EAR1 Represses ABA Signaling. EAR1 Represses ABA Signaling. (A) EAR1 reduces OST1 activity by enhancing the phosphatase activity of ABI1 in vitro. Recombinant proteins with OST1 (4 μg), ABI1 (100 ng is indicated as + and 10 ng as 1/10), and EAR1 (from 4 to 40 ng) were combined for the kinase assay. OST1 was used as a substrate and stained with Coomassie Brilliant Blue (CBB) as a loading control. The experiments were performed three times, with similar results. (B) Quantitative analysis of the OST1 band intensity in (A) with ImageJ software. The OST1 band intensity of ABI1 without EAR1 was set to 1 as a reference compared with the OST1 band intensities of ABI1 with 1×, 5×, or 10× EAR1. Bars are means ± se from three independent experiments. (C) EAR1 inhibits the expression of ABA-responsive genes in a reconstitution assay of the ABA signaling pathway. ProRD29B:LUC was cotransformed with Pro35S:EAR1, Pro35S:ABI1, and Pro35S:SnRK2.6 into snrk2.2/snrk2.3/snrk2.6 (snrk2.2/2.3/2.6) triple mutant protoplasts treated with or without ABA. Luciferase activity was used as an indicator of the expression of the ABA-responsive gene RD29B, and ZmUBQ:GUS was used as an internal control. Bars are means ± se of three biological replicates. Each replicate was independently measured at different times. (D) EAR1 suppresses the core ABA signaling genes in a reconstitution assay of the ABA signaling pathway. ProRD29B:LUC was coexpressed with Pro35S:EAR1, Pro35S:ABI1, Pro35S:ABI2, Pro35S:PYR1, and Pro35S:SnRK2.6 in snrk2.2/snrk2.3/snrk2.6 (snrk2.2/2.3/2.6) triple mutant protoplasts treated with or without ABA. Luciferase activity was used as an indicator of the expression of the ABA-responsive gene RD29B, and ZmUBQ:GUS was used as an internal control. Bars are means ± se of three biological replicates. Each replicate was independently measured at different times. (E) OST1 kinase activity is enhanced in ear1-1 and reduced in the EAR1 overexpression line OE-16# in an in-gel kinase assay. Total proteins (80 μg) were extracted from 10-d-old wild-type, ear1-1, OE-16#, and ost1-3 seedlings treated with or without 60 μM ABA for 30 min. The proteins were separated on a 10% SDS-PAGE gel with GST-ΔABF2 as a substrate. Rubisco was used as a loading control. The experiments were performed three times, with similar results. (F) Quantitative analysis of the band intensity in (E). The band intensity in the wild type was set to 1 as a reference, and the band intensity of ear1-1 or OE-16# was compared with that of the wild type. Bars are means ± se from three independent experiments. (G) OST1 kinase activity is enhanced in ear1-1, 3m, and 4m. Total proteins (80 μg) were extracted from 10-d-old seedlings treated with or without 30 μM ABA for 30 min. The proteins were separated on a 10% SDS-PAGE gel with GST-ΔABF2 as a substrate. Rubisco was used as a loading control. The experiments were performed three times, with similar results. (H) Quantitative analysis of the band intensity in (G). The band intensity in the wild type was set to 1 as a reference. Bars are means ± se (n = 3 three independent experiments). Kai Wang et al. Plant Cell 2018;30:815-834 ©2018 by American Society of Plant Biologists