| While seeds often contain the bulk of a plant's store of fatty acids in the form of triacylglycerols, fatty acids are found throughout plants and fatty acid synthesis (FAS) is one of the primary metabolic pathways. Some plants make "unusual" fatty acids, stored almost exclusively in seeds, which have desirable industrial properties. Factors limiting the use of these oils for industry are (1) plants may not be suitable for crop use and (2) seed may have neither enough total oil, nor a high enough proportion of the unusual fatty acid in the oil, to be cost-effective for industrial use. Current efforts to manipulate both quality and quantity of oil have had mixed results. This is due, in part, to our incomplete knowledge about the components and regulation of FAS in plants. One goal of this thesis has been to extend our understanding of how plants regulate FAS. In plants de novo FAS occurs primarily in plastids and the rate of FAS is regulated in part by acetyl-CoA carboxylase (ACCase). The dicot ACCase investigated in this thesis contains four subunits, one of which, AccD, is encoded on the plastidial genome.;This work presents new data about the regulation of ACCase. In lysates of light-incubated chloroplasts, ACCase activity is at least two-fold higher than from dark-incubated ones. Additionally, acetyl-CoA activates ACCase in a chloroplast lysate. This activation occurs at 10 muM acetyl-CoA, which is similar to the acetyl-CoA concentration found in chloroplasts. When ACCase is assayed in vitro under conditions which simulate the in vivo concentrations of metabolites, ACCase activity is five- to ten-fold lower than that required to sustain known in vivo rates of FAS. Thus, we still have an incomplete understanding of factors which regulate this enzyme.;This thesis also presents a model of the major initial reactions of FAS. This model was built using Stella II software, pool sizes of FAS metabolites, kinetic characterizations of FAS enzymes, and common sense. The simulation allows enzyme expression to be manipulated, either singly or jointly, between 0- and 25-fold. The model's predictions match several results reported from over- and underexpression experiments---even when those results have been non-intuitive. The model predicts that the set of FAS enzymes which limit or co-limit flux vary depending on illumination and products (18:1DeltaA9 or 16:0). The model also predicts a method involving concurrent overexpression of three enzymes by which total FA exported from plastids can be increased.;Finally, this work documents unsuccessful attempts to relocate AccD to the nuclear genome, reports the sequence of Oenothera hookeri AccD, and maps AccD on the plastidial genome of O. hookeri. |
