Figure 1.
Structural analysis of TaNHX2 protein.
Phylogenetic tree of NHX transporter members from wheat, tomato and Arabidopsis. The tree was inferred using the UPGMA method. The alignment is based on the total amino acid sequences. Evolutionary distances were computed by the neighbor joining method. The scale bar indicates the distances as calculated from the multiple alignment. Bootstrap values are indicated at the nodes of the tree and are expressed as percentages.
Figure 2.
Fluorescence patterns of GFP fusion proteins in plant cells.
Arabidopsis protoplasts were transformed with the plasmid pJIT 163 containing GFP alone (A and B), or pJIT 163-TaNHX2-GFP (C and D). The fluorescence images are shown in tridimensional (A and C) and planar (B and D) patterns respectively. Fluorescence microscopic image of GFP (a), fluorescence microscopic image of chloroplasts (b), transmitted light microscopic image of a protoplast (c) and an image of the overlay of the GFP and chloroplast fluorescence and transmitted light (d).
Figure 3.
Relative abundance of TaNHX2 mRNA in wheat plants.
Ten-day-old plants were transferred to nutrient solution containing 200 mM NaCl or 20% PEG. The expression of TaNHX2 was studied in the roots and leaves at different time points (0, 3, 6, 9, 12, 24 and 48 h) of salt treatment (A) or PEG treatment (B) by real-time PCR. The values of expression at the beginning of salt treatment or PEG treatment (0 h) were arbitrarily set to 100. The expression levels of other time points of salt treatment or PEG treatment were expressed as percentages of the value at time zero. Values are the mean ±SE of three replicates.
Figure 4.
Immunodetection of histidine-tagged TaNHX2 in the microsomal membrane fraction.
Microsomal membranes were isolated from yeast cells transformed with empty vector pYES2 and transgenic yeast cells with histidine tagged TaNHX2. 50 µg of microsomal membrane protein was separated electrophoretically and subjected to western blotting with a monoclonal antibody raised against the RGSH4 tag, as described in Materials and Methods. Lane 1, the membrane proteins from transgenic yeast cells with pYES2-TaNHX2-His. Lane 2, the membrane proteins from yeast cells transformed with empty vector pYES2.
Figure 5.
Enhanced salt tolerance of transgenic yeast cells expressing TaNHX2.
Yeast cells of recombinant strains, mutant and wild-type were grown to saturation in APG medium. Ten microliters of serial decimal dilutions were spotted onto plates of APG medium supplemented with 0 (control), 30 or 60 mM NaCl (A) and 0.25 or 0.5 M KCl (B) as indicated. Plates were incubated at 30°C for 3-5d. TaNHX2: mutant strain AXT3K transformed with plasmid pYES2-TaNHX2; pYES2: mutant strain AXT3K transformed with the empty plasmid pYES2; W303: wild-type strain W303.
Figure 6.
K+ and Na+ contents in yeast cells.
K+ (A) and Na+ (B) contents were measured in yeast cells grown in APG medium in the absence (control) or presence of 20 mM NaCl by atomic absorption spectrometry, as described in Materials and Methods. The results were analyzed statistically using the two-tailed Student’s t-test. Values are the mean ± SE of three replicates and differences with a P-value <0.05 were considered significant. AXT3K: untransformed mutant strain AXT3K; pYES2: mutant strain AXT3K transformed with the empty plasmid pYES2; pYES2-TaNHX2: mutant strain AXT3K transformed with plasmid pYES2-TaNHX2.
Figure 7.
Comparison of yeast cell growth upon hygromycin B treatment.
Serial dilutions of the strains were grown on YPD plates containing 0 (control), 30 and 50 µg/ml hygromycin B. Δvnx1: vnx1 mutant yeast strain; TaNHX2: Δnhx1 and Δvnx1 double mutant strain OC02 transformed with plasmid pDR195-TaNHX2; Δnhx1 Δvnx1: Δnhx1 and Δvnx1 double mutant strain OC02 transformed with the empty plasmid pDR195.
Figure 8.
Immunodetection of histidine-tagged TaNHX2 in tonoplasts.
The histidine tagged TaNHX2 cDNA was inserted into yeast expression vector pYes2, and transformed into yeast strain OC02. Tonoplast was isolated from transgenic and untransformed control yeast cells, as described in Materials and Methods. 20 µg of tonoplast protein was separated electrophoretically and subjected to western blotting using a monoclonal antibody raised against the RGSH4 tag, as described in Materials and Methods. Lane 1, the tonoplast proteins from transgenic yeast cells with pYES2-TaNHX2-His. Lane 2, the tonoplast proteins from untransformed yeast cells.
Figure 9.
Cation/H+ antiport activity in tonoplast vesicles.
Fluorescent quenching of quinacrine was used to monitor the acidification of tonoplast vesicles from yeast cells transformed with empty vector pDR195 (pDR195) and transgenic yeast cells expressing TaNHX2 (pDR195-TaNHX2). An inside-acid ΔpH was formed after the addition of ATP to vesicles. Once fluorescence was stabilized, K2SO4 (A) or Na2SO4 (B) was added to the cuvette and fluorescence recovery, indicating proton exchange, was monitored for 3 min, after which ΔpH was disrupted by the addition of 25 mM (NH4)2SO4. Fluorescence is expressed as arbitrary units. C, cation/H+ exchange as a function of the concentrations of cations, values are the mean ± SE of triplicate samples. Cation/H+ exchange activity is given as the proportion of dissipation of the preformed pH gradient per minute and milligram of membrane protein (ΔF%/mg protein min). Na-OC02: Na+/H+ exchange activity in tonoplast vesicles from OC02 cells transformed with empty vector pDR195; Na-TaNHX2: Na+/H+ exchange activity in tonoplast vesicles from OC02 cells transformed with plasmid pDR195-TaNHX2; K-OC02: K+/H+ exchange activity in tonoplast vesicles from OC02 cells transformed with empty vector pDR195; K-TaNHX2: K+/H+ exchange activity in tonoplast vesicles from OC02 cells transformed with plasmid pDR195-TaNHX2.