A voluminous literature documents studies that correlate the catabolism of the polyamines, spermidine and spermine, in plant and animal cells with the process of programmed cell death (apoptosis). Polyamine oxidase (PAO) catalyzes oxygen-dependent cleavage of the polyamines to form hydrogen peroxide (H2O2), among other products. Many studies indicate that the causative agent for eliciting apoptosis is oxidative stress mediated by H2O2. Our project hypothesis is: The reaction catalyzed by the enzyme PAO is the major source of cellular H2O2 that causes H2O2-mediated apoptosis in eukaryotic cells. We have gained strong evidence that PAO catalysis is indeed the major source of H2O2 in a plant model system undergoing the hypersensitive response (HR). HR is a pathogen-elicited process mediated by H2O2, which culminates in apoptosis. The origin of the H2O2 is not known. In preliminary studies, we have applied RNA interference (RNAi) methods, using a newly discovered, rapid technique for introducing double-stranded (dsRNA) into plants during HR, and have succeeded in knocking down PAO enzymatic expression with concurrent abolishment of the H2O2 burst that normally accompanies HR.
The specific aims of this project are: (1) RNA interference (RNAi) methods that exploit our new technique for introducing dsRNA into plant cells will be used to silence (knockdown) PAO gene expression in Avena sativa L. (oat) seedlings. This knockdown will be shown to result in suppression of five cellular parameters: PAO mRNA levels, PAO enzymatic activity, the H2O2 burst in the HR, catabolic loss of cellular polyamines during the HR, and entry into apoptosis. (2) The existence of an NF-kB-like transcription factor in plants will be established, which may have a role in regulating the expression of PAO by pro-apoptotic, H2O2-activated pathways. The DNA nucleotide sequence (gtgaatttcc) was discovered by us in two locations of the 5'-untranslated promoter region of the PAO gene from Oryza sativa (rice). This sequence contains recognition sites for binding transcription factors that link PAO gene transcription to NF-kB-like factors. We will complete the isolation of this protein to confirm its existence in the plant kingdom. (3) Methodologies, plant seed stocks, and plasmid vectors will be developed in order to exploit a synthetic copper(ll) ion-dependent genetic promoter system, produced by our laboratory, for controlling the synthesis of trans genes in plant and yeast hosts.
