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.

Figure 1.

Myoinositol synthesis and metabolism pathway. De novo synthesis of myoinositol (i.e. the Loewus pathway) is catalyzed by myoinositol phosphate synthase (MIPS) and IMP, where its immediate precursor is d-Ins 3-P = l-Ins 1-P. IMP also regenerates myoinositol from the second messenger d-Ins(1,4,5)P₃. Oxidation of inositol by myoinositol oxygenase (MIOX) produces d-GlcUA (d-GlcA), which is a possible entry point into ascorbate synthesis. The major route to ascorbate in plants is the Smirnoff-Wheeler pathway and utilizes GDP-d-Man. VTC4 has homology to the animal IMPs and has been shown to catalyze the conversion of l-Gal 1-P to l-Gal in the Smirnoff-Wheeler pathway. Inositol is also the precursor for the synthesis of several compounds indicated in gray. The asterisk indicates the inositol signaling pathway.

Figure 2.

Gel fractionation of purified VTC4 and IMPLs. The indicated purified recombinant proteins (1.5 μg) were fractionated by 12% SDS-PAGE and stained with Coomassie Brilliant Blue dye. Molecular mass markers, with sizes in kD, are indicated on the right.

Figure 3.

Mg²⁺ and pH dependence of purified VTC4. A, Purified VTC4 (1.5 μg) was incubated in Tris-Cl (pH 7.5) with 0.5 mm d-Ins 3-P for 10 min with varying concentrations of MgCl₂. Enzyme activity was determined by phosphate release. B, As in A, except that pH was varied with Tris-Cl buffers and 4 mm MgCl₂ was present in all assays.

Figure 4.

Kinetic analysis of VTC4 activity with d-Ins 3-P and l-Gal 1-P. Phosphatase activity (solid lines) was plotted versus varying concentrations of d-Ins 3-P (A) or l-Gal 1-P (B) in the standard reaction described in “Materials and Methods.” Data were imported into Kaleidagraph (Synergy Software) and fit to a nonlinear curve with a substrate inhibition equation based on the Michaelis-Menten equation (A) or the Michaelis-Menten equation to calculate apparent K_m and V_max (B). C, Inhibition of VTC4 activity by either LiCl or CaCl₂. VTC4 activity was assayed with d-Ins 3-P (circles) or l-Gal 1-P (squares) in the presence of the indicated concentrations of CaCl₂ (solid lines) or LiCl (dashed lines).

Figure 5.

T-DNA insertions and loss of gene expression in mutant lines. A, Schematic of the T-DNA insertion sites in the vtc4-2, vtc4-3, and vtc4-4 mutants. Exons in At3g02870 are shown as dark gray boxes; the gray arrows indicate primers used to amplify the right border (RB) and left border (LB) of the T-DNA; black arrows indicate the positions of gene-specific primers. B, Verification of the loss of VTC4 expression in mutant lines. Total RNA was isolated from leaves of 14-d soil-grown mutant and wild-type (WT) plants. RT-PCR was carried out with gene-specific primers for VTC4 and actin. Primer sequences can be found in “Materials and Methods.”

Figure 6.

Metabolite levels in control and vtc4 mutant plants. Leaf tissue from 14-d soil-grown plants was frozen in liquid nitrogen, ground, extracted, derivatized, and analyzed by gas chromatography as described in “Materials and Methods.”

Figure 7.

Germination response of mutants to ABA, NaCl, and sorbitol stress. Wild-type CS60000 (WT; solid lines), vtc4-4 and vtc4-2 (dashed lines) seeds were plated on 0.5× MS agar medium with no additions or with 1.25 μm ABA (A), 150 mm NaCl (B), or 300 mm sorbitol (C). Seeds were stratified at 4°C for 3 d and then placed at 23°C under continuous light for the indicated times (hours), and germination was scored. Values represent means ± se of three replicates from one of three independent experiments.

Figure 8.

Cold sensitivity of vtc4 mutants. Wild-type CS60000 (WT; solid lines) and vtc4 mutant (dashed lines) seeds were plated on 0.5× MS agar medium, stratified at 4°C for 3 d, and then placed at 23°C (room temperature [RT]) or 4°C (Cold) under continuous light for the indicated times. Germination (A) and change in root length (B) were scored. Values represent means ± se of three replicates (n = 50). In B, data from day 25 after transfer to the cold are presented. * P < 0.05 compared with the corresponding wild-type sample.