Fig. 5: Genetic interactions among CIPK1 with PYLs.

a ABA treatment inhibits the interaction between CIPK1 and PYL4 in pull-down assays. The experiment was conducted three times, and similar results were obtained. b ABA inhibits the interaction between CIPK1 and PYL4 in Co-IP assays. For ABA treatment, the extracted protein containing 100 μM ABA was incubated overnight with Anti-FLAG antibodies. Three repetitions of the experiment obtained similar results. c The relative density of PYL4 (IP/Input) in (b) reveals an interaction between CIPK1 and PYL4 in vivo that was reduced by ABA. Data were measured using ImageJ and represented as IP/Input. Data are shown as means ± SD. Asterisks indicate significance compared to the IP without ABA according to Student’s t-test (Two-sided, ** P < 0.01). d ABA inhibits the phosphorylation of PYL4 by CIPK1 in vitro. In this assay, indicated concentrations of ABA was added to the reaction system. Two repetitions of the experiment yielded similar results. e Protein kinase assay of CIPK1 with PYL4-His under ABA treatment. Protein kinases were quantified by western blot and shown at the bottom. The experiment was repeated twice and similar results were obtained. f Phosphorylation of PYL4 by CIPK1 requires a moderate amount of Ca²⁺. The experiment was repeated three times with similar results. g CBL1/9-CIPK1 mediates the phosphorylation of PYL4 Ser129 in vivo. The phosphorylation signal of PYL4 was detected by anti-P-Ser antibodies, and the proteins were quantified through western blot and anti-FLAG antibodies. The experiment was conducted two times with similar results. h The 12458/cipk1 mutants showed similar drought sensitivity. Scale bar, 2.0 cm. The experiment was repeated three times with similar results. i 12458 and 12458/cipk1 plants leave temperature imaging. Data were statistical for each replicate of 9 leaves repeated three times. Box plots show the 25% quantile, median (line), and 75% quantile. The whiskers plots (Tukey method) represent minimum and maximum values. Statistical analysis was performed by one-way ANOVA analysis followed by Dunnett’s multiple comparison test. Asterisk indicates significance compared to wild type: **P < 0.01. Scale bar, 2.0 cm.