1a.Objectives (from AD-416)
Understanding plant oil biosynthesis will help to create new oilseed crops with value-added properties to replace petroleum-based compounds in products such as nylons, plastics, inks, and dyes. The biosynthesis and accumulation of biopolymers such as oils is accomplished by highly organized enzyme complexes. The coordination of various components in the complexes is essential for the enzymatic efficiency and specificity. The combinations of various isoforms rather than individual enzymes may be the determining factor for the synthesis of specific components of biopolymers. The concept of enzyme complexes in lipid biosynthesis is supported by the isolation of a cytosolic 10S triacylglycerol biosynthetic multienzyme complex from oleaginous yeast. Extensive attempts to purify oil-synthesizing enzymes have only limited success. The purification difficulty itself may imply that individual enzymes are probably associated with other proteins/enzymes in the native environment. Current studies are focused on the identification and characterization of individual enzymes in plant lipid biosynthesis. However, much needs to be learned about the nature of the oil biosynthetic machinery in plants. The objective of this proposal is to determine the exact nature of protein components in oil bodies and their relevance in the synthesis and accumulation of oils in tung seeds.
1b.Approach (from AD-416)
Enzyme complexes will be identified by a number of approaches including blue native PAGE, size exclusion chromatography, and mass spectrometry. In this study, oil bodies will be isolated from developing seeds of tung tree. The protein profiles will be revealed by electrophoresis. Mass spectrometry will be used to identify individual proteins. Antibodies will be raised against native and/or recombinant proteins of oil bodies. These antibodies will be used to detect the proteins of interest during different stages of seed development and the subcellular localization of the proteins in tung seeds.
Recombinant diacylglycerol transferases, an enzyme involved in tung oil production, from tung tree were over-expressed in the bacterium E. coli. The fusion proteins were partially purified from E. coli and will be further purified and used as antigens for raising antibodies in rabbits (see rationale in approach). The program was monitored via email.