## 4. ascorbate and Glutathione  
### Biosynthesis of ascorbate  
Ascorbate, also known as vitamin C or L-ascorbic acid (Asc), is an important biologically active substance. Despite its importance, the pathway of its biosynthesis in plant cells was described only in 2007 for _A. thaliana_ and many aspects concerning the regulation of the Asc biosynthetic pathway still need clarification. Plants synthesize Asc by four alternative routes: D-mannose/L-galactose (Smirnoff-Wheeler) [254], L-Gulose, Myo-inositol, and D-Galacturonic pathways [255; 256; 257]. The Smirnoff-Wheeler pathway is often referred to as the primary or main pathway of Asc biosynthesis in plants [258]. However, in plant species which produce fruits with high level of vitamin C, the other known pathways for Asc biosynthesis can also be predominant. For example, the L-galactose pathway is the main one in peaches and kiwis, while the D-galacturonic pathway is dominant in grapes and strawberries [259]. Additionally, in a number of plants, such as citrus or tomato, the predominant biosynthetic pathway can shift as the fruits ripen. Most enzymes of the D-mannose/L-galactose pathway are localized in the cytosol. This pathway starts from the conversion of D-glucose into L-galactose through eight stages, which are necessary only to change the spatial position of the hydrogen and hydroxyl groups at the fourth carbon atom in the structure of these carbohydrates (Figure 5). The first step is the transformation of D-glucose to D-glucose-6-phosphate, followed by its transformation into D-fructose-6-phosphate by phospholmannose isomerase (PMI). D-fructose-6-phosphate is further converted into D-mannose-6-phosphate also by phospholmannose isomerase. D-Mannose-6-phosphate is converted into D-Mannose-1-phosphate by phospholmannose mutase (PMM). D-Mannose-1-phosphate is then transformed to GDP-D-Mannose by GDP-D-mannose pyrophosphorylase (GMP). Studies have indicated a correlation between _PMII_ gene expression in Arabidopsis and ascorbate levels [260]. The same authors have shown that the knockdown of _PMII_ has led to decreased Asc levels. In transgenic tobacco overexpressing the _PMM_, _GDP_, and _GMP_ genes derived from _Malpighiq glabra_ (a plant known for its remarkably high vitamin C content), the Asc levels were found to be two-fold higher than in wild-type plants [261; 262; 263]. The information about the genes encoding the main enzymes for Asc biosynthesis in _A. thaliana_ is in Table 4.  
_Antivizati  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Genes** & **Enzyme and Its Alternative Names in Arabidopsis** & **Functions** \\ \hline \multirow{3}{*}{_At4g23100_} & \(\gamma\)-glutamylcysteine synthetase, \(\gamma\)-ECS, ATECS1, ATGSH1, & \multirow{3}{*}{Catalysis of the first, and rate-limiting, step of glutathione biosynthesis.} \\  & & Cinnamyl Alcohol Dehydrogenase Homology 2, & \\  & Glutamate-Cysteine Ligase, GSH1, GSHA & \\ \hline \multirow{3}{*}{_At5g27380_} & \multirow{3}{*}{Glutathione synthetase 2, ATGSH2, GSH2, GSHB} & Binding \(\gamma\)-glutamylcysteine and glycine together to form glutathione \\ \hline \multirow{2}{*}{_At4g29130_} & \multirow{2}{*}{Hexokinase 1, HXK1, ATHXK1, GIN2} & \multirow{2}{*}{Hexose phosphorylation activity} \\ \cline{1-1} \cline{2-2}  & & \\ \cline  
Secondary metabolic reactions that facilitate the transformation of GDP-L-galactose into Asc (Figure 5) are active mainly in mature plants. The rate-limiting step in this metabolic pathway of vitamin C synthesis is the reaction that directly produces Asc, controlled by the enzyme GDP-L-galactose phosphorylase. The enzyme GDP-D-mannose-3\({}^{\prime}\),5\({}^{\prime}\)-epimerase (GME) controls the mutual transformation of GDP-D-mannose and GDP-L-galactose. In young, actively growing plants, most products from this reaction contribute to primary metabolic reactions, specifically, the biosynthesis of cell wall polysaccharides [278; 279]. This suggests that the initial stages of this metabolic pathway are mainly associated with the growth of plant organs.