Figure 5.

CDL1 Activates OST1 by Phosphorylation on Ser7.

(A) In vitro kinase assay shows CDL1 phosphorylates OST1. A kinase-inactive version of MBP-mOST1 (K50R) was used as a substrate.

(B) CDL1 phosphorylates OST1 on Ser-7. MBP-mOST1 (K50R) or MBP-mOST1^S7A harboring the S7A mutation was incubated with GST-CDL1 and [γ-³²P]ATP.

(C) Comparison of the kinase activities of OST1 proteins harboring the Ser-7 mutation. Equal amounts of MBP-OST1^S7A, MBP-OST1, and MBP-OST1^S7D were incubated with MyBP and [γ-³²P]ATP.

(D) CDL1 phosphorylation of OST1 increases OST1 activity and CDL1-mediated OST1 activation is abolished by the S7A mutation. MBP-OST1 or MBP-mOST1^S7A was preincubated with GST, GST-CDL1, or GST-mCDL1 (kinase-inactive CDL1, K102R) with ATP for 2 h. Purified MBP-OST1 or MBP-OST1^S7A (after removal of GST, GST-CDL1, or GST-mCDL1; Supplemental Figure 12B) was incubated further with MyBP and [γ-³²P]ATP. CBB, Coomassie Brilliant Blue.

(E) and (F) Stomatal closure in ost1 overexpressing OST1-YFP or OST1^S7A-YFP. The stomatal apertures in wild-type Col-0, ost1, OST1-YFP ost1, and OST1^S7A-YFP ost1 upon ABA (E) or BL (F) treatment were compared. Result is representative of three independent experiments (n ≥ 30 stomata per genotype). Detached rosette leaves were incubated in stomatal closure solution containing mock, ABA, or BL as indicated for 2 h. Error bars indicate se. Statistical significant differences are indicated by different lowercase letters (two-way ANOVA, P < 0.005).