Date of Award
Doctor of Philosophy
James T. Colbert
The phytochrome plant photoreceptor is important for perceiving the light environment and initiating plant developmental and environmental responses. When dark-grown oat seedlings are treated with light the phytochrome A (PHYA) mRNA abundance rapidly declines due, in part, to the inherent instability of the PHYA mRNA. Messenger RNA stability is an important process in regulating eukaryotic gene expression. My primary goal was to investigate both the mechanism of oat PHYA mRNA degradation and cis-acting sequences involved in regulating oat PHYA mRNA degradation. An oat protoplast system was established to investigate its use as a quick and reliable system to study plant mRNA degradation. The reliability of the oat protoplast system to accurately estimate cytoplasmic mRNA degradation rates could not be unequivocally demonstrated. The abundance and decay rates of distinct oat PHYA mRNAs were determined in RNA gel blots with gene-specific oligonucleotide probes. Two distinct oat PHYA mRNAs (PHYA3 and PHYA4) both had short half-lives, similar to that shown for the average PHYA mRNA half-life. Highly conserved sequences in the 5' and 3' untranslated regions of unstable PHYA mRNAs were identified as candidates for instability determinants. To investigate the mechanisms of PHYA mRNA degradation, putative PHYA mRNA degradation products were analyzed in RNA gel blots and used to predict ribonuclease activities. Polyadenylated RNA fractions contained a significant amount of PHYA mRNA degradation products while ~25% of the apparently full-length PHYA mRNA was poly(A)-deficient, and endoribonucleases do not appear to be involved in PHYA mRNA degradation. Overall, it appears that PHYA mRNA is degraded by two distinct pathways, one pathway appears to degrade polyadenylated PHYA mRNAs by a 5' to 3' exoribonuclease and the second appears to degrades deadneylated PHYA mRNA with a combination of 5' to 3' and 3' to 5' exoribonucleases. Finally, proteins that interact with PHYA mRNA were investigated, and two putative RNA-binding proteins, 25-kD and 20-kD in size, were determined from oat total protein and EDTA-treated polysome extracts, respectively. In the end these proteins appeared to non-specifically interact with PHYA mRNA, reducing the likelihood of them being involved in PHYA mRNA degradation.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
David Charles Higgs
Higgs, David Charles, "Identification of potential oat phytochrome A mRNA degradation mechanisms " (1994). Retrospective Theses and Dissertations. 11263.