Figure 6.

CDL1 Is Required for OST1 Activation in Both ABA- and BR-Induced Stomatal Closure.

(A) CDL1-dependent OST1 activation by BL in plant cells. OST1-myc or OST1-myc cdl1 plants were treated with mock or 0.1 μM BL for 1 h. Next, OST1-myc immunoprecipitated from OST1-myc or OST1-myc cdl1 with anti-myc antibody was incubated with equal amounts of MyBP and [γ-³²P]ATP. Ponceau S was used to estimate protein amounts of input. CBB, Coomassie Brilliant Blue.

(B) An in-gel kinase assay to examine BR or ABA-induced OST1 activation. OST1-myc or OST1-myc cdl1 plants were treated with mock or 0.1 μM BL for 1 h. OST1-myc immunoprecipitated from OST1-myc or OST1-myc cdl1 with anti-myc antibody was loaded onto SDS-PAGE gel containing MyBP. A renatured gel was incubated with [γ-³²P]ATP in the kinase buffer. A small aliquot of immunoprecipitated protein was analyzed by an immunoblot using anti-myc antibody. Ponceau S was used to estimate protein amounts of input.

(C) CDL1 promotes ABA-induced OST1 activation in plant cells. OST1-myc or OST1-myc cdl1 plants were treated with mock or 1 μM ABA for 1 h. Next, OST1-myc immunoprecipitated from OST1-myc or OST1-myc cdl1 with anti-myc antibody was incubated with equal amounts of MyBP and [γ-³²P]ATP.

(D) An in-gel kinase assay to examine ABA-induced OST1 activation. OST1-myc or OST1-myc cdl1 plants were treated with mock or 1 μM ABA for 1 h. OST1-myc immunoprecipitated from OST1-myc or OST1-myc cdl1 with anti-myc antibody was loaded onto SDS-PAGE gel containing MyBP. A renatured gel was incubated with [γ-³²P]ATP in the kinase buffer. A small aliquot of immunoprecipitated protein was analyzed by an immunoblot using anti-myc antibody.

(E) CDL1 phosphorylation of MyBP upon BR treatment. Transgenic CDL1-YFP plants were treated with mock or 0.1 μM BL for 1 h. CDL1-YFP immunoprecipitated with anti-YFP antibody was incubated with equal amounts of MyBP and [γ-³²P]ATP.

(F) ABA as well as BL activates the kinase activity of CDL1 phosphorylating OST1 in Arabidopsis. Transgenic CDL1-YFP plant was treated with mock, 0.1 μM BL, or 1 μM ABA for 1 h. CDL1-YFP immunoprecipitated with anti-YFP antibody was incubated with equal amounts of MBP-mOST1 and [γ-³²P]ATP.

(G) In vitro kinase assay shows OST1 phosphorylates CDL1. A kinase-inactive version of GST-mCDL1 (K102R) was used as a substrate.