Fig. 7: OsSAPK6 phosphorylates IPA1 under chilling stress to upregulate OsCBF3 expression.

a OsSAPK6 kinase activities under chilling stress. Flag-OsSAPK6-OE transgenic seedlings were treated under 4 °C for the indicated time, and Flag-OsSAPK6 protein was then purified with anti-Flag beads, normalized, and incubated with equal amounts of purified GST-IPA1 for 30 min. The phosphorylated GST-IPA1 was detected with a polyclonal antibody against a substrate motif [LXRXX (pS/pT)], and GST-IPA1 was detected with anti-IPA1 monoclonal antibody. b Relative content of phosphorylated IPA1 in ZH11 and ossapk6-2 seedlings under chilling stress. Protein extracts from ZH11 and ossapk6-2 seedlings were separated in a Phos-tag gel, and the phosphorylated and nonphosphorylated IPA1 proteins were detected with anti-IPA1 antibody and quantitated by densitometry. c Relative content of phosphorylated IPA1 in ZH11 and OsSAPK6-OE seedlings under chilling stress. d IPA1 protein levels in ZH11 and ossapk6-2 seedlings under chilling stress. IPA1 protein was detected by anti-IPA1 antibody. Relative amounts of proteins were determined by densitometry and normalized to loadings determined by Ponceau staining (red). e Schematics of constructs used for transcriptional activity assays in rice protoplasts. f Transcriptional activity assay in rice protoplasts showing that IPA1-mediated activation of OsCBF3 expression was promoted by OsSAPK6 and temperature sensitive. g Expression levels of OsCBF3 in ZH11, ossapk6-2, ipa1-3D, and ossapk6-3 ipa1-3D after 4 °C treatment for 6 h. h Expression levels of OsCBF3 gene in ZH11, Ubi:OsSAPK6, ipa1-10, and Ubi:OsSAPK6 ipa1-10 after 4 °C treatment for 6 h. In f–h, values are means ± SD (n = 3 technical replicates). At least three independent experiments were performed with similar results. Different letters indicate significant differences (P < 0.01) according to Tukey’s HSD test. See also Supplementary Figs. S11–13.