9722956 Jones The goals of this project are to characterize the cellular events that accompany programmed cell death (PCD) in the barley aleurone cell. PCD in aleurone cells is controlled by the hormones gibberellic acid (GA) and abscisic acid (ABA). GA induces PCD in barley aleurone and ABA prevents cell death. Experiments are proposed to characterize DNA breakdown, to ask how DNA is degraded, and to identify the enzymes that are involved in this process. Apoptosis in animal cells is accompanied by intranucleosomal cleavage of nuclear DNA resulting in the formation of characteristic DNA ladders. Previously published work by others has suggested that DNA laddering is a feature of PCD in the barley aleurone cell. Preliminary data from the Principal Investigator's laboratory show that whereas PCD is accompanied by DNA breakdown in GA-treated aleurone cells, formation of DNA ladders does not occur. A detailed study is planned of DNA degradation from barley aleurone layers and protoplasts treated with hormones that regulate PCD to characterize the mechanism of DNA breakdown. The nucleases in aleurone cells will be characterized to determine whether these enzymes only possess exonuclease activity, which would account for the absence of DNA laddering in the aleurone cell. Experiments will also be carried out to determine when DNA breakdown occurs in aleurone cells. The Principal Investigator has established that DNA breakdown occurs after GA but not following ABA treatment. Is DNA degraded in living cells or does DNA degradation only occur after PCD? Is the absence of DNA degradation in ABA treated cells a result of the low levels of nuclease activity in these cells, or is DNA modified to be less rapidly degraded in ABA treated cells? Fluorescent dyes that stain DNA in living aleurone cells will be used to monitor DNA degradation and to correlate changes in DNA with PCD. If DNA breakdown occurs after aleurone cells die, dead cells will be purified from prot oplasts to monitor how the DNA is degraded. In the barley seed, there is a starch-rich tissue called the endosperm, which serves as a reservoir for nourishment of the developing plant embryo inside the seed. The aleurone layer is a cellular tissue surrounding the endosperm, which produces and secretes hydrolytic enzymes that digest the starchy endosperm into monosaccharides that are can be used directly as food by the developing embryo. The aleurone can thus be thought of as the digestive organ of the developing embryo within the germinating seed. When the aleurone has finished producing the enzymes, it has no further purpose and the cells of the tissue undergo a programmed cell death. The developmentally regulated life and death of the cells of the aleurone are under the control of two major plant hormones, gibberellic acid and abscisic acid. The results of this work will provide valuable insights into the process of developmentally regulated PCD in plants and the roles that hormones play in regulating this process. ***
