Dissertations, Theses, and Capstone Projects

Date of Degree

9-2016

Document Type

Dissertation

Degree Name

Ph.D.

Program

Chemistry

Advisor

Ruth E. Stark

Committee Members

Akira Kawamura

George John

Subject Categories

Analytical Chemistry | Genomics | Other Plant Sciences

Keywords

Potato Suberin GC/LC-MS SSNMR FHT-RNAi CYP-RNAi

Abstract

Suberin is a biopolyester constituent of specialized plant periderm tissues formed within the phellem cell walls. Suberin and waxes of the periderm layer act to prevent water diffusion, mechanical breakdown and pathogenic invasion in plants. Ferulic esters, ω-hydroxyacids and α,ω-fatty diacids are considered to be the most important linkage between aliphatic and aromatic suberin domains and also linked with cell-wall polysaccharides. The potato gene FHT (fatty ω- hydroxyacid/fatty alcohol hydroxycinnamoyl transferase) esterifies ferulic acid to suberin. Diminished levels of feruloyl transferase activity have been associated with lowered amounts of feruloyl esters of fatty acids in both suberin-associated waxes and suberin breakdown products derived from transesterification. The Cytochrome P450 monooxygenase (CYP) enzyme controls the key step of ω-hydroxylation of fatty acids in the aliphatic suberin biosynthesis of potato suberin. FHT or CYP knockdown by RNAi silencing are accompanied by increases in periderm water permeability. Whereas, FHT has unaltered periderm ultrastructure, CYP86A33 variety loses the characteristic lamellar structure of its suberized tissues.

To investigate how the potato suberin biosynthesis pathway are affected by FHT-RNAi and CYP-RNAi silencing, a bottom-up approach was used to establish metabolite profiles for polar and non-polar periderm tissue extracts by LC/GC-MS and solution NMR measurements.

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Solid suspensions obtained after polar and nonpolar extractions were assessed by solid-state 13C NMR.

Metabolomic analyses (PCA and OPLS-DA) showed consistency among biological replicates and discrimination between FHT-RNAi or CYP-RNAi and wild type samples. Identified biomarkers included alkanes, α,ω-fatty diacids, fatty acids/alcohols with different chain length and glyceryl esters in the non-polar extract. In the polar extract, the phenolic amines (caffeoylputrescine, feruloylputrescine and feruloyltyramine) are highly abundant for FHT-RNAi varieties. The glycoalkaloids (-solanine, -chaconine and solanidine-Glc-Rha) are down- regulated markers for the FHT-RNAi periderm. Reversely, they are up-regulated markers for CYP-RNAi periderm that may function as plant hormones or antimicrobial compounds.

The relative numbers of each major carbon type in the solid residue were estimated from quantitatively reliable DPMAS 13C NMR spectra. The FHT-RNAi and CYP-RNAi varieties have higher oxygenated aromatic-to-(CH2)n and oxygenated-aliphatic-to-(CH2)n ratios than WT. These compositional trends indicate an enhanced hydrophilic-hydrophobic balance in FHT-RNAi suberin samples, consistent with augmented water permeability and reduced relative amounts of fatty acid and fatty alcohol-containing polymeric products deduced from suberin breakdown. These trends were interpreted in terms of the biosynthetic transformations involved in potato cell wall suberization.

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