AUXIN RESPONSE FACTOR Protein Accumulation and Function

Abstract

Auxin is a key regulator of plant developmental processes. Its effects on transcription are mediated by the AUXIN RESPONSE FACTOR (ARF) family of transcription factors. ARFs tightly control specific auxin responses necessary for proper plant growth and development. Recent research has revealed that regulated ARF protein accumulation and ARF nucleo-cytoplasmic partitioning can determine auxin transcriptional outputs. In this review, we explore these recent findings and consider the potential for regulated ARF accumulation in driving auxin responses in plants.

Graphical Abstract

Auxin is important for plant growth and development. The AUXIN RESPONSE FACTOR (ARF) family of transcription factors controls auxin transcriptional output. Post-translational modifications and regulated degradation control ARF activity and accumulation to regulate auxin response and plant growth.

1. Introduction

The phytohormone auxin plays pivotal roles in nearly every plant developmental process, driving cell expansion and differentiation, organogenesis, and tissue development.^[1–4] Over the past two decades, the predominant nuclear auxin signaling pathway was established as the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX PROTEIN (TIR1/AFB) mediated pathway.^[1–5] This pathway involves three major components: the SCF^TIR1/AFB (Skp1-Cullin1-F-box protein) receptor complex, Auxin/INDOLE-3-ACETIC ACID (Aux/IAA) repressor proteins, and AUXIN RESPONSE FACTOR (ARF) transcription factors. In its simplest model, Aux/IAA proteins directly repress ARF transcriptional activity under low auxin concentrations. Elevated auxin levels promote complex formation of SCF^TIR1/AFB and Aux/IAA; auxin acts as a molecular glue to stimulate this association. SCF^TIR1-auxin-Aux/IAA co-receptor formation allows for polyubiquitylation of Aux/IAA proteins to target their degradation through the 26S proteasome. This Aux/IAA protein degradation relieves ARF repression, allowing ARF regulation of auxin-responsive genes.^[1–5] Additional layers of complexity govern this system, as discussed in this review, with a particular focus on emerging roles for ARF protein degradation in regulating auxin responses.

ARFs tightly control auxin transcriptional responses by binding to auxin response elements in promoter regions and either activating or repressing target genes. ARFs fall into three deeply conserved evolutionary classes: A, B and C^[6–8] (Figure 1A). Class-A includes the activator ARFs 5-8 and ARF19. The Class-C ARFs10/16/17 and the Class-B, which encompasses the remaining ARFs, likely function as repressors. The precursors of Class-C ARFs (proto-C-ARFs) exist in charophyte algae, which divided into two classes (A/B and C) in the late-divergent charophyte.^[8–10] A further division of Class-A/B into Class-A and Class-B took place in land plants, giving rise to the three evolutionary classes description today.^[8–10] Class-A ARFs further split into ARF5/7/19 and ARF6/8 in the euphyllophytes, and Class-B ARFs divided into ARF3/4, ARF2, and the remaining Class B-ARFs in the gymnosperms. Key roles of ARF proteins in regulation of developmental processes have been revealed by genetic analysis of the mutants. For example, ARF2 regulates cell division and seed size,^[11] ARF5 affects embryo formation and vascular development,^[12] ARF6 and ARF8 shape floral organ growth,^[13] and ARF7 and ARF19 control leaf cell expansion and lateral root formation.^[6] Phenotypic analyses of arf mutants indicates the importance of ARF proteins in plant growth and development.

Figure 1.

ARF Domains. (A) Cartoon representations of the domain organization of the three classes of ARFs, with DNA-binding domain (DBD), middle region (MR) and PB1 domains indicated. (B) Structure of the ARF1 dimerized DNA-binding domain (DBD). The left ARF1 DBD monomer is shown as a ribbon diagram whereas the right monomer is shown as a surface model. Each ARF1 DBD monomer is composed of three distinct regions: the B3 domain (pink), the dimerization domain (DD, blue), and the ancillary domain (AD, yellow). (C) ARF7 PB1 domain interactions are driven by the conserved residues on opposing positive (+) and negative (−) interaction surfaces. Detail of the ARF7 dimer interaction surface shows the conserved positive K1042 and R1051 (purple) and the negative D1092, E1094, D1096, and D1102 (red) residues that participate in charge–charge interactions.

The ubiquitin-proteasome system (UPS) has a vital role in protein stability regulation. The UPS is generally thought to consist of ubiquitin-activating (E1), ubiquitin-conjugating (E2) and ubiquitin-protein ligase (E3) enzymes. The activating enzyme (E1) activates Ub in an adenosine triphosphate (ATP)-dependent manner to form a thioester bond between a catalytic cysteine on the E1 and the C-terminus of a Ub protein. Then, the E1 transfers Ub to the conjugation enzyme E2 via trans-thioesterification. The E3 ligase simultaneous interaction with a Ub-loaded E2 enzyme and a substrate, facilitating iso-peptide bond formation between the lysine of the substrate and Ub. As interactors with target substrates, E3 ligases are critical UPS components and display the most diversity and specificity in the UPS system.^[14–17] Over 1500 E3 ubiquitin ligases and are encoded in the Arabidopsis genome.^[18] Among the various types of E3 ligases, the SCF-type is one of the best-characterized and extensively-studied in plants. The SCF-type E3 ligase is comprised of an ASK1, Cullin1(Cul1), RING-box protein1(RBX1), and an F-box protein.^[19] The F-box protein recruits the substrate recognition and determine the specificity of target protein degradation. Over 700 F-box proteins have been discovered in Arabidopsis and many of them are involved in numerous plant growth processes.^[18] In particular, SCF E3 ligases control cellular proteostasis to regulate multiple phytohormone responses.

Similar to Aux/IAA proteins, ARF protein accumulation has been reported to be regulated by the 26S proteasome, including the Class-A (transcriptional activator) ARF6,^[20] ARF7,^[21] ARF8,^[20] and ARF19,^[21] the Class-B (transcriptional repressor) ARF1^[22] and ARF2^[23], and Class-C ARF17.^[20] Class-A ARFs are auxin-sensitive transcriptional regulators and can switch from ARF-Aux/IAA repression to activation in an auxin-dependent manner.^[24] Class-B ARFs function independently of auxin and antagonize Class-A ARFs by competing for target sites and, at least in some cases, by recruiting the TOPLESS (TPL) corepressor.^[24] The molecular underpinnings of the regulation of ARF protein accumulation have been largely ignored until recently. The latest discoveries of an F-box that regulates Class-A ARF accumulation,^[21] discovery of regulated ARF nuclear import,^[21] ARF nucleo-cytoplasmic partitioning,^[25] ARF SUMOylation,^[26,27] and ARF phosphorylation^[28–30] have resulted in new insight into post-translational regulation of ARF activity. Here, we review recent advances and discuss the possibilities for regulated ARF accumulation in contributing to auxin response and plant growth.

2. ARF domains

Most ARF proteins contain three regions, consisting of an N-terminal B3-type DNA binding domain (DBD), followed by an intrinsically disordered variable middle region (MR) that functions as either a transcriptional activator or repressor, and a C-terminal type I/II Phox and Bem1 (PB1) protein-protein interaction domain^[31–33] (Figure 1). Each of these regions provides a distinct function to the ARF transcription factor.

2.1. DNA binding domain

The N-terminal ARF DNA binding domain consists of three distinct subdomains: a composite dimerization domain (DD), a B3-type DNA-interaction domain, and an ancillary domain (AD) of unknown function.^[31] The ARF DBD binds the auxin-responsive element (AuxRE) TGTCNN, which displays sequence diversity and ARF specificity to affect auxin responsiveness.^[34] The specific AuxRE sequence, the spacing between AuxREs, and the AuxRE orientation are all important parameters for ARF binding specificity.^[31,35] Structural studies show that the DBD of ARF1 and ARF5 form homodimers through their DD, mediated by hydrophobic interactions^[31,36] (Figure 1B). The DBD provides sequence-specific target recognition and ARF binding to the promoters of auxin response genes.

2.2. The middle region

The ARF intrinsically disordered middle region (MR) displays divergent amino acid composition and low sequence complexity. The MRs from Class-A ARFs are rich in glutamine residues whereas the MRs in Class-B and C ARFs are enriched in serine, proline, and threonine residues.^[37,38] Class-A ARF MRs consist of expanded regions of intrinsic disorder that are hydrophobic and are predicted prion-like domains,^[25] whereas the Class-B/C ARF MRs are typically shorter and lack prion-like domains.^[39] ARF MRs play key roles in ARF function.^[25,40,41] Importantly, ARF transcriptional activation (Class-A) and repression (Class-B) activity is encoded in the MR.^[40,41] In addition, the intrinsically disordered MRs in ARF7 and ARF19 are necessary for condensate formation, which acts to attenuate their activity.^[25] Further analysis of ARF IDR functions may uncover additional roles for these extended regions.

2.3. The C-terminal PB1 domain

The C-terminal ARF PB1 domain (previously named DIII/IV) mediates ARF-ARF and ARF-Aux/IAA interactions. ARF and Aux/IAA PB1 domains exhibit opposing negative and positive faces, which allow head-to-tail PB1-PB1 interactions via electrostatic interactions and hydrogen bonds.^{[32,33,42,43]} Structural and biochemical studies revealed that ARF and Aux/IAA PB1 domains multimerize (Figure 1C). Class-A ARF PB1 interactions with Aux/IAA PB1s are stronger than ARF PB1 or Aux/IAA PB1 homotypic interactions,^[42,44,45] at least with the examined examples. Interactions between Aux/IAAs and Class-B/C ARF-PB1s appear to be limited.^[41,46] Both ARF-Aux/IAA PB1-PB1 heterotypic interactions and ARF-Aux/IAA PB1-PB1 homotypic interactions are important for auxin response and plant growth.^[40,41] Thus, the C-terminal PB1 domain interactions play a central role in regulation of ARF activity.

3. Mechanisms of ARF degradation

Protein degradation plays fundamental regulatory roles and essential housekeeping functions.^[47] Protein degradation also regulates protein fate in response to specific intrinsic and extrinsic cues to regulate signaling events. Depending on the specific roles of the degraded protein, the consequences of target protein degradation are diverse.^[48] Thus, protein degradation pathways, such as the UPS, must be tightly regulated to ensure proper protein homeostasis and to maintain correct cellular function. SCF E3 ubiquitin ligase complex-targeted protein degradation plays important roles in many biological processes, including degradation of ARF proteins. Here, we discuss possible mechanisms of ARF protein degradation (Figure 2).

Figure 2.

Regulation of ARF protein accumulation and nucleo-cytoplasmic partitioning by ubiquitylation, SUMOylation, and phosphorylation. (A) SCF^AFF1 directly interacts with ARF7 and ARF19, and perhaps additional ARFs, to promote ARF degradation and/or nuclear import, likely by promoting distinct ubiquitin moieties to regulate ARF degradation and/or nuclear import. Several possible biological functions for ARF ubiquitylation include: 1) removal of unnecessary ARF protein; 2) regulation of the Class-A and B/C ARF competition; and 3) Environmental responses. (B). SUMOylation of ARF7 and MdARF8 regulate lateral root development. (C) Phosphorylation of ARF2 and SlARF4 block their DNA-binding activity; however, phosphorylation of ARF7 and ARF19 increase the DNA-binding capacity.

3.1. SCF^AFF1 regulates ARF accumulation

Despite multiple reports of ARF protein accumulation regulated by the 26S proteasome,^{[20,22,23,49]} the molecular mechanism regulating this process has remained unknown. Recently, a fluorescence-based forward genetics screen for altered YFP-ARF19 accumulation uncovered a mutant defective in an F-Box protein, which was named AUXIN RESPONSE FACTOR F-BOX1 (AFF1)^[21], that regulates accumulation of the Class-A ARF7 and ARF19. The aff1 mutant displays elevated ARF7 and ARF19 accumulation. Direct interaction of AFF1 with ARF7 and ARF19, the ability of heterologously-expressed AFF1 to promote ARF7 and ARF19 degradation, and the mutant phenotypes lead to a model in which SCF^AFF1 regulates ARF7 and ARF19 accumulation.^[21] Determining whether SCF^AFF1 facilitates the degradation of additional ARFs and/or whether alternative E3 ubiquitin ligases regulate ARF stability will help uncover the mechanisms regulating the complexities of ARF protein accumulation (see section 4).

3.2. Other potential mechanisms to regulate ARF accumulation

ARF7 and ARF19 are two Class-A ARFs that directly interact with SCF^AFF1.^[21] Class-A ARF5 and ARF8 also undergo proteasome-dependent degradation.^[20] Whether ARF5 and ARF8 are also SCF^AFF1 substrates remains unknown. However, it is possible that SCF^AFF1 targets ARFs in addition to ARF7 and ARF19. It seems likely that distinct E3 ligases could differentially regulate the stability of distinct ARFs to aid in specificity of degradation. Indeed, degradation of the Class-B ARF1 is proteasome-dependent, but not dependent on CUL1 (the backbone of SCF complexes),^[22] suggesting that ARF1 degradation is via machinery distinct from SCF^AFF1. In addition to ARF1, the Class-B ARF2^[23] and Class-C ARF17^[20] also undergo proteasome-dependent degradation. Whether Class-B/C ARF stabilities are also regulated through SCF^AFF1 activities or through some other mechanism remains unknown. Additionally, treatment with a proteasome inhibitor results in increased ARF19 protein accumulation in the aff1 mutant,^[21] suggesting additional proteasomal mechanisms exist to contribute to ARF19 stability. Moreover, ARF7 protein accumulation is also regulated by the autophagy pathway.^[50] Finally, the deep evolutionary history of the ARFs, which have existed in three distinct clades early in the plant lineage,^[6–8] raises the possibility that distinct mechanisms could have arisen to differentially regulate members of the three classes of ARF. Taken together, evidence suggests that multiple systems regulate ARF protein accumulation.

Degrons are the substrate recognition sequences identified by E3 ubiquitin ligases. Truncated ARF proteins that lack the DNA bind domain (ARF1)^[22] or the PB1 domain (ARF17)^[20] are still degraded in a proteasome-dependent manner, raising the possibility that the intrinsically disordered middle region might contain the ARF degron. More experiments will be required to determine the extent of various mechanisms regulating ARF stability and the molecular determinants underlying this system.

4. Scenarios for ARF degradation

Regulated protein accumulation is essential to plant signaling processes. Directed protein degradation drives part of this regulation and helps define the half-life of each polypeptide. The degradation of a protein, which is an inherently irreversible process, terminates the process controlled by that protein. Recent studies indicate that ARF protein homeostasis plays important roles in regulation of plant growth and in environmental responses. Here, we discuss possible biological functions for ARF transcription factor degradation (Figure 2).

4.1. Removal of unnecessary or damaged ARF protein

Regulated ARF degradation suggests a possible important point of regulation for auxin-responsive gene transcription. The half-life of ARF1 protein is approximately 3-4 h.^[22] Although the protein half-life was not calculated, ARF2 protein abundance is decreased by ethylene^[23] and enhanced by gibberellin (GA)^[51] treatment. Recently, ARF6,^[20] ARF7,^[21] ARF8,^[20] ARF17,^[20] ARF19,^[21] MpARF1,^[52] and MpARF2^[52] protein accumulation was found to altered by inhibition of the 26S proteasome, suggesting proteasome-dependent degradation of these proteins. Thus, removal of ARF protein may be necessary for remodeling auxin transcriptional complexes for longer-term developmental changes or growth responses. Certainly, regular turnover of these transcription factors allows for resetting of the system and removal of potentially damaged proteins.

4.2. Class-A and B/C ARF competition regulation

Class-A ARFs (5, 6, 7, 8 and 19) are transcriptional activators, whereas Class-B/C ARFs likely function as repressors. The highly conserved ARF N-terminal DBD fulfills the critical DNA binding role through recognition of the AuxRE. Although different ARFs display specificity toward certain AuxRE variants (TGTCNN) and their orientation, a model in which binding competition amongst ARFs serves a regulatory role has emerged. In particular, individual Class A- and Class B-ARFs can antagonize each other through competition for DNA sites in the moss Physcomitrium patens^[53] and in Marchantia polymorpha^[24]. The differential ARF affinity for DNA, combined with Class-A and B/C ARF competition, would be greatly affected by any ARF degradation process. These results raise the possibility that regulated ARF degradation could serve as a modulator of this competition model.

4.3. Environmental responses

Many genetic interaction and transcriptomic analyses reveal ARF roles in a variety of biotic and abiotic stresses.^[54,55] ARFs contribute to salt and drought stress adaptation; altered ARF expression results in improved plant growth under stress conditions in different species.^[56–58] Multiple ARF genes play essential functions in response to heat, flooding, and nutrient stresses.^[55] OsARF19 and OsARF23 are the direct binding target of AET1 (adaptation to environmental temperature1) to participate in temperature responses.^[59] Tomato ARF4 (SIARF4) regulates SCARECROW-LIKE 3 (SCL3) expression to respond to drought stress.^[60] ZmARF2 binds to the potassium (K⁺) transporter gene ZmHAK1 to regulate K⁺ uptake and transport.^[61] ZmARF4 regulate phosphorus deficiency response genes and ZmARF23 overexpression results in phosphorus deficiency tolerance, implicating ZmARF genes in phosphorus deficiency response.^[61,62] In addition to abiotic stress, ARFs are also involved in plant response to pathogens.^[63] OsARF12 and OsARF16 positively regulate immune responses to rice dwarf virus.^[64] Several plant viruses have independently acquired strategies to disrupt OsARF17 activity, indicating the essential OsARF17 roles for antiviral defense.^[65] Moreover, ARF transcriptional activities are impacted by the stress hormone abscisic acid (ABA), indicating a role for ARFs in ABA-mediated regulation of environmental stress.^[54]

Recently, protein accumulation for several ARFs was determined to be stress-responsive. For example, ARF5, ARF6, ARF10, and ARF19 protein levels were affected by temperature, salt, and ABA treatment,^[49] suggesting that the ARF protein levels are regulated by multiple environmental factors. Taken together, ARFs display functional diversity in response to various biotic and abiotic stresses, raising the possibility that stress-regulated ARF degradation could serve as a mechanism to integrate stress and growth responses.

5. Post-translational modifications (PTMs) affecting ARF accumulation

Post-translational modifications (PTMs) are central to many cellular signaling events, affecting protein activity, stability, localization, and interactions.^[66] The most widely investigated PTMs are phosphorylation, ubiquitylation, and SUMOylation,^[66] which have been reported to regulate ARF protein accumulation, activity, and localization.^[40] Here, we explore how PTMs affect ARF function (Figure 2).

5.1. Ubiquitylation

Ubiquitin (Ub) is a highly conserved 76-amino acid protein.^[15,17] Ubiquitylation is the covalent attachment of a Ub moiety to the lysine residue of diverse proteins by the E1-E2-E3 cascade.^[15,16] Ubiquitin itself can be ubiquitylated on seven lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63) or on its N-terminus, leading to isopeptide-linked ubiquitin chains or Met1-linked chains with distinct outcomes^[17]. Single or multiple Lys residues within target proteins can be modified with either a single Ub moiety or with ubiquitin polymers (polyubiquitylation).^[17] Lys48-linked chains occupy more than 50% of all linkages and typically serve to target proteins for proteasomal degradation.^[17]

SCF^AFF1 regulates ARF7 and ARF19 protein accumulation^[21] (Figure 2A). SCF^AFF1 likely facilitates ARF polyubiquitylation and proteasomal degradation, as evidenced by aff1 phenotypes and biochemical activity. Indeed, Class-A ARF6 displayed ubiquitylation and accumulation under different environmental signals^[49], suggesting that the polyubiquitylation of ARFs may contribute to their degradation. Interestingly, in aff1, ARF7 and ARF19 protein is depleted in nuclei and instead localizes to cytoplasmic condensates in cells of the root meristem. A plausible explanation is that SCF^AFF1-mediated ubiquitylation, possibly monoubiquitylation, affects their nuclear import. The different effects on ARF protein accumulation and localization in aff1 raises the possibility that SCF^AFF1 mediates the distinct monoubiquitylation or polyubiquitylation, which promotes ARF7 and 19 nuclear localization and protein accumulation (Figure 2A). More studies will be needed to parse these roles.

5.2. SUMOylation

SUMO (small ubiquitin-like modifier) is an 11-KDa protein that shares a similar structure with other ubiquitin-like modifiers (UBLs).^[67] SUMOylation is the covalent attachment of SUMO molecules to protein substrates, and can act to modulate the transcriptional activity, localization, and stability of target proteins.^[67,68] Like ubiquitylation, SUMOylation mediates the covalent attachment of SUMO moiety via its C-terminal glycine residue to a lysine residue of the target proteins by multiple conjugation enzymes: E1 activating enzymes (SAE1 and SAE2), E2 conjugating enzyme (Ubc9), and a SUMO-protein E3 ligase.^[15] Protein substrates may also be polySUMOylated in a process facilitated by E4 ligases (PIAL1 and PIAL2).^[67]

SUMOylation effects ARF activity and accumulation to regulate lateral root development^[3,26,27] (Figure 2B). Soil-water contact affects root architecture, causing lateral root formation when roots are in direct contact with moisture, a process termed hydropatterning.^[69] ARF7 SUMOylation directly effects the hydropatterning of root adaptive branching response.^[26] SUMOylation has several effects on ARF7, decreasing its DNA binding activity and increasing recruitment of the repressor IAA3. Blocking ARF7 SUMOylation disrupts hydropatterning; water absence stimulates ARF7 SUMOylation and consequent recruitment of IAA3.^[26] This recruitment on the air side of roots reduces auxin-responsive gene expression required for lateral root initiation. Conversely, non-SUMOylated ARF7 on the side of the roots in contact with water is free to induce expression of target genes to promote lateral roots.^[26] Moreover, SUMOylation of MdARF8 regulates lateral root development in apple.^[27] SUMO E3 ligase MdSIZ1 directly SUMOylates MdARF8, resulting in the stabilization of MdARF8 protein, ultimately promoting lateral root development.^[27] Thus, SUMOylation plays critical roles in modulation of ARF transcriptional activity and accumulation to regulate plant growth and development. Whether SUMOylation affects stability of additional ARFs or ARF nucleo-cytoplasmic partitioning, and how it interacts with the ubiquitylation pathway remains to be fully explored.

5.3. Phosphorylation

Protein phosphorylation affects protein surface charge to alter the activity, localization, conformation, and interaction of many components of biological processes.^[70] Phosphorylation is dynamic and reversible, catalyzed by protein kinases and reversed by protein phosphatases. Nearly half of expressed Arabidopsis proteins have been found to be phosphorylated.^[71,72] Protein kinases predominantly catalyze a phosphoryl group transfer from ATP to the hydroxyl groups of specific serine (Ser, S), threonine (Thr, T), or tyrosine (Tyr, R) residues within target proteins.^[70] Multiple kinases and phosphatases are essential for the phosphorylation-based regulation of auxin processes.^[73]

ARF phosphorylation plays regulatory roles to affect plant growth (Figure 2C). ARF7 and ARF19 phosphorylation by BRASSINOSTEROID-INSENSITIVE2 (BIN2) potentiates auxin signal output during lateral root organogenesis by decreasing interaction with Aux/IAAs and increasing DNA-binding capacity and enhance their transcriptional output.^[28] Conversely, ARF2 phosphorylation decreases its ability to bind DNA.^[74] ARF2 is phosphorylated when potassium (K⁺) levels are low, resulting in decreased ARF2 binding to the promoter of the K⁺ transporter gene HAK5 (high affinity K⁺ transporter 5) in Arabidopsis.^[29] Phosphorylation of SlARF4 by SlMAPK8 (mitogen-activated protein kinase 8) inhibits its transcriptional activity to regulate ABA production during fruit development and drought response in tomato.^[30] Taken together, these studies support an important role for phosphorylation of ARFs in regulation of ARF activity (Figure 2C). However, whether phosphorylation is involved in regulation of ARF accumulation needs further exploration.

6. Conclusion and Perspectives

As the first identified and a principal phytohormone, auxin has been and will remain at the forefront of plant development research. Over the past two decades, the enormous success of genetic and molecular approaches delineated the canonical TIR1/AFB-Aux/IAA-ARF auxin signaling module (Figure 2A). ARF transcription factors are the central regulators to control and coordinate auxin-response transcriptional outputs, regulating every aspect of plant development. However, the TIR1/AFB-Aux/IAA-ARF model is not suitable for all ARFs. Only Class-A ARFs elegantly fit into this model, with their transcriptional activity modulated by Aux/IAA interaction. The remaining 17 ARFs are thought to have limited capacity to interact with the repressors, making their regulation by auxin unclear. We are only beginning to understand the multiple ARF regulation layers necessary for generating distinct and dynamic auxin outputs. Here, we have discussed the diverse possibilities for regulated ARF accumulation in driving auxin response and highlighted the unknown aspects of regulatory mechanisms for the stability of ARFs. Other open questions remain to be explored: are all Class-A ARFs degraded by the SCF^AFF1 complex? What is the ARF degron? What are the other mechanisms for regulation ARFs accumulation? Do distinct PTMs affect ARF accumulation? Answering these questions will be an important step to understand the function of ARFs to solve the puzzle that is the complexity of auxin signaling.

Data availability statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.