Chen G , Wang L , Chen Q , Qi K , Yin H , Cao P , Tang C , Wu X , Zhang S , Wang P , Wu J .

2018YFD0201400 National Key R &D Program of China, 2014BAD16B03-4 the National Key Technology R & D Program of the Ministry of Science and Technology of China, KJQN201926 the Fundamental Research Funds for the Central Universities, KYTZ201602 the Fundamental Research Funds for the Central Universities, 31801842 the National Natural Science Foundation of China

	Fig. 1. The determination of S-type anion channels in pear and the expression analysis. a Phylogenetic tree of the Arabidopsis thaliana SLAC/SLAH anion channel family and the pear orthologs. Four putative PbrSLAC/SLAH anion channels were identified in the Pyrus genome database. PbrSLAC1 appears to be related to AtSLAC1, other three homologs were classified into the AtSLAH2/3 group, while AtSLAH1/4 homologs could not be identified in pear. The PbrSLAH2/3–1 sequence was used to clone the ortholog from Pyrus bretschneideri and named PbrSLAH3. b The expression patterns of PbrSLAC/SLAH family genes in the root, leaf, pollen grain and pollen tube of pear were analyzed by qRT-PCR. The values displayed in the figure were the geometric means of the relative expression of PbrSLAC/SLAH when both PbrTUB and PbrUBQ as reference genes. Three biological and three technical replicates were processed for the qRT-PCR assays. The error bars indicate standard deviations. The data are shown as mean values ± SDs. c RT-PCR was used to confirm the expression patterns of the PbrSLAH3 gene and PbrUBQ gene was used as an internal control
	Fig. 2. The subcellular localization of PbrSLAH3 to the plasma membrane, and a histochemical analysis of GUS activity in PbrSLAH3 transgenic Arabidopsis (Col-0) plants. a GFP fluorescence emitted from the leaves of tobacco infected by Agrobacterium harboring PbrSLAH3-GFP was detected under a confocal microscope and the plasma membranes are stained red by FM4–64. GFP: green fluorescent protein; Chl: chlorophyll; Bright: bright-field image of Agrobacterium tumefaciens-infiltrated tobacco leaves; Merge: merged fluorescent images; Bar = 10 μm. b–g GUS-histochemical analysis performed with Arabidopsis plants expressing the reporter gene under the control of the PbrSLAH3 promoter. PbrSLAH3 was expressed in young leaves, mature leaves, roots, stems, epidermal hairs, stomatal and siliques
	Fig. 3. PbrSLAH3 rescues the ammonium toxicity of slah3–3 mutant plants under high ammonium/low-nitrate conditions. a Schematic map of the T-DNA insertion site in the slah3–3 mutant. b RT-PCR analyses of PbrSLAH3 and AtSLAH3 expression levels in transgenic, wild-type (Col-0) and slah3–3 mutant plants. Actin was used as a control. c Growth comparison among 7-d-old transgenic lines 1 and 2, wild-type (Col-0) and slah3–3 mutant seedlings grown on 1/2 N-free Murashige and Skoog medium supplemented with different concentrations of KNO3 (0–20 mM) and 20 mM NH4Cl. d–g The average primary root lengths of seedlings under the same conditions as displayed in (c) were statistical analyzed. Results are averages ± SEs of three independent experiments (n = 15 per experiment). The asterisk represents statistical significance among transgenic lines, wild-type and slah3–3 mutant plant (Student’s t-test, *P < 0.05). Bar = 0.5 cm
	Fig. 4. Interaction between PbrSLAH3 and PbrCPK32 as assessed by yeast two-hybrid and bimolecular fluorescence complementation assays. a Yeast strain NMY51 co-transformed with the PbrSLAH3 bait vector and PbrCPK32 prey vector, along with a positive control (co-transformed NubI vector and pDHB vector) were grown in medium lacking Ade, His, Trp and Leu. The negative control could not grow in the same medium. b YFP indicates yellow fluorescence protein; Chl: chlorophyll; Bright: bright-field image of Nicotiana benthamiana leaves infiltrated with Agrobacterium tumefaciens; Merge: digital merge of bright field and fluorescent images. Bar = 10 μm
	Fig. 5. Electrical properties of PbrSLAH3 co-expressed with AtCPK21 in Xenopus laevis oocytes. a YFP fluorescence was emitted from Xenopus oocytes when PbrSLAH3 was co-expressed with AtCPK21, but no YFP fluorescence was observed after the injection of PbrSLAH3-YFP alone. b Representive macroscopic anion currents were recorded in different concentrations of external NO3− and Cl− when PbrSLAH3 was co-expressed with AtCPK21. Currents were recorded with 1.5-s voltage pulses ranging from − 180 mV to + 40 mV with 20-mV increments, followed by a 0.5-s voltage pulse to − 120 mV, the holding potential was 0 mV (n ≥ 5, means ± SE). c Steady state currents (Iss) plotted against the membrane voltages in the presence of different external NO3− concentrations (n ≥ 5, means ± SE)
	Fig. 6. Electrical properties of PbrSLAH3 co-expressed with PbrCPK32 in Xenopus oocytes. a YFP fluorescence was observed in Xenopus oocytes when PbrSLAH3 was co-expressed with PbrCPK32, while no YFP fluorescence was detected when PbrSLAH3-YFP cRNA alone was injected. b Representive macroscopic anion currents were recorded in different concentrations of external NO3− and Cl− when PbrSLAH3 was co-expressed with PbrCPK32. Currents were recorded with 1.5-s voltage pulses ranging from − 180 mV to + 40 mV with 20-mV increments, followed by a 0.5-s voltage pulse to − 120 mV, the holding potential was 0 mV. (n ≥ 5, means ± SE). c Steady state currents (Iss) plotted against the membrane voltages in the presence of different external NO3− concentrations (n ≥ 5, means ± SE)

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