Rate Motifs Tune Auxin/Indole-3-Acetic Acid Degradation Dynamics

Abstract

Ubiquitin-mediated protein degradation is a common feature in diverse plant cell signaling pathways; however, the factors that control the dynamics of regulated protein turnover are largely unknown. One of the best-characterized families of E3 ubiquitin ligases facilitates ubiquitination of auxin (aux)/indole-3-acetic acid (IAA) repressor proteins in the presence of auxin. Rates of auxin-induced degradation vary widely within the Aux/IAA family, and sequences outside of the characterized degron (the minimum region required for auxin-induced degradation) can accelerate or decelerate degradation. We have used synthetic auxin degradation assays in yeast (Saccharomyces cerevisiae) and in plants to characterize motifs flanking the degron that contribute to tuning the dynamics of Aux/IAA degradation. The presence of these rate motifs is conserved in phylogenetically distant members of the Arabidopsis (Arabidopsis thaliana) Aux/IAA family, as well as in their putative Brassica rapa orthologs. We found that rate motifs can act by enhancing interaction between repressors and the E3, but that this is not the only mechanism of action. Phenotypes of transgenic plants expressing a deletion in a rate motif in IAA28 resembled plants expressing degron mutations, underscoring the functional relevance of Aux/IAA degradation dynamics in regulating auxin responses.

Figure 1.

Sequences flanking the degron are required to recapitulate degradation rates of full-length (FL) Aux/IAAs. A, Schematic of the domain structure of a FL Aux/IAA indicating the location of domain I (repression), the conserved degron (d), and domains III/IV (dimerization). Colored bars indicate the size and position of peptide fragments tested for degradation dynamics in relation to FL protein (black). All peptide fragments and FL proteins were expressed as fusions with an N-terminal fluorescent protein (YFP or VENUS) and a C-terminal 2xSV40 nuclear localization sequence (NLS). B, Raw IAA17 degradation as captured by time course fluorescence flow cytometry. Diploid yeast strains coexpressing TIR1 and YFP-IAA17 fragments were treated with 10 μm auxin (filled circles) or mock (white circles) at time = 0 min. Data from two independent replicates are plotted together, and each point represents 1,000 to 3,000 individual events recorded in the cytometer. Error bars representing the sem are shown for each point, but in most cases the marker size is larger than the error. Bright-yellow circles are data from cells expressing YFP with no protein fusion, whereas dark-yellow circles are data from cells expressing an untagged Aux/IAA. Data were fit to a one-phase decay nonlinear regression with a band representing the 95% confidence intervals across the fit. C, Half-lives for all fusion proteins presented in B and D to F. For all data, half-lives with error were calculated from nonlinear regression fits such as those as shown in B. Error bars represent 95% confidence intervals. D to F, Short peptides (either NdC or dC*) of Arabidopsis IAA1 (D), IAA17 (E), and IAA28 (F) are able to degrade at least as rapidly as their respective FL proteins as assessed by time lapse fluorescence flow cytometry. To facilitate comparison of degradation dynamics, mean fluorescence values were normalized to starting fluorescence. All data represent two independent experiments. NdC and dC* fragments of putative B. rapa orthologs of IAA1 and IAA28 (insets) similarly recapitulated FL degradation dynamics.

Figure 2.

The impact of the N- and C-terminal rate motifs on degradation dynamics is Aux/IAA dependent. A, Colored bars indicate the size and position of Aux/IAA peptide fragments tested for degradation dynamics; colors are used consistently in all sections of this figure and in all following figures. Numbers indicate how many amino acids are found between conserved domains. B to D, Regions N- and C-terminal of the degron influence IAA1 (B) and IAA17 (C) degradation dynamics, whereas only C-terminal regions of the IAA28 (D) degron impact degradation. Auxin-induced degradation in the presence of TIR1 was captured as before; half-lives (inset) are presented with 95% confidence intervals.

Figure 3.

Specific residues in the N terminus contribute to degradation rate, whereas rate determinants in the C terminus are not necessarily sequence specific. A to C, Ala substitution (white circles) of the KR rate motif residues within the NdC fragment (dark-blue) in IAA17 (A) and IAA1 (B) slowed degradation similar to the dC fragment (light-blue), whereas Ala substitution of the KQ residues of IAA28 (C) had no effect on degradation dynamics. D, Replacing the IAA28 KQ with KR (white circles) did not affect half-life but promoted more complete degradation of the Nd fragment (orange). E, Shortening the distance between the IAA17 degron and the KR rate motif by either moving the KR forward 25 residues (diamonds) or deleting 25 residues (triangles) did not lead to faster degradation of IAA17.Nd. F, The C-terminal rate motif of IAA28 displayed no obvious sequence specificity. Ala substitutions and polar→nonpolar mutations (white symbols) of the nine residues immediately terminal of the degron in the IAA28.dC* fragment (bright-blue) had weak effects on degradation. G, Half-lives with 95% confidence intervals for data in A to F. WT, Wild type.

Figure 4.

Aux/IAA rate motifs are functional in plant roots. The C-terminal rate motif predictably influenced IAA28 degradation dynamics in plants. A, When expressed in Arabidopsis roots, an IAA28.Nd fragment (orange) lacking the C-terminal rate motif degrades more slowly in the presence of exogenous auxin than IAA28 fragments with a full (dC, light-blue) or partial (dC*, bright-blue) C-terminal rate motif. Root tips of 7-d-old Arabidopsis seedlings expressing heat shock-induced VENUS-tagged IAA28 reporters or VENUS alone (yellow) were treated with 5 μm auxin and imaged periodically for 1 h to capture degradation profiles. B, Quantification of fluorescence microscopy data from two replicates. Between one and five individuals representing two to four independent insertions were analyzed for each genotype. Mean fluorescence intensity values ±sem are plotted. Data were fit by one-phase decay nonlinear regression, and 95% confidence bands for the fit are shown. Auxin-induced degradation half-lives were calculated through nonlinear regression, and means were plotted with 95% confidence intervals (inset).

Figure 5.

Binding affinity between auxin repressor and receptor is not always correlated with degradation rate. Aux/IAAs with wild-type, mutant, or deleted rate motifs show no differences in ability to interact with TIR1. A, Aux/IAA peptide competition binding assays were used to assess strength of interaction with TIR1. Inhibitory concentration 50% (IC₅₀) values were measured by an AlphaScreen assay using purified TIR1 and IAA7.Nterm conjugated to donor and acceptor beads, respectively. Data shown represent one experiment with n = 6 and are typical of results obtained in additional experiments. B, Interaction between TIR1 and indicated Aux/IAA peptides was assessed by yeast two-hybrid assay in the presence of 10 μm auxin. WT, Wild type.

Figure 6.

Rate motif deletions slow degradation of FL IAA28 and result in phenotypes similar to stabilized IAA28 when expressed in plants. A, Degradation profiles of IAA28 variants were measured in yeast expressing either TIR1 or AFB2 auxin receptors. Partial deletion of the IAA28 C-terminal rate motif (ΔLTAQL, white triangles) in the context of FL IAA28 substantially slowed degradation when compared with the wild-type protein (black circles). As expected, a mutation in the degron (V55G, white squares) completely abolished auxin-induced degradation. Raw mean fluorescence values from two independent experiments are shown. B, Half-lives with 95% confidence intervals for data in A. Half-lives for IAA28.FL-V55G were not applicable (NA) as no auxin-induced degradation was observed. C, Plants expressing IAA28.FL-ΔLTAQL or IAA28.FL-V55G from the IAA28 promoter showed a range of phenotypes, including disruptions in phyllotaxy (gray arrows) and reduced fertility, when compared with plants expressing wild-type (WT) IAA28. Phenotypes of plants expressing IAA28.FL-V55G were more severe than those of plants expressing IAA28.FL-ΔLTAQL, consistent with a stronger effect of the respective mutations on auxin-induced degradation.