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The role and behavior of Arabidopsis thaliana lipid transfer proteins during cuticular wax deposition DeBono, Allan


The primary aerial surfaces of terrestrial plants are coated with a protective hydrophobic layer comprising insoluble and soluble lipids. The lipids are known collectively as cuticular wax. To generate the waxy cuticle during elongative growth, plants dedicate half of the fatty acid metabolism of their epidermal cells. It is unknown how cuticular wax is exported from the plasma membrane into the cell wall, and eventually, to the cuticle at the cell surface. I hypothesized that lipid transfer proteins (LTPs) were responsible for plasma membrane to cell wall transport of cuticular lipids. Using an epidermis-specific microarray, I identified five candidate Arabidopsis LTPs. I discovered that mutations in gene At1g27950 result in a stem wax phenotype: reduced cuticular lipid nonacosane resulting in reduced total wax compared to wildtype. This gene encodes a glycosylphosphatidylinositol (GPI)-linked LTP and thus was named LTPG. In contrast, to LTPG, no detectable wax phenotype was found in mutants for classical LTPs. In phylogenetic analyses, these LTPs clustered into a weakly related group that I named LTPAs. In an attempt to overcome genetic redundancy I made double and triple mutants from the candidate LTPAs. None of these mutants displayed detectable changes in wax compared with wildtype. Using live cell imaging, I showed that LTPG is localized to the epidermal cell plasma membrane and the cell wall and accumulates non-uniformly on the plant surface. I employed fluorescence recovery after photobleaching to demonstrate that, in the plasma membrane, LTPG is relatively immobile and exhibits a complicated recovery, the latter appears linked to the flux of cuticular lipids through the plasma membrane. LTPG accumulates over the long cell walls of stem epidermal cells and this protein moves when observed over 1 min intervals. I created a GPIlinked LTPA and demonstrated that it can rescue the ltpg-1 mutation. I demonstrate that LTPG is required for wax export by associating with the plant cell wall. This is the first experimental evidence linking the lipid transfer function of a plant LTP to a biological role, which in this case is lipid movement through the cell wall to the cuticle.

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