# Vitamin C in Plants: From Functions to Biofortification
## 6 Vitamin C Biofortification
### Manipulation of d-Mannose/t-Galactose Pathway  
Several genes of the d-mannose/t-galactose pathway have been overexpressed in different crops in order to enhance vitamin C levels, but not all have given good results [34; 35]. It has been proved that overexpression of GGP, which represents the bottleneck of vitamin C biosynthesis [137], is a good strategy for biofortification [200]. For instance, in _Arabidopsis_, transient overexpression of GGP leads  
Figure 4: Strategies for vitamin C biofortification. Modulation of light intensity and quality can be used to obtain vitamin C enrichment in crops. The overexpression of single or multiple genes belonging to the biosynthetic and recycling pathways, as well as to the regulatory network, represents a good tool for vitamin C biofortification. Vitamin C accumulation can be controlled by modulating translation by the editing of the uORF in the GGP gene. Finally, vitamin C biofortification can be obtained by plant breeding that exploits candidate genes in the quantitative trait locus (QTL) associated with high vitamin C content (more details are provided in the text).  
to a 2.5-fold increase in vitamin C content, whereas the overexpression of the other genes involved in the same pathway does not cause relevant differences in terms of vitamin C [142]. Similar results were obtained in rice where, among different transgenic lines overexpressing six _Arabidopsis_ genes involved in vitamin C biosynthesis, the highest vitamin C content was found in the line overexpressing GGP [201]. A kiwi gene coding for GGP, initially tested with good results in _Arabidopsis_[138], has been overexpressed in three crops, leading to a vitamin C increase of six-fold in tomato, three-fold in potato, and two-fold in strawberry; however, in tomato, GGP overexpression has led to some morphological fruit alterations, such as seed loss [140]. The GGP gene of acerola, a well-known crop with high vitamin C content, under the control of a leaf-specific promoter, has been overexpressed in rice, increasing the foliar content up to 2.5-fold, which did not cause morphological changes and conferred multistress tolerance [202]. Interestingly, also, the editing of the uORF, which controls the translation of the GGP2 gene in lettuce and tomato, increases the vitamin C content by 150% and confers tolerance to oxidative stress, providing a good strategy to obtain transgene-free lines with improved vitamin C [203; 204]. Moreover, in apple, three paralogs of GGP, colocated in ASC-QTL clusters and, specifically, the GGP1 allele, play a key role in the regulation of vitamin C content in fruits. This suggests that a single-nucleotide polymorphism of this allele is an excellent candidate for breeding in order to improve vitamin C levels in fruits [205].  
The multigenic approach, based on the coexpression of genes of the d-mannose/l-galactose pathway, represents an interesting strategy to obtain high levels of vitamin C in crops. The transient coexpression of GGP and GME in tobacco leaves caused a seven-fold increase in vitamin C content [138]. In _Arabidopsis_, GGP overexpressing lines had a 2.9-fold enhancement of vitamin C, whereas the double-gene transformation with GGP-GPP and GGP-GLDH led to an up to 4.1-fold vitamin C increase [206]. The contemporary overexpression of acerola GGP, GMP, and GME genes in tomato protoplasts caused an increase in vitamin C content, which was approximately four-fold higher than in wild type [207]. A stable transformation with GME, GMP, GGP, and GPP was obtained in tomato through pyramiding, which is a conventional hybridization that is technically achievable and generates stable inherited target genes [208; 209]. Pyramiding transgenic lines GME \(\times\) GMP and GME \(\times\) GMP \(\times\) GGP \(\times\) GPP showed a substantial increase in vitamin C content in leaves and fruits. Moreover, in these lines, vitamin C transport capability, fruit shape and size, as well as stress tolerance were significantly ameliorated [209].