Vacuolar Chloride Fluxes Impact Ion Content and Distribution during Early Salinity Stress

Abstract

The ability to control the cytoplasmic environment is a prerequisite for plants to cope with changing environmental conditions. During salt stress, for instance, Na⁺ and Cl^- are sequestered into the vacuole to help maintain cytosolic ion homeostasis and avoid cellular damage. It has been observed that vacuolar ion uptake is tied to fluxes across the plasma membrane. The coordination of both transport processes and relative contribution to plant adaptation, however, is still poorly understood. To investigate the link between vacuolar anion uptake and whole-plant ion distribution during salinity, we used mutants of the only vacuolar Cl^- channel described to date: the Arabidopsis (Arabidopsis thaliana) ALMT9. After 24-h NaCl treatment, almt9 knock-out mutants had reduced shoot accumulation of both Cl^- and Na⁺ In contrast, almt9 plants complemented with a mutant variant of ALMT9 that exhibits enhanced channel activity showed higher Cl^- and Na⁺ accumulation. The altered shoot ion contents were not based on differences in transpiration, pointing to a vacuolar function in regulating xylem loading during salinity. In line with this finding, GUS staining demonstrated that ALMT9 is highly expressed in the vasculature of shoots and roots. RNA-seq analysis of almt9 mutants under salinity revealed specific expression profiles of transporters involved in long-distance ion translocation. Taken together, our study uncovers that the capacity of vacuolar Cl^- loading in vascular cells plays a crucial role in controlling whole-plant ion movement rapidly after onset of salinity.

Figure 1.

Transcriptional regulation and expression pattern analysis of ALMT9. A to D, qRT-PCR analysis of SOS1 (A) and ALMT9 (B–D) expression in hydroponically grown wild-type shoots and roots after the application of 100 mm NaCl (A and B), 100 mm KCl (C), and 200 mm sorbitol (D) for 0, 6, 24, and 48 h. The data were normalized to expression levels in shoots prior to treatment (0 h). ACT2 served as a reference gene. Data are means ± sd of n = 3 biological replicates. E to J, ALMT9 expression pattern revealed by histochemical localization of GUS activity directed by the ALMT9 promoter. E, Mesophyll cell and vasculature expression in the third rosette leaf. F, Vasculature expression in the sixth rosette leaf. G, Expression in guard cells. H, Expression in root stelar cells. I, Expression in response to 100 mm NaCl for 24 h was enhanced but remained restricted to the root stele. J, In cross-sections of roots, no expression was detected in cortex cells (c), but in the endodermis (e), the pericycle (p), and the vasculature (v). Scale bars represent 0.2 mm in E and F, 10 µm in G and J, and 100 µm in H and I.

Figure 2.

Ion content analysis in shoots and roots of wild-type and almt9 mutants upon 24-h salt stress. The two knock-out alleles almt9-1 and almt9-2 and the corresponding wild types (wt-1 and wt-2) were grown in hydroponics, and Na⁺ (A), Cl⁻ (B), K⁺ (C), and NO₃⁻ (D) contents were determined prior to (0 mM) and after NaCl treatment (100 mM). Data are means ± sd of n ≥ 5 biological replicates derived from two independent experiments. One-way ANOVA of each tissue and treatment and a pairwise comparison was used for statistical analysis. Asterisks indicate significant differences from the corresponding wt (*P < 0.05, **P < 0.01, ***P < 0.001). DW, Dry weight; FW, fresh weight.

Figure 3.

Electrophysiological properties of the mutant channel ALMT9_E196A. A, Fluorescence and transmission images of vacuoles released from lysed tobacco protoplasts that transiently overexpress ALMT9-GFP (left) and ALMT9_E196A-GFP (right). Auto-fluorescence of chloroplasts is shown in magenta. Scale bars = 20 µm. B, Patch-clamp experimental procedure. Vacuoles were patched in excised cytosolic-side-out configuration under symmetric ionic conditions (100 mm Cl⁻_vac/ 100 mm Cl⁻_cyt). The cytosolic buffer was sequentially exchanged (100 mm Cl⁻; 100 mm Cl⁻ + 1 mm MA²⁻; 100 mm MA²⁻) on the same membrane patch. C, Representative currents of ALMT9 (left) and ALMT9_E196A (right) in presence of 100 mm Cl⁻ (Cl⁻) and 100 mm Cl⁻ + 1 mm MA²⁻ (Cl⁻ + MA²⁻) in the cytosolic buffers. Currents were evoked by a 2.5-s voltage ramp ranging from +40 mV to −100 mV. D, Relative Cl⁻ and Cl⁻ + MA²⁻ currents mediated by ALMT9 and ALMT9_E196A. Currents were normalized to the current amplitude measured at −100 mV in presence of 100 mm MA²⁻ in the cytosolic solution. E, Level of MA²⁻-activation (I_Cl⁻ _{+ MA}²⁻/ I_Cl⁻) of ALMT9- and ALMT9_E196A-mediated Cl⁻ currents at −100 mV. Data are means ± sd. Asterisks indicate statistically significant differences between ALMT9 (n = 5) and ALMT9_E196A (n = 6) currents (*P < 0.05, **P < 0.01, ***P < 0.001; two-tailed Student’s t test).

Figure 4.

Na⁺ and Cl⁻ content analysis in the transgenic line E196A. In plants of E196, the point-mutated channel variant ALMT9_E196A is expressed under the control of the native ALMT9 promoter in the genetic background of almt9-2. Na⁺ (A) and Cl⁻ (B) measurements were performed in shoot and root tissue of hydroponically grown wt-2, almt9-2, and E196A plants upon treatment with control (0 mM) or NaCl (100 mM) solutions for 24 h. The results are shown as mean ± sd of n ≥ 5 biological replicates derived from two independent experiments. For statistical analysis, one-way ANOVA of each tissue and treatment and a Tukey-Kramer multiple comparison posttest was used. Different lowercase letters indicate significant differences in ion content (P < 0.05) under control conditions, capital letters under salinity. DW, Dry weight. C, Expression analysis of ALMT9 in wt-2 and E196A using qRT-PCR. ACT2 served as a reference gene. The expression levels were normalized to wt-2. Data are means ± sd from n = 2 biological replicates.

Figure 5.

Investigation of stomatal movement and ion content in a guard cell-specific complementation line of almt9-2. A, In situ assay of native stomatal apertures (see “Materials and Methods”) using hydroponically grown plants of the almt9-1 and almt9-2 mutant alleles and the corresponding wild types (wt-1 and wt-2). Roots were exposed to control (0 mM) or NaCl (100 mM) solutions, and the stomatal aperture was measured before (0 h) and after (24 h) treatment. Error bars correspond to SEM, which was calculated from averages of at least four biological replicates. Asterisks indicate statistically significant differences in stomatal aperture from the corresponding wt (*P < 0.05, **P < 0.01, ***P < 0.01; two-tailed Student’s t test). B, In situ assay of native stomatal apertures of wt-2, almt9-2, and the complemented line GC:ALMT9 that guard cell-specifically expresses ALMT9 in control conditions. Error bars correspond to SEM, which was calculated from n ≥ 4 biological replicates. C and D, Contents of Na⁺ (C) and Cl⁻ (D) were measured in shoots and roots of hydroponically grown wt-2, almt9-2, and GC:ALMT9 plants after 24-h treatment with control (0 mM) or NaCl (100 mM) solutions. The combined results from two independent experiments are shown as mean ± sd (n ≥ 5). For statistical analysis in B to D, one-way ANOVA and a Tukey-Kramer multiple comparison were used. Different letters indicate significant differences in ion content (P < 0.05) within each tissue (lowercase in control conditions, capital letters under NaCl stress). DW, Dry weight.

Figure 6.

Transcriptome analysis in wild-type and almt9 shoots and roots upon salinity. A and B, RNA-seq analysis of hydroponically grown wt-1 and almt9-1 plants upon exposure to control (0 mM) or NaCl (100 mM) conditions for 24 h. A, Venn diagram showing the number of differentially expressed genes between wt-1 and almt9-1 within each tissue and treatment. The overlap between the ovals represents genes that have significant changes in gene expression under both treatments. B, Number of genes that are significantly differentially expressed between both genotypes exclusively under salinity. For selection requirements, see “Materials and Methods.” up, up-regulated genes in almt9-1; down, down-regulated genes in almt9-1. C to E, The expression levels of candidate genes were determined in wt-2, almt9-2, and the complemented lines E196A and GC:ALMT9 by qRT-PCR. The same experimental set-up as for the RNA-seq analysis was used. Transcript abundance of CHX21 (C) and DTX1 (D) was determined in shoots, transcript abundance of HKT1;1 (E) in roots. The data were normalized to the expression level of the respective gene in wt-2 under control conditions. ACT2 served as a reference gene. Each data point was derived from n ≥ 3 biological replicates and is shown as mean ± sd. In C to E, significances at P < 0.05 were analyzed by one-way ANOVA and Tukey-Kramer multiple comparison posttest for each treatment and are indicated by lettering (lowercase for control conditions, capital letters for salinity).

Figure 7.

Proposed model for the transcriptional regulation of transporter genes involved in whole-plant ion distribution by vacuolar ion uptake during early salinity. The expression of plasma membrane-localized transport proteins involved in long-distance ion transport is tuned by the efficiency of vacuolar Cl⁻ fluxes in the vasculature at the onset of salinity stress. The reduced Cl⁻ storage in the vacuoles of almt9 mutants might perturb the intracellular homeostasis of Na⁺ and Cl⁻ (black circles represent positively and negatively charged ions). This might signal an enhanced NaCl stress and initiate elevated expression levels of transporters such as the Na⁺ transporter HKT1;1 (HKT). In accordance, wild-type and E196A plants have more efficient vacuolar Cl⁻ uptake, and exhibit lower salt-induced HKT1;1 transcript increase. Similarly, transcript levels of other genes encoding for transporters (x) involved in the regulation of long-distance ion translocation in shoots and roots might be up- or down-regulated (dashed lines) in response to the capacity of storing Cl⁻ in the vacuoles of the vascular system.