myo-Inositol Oxygenase Offers a Possible Entry Point into Plant Ascorbate Biosynthesis

Abstract

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...

The inositol oxygenase gene family of Arabidopsis is involved in the biosynthesis of nucleotide sugar precursors for cell-wall matrix polysaccharides

“…In addition, the overexpression of a single MIOX gene has been reported to result in elevated levels of ascorbic acid in plants (Lorence et al 2004). The amounts of ascorbate in wild-type and DMIOX2 or DMIOX5 knock-out plants were between 2 and 3.5 lmol g À1 fresh weight in individual experiments, depending on the environmental conditions, and differed less than 5% in several independent measurements.…”

“…Ascorbic-acid content was measured as previously described (Lorence et al 2004). Leaves from 3-or 5-weekold Arabidopsis plants, grown in the greenhouse (16-h light period, $23°C), were collected at noon on either a sunny or a cloudy day.…”

“…The amino acid sequence of the yeast MIOX is highly similar to a family of putative proteins in Arabidopsis. A BLAST search identified four MIOX-like proteins from Arabidopsis (P-value < 1 e À65 )(see also Lorence et al 2004). The hits also include a MIOX gene from pig, for which the enzyme was recently purified (Arner et al 2001).…”

“…It is important to note that the BLASTP search does not identify any other similar protein from Arabidopsis (using a cutoff of P<0.01), indicating that the MIOX sequences represent a unique class of proteins with no evolutionary close relatives. The amino acid sequences of the four identified MIOX proteins, which were named according to an earlier suggestion (Lorence et al 2004), were aligned with the CLUSTALW program. The amino acid sequence varies between the four Arabidopsis isoforms, particularly within the N-terminal 50 amino acids, but is highly conserved for the large C-terminal part ($270 amino acids).…”

“…These sequences now provide molecular tools to analyze the role of the MIOX pathway for nucleotide sugar biosynthesis. Using a similar approach, Lorence et al (2004) investigated the role of MIOX for ascorbate biosynthesis in transgenic Arabidopsis plants. They isolated the Miox4 gene by a homology-based PCR approach and found a flower-specific gene expression for the Miox4 gene.…”