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.

Recapitulation of the forward nuclear auxin response pathway in yeast

“…A freshly grown colony for each strain was inoculated in SC and the cell density (in events per microliter) was estimated using cytometry data gated for yeast by a custom R script (27). Each culture was then diluted to 0.25 events per microliter in 12 mL of SC.…”

“…Fluorescence measurements were taken with a BD Accuri C6 flow cytometer and CSampler plate adapter using an excitation wavelength of 488 and an emission detection filter at 533 nm. A total of 10,000 events above a 400,000 FSC-H threshold (to exclude debris) were measured for each sample and data exported as FCS 3.0 files for processing using the flowCore R software package and custom R scripts (27).…”

“…A library of IAAs was cloned downstream of a GPD promoter in TRP1 integration vectors using a standard Gateway LR reaction (LRClonase II; Life Technologies) and transformed into W814-29B MATa strains (YKL381) containing pGP5G-AFB2 [AFB2 integration at LEU2, expression from the GPD promoter (27)]. ARFs were amplified from Arabidopsis cDNA and subcloned into the Gateway pDONR221 plasmid using a standard Gateway BP reaction (BP Clonase II; Life Technologies).…”

“…S5 and Table S2). TRP1 targeting single integrating Gateway vectors (27) were further modified to contain an N-terminal TPL repression domain (RD) for auxin response assays, or an N-terminal YFP-RD tag to monitor the degradation of RD-IAA fusions. Repression domains TPL-N100 and TPL-N300 were amplified from A. thaliana Columbia ecotype cDNA.…”

“…To quantify the differences in the behavior of ARC Sc IAA variants, we developed a small model that expands upon the model used to quantify our degradation system (27). The present model captures the overall flow of information while avoiding reference to specific molecules and kinetic parameters that cannot be measured and limiting the potential for overfitting (28,29).…”