The primary mechanism of action of the plant hormone auxin is poorly understood; however, recent experimental evidence indicates that the hormone has the capacity to act very rapidly at the transcriptional or post-transcriptional level. Studies on the structure and regulation of the auxin-inducible genes in pea stem tissue 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 this proposal are: 1. Isolation and structural characterization of genomic sequences to early auxin-inducible mRNAs from pea tissue. 2. Regulation of expression of the hormonally regulated genes in vivo and in isolated pea nuclei. 3. Purification and subcellular localization of the proteins coded by the auxin-regulated genes. To achieve the above goals the following experiments are proposed. Genomic libraries will be constructed into the cloning vector EMBL 3 using DNA from etiolated pea seedlings. The genomic sequences of the early auxin-regulated mRNAs will be isolated by plaque filter hybridization using the already isolated cDNA clones pIAA4/5 and pIAA6 as probes. The organization and structural analysis of the genes will be investigated in detail. To determine whether auxin acts at the transcriptional or post-transcriptional level, the stability of the inducible mRNAs, will be examined in vivo. These experiments will be supplemented with in vitro transcription in isolated nuclei. Finally, the auxin cDNA clones will be introduced into Lambda expression vectors to produce fused proteins with Beta-galactosidase. Antibodies directed toward the hybrid proteins will then be used to purify the proteins, localize them at the subcellular level and determine the kinetics of their accumulation during cell growth.
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