## 4. ascorbate and Glutathione
### Biosynthesis of ascorbate
Knockouts of the _VTC4_ gene in Arabidopsis resulted in only a partial decrease in GPP activity and vitamin C content [271; 272]. This unexpected retention of activity can be attributed to the fact that, in Arabidopsis, this reaction is also catalyzed by purple acid phosphatase AtPAP15 (Table 4) [273]. The conversion of L-galactose to L-galacton-1,4-lactone is catalyzed by L-galactose dehydrogenase (GDH), which is an NAD-dependent enzyme [254]. Overexpression of GDH in Arabidopsis did not change the content of vitamin C, and in antisense plants, the content of vitamin C decreased only under bright light [283], which suggests that it is not a crucial stage of Asc biosynthesis. The last step of the L-galactose pathway is the oxidation of L-galactono-1,4-lactone dehydrogenase (GLDH) into Asc (Figure 5). This step takes place in mitochondria, and is coupled with the cytochrome \(c\) pathway that is present there [284]. GLDH was identified as one of the proteins in mitochondrial complex I [285]. It acts as an essential plant-specific factor for complex I assembly [286]. Thus, stresses that disrupt electron flow have significant effects on ascorbate biosynthesis [284]. The second important pathway for Asc biosynthesis in plants is the D-galacturonate pathway, which involves methyl-D-galacturonate from pectin of cell walls (Figure 5). In the first step of this pathway, methyl esterase catalyzes the conversion of methyl-D-galacturonate into D-galacturonate, which is then transformed into L-galacturonate by D-galacturonate reductase. It has been shown that D-galacturonate methyl ester supplied exogenously to cell cultures, including those of Arabidopsis, caused an increase in ascorbate level [287; 288]. The _GalLIR_ gene, which expression correlates with the increase in Asc during fruit ripening, was cloned, and the recombinant enzyme was shown to have NADPH-dependent D-GalUA reductase activity [257]. Its role in ascorbate biosynthesis was confirmed by overexpression in Arabidopsis, which resulted in a several-fold increase in foliar ascorbate. However, there is no evidence for this pathway operating under normal conditions; it takes place only under conditions of cell wall breakdown [289]. Exogenous L-glucose addition also increased the Asc content of Arabidopsis cell cultures but much less effectively than the addition of methyl galacturonate [287]. In the L-glucose pathway of Asc biosynthesis, the precursor of L-glucose is GDP-D-mannose, a compound also found in the Smirnoff-Wheeler pathway (Figure 5). The first step of the L-glucose pathway is the conversion of GDP-D-mannose into GDP-L-glucose by GDP-D-mannose-3,5\({}^{\prime}\)-epimerase (GME), followed by the formation of L-glucose-1-phosphate by GDP-L-glucose-1-phosphate phosphatase. In the last step of this pathway, AsA is formed from L-gallono-1,4-lactone by L-gallono-1,4-lactone dehydrogenase in mitochondria. The myo-inositol pathway includes conversion of myo-inositol into L-gulonic acid, with its subsequent lactonization to L-gallono-1,4-lactone by aldono lactonase (Figure 5). Then L-gallono-1,4-lactone dehydrogenase converts L-gallono-1,4-lactone into Asc, a step also seen in the gulose and the Smirnoff-Wheeler pathways [264; 290]. It was also shown that the transformation of lettuce and tobacco by constitutive expression of the rat cDNA encoding L-gallono-1,4-lactone oxidase resulted in a four- to seven-fold increase in the content of vitamin C in the leaves of these plants [291]. Experimental data demonstrating the significance of the myo-inositol metabolic pathway for Asc synthesis are contradictory. The main enzyme in this pathway is myo-inositol oxygenase (MIOX). Overexpression of the gene encoding this enzyme (_At4g26260_) in Arabidopsis was reported to increase the content of foliar ascorbate [256]. Later, [PERSON] and [PERSON] [292] showed a decrease in myo-inositol with no changes in the Asc level in the same transgenic plants.