Metabolic Regulation Of Vitamin E And Vitamin C In Rice

Both vitamin E and C are the mian parts of the plant antioxidant systerm, and play important regulatory roles in plants. Vitamin E is lipid-soluble antioxidant, which can be separated into two groups, tocopherols and tocotrienols. Tocopherols are widespread in the plant kindom. Tocotrienols, however, not occur so widely in plants than tocopherols and are the primary form of vitamin E in seeds of most monocot species. Vitamin C (ascorbic acid, AsA) is water-soluble antioxidant, and it can only be synthesized by photo synthetic organism including higher plants and cyanobacteria. Humans have lost the ability to synthesize vitamin E and vitamin C, and the primary source of them was provided by plants. Rice is one of the leading food crops worldwide, so it is very important to use genetic engeneering strategy to improve vitamin E and vitamin C content in rice.In this study, with the aim to improve the content or activity of both vitamins, we firstly analyzed the variation and distribution of vitamin E and composition in the seeds among different rice varieties, as well as the expression parterns of vitamin C synthesis related genes in rice. Then, some key genes involved in vitamin E and C metabolic pathway were over expressed in transgenic rice. The main results are as followings.1. Variation and distribution of vitamin E and composition in the seeds among different rice varieties. In this study, a reverse-phase HPLC method was used to simultaneously measure the contents of either tocopherols or tocotrienols in the brown rice, and their contents were compared among18indica and16japonica cultivars. The results showed that the contents of these vitamin E isomers were extremely different between indica and japonica rice, and the mean content of total tocopherol or vitamin E was significantly higher (P<0.01) in japonica rice than in indica rice, while the total tocotrienol content showed no differences between them. The principal isomer of vitamin E was also different between the two subspecies, for example, y-tocotrienol was the predominant compound in indica rice, on the contrary, in japonica rice, the most abundant isomer was a-tocopherol. The ratio of total tocotrienols to tocopherols was also significantly higher (P<0.01) in indica (1.61) than in japonica rice (0.95). Pearson’s correlation analysis demonstrated that a-tocopherol content was positively correlated with a-tocotrienol content in rice. The same relationship was also presented between the contents of γ-tocopherol and y-tocotrienol, but there was a negative correlation between contents of a-and y-isomers. Taking together, all the present results provide some useful information for research on vitamin E metabolism or nutritional improvement in rice.2. Transgenic expression of key genes involved in vitamin E metabolic pathway in rice. OsHGGT, OsHPT and AtTMT were over expressed in an eilte japonica cultivar Wuyujing3(WY3), vitamin E content and compositions were detected by HPLC. In OsHGGT transgenic lines, total tocotrienol contents increased to1.52and1.67folds compared to transgenic controls derived from the empty vectors in brans and endosperms, respectively. The increased tocotrinol contents main due to the increase of y-tocotrienol, while tocopherol contents were not influenced. In OsHPT transgenic lines, total tocopherol contents increased to1.44and2.13folds compared to those of transgenic controls in brans and endosperms, respectively, which mainly due to the increase of a-tocopherol. We also found that the total tocotrienol content was increased slitely in some transgenic lines; Over expression of AtTMT could shif tocopherol and tocotrienol composition in favor of a-tocopherol and a-tocotrienol. As a result, the ratio of a-tocopherol to total tocopherol was0.95and0.98in transgenic brans and endosperms, respectively, while which were0.86and0.89in brans and endosperms from transgenic control with empty vector, respectively.3. Expression analysis of vitamin C synthesis related genes in rice. The Smirnoff-Wheeler (SW) pathway has been proven to be the only significant source of AsA in higher plants. In this study, the coding sequences of AsA biosynthesis and metabolic related genes from Arabidopsis were used as query sequences to search in the rice genome database and we got a series of corresponding homologous genes. Real-time PCR analysis was done to examine the expression patterns of these genes in different tissues as well as in grains at different developing stages in ajaponica rice. The results revealed that these genes expressed in all tested tissues, but their expression was highest in leaves, lower in developing seeds as well as in sheaths, and lowest in roots and stems. Their expressions in young leaves were much higher than in old leaves.4. Transgenic expression of key genes involved in vitamin C metabolic pathway in rice. The genes encoding the enzymes involved in AsA biosynthesis and recycling were over expressed in transgenic rice, and there are many transgenic plants for each construct. The HPLC method was used to detect the AsA content in transgenic rice leaves. The results showed that the AsA content increased up to2.52folds in the leaf of GMP transgenic lines, when compared with the control lines derived from empty vectors. In the GME, GGP, GPP, GDH or DHAR transgenic lines, the AsA content increased to1.69,1.83,1.41,1.8and1.7folds, respectively, when compared with that of transgenic control derived from the empty vectors. GMP was the first key enzyme in L-Galactose pathway, which was regarded as the main biosynthetic pathway of AsA, and our result supported that it plays a more significant role over other genes in AsA metabolism in rice. The present results were useful for us to understand the regulation and function of vitamin C in rice.