| All higher plants depend on extra cellular lipophilic barriers that control the movement of water, solutes, and gases, and that provide an interface with the biotic and abiotic environment. Cutin and suberin are polyesters of fatty acids that constitute the polymeric matrices of these barriers. Because of their structural complexity and intractability, progress in understanding the structure, enzymology and genetics of lipid polyesters has been limited. Overall, the goal of this study was to identify genes and enzymes involved in the biosynthesis of these biopolymers using the model plant, Arabidopsis thaliana. To aid this goal, a new method was developed for the quantitative analysis of lipid polyester monomers in seeds. Monomers typical of both cutin and suberin were found and most of these monomers are deposited in the seed coat. Distinct temporal patterns of accumulation of individual monomers were observed at different stages of Brassica seed development. Promoter-YFP fusions of cutin and suberin associated genes revealed distinct spatial patterns of expression in seed coat cell layers. Evidence from these studies support the presence of a suberized layer deposited on the outer integument, and of a cutin-like polyester layer associated with the inner integument of the seed coat.;The method developed for seed polyester analysis allowed a reverse-genetics approach that was suitable for screening for genes involved in either cutin or/and suberin biosynthesis. Candidate genes were selected using bioinformatic approaches including transcript co-expression analysis. Among the mutants of preferred genes, a knockout mutant of a cytochrome P450-dependent o-oxidase gene, cyp86A1, showed major reductions in o-oxidized suberin monomers thereby demonstrating its involvement in suberin biosynthesis. Furthermore, this approach was useful to identify a novel acyl-transferase of the BAHD family that is involved in the deposition of ferulate in Arabidopsis suberin. The monomer phenotype of this mutant suggests that the disrupted gene encodes a feruloyl-CoA transferase, the first such enzyme discovered in plants.;In addition, this research work used in planta overexpression approaches to explore the function of two orthologous P450 enzymes; ATT1 from Arabidopsis and PH1 from petunia stigmas. The interchange of these genes was expected to complement the att1 mutant and increase the production of o-hydroxy fatty acids in tobacco stigma polyesters. However, transgenic plants failed to accumulate higher levels of o-hydroxy fatty acids. Although the current assumption is that ATT1 catalyzes the o-hydroxylation step leading to synthesis of cutin precursors, results suggest that they may be able to catalyze further oxidation steps to produce dicarboxylic acids. The hypothesis of the existence of a "metabolon" for polyester synthesis was tested. The combination of these results has provided new insights on how CYP86A enzymes may function in vivo. |
