A9C sensitive Cl⁻-accumulation in A. thaliana root cells during salt stress is controlled by internal and external calcium

Abstract

The involvement of chloride in salt stress symptoms and salt tolerance mechanisms in plants has been less investigated in the past. Therefore, we studied the salt-induced chloride influx in Arabidopsis expressing the GFP-based anion indicator Clomeleon. High salt concentrations induce two phases of chloride influx. The fast kinetic phase is likely caused by membrane depolarization, and is assumed to be mediated by channels. This is followed by a slower "saturation" phase, where chloride is accumulated in the cytoplasm. Both phases of chloride uptake are dependent on the presence of external calcium. In general: with high [Ca (2+)] less chloride is accumulated in the cytoplasm. Surprisingly, also the internal calcium availability has an impact on chloride transport. A complete block of the second phase of chloride influx is achieved by the anion channel blocker A9C and trivalent cations (La (3+), Gd (3+), and Al (3+)). Other channel blockers and diuretics were found to inhibit the process partially. The results suggest that several transporter species are involved here, including electroneutral cation-chloride-cotransporters, and a part of chloride possibly enters the cells through cation channels after salt application.

Keywords: calcium; cation-chloride-cotransporter; channels; chloride; salinity; transport.

Figure 1. Kinetics of chloride influx at two different NaCl concentrations. Flow-through in vivo experiments with Arabidopsis thaliana plants grown on full MS were performed in a buffer system consisting of 5 mM MES/KOH (pH = 6.0). 100 mM and 150 mM NaCl were applied after 120 min. Curves are averages from 4 independent experiments. The data were normalized by the mean of the time interval 5 min ≤ t ≤ 15min. Error bars represent StDv. The the two distinct phases are indicated by red lines at the bottom: depolarization phase (DP, full line), saturation phase (SP, dotted line).

Figure 2. Two phases of salt-induced chloride influx kinetics are affected by external calcium. Superfusion experiments with Arabidopsis thaliana plants grown on half strength MS and treated with MES-buffer (5 mM MES/KOH; pH 6.0). CaCl₂-concentrations of the flow-through medium are indicated in the Fig. legend. Additionally 150 mM NaCl were applied after 40 min. All curves are averages from at least 3 independent experiments. The data were normalized by the mean of the time interval 5 min ≤ t ≤ 15 min. Error bars represent StDv. The duration of the two distinct phases is indicated by red lines at the chart bottom: depolarization phase (DP, full line), saturation phase (SP, dotted line).

Figure 3. Internal calcium alleviates the salt induced chloride influx. Arabidopsis thaliana plants grown on MS medium with different calcium concentrations (as indicated in the Fig. legend) were treated with salt in absence of external calcium. 150 mM NaCl were applied after 40 min. All curves are averages from at least 3 independent experiments. The data were normalized by the mean of the time interval 5 min ≤ t ≤ 15min. Error bars represent StDv

Figure 4. Ca-inhibition of chloride flux is reversed by nifedipine. Flow-through experiment with Arabidopsis root cells expressing Clomeleon, superfused with SM-medium; treated with NaCl (t = 40 min) and 100 µM nifedipine (t = 45 min, red bar). In the respective control experiment, plants were treated only with salt no Nif. Plants were grown on 1× MS for 6–12 d. The graphs represent an average of at least 4 experiments. The data were normalized by the mean of the time interval 5 min ≤ t ≤ 15 min. Error bars represent StDv.

Figure 5. Effect of the channel blocker A9C on NaCl-induced chloride influx. Flow-through experiments with Arabidopsis root cells expressing Clomeleon, perfused with MES buffer; treated with NaCl (t = 30 min) and 100 µM A9C (t = 45 min). Plants had been grown on 0.5× MS for 6–12 d. The graph represents an average of at least 4 experiments. The data were normalized by the mean of the time interval 5 min ≤ t ≤ 15 min. Error bars represent StDv.

Figure 6. Inhibition rate of different effectors on salt induced chloride influx. The inhibition rate was calculated as a percentage decrease in the slope of the emission ratio of control experiments (without inhibitor) for the time interval 45 min ≤ t ≤ 60 min). Data obtained from 5 experiments each, as shown in Figure 5. The CCC inhibitors are given as gray columns.

Figure 7. Trivalent cation block of NaCl-induced chloride entry. Arabidopsis thaliana root cells expressing Clomeleon were perfused with 5 mM MES/KOH, pH6 without SM; treated with 150 mM NaCl (t = 30 min) and 2 mM AlCl₃, GdCl₃ and LaCl₃ (40 min ≤ t ≤ 75 min), respectively. The plants had been grown for 6–14 d on 0.5× MS. Each curve is an average of 4 independent experiments. The data were normalized by the mean of the time interval 5 min ≤ t ≤ 15min. Error bars represent StDv.