L-ascorbic acid (vitamin C) is a powerful reducing agent found in millimolar concentrations in plants, and is proposed to play an important role in scavenging free radicals in plants and animals. However, surprisingly little is known about the role of this antioxidant in plant environmental stress adaptation or ascorbate biosynthesis. We report the isolation of sozi, a semi-dominant ozone-sensitive mutant that accumulates only 30% of the normal ascorbate concentration. The results of genetic approaches and feeding studies show that the ascorbate concentration affects foliar resistance to the oxidizing gas ozone. Consistent with the proposed role for ascorbate in reactive oxygen species detoxification, lipid peroxides are elevated in sozi, but not in wild type following ozone fumigation. We show that the sozi mutant is hypersensitive to both sulfur dioxide and ultraviolet B irradiation, thus implicating ascorbate in defense against varied environmental stresses. In addition to defining the first ascorbate deficient mutant in plants, these Free radicals can damage macromolecules by oxidative processes, leading to cancer and other diseases associated with aging (1). Antioxidants act to detoxify reactive oxygen species (ROS) such as hydrogen peroxide (H202), superoxide (02-), hydroxyl radical (-OH-), and organic hydroperoxides. Enzymes active in ROS removal include superoxide dismutases, catalases, peroxidases, and glutathione S-transferases (1). Plants also synthesize abundant small molecule antioxidants including AsA (L-ascorbic acid or Vitamin C), glutathione, a-tocopherol (Vitamin E), and carotenoids (2). Although there is increasing evidence that these plant-derived antioxidants are important components of the human diet, relatively little is known about their specific functions in plants.Microbial and animal mutants altered in antioxidant enzymes and signal transduction processes played a pivotal role in testing the importance of specific antioxidant detoxification pathways, evaluating the proposed role of ROS in cellular processes and dissecting ROS signal transduction. For example, bacterial mutants altered in response to growth under enhanced active oxygen conditions identified key antioxidant enzymes and proteins that regulate their synthesis (3). Yeast mutants deficient in glutathione are hypersensitive to H202, implicating glutathione in ROS detoxification (4). A mammalian cell line expressing a mutant p2lras was recently used to demonstrate a key role for this G protein in ROS signal transduction (5).A collection of oxidative stress-sensitive plant mutants would permit a critical assessment of the roles of antioxidant systems and elucidation of ROS signal transduction pathways. With this goal in mind, Arabidopsis thaliana mutants with altered sensitivity to the anthropogenic oxidizing air pollutant 03 (ozone) (6) are being identified. We describe here a semi-dominant monogenic 03 sensitive mutant (sozl, sensitive to ozone), which is deficient in ascorbic acid (AsA). This deficiency also causes...

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 AsAdeficient mutants of Arabidopsis thaliana that are valuable tools for testing of an AsA biosynthetic pathway. The bestcharacterized of these mutants (vtc1) contains Ϸ25% of wildtype 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.

Ascorbic acid is a well-known antioxidant and cellular reductant with an intimate and complex role in the response of plants to ozone. It is clear from a number of studies that sensitivity to ozone is correlated with total ascorbic acid levels, and that a first line of defence against the reactive oxygen species generated in the apoplastic space by ozone is ascorbic acid. For activity, ascorbic acid must be in the fully reduced state. Therefore, both the rate of ascorbic acid synthesis and recycling via dehydroascorbate and monodehydroascorbate reductases are critical in the maintenance of a high ascorbic acid redox state. Active transport of ascorbic acid across the plasma membrane is necessary to achieve reduction of oxidized ascorbic acid by cytoplasm-localized reductases. It has been known for some time that the chlorotic lesions produced by exposure to ozone are not unlike lesions produced by the hypersensitive response to avirulent pathogen attack. Surprisingly, activation of a defence gene-signalling network by both ozone and pathogens is influenced by the level of ascorbic acid. Indeed, in addition to acting simply as an antioxidant in the apoplastic space, ascorbic acid appears to be involved in a complex phytohormone-mediated signalling network that ties together ozone and pathogen responses and influences the onset of senescence.

The structure of the familiar antioxidant L-ascorbic acid (vitamin C) was described in 1933 yet remarkably, its biosynthesis in plants remained elusive until only recently. It became clear from radioisotopic labeling studies in the 1950s that plant ascorbic acid biosynthesis does not proceed in toto via a route similar to that in mammals. The description in 1996 of an Arabidopsis thaliana mutant deficient in ascorbic acid prompted renewed research effort in this area, and subsequently in 1998 a new pathway was discovered that is backed by strong biochemical and molecular genetic evidence. This pathway proceeds through the intermediates GDP-D-mannose, L-galactose, and L-galactono-1,4-lactone. Much research has focused on the properties of the terminal enzyme responsible for conversion of the aldonolactone to ascorbate, and on related enzymes in both mammals and fungi. Two of the plant biosynthetic genes have been studied at the molecular level and additional ascorbate-deficient A. thaliana mutants may hold the key to other proteins involved in plant ascorbate metabolism. An analysis of the biosynthesis of ascorbate and its analogues in algae and fungi as well as the study of alternative proposed pathways should broaden our understanding of ascorbate metabolism in plants. With a biosynthetic pathway in hand, research on areas such as the control of ascorbate biosynthesis and the physiological roles of ascorbate should progress rapidly.

Arabidopsis cyt1 mutants have a complex phenotype indicative of a severe defect in cell wall biogenesis. Mutant embryos arrest as wide, heart-shaped structures characterized by ectopic accumulation of callose and the occurrence of incomplete cell walls. Texture and thickness of the cell walls are irregular, and unesterified pectins show an abnormally diffuse distribution. To determine the molecular basis of these defects, we have cloned the CYT1 gene by a map-based approach and found that it encodes mannose-1-phosphate guanylyltransferase. A weak mutation in the same gene, called vtc1, has previously been identified on the basis of ozone sensitivity due to reduced levels of ascorbic acid. Mutant cyt1 embryos are deficient in N-glycosylation and have an altered composition of cell wall polysaccharides. Most notably, they show a 5-fold decrease in cellulose content. Characteristic aspects of the cyt1 phenotype, including radial swelling and accumulation of callose, can be mimicked with the inhibitor of N-glycosylation, tunicamycin. Our results suggest that N-glycosylation is required for cellulose biosynthesis and that a deficiency in this process can account for most phenotypic features of cyt1 embryos.