9303877 Jones The objectives of this research are to localize the receptors for gibberellins (GAs) and abscisic acid (ABA) in the aleurone layer of barley and to identify and test the roles of the molecules that transduce hormonal signals in this cell. Alpha amylase secretion can be visualized when single, secreting protoplasts are embedded in an agarose matrix containing starch. This secretory response provides a rapid assay for the effects of GA and ABA. Preliminary experiments show that cells microinjected with GA or ABA do not respond to these hormones by changing their rate of secretion, but when GA or ABA are presented to the cell surface, GA stimulates alpha-amylase secretion and ABA inhibits it. The cellular location of the receptors for these hormones will be determined. Aleurone protoplasts will also be used to examine the events that lead from hormone perception to cellular responses. One of the first detectable responses to GA in the aleurone cells is proton extrusion. Since proton extrusion via the plasma membrane proton pump is associated with hyperpolarization of plant cells, the hypothesis that membrane potential measurements can be used as an assay for the early response of aleurone cells to GA and ABA will be tested. The pH of the cytosol will be mapped and quantitated fluorescent dyes and confocal microscopy to determine whether pH plays a role in signal transduction. The possibility that protons and membrane potential act to gate ion channels in the plasma membrane will be explored using patch clamp. Calmodulin has been implicated in signal transduction in the aleurone cell. The relationship between calmodulin and GA and ABA will be studied, and the distribution of activated calmodulin in aleurone cells will be mapped using a new fluorescent probe. These experiments should establish whether the calmodulin gene, like the genes for secretory proteins, are regulated by GA and ABA at the transcriptional level in aleurone cells. The m apping of calmodulin activity should clarify the relationship between calmodulin and membrane transporters whose activities are regulated by calmodulin. The roles that molecules such as calcium, calmodulin, and protons play in signal transduction will be tested by asking whether the signal transduction pathway from GA or ABA can be mimicked in aleurone cells by the introduction of components of the signaling pathway that act as agonists or antagonists of the hormones. %%% The goals of this research are to understand the perception and transduction of hormonal signals in plants. Techniques have been developed to test the hypothesis that some hormones are perceived at the cell membrane, and perception generates signals that affect the activity of proteins within the cell. The roles that molecules such as calcium, calmodulin, and protons play in signal transduction will be tested using a variety of biochemical and biophysical techniques. The results will provide fundamental new knowledge about the nature of the receptors and signal transduction pathways linking GA and ABA to the response of the cereal aleurone cell. ***
