Doctor of Philosophy (PhD), Ohio University, 2020, Molecular and Cellular Biology (Arts and Sciences)

Arabinogalactan-proteins (AGPs) are a diverse family of plant hydroxyproline-rich glycoproteins implicated to function in a number of physiological processes including growth, development, cellular signaling, somatic embryogenesis, programmed cell death, and wounding. AGPs are known for the abundance of sugars present on their molecular surface. Addition of the various sugars to AGPs requires the action of numerous distinct enzymes called glycosyltransferases (GTs). Glucuronic acid (GlcA), which is the only negatively charged sugar on AGPs, is added by the action of three glucuronic acid transferases (GLCATs), namely GLCAT14A, GLCAT14B, and GLCAT14C. Hydroxyproline-Galactosyltransferases (GALTs) are responsible for initiating sugar addition to AGPs by adding galactose (Gal) to hydroxyproline residues in the AGP core protein. To date, eight GALTs, namely GALT2-6 and Hyp-O-galactosyltransferases 1-3 (HPGTs 1-3) have been identified. To overcome gene redundancy within these two GT families, I applied a cutting-edge clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9 ) gene multiplexing approach to produce higher order genetic mutants (i.e., mutants with multiple gene family members being mutated). Seven CRISPR mutants were generated including glcat14b, glcat14c, glcat14a glcat14b, glcat14b glcat14c, and glcat14a glcat14b glcat14c for the GLCAT gene family and galt3 galt4 galt6 and galt2 galt3 galt4 galt5 galt6 for the GALT gene family. These CRISPR mutants along with two existing T-DNA mutants, namely galt2 galt5 and hpgt1 hpgt2 hpgt3, were subjected to extensive biochemical and physiological phenotypic characterization. Biochemical analysis of the glcat mutants revealed that the double and triple mutants generally had small increases of Ara and Gal and concomitant reductions of GlcA, particularly in the glcat14a glcat14b and glcat14a glcat14b glcat14c mutants. Moreover, all the glcat mutants displayed significant reducti (open full item for complete abstract)

Committee: Allan Showalter (Advisor) Subjects: Biochemistry; Genetics; Molecular Biology; Plant Biology