POT proteins are important for chloride transport in Arabidopsis.

Chloride (Cl⁻) is an essential plant micronutrient, but is toxic when accumulated to high concentrations within the cytoplasm, especially in the shoot. Exclusion of Cl⁻ from the shoot is an important trait contributing to salinity tolerance of plants, particularly for Cl⁻ sensitive woody perennials (e.g. grapevine, citrus and avocado) and legumes (e.g. soybean and lotus), where Cl⁻ is considered to be more toxic than the sodium ion (Na⁺). To enhance plant salinity tolerance, it is necessary to understand the mechanisms of Cl⁻ transport through the plant and how it is regulated in response to salinity stress. However, when compared with Na⁺, much less is known about the transport processes involved in controlling Cl⁻ accumulation in the shoot. Two candidate genes encoding putative Cl⁻ transporters in Arabidopsis, proton dependent oligo-peptide transporter 1 (AtPOT1) and AtPOT2 were investigated to examine their role in controlling the loading of Cl⁻ into the apoplastic vessels of root xylem, and therefore Cl⁻ accumulation in the shoot. Transient expression of yellow fluorescent protein (YFP)::AtPOT1 or YFP::AtPOT2 in Arabidopsis mesophyll protoplasts, along with stable expression of green fluorescent protein (GFP)::AtPOT1 or GFP::AtPOT2 determined that both AtPOT1 and AtPOT2 are targeted to the plasma membrane, a location necessary for both POTs to be involved in facilitating Cl⁻ efflux from a cell. Promoter:UidA fusions showed that pAtPOT1 drives expression of the AtPOT1 predominantly in the root stelar cells, suggesting the involvement of AtPOT1 in long distance transport in vasculature tissue. In contrast, AtPOT2 was shown to be located in the cortex of the mature root. Use of quantitative real-time PCR to determine the levels of mRNA transcripts in response to salt stress demonstrated that AtPOT1 transcripts are significantly reduced by both salt and ABA treatments, whereas AtPOT2 transcripts are increased by salt stress. As AtPOT1 transcripts are reduced by ABA and as AtPOT1 encodes an anion transporter located at the plasma membrane of the cells bordering root xylem vessels, it is hypothesised that AtPOT1 is responsible, at least partially, for loading of Cl⁻ into the conductive cells of xylem in roots. Electrophysiological characterisation of AtPOT1 in Xenopus laevis oocytes showed that AtPOT1 is able to facilitate Cl⁻ efflux across the cell membrane at negative membrane potentials, suggesting a role of AtPOT1 in the efflux of Cl⁻ across the plasma membrane of xylem parenchyma cells into the apoplastic xylem transpiration stream. This flux was not affected by the changes in external pH, consistent with the Cl⁻ transport being a uniport, independent of the movement of H⁺. There were no knockout mutants of AtPOT1 available. Therefore, in order to test the effect of alterations of AtPOT1 expression on Cl⁻ accumulation in the shoot, artificial microRNA knockdown constructs were designed and used to transform Arabidopsis Col-0 plants. AtPOT1 transcripts were shown to be reduced by up to 80% in the knockdown lines when compared with nulls, which resulted in a reduction in shoot Cl⁻ concentration by up to 60%. AtPOT1 expression was found to be negatively correlated with shoot Cl⁻ concentration (R² = 0.77). Conversely, constitutive over expression of AtPOT1 increased shoot Cl⁻ accumulation, indicating the important role that AtPOT1 plays in facilitating Cl⁻ xylem loading in Arabidopsis. It is concluded that AtPOT1 mediates Cl⁻ flux into the conductive cells of root xylem in Arabidopsis and the expression of the AtPOT1 is down-regulated during salinity stress. Manipulations of AtPOT1 transcript levels altered shoot Cl⁻ concentrations, which could be utilised for enhancing shoot exclusion of Cl⁻, and hence plant salinity tolerance. Although more functional data is required, AtPOT2 might be involved in the efflux of Cl⁻ from the root to the soil. Therefore, reducing AtPOT1 expression and increasing AtPOT2 expression, may be two strategies for excluding Cl⁻ from the shoot under saline conditions.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 201