| Popis: |
Changes in the level of polyamines enable plants to respond optimally to both biotic and abiotic stresses such as increased salinity, drought, chilling, flooding, heavy metals and increased concentrations of reactive oxygen species. To identify new classes of transporters that mediate movement of polyamine between cells and within organelles, this study focused on the OCT family in the model plant Arabidopsis thaliana. The A. thaliana OCT family contains six members with no splice forms and have been less studied. Because the expression of OCT5 is upregulated during salt stress assays of seedlings, we tested the response of oct5 seedlings under these conditions. In the salt assays, oct5Δ mutants showed faster bleaching of leaves than the wild type, a common phenotype associated with increased sensitivity to salt stress. To determine whether OCT5, could function as a polyamine transporter, OCT5 was heterologously expressed in WT yeast and in tpo5Δ, a yeast strain deficient in polyamine export. The dilution of rapidly growing yeast cells in liquid culture to fresh media containing elevated levels of putrescine, resulted in an inhibition of growth for all strains in response to 150 or 200 mM putrescine. WT cells expressing OCT5 were more sensitive to exogenous putrescine than WT cells alone. As expected, the tpo5Δ yeast strain was more sensitive to exogenous putrescine than WT cells, but the heterologous expression of OCT5 in this strain did not result in further growth inhibition. The inhibition of growth in WT cells expressing OCT5 was unexpected, since this gene is localized to the vacuole in plants and increased expression of this gene was expected to have a protective effect on cell growth by having this transporter sequester putrescine from the cytoplasm in the vacuole. However, in humans, OCT transporters are polyspecific and are believed to mediate substrate transport in either direction. Thus the increased sensitivity to putrescine when this gene was expressed in yeast could have been due to export from the vacuole to the cytoplasm. Future experiments will determine whether OCT5 is also localized to the yeast vacuolar membrane and whether OCT5 could also function as a vacuolar exporter of putrescine. To address the substrate specificity of OCT1, in future work, this gene has been expressed in an E. coli strain deficient in the polyamine exchangers CadB and PotE. |
