| Ethylene regulates fruit ripening in tomato, partly through transcriptional activation. I have studied the function of one such gene product, the E8 protein. E8 expression is regulated by ethylene-dependent and-independent factors in ripening tomato fruits, and E8 has a negative effect on ethylene evolution in fruit. Sequence analysis showed that the E8 protein belongs to a family of enzymes termed 2-oxoglutarate-dependent dioxygenases (2-ODD's).;E8's affect on ethylene biosynthesis was studied using transgenic tomato plants exhibiting altered levels of E8 protein. Reduced E8 protein levels resulted in a 7-fold increase in ethylene evolution, the level of 1-aminocyclopropane-1-carboxylic acid (ACC) and ACC oxidase mRNA during fruit ripening. ACC synthase 2 (ACS2) mRNA was increased in these plants, 2.5-fold; ACS4 mRNA was not affected. E8 was found to be expressed in the pollen and anthers of tomato. In anthers, a reduction in E8 protein had no effect on ethylene biosynthesis.;Using size-exclusion chromatography and the yeast two-hybrid assay, I examined the possibilities that E8 interacted with ACO or as a homodimer. These experiments did not support these hypotheses.;Experiments were performed to immunolocalize the E8 protein, and progress was made towards this goal.;Examination of two novel E8 homologs in Arabidopsis thaliana suggested that there are at least three such homologs in the Arabidopsis genome, which share sequence similarity with E8-9 and E8-6 tomato cDNAs. The mRNA encoding one of these, 40F, appears to be increased 2-fold by treatment with ACC in shoots. Both genes are expressed in flowers, shoots and siliques. Ethylene evolution in Arabidopsis siliques increased as the siliques matured, suggesting a role for ethylene in Arabidopsis silique maturation.;Taken together, these data suggest that the E8 protein has a broader role than originally thought. E8 protein is expressed in several tissues in tomato, and may function in non-climeracteric species such as Arabidopsis. The affect of E8 on ethylene biosynthesis in tomato fruits either requires factors specific to ripening fruits or is secondary to its primary function. |
