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

Doctor of Philosophy (PhD), Ohio University, 2015, Chemistry and Biochemistry (Arts and Sciences)

The regulation of plant cell growth and defense involves the insolubilization of hydroxyproline-rich glycoproteins (HRGPs), such as extensin, in the primary cell wall. In tomato (Solanum lycopersicum), insolublization occurs by the formation of tyrosylcrosslinks catalyzed specifically by the pI 4.6 extensin peroxidase (EP). To date, neither the gene encoding EP nor the protein itself has been identified. Here, tomato EP candidates were identified using both proteomic and bioinformatic approaches. Bioinformatic screening of the tomato genome yielded eight EP gene candidates, which encoded a putative signal sequence and a protein with a predicted pI near 4.6. Biochemical fractionation of tomato culture media followed by proteomic detection further refined our list of EP candidates to three, with the lead candidate designated a "CG5". The CG5 open-reading frame from a tomato cDNA was then cloned into a bacterial expression vector to test for EP crosslinking activity. CG5 was expressed in E. coli, fractionated from inclusion bodies, and folded in vitro. Peroxidase activity of CG5 was assayed and quantified by the ABTS (2,2'- zinobis(3-ethylbenzothiazoline-6-sulphonic acid)) assay. Subsequent extensin crosslinking assays showed that CG5 can covalently crosslink authentic tomato P1 extensin and P3-type extensin analogs in vitro supporting the hypothesis that CG5 encodes a tomato EP. Recombinant CG5 was then purified for enzyme kinetics study which gave out a Km of 0.105 mM. A computational 3D model was generated for CG5 with highlighted active site and predicted heme group binding.

Committee: Marcia Kieliszewski (Advisor); Micheal Held (Advisor) Subjects: Biochemistry; Plant Biology

Doctor of Philosophy (PhD), Ohio University, 2014, Chemistry and Biochemistry (Arts and Sciences)

The Hydroxyproline Rich GlycoProtein (HRGPs) superfamily, which includes the Arabinogalactan proteins (AGPs), Proline-rich Proteins (PRPs) and Extensins (EXTs), contributes crucially to cell wall architecture. EXT3 (AT1G21310, RSH) is an extensin required for the correct positioning of the cell plate during cytokinesis in the developing mbryo root. A RSH analog lacking an N-terminus (13.5Ct) was expressed in tobacco cells, and after purification and chemical characterization exhibited a slower (about 0%) crosslinking rate catalyzed by tomato extension peroxidase (EP) than wild type RSH. The self-assembly of 13.5Ct was monitored by Atomic Force Microscope (AFM) imaging and no special pattern was evident compared to native RSH, indicating the N-terminus of extensin was related to the crosslinking efficiency but not the self-assembly process. The analog FK9 (“SOOOOSOSOOOOFFFK”, expressed in tobacco) inhibited the crosslinking reaction when mixed with extensin mononers. The crosslinking of RSH, tomato P1 and extensin analog YK8 (“SOOOOSOSOOOOYYYK”) were inhibited to different degrees by FK9. The 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) activity of extensin peroxidase (EP) was also inhibited by FK9. The cell walls of rsh knock out mutant seedling roots were analyzed proteomically. In the samples of the abnormal phenotype (ANP) cell lines, which were rsh knock-out mutants (confirmed by qRT-PCR) but exhibited normal phenotypes, LRX1 (AT1G12040), LRX2 (AT1G62440) and EXT4 (AT1G76930) were detected but no other extensin or extensin-like proteins, indicating the LRX1/2 or EXT4 might compensate theabsence of RSH. The EP in Arabidopsis was partially characterized. One basic peroxidase fraction was isolated from the salt eluate of Arabidopsis cultured cells, and it exhibited extensin crosslinking ability. The proteomic analysis indicated three candidate peroxidases (AT5G05340, AT5G64120 and AT3G49120) might be responsible for the crosslinking.

Committee: Marcia Kieliszewski (Advisor); Michael Held (Committee Member); Allan Showalter (Committee Member); Hao Chen (Committee Member) Subjects: Biochemistry; Plant Biology; Plant Sciences