In a groundbreaking study that reshapes our understanding of plant hormone signaling, researchers have uncovered a conserved molecular mechanism governing the stability and degradation of AUXIN RESPONSE FACTOR (ARF) proteins across diverse land plants. This discovery not only elucidates long-standing enigmas surrounding ARF regulation but also paves new avenues for agricultural innovation by manipulating key transcriptional regulators of auxin signaling pathways.
Auxin, a phytohormone central to plant growth and development, exerts its effects through the activity of ARFs—transcription factors that orchestrate gene expression in response to auxin stimuli. Despite their crucial role, the post-translational regulation of ARFs has remained murky, particularly the mechanisms underlying their stability and turnover. In this illuminating study, Prigge et al. employed a comparative mutant analysis involving maize (Zea mays), the moss Physcomitrium patens, and Arabidopsis thaliana to reveal a novel, evolutionarily conserved pathway that targets class-B ARF proteins for proteasome-dependent degradation.
The investigators focused on class-B ARFs—an important subclass with distinct DNA-binding and regulatory functions—investigating how mutations in different plant species impact the abundance of these proteins. Initial observations in maize and moss mutants indicated increased accumulation of ZmARF28 and PpARFb proteins, respectively. Intriguingly, despite no significant changes at the transcript level, these mutant lines exhibited hyperaccumulation of the corresponding ARF proteins, suggesting post-translational stabilization.
To meticulously quantify and visualize protein abundance and distribution, the researchers employed advanced live-cell imaging techniques, tagging the endogenous PpARFb loci with the mYPet yellow fluorescent protein (YFP). This innovative approach enabled direct measurement of protein levels in wild-type and mutant moss lines. Confocal microscopy revealed a dramatic intensification of YFP fluorescence in nuclei of mutant plants compared to sporadic, weak signals in wild-type counterparts, affirming that the mutations conferred increased ARF protein stability without altering subcellular localization.
Recognizing that protein turnover often involves targeted degradation by the ubiquitin-proteasome system, Prigge et al. investigated whether class-B ARFs undergo proteasomal degradation. Wild-type moss lines treated with the proteasome inhibitor bortezomib showed significant elevation in fluorescence levels, providing compelling evidence that PpARFb proteins are normally subjected to degradation via the 26S proteasome. This key finding complemented the mutant phenotype data and confirmed the regulatory significance of ARF protein stability in controlling auxin-mediated responses.
Extending their analysis to maize, the team developed a specific antibody against ZmARF28 to evaluate protein accumulation in mutant lines. Western blot assays demonstrated a statistically significant increase in ZmARF28 abundance in Trf mutant seedlings compared to normal siblings. These results underscored that the maize Trf mutations lead to ARF protein stabilization in vivo, corroborating observations in moss and supporting the hypothesis of a conserved regulatory mechanism.
To probe the dynamics of proteasome-mediated degradation in a heterologous system, wild-type and Trf-mutant versions of ZmARF28 fused to green fluorescent protein (GFP) were transiently expressed in Nicotiana benthamiana leaves and maize protoplasts treated with MG132, another proteasome inhibitor. Immunoblotting revealed accumulation of ZmARF28-GFP upon proteasome inhibition, especially pronounced in the wild-type samples, indicating that both wild-type and mutant proteins are subject to degradation pathways, albeit with altered kinetics or efficiency.
Remarkably, the study highlighted a critical domain within class-B ARFs that is essential for their degradation. Mutations clustered in this specific region rendered the proteins more stable, indicating that it functions as a degron—a peptide motif recognized by the ubiquitin machinery. The conservation of this domain was substantiated by experiments in Arabidopsis, wherein orthologous AtARF2 and its mutant variant harboring the corresponding Trf mutation exhibited significant differences in protein half-life.
Using ratiometric reporter assays in Arabidopsis mesophyll protoplasts, the team observed that AtARF2 displayed a half-life of approximately four hours, consistent with prior studies on ARF stability. In contrast, the Trf-mutant AtARF2 (arf2^T298N) showed markedly delayed degradation, retaining 77% of its initial protein levels after four hours of cycloheximide treatment, which inhibits new protein synthesis. These results confirm that the Trf mutation prolongs protein residence time by hampering degradation rather than enhancing synthesis, underscoring the functional importance of this conserved domain in regulating ARF turnover.
To directly investigate the involvement of ubiquitylation—a prerequisite for proteasomal targeting—the researchers performed immunoprecipitation of the DNA-binding domain (DBD) of PpARFb2 fused to YFP from proteasome-inhibited moss lines. Western analysis with anti-ubiquitin antibodies revealed robust polyubiquitylation of the wild-type DBD but a stark absence in the mutant E266K/R269Q double mutant, establishing that the identified mutations impair ubiquitylation and subsequent degradation.
Collectively, these multidimensional data demonstrate that dominant mutations in class-B ARF proteins disrupt a regulatory domain critical for their ubiquitylation and proteasomal degradation. This degradation pathway is conserved across land plants from bryophytes to angiosperms, highlighting an ancient and universal mechanism modulating auxin response through fine-tuning ARF protein stability.
The implications of this discovery extend beyond fundamental plant biology. By elucidating a key proteolytic regulation node of ARF transcription factors, it opens new strategies to engineer plants with tailored growth and developmental profiles. Modulating ARF degradation could selectively alter auxin signaling outputs, potentially leading to crops with enhanced stress resilience, optimized architecture, or improved yield.
Moreover, the innovative use of fluorescent protein knock-ins and proteasome inhibitors in moss and crop species illustrates a powerful methodological framework to dissect dynamic protein regulation in vivo. This cross-species approach elegantly bridges evolutionary biology and molecular genetics, reinforcing the universality of proteasome-mediated control in plant signaling networks.
While the authors focus primarily on class-B ARFs, their findings raise intriguing questions about whether similar post-translational regulatory mechanisms operate across other ARF classes or transcription factor families. Future studies will no doubt explore the broader landscape of protein turnover in hormone signaling pathways, potentially revealing new degrons and ubiquitin ligases involved.
Furthermore, the identification of a distinct degron motif governing ARF stability invites detailed biochemical and structural characterization, which could enable the development of small molecules to modulate ARF degradation artificially. Such tools would be invaluable for precision agriculture and fundamental research alike.
In conclusion, the research by Prigge and colleagues represents a significant leap forward in our comprehension of auxin signaling regulation. By uncovering a conserved degron domain essential for ARF degradation and demonstrating its evolutionary conservation and functional importance, this study illuminates an underappreciated layer of transcriptional control. It heralds a paradigm shift in understanding how plants dynamically regulate developmental signals, with promising translational prospects.
This seminal work not only solves fundamental puzzles about ARF protein homeostasis but also exemplifies the power of integrative plant molecular biology, combining genetics, live-cell imaging, proteomics, and cross-species analyses to reveal hidden universality in cellular control mechanisms. As plant scientists continue to unravel the exquisite regulation of hormone-responsive transcription factors, insights gleaned here will resonate across fields ranging from evolutionary biology to crop improvement.
Subject of Research: Regulation of AUXIN RESPONSE FACTOR (ARF) protein stability and degradation mechanisms in land plants.
Article Title: Comparative mutant analyses reveal a novel mechanism of ARF regulation in land plants.
Article References:
Prigge, M.J., Morffy, N., de Neve, A. et al. Comparative mutant analyses reveal a novel mechanism of ARF regulation in land plants. Nat. Plants 11, 821–835 (2025). https://doi.org/10.1038/s41477-025-01973-3
Image Credits: AI Generated
