Fig. 2: Three PYL homologs from Zygnematales exhibit PP2C inhibitory activity.
From: ABA-independent PP2C-binding in PYLs traces to bacterial origins and persists in land plants
a Sequence alignment of PYL homologs from algae (brown) and land plants. At, Arabidopsis thaliana; Sm, Selaginella moellendorffii; Meen, Mesotaenium endlicherianum; Mekr, Mesotaenium kuamstae; Zysp, Zygnemopsis sp.; Zc, Zygnema circumcarinatum; Sppr, Spirogyra sp.; Mp, Marchantia polymorpha; Pp, Physcomitrella patens. Two key residues for ABA binding are indicated by red rectangles. b RD29Bpro:LUC expression in pyl duodecuple mutant protoplasts transformed with ancestral Zygnematophyceae PYLs. Transformation with SnRK2.6, ABF2, and RD29Bpro:LUC served as control. Error bars, SEM (n ≥ 3 biological repeats). Two-way ANOVA, ****p < 0.0001. Right: the two residues critical for ABA-binding (black) and the corresponding variant residues (red). Expression data of control and PYR1 were shared by fig. 1b. c Interactions between MeenPYL3 and Arabidopsis PP2Cs in Y2H assay. Yeast cells were grown on nonselective SD/−LW and selective SD/−LWH or SD/−LWHA media, without or with 10 µM ABA. d Interactions between MeenPYL3 and Arabidopsis PP2Cs in N. benthamiana leaves assessed by firefly luciferase complementation imaging (LCI). e, f In vitro kinase assays testing whether ZcPYL8 or MeenPYL3 could relieve PP2C-mediated inhibition of SnRK2.6 with or without 100 μM ABA. ZcPYL8, but not MeenPYL3, released SnRK2.6 from HAB1-mediated inhibition (e). In contrast, MeenPYL3, but not ZcPYL8, released SnRK2.6 from PP2CA-mediated inhibition (f). SnRK2.6 autophosphorylation was detected by autoradiography (upper panels). g–j PP2C enzyme activity assays using pNPP as substrate. MeenPYL3 inhibited HAB1 (h), whereas ZcPYL8 inhibited PP2CA (i), both irrespective of 10 µM ABA. Error bars, SD (n ≥ 6 biological repeats). k Predicted structure of MeenPYL3 (purple, Alphafold3) aligned with the reported complex (PDB ID: 3KDJ) containing ABA (green/red), AtPYL1 (gold), and AtABI1. Residues within a 5 Å radius of ABA are shown (right). AtPYL1 (K86/L114) and corresponding residues in MeenPYL3 (R63/I93) are labeled. Cyan dashed lines represent hydrogen bonds between ABA and K86 in AtPYL1, while the black dashed line indicates steric hindrance (<2 Å) between ABA and R63 in MeenPYL3. l Model depicting that certain Zygnematophyceae PYLs gained PP2C inhibitory activity prior to the last common ancestor of Zygnematophyceae and land plants. Exact p values for (b, e, f) are provided in Source Data.
