Lack of Hydroxyproline O-Arabinosyltransferases Does Not Cause Over Deposition of Cellulose in the Moss Physcomitrella patens
Gribbin, William T.
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The plant cell wall is a highly dynamic structure necessary for structural stability in the plant cell. Hydroxyproline O-Arabinosyltransferase (hpat) are genes which code for the proteins responsible for the arabinosylation of extensins and Leucine Rich Repeat Extensins (LRX). In higher plants, extensins are vital in cell wall development and structure and in both higher and basal plants, LRX's are important for cell wall development. Our lab had previously generated an hpata/hpatb double knockout mutant (AB5) of P. patens, and noted its enhanced growth rate and colony size over wildtype. The larger growth phenotype indicated that the cell wall may have been modified in the mutant. We hypothesized that the cellulose may have been acting in a compensatory manner because LRX's are associated with cell wall stability. Additionally, we sought to create a timeline of cell wall regeneration in P. patens protoplast. We generated protoplasts of P. patens by digesting cell walls with driselase and then we monitored cell wall regeneration via fluorescence microscopy. Protoplasts were also treated with a hypotonic buffer at regular intervals into regeneration and observed for colony formation. I found that there was no difference in the level of cellulose deposited in AB5 line regenerating protoplasts and wildtype. I also found that wildtype protoplasts treated with hypotonic buffer showed enhanced branching and malformed cells. Observation of protoplast recovery from hypoosmotic bursting treatment proved to be too insensitive for meaningful analysis. Cell wall regeneration proved to be a highly variable process for regenerating protoplasts. In the future, a more sensitive method of quantifying viability of protoplasts is recommended.