Date of Award

2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cell & Molecular Biology

First Advisor

Alison Roberts

Abstract

A detailed analysis of cellulose synthesis in nonvascular plants can contribute to a better understanding of the evolution of this important process. In this study, the nonvascular plant Physcomitrella patens was used as a model system to investigate the roles of the different isoforms of cellulose synthase (CESA). PpCESA gene expression was quantified through Reverse Transcription quantitative (RT-q) PCR and localized through construction and analysis promoter::reporter lines to determine the roles of the PpCESAs throughout development. Physcomitrella patens CESA genes are ubiquitously expressed in the filamentous protonema stage. All of the PpCESAs are expressed in the gametophore as well, with PpCESA4 and PpCESA10 mainly expressed in the axillary hairs. This broad expression is unique to non-vascular plants, in contrast to vascular plants in which CESA expression is restricted to cells depositing either primary cell walls or secondary cell walls during development.

Upregulation under osmotic stress induced by mannitol may indicate a role for cellulose under high osmotic stress. PpCESA6, PpCESA7, and PpCESA8 was hypothesized to be responsible for osmotic stress-induced cellulose synthesis based on mannitol-induced upregulation of expression as indicated by analysis of microarray data. The roles of CESAs in development and stress tolerance were assessed by producing knockout mutants of PpCESA6, PpCESA7, and PpCESA8. Ppcesa8 knockout (KO) and ppcesa6/7 KO mutants do not have dramatic developmental phenotypes. However, ppcesa6/7KO mutants show sensitivity towards high salinity, indicating that cellulose is important under abiotic stress.

Currently, only ppcesa5 KO mutants show a phenotype in the gametophore and no single KO mutants have phenotypes in the protonema. Cellulose synthesis inhibitors were used to examine the role of cellulose in the protonema. Results show that protonemal tissue is relatively insensitive to cellulose inhibitors, since only high concentration of the cellulose synthesis inhibitor DCB had any effect. DCB caused rupturing of tips, indicating that cellulose is necessary in tip growth. Results also indicate that cellulose synthase-like D (CSLD) proteins may contribute to the synthesis of cellulose in moss protonema.

Since single and double KO mutants of PpCESA6, PpCESA7, and PpCESA8 do not produce a phenotype and PpCESA expression is ubiquitous, PpCESAs maybe be redundant in function such that another PpCESA may compensate loss of a single PpCESA. PpCESAs are highly similar in sequence and may have not fully sub-functionalized (A. W. Roberts, Roberts, & Haigler, 2012) and therefore, the PpCESAs isoforms may be more interchangeable than those of seed plants. Other cell wall components, such as hemicelluloses, pectins, and arabinoproteins, may also compensate for lack of cellulose. These cell wall components were also examined through immunolabeling of regenerating protoplasts. The results showed the highest abundance of crystalline cellulose and moderate levels of callose, mannan, 1,5-α-L-arabinan and arabinogalactan proteins. Very low levels of 1,4-β-D-galactan and no homogalacturonans were detected.

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