Li B , Qiu J , Jayakannan M , Xu B , Li Y , Mayo GM , Tester M , Gilliham M , Roy SJ .

	Figure 1. In silico analysis of NPF2.5. (A) Protein sequence alignment of NPF2.5 to NAXT family members with known functions (NPF2.4, NPF2.3, and NPF2.7/NAXT1). Sequence alignment was performed using ClustalW2 with a gap open of 10 and a gap extension of 0.1. Black and shaded regions represent identical residues and conservative substitutions respectively. (B) NPF2.5 protein was predicted to have 12 trans-membrane domains and a hydrophobic loop. Trans-membrane domain prediction of NPF2.5 was performed using TMHMM 2.0 with default settings.
	Figure 2. NPF2.5 is predominantly expressed in the root and is salt-inducible. (A) NPF2.5 transcript abundance in the root and the shoot. Col-0 wild type Arabidopsis plants were grown hydroponically for 4 weeks before whole root and shoot were harvested separately for quantitative RT-PCR. (B) Transcript abundance of NPF2.5 was normalized relative to control group (2 mM NaCl) to show expression of NPF2.5 in response to salt stress. Four-week old Col-0 Arabidopsis plants were grown hydroponically before application of 2 mM, 50 mM or 100 mM NaCl for 72 h. Whole root was harvested for quantitative RT-PCR. Results are presented as mean ± SEM (n = 4); significance is indicated by the asterisks (one way ANOVA and Tukey test, **P ≤ 0.01, ***P ≤ 0.001).
	Figure 3. NPF2.5 is preferentially expressed in the cortical cells in the root. T2 proNPF2.5:uidA plants were grown on ½ MS media for 2 weeks. Plants were harvested and stained for GUS activity for 1 h. (A) No GUS activity in a wild type Col-0 plant. (B) GUS activity predominantly in the root of proNPF2.5:uidA plants. (C) GUS activity in the root of proNPF2.5:uidA plants. (D) GUS activity in the root-to-shoot junction of proNPF2.5:uidA plants. (E) Root cross-section of non-transformed Col-0 plant. (F) Root cross-section of proNPF2.5:uidA plant. Scale bars = 4 mm in (A,B), Scale bars = 0.3 mm in (C,D), Scale bars = 0.05 mm in (E,F).
	Figure 4. NPF2.5 is localized to the plasma membrane in Arabidopsis. (A) Green fluorescence detected in root cells of 2-week old GFP::NPF2.5 plants. (B) Transmitted light image of root cells of 2-week old GFP::NPF2.5 plants. (C–E) Confocal image of plasmolysis performed on root cells of 2-week GFP::NPF2.5 plants. (F) Yellow florescence of YFP::NPF2.5 fusion protein observed in an Arabidopsis mesophyll protoplast. (G) Cyan fluorescence of plasma membrane marker eCFP::ROP11 observed in the protoplast. (H) Transmitted light image of the protoplast. White arrow points to the separation of the tonoplast from the plasma membrane. (I) Merged image showing co-localisation of yellow fluorescence of YFP::NPF2.5 fusion protein and cyan fluorescence of plasma membrane marker. Scale bars = 50 μm in (A,B); Scale bars = 10 μm in (C–E); Scale bars = 20 μm in (F–I).
	Figure 5. Growth of NPF2.5 transformed yeast was inhibited by high levels of external Br−. Yeast was transformed with pYES2-DEST52/NPF2.5, or empty vector. Five 10 × serial dilutions of liquid culture were spotted and incubated at 28°C for 2 days on SD medium (-uracil) containing 2% (w/v) galactose, 1.67% (w/v) agar and salts as indicated. (A) 500 mM KCl; (B) 400 mM NaCl; (C) 400 mM KBr; (D) 300 mM NaBr.
	Figure 6. NPF2.5 encodes a protein that is able to facilitate Cl− efflux. MIFE and TEVC were performed to study putative Cl− transporter NPF2.5 both in planta and in heterologous system. (A) Inward currents of oocytes expressing NPF2.5 when membrane potential was clamped at −120 mV. Oocytes were incubated in a solution of ND50 (50 mM Cl−). Results are presented as mean ± SEM (n = 7); significance is indicated by asterisks (Two-tailed t-test, **P ≤ 0.005) (derived from Supplementary Figure 2A). (B) MIFE measurement of X. laevis oocytes for Cl− efflux. Oocytes were incubated in ND50 solution. Flux measurements were made for 15 min. Net fluxes of 5 or 7 individual oocytes were averaged for each treatment. Results are presented as mean ± SEM (n = 7 for NPF2.5 injected ones; n = 5 for controls; Two-tailed t-test, ***P ≤ 0.001). (C) MIFE measurement of plants for Cl− efflux. 2-week old npf2.5 and Col-0 plants were transferred to 48 mM NaCl solution for 30 min before a 15-min long measurement. Net fluxes of 6 or 9 individual seedlings were averaged for each genotype. Results are presented as mean ± SEM (n = 9 for npf2.5; n = 6 for Col-0; Two-tailed t-test, ***P ≤ 0.001). (D) NPF2.5 knockout mutant plants accumulated higher Cl− in the shoot. Four-week old T4 npf2.5 and Col-0 plants were grown hydroponically before being treated with 75 mM NaCl for 5 days. Cl− accumulation in the shoot of npf2.5 after salt treatment is presented as mean ± SEM (n = 9 for npf2.5; n = 4 for Col-0); significance is indicated by asterisks (Two-tailed t-test, *P ≤ 0.05).
	Figure 7. NPF2.5 amiRNA lines treated with 75 mM NaCl. Four-week old hydroponically grown T2 NPF2.5 amiRNA lines were treated with 75 mM NaCl for 5 days before harvest. (A) NPF2.5 transcript abundance detected in roots of knockdown lines and null segregate controls. (B) Shoot Cl− accumulation in knockdown lines and null segregate controls. (C) Shoot NO3− accumulation in knockdown lines and null segregate controls. (D) Shoot Na+ accumulation in knockdown lines and null segregate controls. (E) Shoot K+ accumulation in knockdown lines and null segregate controls. Results are presented as mean ± SEM (n = 4); significance is indicated by asterisks (one way ANOVA and Tukey test, *P ≤ 0.05; **P ≤ 0.01).
	Figure 8. NPF2.5 over-expression lines treated with low/high salt. Four-week old hydroponically grown T3 NPF2.5 over-expression lines were treated with 2 mM (low salt) or 75 mM NaCl (high salt) for 5 days before harvest. (A) NPF2.5 transcript abundance detected in roots of mutant lines and null segregant control plants. Columns with different letters indicate statistically significant difference (P ≤ 0.05). (B) Shoot Cl− accumulation in over-expressing lines and null segregate controls after low salt treatment. (C) Shoot NO3− accumulation in over-expressing lines and null segregate controls after low salt treatment. (D) Shoot Cl− accumulation in over-expressing lines and null segregate controls after high salt treatment. (E) Shoot NO3− accumulation in over-expressing lines and null segregate controls after high salt treatment. Results are presented as mean ± SEM (n = 4); significance is indicated by asterisks (one way ANOVA and Tukey test, *P ≤ 0.05).

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