VTC4 Is a Bifunctional Enzyme That Affects Myoinositol and Ascorbate Biosynthesis in Plants      

Abstract

Myoinositol synthesis and catabolism are crucial in many multiceullar eukaryotes for the production of phosphatidylinositol signaling molecules, glycerophosphoinositide membrane anchors, cell wall pectic noncellulosic polysaccharides, and several other molecules including ascorbate. Myoinositol monophosphatase (IMP) is a major enzyme required for the synthesis of myoinositol and the breakdown of myoinositol (1,4,5)trisphosphate, a potent second messenger involved in many biological activities. It has been shown that the VTC4 enzyme from kiwifruit (Actinidia deliciosa) has similarity to IMP and can hydrolyze L-galactose 1-phosphate (L-Gal 1-P), suggesting that this enzyme may be bifunctional and linked with two potential pathways of plant ascorbate synthesis. We describe here the kinetic comparison of the Arabidopsis (Arabidopsis thaliana) recombinant VTC4 with D-myoinositol 3-phosphate (D-Ins 3-P) and L-Gal 1-P. Purified VTC4 has only a small difference in the V max /K m for L-Gal 1-P as compared with D-Ins 3-P and can utilize other related substrates. Inhibition by either Ca 2+ or Li + , known to disrupt cell signaling, was the same with both L-Gal 1-P and D-Ins 3-P. To determine whether the VTC4 gene impacts myoinositol synthesis in Arabidopsis, we isolated T-DNA knockout lines of VTC4 that exhibit small perturbations in abscisic acid, salt, and cold responses. Analysis of metabolite levels in vtc4 mutants showed that less myoinositol and ascorbate accumulate in these mutants. Therefore, VTC4 is a bifunctional enzyme that impacts both myoinositol and ascorbate synthesis pathways.

TheArabidopsis thaliana Myo-Inositol 1-Phosphate Synthase1 Gene Is Required forMyo-inositol Synthesis and Suppression of Cell Death

“…Evidence exists that a minor pathway to ascorbic acid synthesis could proceed via myo-inositol synthesis (Baig et al, 1970;Radzio et al, 2003;Lorence et al, 2004;Torabinejad et al, 2009). We have previously shown that the vtc4 mutant (Conklin et al, 2006) has reduced myo-inositol and ascorbic acid levels, suggesting such a connection between these pathways (Torabinejad et al, 2009).…”

“…We have previously shown that a loss of function in another gene required for myo-inositol synthesis, Vitamin C-4 (VTC4), results in sensitivity to abscisic acid (ABA), salt, and cold (Torabinejad et al, 2009). To test whether known stress physiological pathways that use myo-inositol signaling (Xiong et al, 2001;Taji et al, 2006) were altered in mips mutants, we produced age-matched seed populations that had been harvested from plants grown at the same time.…”

“…To determine if a loss in MIPS1, MIPS2, or MIPS3 function impacts myo-inositol synthesis, we used our previously established gas chromatography (GC) assay (Torabinejad et al, 2009) to quantify myo-inositol levels in the wild type and mips mutants ( Figure 8A). To investigate the timing of myo-inositol changes in mips mutants, we analyzed leaves of 12-, 19-, and 60-d-old wildtype, mips1-2, mips2-2, and mips3-2 mutant plants ( Figure 8B).…”

“…This reaction is followed by dephosphorylation of L-myo-inositol 1-phosphate to myoinositol, which is catalyzed by the myo-inositol monophosphatase (IMP; EC 3.1.3.25) (Sherman et al, 1981;Loewus and Loewus, 1983;Torabinejad and Gillaspy, 2006;Torabinejad et al, 2009). These two reactions together are known as the Loewus pathway, which was first studied in plants and is the only known route for myo-inositol synthesis.…”

KONJAC1 and 2 Are Key Factors for GDP-Mannose Generation and Affect l-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis

“…The main synthetic pathway for AsA in vascular plants is Man (sugars in this study are D series unless designated otherwise)/L-galactose pathway (Smirnoff-Wheeler pathway) (Wheeler et al, 1998), in which the conversion of GDP-Man from Man 1-phosphate (Man 1-P) catalyzed by GDP-Man pyrophosphorylase (GMPP) is a rate-limiting step affecting AsA quantity, as well as several downstream steps (Supplemental Figure 1) (Dowdle et al, 2007;Linster et al, 2007;Torabinejad et al, 2009). VITAMIN C DEFECTIVE1 (VTC1)/CYTOKINESIS DEFECTIVE1 (CYT1; hereafter, VTC1 indicates VTC1/CYT1 protein) has been identified as the major form of GMPP in Arabidopsis, and evidence for its key role in AsA synthesis comes from a number of different studies: In Arabidopsis thaliana, the vtc1-1 mutant, a leaky mutant for the VTC1 gene, exhibits a 70% decrease in AsA level (Conklin et al, 1997(Conklin et al, , 1999) and a CONSTITUTIVE PHO-TOMORPHOGENESIS9 (COP9) signalosome subunit, CSN5B, downregulates AsA level through the degradation of VTC1 in the dark (Wang et al, 2013b).…”