A Guide to the Continuing Investigation of the Relationship between the Cytoskeleton and Cell Wall in Developing Buds of Physcomitrella patens

Persistent questions in biology address the nature of tissue organization and how information encoded in the genome can be manifested as a physical form. While the ‘final’ product of gene expression is a protein, science has yet to elucidate how those proteins are able to interact with other cellular components and external forces to generate a specific cell shape. This complex process is critical in determining not only the shape of an individual cell but also that of an entire organ. In plants, cell shape is controlled by cellulose microfibrils of the cell wall, which are typically oriented perpendicular to the direction of cell growth. The deposition of cellulose microfibrils is believed to be directed by the orientation of cortical microtubules. What controls the orientation of those microtubules remains unknown. Moreover, there is evidence that physical forces acting on the cell are responsible for cell shape as much as internal cell chemistry. How all of these factors interact to generate the final cell and organ shape in plants is the subject of this project. To investigate these questions, the moss Physcomitrella patens was used as a model organism to study leafy shoot morphogenesis. Molecular techniques were employed to label the microtubules with green fluorescent protein (GFP) in two strains of the moss – the wild type and a cellulose synthase mutant with reduced cell wall synthesis in the buds that produce leafy shoots. Microscopy methods were then developed to view microtubule and cellulose microfibril behavior while the cells divide and the buds develop into leafy shoots. More work remains to be done and this manuscript is intended to serve as a guide for the student who wishes to complete this project. The data gained from these experiments can help to clarify the relationship between the major factors controlling cell and organ shape. Particularly, this data can help reveal the extent to which external physical forces play a role in organ development. The strains of moss with labeled cytoskeletons that were produced will also be useful for further studies in developmental cell biology