ABSTRACT
The moss Physcomitrella patens has become established as a model for investigating plant gene function due to the feasibility of gene targeting. The chemical composition of the P. patens cell wall is similar to that of vascular plants and phylogenetic analyses of glycosyltransferase sequences from the P. patens genome have identified genes that putatively encode cell wall biosynthetic enzymes, providing a basis for investigating the evolution of cell wall polysaccharides and the enzymes that synthesize them. The protocols described in this chapter provide methods for targeted gene knockout in P. patens, from constructing vectors and maintaining cultures to transforming protoplasts and analysing the genotypes and phenotypes of the resulting transformed lines.
Subject(s)
Bryopsida/genetics , Cell Wall/genetics , Gene Targeting , Bryopsida/anatomy & histology , Genetic Vectors/metabolism , Genotype , Polymerase Chain Reaction , Transformation, GeneticABSTRACT
In seed plants, different groups of orthologous genes encode the CELLULOSE SYNTHASE (CESA) proteins that are responsible for cellulose biosynthesis in primary and secondary cell walls. The seven CESA sequences of the moss Physcomitrella patens (Hedw.) B. S. G. form a monophyletic sister group to seed plant CESAs, consistent with independent CESA diversification and specialization in moss and seed plant lines. The role of PpCESA5 in the development of P. patens was investigated by targeted mutagenesis. The cesa5 knockout lines were tested for cellulose deficiency using carbohydrate-binding module affinity cytochemistry and the morphology of the leafy gametophores was analyzed by 3D reconstruction of confocal images. No defects were identified in the development of the filamentous protonema or in production of bud initials that normally give rise to the leafy gametophores. However, the gametophore buds were cellulose deficient and defects in subsequent cell expansion, cytokinesis, and leaf initiation resulted in the formation of irregular cell clumps instead of leafy shoots. Analysis of the cesa5 knockout phenotype indicates that a biophysical model of organogenesis can be extended to the moss gametophore shoot apical meristem.
Subject(s)
Bryopsida/enzymology , Bryopsida/genetics , Genes, Essential/genetics , Genes, Plant/genetics , Germ Cells, Plant/growth & development , Glucosyltransferases/genetics , Morphogenesis/genetics , Bryopsida/growth & development , Cellulose/metabolism , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Gene Knockout Techniques , Genetic Complementation Test , Genetic Vectors/genetics , Genotype , Germ Cells, Plant/cytology , Germ Cells, Plant/metabolism , Glucosyltransferases/metabolism , Microscopy, Confocal , Reproducibility of Results , Restriction Mapping , Reverse Transcriptase Polymerase Chain Reaction , Transformation, GeneticABSTRACT
The moss Physcomitrella patens has become established as a model for investigating plant gene function due to the feasibility of gene targeting. The chemical composition of the P. patens cell wall is similar to that of vascular plants and phylogenetic analyses of glycosyltransferase sequences from the P. patens genome have identified genes that putatively encode cell wall biosynthetic enzymes, providing a basis for investigating the evolution of cell wall polysaccharides and the enzymes that synthesize them. The protocols described in this chapter provide methods for targeted gene knockout in P. patens, from constructing vectors and maintaining cultures to transforming protoplasts and analyzing the genotypes and phenotypes of the resulting transformed lines.