Enzymatic Mechanisms And Regulatory Pathways Underlying The Formation Of The Tomato (Solanum Lycopersicum) Fruit Cuticle

Abstract

The plant cuticle, a complex hydrophobic membrane covering the aerial surfaces of land plants, has many critical functions, including limiting desiccation and preventing microbial infection. The cuticle consists of a polyester matrix of fatty acids, termed cutin, which is covered by and impregnated with a range of organic solvent soluble waxes. Genetic and biochemical strategies have uncovered many of the genes involved in wax and cutin precursor biosynthesis, but far less is known about the mechanisms that mediate the regulation, trafficking and subsequent assembly of cuticle components. This dissertation describes the results of three studies that focused on cuticle regulation and assembly, using tomato (Solanum lycopersicum) as an experimental system. In the first study, characterization of the cutin deficient 1 (cd1) tomato mutant revealed that CD1 encodes for an extracellular GDSL-motif lipase/hydrolase family protein that is preferentially expressed in developing organs with a high rate of cuticle production. Biochemical analysis demonstrated that CD1 polymerizes the cutin monomer 2-mono(10,16-dihydroxyhexadecanoylglycerol) (2MHG) in vitro. Collectively these results suggest that CD1 catalyzes cutin polymerization and is a cutin synthase. The second study focused on tomato Cutin Deficient 2 (CD2), a putative HD-ZIP IV transcription factor. CD2 is widely expressed in various organs and tissues; however, the preferential accumulation of CD2 in the fruit epidermis is consistent with a role in regulating the expression of cuticle biosynthesis related genes. Indeed, an analysis of genes that are differentially expressed in the epidermis of cd2 mutant and wild type fruit revealed numerous genes implicated in cuticle biosynthesis, transport and assembly. CD2 is also potentially important for other aspects of plant development as genes involved in lipid metabolism, anthocyanin biosynthesis, fruit ripening and stress responses were also differentially expressed in cd2 fruit. The third study focused on another potential regulator of cuticle biosynthesis, the hormone abscisic acid (ABA). The leaf cuticle of ABA-deficient tomato mutants was observed to be thinner and to have structural abnormalities. Additionally, the cuticular wax composition was altered in the mutant genotypes compared to the corresponding wild types, with a relative increase in levels of alkanes shorter than C31 and isoalkanes, relative decreases in the abundance of alkanes  C31, the anteisoalkane C32, amyrins and taraxasterol. Taken together, these results show that ABA is necessary for cuticle formation during leaf ontogeny.