Auxin Response Factor Degradation: Understanding Hormone Regulation in Plants

Abstract

The regulated degradation of Auxin Response Factors (ARFs) is central to the dynamic control of auxin signaling, a hormone pathway critical for plant development. However, the specific degron sequences that signal ARF proteins for degradation remain poorly defined. This thesis focuses on identifying the degron region within ARF2, a transcriptional repressor and key regulator of auxin responses in Arabidopsis thaliana.To localize the degron, the ARF2 gene was divided into 28 overlapping segments, each cloned into a dual-fluorescence vector containing mNeonGreen and mScarlet. These constructs were transformed into protoplasts (plant cells with a dissolved cell wall), and fluorescence ratios were analyzed to infer degradation activity. Due to inconclusive results from the segmented approach, a second strategy was employed that split ARF2 into three functional regions: the DNA-binding domain (DBD), the intrinsically disordered middle region (MR), and the PB1 domain. Fluorescence assays were used to compare stability across these larger domains, supported by colony PCR and sequencing for construct validation. Results indicated region-specific differences in degradation likelihood and demonstrated that the protoplast assay system is sensitive to degradation-dependent fluorescence changes. These findings lay a methodological foundation for more refined biochemical and genetic studies. Future work could apply targeted mutagenesis, hormone treatments, and protein-interaction analyses to test hypotheses about signal-induced degron exposure, interaction-based stabilization, and post-translational modification. This study establishes a tractable system for probing the post-translational regulation of ARFs and proposes a framework for continued investigation into the molecular logic of auxin signaling.