Polyamine oxidase (PAO) catalyzes oxidative cleavage of polyamines spermine (spd) or spermine (spm) to produce diaminopropane (dap), H2O2, and an aminoaldehyde derivative. In plants, dap is the precursor for biosynthesis of norspermidine (nspd) and norspermine (nspm) via the enzyme, Schiff base reductase decarboxylase (SBRD). Recently, the catabolism of spd and spm by PAO has been proposed. By several investigators to be a causative agent, through product H2O2 and ensuing oxidative stress, which forces animal cells into programmed cell death (apoptosis). The gene for PAO from oat seedlings was recently cloned, sequenced, and characterized in this laboratory. The availability of this newly isolated gene offers unique opportunities to gain genetic evidence for a potential role(s) of PAO and polyamine-catabolism in apoptosis in a plant model test system. The hypothesis of this application is: (i) PAO has a causative role in apoptosis in cells through H2O2 produced by its oxidation of spd or spm, and (ii) the nspd and nspm produced from dap serve as suppressors of apoptosis through feedback inhibition of PAO and reduction in H2O2 synthesis.
The specific aims of this proposal are: (1) The PAO cDNA gene sequence will be used to transform alfalfa plants with Ti-plasmid methods to analyze the consequences of controlled PAO- deficiency on apoptosis in plant tissues. (2) The cDNA gene sequence for the enzyme, SBRD, which catalyzes nspd and nspm biosynthesis from dap, will be used to transform alfalfa plants in order to analyze the consequences of uncoupling generation of H202 by PAO from the biosynthesis of nspd and nspm derived from dap by SBRD.
Aims (1) and (2) are direct tests of parts (i) and (ii) of the hypothesis. (3). The PAO cDNA gene sequence from oat will be used as a gene probe to attempt the isolation of the human PAO gene from kidney and liver cDNA libraries. A characterized human PAO gene will make possible the development of a molecular biological approach to investigate the role of PAO in generating H202 and its alleged role to elicit animal apoptosis. (4) The cDNA gene sequence of a signal peptide for the PAO gene will be ligated to a gene coding for a fluorescent protein (GFP). The fusion protein produced from this construction will be analyzed by fluorescent imaging techniques in tissue of alfalfa and out plants transformed with this construction in order to identify the subcellular localization of PAO. In situ labeling of PAO by immunogold antibody reagents and electron microscopic analysis, will corroborate the localization studies by the GFP protein technique. These results will aid in localizing the origin of events that initiate PAO-dependent apoptosis.
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