Article Figures & Data
Figures
- Fig. 1.
AtHKT1 sequence analysis. A, Nucleotide sequence of AtHKT1. The deduced amino acid sequence of AtHKT1 is shown below the nucleotide sequence. Putative TATA and CAAT boxes are boxed. The primer HKTSAL used for RACE-PCR and TAIL-PCR is underlined. The dashed underlines with arrowheads mark other primers used for RACE-PCR. The arrows indicate primers for TAIL-PCR. The thick underline shows the primer used to confirm the promoter sequence ofAtHKT1. The end of the cDNA isolated from the cDNA library is indicated by a white arrowhead. The vertical bar indicates the end of nucleotides extended by RACE-PCR. B, Hydropathy plots of AtHKT1 and HKT1. The plots were performed according to the method ofKyte and Doolittle (1982). Hydrophobic amino acids have positive values. The homologous regions (I, II, and III in C) are underlined. C, Homologous regions in the deduced amino acid sequences of AtHKT1, HKT1, TRK1, and TRK2. The first amino acid residue of the translation start site is designated position 1. Identical amino acids are boxed. Conserved amino acids are indicated by asterisks.
- Fig. 2.
Genomic DNA gel-blot analysis ofAtHKT1. Arabidopsis genomic DNA digested with the indicated restriction enzymes was probed with the AtHKT1cDNA. DNA size markers are indicated on the left.
- Fig. 3.
Competitive RT-PCR analysis ofAtHKT1 expression in Arabidopsis. The ethidium bromide-stained agarose gel shows the products obtained by competitive RT-PCR products after amplification of 80 pg of total RNA and 16 pg of heterologous competitor DNA using AtHKT1-specific primers. Competitive DNA, 432-bp heterologous competitor DNA;AtHKT1, 843-bp DNA fragment amplified from theAtHKT1 cDNA.
- Fig. 4.
AtHKT1 expressed in oocytes exhibits selectivity for Na+. A and B, AtHKT1 expressed in oocytes causes inward Na+ currents. cRNA-injected oocytes and uninjected oocytes were maintained in standard Barth's solution (containing 88 mmNaCl) and later used for recordings. The membrane potential of uninjected oocytes (A) and AtHKT1-expressing oocytes (B) was held at −120 mV. The bath solution contained 0.3 mmK+ Glu, 1 mm Na+ Glu, or 0.3 mm K+Glu plus 1 mm Na+Glu with Tris-Glu to balance varying K+ and Na+concentrations. C and D, The Na+ conductance of AtHKT1 does not depend on external K+, but on external Na+. Steady-state current-voltage curves were obtained from anAtHKT1-expressing oocyte exposed to 0.3 mmNa+ plus varying concentrations of K+ (0.3, 1, 3, and 10 mm) (C) and to 0.3 mm K+plus varying concentrations of Na+ (0.3, 1, 3, and 10 mm) (D).
- Fig. 5.
Average steady-state current magnitudes recorded at −120 mV from AtHKT1-expressing oocytes with 100 mm Li+, Na+, K+, Rb+, or Cs+ in the bath solution.
- Fig. 6.
Na+-induced growth inhibition ofS. cerevisiae G19 expressing AtHKT1. A, Growth inhibition of S. cerevisiae G19 expressingAtHKT1, HKT1, KAT1, and empty vector. The agar medium contained 250 mmNa+. B, Complementation of the K+uptake-deficient yeast mutant (S. cerevisiae strain CY162) with AtHKT1, HKT1,KAT1, and empty vector. C, Growth curve of the S. cerevisiae G19 expressing AtHKT1 (■),HKT1 (▵), and pPAB404 (○) in liquid culture containing 250 mm Na+. Note that higher Na+ concentrations are needed to cause Na+toxicity via wheat HKT1 in the G19 line.
- Fig. 7.
Complementation of the K+-uptake-deficient E. coli strain by AtHKT1 and K+ uptake rate of AtHKT1-expressingE. coli cells. A, Complementation of LB2003 cells by AtHKT1. E. coli LB2003 was transformed with plasmids containing AtHKT1 or with the empty vector. Transformants were grown on medium supplemented with 10 mmK+. B, Growth curve of transformants containingAtHKT1 (○) or the empty vector (●) in liquid medium. The initial K+ concentrations were measured by flame photometry (8.7 mm). C, K+ uptake by K+-depleted E. coli containingAtHKT1. Net K+ uptake byAtHKT1-expressing LB2003 (○) and control cells with empty vector (●) were measured at 20 mm KCl. D, Lineweaver-Burk plot of K+ uptake data. The results of experiments with six different cell preparations ofAtHKT1-expressing cells and control cells are compiled. The lines represent linear regression of the data given in D. Experimental conditions and symbols as in C.
Tables
Cation Percentage of Na+ Current Amplitude % Li+ 1.6 ± 0.25 Na+ 100 K+ 3.3 ± 0.66 Rb+ 3.5 ± 0.42 Cs+ 1.6 ± 0.60 Values represent means ± sd of currents measured at −120 mV and relative to the Na+ current amplitude, which was defined as 100% for each oocyte. Data obtained from four oocytes were averaged.
