Autoregulation of pokeweed antiviral protein mRNA and genetic control of enzymatic browning in potato

Pokeweed antiviral protein (PAP), a ribosome inactivating protein (RIP) from Phytolacca americana, removes specific adenine and guanine residues from the conserved, alpha-sarcin/ricin loop in rRNA. PAP is an inhibitor of viral replication in plants and animals. Due to its cytotoxicity to dividing cells, PAP is currently used in clinical trials as an anticancer agent. However, the mechanism of antiviral activity and cytotoxicity of PAP is not understood. Accumulation of wild-type PAP protein in transgenic plants and Sacchromyces cerevisiae led to down-regulation of PAP mRNA accumulation, indicating that PAP autoregulates its expression. Using different PAP mutants, we present evidence that depurination of rRNA is required for autoregulation of PAP mRNA. PAP expression had no effect on accumulation of mRNA from endogenous yeast genes examined. PAP mRNA levels decreased post-transcriptionally and turnover of PAP mRNA was blocked when translation was inhibited by cycloheximide. This shows that PAP autoregulates its mRNA stability by a post-transcriptional mechanism that requires ongoing translation and ribosome depurination.; Browning of fruits and vegetables during storage and processing is of major importance in the food industry. The limitations and potential adverse health effects of antibrowning agents such as sulfites necessitate development of safer and more effective methods for the prevention of enzymatic browning. Enzymatic browning is caused by polyphenol oxidase (PPO) which catalyzes the oxidation of phenolic substrates to quinones that polymerize into melanin. PPO activity of Russet Burbank potato was inhibited by sense and antisense PPO RNA expressed from a tomato PPO cDNA under the control of the 35S promoter from cauliflower mosaic virus. Transgenic tubers from 37 different lines were harvested during 1997 and 1998. PPO activity, and the level of enzymatic browning were measured in these tubers. 28 lines that were sampled, five had reduced browning. The analysis of PPO activity correlated with reduction in enzymatic browning. These results indicate that expression of tomato PPO RNA in sense or antisense orientation limits PPO activity in tubers. Moreover, expression of a closely related heterologous gene can potentially be used to control enzymatic browning without the use of sulfiting agents.