Cryptochromes are blue-light photoreceptors found in plants and other organisms that control important aspects of growth and development. In animals, cryptochromes function as components of the circadian clock and are therefore of potential interest for treating jet lag and depression. They are also important in many plant processes. This research involves screening Arabidopsis plants for novel mutations within cryptochrome photoreceptors, followed by detailed structure/function analysis of the mutated proteins. A saturation mutagenesis approach will use simple, classic genetic screens available for detecting plant cryptochrome mutations. Mutant plants that are insensitive to blue light will be selected in populations of chemically mutagenized Arabidopsis seedlings followed by sequencing of mutant alleles by PCR-based methods. Expression of the cryptochrome protein in the mutant seedlings will be verified by Western Blot analysis. The mutant photoreceptor proteins will then be expressed in a recombinant baculovirus expression system, from which large amounts of virtually pure receptor protein can be prepared. Mutant cryptochrome proteins will be compared to wild type proteins via both spectroscopic (absorption and fluorescence) and autophosphorylation assays. It will therefore be possible to relate the effect of mutations that partially or totally inactivate the cryptochrome photoreceptor in vivo with biochemical and biophysical properties of the photoreceptor in vitro. These data should help to provide insight into the primary mechanism of action of cryptochrome, and will complement several site-specific mutagenic approaches being undertaken simultaneously by the investigators.
Broader Impact: The broader impacts of this research are the development of the infrastructure of the university and the stimulation of student interest in molecular biology at the regional campus. Equipment for advanced molecular biology techniques will be shared with the teaching laboratories for use in designing more advances laboratory courses. The intensive summer research program in Paris will provide students with direct experience working in a foreign country and engender an appreciation of the international nature of the scientific community as well as providing a full-time exposure to a research environment of advanced standing. Based on the record of past students who have participated in this program, the potential for stimulating student interest in a scientific career is significant.
