ABSTRACTCHLOROPLAST LIPID IMPORT AND METABOLISM, AND THEIR EFFECT ON CHLOROPLAST STABILITYByAnna HurlockMuch is already known about plant lipid metabolism and the basic enzymes required for lipid assembly. However, lipid exchange between subcellular compartments remains a challenging area, which this thesis helps to address. Currently, it is known that the chloroplast synthesizes fatty acids that are subsequently used by lipid biosynthetic pathways in both the chloroplast and the endoplasmic reticulum. Fatty acid export to the endoplasmic reticulum (ER) and lipid transport from the ER to the chloroplast are both known to occur, but the participating membrane proteins and transporters have proven to be difficult to work with due to their hydrophobic nature. Many in vivo, genetic, and in vitro techniques have been employed to carry out these studies, but the field has reached a point at which new creative techniques need to be developed in order to answer the remaining questions regarding the nature of transported lipid substrates and the mechanisms of intermembrane lipid transfer. In this thesis, both in vivo and in vitro methods were developed in order to study lipid transport, acyl editing, and other aspects of lipid metabolism for which our knowledge is incomplete. An in vivo method using a "tag and track" approach introducing an ER-located fatty acid desaturase into Arabidopsis, which is distinct from any of the native fatty acid desaturases, led to a subtle modification of the acyl group composition of phosphatidylcholine. This "tag and track" approach proved useful in studying lipid trafficking with high sensitivity in different lipid mutant backgrounds affected in lipid trafficking and assembly. It clearly demonstrated the import of lipid precursors from the ER for phosphatidylglycerol biosynthesis and extensive acyl editing of chloroplast lipids as described in Chapter 2. In vitro methods to study lipid exchange between different membranes using isolated chloroplasts and envelope membranes from different lipid trafficking mutants with either radio- or fluorescently labeled lipids as described in Chapter 3, yielded promising results, further suggesting candidates for lipid molecular species that might be transported from the ER to the chloroplast. However, there were many caveats with the interpretations of the results using these methods that will require further consideration. One example is the differing stability of isolated chloroplasts from different lipid mutants which affected the interpretation of the results. On the other hand, the discovery of differences in the stability of isolated chloroplasts for different lipid mutants, as described in Chapter 4, provides a new avenue to study the specific physiological effects of altered chloroplast lipid composition. As a whole, the work presented here not only yielded new information about plant lipid metabolism, but it also provides a new tool set for further studies of lipid trafficking and lipid metabolism in general.
Read
