**2. Increasing Vitamin C Content through Improved Biosynthesis**

The pathway of vitamin C synthesis in mammals begins with D-glucose and proceeds through D-glucose-1-P, UDP-glucose, UDP-D-glucuronic acid, UDP-D-glucuronic acid-1-P, D-glucuronic acid, L-gulonic acid, and finally gulono-1,4-lactone (Figure 1). Gulono-1,4-lactone oxidase then converts gulono-1,4-lactone into 2-keto-gulono-γ-lactone which spontaneously converts to L-ascorbic acid [8].

In contrast to this single pathway, there are at least four biosynthetic pathways suggested to date in plants. The first discovered was the Smirnoff-Wheeler pathway in which Asc synthesis originates with L-galactose [9] (Figure 1). L-Galactose is produced from mannose-1-phosphate through the intermediates guanosine diphosphate (GDP)-mannose and GDP-L-galactose [10]. L-Galactose then undergoes oxidation to L-galactono-1,4-lactone catalyzed by the NAD-dependent L-galactose dehydrogenase followed by oxidation to L-ascorbic acid by the mitochondrial-localized L-galactono-1,4-lactone dehydrogenase [11,12].

Feeding experiments provided support for the Smirnoff-Wheeler pathway. For example, feeding leaf tissue with the Asc precursors L-galactose or L-galactono-1,4-lactone resulted in their conversion to Asc and therefore increased Asc content [9,13,14]. In another study, exogenous application of L-galactono-1,4-lactone to *Arabidopsis* or *Medicago sativa* leaves increased foliar Asc content up to 8-fold and was proportional to the amount applied [15]. Application of L-galactono-1,4-lactone or L-galactose to source potato leaves also increased the Asc content of these leaves as well as in sink organs, e.g., flowers and developing tubers [16].

*Arabidopsis* mutants affected at different steps in the Smirnoff-Wheeler pathway resulted in substantial reductions in Asc content, supporting the conclusion that this pathway is responsible for much of the Asc biosynthetic capacity in this species. For example, the *vtc1* mutant lacks GDP-mannose pyrophosphorylase expression whereas the *vtc2* and *vtc5* mutants lack GDP-L-galactose phosphorylase expression. The *vtc1* mutant exhibits a 70%–75% reduction in Asc content while the *vtc2* mutant contains just 10%–20% of the wild-type level of Asc, *vtc5* contains 80% of the wild-type level, and the *vtc2*/*vtc5* double mutant bleaches in the absence of exogenous Asc or L-galactose which overcomes the block in the pathway [13,17,18]. The *vtc4* mutant results from a mutation in L-galactose-1-P phosphatase [19,20].

Attempts to increase Asc content through increasing its biosynthesis have achieved some success. Overexpression of GDP-L-galactose phosphorylase from kiwifruit (*Actinidia chinensis*) increased Asc content in tobacco leaves by more than 3-fold with an accompanying 50-fold increase in enzyme activity [21]. Although the agroinfection approach employed resulted in only a transient increase in enzyme expression, up to a 4-fold increase in Asc content was observed in stably-transformed *Arabidopsis* where the enzyme was overexpressed [21,22]. Stable transformation of GDP-L-galactose phosphorylase into potato, tomato, and strawberry resulted in up to a 3, 6, and 2-fold increase in Asc, respectively, in tubers and fruits, although some loss of seed and the jelly of locular tissue surrounding the seed were observed in tomato and an increase in polyphenolic content was observed in strawberry and tomato [23]. A combinatorial approach in which kiwifruit GDP-L-galactose phosphorylase and GDP-mannose-3′,5′-epimerase were transiently overexpressed in agroinfected tobacco leaves increased Asc content up to 7-fold [22]. Overexpression of L-galactose dehydrogenase, which catalyzes the conversion of L-galactose to L-galactono-1,4-lactone (Figure 1), however, failed to increase foliar Asc content in tobacco despite a 3.5-fold increase in the activity of the enzyme [24], suggesting that the endogenous level of L-galactose dehydrogenase is not rate-limiting. Transformation of *Arabidopsis with* GDP-galactose guanylyltransferase resulted in a 2.9-fold increase in Asc and co-transformation with either L-galactose-1-phosphate phosphatase or L-galactono-1,4-lactone dehydrogenase increased Asc content up to 4.1-fold [25]. Overexpressing multiple enzymes within the Smirnoff–Wheeler pathway, particularly those whose endogenous level

is closest to being rate-limiting, may offer more promise to achieving substantial increases in Asc rather than the overexpression of any one enzyme. The choice of which enzymes to overexpress may be species-dependent as the level of expression for each enzyme in the pathway may differ among species.

Evidence for other biosynthetic pathways has suggested three alternative routes for the synthesis of Asc. In the first of these alternative pathways, D-galacturonic acid, generated from the breakdown of pectin during fruit ripening, serves as the starting point for Asc synthesis and is reduced to L-galactonic acid as catalyzed by the NADPH-dependent D-galacturonic acid reductase (GalUR) [26] (Figure 1). L-Galactonic acid spontaneously converts to L-galactono-1,4 lactone which L-galactono-1,4-lactone dehydrogenase converts to Asc [26]. Early support for this pathway came from the observation that D-galacturonic acid-1-14C was metabolized to L-ascorbic acid-6-14C through an inversion pathway in detached ripening strawberry fruit [27]. Supplying a methyl ester of D-galacturonic acid to cress seedlings and *Arabidopsis* cell cultures also increased Asc [28,29], suggesting that GalUR expression was not confined to fruits. Expression of the GalUR gene from strawberry increased whole-plant Asc content 2- to 3-fold in *Arabidopsis* [30], supporting the existence of this pathway. Demonstration that GalUR can increase foliar Asc biosynthesis through D-galactonic acid and D-galacturonic acid suggests that the substrates for this pathway are present in leaves. The potential for manipulating this pathway to achieve increased Asc content will depend on whether the enzymes of the pathway are expressed and whether D-galacturonic acid is present, e.g., following pectin degradation.

An example of the contingent basis of this pathway was observed in developing tomato fruit. Feeding tomato plants with D-galacturonate failed to increase Asc content in immature green tomato fruit while feeding with L-galactose, representing the D-mannose/L-galactose (or Smirnoff–Wheeler) pathway, did increase Asc content [31]. In contrast, feeding of either precursor increased Asc content of red ripened fruits, correlating with the increase in activity of D-galacturonate reductase and aldonolactonase, the last two enzymes of the D-galacturonate pathway in ripe fruits [31]. These observations suggest that the D-galacturonate pathway is not operative prior to ripening during which pectin is degraded. Thus, the contribution that the D-galacturonate pathway makes to Asc biosynthesis in tomato fruit may be limited to the ripening stage while the Smirnoff-Wheeler pathway is operative throughout fruit development (e.g., [23]). In addition, tracer studies have suggested that the D-galacturonate pathway may contribute only moderately to fruit Asc content [32] while its contribution in other organs has not been examined. In the second alternative pathway, GDP-mannose 3′,5′-epimerase, which catalyzes conversion of GDP-D-mannose to GDP-L-galactose in the L-galactose pathway [10], also catalyzes the 5′-epimerization of GDP-D-mannose to produce GDP-L-gulose [33] (Figure 1). Conversion of GDP-L-gulose to L-gulonic acid allows Asc synthesis essentially as described in the animal pathway although evidence for this is still lacking. The presence of L-gulonic acid and L-gulono-1,4-lactone dehydrogenase activity [33,34] supports the existence of this pathway in plants. The expression of L-gulono-1,4-lactone oxidase (GulLO) from rat in lettuce and tobacco increased Asc content up to 7-fold [35] and reversed the reduction in Asc content in *Arabidopsis* mutants affected in the Smirnoff–Wheeler pathway [36] although it is not known whether L-gulono-1,4-lactone or L-galactono-1,4-lactone served as the substrate as the possibility that L-galactono-1,4-lactone served as the substrate was not examined. Although feeding

with L-gulono-1,4-lactone did not increase the Asc content of tomato fruit at any developmental stage [31], its conversion to Asc has been reported for several plant species [28,37,38], supporting the presence of this pathway in plants. Expression of a foreign gene, however, can result in the ectopic expression of a pathway or the introduction of a novel pathway. Therefore, the degree to which this pathway functions in plants remains to be determined. Demonstrating that a labeled precursor directly labels Asc or that mutating a specific enzyme decreases Asc would provide more compelling evidence for such pathways.

The third alternative pathway involves D-glucuronic acid, an intermediate of the animal pathway which in plants can be generated by *myo*-inositol oxygenase (Figure 1). Support for this pathway in plants comes from the observation that overexpressing an *Arabidopsis* gene having homology to a porcine *myo*-inositol oxygenase increased Asc content [39]. As *myo*-inositol does not function as a precursor of Asc in strawberry fruit or in parsley leaves [32], this raises the question of the extent to which this pathway contributes to Asc content in plants. Nevertheless, the ability to increase Asc through the overexpression of this putative *myo*-inositol oxygenase gene may provide another strategy for increasing Asc biosynthesis.

Although multiple Asc biosynthetic pathways may exist in plants, the observation that mutants affected in the Smirnoff–Wheeler pathway result in substantial reductions in Asc content does indicate that the alternative pathways are unable to compensate for the loss in Asc biosynthetic capacity in Smirnoff–Wheeler pathway mutants. Thus, these alternative pathways may make only minor contributions to Asc biosynthesis and strategies focusing on these other pathways may be limited to increasing Asc in specific organs or at specific developmental stages.
