The miox4 message is present in flowers and leaves, tissues with high concentration of AsA. A, RNA expression of miox4 in leaf, flower, and root tissues of 6-week-old Arabidopsis wild-type plants. rRNA of the samples stained with ethidium bromide is shown as a loading control in the lower panel. B, Mean ascorbic acid content of the corresponding plant tissues (n = 3). Bars = one sd. L, Leaf; S, stem; F, flower; R, root; Si, silique.

Proposed biosynthetic network for AsA synthesis in plants (reactions 1–12), and animals (reactions 13–20). Four potential branch pathways operating in plants, the Man pathway, galacturonate pathway, the gulose pathway, and the MI pathway, are shown. Enzymes catalyzing the numbered reactions are: 1, methyl-esterase; 2, d-galacturonate reductase; 3, aldono-lactonase; 4, Glc-6-phosphate isomerase; 5, Man-6-phosphate isomerase; 6, phosphomannomutase; 7, GDP-Man pyrophosphorylase; 8, GDP-Man-3,5-epimerase; 9, phosphodiesterase; 10, sugar phosphatase; 11, l-Gal-1-dehydrogenase; 12, l-galactono-1,4-lactone (l-GalL) dehydrogenase; 13, phosphoglucomutase; 14, UDP-Glc pyrophosphorylase; 15, UDP-Glc dehydrogenase; 16, glucuronate-1-phosphate uridylyltransferase; 17, glucurono kinase; 18, glucuronate reductase; 19, aldono lactonase, and 20, guluno-1,4-lactone dehydrogenase. The reaction catalyzed by MIOX, and the possible pathway from MI to AsA (dashed arrows), are also shown. Question marks indicate enzymatic reactions that have not been demonstrated in plants.

Constitutive expression of miox4 in Arabidopsis increases the ascorbic acid content of the leaves. A, RNA expression of miox4 in control (labeled as C, pCAMBIA1380), and three homozygous transgenic lines, numbered L1 to L3. rRNA of the samples stained with ethidium bromide is shown as a loading control in the lower panel. B, Mean ascorbic acid content in the leaf extract of the corresponding control (C) and transgenic lines L1 to L3 (n = 3). Bars = one sd.