Date of Award
2025
Degree Type
Thesis
Degree Name
Master of Science in Biological and Environmental Sciences (MSBES)
Specialization
Cell and Molecular Biology
Department
Biological Sciences
First Advisor
Alison Roberts
Abstract
Cellulose Synthase-Like Ds (CSLDs) are cellulose synthase known to participate in cell plate formation and cell wall remodeling. In the moss model, Physcomitrium patens, double csld2/6 knockout (KO) mutants were previously shown to have gametophore leaf phenotypes that are consistent with defects in cytokinesis, including irregular cell shape, cell separations, and incomplete midrib formation (Wu et al., 2023a). When these mutants were grown on ammonium-free medium to promote gametophore development, the severity of the phenotype was more extreme, including incompletely formed cell plates and multinucleated cells (A. Roberts, personal communication). It was also reported that expression of several enzymes promoting cell wall expansion, including three Pectin Methylesterases (PMEs), are downregulated by ammonium, possibly explaining this observation.
To investigate the roles of PMEs in regulating cell expansion and cell division in P. patens gametophores, I knocked out the PME genes that are downregulated by ammonium in a csld2/6KO background line by transforming with CRISPR-Cas9 vectors designed to target each of the three PME genes. Colonies selected for transient antibiotic resistance were PCR genotyped to identify lines with frame-shift mutations and the PCR products were sequenced to confirm the mutations. Confirmed KO lines were grown with and without ammonium to compare the phenotypes with the csld2/6KO lines. No obvious phenotypic differences in colony or gametophore cell morphology were observed compared to the control line, suggesting that upregulation of PME genes is not responsible for the enhancement of the csld2/6KO phenotype in the absence of ammonium.
The P. patens genome encodes eight CSLDs and CSLD2 and CSLD6 are required for normal cell patterning in the gametophore. The others are more highly expressed in the protonema, but also localize to cell plates (Wu et al., 2023a). I investigated whether any of these six CSLDs are involved in cytokinesis in gametophores by transforming a csld2/6KO starting line sequentially with vectors targeting each remaining CSLD and selecting for mutations as described above. I hypothesized that knocking out these genes would increase the severity of the csld2/6KO phenotype with and without ammonium. No obvious phenotypic differences were observed between the background csld2/6KO and the csld2/6/1KO and csld2/6/1/3/5KO lines. The generation of a csld2/6/1/3/5/8KO resulted in a new phenotype characterized by few gametophores and small colony size. The resulting phenotypes suggest that CSLD5 and CSLD8 may participate in tip growth of protonemal filaments. Although lines transformed with CSLD4 and CSLD7 vectors have yet to be assessed for mutations, now that we have the tissue, we can answer an interesting question in the future: Can a plant live with only one CSLD?
Recommended Citation
Bozza, Lia, "INVESTIGATION OF CSLD AND PME GENES IN CELL DIVISION AND EXPANSION IN PHYSCOMITRIUM PATENS" (2025). Open Access Master's Theses. Paper 2656.
https://digitalcommons.uri.edu/theses/2656