The Arabidopsis Vitamin C Content Of Metabolic Network Regulation

Vitamin C (ascorbic acid) is a key component of the plant antioxidant system and it has a number of other proposed physiological roles. Although plants and most animals can synthesize ascorbic acid (AsA), humans lack L-gulono-1,4-lactone oxidoreductase, which is required for the final step in AsA synthesis. Because AsA cannot be stored in the body, the vitamin must be acquired regularly from dietary sources, plants provide the major source of dietary vitamin C for humans. Engineering plant AsA metabolism has now become feasible because of the increased understanding of its biosynthetic pathway and key enzymes.The genes which involved in AsA metabolic pathway, including biothynsis, catabolism and recycling pathway, were cloned with PCR from Arabidopsis thaliana cDNA. Plant expression vectors which harboring these genes were transformed into Arabidopsis thaliana to evaluate their contribution in increasing AsA content.In most of the biothyntic gene transgenic lines, the expression of the target gene was increased on RNA level(real-time PCR). The AsA content was detected using HPLC. The gdpgalppase transgenic lines increased the AsA content to 2.9-fold compared with the blank transformation(CK); the gdpmppase, gdpme, galppase, galdh, gldh transgenic lines increased the AsA conent to 1.32-fold, 1.38-fold, 1.45-fold, 1.21-fold, 1.81-fold compared with CK respectively. This result support that gdpgalppase encode the enzyme which catalyze the first committed step in L-galactose pathway. It is important in AsA metabolic engineering.To depress the catabolic pathway using RNAi, the expression of ao in the transgenic lines was down to 10%-29% compared with CK. The AsA content increased to 1.5-fold, which was similar with most lines above. To enhance the recycling pathway by over-expressing dhar, the AsA content increased to 1.39-fold compared with CK.All aspects of AsA metabolic network were involved in this research. The genes and pathway were evaluated systematically. The existing and potential achievements in increasing AsA production would present a better understanding of the mechanisms internal and external to plants that control AsA accumulation and provide the opportunity for enhancing nutritional quality and stress tolerance of crop plants.