The Function Of GDP-mannose Pyrophosphorylase OsVTC1-1 And OsVTC1-3 In The Biosynthesis Of AsA And The Response To Stress In Rice

The D-Man/L-Gal pathway is recognized as the predominant pathway of L-ascorbic acid(AsA) biosynthesis in plants. GDP-mannose pyrophosphorylase(GMPase) is the first key enzyme in L-galactose pathway and plays an important role in regulating AsA biosynthesis. The biological information analysis of rice genome revealed that there were three different GMPase genes in rice genome, named OsVTC1-1, OsVTC1-3 and OsVTC1-8. Our previous studies had demonstrated that three rice GMPase homologous genes had different roles in AsA biosynthesis. OsVTC1-1 played a main role in regulating the synthesis of AsA in rice; the function of OsVTC1-3 in AsA synthesis was not clear; while OsVTC1-8 did not participate in AsA biosynthesis. These results further suggested that there should be different mechanisms in the regulating AsA synthesis between rice and Arabidopsis thaliana. To illuminate the mechanism of rice GMPase genes in regulating AsA biosynthesis, we made a further study on the function of OsVTC1-1 and OsVTC1-3 in rice AsA synthesis and stress responses.We found AsA content was significantly decreased in the leaves of OsVTC1-1 interference plants, while AsA content in roots of OsVTC1-1 interference plants had no significant difference with the wild type. On the contrary, AsA content was obviously decreased in the roots of OsVTC1-3 interference plants, while the AsA content in leaves of OsVTC1-3 interference plants did not show difference with the wild type. These results showed that different to Arabidopsis, GMPase homologous genes in rice were responsible for AsA synthesis in different parts of rice. In plants, light can regulate the biosynthesis of AsA. Studies showed that continued lighting could promote AsA synthesis in leaves of plants, and continuous dark inhibited AsA synthesis in leaves of plants. We also found that decreasing the transcript level of OsVTC1-3 did not significantly affect the synthesis of AsA induced by light and dark treatments; on the contrary, interfering the expression of OsVTC1-1 significant inhibited the biosynthesis of AsA regulated by continuous lighting and darkness. The above results suggested that OsVTC1-1 was involved in the synthesis of AsA in rice leaves, depending on lighting; OsVTC1-3 was mainly involved in the regulation of AsA synthesis in rice roots.GMPase has an important role in the plant in response to stress responses, such as high salt, drought or high/low temperature. Plants can adjust the responses by regulating the enzyme activity of GMPase in transcript level and post-transcript level. Here, we found that the expression of OsVTC1-1 was induced by salt(NaCl), osmotic stress(PEG) and oxidative stress(MV), but the expression of OsVTC1-3 was only induced by osmotic stress(PEG), suggesting that OsVTC1-1 and OsVTC1-3 also play different roles in the response to environment stresses.The further study was found that impairing the expression level of OsVTC1-1 reduced salt tolerance of rice, and overexpression of OsVTC1-1 in Arabidopsis vtc1-1 significantly improved the salt tolerance of vtc1-1. In addition, OsVTC1-1 RNAi lines decreased the production of rice due to reduced number of tillering and panicle, and seed setting rate under salt stress. The effects of OsVTC1-1 on the redox state during the salt response were also examined. We found that OsVTC1-1 RNAi lines accumulated more ROS and MDA, decreased chlorophyll content after salt stress. These results showed that the regulation of OsVTC1-1 in response to salt stress was involved in scavenging excess ROS accumulated under salt stress to improve the salt tolerance of rice. Beyond that, we found that the supplement of exogenous AsA restored the salt tolerance of OsVTC1-1 RNAi lines, suggesting that OsVTC1-1 regulate the tolerance of rice to salt stress relying on AsA synthesis.The study about the function of OsVTC1-3 in response to osmotic stress showed that the expression of OsVTC1-3 was induced by osmotic stress. The growth of roots of OsVTC1-3 RNAi plants and mutant osvtc1-3 were more obviously inhibited than that of wild type, while the growth of roots of OsVTC1-3 overexpression plants was better than that of wild type under osmotic stress. In addition, we also found that the supplement of exogenous AsA partly restored the tolerance of the roots of OsVTC1-3 RNAi lines and osvtc1-3 to osmotic stress, indicating that OsVTC1-3 was involved in the regulatory to growth of rice through AsA pathway and non AsA biosynthesis pathway in response to osmotic stress.