Organisation and control of ascorbate biosynthesis pathway in plants

Abstract

Ascorbate, also known as vitamin C, plays fundamental roles in human health. However, we are unable to synthesise ascorbate due to a series of accumulated mutations in the last enzyme of the pathway. Therefore, fruits and vegetables become the dietary source of ascorbate, hence being called a vitamin. In plants, ascorbate plays a plethora of roles provinding both biotic and abiotic stress resistance in plants. Although it is known that VTC2 (GDP-L-Galactose Phosphorylase) is the bottleneck of the pathway, little information is available on the basis of how it happens. In this thesis, I confirmed it by several two-by-two combinations as well as expressing the whole pathway. Furthermore, I have shown that this translation fusions are functional and locate in cytosol and nuclei, with the exception of GME and GLDH. Since ascorbate is essential in the response to stress in plants, and the enzymes involved in its biosynthesis locate in the same subcellular region, it was tempting to suggest that they may associate like in other pathways. Our protein-protein association assays suggest that they associate, further supported by the fact that the first and the last cytosolic enzymes immunoprecipitate together, although it deserves further investigation. Since VTC2 was found to be limiting ascorbate biosynthesis, we sought to understand the basis behind this fact. We found that the level of VTC2 expression is very low compared to the expression of the other components of the pathway. However, it was high enough to complement vtc2 phenotype therefore suggesting that keeping its protein amount little expressed in the first control point of the pathway. In addition, we observed that VTC2 is degraded rapidly just after entering the night period, which was further elucidated to be controlled by 26S proteasome.