Reduced tonoplast fast-activating and slow-activating channel activity is essential for conferring salinity tolerance in a facultative halophyte, quinoa

Abstract

Halophyte species implement a "salt-including" strategy, sequestering significant amounts of Na(+) to cell vacuoles. This requires a reduction of passive Na(+) leak from the vacuole. In this work, we used quinoa (Chenopodium quinoa) to investigate the ability of halophytes to regulate Na(+)-permeable slow-activating (SV) and fast-activating (FV) tonoplast channels, linking it with Na(+) accumulation in mesophyll cells and salt bladders as well as leaf photosynthetic efficiency under salt stress. Our data indicate that young leaves rely on Na(+) exclusion to salt bladders, whereas old ones, possessing far fewer salt bladders, depend almost exclusively on Na(+) sequestration to mesophyll vacuoles. Moreover, although old leaves accumulate more Na(+), this does not compromise their leaf photochemistry. FV and SV channels are slightly more permeable for K(+) than for Na(+), and vacuoles in young leaves express less FV current and with a density unchanged in plants subjected to high (400 mm NaCl) salinity. In old leaves, with an intrinsically lower density of the FV current, FV channel density decreases about 2-fold in plants grown under high salinity. In contrast, intrinsic activity of SV channels in vacuoles from young leaves is unchanged under salt stress. In vacuoles of old leaves, however, it is 2- and 7-fold lower in older compared with young leaves in control- and salt-grown plants, respectively. We conclude that the negative control of SV and FV tonoplast channel activity in old leaves reduces Na(+) leak, thus enabling efficient sequestration of Na(+) to their vacuoles. This enables optimal photosynthetic performance, conferring salinity tolerance in quinoa species.

Figure 1.

A and B, Quinoa, a facultative C3 halophytic plant capable of producing grain even when irrigated with high (seawater) levels of salinity. C and D, To a large extent, the remarkable salinity tolerance of quinoa plants is attributed to salt bladders (EBC) located at both the adaxial and abaxial leaf surfaces. Salt bladder density, however, is very different between young (C) and old (D) leaves. [See online article for color version of this figure.]

Figure 2.

Photosynthetic characteristics of quinoa leaves grown under optimal (50 mm; control) and saline (400 mm NaCl for 3 weeks) growth conditions. Values are means ± se (n = 8–10). A, Chlorophyll fluorescence F_v/F_m value. B, Chlorophyll content (SPAD readings). arb., Arbitrary.

Figure 3.

Leaf sap sodium content in brushed (A) and nonbrushed (B) quinoa leaves of a different physiological age (young and old) from control and salt-grown (400 mm NaCl for 3 weeks) plants. Values are means ± se (n = 6).

Figure 4.

A, Relative vacuole volume (percentage of the total cell volume) in old (O) and young (Y) quinoa leaf cells grown under control (50 mm NaCl) and saline (400 mm NaCl) conditions. Values are means ± se (n = 40 cells from five plants). Data with different lowercase letters are significantly different at P < 0.05. B, Vacuolar Na⁺ content in quinoa mesophyll vacuoles, quantified using corrected total vacuole fluorescence of CoroNa Green, expressed in arbitrary (arb.) units. Values are means ± se (n = 55–97).

Figure 5.

Sodium sequestration in quinoa leaf vacuoles imaged by CoroNa Green dye. One (of six to 10) representative images is shown for each treatment. A, Old leaf in control plant. B, Old leaf grown at 400 mm NaCl salinity. C, Young leaf in control plant. D, Young leaf at 400 mm NaCl.

Figure 6.

The FV current undergoes run-up in the whole-vacuole configuration. A, Typical FV current recordings from a small (approximately 12 pF) vacuole, isolated from a young quinoa leaf, shortly (1 min) and 30 min after the whole-vacuole configuration was obtained. The instantaneous current amplitudes are plotted against voltage in both cases. B, I/V relations for FV current records presented in A. C, Typical time course of run-up transition from the low- to the high-conductance state for the tonoplast FV current in vacuoles from young (same sample as in A) and old quinoa leaves. Steady-state FV currents at +140 (white symbols) and −140 mV (black symbols) are plotted against the time spent in the whole-vacuole configuration. Symmetric 100 mm KCl, standard bath, and pipette solution were used for all FV current recordings (see “Materials and Methods”).

Figure 7.

The FV current poorly discriminates between Na⁺ and K⁺. I/V relations in a high-conductance stable state, 100 mm KCl in the bath, and either 100 mm KCl or 500 mm NaCl in the pipette (the arrow indicates the reversal potential of the whole-vacuole current for the latter case). A and B, Young (A) and old (B) quinoa leaves. Values are means ± se (n = 5–6 mesophyll vacuoles). C, Plot of conductance versus voltage. Symbols are as in A and B. Measurements were taken 30 min after vacuole perfusion, when currents reached their maximum values.

Figure 8.

Saline conditions reduce FV currents in vacuoles from old quinoa leaves. Pipette and bath solutions contained 100 mm KCl. A and B, Whole vacuolar FV conductance was evaluated by taking the first derivative of the whole-vacuole I/V relation, measured immediately after achieving the whole-vacuole configuration. Vacuoles were isolated from either young or old quinoa leaves, grown in the presence of 50 mm NaCl (control) or saline (400 mm NaCl for 3 weeks; salt). Data are means ± se (n = 5–8 vacuoles). Solid lines are best fit to a three-state (open1-closed-open2) model (Pottosin and Martínez-Estévez, 2003). C, Same data as in A and B, but only the points within the physiological tonoplast potential range (±20 mV) are considered. O, Old leaves; Y, young leaves.

Figure 9.

Effects of salinity and leaf age on the FV channel activity in quinoa mesophyll vacuoles. A and B, Whole vacuolar FV conductance versus voltage, the first derivative of the whole-vacuole I/V relation, measured upon establishment of the high-conductance state. C, Same data as in A and B, but only the points within the physiological tonoplast potential range (±20 mV) are considered. Data with different lowercase letters are significantly different at P < 0.05. O, Old leaves; Y, young leaves. Data are means ± se (n = 3–7 vacuoles for each condition). Other conditions are as in Figure 8.

Figure 10.

Selectivity of the SV current in quinoa mesophyll vacuoles to Na⁺ and K⁺. Typical whole-vacuole current recordings are shown. SV currents were activated by a prepulse to +140 mV, followed by a series of test steps to less positive voltages, resulting in a complete or partial closure of SV channels (tail currents). Vacuole (pipette solution) contains 500 mm NaCl, and bath contains 100 mm KCl. Reversal potential (approximately 50 mV) is indicated by the arrowhead. At right is a summary of tail current I/V relationships obtained with a standard 100 mm KCl bath and either 500 mm (n = 8) or 100 mm (n = 4) NaCl in the pipette (vacuolar side).

Figure 11.

Effects of salinity and leaf age on the SV channel activity in quinoa mesophyll vacuoles. A, Typical whole-vacuole records of time- and voltage-dependent SV currents in vacuoles of young and old leaves from quinoa plants grown under control conditions (50 mm NaCl) and of old leaves from plants grown under saline conditions (400 mm NaCl). For symmetric 100 mm KCl, 1 mm free Ca²⁺ was added to the cytosolic side to activate SV and inhibit FV currents. B and C, Mean numbers of open SV channels in quinoa tonoplast as a function of membrane voltage. Solid lines are the best fits to the Boltzmann equation. D, Same data as in B and C, but only the points within the physiological tonoplast potential range (±20 mV) are considered. Data with different lowercase letters are significantly different at P < 0.05. O, Old leaves; Y, young leaves. Values are means ± se (n = 4–6 vacuoles).