Fig. 4.

ABA-activated SnRK2 activity of P. patens depends on the evolutionarily conserved ARK phosphorylation motif in the activation loop. a Protonemata of wild-type (WT) or transgenic plants expressing SnRK2B-GFP or mutated SnRK2-GFP (S165/169 A) under the control of the rice actin promoter were treated with or without 10 µM ABA for the indicated periods in minutes, then the protein extracts were subjected to in-gel kinase assay. Arrows indicate the position of recombinant SnRK2B (SnRK2-GFP) and endogenous SnRK2. b Constitutive rice actin promoter–driven SnRK2B-GFP or mutated SnRK2-GFP (S165/169 A) in the predicted phosphorylation sites was introduced into QKO protonemata with Em-GUS and Ubi-LUC by particle bombardment. Protonemal cells were incubated with or without 10 µM ABA for 1 day, then subjected to GUS and LUC assays. Promoter activity of the Em promoter in WT and QKO in the absence of the effector construct is shown as controls. Levels of gene expression are represented by GUS per LUC ratio. Error bars indicate the standard error (SE; n = 4). c, d PpSnRK2D or its mutant variants fused to PpSnRK2D gene native promoter were introduced into QKO protonemata with PpLEA1-GUS for 10 µM ABA treatment for 1 d (c), or with Em-GUS for 0.4 M mannitol treatment for 1 d (d). Promoter activity of promoters in WT and QKO in the absence of the effector construct are shown as controls. Levels of gene expression are represented by the GUS per LUC ratio. Error bars indicate the SE (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 compared with the PpSnRK2D effector of QKO with ABA