## 4 Discussion  
AsA biosynthesis in plants predominantly involves the conversion of D-glucose-6-phosphate to AsA via GDP-mannose and GDP-L-galactose [5]. Evidence for the importance of the L-galactose pathway in AsA production has been studied in several mutants. Many studies showed that the mutants affected the AsA levels, plant growth and development and abiotic stress tolerance. _vtc1_ mutant, which is a knock-down mutant of _GDP-mannose pyrophosphylase_ (_VTC1_) gene, contained 25% AsA levels compared to wild type [7]. Mutants in _VTC2_ (_vtc2-1_ and _vtc2-2_), which encodes GDP-L-galactose phosphorylase, contain \(\sim\)20% of wild type AsA and are sensitive to ozone in Arabidopsis [43]. Similarly, the AsA content and seed germination rate in Arabidopsis was decreased in the T-DNA knockout _vtc4_ mutant (_L-galactose-1-phosphate phosphatase_). Although the L-galactose pathway was controlled by several genes, _VTC2_ gene was identified as the first rate-limiting step of the L-galactose pathway [14]. However, _VTC1_ gene is also required for ascorbate production in plants. For example, the AsA production of whole rice plants reduced \(\sim\)30-60% in _OsVTC1-1_ RNAi lines and decreased salt tolerance under salt stress [8; 11]. In potatoes, antisense down-regulation of _VTC1_ gene reduced AsA content and GMPase activity [44]. Overexpression of _GMP_ or _VTC1_ gene increases the GMPase activity and leads to a two- to four-fold increase in the AsA content in leaves [45]. In this study, expression levels of eight genes in the L-galactose pathway and glutathione peroxidase (_OsGPX1_) gene in wild type and _OsVTC1-1_ RNAi lines were examined. Our results showed that _OsVTC1-1_ RNAi lines led to a reduced expression level of _OsVTC1-1_ gene, but it did not affect the expression of _OsVTC1-3_ and _OsVTC1-8_ genes. This result is consistent with a previous study [8]. The mRNA transcript levels of _OsVTC1-1_ and _OsVTC1-3_ genes showed the highest expression in leaves and roots, respectively, while _OsVTC1-8_ expression level was low in both leaves and roots. The transcript levels of _GDP-mannose-3',5'-epimerase_ (_OsGME1_, _OsGME2_) and _OsVTC2_ also decreased in RNAi lines. There have been several studies on the mutation of AsA-related genes. _vtc1_ mutant had decreased AsA to 70% of wild type levels in Arabidopsis [10]. Overexpression of _GMP_ gene has resulted in higher AsA levels in several plants species, including 2.0-fold in tobacco [46], 1.3-fold in Arabidopsis [47] and 1.7-fold in tomato [48]. On the other hand, the AsA content of _GME_ RNAi lines in tomatoes was decreased by 40-60% [49]. Similarly, AsA content in Arabidopsis _vtc2-4_ and _vtc2-1_ mutant lines was 70% lower than in wild type [50]. There was no effect on the expression of _L-galactose-1-phosphate phosphatase_ (_OsVTC4_) and _L-galactose dehydrogenase_ (_OsGDH_) genes. _VTC4_ and _GDH_, which are downstream genes in the L-galactose pathway, are not a major rate-limiting step in the L-galactose pathway in rice. VTC4 has a wide substrate specificity and is involved in the synthesis of both myo-inositol and AsA [16]. In tomatoes, the AsA content was unchanged in _GPP_ (_VTC4_) overexpression line [51]. The AsA concentration in leaves was not affected in _GDH_ over-expression lines of tobacco, although the transgenic lines exhibited a 3.5-fold increase in GDH activity [52]. _GPX_ gene expression also decreased in _OsVTC1-1_ RNAi lines. GPX is an important enzymatic antioxidant to scavenge hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)) and peroxide radicals against oxidative damage. AsA is involved in the detoxification of ROS either directly or through the ascorbate-glutathionee (AsA-GSH) pathway [53; 54]. Previously, [PERSON] et al. reported that a tomato ethylene response factor, also called TERF1, plays a role in regulating ROS