The VTC4 gene has been reported to be a bifunctional enzyme, also catalysing conversion of D-myoinositol 3-phosphate to myoinositol in myoinositol biosynthesis . The myoinositol can be converted to UDP-glucuronic acid , which is a common but cell wall-specific biochemical precursor for cell wall bio-genesis . The bifunctional VTC4 enzyme facilitates formation of AsA and cell walls . Therefore, the VTC4 may be a candidate gene for enhancing nutrition and delaying softening via influencing of the AsA production and cell wall formation in tomato fruit. Tomato has three VTC4 homologue genes SlIMP1, SlIMP2, and SlIMP3 . The SlIMP3, which has the highest expression level in tomato fruit, was selected for this investigation as it also has bifunctional enzyme activity, similar to that of the VTC4 in Arabidopsis. Overexpression of SlIMP3 in tomato increases the AsA content in multiple tissues. Overexpression of SlIMP3 increased the myoinositol accumulation, cell wall thickness, and altered cell-wall composition. Overexpression of SlIMP3 markedly delayed fruit softening and enhanced fruit resistance to Botrytis cinerea. The results demonstrate a critical role for SlIMP3 in AsA biosynthesis and cell wall bio-genesis and provide new method of delaying fruit softening and extending shelf-life of tomato.Three IMP genes have been identified in tomato . Alignment and sequence analysis revealed that the IMP amino acid sequences contained signature motifs . A phylogenetic analysis of tomato IMP sequences, together with Arabidopsis and tobacco IMP-related genes, large plastic planting pots was carried out using the neighbour-joining method on mega6. The results indicated that SlIMP3 was most closely related to NtIMP3 and clustered with SlIMP1, NtIMP1, and AtVTC4 into one subfamily .
The expression pattern of three SlIMPs in vegetative and reproductive tissues was carried out using the online TomExpression platform. The SlIMPs had ubiquitous expression in all tested tissues, including roots, shoots, leaves, flowers, and fruits. It was interesting that SlIMP3 gene had highest expression levels during fruit development and ripening . A qRT-PCR test was performed to confirm the expression patterns of SlIMPs in tomato plants. The results were consistent with the TomExpression data, with the highest expression levels of SlIMP3 in fruits . The expression pattern of SlIMP3 was also explored through a transgenic tomato plant in which GUS reporter gene was driven by the SlIMP3 promoter. Consistent with the qRT-PCR results, the GUS staining revealed the ubiquitous expression pattern of SlIMP3 in leaves, stems, buds, flowers, and fruits at different developmental stages, with strong expression in immature green fruit. The expression of SlIMP3 was also decreased in the ripening stages, though weakly expression in orange fruits .It is established that the VTC4 gene encodes a bifunctional enzyme that influences myoinositol and ascorbate biosynthesis . We expressed and purified recombinant SlIMP3 to analyse the catalytic character. The open reading frame of the SlIMP3 gene was cloned into a pGEX-4T-1 vector to generate translational fusion with glutathione-S-transferases . The recombinant protein was expressed in E. coli and purified with affinity chromatography. The molecular mass of the GST-SlIMP3 fusion protein was estimated to be 55 kD , similar to the predicted molecular mass. Mg2+ is necessary for myoinositol monophosphatase activity.
Optimal SlIMP3 activity was obtained by examining the optimum MgCl2 concentration for enzyme activity from 1 to 30 mM of MgCl2. The 3.5 mM of MgCl2 concentration was the most effective at a pH of 7.0 . The Arabidopsis VTC4 enzyme has been reported to use L-Gal 1-P and D-Ins 3-P as substrates . Therefore, the catalytic properties of SlIMP3 enzyme were analysed using L-Gal 1-P and D-Ins 3-P as substrates. In the reaction mixtures of 3.5 mM of MgCl2, pH 7.0, and 2 mg of enzyme, the apparent Km for L-Gal 1-P was 0.29 mM and that for D-Ins 3-P was 0.28 mM . The apparent Vmax values of SlIMP3 for L-Gal 1-P and D-Ins 3-P calculated were 6.0 and 8.0 units, respectively. LiCl was an important inhibitor, which inhibited the catalytic effect of IMPs on the substrate. In previous studies, LiCl inhibited the catalytic activity of VTC4 for D-Ins 3-P, and the half-maximal inhibitory concentration was 0.08 to 0.1 mM . The inhibition of SlIMP3 for D-Ins 3-P by LiCl was also tested . The IC50 of SlIMP3 with LiCl was 0.03 to 0.05 mM when the reaction contained 0.5 mM of substrate. The relationship between AsA biosynthesis and SlIMP3 gene function, transgenic lines expressing either sense or anti-sense SlIMP3 constructs under the control of the cauliflower mosaic virus 35S promote were further characterized. A qRT-PCR was conducted to analyse the expression levels. The independent overexpressed or anti-sense lines, which displayed substantial altered gene expression by comparison with the wild type plants, were used for further analysis . Total AsA and reduced AsA content quantification were conducted using these transgenic plants. Interestingly that altered SlIMP3 expression led to dramatic AsA content change in different tissues of the transgenic lines .
The AsA content quantification in leaf, stem, and different fruit developmental stages indicated that SlIMP3-overexpressed fruits accumulated higher amounts of AsA than the WT plants, whereas the downregulated fruits had lower AsA content than the WT plants. In addition, qRT-PCR results indicated that SlPGI, SlGMP1, SlGMP3, SlGGP1, SlGGP2, and SlGalLDH were significantly upregulated in SlIMP3-overexpressed fruits . We also found that SlGalLDH was significantly down-regulated in downregulated fruits . These results indicated that the SlIMP3 gene regulated AsA biosynthesis in tomato plants.Postharvest fruit storage was conducted to test the influence of SlIMP3 overexpression on fruit softening. The WT and SlIMP3- downregulated fruits were shrivelled after 26 d of storage versus the well maintained pericarp quality of the SlIMP3-overexpressed fruits . The fruit water loss of the SlIMP3-overexpressed fruits was less than that of WT and SlIMP3-downregulated fruits . The SlIMP3-overexpressed fruits also were firmer throughout fruit development versus the downregulated fruits and WT fruits, which were again similar . Ethylene production in SlIMP3-overexpression and SlIMP3-downregulated fruits did not change significantly compared with the WT fruits . Overexpression of SlIMP3 had no effect on fruit yield and weight . In addition, both the up-regulation and down-regulation of SlIMP3 did not affect the fruit development and maturation . Transmission electron microscopy was used to examine the cell wall thickness to determine the effects of altered SlIMP3 expression on cell walls. The SlIMP3-overexpression fruits had thicker cell walls than those of WT fruit but not of those of SlIMP3-downregulation fruits . At the same time, we measured the expressions of cell wall metabolism-related genes as previously described . qRT-PCR results indicated that SlEXP1, SlPG2, SlPL, SlTBG4, SlXYL1, and SlXTH5 were significantly down-regulated in SlIMP3-overexpressed fruits . Moreover, like fruits, the cell wall in SlIMP3- overexpression leaves and stems were also increased . Collectively, these results suggested overexpression of SlIMP3 increased the cell wall thickness, delayed the fruit softening, and enhanced tomato shelf-life.To gain more insight into the mechanism by which cell wall thickness was impacted in SlIMP3-overexpressed plants, biochemicals related to cell wall bio-genesis was analysed. Measurement demonstrated overexpression of the SlIMP3 gene increased myoinositol in mature green and red fruits versus no effect of the downregulation of SlIMP3 gene . Uronic acid content was measured in the cell wall of mature green and red fruits by high-performance liquid chromatography . The uronic acid content increased markedly in SlIMP3-overexpressed red fruits but was unchanged in SlIMP3-downregulated fruits and WT fruits . Various neutral sugars, rhamnose, fucose, arabinose, xylose, mannose, galactose, and glucose were also measured. The rhamnose, xylose, mannose, black plastic planting pots and galactose content of SlIMP3-overexpressed red fruits were significantly higher than that of WT . Collectively, these results suggested that overexpression of SlIMP3 improved cell wall bio-genesis in tomato fruit.Because myoinositol is related to cell wall bio-genesis, the WT tomato plants were treated with 10 mg/L, 100 mg/L, and 1 g/L of myoinositol to examine the role myoinositol in cell wall bio-genesis and fruit softening. When treated with 10 mg/L of myoinositol, the fruit cell wall thickness increased slightly, but the fruit softening and water loss were unchanged versus the control.
When treated with 100 mg/L, myoinositol fruit softening was delayed, cell wall thickness increased, and water loss decreased significantly . However, after treatment with 1 g/L of myoinositol, fruit cell wall thickness, storage life, and water loss were unaffected . The 10 mg/L and 100 mg/L of myoinositol treatments sharply increased fruit AsA concentration, but 1 g/L of myoinositol did not modulate fruit AsA production . These experimental results demonstrated exogenous myoinositol application 100 mg/L enhanced the cell wall bio-genesis and delayed softening in tomato fruits. A B. cinerea spore suspension surface applied to injured tomato transgenic tomato fruits produced significantly smaller lesion diameters in SlIMP3-overexpressed fruits versus those in WT and SlIMP3-downregulated fruits, which were approximately equal . Similarly, the biomass of B. cinerea detected with qRT-PCR was significantly lower in the SlIMP3-overexpressed fruits versus the WT and downregulated fruit, which were again approximately equal . Moreover, the expression levels of pathogen-related genes were significantly up-regulated in SlIMP3-overexpressed fruits but were unchanged in SlIMP3-downregulated fruits and WT fruits. This demonstrated overexpression SlIMP3 in tomato significantly improved the tolerance to B. cinerea.In Arabidopsis, the VTC4 gene encodes a bifunctional enzyme that catalyses conversion of L-Gal 1-P to L-galactose in AsA biosynthesis and catalyses conversion of the D-Ins 3-P to myoinositol. In tomato, three SlIMP isoforms are lithium-sensitive enzymes that catalyse the myoinositol biosynthesis from myoinositol monophosphate. In this study, the SlIMP3 gene that had highest expression level among the SlIMP genes was expressed and the SlIMP3 protein purified. The SlIMP3 showed high affinity with the L-Gal 1-P and D-Ins 3-P, and was sensitive to lithium, consistent with enzymatic properties of the Arabidopsis VTC4 . Overexpression of the SlIMP3 gene increased the AsA content, while downregulation of the SlIMP3 gene decreased the AsA content in tomato .Overexpression of the SlIMP3 gene also increased the myoinositol content in tomato fruit . Our data indicated the SlIMP3, which functions like the VTC4, is involved in the biosynthesis of AsA and myoinositol in tomato. But downregulation of the SlIMP3 did not decrease the myoinositol in tomato fruit , which may indicate a redundancy in myoinositol biosynthesis, and suggests the SlIMP1 and SlIMP2 may supplement the enzymatic activity of SlIMP3 in tomato. Interestingly, we found that the expression level of SlIMP2 was significantly up-regulated in SlIMP3-downregulated fruits, which may compensate myoinositol biosynthesis. However, the relationship between SlIMP2 and SlIMP3 remains unknown. Perhaps, in future studies, a tertiary mutant of SlIMPs can be generated using CRISPR/Cas9 methods and used to study their precise function in the biosynthesis of AsA and myoinositol in tomato. In Arabidopsis, overexpression of the myoinositol oxygenase increased AsA content, suggesting that myoinositol can act as a precursor for ascorbate biosynthesis. However, it has also been reported that the MIOS controls the myoinositol level, but does not contribute to AsA biosynthesis . In this study, treating fruits with 100 mg/l of myoinositol not only increased the cell wall bio-genesis, but also increased the fruit’s AsA content . The myoinositol was converted into D-Glucuronic acid by MIOS and the D-Glucuronic acid was catalysed into L-gulonate, which can be converted into the AsA by a multistep process in animals. Our results support that the hypothesis that the myoinositol oxidation pathway contributes to AsA biosynthesis in tomato fruit. Perhaps rigorous radiotracer experiments could elucidate the pathway of myoinositol conversion into AsA in tomato fruits.Myoinositol has been reported to play an important role in cell wall formation . Myoinositol is catalysed by inositol oxidase to D-glucuronic acid, a precursor of pectin and hemicellulose in cell wall biosynthesis . Radioactive myoinositol injected into ripening strawberry fruits was converted to the Dgalacturonosyl residue of pectin and D-xylosyl residues of hemicellulose . In this study, overexpression of SlIMP3 markedly increased the myoinositol, uronic acid and neutral sugar content, and fruit cell wall thickness . Simultaneously, treating fruits with 100 mg/L of myoinositol produced a similar phenotype as that of SlIMP3-overexpressed fruits . The data presented here proved that myoinositol participates in tomato fruit cell wall bio-genesis. Silencing of the PL gene reinforced the tricellular junction in the fruit cell wall and delayed the tomato fruit softening . Mutations in the GA2-oxidase gene improves cutin and wax biosynthesis and increases tomato fruit firmness and shelf-life . In this study, overexpression of SlIMP3 gene increased cell-wall thickness and fruit firmness, delayed fruit softening, and prolonged tomato fruit shelf-life . These results support that improving cell wall bio-genesis is an effective strategy for delaying fruit softening and extending fruit shelf-life.