The Myo-inositol pathway does not contribute to ascorbic acid synthesis

Abstract

Ascorbic acid (AsA) biosynthesis in plants predominantly occurs via a pathway with d-mannose and l-galactose as intermediates. One alternative pathway for AsA synthesis, which is similar to the biosynthesis route in mammals, is controversially discussed for plants. Here, myo-inositol is cleaved to glucuronic acid and then converted via l-gulonate to AsA. In contrast to animals, plants have an effective recycling pathway for glucuronic acid, being a competitor for the metabolic rate. Recycling involves a phosphorylation at C1 by the enzyme glucuronokinase. Two previously described T-DNA insertion lines in the gene coding for glucuronokinase1 show wild type-like expression levels of the mRNA in our experiments and do not accumulate glucuronic acid in labelling experiments disproving that these lines are true knockouts. As suitable T-DNA insertion lines were not available, we generated frameshift mutations in the major expressed isoform glucuronokinase1 (At3g01640) to potentially redirect metabolites to AsA. However, radiotracer experiments with ³ H-myo-inositol revealed that the mutants in glucuronokinase1 accumulate only glucuronic acid and incorporate less metabolite into cell wall polymers. AsA was not labelled, suggesting that Arabidopsis cannot efficiently use glucuronic acid for AsA biosynthesis. All four mutants in glucuronokinase as well as the wild type have the same level of AsA in leaves.

Keywords: Alternative myo-inostiol pathway; ascorbic acid; glucuronic acid; mammalian-like pathway; myo-inositol.

Figure 1

Pathway overview for AsA biosynthesis in plants. The d‐Man/l‐Gal pathway (left) is the dominant route to AsA in plants. The mammalian‐like pathway (middle) has been debated for many years and is addressed in this paper. The salvage pathway (right) contributes to cell wall polymers in many plants. ①: myo‐inositol oxygenase (MIOX); ②: glucuronokinase; ③: glucuronate reductase; ④: l‐galactono‐1,4‐lactone dehydrogenase (GLDH); ⑤: gulono‐1,4‐lactone oxidase.

Figure 2

Gene structure, position of T‐DNA insertion in GlcAK1 and expression of the GlcAK1‐gene. A: T‐DNA position for both SALK lines was determined by sequencing and found to be 197 bp upstream of the start ATG. The T‐DNA position according to Xiao et al. (2017) is shown with dashed lines. The position and the sequences of the frame shift mutants glcak1‐3 and glcak1‐4 in exon1, generated by CRISPR/Cas9, is also shown. UTRs are indicated in black and exons in grey boxes, introns are represented by black lines. B: Expression of GlcAK1 was measured by qPCR for the four glcak1 mutants. The data show average expression from three biological independent experiments. Statistical differences were evaluated using anova (Tukey's test, P < 0.01), different letters display significant differences between lines.

Figure 3

Distribution of radiolabelled compounds after ³H‐myo‐inositol feeding. Seedlings were grown in 0.5 × MS medium and ³H‐myo‐inositol was added for 3 days. A: Radioactivity from insoluble cell wall, and B: ethanol soluble metabolites determined by scintillation counting. Statistically significant differences were determined using anova (Tukey's test, P < 0.01), different letters display significant differences (n = 6).

Figure 4

Profile of ³H‐labelled compounds after separation on a Hilic HPLC column. Soluble metabolites after ³H‐myo‐inositol feeding (as described in Fig. 3) were subjected to HPLC separation. Fractions of 0.33 ml were collected in a microtiter plate and counted. The experiment was done with glcak1‐3 and glcak1‐4 mutants, showing the same metabolite pattern. The elution profile of AsA was determined by UV absorption. GlcA was measured by aldehyde colour assay and myo‐inositol was quantified by an enzymatic reaction.

Figure 5

Metabolite analysis of WT, glcak1‐3 and glcak1‐4 mutants. Metabolites were separated on a CarboPac PA20 column to quantify GlcA. The data show average ± SD of three independent biological experiments. Mutants were compared to WT using t‐test (two‐tailed, unpaired, P < 0.01), significant differences are displayed with *.

Figure 6

Measurement of AsA from WT and glcak1 mutants. Leaves were taken from plants at different times of day and immediately measured for AsA. The data show average values ± SD from eight samples. There were no statistical differences between lines using anova (Tukey's test, P < 0.05).