| Suberin is a biopolymer deposited in the cell walls of specific internal and external tissues layers of plants, such as root endodermis, root and tuber peridermis, and seed coats. Suberin serves as a hydrophobic barrier controlling water and ion transport in these tissue layers. It is also a wound-induced antimicrobial barrier. The suberin polymer consists of phenolics, glycerol, and a variety of fatty acid derivatives, including C18:0-C22:0 primary fatty alcohols. In this thesis, I report on the identification and characterization of fatty acyl reductase (FAR) enzymes that synthesize primary alcohols associated with suberin. Three genes encoding alcohol-forming fatty acyl reductases (FARs) from the model plant Arabidopsis thaliana, FAR1, FAR4 and FAR5, were found to be expressed at sites of suberin deposition. Suberin composition in loss-of-function far mutants was uniquely modified; specifically, C18:0-0H was reduced in far5, C20:0-0H was reduced in far4, and C22:0-0H was reduced in far1, indicating that FAR1, FAR4, and FARS each generates a distinct chain-length of fatty alcohol found in root, seed coat, and wound-induced suberin. Heterologous expression of FAR1, FAR4, and FARS in yeast and Nicotiana benthamiana leaves confirmed their preferred substrate specificity to C22:0, C20:0, and C18:0 acyl chains, respectively. Double and triple far mutant lines were developed using conventional genetic crossing and artificial microRNA (amiRNA)-mediated gene silencing. The double mutants far1 far4, fan far5, and far4 far5* (*denotes amiRNA line) had reduced levels of both respective chain lengths of fatty alcohols in root and seed coat suberin, and the triple mutant far1 far4 far5* had reduced levels of all three C18:0 to C22:0 primary fatty alcohols.;The C18:0-C22:0 alkyl hydroxycinnamates of suberin-associated root waxes were also significantly reduced in these lines. The triple mutant seeds were found to have increased seed coat permeability and increased sensitivity to abscisic acid (ABA) hormone during germination. Domain swaps of the FAR1, FAR4, and FARS proteins and their heterologous expression in yeast showed that the substrate specificities of these enzymes are largely governed by the region between amino acid positions 225-385. Transient expression of a fusion of Arabidopsis FAR1 with green fluorescent protein in Nicotiana benthamiana leaves revealed localization to the endoplasmic reticulum. FAR1, FAR4, FARS and other suberin biosynthetic genes showed differential inductions at the transcript level when subjected to various abiotic stresses (ABA, cold, salt, osmotic, and wounding). All the genes were highly induced by abscisic acid, while they showed the least amount of induction by cold stress. Altogether, the results reported in this thesis have advanced our understanding of the suberin biosynthetic pathway and its regulated deposition. This knowledge may lead to novel approaches for the development of drought, salt, and pathogen resistant crop plants. Furthermore, the engineering of FAR enzymes with desired substrate specificities would enable the production of high-value fatty alcohols or wax esters in an industrial oilseed crop or engineered microbe. |
