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Identification and Characterization of Five Arabidopsis Hydroxyproline Galactosyltransferases and Their Functional Roles in Arabinogalactan-Protein Glycosylation, Growth, Development, and Cellular Signaling
Basu, Debarati

2015, Doctor of Philosophy (PhD), Ohio University, Molecular and Cellular Biology (Arts and Sciences).
Arabinogalactan-proteins (AGPs) are a class of highly glycosylated ubiquitous plant cell wall glycoproteins, implicated in diverse biological roles. Although plants contain substantial amounts of AGPs, enzymes responsible for glycosylation of AGPs are largely unknown. Given that sugar side chains account for about 90% of the molecular mass of AGPs, sugars likely define the interactive surface of the molecule and hence its function. Bioinformatic analysis indicated that hydroxyproline (Hyp)-O-galactosyltransferases (GALTs) for AGPs might belong to the Carbohydrate-Active enZYme (CAZY) glycosyltransferase 31-family, which includes ß-GALTs involved in the synthesis of glycoproteins. Here, five GT 31 Hyp-O-GALTs, namely AtGALT2 (At4g21060), AtGALT3 (At3g06440), AtGALT4 (At1g27120), AtGALT5 (At1g74800) and AtGALT6 (At5g62620), were characterized. Detergent permeabilized microsomes obtained from Pichia pastoris expressing AtGALT2 and AtGALT5 and from tobacco epidermal cells expressing AtGALT2, AtGALT3, AtGALT4, AtGALT5 and AtGALT6 specifically catalyzed the transfer of [14C]Gal from UDP-[14C]Gal to Hyp in the chemically synthesized AGP peptide substrate acceptor, [AO]7. Furthermore, these Hyp-O-GALTs exhibited similar biochemical properties. Confocal microscopic analysis of fluorescently tagged AtGALT2-6 indicated AtGALT2 was localized in the ER and Golgi, while the other four proteins were localized exclusively in Golgi vesicles. Additional support that these five GALTs encode AGP-specific Hyp-O-GALTs was provided by analysis of allelic knockout mutants of the five GALT genes. These mutants demonstrated significantly lower Hyp-O-GALT activities, reduced ß-Yariv-precipitated AGPs and pleiotropic growth and developmental phenotypes compared to wild type plants with increasing severity in galt2galt5 double mutants. To varying degrees, all five Hyp-O-GALTs were essential for tip growth and involved in root development in response to salt stress. Interestingly, the galt2galt5 double mutant phenocopied the root swelling phenotypes as well as the seed coat mucilage and cellulose-deficient phenotypes of previously characterized mutants, namely sos5(a GPI-anchored fasciclin-like AGP), fei1fei2 (a pair of leucine rich cell wall receptor-like kinases), and sos5fei1fei2. These findings indicated that the arabinogalactan (AG) polysaccharides of SOS5 are likely critical for cellular signaling and in stimulating cellulose biosynthesis. In summary, this dissertation contributes to an understanding of AGP biosynthesis, particularly with respect to Hyp-galactosylation, and to the functional roles which AG polysaccharides play in plants.
Allan M Showalter (Advisor)
Sarah Wyatt (Committee Member)
Marcia Kieliszewski (Committee Member)
Ahmed Faik (Committee Member)
365 p.

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Basu, D. (2015). Identification and Characterization of Five Arabidopsis Hydroxyproline Galactosyltransferases and Their Functional Roles in Arabinogalactan-Protein Glycosylation, Growth, Development, and Cellular Signaling. (Electronic Thesis or Dissertation). Retrieved from https://etd.ohiolink.edu/

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Basu, Debarati. "Identification and Characterization of Five Arabidopsis Hydroxyproline Galactosyltransferases and Their Functional Roles in Arabinogalactan-Protein Glycosylation, Growth, Development, and Cellular Signaling." Electronic Thesis or Dissertation. Ohio University, 2015. OhioLINK Electronic Theses and Dissertations Center. 22 Nov 2017.

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Basu, Debarati "Identification and Characterization of Five Arabidopsis Hydroxyproline Galactosyltransferases and Their Functional Roles in Arabinogalactan-Protein Glycosylation, Growth, Development, and Cellular Signaling." Electronic Thesis or Dissertation. Ohio University, 2015. https://etd.ohiolink.edu/

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