Suberin-associated fatty Acyl-CoA reductases from Arabidopsis thaliana

Suberin is a biopolymer deposited in the cell walls o f 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 anti­ microbial barrier. The suberin polymer consists o f phenolics, glycerol, and a variety o f fatty acid derivatives, including C18:0-C22:0 prim ary fatty alcohols. In this thesis, I report on the identification and characterization o f fa tty acyl reductase (FAR) enzymes that synthesize prim ary alcohols associated w ith 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 fa r mutants was uniquely modified; specifically, C18:0-OH was reduced in far5, C20:0-OH was reduced in far4, and C22:0-OH was reduced in fa r l , indicating that FAR1, FAR4, and FARS each generates a distinct chain-length o f fatty alcohol found in root, seed coat, and wound-induced suberin. Heterologous expression of FAR1, FAR4, and FAR5 in yeast and Nicotiana benthamiana leaves confirmed th e ir preferred substrate specificity to C22:0, C20:0, and C18:0 acyl chains, respectively. Double and trip le fa r mutant lines were developed using conventional genetic crossing and artificial microRNA (amiRNA)-mediated gene silencing. The double mutants f a r l far4, fa r l far5, and fa r4 fa r5 * (*denotes amiRNA line) had reduced levels of both respective chain lengths of fatty alcohols in root and seed coat suberin, and the trip le mutant fa r l fa r4 fa r5 * had reduced levels o f all three C18:0 to C22:0 prim ary fatty alcohols. The C18:0-C22:0 alkyl hydroxycinnamates o f suberin-associated root waxes were also significantly reduced in these lines. The trip le 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 FAR5 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 w ith green fluorescent protein in Nicotiana benthamiana leaves revealed localization to the endoplasmic reticulum. FAR1, FAR4, FAR5 and other suberin biosynthetic genes showed differential inductions at the transcript level when subjected to various abiotic stresses (ABA, cold, salt, osmotic, and wounding). A ll 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 w ith desired substrate specificities would enable the production of high-value fatty alcohols or wax esters in an industrial oilseed crop or engineered microbe. PREFACE This thesis follows the integrated thesis format and, as such, the main chapters represent work that has been published in a peer-reviewed journal at the time of submission of this thesis (Chapter 2), or w ill soon be submitted for publication. I formally acknowledge the contributions o f the co-authors o f the manuscript that comprise the research chapters o f my thesis. My supervisor, Dr. 0. Rowland contributed his expertise by guiding me in the formulation of the research questions and experimental design, and by assisting w ith the analysis and interpretation of data, and the preparation o f manuscripts fo r each of the research chapters o f my thesis. Chapter 2 is part o f a published research paper: Domergue, F. *, Vishwanath, S.J.*, Joubes, J., Ono, J., Lee, J., Bourdon, M. , Alhattab, R. , Lowe, C. , Pascal, S. , Lessire, R. , and Rowland, 0. (2010). Three Arabidopsis Fatty AcylCoA Reductases, FAR1, FAR4, and FAR5, Generate Primary Fatty Alcohols Associated w ith Suberin Deposition. Plant Physiology, 153: 1539-1554. (*These authors contributed equally to this work). All data including figures and tables are extracted from the above paper and reprinted w ith permission of the American Society of Plant Biologists. Statement of contribution: The experiments reported in the Results section of this chapter were conceived and designed by me, F. Domergue, and 0. Rowland. The manuscript was written by me, F. Domergue, and 0. Rowland, and then updated and modified for thesis format by me. I generated the following data reported in this chapter: ■ FAR promoter: GUS expression analysis of various tissues under normal and wound stress conditions, and root sectioning presented in Figure 2.3. ■ Semi-quantitative RT-PCR analysis o f FAR1, FAR4 and FARS gene induction under wound and salt stress conditions presented in Figure 2.4. ■ Confirmation of T-DNA single mutant lines by semi-quantitative RT-PCR and identification of T-DNA insertion sites by DNA sequencing analysis presented in Figure 2.5. ■ Analysis o f fatty acyl chain profiles o f yeast heterolgously expressing FAR1, FAR4 and FAR5 by GC-FID, presented in Figure 2.10. The other authors' contributions were: ■ J. Ono and J. Lee were Carleton University undergraduate students who first identified the T-DNA fa r mutant lines, which were subsequently characterized by me. ■ J. Ono, J. Lee, and C. Lowe were Carleton University undergraduate students who generated the FAR promoter:GUS constructs and transformed Arabidopsis plants, and then the reported GUS assay and microscopic analyses were performed by me. ■ R. Alhattab, a Carleton University undergraduate student, cloned the FAR4 cDNA and generated the FAR4 yeast expression construct, along w ith prelim inary experimental data on fatty alcohol production in yeast.