| Polyamine oxidase (PAO) catalyzes the oxidative cleavage of the polyamines spermine and spermidine. Hydrogen peroxide is one molecule produced by the oxidative catabolism of polyamines. In plants, it is hypothesized that under stress conditions, the hydrogen peroxide produced by PAO is a major contributor to protective responses such as the hypersensitive response (HR), which leads to programmed cell death.; For the purpose of this research, this broad hypothesis was focused to the following: Through the catabolism of polyamines, PAO significantly contributes to the levels of hydrogen peroxide responsible for the oxidative burst during HR. The first approach to test this hypothesis involved silencing PAO using RNA interference. It was shown that lowering cellular levels of PAO enzymatic activity in this fashion resulted in an approximately 50% reduction in the levels of hydrogen peroxide during the late HR. This indicated that the hydrogen peroxide produced by PAO significantly contributes to the oxidative burst of HR.; The second approach to link PAO with HR involved exploring the promoter region of PAO for transcription factor binding sites related to defense responses. Using bioinformatics tools, a 500-bp repeat within the promoter region of rice PAO was discovered. Within this region was a 32-nt cluster of five stress-related transcription factor binding sites, including the mammalian transcription factor NF-kappaB. The PAO promoter region was also compared to the promoter region of PR-1, a gene that is essential in the induction of HR. Several similarities were discovered, including a phosphate starvation binding site and an NF-kappaB binding site.; During the course of this research, a copper-inducible gene promoter was constructed. This promoter, called ABS2, has been shown to drive extremely high levels of transcription and exhibit very low background levels of transcription in the absence of inducer. This promoter was also tested transiently in Nicotiana tabacum, a plant system. In this system, ABS2 was shown to drive higher levels of expression than ABS4, the promoter that it was based on (Mett, 1993). ABS2 was also shown to have lower background levels of transcription in the absence of inducer than the original ABS4 promoter. |
