Expansins are plant cell wall-loosening proteins with the unique ability to catalyze the relaxation and extension of plant cell walls in a pH-dependent manner. These proteins were originally discovered in the course of a biochemical dissection of the mechanism of wall growth (extension) in cucumber hypocotyl walls. In the interim, expansins have been identified in many plant species and are believed to function in cell enlargement and perhaps in other processes where cell wall loosening may be important, e. g. cell separation and cell wall dissolution. Work published last year identified a second family of expansins with different substrate specificity and our analysis of data from the Arabidopsis genome project indicates that expansins comprise a large multigene superfamily. This proposal outlines an ambitious research plan aimed at two broad questions: (A) what is the molecular mechanism by which expansins increase wall extensibility, and (B) what are the specific developmental functions of the individual members of this superfamily of genes. To answer question (A), we will make use of recombinant expansins expressed in the Baculovirus/insect cell culture system. These will be used in biochemical assays designed to identify the specific substrate that expansins work on and the specific biochemical nature of their wall loosening action. Site-directed mutagensis will be used to modify specific amino acid residues hypothesized to form the catalytic site of the protein. The PI will also use epitope tagging to examine the mobility of expansins within the wall - potentially an important issue for cell-specific control of cell enlargement and for the interpretation of experiments requiring extraction of expansins to assay their activity. To answer question (B), the PI will take advantage of the wealth of sequence information and other tools generated from the Arabidopsis genome project. By a combination of methods we plan to identify expression patterns for all the members of the expansin superfamily and to identify and characterize expansin null mutants from the growing collection of T-DNA and transposon insertional mutants. Experiments to test for regulation of specific expansins by hormones, light and other growth stimuli are also outlined. These experiments should shed light on the involvement of expansins in the control of plant cell wall rheology, growth and other morphogenetic processes.
