Figure 1: IAA6 and IAA19 ohnologues exhibit different selection pressure signatures and their TIR1-containing receptor complexes show dissimilar auxin binding properties.
From: Variation in auxin sensing guides AUX/IAA transcriptional repressor ubiquitylation and destruction
(a) Comparison of available A. thaliana IAA6 (green) and IAA19 (blue) expression data. Box plots depict IQR and the median of three different datasets obtained from the Arabidopsis eFP browser78. Outliers were defined as 1.5 × IQR. Graphs for different conditions in each data set are shown in Supplementary Fig. 2. IQR, interquartile range. (b) Sliding window plots of nucleotide sequence divergence between IAA6 and IAA19 in A. thaliana, A. lyrata, A. halleri and C. rubella. dN/dS ratios are plotted against the midpoint position of each 50 bp window. Black bars (top) display positions of the different protein domains. See Supplementary Data 1 for detailed AUX/IAA domain structure. (c) Y2H interaction matrix for TIR1, AFB1, AFB2 with IAA6, IAA19, and their putative dominant mutant versions32,33,35: IAA6C78R/suppressor of hy2 (shy1-1), IAA6P76S, IAA19P76S/massugu (msg2-1), carrying mutations in the degron (right). Yeast diploids containing LexA DBD-TIR1/AFBs and AD-AUX/IAAs were spotted on selective medium with increasing IAA concentrations, and β-galactosidase reporter expression indicates IAA-induced TIR1/AFB1/2-AUX/IAA interactions. EV, empty vector. (d) One-point saturation binding assays using 200 nM [3H]IAA to recombinant ASK1-TIR1-AUX/IAA ternary complexes. IAA6C78R/shy1-1 and IAA19P76S/msg2-1 mutants that mimic stabilized version of the proteins affect significantly specific auxin binding. (e,f) IAA6 and IAA19 provide ASK1-TIR1-containing complexes different auxin-sensing capabilities. (e) Representative saturation binding curves for IAA6 and IAA19 (left). ASK1-TIR1-IAA19 complexes bind auxin with high affinity (Kd=15.6±2.00 nM), whereas IAA6-containing co-receptor complexes provide fivefold lower affinity for auxin (Kd=72.0±10.5 nM) (right). (f) Homologous competition experiments were performed using 50 or 25 nM [3H]IAA for ASK1-TIR1-IAA6 or -IAA19, respectively. IC50s were obtained from curve fitting, and Ki values were calculated using the Cheng–Prusoff equation given the observed dissociations constants from saturation binding experiments. Error bars, s.d. (d) or minimum and maximum values (e,f) of three independent biological replicates. Asterisks denote significant statistical differences (P<0.001 (***), and P<0.0001 (****)) calculated using either two-tailed Student’s t-test (e,f), or one-way ANOVA (a,d) followed by Tukey’s honest significant difference test. ANOVA, analysis of variance.
