Decoding the Transcriptional Specificity of Auxin Signaling: A Synthetic Biology Approach

Abstract

Auxin is a central regulator of nearly all aspects of plant growth, development, and environmental response. Despite its broad roles, the mechanisms by which a single hormone elicits such diverse and context-specific transcriptional responses remain unclear. These responses are primarily mediated by the nuclear auxin signaling pathway, which consists of ARF transcriptional activators, Aux/IAA repressors, and TIR1/AFB auxin receptors that work together to modulate gene expression. Functional diversity within these modular components, such as differences in DNA binding, protein-protein interactions, subcellular localization, and co-expression patterns is believed to act in concert to tune auxin signaling specificity. In support of our efforts to decode auxin signaling specificity, we developed an open-source, R-based analysis pipeline to process flow cytometry data generated by protoplast transient expression assays. This reproducible workflow automates transformation-based reporter analysis, streamlining quantification of gene expression and effector function in high-throughput experiments. To understand the functional diversity in ARF-mediated transcriptional responses, we performed RNA sequencing of Arabidopsis protoplasts transiently expressing irrepressible ARF variants to determine if they have distinct downstream regulatory targets. Transcriptomic analysis revealed that while each ARF appears to regulate largely overlapping sets of genes, they do so with varying efficiencies. Gene clusters preferentially regulated by specific ARFs were associated with distinct biological processes, aligning with the known developmental roles of those specific ARFs. While our transcriptomic analysis suggested that ARFs can drive distinct transcriptional outputs independently of Aux/IAAs, the extent to which specific ARF-Aux/IAA interactions contribute to this specificity remained unclear. To determine whether specific ARF-Aux/IAA interactions also contribute to transcriptional specificity, we developed a synthetic system that isolates these interactions using a recombinant protein-interaction domain from an animal ortholog. The system was validated through structural prediction, yeast two-hybrid assays, and protoplast transient expression assays. This system serves as the closest approximation to date of native ARF-Aux/IAA interactions, providing a powerful tool to dissect signaling specificity at the level of individual protein pairings. Together, the development of a streamlined data analysis pipeline, transcriptional profiling, and a synthetic interaction system provides both computational and experimental tools that advance our understanding of auxin signaling specificity. Ultimately, these insights will help explain how a single hormone can direct a vast array of developmental and environmental responses in plants.