A synthetic approach reveals extensive tunability of auxin signaling

Abstract

Explaining how the small molecule auxin triggers diverse yet specific responses is a long-standing challenge in plant biology. An essential step in auxin response is the degradation of Auxin/Indole-3-Acetic Acid (Aux/IAA, referred to hereafter as IAA) repressor proteins through interaction with auxin receptors. To systematically characterize diversity in degradation behaviors among IAA|receptor pairs, we engineered auxin-induced degradation of plant IAA proteins in yeast (Saccharomyces cerevisiae). We found that IAA degradation dynamics vary widely, depending on which receptor is present, and are not encoded solely by the degron-containing domain II. To facilitate this and future studies, we identified a mathematical model able to quantitatively describe IAA degradation behavior in a single parameter. Together, our results demonstrate the remarkable tunability conferred by specific configurations of the auxin response pathway.

Figure 1.

IAA degradation is highly variable. A, Plant auxin receptors (TIR1 or AFB2) and YFP-tagged IAA repressors were integrated into the yeast ubiquitin pathway, shown in gray. B, Yeast cells were imaged while exposed to a square wave of auxin. Auxin leads to a rapid decrease in YFP (fluorescence of individual microcolonies in blue, average value in black), which can be recovered with auxin removal. C, A range of IAA|receptor degradation rates were obtained using time-lapse flow cytometry. Degradation curves were normalized to starting fluorescence. IAAs are listed in order of the relative difference in degradation in the presence of TIR1 versus AFB2. Strains expressing the F-box-deficient mTIR1 show no auxin-dependent degradation. [See online article for color version of this figure.]

Figure 2.

Degradation dynamics can be described using few parameters. A, Our model is described by two ordinary differential equations. Degradation curves for AFB2 strains expressing IAA1 or yeast codon-optimized IAA1.1 are shown. B, k₅ is largely independent of expression levels. IAA1 and IAA1.1 degradation curves overlap after normalization, although there is an approximately 2-fold difference in k₃ values. C, IAA|AFB2 pairs have increased degradation rates (k₅), a different rank order when compared with IAA/TIR1 pairs, and an increased dynamic range between the slowest and fastest pairs. Parameters were estimated for two independent replicates. All error bars represent 1 sd. Additional parameters are listed in Supplemental Tables S4 and S5. a.u., Arbitrary units. [See online article for color version of this figure.]

Figure 3.

Residues outside of domain II contribute to auxin-induced degradation rates. A, Schematic of IAA truncations. B and C, Degradation dynamics of full-length proteins are not maintained in truncations. B, Degradation rates of truncations expressed with TIR1 or AFB2, normalized to the starting fluorescence for each strain. C, Parameters k₃ and k₅ were determined using parameters k₁, k₂, and k₄ from previous model fitting. Additional parameters are listed in Supplemental Table S6.