# Metabolism and Regulation of Ascorbic Acid in Fruits
## 2 Metabolic Pathway of Ascorbic Acid
### Biosynthesis of Ascorbic Acid  
The AsA synthesis has been demonstrated to occur in the mitochondria via several proposed routes. The L-galactose pathway, also called the Smirnoff-Wheeler pathway, is the major biosynthesis pathway [31]. _Arabidopsis_ is a model plant for gene function research, and most genes and enzymes involved in AsA biosynthesis have been well characterized and described. In the L-galactose pathway, D-glucose-6-phosphate is first converted to D-fructose-6-phosphate by phosphoglucose isomerase (PGI) [4]. Then phosphomannose isomerase (PMI) catalyzes the conversion of D-fructose-6-phosphate to D-Mannose-6-phosphate, which is then converted to D-mannose-1-phosphate by phosphomannoutatase (PMM) [32; 33]. GDP-mannose pyrophosphorylase (GMP), named _VTC1_ in _Arabidopsis_, catalyzes a rate-limiting step in AsA synthesis that converts D-mannose-1-phosphate to GDP-D-mannose [34]. GMP gene family has been identified in several horticultural crops, including tomato, apple, and pear [35; 36; 37]. In the next step, GDP-D-mannose3\({}^{\prime}\), 5\({}^{\prime}\)-epimerase (GME) catalyzes GDP-D-mannose conversion to GDP-L-galactose [38]. The first dedicated step in the L-galactose pathway is accomplished by GDP-L-galactose-phosphorylase (GGP), which converts GDP-L-galactose to L-galactose-1-phosphate [39]. In _Arabidopsis_, GGP is encoded by the _VTC2_ and _VTC5_ gene and low content of AsA is presented in _vtc2_ and _vtc5_ mutants [40]. Then the removal of the phosphate group by L-galactose-1-phosphate phosphatase (GPP) to L-galactose-1-phosphate produces L-galactose [41]. Although GPP in _Arabidopsis_ (encoded by the _VTC4_) has phosphatase activity, AsA is still accumulated in vtc4  
Figure 1: The AsA metabolic pathway in plants. The main pathways for ascorbic acid synthesis in plants are L-galactose pathway, Myoinositol pathway, L-glucose pathway, and D-galacturonate pathway. Alase, aldono-lactamase; AO, ascorbate oxidas; APX, ascorbate peroxidase; DHA, dehydro-drosacorbic acid; DHAR, dehydrodrosacorbate reductase; GalIUR, D-galacturonate reductase; GDH, L-galactose dehydrogenase; GGP, GDP-L-galactose-phosphorylase; GLDH, L-galactono-1,4-lactone dehydrogenase; GME, GDP-D-mannose3\({}^{\prime}\), 5\({}^{\prime}\)-epimerase; GMP, GDP-mannose pyrophosphorylase; GPP, L-galactose-1-phosphate phosphatase; GSH, glutathione; GSH, oxidized glutathione; GuILO, L-gallono-1,4-lactone oxidase; IMP, myoinositol monophosphate; MDHA, monodehydroacorbic acid; MDHAR, monodehydroacorbate reductase; MIOX, myoinositol oxygenase; MIPS, myoinositol phosphate synthase; PGI, phosphoglucose isomerase; PMI, phosphomannose isomerase; PMM, phosphomannomutase. mutants, suggesting that GPP is not a key rate-limiting enzyme in AsA biosynthesis [42; 43]. Lastly, L-galactose dehydrogenase (GDH) converts L-galactose to L-galactono-1,_A_-lactone, which is finally restored to AsA by L-galactono-1,_A_-lactone dehydrogenase (GLDH) [41; 44]. Almost all enzymes involved in AsA biosynthesis are located in the cytosol, but GLDH is located in the mitochondrial inner membrane, indicating that AsA is finally synthesized in the mitochondria [45; 46]. In plants, the L-galactose pathway is the major but not the only pathway leading to AsA synthesis. Studies have found that, in addition to catalyzing GDP-D-mannose to produce GDP-L-galactose, GME can also catalyze GDP-D-mannose to GDP-L-glucose [47]. In the L-glucose pathway, GDP-L-glucose is successively converted to L-Gulose-1-phosphate, followed by L-Gulose, which can be converted into L-gallono-1,_A_-lactone [48]. The L-gallono-1,_A_-lactone is finally converted to AsA by L-gallono-1,_A_-lactone oxidase (GuILO), a paralogous gene of GLDH [49]. Two GuILO genes in _Arabidopsis_ (_AtGuILO3_ and _AtGuILO5_) have been characterized [50]. However, there are almost no reports on the characterization of GuILO in fruits. L-gallono-1,_A_-lactone oxidase is not only the last precursor of AsA biosynthesis in L-glucose pathway, but also the last precursor of AsA biosynthesis in the myoinositol pathway [51].