Journal: Nature Communications
Article Title: Reciprocal control of metabolic and chromatin regulators improves rice tolerance to heat
doi: 10.1038/s41467-025-66406-3
Figure Lengend Snippet: a Phos-tag analysis of GCN5 phosphorylation in 14-day-old Ubipro::GCN5-2×FLAG-2×HA transgenic rice seedlings grown at 25 °C. GCN5-FLAG-HA was enriched with anti-FLAG beads and treated with or without phosphatase (CIAP). Immunoblotting with anti-HA antibody was used as controls of loading and input. b Immunoblot analysis of GCN5 phosphorylation levels in wild type (WT) and pk1 ( pk1 #1) protoplasts transfected with GCN5-FLAG fusion plasmid. The fusion protein was purified with anti-FLAG beads and treated with or without phosphatase (CIAP). c In vitro kinase assays of E.coli -produced PK1-His protein with GCN5-GST or GCN5 T271A -GST as substrates and phosphoenolpyruvate (PEP) as phosphate donor. GCN5 phosphorylation was detected by immunoblotting with anti-Phospho-threonine antibody. Immunoblotting with anti-GST was used as loading control. d Immunoblot analysis of GCN5 phosphorylation in Ubipro::GCN5-2×FLAG-2×HA transgenic rice plants treated with heat stress at the indicated time points. GCN5 proteins were precipitated from nuclear extracts using anti-FLAG beads and treated with or without phosphatase (CIAP). Error bars represent means ± SD. Two-tailed Student’s t tests or one-way ANOVA with Tukey’s multiple comparison test were performed with measures from 3 repetitions of the immunoblots in ( b–d ). e PK1 stimulated the GCN5 histone acetyltransferase activity at H3K9 site. FLAG-tagged H3 protein was produced together with or without PK1-GFP and GCN5-mCherry in transfected tobacco leaf cells. The acetylation and phosphorylation level of purified H3-FLAG protein was analyzed by immunoblotting using the H3K9ac, H3K14ac and H3T11p antibodies. The phosphorylation levels of purified GCN5 protein were analyzed by immunoblotting using the anti-Phospho-threonine antibody. Tagged H3, PK1, and GCN5 proteins were detected by anti-FLAG, anti-GFP, and anti-mCherry antibodies, respectively. Anti-FLAG antibody was used as loading controls. Error bars represent means ± SD. Two-tailed Student’s t tests or one-way ANOVA with Tukey’s multiple comparison test were performed with measures from 3 repetitions of the immunoblots. Significant difference (p < 0.05) between bars are indicated by different letters. The exact p values for each comparison are provided in the Source Data. Immunoblotting bands in ( b–e ) were quantified using ImageJ. Signals relative to the respective controls (set at 1) are indicated below the bands. Source data are provided in the file.
Article Snippet: For GCN5 phosphorylation assays in rice protoplasts, 35Spro::GCN5-FLAG construct was transfected into rice protoplasts isolated from wild type (WT) and pk1 mutants for 14 h, then the proteins were extracted using lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40) and incubated with anti-FLAG M2 magnetic beads (Sigma, M8823) at 4 °C for 5 h. After three washes with washing buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA), the immunoprecipitated proteins were treated with CIAP (Takara 2250 A) with their reaction buffer as described by the manufacturer and then separated by SDS-PAGE with anti-FLAG (1:1000, Sigma, F3165) and anti-Phos-threonine (1:1000, ABclonal, AP1422) antibodies.
Techniques: Phospho-proteomics, Transgenic Assay, Western Blot, Transfection, Plasmid Preparation, Purification, In Vitro, Produced, Control, Two Tailed Test, Comparison, Activity Assay
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