# Vitamin C in Plants: From Functions to Biofortification
## 4 Vitamin C Biosynthesis in Plants
### d-Mannose/l-Galactose Pathway  
In the Smirnoff-Wheeler pathway, d-glucose-6-phosphate is transformed into d-fructose-6-phosphate by phosphoglucose isomerase (PGI) and then is directed into d-mannose metabolism by phosphomannose isomerase (PMI), which produces d-mannose-6-phosphate. In _Arabidopsis_, PMI1 expression increases concomitantly with vitamin C levels under continuous light, and knockdown _pmi1_ plants showed decreased levels of this metabolite [125]. d-Mannose-6-phosphate is then converted into p-mannose-1-phosphate by phosphomannose mutase (PMM). Genetic evidence for the involvement of PMM in vitamin C biosynthesis has been obtained in _Nicotiana benthamiana_ and _Arabidopsis_[126; 127]. GDP-d-mannose pyrophosphorylase (GMP) transfers guanosine monophosphate from GTP to give GDP-d-mannose. In _Arabidopsis_, GMP is coded by VTC1; the _vtc1_ mutants accumulate -25-30% of vitamin C levels of wild type and are hypersensitive to ozone [128; 129]. _vtc1_ mutants also have altered sensitivity to other ROS-generating conditions, including H2O2, UV-B, SO2, and combined high light and salt stress [61]. Additional support for the involvement of GMP in vitamin C biosynthesis was obtained in potato plants constitutively expressing the antisense GMP gene. These plants showed a significant decrease in the activity of the enzyme and a significant reduction of vitamin C in leaves and tubers [130]. GDP-l-galactose is produced directly by GDP-d-mannose through a 3'5' \(\!\!^{\prime}\) epimerization catalyzed by GDP-d-mannose epimerase (GME). GME has been characterized in _Chorella_, flax, and _Arabidopsis_[131; 132]. The enzyme belongs to the extended short-chain dehydratase/reductase protein family, with a modified NAD\({}^{+}\) binding Rossman fold domain [133]. GME is also able to catalyze the 5' \(\!\!^{\prime}\) epimerization of GDP-mannose, giving GDP-l-glucose, which is the precursor of a possible side-branch biosynthetic pathway (the gulose pathway) for vitamin C synthesis [134; 132; 122]. GDP-d-mannose and GDP-l-galactose are substrates for the synthesis of glycoproteins and polysaccharides of cell walls [135; 136]. Thus, the first dedicated step for vitamin C synthesis in the d-mannose/l-galactose pathway is the conversion of GDP-l-galactose into l-galactose-1-phosphate, catalyzed by GDP-l-galactose-phosphorylase (GGP) [137]. In _Arabidopsis_, GGP is encoded by the VTC2 and VTC5 genes [121]. The VTC2 expression levels are significantly higher (100-1000 times) than that of VTC5; moreover, T-DNA insertion mutants of VTC2 and VTC5 have 20% and 80% of the vitamin C content of wild-type plants, respectively. The double _vtc2 vtc5_ mutants are unable to grow after cotyledon expansion, unless there is feeding of galactose or ASC, suggesting that at least in _Arabidopsis_, the d-mannose/l-galactose pathway is the substantial font of vitamin C [121]. The key role of GGP as a control point in vitamin C biosynthesis has been shown not only in _Arabidopsis_ but also in tobacco, tomato, kiwifruit, strawberry, potato, citrus, and blueberry [138; 139; 140; 141; 142; 143]. The transcript levels of VTC2 and VTC5 strongly correlate with the vitamin C content and the increase with light irradiation [144; 121]. Moreover, VTC2, as well as other orthologogene genes, can be controlled at the translation level by a noncanonical upstream open reading frame (uORF). In the presence of a high amount of ASC, the uORF encodes for a peptide, which acts as an inhibitor of translation, whereas under a low amount of ASC, the uORF is bypassed and GGP is translated [145]. l-Galactose-1-phosphate is converted into l-galactose by l-galactose-1-phosphate phosphatase (GPP), which is encoded by VTC4 in _Arabidopsis_[146; 147]. However, _vtc4_ mutants have a partial decrease of GPP activity and vitamin C content [148; 147]. Accordingly, in _Arabidopsis_, the reaction can also be catalyzed by the purple acid phosphatase AtPAP15 [149].