The Analysis About The Role Of Some Related Genes For High Efficiency Synthesis Of Tocopherol In Castor Bean

Tocopherol is one of the two major categories of vitamin E.The other is called tocotrienol.Tocopherols are an important antioxidant organic substance in plants and animals,but they can only be synthesized by photosynthetic organisms.Tocopherols are synthesized from the initial condensation of the substrates phytl diphosphate?PDP?and homogentisate?HGA?.The studies described here were intended to develop novel ways to increase the amounts of each substrate in oilseeds for increased tocopherol production.It has been estimated that⁸0%of PDP substrate needed for tocopherol synthesisin photosynthetic green seeds?e.g.,rapeseed,Arabidopsis thaliana?is derived from the chlorophyll degradation pathway,and²0%is derived by the direct conversion of geranylgeranyl diphosphate?GGDP?to PDP by geranylgeranyl reductase?GGR?.In this research,we hypothesized that non-photosynthetic?white?seeds?eg.castor bean,peach kernels?primarily produce PDP for tocopherol synthesis by GGDP conversion to PDP by GGR activity rather than through the chlorophyll degradation pathway.We conducted experiments to test this hypothesis with castor bean?Ricinus communis?.This plant is an important oilseed that has white endosperm and embryo during seed development.The content of vitamin E in non-photosynthetic castor bean seeds is at least 1.7 times than photosynthetic soybean seeds and³2%higher than that in green rapeseed.Therefore,we speculated that the castor bean GGR enzyme should have a stronger ability to generate PDP.We studied the biochemical characteristics of GGR enzyme in non-photosynthetic castor bean seeds to understand the metabolic mechanism of its efficient accumulation of tocopherols and to use this information to increase tocopherol content in other oilseeds,such as rapeseed.We also studied hydroxyphenylpyruvate dioxygenase?HPPD?the enzyme that synthesizes HGA and HGA phytyltransferase?HPT?that catalyzes the HGA and PDP condensation as the first step in tocopherol biosyntheseis.Collectively,the results obtained from my research provides new insights into tocopherol biosynthesis that can be used for vitamin E biofortification of Brassica crops.The main results obtained from my research are as follows:1.The whole development period of castor bean from flowering to maturity was marked and the embryos,endosperm and leaves were sampled.The determination of vitamin E was performed on all samples.The results showed that vitamin E was accumulated during the entire development of castor seeds.2.Two cDNAs were isolated from mRNA of developing castor bean seeds for geranylgeranyl reductase,designated RcGGR1 and RcGGR2.Expression of these cDNAs under control of the constitutive cauliflower mosaic virus 35S failed to complement the Arabidopsis ggr mutant in 60 independent independent lines for35S-RcGGR1 and 20 independent lines for 35S-RcGGR2 recovered,whereas expression of the Arabidopsis GGR cDNA was able to complement the mutant while the RcGGR1and RcGGR2 wasn‘t complement the mutant.3.The castor bean RcHPT and Arabidopsis thaliana AtHPT cDNAs were isolated,placed under control of seed-specifc promoters,and transformed into Arabidopsis.Seeds from transformed lines with the AtHPT had increased tocopherol content,but little or no changes in tocopherol content were detected in seeds from lines expressing RcHPT.4.Expression of cDNAs for the castor RcHPPD and Arabidopsis AtHPPD genes in Arabidopsis under control of seed-specific promoters resulted in increased vitamin E tocopherol content in seeds from the transformed plants.5.Based on the previous studies,we also co-expressed AtHPT,RcHPT,AtHPPD,RcHPPD,AtGGR,and RcGGR in different combinations in Arabidopsis under control of seed-specific promoters.We are currently awaiting the collection of T₂ seeds from these plants to measure tocopherol concentrations.