Date of Award

8-2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Plants, Soils, and Insects

Major Professor

C. Neal Stewart

Committee Members

Scott C. Lenaghan, Juan-Luis Jurat-Fuentes, Terek Hewezi

Abstract

English ivy naturally produces organic nanoparticles from its adventitious root hairs, and possesses characteristics that may allow them to replace metal-based nanoparticles in common applications, such as sunscreen. At the onset of the research, it was hypothesized that a physical mechanism of attachment, similar to the gecko footpad, was used to generate the adhesive force for attachment; however, through the results obtained from recent work, it is clear that a biochemical mechanism is involved in the generation of the strength of adhesion. Therefore, the goal of this research was to provide a better understanding of the genetic basis of nanoparticle biosynthesis and identify the putative gene(s) and encoded proteins that are involved in the formation or biosynthesis of the nanoparticle.

It was first necessary to develop a biofabrication system that produced large quantities of adventitious roots. This enhanced system was achieved by modifying GA7 Magenta boxes and identifying the optimal concentration of the auxin indole-3 butyric acid (IBA) for adventitious root growth. Maximum adventitious root production was achieved by a 4 h application of 1 mg/ml IBA to juvenile English ivy shoot segments cultured in custom vessels. This method produced 90 mg of dry weight nanoparticles, confirmed by atomic force microscopy, from 12 g adventitious roots after 2 wk.

Following bulk production, I utilized a proteomic- and transcriptomic-based approach to identify and analyze the specific proteins involved in the formation of the nanoparticle adhesive complex from adventitious root samples. A 10 mM dithiothreitol (DTT)-based extraction buffer allowed the separation of a single high molecular weight band (> 460 kDa) from nanoparticles into 9 bands ranging in molecular weight from 25-130 kD, with no effects on the size or stability of the nanoparticles. The results of the omics analysis identified 11 protein candidates from the English ivy adventitious root transcriptome, and 9 candidates from the UniProt (all plant) database for a combined total of 20 individual putative proteins that comprise ivy nanoparticles. These studies provide a reasonable starting point in the identification of those proteins involved in nanoparticle formation and function.

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