Fig. 2: AtCLCf expression is induced by NaCl treatment.

a Tissue-specific expression of AtCLCf n = 3 (3 biological replicates, each with 3 technical replicates). Data are mean ± SE, means with different letters within a data set are significantly different, P < 0.05 (one-way ANOVA followed by Tukey’s test). pAtCLCf::GUS expression in one-month-old Arabidopsis (b) root, (c) flower, (d) leaf, (e) silique, and (f) leaf vasculature. Scale bar in b and f = 100 µm, c–e = 500 µm, data were obtained from three independent experiments, and representative images are shown in b–f. g Time-course analysis of AtCLCf expression in the shoots and roots of one-week-old Arabidopsis seedlings after 100 mM NaCl treatment. Data are mean ± SE, n = 3 (3 biological replicates, each with 3 technical replicates). Asterisks indicate statistically significant differences (*P < 0.05, **P < 0.01) between 0 h and other time points, as measured by unpaired Student’s t test (two-tailed). h pAtCLCf::GUS expression in one-week-old untreated and salt-treated (100 mM NaCl, 6 h) Arabidopsis seedlings. Scale bar = 1 mm, data were obtained from three independent experiments, and representative images are shown here. i Close-up images of the roots in h Scale bar = 100 µm. j, k Growth responses of one-month-old WT, atclcf, and 35S::AtCLCf plants grown in soil (j) without NaCl treatment and (k) after one week of 150 mM NaCl treatment. Scale bar = 1 cm, (n = 3). l Cl⁻ contents in the shoots and roots of WT, atclcf, and 35S::AtCLCf plants under control (untreated) and salt-treated conditions (n = 5). Data are mean ± SE, means with different letters within a data set are significantly different, P < 0.05 (one-way ANOVA followed by Tukey’s test). The box plots with whiskers show all the data points and their distribution. Horizontal lines within the boxes indicate the median. Minimal and maximal data points are indicated by the lower and the upper whiskers, respectively, untr untreated, sh shoot, R root. m ChIP-qPCR analysis shows enrichment of AtCLCf promoter fragment in WRKY9 ChIP sample. Data are mean ± SE, n = 3 (3 biological replicates, each with 3 technical replicates). n Luciferase assay carried out using the mesophyll protoplasts obtained from leaves of 4-week-old atwrky9 mutants. pAtCLCf::LUC was used as the control and 35S::AtWRKY9 was used as the test. The AtCLCf promoter fragments with mutated WRKY binding sites were used as additional controls. Firefly luciferase activity was normalized to Renilla luciferase activity and plotted (Mean ± SE, n = 5). Asterisks in m and n indicate statistically significant differences (*P < 0.05, **P < 0.01) between control and test, as measured by Student’s t test.