Analysis of structure-function of plant seed oleosins

Seed oil bodies are organelles composed of a matrix of triacylglycerol surrounded by a phospholipid monolayer embedded with proteins called oleosins. Oleosins are uniquely targeted to oil bodies and once inserted in these organelles, they are incredibly stable. Oleosins contain a conserved central hydrophobic domain with 72 residues, composing the longest lipophilic stretch reported for a natural protein. Although it is clear that this domain is crucial for the targeting to oil bodies, the significance of its length or primary structure has never been explored.; Oleosins are present nearly in all angiosperms, suggesting that during evolution they provide a survival advantage. It has been tacitly assumed that these proteins maintain oil bodies as small single units preventing their coalescence, especially during seed desiccation. This assumption was based on indirect observations on the size of oil bodies in different species and on in vitro experimentation. However, no direct evidences have been reported to support this theory.; The main purposes of this thesis were: (i) to explore the structural requirements of the hydrophobic domain to provide correct targeting and stability of oleosins in the oil bodies; (ii) to provide direct evidences that oleosins are the primary determinants of the size of oil bodies in seeds and evaluate the importance of maintaining these organelles as small individual units.; The major findings include the detection of a small region in the hydrophobic domain responsible for efficient targeting to oil bodies. This result highlights the importance of the primary structure and not the length or hydrophobicity of the central domain. Furthermore it was established for the first time a direct relationship between the length of the hydrophobic domain and the affinity of oleosins to oil bodies. It was also directly demonstrated that the accumulation of oleosins dictates the size of oil bodies in seeds. Changes in the size of oil bodies caused disruption of storage organelles altering accumulation of seed lipids and proteins, causing also a delay in germination. In conclusion, these findings provide a better understanding of the structure-function of seed oleosins.