Fig. 6: IPA1 directly binds to and activates OsCBF3 promoter to regulate chilling stress response.
From: Chilling-induced phosphorylation of IPA1 by OsSAPK6 activates chilling tolerance responses in rice
a–c Expression levels of OsCBF1 (a), OsCBF2 (b), and OsCBF3 (c) in ZH11, ipa1-3D, and ipa1-10 plants under 25 °C (CK) or 4 °C for 6 h. d Schematics showing the promoter structure of OsCBF3. Solid arrowheads indicate GTAC motifs in the OsCBF3 promoter. Hatched box P1 represents the fragment amplified in the ChIP-qPCR assay, and P2 represents the 42-bp fragment for EMSA. e ChIP-qPCR analysis of IPA1 binding sites (P1 in d) in the OsCBF3 promoter with ubiquitin as a control. f Direct binding of IPA1 to the OsCBF3 promoter in the EMSA assay. Biotin-labeled 42-bp fragment of the OsCBF3 promoter (P2 in d) was incubated with GST or GST-IPA1 protein. g Transcriptional activity assay in rice protoplasts shows that IPA1 could activate the expression of OsCBF3. ProOsCBF3:LUC was co-transformed with GFP or GFP-IPA1 effector for 12 h and then incubated at 28 °C (CK) or 4 °C for 1 h. h Plant morphologies of 2-week-old ZH11, ipa1-10, Ubi:OsCBF3, and ipa1-10 Ubi:OsCBF3 seedlings before treatment, after 4 °C treatment for 7 days, and subsequent recovery for 7 or 9 days. i Survival rates of ZH11, ipa1-10, Ubi:OsCBF3, and ipa1-10 Ubi:OsCBF3 after 4 °C treatment as shown in h. In a–c, g and i, values are means ± SD (n = 3 biological replicates). Different letters indicate significant differences (P < 0.01) according to Tukey’s HSD test. In e, values are means ± SD (n = 3 technical replicates). At least three independent experiments were performed with similar results, the asterisks indicate significant differences compared with the control (**P < 0.01, Student’s t-test). Bars = 5 cm in h. See also Supplementary Figs. S7–S10 and Table S2.
