Investigation of chloride transport mechanisms in Arabidopsis thaliana root

Abstract

Salinity tolerance is correlated with shoot chloride (Cl⁻) exclusion in many horticultural and crop species (e.g. grapevine, soybean). It is hypothesized that the key regulatory step in root-to-shoot transfer of Cl⁻ is conferred by plasma membrane-localised anion transporters associated within the root vasculature. Reducing long-distance Cl⁻ transport by manipulating the regulation of anion transporters in the root vasculature is therefore a strategy that promises to increase plant tolerance to saline environments. However, the information of which candidate genes are responsible for this process is limited. To gain a greater knowledge of the long distance Cl⁻ movement from a molecular aspect, a number of candidate anion transporters from Arabidopsis thaliana were identified from a preliminary microarray study. Quantitative PCR was used to indicate transcriptional levels of candidate anion transporters that decreased upon NaCl and ABA treatment. Based on this analysis, AtSLAH1, AtSLAH3 and AtNRT1.5 were selected as genes of interest (GOI) that were likely to be involved in the Cl⁻ movement between the root stele symplast and the xylem vessels. To functionally characterize the transport properties of all GOIs at a protein level, various heterologous systems were used to investigate the anion (Cl⁻ and NO₃⁻) transport capacity. Two-electrode voltage clamp electrophysiology was used to measure the currents that were generated by the target anions crossing oocyte membranes. A yeast expression system was also used to further study the anion transport properties in vitro. AtSLAH1 cRNA injected oocytes were not able to produce significant anion currents. Also, no evident anion currents were generated from a site-directed mutant of AtSLAH1 in a putative phosphorylation site injected into oocytes. Although there was evidence that anion currents were elicited from AtSLAH1 and AtSnRk2.3 co-injected oocytes, due to difficulties in the ability to reproduce these results, it is uncertain whether AtSLAH1 can function as an anion transporter in the conditions tested. Both wild type and site-mutated AtSLAH1 was also separately transformed into yeast for further examination without an observable phenotype. In order to examine the effect of altered AtSLAH1 expression on shoot anion accumulation, AtSLAH1 amiRNA knockdown and constitutive over expression of AtSLAH1 mutant plants were generated. AtSLAH1 knockdown lines (T2) exhibited strong repression in transcript abundance in low salt environments and resulted in a significant reduction in shoot Cl⁻ when compared to nulls. Constitutive over expression of AtSLAH1 showed increased shoot Cl⁻ contents under high salt stress. These results indicated the potential role of AtSLAH1 in Cl⁻ transport in plants. Electrophysiological characterization of AtSLAH3 in oocytes showed that AtSLAH3 was able to produce significant NO₃⁻ but not Cl⁻ currents suggesting a role in the efflux of NO₃⁻ out of cells in most of circumstances. Similar results were gained in AtSLAH3- transformed yeast. However, AtSLAH3 over-expression lines showed a decreased shoot Cl⁻ without an effect on shoot NO₃⁻ under high salt stress compared to null plants. The potential reasons for this are discussed and further experiments are proposed to test these hypotheses. Although AtNRT1.5 has been reported to transport NO₃⁻, electrophysiological characterization of AtNRT1.5 in X. Laevis oocytes was not able to detect any anion currents induced by the gene. Interestingly, AtNRT1.5 transformed yeast showed a significant inhibited phenotype (grow less well than empty vector control) when challenged with high concentration of Cl⁻ and NO₃⁻ within the growth media, indicating a role the transport of both anions. Constitutive over-expression lines showed a potent shoot Cl⁻ reduction under high salt stress compared to nulls. Interestingly, no significant NO₃⁻ accumulation in shoot was identified. These results might suggest that AtNRT1.5 was able to regulate both Cl⁻ and NO₃⁻ transport from root to shoot; however, the mechanism by which this occurs is unclear. Previous findings indicated the possibilities that Cl⁻ and NO₃⁻ can be transported through the same anion channel/transporter. To further study the regulation of Cl⁻ and NO₃⁻ uptake, an anion blocker (DIDS) was used to test the anion shoot accumulation under different salt conditions. Under high salt stress, DIDS was able to reduce the Cl⁻ accumulation and increase the NO₃⁻ contents in shoots. Further experiments are required at both a physiological and molecular level to further understand how plants recognize and respond to this blocker, as the molecular targets of this blocker are a potential way to improve the plant salt tolerance and nitrogen use efficiency under high salt stress. In summary, new information was revealed on several candidates that affect root-to-shoot loading of chloride and new research avenues have been proposed based on the findings of this study.