Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biology

Supervisor

Dr. Mark Bernards

Abstract

Suberin is a complex biopolymer composed of two distinct but covalently-linked domains. The first domain is composed of polymerized phenolic monomers, whereas the second domain is predominately fatty acid derivatives esterified with glycerol. Deposited in specialized cells during development or in response to abiotic stress, suberin functions as a barrier to water loss and pathogen attack. In potato, more than 65% of suberin monomers undergo ω-hydroxylation, representing a major class of fatty acids in the final biopolymer. The ω-hydroxylation reaction is catalyzed by Cytochrome P450 (CYP) proteins, of which few have been characterized to date. In 2009, CYP86A33 from potato was identified and implicated as the main suberin-associated ω-hydroxylase by another research group through RNAi gene silencing; although functional characterization of in vitro protein was unsuccessful. Simultaneously, I identified and characterized gene expression for three CYP86A and CYP94A ω-hydroxylase genes in potato. From this expression analysis, CYP86A33 was also identified as the primary candidate for a suberin-associated ω-hydroxylase, from which ω-hydroxylase activity was confirmed through an in vitro enzyme assay with recombinant protein. Following an in silico analysis of the CYP86A33 promoter region, which identified many ABA-responsive promoter elements, an extensive analysis of the effects of ABA on gene expression and suberin biosynthetic regulation was conducted. Using the biosynthetic inhibitor of ABA production, fluridone, I was able to investigate the effects of ABA on suberin regulation by inhibiting ABA de novo biosynthesis with or without the addition of exogenous ABA. Using wounded potato tubers, three parallel timecourse experiments were conducted with different treatments to quantify ABA tissue concentration, suberin-associated gene expression, and soluble and insoluble aliphatic monomer deposition into the suberin biopolymer. Expression of suberin-associated genes, including CYP86A33, was reduced post-wounding with fluridone treatment. Similarly, insoluble aliphatic monomer accumulation was nearly eliminated from suberin in fluridone-treated tissues, exhibiting both chain length and monomer class specific effects. These fluridone effects to gene expression and suberin deposition were rescued through the addition of exogenous ABA. Overall, ABA was shown to have a regulatory effect post-wounding on the gene expression of key suberin-associated genes, with concomitant downstream impact on aliphatic suberin deposition.

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