Root plasma membrane transporters controlling K+/Na+ homeostasis in salt-stressed barley

Abstract

Plant salinity tolerance is a polygenic trait with contributions from genetic, developmental, and physiological interactions, in addition to interactions between the plant and its environment. In this study, we show that in salt-tolerant genotypes of barley (Hordeum vulgare), multiple mechanisms are well combined to withstand saline conditions. These mechanisms include: (1) better control of membrane voltage so retaining a more negative membrane potential; (2) intrinsically higher H(+) pump activity; (3) better ability of root cells to pump Na(+) from the cytosol to the external medium; and (4) higher sensitivity to supplemental Ca(2+). At the same time, no significant difference was found between contrasting cultivars in their unidirectional (22)Na(+) influx or in the density and voltage dependence of depolarization-activated outward-rectifying K(+) channels. Overall, our results are consistent with the idea of the cytosolic K(+)-to-Na(+) ratio being a key determinant of plant salinity tolerance, and suggest multiple pathways of controlling that important feature in salt-tolerant plants.

Figure 1.

A, Contrasting barley genotypes grown under 320 mm NaCl for 4 weeks in the glasshouse experiment. Salt-tolerant (T) and salt-sensitive (S) varieties are easily distinguished. B, Steady-state net K⁺ fluxes (inward positive). C, Effects of different external Ca²⁺ (0.1 and 1 mm) on NaCl-induced K⁺ flux measured from 3-d-old roots of barley genotypes contrasting in their salinity tolerance after 1 h of 80 mm NaCl treatment. Results in C are averaged over 15 min of K⁺ flux measurement. Means ± se (n = 7–10). [See online article for color version of this figure.]

Figure 2.

Pharmacology of K⁺ flux responses. Net K⁺ fluxes were measured in response to 20 mm TEA⁺, applied at arrow, from roots of two contrasting barley genotypes (salt-tolerant ‘ZUG293’; salt-sensitive ‘Gairdner’) preincubated in 80 mm NaCl for 1 h. Means ± se (n = 7–8).

Figure 3.

A, Membrane potential of epidermal root cells of salt-tolerant ‘ZUG293’ and salt-sensitive ‘Gairdner’ measured in response to 80 mm NaCl treatment (at arrow). Means ± se (n = 6). B, Steady-state membrane potential (E_m) values in control (prior to NaCl treatment) and after 20 min root exposure to 80 mm NaCl. Means ± se (n = 10).

Figure 4.

A, ATP hydrolytic activity of PMs isolated from the microsomal fraction of roots of barley genotypes contrasting in salinity tolerance. Means ± se (n = 6). The statistics are based on two independent PM preparations and each of the preparations was tested three times with reproducible results. B, Western-blot results demonstrating that the observed difference in the H⁺-ATPase activity is not due to a difference in the enzyme level.

Figure 5.

Unidirectional ²²Na⁺ uptake, at times up to 20 min, into excised roots of four barley cultivars contrasting in their salinity tolerance. A, Immediately after 80 mm NaCl treatment. B, After 24 h incubation in 80 mm NaCl. C, For the immediate treatment, the ²²Na⁺ uptake during 5 min with two levels of external Ca²⁺ (0.1 as in A and 10 mm in C). Means ± se (n = 8–13).

Figure 6.

K⁺ loss (A) and Na⁺ uptake (B) by barley roots measured in depletion experiments. In each treatment, roots of ten 3-d-old seedlings were immersed in 10 mL saline solution (80 mm NaCl, 0.5 mm KCl, 0.1 mm CaCl₂) in a plastic test tube and aerated for 24 h in the dark at 25°C. Two individual measurements were performed with three replicates for each genotype. Means ± se (n = 6).

Figure 7.

Leaf sap Na⁺ concentrations of six barley genotypes in both control (A) and 320 mm NaCl treatment (B). Flag leaf samples of all cultivars were collected 8, 18, and 28 d after the imposition of salinity. Means ± se (n = 4).

Figure 8.

Time- and voltage-dependent TEA-sensitive KOR currents in barley epidermal protoplasts. K⁺ concentration in bath/pipette was 5/100 mm (see “Materials and Methods” for detailed solution composition). A, Typical record of KOR currents in salt-sensitive ‘Gairdner’. Voltage was stepped from −100 mV (holding) in 20 mV increments up to +100 mV for 1.4 s and returned to −100 mV at the end of episode. B, I/V relation for the time-dependent component of the depolarization activated current; equilibrium potentials for K⁺ and Cl⁻ are indicated by arrows. Inset shows amplitude of the tail currents (prepulse to +80 mV, subsequent test pulses to voltages between −100 and +20 mV) as a function of test voltage. C, KOR currents in two contrasting barley cultivars (salt-tolerant ‘CM72’ and salt-sensitive ‘Gairdner’) show similar sensitivity to external TEA⁺. Shown are normalized I/V relations of the time-dependent current component in control conditions and after external application of 20 mm TEACl. Means ± se (n = 3 and 4 protoplasts for ‘CM72’ and ‘Gairdner’, respectively). Note that in some cases error bar is smaller than the symbol size.

Figure 9.

Comparison of KOR-mediated currents in two contrasting barley cultivars: salt-tolerant ‘CM72’ and salt-sensitive ‘Gairdner’. A, Frequency of detection (successful/total records) of KOR channels, average current densities at +60 mV, and average KOR current per protoplast. Means ± se (n = 36 for ‘CM72’; 70 for ‘Gairdner’). B, Voltage dependence of KOR-mediated conductance. Solid lines are best fits to Boltzmann equation, with midpoint potential values of 6.4 ± 1.4 mV and 9.6 ± 2.4 mV, and the slope factor (membrane depolarization that increases open/closed states ratio e times) of 18.6 ± 0.8 mV and 18.8 ± 1.2 mV for ‘CM72’ and ‘Gairdner’, respectively.