A combinatorial TIR1/AFB-Aux/IAA co-receptor system for differential sensing of auxin

Abstract

The plant hormone auxin regulates virtually every aspect of plant growth and development. Auxin acts by binding the F-box protein transport inhibitor response 1 (TIR1) and promotes the degradation of the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) transcriptional repressors. Here we show that efficient auxin binding requires assembly of an auxin co-receptor complex consisting of TIR1 and an Aux/IAA protein. Heterologous experiments in yeast and quantitative IAA binding assays using purified proteins showed that different combinations of TIR1 and Aux/IAA proteins form co-receptor complexes with a wide range of auxin-binding affinities. Auxin affinity seems to be largely determined by the Aux/IAA. As there are 6 TIR1/AUXIN SIGNALING F-BOX proteins (AFBs) and 29 Aux/IAA proteins in Arabidopsis thaliana, combinatorial interactions may result in many co-receptors with distinct auxin-sensing properties. We also demonstrate that the AFB5-Aux/IAA co-receptor selectively binds the auxinic herbicide picloram. This co-receptor system broadens the effective concentration range of the hormone and may contribute to the complexity of auxin response.

Figure 1. The Auxin Receptor Is a Co-Receptor System

a.In vitro binding of 200 nM [³H] IAA to recombinantly expressed TIR1 and/or IAA7 full-length or a peptide corresponding to the DII, degron motif. Together, the TIR1-IAA7 pair constitutes an auxin co-receptor. A mutation that mimics a gain of function allele in the degron of IAA7 (IAA7^axr2-1) abolishes auxin binding. B. and c. Saturation binding experiments of [³H] IAA to b. TIR1-IAA7 and c. TIR1-DI-DII (left) and TIR1-IAA7 DII co-receptor complexes (right). b. TIR1-IAA7 constitutes a high-affinity auxin co-receptor with a K_D in the low nanomolar range. c. TIR1-DI-DII but not TIR1-IAA7 DII binds auxin with high affinity. d. [³H]-IAA binding at 200 nM to TIR1-IAA7 compared to TIR1-IAA7 K35Q and TIR1-IAA7 K35Q R36Q. Values are determined as mean ± SEM of either two or three independent experiments carried out in triplicate.

Figure 2. Differences in Auxin Dependent TIR1/AFB-Aux/IAA Interaction Are Not Exclusively Determined by the Degron Domain

a. Yeast-two hybrid interaction experiments of TIR1, AFB1, AFB2 and AFB5 with IAA3, IAA5, IAA7, IAA8, IAA12, IAA20, IAA28, IAA29, IAA31, which represent the different subclades of Arabidopsis Aux/IAAs. Diploids containing LexA DBD-TIR1/AFBs and ADAux/IAAs were generated and spotted in selective media (Gal/Raff -Ura-His-Trp + X-Gal) containing increasing concentrations of IAA. β-galactosidase reporter expression evidenced IAA-induced protein-protein interactions 4 days after spotting. b. Aux/IAA proteins with a very similar DII domain interact differentially with TIR1/AFBs suggesting that regions outside of DII contribute to binding. IAA7 DII depicted as stick (yellow), with conserved tryptophan and second proline residues, which interact with the surrounding hydrophobic wall in the TIR1 pocket and stack against the auxin molecule lying underneath. DII sequences of the selected Aux/IAAs are shown for comparison (right).

Figure 3. Aux/IAA Proteins Determine the Affinity of the Co-receptor Complex for Auxin and, Together with TIR1, Form a Series of Co-Receptor Complexes With a Range of Auxin Sensing Properties

Homologous competitive binding experiments for a. TIR1-IAA14, b. TIR1-IAA1 and c. TIR1-IAA28 co-receptor complexes. Specific binding of a constant concentration of [³H] IAA by the different co-receptors in the presence of various concentrations of unlabeled IAA was measured. IC50 values were determined using appropriate concentrations (5, 10, 25 or 100 nM) of radiolabeled auxin, so that the concentration of hot IAA was less than half the IC50. K_D was then calculated as the IC₅₀- [[³H]-IAA] fitted to a built-in equation of one-site competition. d. Saturation binding of the TIR1-IAA12 co-receptor complex, which exhibits low auxin binding affinity in the high nanomolar range (left). The binding affinity of the TIR1-IAA12 co-receptor for IAA increases when the canonical degron motif GWPPVR is incorporated in IAA12 (right). e. Mutated versions of Aux/IAA proteins that mimic the stabilized versions of the proteins (solitary root (slr-1) and bodenlos (bdl), abolish specific auxin binding by the different TIR1-Aux/IAA co-receptor complexes. Binding of [3H] IAA (200 nM) to recombinant TIR1-ASK1 and IAA14, IAA12, slr and bdl, where error bars correspond to SEM of four replicates.

Figure 4. Mutations at Auxin, Aux/IAA and InsP₆ Binding Sites Impair Auxin-Dependent TIR1-Aux/IAA Interaction And Compromise TIR1 Function In Vivo

a. Side view of auxin (green) and IAA7 peptide (yellow) (left), auxin and InsP₆ (rainbow) (middle), and InsP₆ (right) binding sites in TIR1 (grey). Selected ligand-interacting residues in TIR1 are shown in mostly white stick representation. b. Yeast-two hybrid ASK1, and auxin-induced IAA7 interactions with wild type (WT) TIR1 or TIR1 carrying mutations on ligand-binding sites. c. Five-day-old seedlings grown on MS media were transferred to media containing either 40 or 80 nM 2,4-D. Root elongation was measured after additional 4 days and expressed as a proportion of growth in the absence of auxin (scale bars represent STDEV). Auxin inhibits root growth elongation in wild type plants (Col-0) but tir1-1, as well as the tir1-1afb2–3 auxin-receptor mutants are resistant to auxin treatment. Auxin resistance in tir1-1 mutants is reverted by introducing TIR1 wild type fused to the β-glucuronidase (GUS) reporter gene, under the TIR1 promoter. Interrupted lines indicate that unlike TIR1p:TIR1:GUS, versions of TIR1 that carry mutations in ligand binding sites are unable to restore auxin sensitivity in tir1-1.

Figure 5. Auxin Agonists Differentially Stabilize TIR1-Aux/IAA Complexes

a. IAA, 1-NAA, and 2,4-D enhance TIR1-IAA7 interaction in yeast. Diploids carrying LexA DBD-TIR1 and AD-IAA7 were spotted on selective media containing 50 μM auxins. β-galactosidase expression is a measure of TIR1-IAA7 interaction. b. Surface Plasmon Resonance analysis of auxin-dependent TIR1-IAA7 DII interaction. Sensorgram shows the effect of no auxin (brown) and various auxinic compounds (500 μM IAA (dark blue), 50 μM IAA (light green), 50 μM 1-NAA (light blue), 50 μM 2,4-D (purple), 50 μM picloram (pink), 50 μM tryptophan (orange)) on TIR1-DII peptide association and dissociation. Auxins were mixed with TIR1 in solution prior to injection over DII peptide. c and d. Competitive [³H] IAA binding assays using 1-NAA and 2,4-D cold competitors. TIR1-IAA7 co-receptor complex binds 1-NAA and 2,4-D with a K_i (inhibition constant) of 113.50 ± 3.51 nM and > 1 μM, respectively.

Figure 6. TIR1-IAA7 And AFB5-IAA7 Co-Receptor Complexes Exhibit Differential Auxin Binding Affinities

a. Heterologous competition binding experiments of TIR1-IAA7 and AFB5-IAA7 co-receptor complexes. Picloram displaces efficiently [³H] IAA binding by AFB5-IAA7, but not by the TIR1-IAA7 complex. b–c. 3D-structures of TIR1 and AFB5 show an almost identical fold with regard to secondary structure arrangements. b. Docking arrangements of IAA to TIR1 (left) and AFB5 (right) and c. of picloram to TIR1 (left) and AFB5 (right). IAA and picloram are stabilized in the TIR1 auxin binding pocket by hydrogen bonds to Arg403 (b and c left). IAA and picloram form strong hydrogen bonds (salt bridges) to Arg123 and Arg449 and to the backbone carbonyl group of Val485 in AFB5 (b and c (right)). b. Picloram lacks the hydrogen bond to the backbone carbonyl group of Leu404 in TIR1. The replacement of Arg123 (in AFB5) by His78 (in TIR1) is likely responsible for the reduced affinity of picloram to TIR1 compared to AFB5. c.