Molecular Genetic Analysis of det Loci of Arabidopsis thaliana

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The purpose of this proposal is to begin to unravel the control circuits of light-regulated seedling development using Arabidopsis thaliana mutants, the de-etiolated mutants (det). Previous work in my laboratory has shown that recessive mutations in any one of four DET genes uncouple light signals from a number of light-dependent processes, resulting in dark-grown plants that grow as light-grown seedlings. Because det mutations are both recessive and pleiotropic, we have proposed that the wild-type DET proteins play a negative regulatory role in light-regulated gene expression and development in Arabidopsis. The genetic, biochemical, and molecular experiments proposed here seek to further define these genes, with emphasis placed on the DET1 gene and its protein product, DET1. A major objective of the proposed experiments is to complete the cloning the DET1 locus. We are cloning DET1 by chromosome walking from a nearby RFLP marker. We have cloned over 1 megabase of DNA around the chromosome walk is almost finished. The DET1 gene will be used in biochemical and genetic experiments to begin analysis of the molecular mechanisms of how light signals are transduced to developmental strategies. To further elucidate the role of DET1 in the negative regulation of the light response, we have isolated extragenic suppressors of the det1-1 mutation. These suppressors will be mapped and extensively characterized in the det1- and DET+ backgrounds with respect to light-regulated gene expression, pigment synthesis, and leaf and chloroplast development. Finally, we plan to investigate the functional interactions between the various det genes and other genes involved in light signal transduction by constructing doubly mutant strains carrying both det and various other photomorphogenetic mutations that have been previously isolated by ourselves and others. The interactions between the various det genes will also be determined by analysis of doubly, triply, and quadruply mutant lines. %%% The date from these experiments will allow us to construct models of the mechanisms by which light and other signals control chloroplast development in plants. Arabidopsis thaliana is uniquely suited for these studies due to its small genome size, high seed yield, low level of interspersed repetitive DNA, and transformability.