Dissertations, Theses, and Capstone Projects

Date of Degree

9-2016

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Ruth E. Stark

Committee Members

Urs Jans

Ana Carnaval

Edward J. Kennelly

Emmanuel Chang

Subject Categories

Analytical Chemistry | Food Chemistry

Abstract

Potato tubers are protected from dehydration and pathogens by a covering peel (periderm) impregnated with suberin, a complex cross-linked biopolymer that contains both polyaliphatic and lignin-like aromatic domains. Current models describing the macromolecular structure of suberin assume that ferulic acid cross-links both domains as it may form carboxyl ester bonds with aliphatic monomers and non-ester radical-coupled bonds with phenolics. Ferulic acid also links by ester bonds to glycans and acts in cross-linking polysaccharides and lignin. Fatty alcohol/ω-hydroxyacid hydroxycinnamoyl transferase (FHT) is a BADH acyltransferase responsible for the synthesis of akyl-ferulates that is necessary for suberin biosynthesis. Periderm from FHT-RNAi tubers showed a significant decrease in suberin ferulate esters and an increase in soluble phenolic breakdown products. This periderm also showed changes in texture and water permeability but, surprisingly, the suberin lamellar structure was not altered. The CYP86A gene is a strong candidate for aliphatic suberin biosynthesis. CYP86A33-RNAi potato periderm had a striking loss of suberin lamellar structure and diminished w-hydroxyacids and a,w-diacids.

To obtain a more comprehensive molecular view of the consequences of gene silencing on potato periderm composition, metabolite profiling was conducted on soluble polar and nonv polar extracts from native and wound periderms of wild-type (WT) and FHT-RNAi silenced potato samples using liquid-chromatography mass-spectrometry (LC-MS), gas-chromatography mass-spectrometry (GC-MS) and 1H nuclear magnetic resonance spectroscopy (NMR). The solid interface was analyzed by solid-state NMR. FHT deficiency induced significant changes in the metabolite pool of both polar and non-polar extracts from native periderms. In particular, FHT silencing induced accumulation of phenolic amides such as caffeoylputrescine, feruloylputrescine, feruloyltyramine, and amide dimers in the polar extracts and long-chain saturated fatty acids (C22, C23, C24, C27), long-chain primary alcohols (C22, C26, C27, C28, C29), methyl esters (C17:0, C19:2) and 1-monohexadecanoyl glycerol in the non-polar extracts. Phenolic acids, glycoalkaloids, alkanes (C21, C23, C25, C27, C29) and fatty acids (C16:0, C18:0, C18:2) were down-regulated in FHT-RNAi native periderms. For the solid interfacial residue, the FHT-RNAi variety displayed higher arene-to-(CH2)n and oxygenated alkoxy-to-(CH2)n ratios compared to WT. In contrast to the native tissues, the WT and FHT-RNAi-silenced tissue extracts and solid interfacial residues from developing wound periderms each exhibited similar compositional progressions with time; their initially distinct metabolic profiles became overlapped and underwent a striking convergence. An analogous analysis was conducted on CYP86A33-RNAi native periderm. In CYP86A33 silenced native potato periderms, glycoalkaloids were highly abundant in polar extracts while polyamides were down-regulated compared with WT. Non-polar extracts are to be analyzed with GC-MS and LC-MS.

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