Polyamine oxidase (PAO) catalyzes oxidative cleavage of polyamines spermidine (spd) or spermine (spm) to yield diaminopropane, H202, and an aminoaldehyde derivative. A voluminous literature correlates the catabolism of spd and spm by PAO in plant and animal cells with the process of programmed cell death (apoptosis). Numerous studies have suggested that the causative agent for eliciting apoptosis is oxidative stress mediated by H202. The gene for PAO was first cloned, sequenced, and characterized in this laboratory from oat seedlings. The availability of this isolated gene offers unique opportunities to gain genetic evidence for potential roles of PAO and polyamine-catabolism in apoptosis in a plant model test system. The hypothesis of this application is: PAO has a causative role in programmed cell death (apoptosis) through H202 produced by oxidation of spd or spm.
The specific aims of this proposal are: (1) To define the potential role of PAO as a major generator of cellular H202, and hence as a determinant of apoptosis. The PAO cDNA gene sequence has been used to generate antisense PAO gene segments. Plasmid constructs of these antisense and corresponding sense sequences, ligated to chemically controllable promoters, will be used to produce dsRNA in transformed maize, in order to analyze the consequences on apoptosis of post-translational gene silencing of PAO by RNAi; (2) To exploit the biochemical properties of PAO for depleting cellular levels of polyamines in recombinant cells by controlled expression of a trans-gene for PAO using chemically-activated promoters.
This aim seeks to construct gene vectors that can be applied to a spectrum of cells, in order to delineate functions for the polyamines; (3) To confirm that a putative promoter sequence -AGGGACTTTCCG-, which we have discovered in the 5C-promoter region of the PAO gene, is a recognition signal for a pro-apoptotic, H202-activated NF-kappaB-like transcription factor. We also seek to identify an NF-kappaB-like protein in plants which binds to this region; (4) To localize the cellular compartment where PAO resides in natural and recombinant cell systems. In situ labeling of PAO by immunogold antibody reagents, followed by electron microscopy will corroborate localization studies by the GFP fusion protein technique. This information will localize the origin of events that initiate programmed cell death with participation of PAO.
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