Date of Award

2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Biological and Environmental Sciences

Department

Biological Sciences

First Advisor

Alison Roberts

Abstract

Due to the enormous economic value and significance of cellulose in human consumption and plant cell walls, production of cellulose microfibrils is considered to be one of the most critical biochemical processes in plant biology. In the past decades, cellulose biosynthesis has been extensively studied in vascular plants. More and more fundamental questions related to this key process are being answered. One such question is: What are the protein components of the enzymatic complex for cellulose synthesis? In seed plants, membrane-embedded rosette Cellulose Synthesis Complexes (CSCs) producing cellulose microfibrils are obligate hetero-oligomeric, being assembled from three functionally distinct and non-interchangeable cellulose synthase (CESA) isoforms. For instance, Arabidopsis has two types of CSCs. One contains AtCESA1, AtCESA3, and AtCESA6, involved in cellulose synthesis in primary cell walls; the other consists of AtCESA4, AtCESA7, and AtCESA8, specialized for secondary cell wall deposition. Recently, the stoichiometry for the three Arabidopsis CESAs forming a CSC was determined to be a 1:1:1 molecular ratio. The constructive neutral evolution hypothesis has been proposed as a mechanism for evolution of these hetero-oligomeric complexes.

Physcomitrella patens, a non-vascular plant, is one of the most popular models for genetics studies. A relatively small genome, dominant haploid phase, and high rate of homologous recombination make P. patens a simple and efficient system for genetic manipulation. Seven CESA genes (PpCESA3, PpCESA4, PpCESA5, PpCESA6, PpCESA7, PpCESA8, and PpCESA10) were identified in the P. patens genome, but proteins encoded by these genes are not orthologs of functionally distinct seed plant CESAs according to phylogenetic studies. The similar rosette-type of CSCs were observed in P. patens by freeze-fracture electron microscopy. It is not yet known whether the P. patens CSCs are homo-oligomeric complexes consisting of only a single type of CESA, or hetero-oligomeric complexes assembled by different CESAs like those in seed plants. Knowing this information would be helpful for understanding the roles of different CESAs that compose seed plant CSCs. Furthermore, answers to this question potentially will be useful for testing the constructive neutral evolution hypothesis, since moss CESAs diversified independently from seed plant CESAs.

In this study, I generated PpCESA knock out (KO) mutants. Morphological analyses were carried out to identify mutant phenotypes of these KOs together with several previously made KO mutants. Cellulose defects in these mutants were also analyzed using quantitative methods. Reverse transcriptase PCR (RT-qPCR) was performed to examine the expression of all seven PpCESAs in KO lines to identify co-expressed PpCESAs that potentially reside within the same CSCs as the deleted PpCESA. Immunoblot analysis using specific monoclonal antibodies was used as an additional method to detect co-expression based on the accumulation of the protein products of these PpCESA genes. Finally, I carried out Co-immunoprecipitation (Co-IP) assays to identify potential physical interactions between different PpCESA isoforms. The results show that functionally distinct CESA isoforms have evolved in the moss P. patens independently from seed plants, and CSCs synthesizing cellulose microfibrils in secondary cell walls of P. patens gametophore leaves are obligate hetero-oligomeric complexes. Meanwhile, our research also suggests that PpCESA5 alone is able to form homo-oligomeric CSCs, making P. patens an intriguing model in which to study the evolution of cellulose synthase.

Available for download on Wednesday, November 28, 2018

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