Exploring the Function of GT2 in Physcomitrella patens

Plant cell walls are composed of a variety of carbohydrates, among them cellulose, pectin and hemicellulose. Cellulose is deposited in the cell wall as microfibrils via cellulose synthesis complexes (CSCs). These complexes contain the cellulose synthase proteins (CESAs) and come in two different morphological forms: rosettes and linear complexes. Rosette shaped cellulose synthesis complexes occur in land plants, whilst linear complexes are commonly found in red algae. However, some land plants, notably bryophytes (mosses) and seedless vascular plants, contain genes that encode both CESAs of the type that form rosette CSCs and also genes similar to those found in red algae. The moss Physcomitrella patens contains one gene of the latter type that has been named GT2. GT2 may represent a gene that mosses and seedless vascular plants inherited from their algal ancestors and that has been lost in seed plants. This suggests an evolutionary divergence between mosses and seedless vascular plants and seed plants. Although the precise function of the gene is unknown, its similarity to red algal CESAs suggests that it plays a part in cellulose microfibril synthesis in the plant cell wall. The goal of this research project is to investigate the particular function of the gene GT2 in Physcomitrella patens using targeted gene replacement techniques. During the course of this project, mutant lines of P. patens were created by removing GT2 from their genome. The removal of GT2 was performed by using targeted gene replacement techniques and it was replaced with a gene encoding antibiotic resistance. Proper integration of the vector encoding antibiotic resistance was tested using PCR and agarose gel electrophoresis. A promoter construct is being created and will be inserted into P. patens lines to see where GT2 is expressed. Phenotypic analysis will be performed on the mutant lines to gain insight into the function of GT2.