1. Volume 26, Issue 16, Pages 2213-2220 (August 2016)
Silent S-Type Anion Channel Subunit SLAH1 Gates SLAH3 Open for Chloride Root-to- Shoot Translocation 
Paloma Cubero-Font, Tobias Maierhofer, Justyna Jaslan, Miguel A. Rosales, Joaquín Espartero, Pablo Díaz-Rueda, Heike M. Müller, Anna-Lena Hürter, Khaled A.S. AL-Rasheid, Irene Marten, Rainer Hedrich, José M. Colmenero-Flores, Dietmar Geiger 
Current Biology 
Volume 26, Issue 16, Pages (August 2016)
DOI: /j.cub
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2. Figure 1
AtSLAH1 and AtSLAH3 Are Co-expressed in the Xylem-Pole Pericycle
Localization of AtSLAH1 (A–D) and AtSLAH3 (E–H) gene expression in transgenic Arabidopsis plants expressing the GFP::GUS or GUS reporter genes under the control of the AtSLAH1 and AtSLAH3 promoter regions, respectively. Tissue- and cell-specific expression of AtSLAH1 gene (A, C, and D) or AtSLAH3 gene (E–H) was obtained through histochemical localization of GUS activity. Localization of SLAH1-regulated GFP fluorescence in pericycle cells was observed through confocal microscopy (B). E, epidermis; C, cortex; asterisk, endodermis; red spot, protoxylem; blue spot, metaxylem. Scale bars, 100 μm (A and E), 20 μm (C and G) and 10 μm (B, D, F, and H).
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3. Figure 2
AtSLAH1 and AtSLAH3 Regulate Root-to-Shoot Translocation of Cl−
(A and B) Percentage of Cl− (A) or NO3− (B) xylem sap concentration, relative to the wild-type (WT; 100%), in the slah1-2 mutant line and in the two slah1-2/SLAH1 complemented lines (P1-12 and P1-31). For the slah1-2 line, values are the average (±SE) of 12 biological replicates obtained from 12 independent experiments. For slah1-complemented lines, values are the average (±SE) of four biological replicates obtained from four independent experiments. Different letters indicate statistically significant differences (p < 0.05, Student’s t test).
(C and D) Percentage of Cl− (C) or NO3− (D) xylem sap concentration relative to the WT line (100%) in the slah3-1 and slah3-4 mutant lines. For the slah3-1 line, values are the average (±SE) of nine biological replicates from three independent experiments. For the slah3-4 line, values are the average (±SE) of three biological replicates from one experiment. Different letters indicate statistically significant differences (p < 0.05, Student’s t test).
(E) Chloride concentration in the xylem sap of the slah1-2 mutant line and the WT lines 3 hr after high salt load treatments (15, 30, 60, and 120 mM Cl−). Values are the average (±SE) of three biological replicates from one experiment. Asterisks indicate statistically significant differences (p < 0.05, Student’s t test).
(F) Quantification of the abundance of AtSLAH1 and AtSLAH3 transcripts 3 hr and 24 hr after treatments. Transcript abundance was quantified in plants growing under control conditions (CTR) and 3 hr and 24 hr after the application of abiotic stress treatments consisting of salinity induced with NaCl 150 mM (NaCl), water deficit induced with PEG  g L−1 (PEG), or exogenous application of 100 μM abscisic acid (ABA). Transcript abundance was quantified with standard curves calculated for the individual PCR products and normalized with the housekeeping translation initiation factor AteIF4A1 gene (At3g13920). Results are the mean (±SE) of three biological replicates.
(G) Histochemical localization of AtSLAH1 expression in transgenic Arabidopsis plants expressing the GUS reporter genes under the control of the AtSLAH1 promoter (PSLAH1-GUS) either treated with 100 μM ABA or without the phytohormone (control).
See also Figures S1 and S2 and Table S1.
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4. Figure 3
Activation of S-Type Anion Currents via Co-expression of SLAH3 and SLAH1 in Oocytes and Guard Cell Protoplasts
(A) Whole-oocyte currents of Xenopus oocytes in response to the standard voltage protocol co-expressing either SLAH3 with CPK21ΔEF or SLAH3 with SLAH1 were recorded in chloride- or nitrate-based buffers (30 mM). Representative cells are shown.
(B) Instantaneous currents (Iinst) of oocytes co-expressing SLAH1 with different members of the Arabidopsis S-type anion channel family recorded at −100 mV in the presence of either 100 mM chloride or 100 mM nitrate (n ≥ 4; mean ± SE).
(C) Pixel-by-pixel FRET-FLIM analysis of TSapphire:SLAH3 (TS:SLAH3) in the presence (left) or in the absence (right) of SLAH1:mOrange (SLAH1:mO) expressed in epidermal cells of N. benthamiana. The false color code depicts the lifetime window from 2 ns (blue) to 3 ns (red). Representative images are shown.
(D) TS:SLAH3 lifetime histogram in the presence (Gaussian fit, solid line; n = 9 experiments) or absence (Gaussian fit, dotted line; n = 11 experiments) of SLAH1:mO (mean ± SE).
(E) Relative Expression of the SLAC1 homologs in guard cells of slac1-2 compared to the complementation line slac1-2/SLAH1. Transcript numbers per 10,000 act2/8 molecules are presented as mean ± SE (n ≥ 4 experiments; n. d., not detectable).
(F) Representative macroscopic currents monitored from slac1-2 and slac1-2/SLAH1 in response to the standard patch-clamp voltage protocol.
(G) Steady-state current densities (Iss/Cm) are shown as a function of the clamped voltage (slac1-2, n = 7 from four independent experiments; slac1-2/SLAH1, n = 6 independent experiments; mean ± SE).
See also Figure S3.
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5. Figure 4 SLAH1 Modifies the Activation Properties of SLAH3
(A) Instantaneous currents (Iinst) recorded at −100 mV in nitrate-based solutions (100 mM). Currents were recorded from oocytes expressing different combinations of SLAH1 WT or mutants and SLAH3 WT or mutants as indicated in the figure.
(B) Chord conductance of Xenopus oocytes co-expressing SLAH3 either with CPK21ΔEF or with SLAH1. Standard bath solution contained 100 mM chloride or nitrate (n = 4 experiments; mean ± SE).
(C and D) Relative voltage-dependent open probabilities (rel. PO) derived from oocytes co-expressing SLAH3 with CPK21ΔEF (C) or SLAH3 with SLAH1 (D) in the presence of varying Cl− concentrations (black) or 30 mM NO3− (red). Data points were fitted by a Boltzmann equation (continuous line) (n = 4 experiments; mean ± SE).
See also Figure S4.
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