Doctor of Philosophy (PhD), Ohio University, 2020, Chemistry and Biochemistry (Arts and Sciences)

Extensin peroxidases play a critical role in plant cell growth and are believed to play equally important roles in defense from pathogenesis and mechanical stress. By catalyzing the covalent polymerization of extensin proteins, they participate in the formation of the cell plate for cell division and help to reinforce the wall—preventing pathogen infection. Due to it's anionic character and catalytic processivity, TomEP is a particularly unique extensin peroxidase that requires much less time and enzyme than other extensin peroxidases to crosslink extensin substrate. Previous work identified the TomEP gene, and established methods to produce functional enzyme through heterologous expression in E. coli. This work aimed to expand upon these previous efforts by characterizing TomEP expression, TomEP function in vivo, and design a purification scheme to produce milligram-level quantities of pure enzyme for crystallization. An expression profile of TomEP was compiled using both qPCR analysis and promoter-GUS fusion experiments to provide data describing normal expression and response to wounding. Basal TomEP expression was demonstrated to be significantly higher in roots than in flowers, stems, or leaves. Through the same methods, wounding treatments were shown to increase TomEP expression in tomato roots from one to four hours, followed by attenuation for the following sixteen hours. The foundations of gain and loss-of-function experiments were pursued in an attempt to discern TomEP's influence on di-isodityrosine and pulcherosine content in tomato cell walls, using overexpression and CRISPR knock-out strategies. Overexpression lines of tomato and Arabidopsis were generated using Agrobacterium mediated methods, though these efforts failed to produce verifiable protein product, despite expression being observed on the RNA level. Transient expression in tobacco epidermal cells was successful however, allowing for in vivo analysis of TomEP activity, though no clea (open full item for complete abstract)

Committee: Michael Held II (Advisor); Marcia Kieliszewski (Committee Member); Showalter Allan (Committee Member); McMills Lauren (Committee Member) Subjects: Biochemistry; Botany; Plant Biology; Plant Sciences