Fig. 3: Two invariant residues are crucial for the ABA dependence of plant PYLs.
From: ABA-independent PP2C-binding in PYLs traces to bacterial origins and persists in land plants
a, c, h RD29Bpro:LUC expression in pyl duodecuple mutant protoplasts transformed with Arabidopsis PYLs (a), AtPYL13-Q38K/F71L (c), and variants of AtPYLs (h). Transformation with SnRK2.6, ABF2 and RD29Bpro:LUC served as control. Error bars, SEM, n ≥ 3 biological repeats in (a, h), and n ≥ 6 biological replicates in (c). Two-way ANOVA, *p < 0.05, **p < 0.01, ****p < 0.0001. Expression data of control was shared among figs. 3a, c, h and 4c. b Sequence alignment of ABA-binding regions in AtPYLs. The two residues in PYL13 that differ from other PYLs are labeled with red asterisks and rectangles. d, e In vitro kinase assays assessed whether wild-type or mutated AtPYL13 could relieve PP2C-mediated inhibition of SnRK2.6 with or without 100 μM ABA. SnRK2.6 activity was repressed by AtABI1 (d) and AtPP2CA (e). SnRK2.6 autophosphorylation (autoradiography, lower panels) and protein loading (Coomassie blue staining, upper panels) are shown. Radioactive intensity of the bands was normalized to that of the AtPYL13-Q38K/F71L sample without ABA. Error bars, SD (n = 3 biological replicates). Two-sided Student’s t test, *p < 0.05. f, g The ABA-hyposensitive phenotype of the pyr1pyl124 quadruple mutant was complemented by 35Spro:AtPYL13-Q38K/F71L but not by 35Spro:AtPYL13 (f). Seedling establishment was quantified (g). Error bars, SD (n = 3 biological replicates). Two-way ANOVA followed by a Tukey test. i Release of SnRK2.6 from ABI1 inhibition by AtPYL9 and AtPYL9-K63Q/L89F, with or without 100 µM ABA, during in vitro kinase assays. SnRK2.6 autophosphorylation was detected by autoradiography (lower panel). Radioactive intensity of the bands was normalized to that of the AtPYL9 sample (2×) with ABA. Error bars, SD (n = 3 biological replicates). Two-sided Student’s t test, **p < 0.01, ****p < 0.0001. j, k The ABA-hyposensitive phenotype of pyr1pyl124 was complemented by AtPYL1pro:AtPYL1 but not by AtPYL1pro:AtPYL1-K86Q/L114F (j). Seedling establishment was quantified (k). Error bars, SD (n = 3 biological replicates). Two-way ANOVA followed by a Tukey test. Exact p values for (a, c, d, e, g–i, and k) are provided in Source Data. For figures (d, e, and i), images shown are representative of three independent experiments.
