The plant hormone auxin regulates various aspects of plant growth and development such as cell division, differentiation, morphogenesis, oncogenesis, and is generally considered to be responsible for regulating plant cell growth. The primary mechanism of action of the hormone is poorly understood; however, recent experimental evidence indicates that the hormone has the capacity to act very rapidly at the transcriptional level. Biochemical and genetic studies on the regulation of the auxin inducible genes in Pisum sativum (pea) and Arabidopis thaliana are proposed herein to gain insight into the details of the biochemical machinery that regulates their expression and their role in plant cell growth.
The specific aims of the proposal are: 1. To isolate and structurally characterize the rest of the auxin regulated genes in pea for identifying the presence of common DNA sequences in their promoter region. 2. To define the Cis-acting elements in the PSIAA4/5 gene responsible for auxin and cycloheximide inducibility. The elements will be defined by BAL-31 deletion analysis of the promoter region. Promoter constructs with appropriate reporter genes will be introduced into plant tissue by, a) high velocity microprojectiles, b) electroporation, and, c) Ti-mediate transformation. 3. To purify by DNA affinity chromatography transcriptional factors interacting with the PSIAA4/5 promoter and isolate complementary DNA sequences to some of these factors by screening lambda gtll expression libraries with DNA specific probes. 4. To isolate mutations in Arabidopis thaliana for genes acting in trans on the auxin regulated genes. 5. To isolate complementary DNA sequences to the putative repressor of the auxin regulated genes by complementation experiments in Saccharomyces cerevisiae. 6. To define the cell types that respond to auxin by RNA in situ hybridization techniques and to subcellularly localize the proteins encoded by some of these genes with antibodies made to synthetic peptides by means of the electron microscopy.
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