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

A Complementation Assay for in Vivo Protein Structure/Function Analysis in \u3cem\u3ePhyscomitrella patens\u3c/em\u3e (Funariaceae)

Premise of Study: A method for rapid in vivo functional analysis of engineered proteins was developed using Physcomitrella patens. Methods and Results: A complementation assay was designed for testing structure/function relationships in cellulose synthase (CESA) proteins. The components of the assay include (1) construction of test vectors that drive expression of epitope-tagged PpCESA5 carrying engineered mutations, (2) transformation of a ppcesa5 knockout line that fails to produce gametophores with test and control vectors, (3) scoring the stable transformants for gametophore production, (4) statistical analysis comparing complementation rates for test vectors to positive and negative control vectors, and (5) analysis of transgenic protein expression by Western blotting. The assay distinguished mutations that generate fully functional, nonfunctional, and partially functional proteins. Conclusions: Compared with existing methods for in vivo testing of protein function, this complementation assay provides a rapid method for investigating protein structure/function relationships in plants

Cellulose synthase ‘class specific regions’ are intrinsically disordered and functionally undifferentiated

Cellulose synthases (CESAs) are glycosyltransferases that catalyze formation of cellulose microfibrils in plant cell walls. Seed plant CESA isoforms cluster in six phylogenetic clades, whose non‐interchangeable members play distinct roles within cellulose synthesis complexes (CSCs). A ‘class specific region’ (CSR), with higher sequence similarity within versus between functional CESA classes, has been suggested to contribute to specific activities or interactions of different isoforms. We investigated CESA isoform specificity in the moss, Physcomitrella patens (Hedw.) B. S. G. to gain evolutionary insights into CESA structure/function relationships. Like seed plants, P. patens has oligomeric rosette‐type CSCs, but the PpCESAs diverged independently and form a separate CESA clade. We showed that P. patens has two functionally distinct CESAs classes, based on the ability to complement the gametophore‐negative phenotype of a ppcesa5 knockout line. Thus, non‐interchangeable CESA classes evolved separately in mosses and seed plants. However, testing of chimeric moss CESA genes for complementation demonstrated that functional class‐specificity is not determined by the CSR. Sequence analysis and computational modeling showed that the CSR is intrinsically disordered and contains predicted molecular recognition features, consistent with a possible role in CESA oligomerization and explaining the evolution of class‐specific sequences without selection for class‐specific function

Immuno and Affinity Cytochemical Analysis of Cell Wall Composition in the Moss Physcomitrella patens

In contrast to homeohydric vascular plants, mosses employ a poikilohydric strategy for surviving in the dry aerial environment. A detailed understanding of the structure, composition, and development of moss cell walls can contribute to our understanding of not only the evolution of overall cell wall complexity, but also the differences that have evolved in response to selection for different survival strategies. The model moss species Physcomitrella patens has a predominantly haploid lifecycle consisting of protonemal filaments that regenerate from protoplasts and enlarge by tip growth, and leafy gametophores composed of cells that enlarge by diffuse growth and differentiate into several different types. Advantages for genetic studies include methods for efficient targeted gene modification and extensive genomic resources. Immuno and affinity cytochemical labeling were used to examine the distribution of polysaccharides and proteins in regenerated protoplasts, protonemal filaments, rhizoids, and sectioned gametophores of P. patens. The cell wall composition of regenerated protoplasts was also characterized by flow cytometry. Crystalline cellulose was abundant in the cell walls of regenerating protoplasts and protonemal cells that developed on media of high osmolarity, whereas homogalactuonan was detected in the walls of protonemal cells that developed on low osmolarity media and not in regenerating protoplasts. Mannan was the major hemicellulose detected in all tissues tested. Arabinogalactan proteins were detected in different cell types by different probes, consistent with structural heterogneity. The results reveal developmental and cell type specific differences in cell wall composition and provide a basis for analyzing cell wall phenotypes in knockout mutants

Cell Wall Compositions of Sorghum bicolor Leaves and Roots Remain Relatively Constant Under Drought Conditions

International audienceScavuzzo-Duggan et al. Cell Walls in Droughted Sorghum on the transcriptomic level and see whether those changes translate to compositional or biomass conversion differences. Our results bolster the conclusion that drought stress does not substantially affect the cell wall composition of specific aerial and subterranean biomass nor impede enzymatic hydrolysis of leaf biomass, a positive result for biorefinery processes. Coupled with previously reported results on the root microbiome and rhizosphere and whole transcriptome analyses of this study, we can formulate and test hypotheses on individual gene candidates' function in mediating drought stress in the grass cell wall, as demonstrated in sorghum