|
Figure 1. Phylogenetic analysis of high-affinity potassium transporters (HKTs). A phylogenetic analysis of the selected HKT amino-acid sequences was performed using the neighbor-joining method in the MEGA-X [39] software package. Accession numbers of amino-acid sequences used are listed in Supplementary Figure S1. The branch length is proportional to the evolutionary distance between the HKTs, indicating the number of amino-acid changes per site. The scale bar shows a length corresponding to 0.10 of the value.
|
|
Figure 2. Expression profile of SvHKT1;1 gene and Na+ concentrations in Sporobolus virginicus under salt stress. The expression profile of SvHKT1;1 gene and Na+ concentrations in roots and shoots of hydroponically grown S. virginicus were determined. (A–D) Expression levels of SvHKT1;1 gene determined by qRT-PCR under different NaCl concentrations (A,B) or at different time points after salt treatment (C,D). Plants grown in 1/2 Murashige and Skoog (MS) medium were transferred to 1/2 MS medium supplemented with 0, 100, 300, or 500 mM NaCl, and the roots (A) and shoots (B) were harvested at 48 h after the treatment. Plants grown in 1/2 MS medium were transferred to 1/2 MS medium supplemented with 500 mM NaCl, and the roots (C) and shoots (D) were harvested at indicated time points. Expression levels relative to that in roots at 0 h after treatment (1.0) are shown. eIF3 was used as a reference gene. (E–H) Na+ (E,F) and K+ (G,H) concentrations in roots (E,G) and shoots (F,H) of hydroponically grown S. virginicus under different NaCl concentrations. The roots and shoots were harvested at 48 h after the treatment. Data are presented as means ± SE (n = 3 biological replicates). Single and double asterisks denote significant differences compared with the values of WT plants of the same conditions at p < 0.05 and p < 0.01, respectively, determined using the Student’s t-test.
|
|
Figure 3. Subcellular localization of EGFP-fused SvHKT1;1 protein in Nicotiana benthamiana leaves. Confocal fluorescence images of EGFP (AâC), differential interference contrast images (DâF), and merged images (GâI) of N. benthamiana leaf cells expressing EGFP control (A,D,G) and EGFPâSvHKT1;1 (B,C,E,F,H,I). Images of non-plasmolyzed (AâF) and plasmolyzed (GâI) cells. Scale bar represents 20 μm and is applicable to all panels in this figure.
|
|
Figure 4. Analyses of SvHKT1;1-mediated ion transport by two electrode voltage clamp experiments using Xenopus laevis oocytes. Currentâvoltage relationship of oocytes injected with 12.5 ng of SvHKT1;1 complementary RNA (cRNA) (A) or water (B) bathed in solutions containing an indicated amount of NaCl, KCl, or Na-gluconate. Voltage steps ranged from â150 to +30 mV with 15-mV increments. Data are presented as means ± SD (n = 3â7).
|
|
Figure 5. Complementation test of athkt1;1 mutant Arabidopsis with AtHKT1;1pro::SvHKT1;1 construct. Transcripts of AtHKT1;1 or SvHKT1;1 were detected in WT, mutant, and two independent lines of transformed Arabidopsis plants (three biological replicates) by RT-PCR (A). The appearance (B) and fresh weight (C) of the plants grown for two weeks on 100 mM NaCl medium. Na+ (D,F) and K+ (E,G) concentrations in the shoots (D,E) and roots (F,G) of the plants. Data are presented as means ± SD (n = 9 (B,C) and n = 3 (DâG)). Please note that each panel has a different Y-axis scale. Single and double asterisks denote significant differences compared with the values of WT plants of the same conditions at p < 0.05 and p < 0.01, respectively, determined using the Studentâs t-test.
|
|
Figure 6. Expression level of the transgene, root growth, and salt tolerance of transgenic Arabidopsis plants expressing SvHKT1;1. (A) Expression levels of transgenes in the SvHKT1;1 transgenic lines. Actin was used as an internal standard. ND; not detected. Data are presented as means ± SE (n = 3 biological replicates). (B) Root elongation of transgenic and WT seedlings grown on 0.1 mM K+ medium. Data are presented as means ± SE (n = 3 biological replicates). (C) The appearance of transgenic lines and WT seedlings on 0.1 mM K+ medium examined in panel B. (D) Fresh weight (FW) of WT and the transgenic lines. One-week-old seedlings germinated on 1/2 MS agar medium were transplanted onto 1/2 MS agar medium supplemented with 50 mM NaCl, and their FW was determined after another two weeks of incubation. Data are presented as means ± SE (n = 10 biological replicates). (E) The appearance of plants examined in panel D. Single and double asterisks denote significant differences compared with the values of WT plants of the same conditions at p < 0.05 and p < 0.01, respectively, determined using the Studentâs t-test. Single and double asterisks denote significant differences compared with the values of WT plants of the same conditions at p < 0.05 and p < 0.01, respectively, determined using the Studentâs t-test.
|
|
Figure 7. Shoot fresh weight and ion concentrations of SvHKT1;1 transgenic lines and WT plants. (A,B) Shoot fresh weight of WT plants and the transgenic lines. Two-week-old seedlings germinated on 1/2 MS agar medium were hydroponically cultured in 1/2 MS liquid medium for another one week, and then cultured in 1/2 MS medium supplemented with 0 (A) or 100 mM (B) NaCl, and their FW was determined after one week. (CâJ) Ion concentrations in WT plants and the transgenic lines. Na+ (C,E,G,I) and K+ (D,F,H,J) concentrations in their shoots (C,D,G,H) and roots (E,F,I,J) were determined. Data are presented as means ± SE (n = 3â4 biological replicates). Please note that each panel has a different Y-axis scale. Single and double asterisks denote significant differences compared with the values of WT plants of the same conditions at p < 0.05 and p < 0.01, respectively, determined using the Studentâs t-test.
|
|
Figure 8. Na+ and K+ concentrations in the xylem and phloem saps from SvHKT1;1 transgenic lines and WT plants. (AâD) Ion concentrations in the xylem and phloem saps of WT plants and the transgenic lines under nonstress condition. Plants were hydroponically cultured in 1/2 Hoagland liquid solution until the bolting stage, and Na+ (A,C) and K+ (B,D) concentrations in their xylem (A,B) and phloem (C,D) saps were determined. (E,F) Ion concentrations in the phloem saps of WT plants and the transgenic lines under 100 mM NaCl. ND; not determined. Three-week-old plants were subjected to 1/2 Hoagland liquid solution supplemented with 100 mM NaCl for seven days, and Na+ (E) and K+ (F) concentrations in their phloem saps were determined. Xylem saps were not obtained from salt-treated plants. Data are presented as means ± SE (n = 3â4 biological replicates). Please note that each panel has a different Y-axis scale. Single and double asterisks denote significant differences compared with the values of WT plants of the same conditions at p < 0.05 and p < 0.01, respectively, determined using the Studentâs t-test.
|
|
Figure 9. Na+ and K+ uptake and translocation rates in SvHKT1;1 transgenic lines and WT plants under salt stress. Twelve-day-old Arabidopsis seedlings, which were pre-incubated in 1/2 MS liquid medium for 24 h to adapt to liquid medium, were transferred into micro cuvettes filled with 1/2 MS liquid medium supplemented with 100 mM NaCl and further incubated for one hour. Their roots and shoots were separately harvested before and after the treatment, dried overnight, and weighted. Na+ (A) and K+ (B) uptake and translocation rates in the roots, shoots, and whole plants under salt stress were calculated and expressed as mmol per g of dry weight per hour of salt treatment (mmol·g DWâ1·hâ1). Ten seedlings were pooled and used as one sample. Data are presented as means ± SE (n = 3 biological replicates). Single and double asterisks denote significant differences compared with the values of WT plants at p < 0.05 and p < 0.01, respectively, determined using the Studentâs t-test.
|
