RUI: Cell Wall Associated Protein Kinases and Cell Elongation

Bowdoin College, Brunswick ME

Investigators

Abstract

The plant Wall Associated Kinases,WAKs, which were discovered by Dr. Kohorn, are phosphorylating enzyme proteins that physically link the plasma membrane to the carbohydrate and protein matrix that comprises the plant cell wall. WAKs are of importance as they have the potential to directly signal cellular events through their cytoplasmic kinase domain. There are five Arabidopsis WAKs that vary primarily in their extracellular domain and that collectively are expressed in most tissues. WAK mRNA and protein are present in vegetative meristems, junctions of organ types and areas of cell expansion. They are also induced by pathogen infection and wounding. Recent experiments indicate that WAKs are also involved in pollen tube growth in the gynoecium, and may possibly be involved in pollen tube orientation. WAKs can be found bound to pectin in the cell wall, and at least one isoform is associated with a secreted glycine rich protein (GRP). Disruption by antisense and RNAi of WAK expression in leaves leads to the loss of cell expansion, indicating that WAKs play an important role in plant growth and development. It remains to be discovered how WAKs, pectins and GRPs combine to regulate the patterns of cell expansion in coordination with the complex architecture and mechanical constraints of the plant cell wall. This project will explore the role WAKs play in cell expansion and in pollen tube growth. A set of WAK specific antibodies will be generated to allow a thorough characterization of the protein expression of each family member and to serve as tools in the analysis of plants with mutations in individual WAKs. Arabidopsis lines carrying an insertion mutation in a WAK gene are unable to produce progeny homozygous for the mutation because fertilization is affected (mutant pollen grains are able to germinate but do not reach the egg). The exact nature of the defect will be determined using microscopy, antibodies and markers specific for the mutant pollen. The pollen mutant phentoype will be complemented such that the progeny can be made homozygous for the WAK mutation. The resulting vegetative mutant tissue will be analyzed to understand how WAKs mediate cell expansion. Tests for altered turgor, cell wall extension and altered expansin and cell wall enzyme expression will be performed. Application of Gibberellin (GA) rescues plants that are rendered dwarf by WAK mutations. Therefore, the relationship between the GA and WAK signaling pathways will be explored through a series of crosses with mutants in the GA pathway and through the analysis of GA biosynthetic enzyme expression. Finally, the interaction of WAKs with both extracellular glycine rich proteins and with cytoplasmic transcriptional adaptor proteins will be explored using genetics and biochemical analysis. The broader impacts of this project will be manifested primarily through the teaching and training of undergraduates. Bowdoin College is a predominantly undergraduate institution, and undergraduate student researchers will be extensively involved in performing these experiments.