Biochemical Characterization of Hydroxyproline-rich Glycoproteins in the Arabidopsis Root Cell Wall

RSH (AtEXT3) is an Arabidopsis extensin that plays a crucial role in root cell wall integrity and embryogenesis. It is a member of the plant cell wall hydroxyprolinerich glycoproteins (HRGPs) superfamily and presumably functions by self-assembling into a scaffold that forms a covalently cross-linked wall network. Here, the self-assembly of extensins from Arabidopsis, tomato, tobacco and maize and extensin analogs from tobacco was examined by AFM and they all formed a dendritic scaffold in water or in pH 5.2 buffer. Three different approaches were taken to obtain biochemical evidence for the covalent association of RSH with the cell wall and determine the mechanism for its crosslinking: (1) Peptides were released by trypsin from cell wall covalently cross-linked protein networks after deglycosylation of the wall by anhydrous hydrogen fluoride followed by MS/MS sequencing to identify wall-associated proteins. The extensins known as EXT1 and RSH were found in wild type Arabidopsis root cell walls. This result suggests that RSH and EXT1 are covalently cross-linked in the cell wall; (2) RSH analog genes with Tyr to Phe mutations in the isodityrosine (Idt) crosslinking motifs were constructed but they failed to complement an rsh knockout mutant identified earlier, suggesting that the Idt motifs are important for RSH cross-linking; and (3) The arabinooligosaccharide structures of RSH were determined by NMR, and in vitro cross-linking of dearabinosylated extensins showed that extensin initial cross-linking rates are positively correlated with the presence of the 4th Araf residue on the oligotetraarbinosides in extensins. Altogether, these results corroborate the hypothesis that RSH is covalently cross-linked into the wall by Tyr derivatives and that the glycosylation of extensin plays an important role in their function.

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Chemistry and Biochemistry (Arts and Sciences)

Grantor dc:publisher

Ohio University

Year dc:date

2012

Author dc:creator

• Chen, Yuning

Contributors dc:contributor

• Kieliszewski, Marcia

• Biochemistry
• RSH
• HRGP
• EXT1
• extensin cross-linking

Statement dc:rights

• unrestricted
• This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.

Language dc:language

English

Repository record dc:identifier

http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1352912989

OAI identifier oai:identifier

oai:etd.ohiolink.edu:ohiou1352912989