynthetic pathway of L-ascorbic acid in animals is well understood, the plant pathway has remained unknown-one of the few primary plant metabolic pathways for which this is the case. L-ascorbate is abundant in plants (found at concentrations of 1-5 mM in leaves and 25 mM in chloroplasts) and may have roles in photosynthesis and transmembrane electron transport. We found that D-mannose and L-galactose are efficient precursors for ascorbate synthesis and are interconverted by GDP-D-mannose-3,5-epimerase. We have identified an enzyme in pea and Arabidopsis thaliana, L-galactose dehydrogenase, that catalyses oxidation of L-galactose to L-galactono-1,4-lactone. We propose an ascorbate biosynthesis pathway involving GDP-D-mannose, GDP-L-galactose, L-galactose and L-galactono-1,4-lactone, and have synthesized ascorbate from GDP-D-mannose by way of these intermediates in vitro. The definition of this biosynthetic pathway should allow engineering of plants for increased ascorbate production, thus increasing their nutritional value and stress tolerance.

Arabidopsis↗
PubMed1998

Plant resistance to environmental stress

A common effect of many environmental stresses is to cause oxidative damage; consequently, the antioxidant system is being intensively investigated. The use of transgenic plants to probe the role of the antioxidant system continues to be an important approach. The uncharted area of signal transduction in relation to oxidative stress is beginning to attract attention. Studies of drought response at the cellular level have focused on the role of compatible solutes (osmolytes) in acclimation to water stress. Information on signal transduction processes during drought is beginning to appear. As with the antioxidant system, there is increasing use of metabolic engineering in transgenic plants to introduce exotic compatible solutes. It is concluded that these potentially have a use in understanding, or even improving, drought resistance; however, there is a need for the assessment of stress tolerance of transgenics to be carried out at a more sophisticated level and for a critical analysis of the relevance for crop yield of the genes currently being manipulated.

Journal Article↗
PubMed1997

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.

Arabidopsis↗