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Functional Characterization of Beta-Glucuronosyltransferases (GLCATs) and Hydroxyproline-Galactosyltransferases (GALTs) Involved in Arabinogalactan-Protein (AGP) Glycosylation Using CRISPR/Cas9 Gene Editing Technology In Arabidopsis

Zhang, Yuan
http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1588687871450172

Abstract Details

2020, Doctor of Philosophy (PhD), Ohio University, 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 reductions in calcium binding by their AGPs compared to wild type (WT) plants. Physiological analyses revealed that both the glcat14a glcat14b and glcat14a glcat14b glcat14c mutants exhibited significant delays in seed germination, reductions in root hair length, reductions in trichome branching, and accumulation of defective pollen grains. Additionally, both the glcat14b glcat14c and glcat14a glcat14b glcat14c mutants displayed significantly shorter siliques and reduced seed set. Finally, all higher-order mutants exhibited significant reductions in adherent seed coat mucilage. Biochemical analysis of the higher-order galt mutants showed that all the galt mutants (galt2 galt5, galt3 galt4 galt6, galt4 galt6, and galt2 galt3 galt4 galt5 gal6) contained much less glycosylated AGPs in rosette leaves, stems, and siliques compared to WT. Monosaccharide compositional analysis revealed significant decreases of arabinose (Ara) and galactose (Gal) in all the higher order galt mutants. Further biochemical analysis found that loss of two or more GALTs were able to overcome the growth inhibitory effect of β-D-Gal-Yariv reagent. Physiological analysis revealed that the galt2 galt3 galt4 galt5 gal6 mutant exhibited a delay in overall aboveground growth and development, impaired root growth, abnormal pollen, shorter siliques, and reduced seed set. Higher order genetic mutants of GTs generated in these two research projects facilitated the understanding of functional importance of the GlcA residues (added by the GLCATs) and Gal residues (added by the GALTs) and revealed sugar structure/function relationships for AGPs. The CRISPR/Cas9 gene editing approach described here provides a simpler and faster way to generate higher order plant mutants for functional characterization compared to conventional genetic crossing of T-DNA mutant lines. This CRISPR/Cas9 mediated multiplexing approach will be especially useful for editing multigene families with members having redundant functions as is the case for many GT families and other cell wall related gene families.
Allan Showalter (Advisor)
205 p.
Biochemistry; Genetics; Molecular Biology; Plant Biology
Arabidopsis; Arabinogalactan-proteins; CRISPR-Cas9; calcium; development; glucuronic acid; glucuronosyltransferase; Hydroxyproline-Galactosyltransferases; pollen; root hairs; seed coat mucilage; seed germination; siliques; trichomes; reproduction

Recommended Citations

Citations

  • Zhang, Y. (2020). Functional Characterization of Beta-Glucuronosyltransferases (GLCATs) and Hydroxyproline-Galactosyltransferases (GALTs) Involved in Arabinogalactan-Protein (AGP) Glycosylation Using CRISPR/Cas9 Gene Editing Technology In Arabidopsis [Doctoral dissertation, Ohio University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1588687871450172

    APA Style (7th edition)

  • Zhang, Yuan. Functional Characterization of Beta-Glucuronosyltransferases (GLCATs) and Hydroxyproline-Galactosyltransferases (GALTs) Involved in Arabinogalactan-Protein (AGP) Glycosylation Using CRISPR/Cas9 Gene Editing Technology In Arabidopsis. 2020. Ohio University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1588687871450172.

    MLA Style (8th edition)

  • Zhang, Yuan. "Functional Characterization of Beta-Glucuronosyltransferases (GLCATs) and Hydroxyproline-Galactosyltransferases (GALTs) Involved in Arabinogalactan-Protein (AGP) Glycosylation Using CRISPR/Cas9 Gene Editing Technology In Arabidopsis." Doctoral dissertation, Ohio University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1588687871450172

    Chicago Manual of Style (17th edition)

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This open access ETD is published by Ohio University and OhioLINK.