Identification and characterization of the xyloglucan endotransglycosylases of Arabidopsis thaliana
Campbell, Paul Harwell
Doctor of Philosophy
The plant cell wall is a fundamental determinant of cell shape, and therefore plant form. Although the molecular basis of plant morphology is unknown, it most likely involves enzymes capable of modifying different components of the cell wall. One of the major components of the plant cell wall, the polysaccharide xyloglucan, is capable of hydrogen bonding to cellulose microfibrils, potentially acting as a molecular tether between adjacent microfibrils. Therefore, enzymes capable of modifying xyloglucan may play a role in the development of cell shape and plant form. Xyloglucan endotransglycosylases (XETs) are enzymes capable of cleaving xyloglucan polymers endolytically and transferring one of the newly-generated free ends of the polymer to the free end of another xyloglucan chain. The genome of Arabidopsis thaliana encodes at least sixteen XET and XET- related (XTR) genes. I have sequenced 6 novel XTR genes. Several members of this gene family are differentially regulated by environmental stimuli (such as touch, darkness, and temperature extremes) and the growth-promoting hormones auxin and brassinosteroids. The putative XTR proteins share from 34 to 89% identity at the amino acid level. Like XETs from other plant species, the XTRs are predicted to encode signal peptides, N-linked glycosylation motifs, several cysteines with the potential to form disulfide bonds, and a conserved motif related to the proposed active site of the Bacillus beta-glucanases. Four of the Arabidopsis XTRs, TCH4, Meri-5, EXGT, and XTR9, were produced using the baculovirus/insect cell expression system. All four recombinant proteins catalyzed the transglycosylation of xyloglucan in vitro, demonstrating that the proteins are XETs. The isozymes were characterized biochemically for differences in Kms, pH optima, temperature optima, glycosylation, glucanase activity, and the ability to utilize different xyloglucan substrates. In addition, site-directed mutagenesis of TCH4 revealed that the conserved motif shared with Bacillus beta-glucanases encodes at least one amino acid, glutamate 97, essential for XET activity in TCH4.
Molecular biology; Biochemistry