Two biosynthetic pathways for ascorbate (l-ascorbic acid [AsA]; vitamin C) in plants are presently known, the mannose/ l-galactose pathway and an l-GalUA pathway. Here, we present molecular and biochemical evidence for a possible biosynthetic route using myo-inositol (MI) as the initial substrate. A MI oxygenase (MIOX) gene was identified in chromosome 4 (miox4) of Arabidopsis ecotype Columbia, and its enzymatic activity was confirmed in bacterially expressed recombinant protein. Miox4 was primarily expressed in flowers and leaves of wild-type Arabidopsis plants, tissues with a high concentration of AsA. Ascorbate levels increased 2-to 3-fold in homozygous Arabidopsis lines overexpressing the miox4 open reading frame, thus suggesting the role of MI in AsA biosynthesis and the potential for using this gene for the agronomic and nutritional enhancement of crops.l-Ascorbic acid (AsA) is the major antioxidant in plant cells. Since AsA was first isolated, there have been numerous reports on its role regulating redox potential during photosynthesis, environmentinduced oxidative stress (ozone, UV, high light, SO 2 , etc), and wound-and pathogen-induced oxidative processes. In both plants and animals, AsA is important as a cofactor for numerous key enzymes such as hydroxylases and dioxygenases, some of which are involved in the biosynthesis of phytohormones and secondary metabolites or in the hydroxylation of specific peptidyl-prolyl and peptidyl-lisyl residues (Loewus and Loewus, 1987;Smirnoff et al., 2001;Arrigoni and De Tullio, 2002). Current data indicate that AsA is a major substrate for synthesis of l-tartaric, l-threonic, l-glyceric, and l-oxalic acids used for calcium regulation via calcium oxalate formation (Loewus, 1999). There is emerging evidence that AsA is involved in photoprotection, metal and xenobiotic detoxification, the cell cycle, cell wall growth, and cell expansion (Smirnoff, 2000;Smirnoff and Wheeler, 2000;Franceschi and Tarlyn, 2002). Interestingly, a recent study indicates that leaf AsA content can also modulate the expression of genes involved in plant defense and regulate genes that control development through hormone signaling (Pastori et al., 2003). The antioxidant property of AsA also is one of its major functions in humans (Homo sapiens), who are unable to synthesize their own vitamin C. Because AsA can neither be produced nor stored in the body, the vitamin must be acquired regularly from dietary sources, primarily from plants rich in AsA. Because ascorbate levels in plants vary widely, the ability to increase the level of this vitamin in crops would increase their nutritive value, shelf life, and ability to withstand oxidative stress during the growing season.AsA biosynthetic pathways differ between animals and plants (Fig. 1). In animals, d-Glc is converted to AsA via d-glucuronate, l-gulonate, and l-gulono-1,4-lactone (l-GulL), which is then oxidized to AsA. In plants, AsA biosynthesis proceeds via GDP-Man, GDP-l-Gal, l-Gal-1-phosphate, l-Gal, and l-GalL (Wheeler et al., 1998). Althoug...

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Ascorbate (AsA) is the most abundant antioxidant in plant cells and a cofactor for a large number of key enzymes. However, the mechanism of how AsA levels are regulated in plant cells remains unknown. The Arabidopsis (Arabidopsis thaliana) activationtagged mutant AT23040 showed a pleiotropic phenotype, including ozone resistance, rapid growth, and leaves containing higher AsA than wild-type plants. The phenotype was caused by activation of a purple acid phosphatase (PAP) gene, AtPAP15, which contains a dinuclear metal center in the active site. AtPAP15 was universally expressed in all tested organs in wild-type plants. Overexpression of AtPAP15 with the 35S cauliflower mosaic virus promoter produced mutants with up to 2-fold increased foliar AsA, 20% to 30% decrease in foliar phytate, enhanced salt tolerance, and decreased abscisic acid sensitivity. Two independent SALK T-DNA insertion mutants in AtPAP15 had 30% less foliar AsA and 15% to 20% more phytate than wild-type plants and decreased tolerance to abiotic stresses. Enzyme activity of partially purified AtPAP15 from plant crude extract and recombinant AtPAP15 expressed in bacteria and yeast was highest when phytate was used as substrate, indicating that AtPAP15 is a phytase. Recombinant AtPAP15 also showed enzyme activity on the substrate myoinositol-1-phosphate, indicating that the AtPAP15 is a phytase that hydrolyzes myoinositol hexakisphosphate to yield myoinositol and free phosphate. Myoinositol is a known precursor for AsA biosynthesis in plants. Thus, AtPAP15 may modulate AsA levels by controlling the input of myoinositol into this branch of AsA biosynthesis in Arabidopsis.

Antioxidant metabolites in eastem white pine (Pinus strobus L.) needles increased two-to fourfold from the summer to the winter season. Antioxidant enzymes in needle tissue increased between 2-and 122-fold during this same period. These seasonal changes were determined by monitoring ascorbate and glutathione concentrations and the activity of ascorbate peroxidase, glutathione reductase (GR), and superoxide dismutase. Levels of antioxidant metabolites and enzymes were observed always to be lowest during the summer, or active growing season, and highest during the winter, or dormant season. These data correlated well with the thermal kinetic window for purified GR obtained from summer needles. The minimum, apparent KmDm for two isoforms of GR (GRA and GRo) occurred at 5 and 10°C, respectively. The upper limit of the thermal kinetic window (200% of the minimum K.) for GRA and GRB was 20 and 250C, respectively, indicating that needle temperatures exceeding 250C may result in impairment of antioxidant metabolism. The needle content and kinetic properties of GR, the increased activities of other enzymes, and the high substrate concentrations observed during the winter are consistent with the protective function this pathway may provide against photooxidative, winter injury.

Ascorbic acid (AsA) biosynthesis in plants occurs through a complex, interconnected network with mannose (Man), myoinositol, and galacturonic acid as principal entry points. Regulation within and between pathways in the network is largely uncharacterized. A gene that regulates the Man/L-galactose (L-Gal) AsA pathway, AMR1 (for ascorbic acid mannose pathway regulator 1), was identified in an activation-tagged Arabidopsis (Arabidopsis thaliana) ozone-sensitive mutant that had 60% less leaf AsA than wild-type plants. In contrast, two independent T-DNA knockout lines disrupting AMR1 accumulated 2-to 3-fold greater foliar AsA and were more ozone tolerant than wild-type controls. Real-time reverse transcription-polymerase chain reaction analysis of steady-state transcripts of genes involved in AsA biosynthesis showed that AMR1 negatively affected the expression of GDP-Man pyrophosphorylase, GDP-L-Gal phosphorylase, L-Gal-1-phosphate phosphatase, GDP-Man-3#,5#-epimerase, L-Gal dehydrogenase, and L-galactono-1,4-lactone dehydrogenase, early and late enzymes of the Man/L-Gal pathway to AsA. AMR1 expression appears to be developmentally and environmentally controlled. As leaves aged, AMR1 transcripts accumulated with a concomitant decrease in AsA. AMR1 transcripts also decreased with increased light intensity. Thus, AMR1 appears to play an important role in modulating AsA levels in Arabidopsis by regulating the expression of major pathway genes in response to developmental and environmental cues.