Journal: Plant Communications

Article Title: The ZAT7–OTU11–DOA9–CYS13 module enhances salt resistance by suppressing NPF2.4-mediated chloride transport in Arabidopsis

doi: 10.1016/j.xplc.2026.101962

Figure Lengend Snippet: DOA9 interacts with ZAT7 and reduces its protein stability. (A) Protein levels of ZAT7 in 8-day-old Pro35S : ZAT7-HA #9 seedlings treated with 200 μM cycloheximide (CHX), 200 μM CHX + 200 mM NaCl, or 200 μM CHX + 100 μM MG132 (a specific 26S proteasome inhibitor) for the indicated times. Similar results were obtained in three independent experiments, and the averages are indicated. (B) Protein levels of ZAT7 in Arabidopsis protoplasts expressing ProZAT7 : ZAT7-GFP . Cells were treated with 100 μM CHX, 100 μM CHX + 150 mM NaCl, or 100 μM CHX + 50 μM MG132 for the indicated times. Similar results were obtained in three independent experiments, and the averages are indicated. (C–F) Interactions of DOA9 and ZAT7 in Y2H assays (C) , AlphaFold 3 prediction (D) , pull-down assays (E) , and LCI assays (F) . Y2H, yeast two-hybrid; LCI, luciferase complementation imaging; −WL, without leucine and tryptophan; −WLAH, without leucine, tryptophan, adenine, and histidine. In the pull-down assay, His-ZAT7 proteins were incubated with immobilized glutathione S-transferase (GST)-DOA9 or GST, and the proteins immunoprecipitated with GST beads were detected using an anti-His antibody. (G) Phenotypes of 4-day-old WT and doa9-1 seedlings grown on 1/2 MS medium with or without 150 mM NaCl, 125 mM KCl, or 125 mM NaNO 3 for 14 days. (H) Root growth of the seedlings shown in (G) . Mean ± SD ( n > 10). Student’s t -test; ∗∗ p < 0.01; ns, not significant. (I) Phenotypes of 4-day-old WT, doa9-1 , zat7-1 , and doa9-1 zat7-1 seedlings grown on 1/2 MS medium with or without 150 mM NaCl or 125 mM KCl for 14 days. (J) Root growth of the seedlings shown in (G) . Mean ± SD ( n > 10). Significance was determined by one-way ANOVA followed by Tukey’s HSD test; p < 0.05. (K) ZAT7 protein levels in 10-day-old Pro35S : ZAT7-HA #9 and Pro35S : ZAT7-HA #9; doa9-1 seedlings treated with 200 μM CHX or 200 μM CHX + 200 mM NaCl for the indicated times. Similar results were obtained from three independent experiments, and the averages are indicated below the figure. (L) The degradation rate of ZAT7 in Arabidopsis protoplasts isolated from WT, Pro 35S: DOA9 #1, and doa9-1 seedlings expressing ProZAT7 : ZAT7-GFP . Cells were treated with 100 μM CHX or 100 μM CHX + 150 mM NaCl for the indicated times. Similar results were obtained from three independent experiments, and the averages are indicated.

Article Snippet: To examine the effect of salt stress on ZAT7 protein stability and its degradation pathway, 8-day-old Pro35S : ZAT7-HA #9 seedlings were treated with 200 μM cycloheximide (CHX), 200 μM CHX + 200 mM NaCl, or 200 μM CHX + 100 μM MG132 (Selleck, Houston, TX, USA) for 6, 12, or 24 h. To assess how DOA9 influences ZAT7 protein stability, 8-day-old Pro35S : ZAT7-HA and Pro35S : ZAT7-HA ; doa9-1 seedlings were treated with 200 μM CHX for 6, 12, or 24 h. To investigate the effect of salt stress on OTU11 protein stability, 8-day-old Pro35S : OTU11-GFP #1 seedlings were treated with 200 μM CHX or 200 μM CHX + 200 mM NaCl for 6, 12, or 24 h. To determine the impact of salt stress on DOA9 stability and its degradation pathway in a cell-free system, recombinant GST-DOA9 was incubated with extracts from WT seedlings treated with 200 mM NaCl or 200 mM NaCl + 100 μM MG132 for 30, 60, or 90 min.

Techniques: Stability, Expressing, Pull Down Assay, Yeast, Luciferase, Imaging, Incubation, Immunoprecipitation, Isolation

Journal: Plant Communications

Article Title: The ZAT7–OTU11–DOA9–CYS13 module enhances salt resistance by suppressing NPF2.4-mediated chloride transport in Arabidopsis

doi: 10.1016/j.xplc.2026.101962

Figure Lengend Snippet: ZAT7 represses NPF2 . 4 expression to enhance plant Cl − tolerance. (A) NPF2.4 t ranscript levels in 8-day-old seedlings treated with 200 mM NaCl or 200 mM KCl for 6 h. Mean ± SD ( n = 3). Significance was determined by one-way ANOVA followed by Tukey’s HSD test; p < 0.05. (B) Putative AGT motifs in the NPF2 . 4 promoter. (C) Phenotypes of 4-day-old wild-type (WT) and zat7-1 seedlings grown on 1/2 MS medium with or without 125 mM NaCl, 125 mM KCl, or 125 mM NaNO 3 for 14 days. (D) Root growth of the seedlings shown in (C) . Mean ± SD ( n > 10). Student’s t- test; ∗∗∗ p < 0.001; ns, not significant. (E) Phenotypes of 4-day-old WT, Pro35S : ZAT7-HA #3, Pro35S : ZAT7-HA #9, and Pro35S : ZAT7-HA #10 seedlings grown on 1/2 MS medium with or without 150 mM NaCl, 125 mM KCl, or 125 mM NaNO 3 for 14 days. (F) Root growth of the seedlings shown in (E) . Mean ± SD ( n > 10). One-way ANOVA followed by Tukey’s HSD test; p < 0.05. (G) NPF2.4 t ranscript levels in 8-day-old WT, zat7-1 , Pro35S : ZAT7-HA #3, and Pro35S : ZAT7-HA #10 seedlings treated with or without 200 mM NaCl for 6 h. Mean ± SD ( n = 3). One-way ANOVA followed by Tukey’s HSD test; p < 0.05. (H) Dual-luciferase reporter assay assessing the ability of ZAT7 to bind fragments F1–F3 of the NPF2 . 4 promoter. GFP served as a negative control. Mean ± SD ( n > 3). Student’s t- test; ∗ p < 0.05; ns, not significant. (I) Yeast one-hybrid (Y1H) assay assessing the ability of ZAT7 to bind the full-length (FL) NPF2.4 promoter and various promoter fragments. (J) Electrophoretic mobility shift assay (EMSA) showing binding of ZAT7 to unlabeled F2, labeled F2, or unlabeled mF2 probes.

Article Snippet: To examine the effect of salt stress on ZAT7 protein stability and its degradation pathway, 8-day-old Pro35S : ZAT7-HA #9 seedlings were treated with 200 μM cycloheximide (CHX), 200 μM CHX + 200 mM NaCl, or 200 μM CHX + 100 μM MG132 (Selleck, Houston, TX, USA) for 6, 12, or 24 h. To assess how DOA9 influences ZAT7 protein stability, 8-day-old Pro35S : ZAT7-HA and Pro35S : ZAT7-HA ; doa9-1 seedlings were treated with 200 μM CHX for 6, 12, or 24 h. To investigate the effect of salt stress on OTU11 protein stability, 8-day-old Pro35S : OTU11-GFP #1 seedlings were treated with 200 μM CHX or 200 μM CHX + 200 mM NaCl for 6, 12, or 24 h. To determine the impact of salt stress on DOA9 stability and its degradation pathway in a cell-free system, recombinant GST-DOA9 was incubated with extracts from WT seedlings treated with 200 mM NaCl or 200 mM NaCl + 100 μM MG132 for 30, 60, or 90 min.

Techniques: Expressing, Luciferase, Negative Control, Yeast, Y1H Assay, Electrophoretic Mobility Shift Assay, Binding Assay, Labeling