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Identification and characterization of a cellulose synthase from the calcifying red alga Calliarthron tuberculosum

University of British Columbia

In land plants and algae, cellulose, a glucose polymer, is important for strengthening tissues and preventing breakage in the face of physical forces. Cellulose synthase enzymes (CESA) are responsible for producing cellulose. While our understanding of land plant CESAs has advanced for several species, it is unclear whether the systems surrounding cellulose synthesis are the same in all plant lineages. For example, no red algal CESAs have been functionally demonstrated. The objective of this thesis is to discover and characterize putative CESA encoding genes from the calcifying red alga Calliarthron tuberculosum and compare their function to those from the land plant eudicot Arabidopsis thaliana. Using a bioinformatics approach, I identified three candidate CESAs from Calliarthron tuberculosum’s transcriptome dataset (CtCESA1, CtCESA2, and CtCESA3). I explore the evolution of CESAs in gene tree analysis and find that while CtCESA1 was closely related to other red algal CESA sequences, CtCESA2 and 3 were more closely related to bacterial cellulose synthases (Ch 2). Using yeast and insect cell expression systems, I heterologously express, purify, and test the CtCESA1 protein in glucose tracer assays to look for polymer formation. CtCESA1 showed evidence of glucan synthase activity that was comparatively lower than plant (PttCESA8) and bacterial (BCSA/BCSB) cellulose synthases (Ch 3). Finally, I test for functional compatibilities between the land plant (A. thaliana) and red algal (C. tuberculosum) CESAs. A. thaliana encodes multiple non-redundant CESAs that function in primary cell wall and secondary cell wall regions as well as several other accessory proteins critical to cellulose synthesis. However, only some accessory proteins were recovered from C tuberculosum’s transcriptome in bioinformatics analyses (Ch 2). To ultimately test for functional differences, I introduced the CtCESA1 gene into A. thaliana cesa mutants deficient in cellulose production (Ch 4). The red algal CtCESA1 partially rescued the A. thaliana primary cell wall cesa6 mutant but not cesa3 or the secondary cell wall cesa7 mutant. This thesis collectively presents the first functional evidence of a red algal CESA and demonstrates a combination of both deeply conserved and largely distinct aspects of cellulose production between the red algal and land plant lineages.

Text

2021-01-06

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/76971

Botany

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/