Mutational analyses of an instability domain reveal its conserved role in the regulation of class-B ARF levels in <i>Arabidopsis</i>

Abstract

The plant hormone auxin is a central regulator of plant growth and development. The canonical nuclear auxin signaling pathway acts through the regulation of gene transcription, and the protein levels of core component AUXIN RESPONSE FACTORS (ARFs) are key to this regulation. Here, we investigate the physiological role of a conserved instability (INS) domain in two class-B ARFs, ARF2 and ARF3, in Arabidopsis thaliana . Using native-promoter transgenic lines expressing different ARF2 variants, we show that T298N, T298D, and T298E mutant versions of the INS domain cause pronounced auxin-related phenotypes, including male sterility and defective root hair development. Notably, the T298D and T298E ARF2 variants, which exhibit increased protein stability, persist in epidermal nuclei within the root differentiation zone and are associated with shorter, branched root hairs, whereas wild-type ARF2 levels decline in this region coincident with root hair elongation. These lines also display auxin-resistant primary root elongation and lateral root promotion, along with reduced DR5:Luciferase responses, correlating with impaired ubiquitylation and enhanced ARF2 stability. Similarly, stabilized ARF3 variants (S293E and S293N) exhibit auxin resistance and severe developmental defects, indicating a shared regulatory mechanism among class-B ARFs. Our findings highlight critical physiological roles for class-B ARFs and verified the conserved function of the instability domain in controlling protein stability in Arabidopsis . We also reveal that INS domain-mediated turnover of ARF2 restricts their spatial accumulation and is essential for root hair elongation, providing insight into how signaling specificity is achieved through posttranslational control of ARF activity.