Avena phytochrome mRNA: in vivo localization, degradation products, and half-life
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The purpose of this research is to determine the role that post-transcriptional processes play in the regulation of phytochrome (phy) steady-state messenger RNA (mRNA) levels. The control of etiolated phytochrome (phyA) and chlorophyll a/b binding protein (cab) gene transcription and steady-state mRNA levels are known to be regulated by the phyA signal transduction pathway. I performed a number of control experiments that indicate the degradation products detected in northern analysis of phyA mRNA were produced in vivo and were not an artifact of RNA isolation techniques. I have also shown that infrared (IR) irradiation has no appreciable effect on phyA steady state mRNA abundance, which allowed us to use IR radiation to observe while manipulating the seedlings in otherwise total darkness. I have analyzed the steady-state levels of phyA and cab mRNA in different tissues in oat seedlings using northern hybridization analysis and found that most of the phyA mRNA in the oat seedling is localized in the coleoptile sheath and mesocotyl node, with very low levels found in the enclosed primary leaf. In contrast, cab mRNAs are found almost exclusively in the enclosed primary leaf. Experiments with excised primary leaves and coleoptiles show that cab can be induced in the leaf in the absence of the coleoptile, which contains the majority of the phyA message and the phytochrome protein. Experiments using cordycepin (an adenosine analog) as a transcription inhibitor indicate that the phyA mRNA has a 1 hr half-life in dark grown seedlings. This is similar to the 60-90 min half-life seen following red light induced phyA transcription termination. Control experiments showed that the phyA mRNA levels were not affected by the abrasion or buffers involved in the inhibitor experiments. Although more inhibitor data are needed, it appears that phyA mRNA is continuously turned over at a rate that is substantially higher than that of the average plant mRNA, independent of light treatment. In addition, a large part of the phyA mRNA and protein found in the seedling is not involved in the light-induced increase in cab mRNA levels.