Transcription factors are universal master regulators of gene expression that bind to unique DNA sequences in the promoter of their target genes to regulate their expression (Suter, 2020). A critical, yet unresolved in aspect of auxin biology is the mechanism by which the simple tryptophan-like indole-3-acetic acid triggers a wide range of cellular responses. During the last step of auxin signaling prior to gene regulation, the ARFs confer specificity to auxin response through selection of target genes. ARF TFs possess typical B3 DBD at their N-terminus, which allows them to bind to DNA motifs called Auxin Response Elements (AuxREs) (Boer et al., 2014; Weijers and Wagner, 2016). The first AuxRE was identified in pea (Ballas et al., 1993) and soybean (Ulmasov et al., 1995) in the promoters of auxin-responsive genes as TGTCTC (Liu et al., 1994). The identification of AuxRE is one of the most significant events that has enhanced the understanding of auxin-mediated regulation of gene expression and the creation of auxin-reporter systems (Hagen and Guilfoyle, 2002), and the identification of the first ARF protein (Ulmasov et al., 1997). The crystal structures of the DBD of ARF1 and ARF5/MONOPTEROS (MP) homodimers, as well as complex of ARF1 DBD with DNA has permitted visualization of protein-DNA interaction (Roosjen et al., 2018), and depicts how amino acids in the DBD interact with the DNA-binding motif TGTCTC (Freire-Rios et al., 2020). The higher affinity of ARFs to the TGTCGG element is because of deeper rotation of H136 into the major DNA groove, which forms additional hydrogen bonds with G5 and G6 in the TGTCGG structure (Boer et al., 2014; Freire-Rios et al., 2020). Mutations in these DNA-interacting amino acids interfered with the DNA binding properties of these ARFs and their biological functions. The TGTC serves as the invariable core element crucial for auxin response, while the final two nucleotides are variable (Boer et al., 2014). In recent years, adoption of advanced techniques has contributed to the identification of other AuxREs and the revelation that variation in the last two nucleotides of an AuxRE were permitted and could play a role in the affinity of ARFs for DNA binding. Although TGTCTC DNA-binding motif was the first to be identified, protein-binding microarrays (PBMs) has revealed that TGTCGG motif possesses relatively higher ARF binding affinity than the TGTCTC motif (Boer et al., 2014). It has been revealed through a ‘cistrome’ analysis that ARF2 and ARF5/MP have higher affinity for TGTCGG than the classical TGTCTC (O’Malley et al., 2016).

Through crystal structures, in vitro, and heterologous studies, a model in which ARF dimers bind with high affinity to distinct repeats of canonical AuxRE motifs has been unraveled. Like all TFs, ARFs bind to DNA as dimers and can homodimerize through their DBD by binding to tandem repeat motifs of TGTCNN elements. Configurations of the tandem repeat and the number of bases between the individual motifs determine their nomenclature: Inverted repeats (IR) where two AuxREs are oriented towards each other in different strands of DNA, direct repeat (DR) where two AuxREs follow each other in the same DNA strand and everted repeat (ER) where two AuxREs orient back to back in different strands of DNA (Freire-Rios et al., 2020). Yeast synthetic auxin signaling system suggest that some ARFs may activate transcription on a single AuxRE, but dimerization between the ARFs is necessary for transcription to occur (Lanctot et al., 2020). Enrichment for single AuxREs upstream of auxin-responsive genes has also been detectable (Freire-Rios et al., 2020), in affirmation to the yeast synthetic auxin signaling system. The biochemical mechanism underlying the differences in DNA-binding specificity of ARFs to single AuxRE binding sites is yet to be proven. Genome-wide DNA binding by ARFs has revealed both overlapping and distinct motif preferences for class A and B ARFs (Galli et al., 2018; Stigliani et al., 2019). DNA affinity purification and sequencing (DAP-seq) experiments performed on maize and Arabidopsis revealed that both class A and class B ARFs can bind IR7/8 motifs, while class A ARFs are additionally capable of binding to several DR and ER motifs (O’Malley et al., 2016; Galli et al., 2018; Stigliani et al., 2019). Although C-ARFs have been proven not to be involved in auxin-dependent transcriptional responses, at least in Marchantia (Mutte et al., 2018), one algal ARF related to the class C ARFs bind to the TGTCNN motifs (Carrillo-Carrasco et al., 2023).

Another element that determines the specificity of the DBD binding is the spacing between both sites of the AuxRE. The binding affinity of two ARFs differ significantly based on spacing between the AuxRE repeats, which dictates the formulation of a caliper model that determine specificity of ARFs binding sites (Boer et al., 2014). The dimerization ability of ARFs through their DBD or C-terminal PB1 domain permits strong binding to double-stranded DNA (dsDNA) carrying a pair of AuxREs with a spacer of a specific length (Boer et al., 2014; Pierre-Jerome et al., 2016). It has been reported that spacing of 7 or 8 bp in ARF1 and 5 to 9 bp in ARF5/MP is required between AuxRE repeats to enhance the interaction between these ARFs and their targeted AuxRE (Boer et al., 2014). Fluorophore or enzyme reporter genes under the control of synthetic promoters including DR5 promoter, characterized by tandem direct repeat of TGTCTC spaced at 5-bp intervals, has often been used for visualizing the distribution pattern of auxin signal in many plant species (Goldental-Cohen et al., 2017), suggesting that this repeat constellation is biologically meaningful.

The C-terminal of ARFs is a classical type -I/II PB1domain of 80-100 amino acids, which was previously named domain III/IV for ARFs and Aux/IAAs (Guilfoyle and Hagen, 2007). Besides the DBD, the PB1 domain is also an ARF interacting domain. Structural analysis on the C-terminal domain of ARFs revealed the structural basis of such heterotypic interaction of ARF5/MP (Nanao et al., 2014), ARF7 (Korasick et al., 2014), IAA17 (Han et al., 2014), and PsIAA4 (Dinesh et al., 2015). PB1 domains are also present in fungi, animals, amoeba, and in several protein families in plants. Characteristic of the type -I/II PB1 domains, the ARF PB1 domain permits for head to tail oligomerization, such that the positive face of one PB1 domain interacts with the negative face of another PB1 domain (Korasick et al., 2014). ARFs and Aux/IAAPB1s interact due to similarity in their 3D structure, such that one negative and one positive face will permit ARF-PB1 interact with AUX/IAA-PB1 in a head-to-tail manner through electrostatic interactions and hydrogen bonds (Vernoux et al., 2011; Piya et al., 2014). The positive face is characterized by an invariant lysine residue that interacts with an array of conserved aspartic and glutamic acids (Korasick et al., 2014), such that alteration in the lysine residue of the positive face hinders interactions with the negative face and preventing oligomerization (Powers et al., 2019).

The PB1 domain of ARFs contributes to their functioning in numerous ways. The PB1 domain mediates the interaction between ARFs and the AUX/IAA proteins, which is required for appropriate canonical auxin signal transduction, which will be discussed briefly. Mutation on the positive face of ARF19 that ablates oligomerization resulted in increased transcription of both auxin-responsive genes and novel targets in the absence of auxin (Powers et al., 2019), suggesting that the ARF19 PB1 mutant is acting as a constitutive auxin signaling factor probably due to its lack of interaction with its transcriptional corepressor Aux/IAAs. Further in vivo oligomerization assay revealed that ARF19 PB1 mutant did not display nuclear dimerization (Powers et al., 2019), which could be inferred that the ARF PB1 domain rather than the DBD primarily promotes ARF homodimerization. Besides the Aux/IAA-ARF interaction, the PB1 domain of ARFs is involved in transcriptional regulations. For example, ARF19 with a mutant PB1 domain that inhibits dimerization did not activate transcription of single AuxRE, but activated paired AuxRE without any hindrance. In the case of the DBD, DBD dimerization is required for both single and paired AuxRE. This data outlines the possibility that the PB1 domain confers on ARFs the ability to activate transcription of AuxREs and could stabilize ARF dimerization under less ideal AuxRE numbers.

Interestingly, the PB1 domain seems to have diverse effects on different class A ARFs, as its deletion in Marchantia polymorpha ARF1 generates a loss-of-function mutant (Kato et al., 2020), whereas in A. thaliana ARF5/MP, the mutant maintains its function and is hyperactive (Krogan et al., 2012). Although heterotypic interactions are stronger than ARF or Aux/IAA homotypic interactions, most PB1s of class A ARFs interact with Aux/IAAs. The disparity between the strength of heterotypic and homotypic interactions result from higher number of electrostatic bonds between ARF and Aux/IAA-PB1s (Parcy et al., 2016; Kim et al., 2020). However, a limited set of interactions between Aux/IAAs and Class B or C ARFs have been identified (Vernoux et al., 2011; Piya et al., 2014), which suggest that the repressor ARF proteins function independently of auxin regulation, and instead compete for DNA binding sites or heterodimerize with other ARF proteins to block transcription (Lavy et al., 2016).

Between the N-terminal DBD and C-terminal of ARFs is the middle region (MR), which is highly variable among ARF TFs. Functional characterization of the middle region thus far has been quite elusive owing to its variability. Nonetheless, the middle region provides the framework for classifying the ARF family proteins. The amino acid composition of the middle region is critical in determining an ARF’s function, with glutamine-rich ARFs acting as transcriptional activators (Wu et al., 2015), whiles those enriched in serines, prolines, and threonines functioning as transcription repressors (Tiwari et al., 2003; Guilfoyle and Hagen, 2007). The activator/repressor classification correlates with the division in subgroups A/B/C, such that those ARFs tested as activators belong to class A, while class B and C ARFs encompass those tested as repressors (Tiwari et al., 2003). The activation and repressive activity of ARFs was decoupled from auxin induction by expressing the MR alone in a synthetic transcription factor assay in carrot protoplasts (Tiwari et al., 2003).

In contrast to the ARF repressor domains, the ARF activation domain remains unknown. This occurrence is probably due in part to the intrinsic disorder in the middle region of class A ARFs. Most activation domains are not characterized by semblance in their sequence, but by sequence characteristics such as hydrophobicity and negative charge (Erijman et al., 2020). It is however worth mentioning that the intrinsic disorder predominantly found in the MR of class A ARFs does not only dictate transcription potential but extends to other cellular features. For example, the MR of ARF7 and ARF19 dictates their subcellular localization (Powers et al., 2019), which is significantly influenced by the C-terminal PB1 domain. ARF19 is differentially localized to the nucleus of young roots and cytoplasm of matured roots. This tissues specific localization of ARF19 is altered by mutation in the PB1 domain, such that more ARF19 is driven to the nucleus of matured roots compared to wild-type. This cooperative relationship between ARF MR and the PB1 is believed to drive the nucleocytoplasmic partitition of ARFs through protein condensation. The PB1 domain probably increases the local concentration of ARF19 and that the intrinsic disorder of the MR contributes to phase separation and protein condensation (Powers and Strader, 2020). Just like other transcription factors, the relationship between ARF localization and transcriptional activity provides further insight into the regulatory mechanism governing the auxin signaling cascade. It is instructive to unravel the mechanisms that drive ARF condensation and the level of participation of other ARFs in this regulatory process, which will significantly broaden our understanding of auxin signaling specificity. The MR also acts as an interaction domain for the recruitment of different types of cofactors such as chromatin remodelers that aid ARFs to carry out their functions. It however remains unknown whether class B ARFs can function as transcriptional activators at certain loci or in the presence of other unknown cofactors.

Transcription activators belonging to the class A ARFs may also induce transcription indirectly by recruiting the SWITCH/SUCROSE NONFERMENTING (SWI/SNF) chromatin-remodeling complex (Clapier and Cairns, 2009). For example, the MR of ARF5/MP increases chromatin accessibility at its binding sites by recruiting the SWI/SWF complex through interactions with BRAHMA and SPLAYED, respectively (Wu et al., 2015). This result reveals a mechanism in which ARF5/MP, and most likely other activator ARFs, alter nucleosome positioning to make more transcription factor-binding sites accessible (Wu et al., 2015; Weijers and Wagner, 2016). In contrast, Arabidopsis class B ARF harbor a conserved TPL-binding motif (RLFGV), and may additionally encode a canonical ethylene-responsive element binding factor (EAR motif), which act as repressor domains in vivo (Choi et al., 2018). For example, both the conserved RLFGV motif and the additional EAR motif are needed for ARF2 to function as a transcriptional corepressor, but only the RLFGV motif is required for TPL interactions in yeast two-hybrid experiments (Choi et al., 2018). These evidences suggest that class B ARFs act as auxin-insensitive negative regulators of auxin-responsive genes (Kato et al., 2020). Additionally, the MR of AtARF2 also harbors the EAR motif (Causier et al., 2012) which bears semblance to that found on Aux/IAAs and which permits interaction with the N-terminal part of TPL/TPRs (Ke et al., 2015). Class C ARFs possess a BRD-like domain with a slightly different sequence (VLFG).

Auxin regulates multiple outputs in plants primarily by controlling the activity of thousands of genes through the nuclear auxin pathway. The canonical auxin transcriptional response system was originally characterized in flowering plants. The nuclear auxin signaling pathway consists of a small number of core components which are represented by a large gene family. Changes in cellular auxin concentrations trigger transcriptional responses of numerous genes, mediated by ARF transcription factors (Weijers and Wagner, 2016). Significant advancement in understanding the auxin signaling machinery has been achieved in recent years (Weijers and Friml, 2009). The core components of the auxin signaling pathway comprises the F-box-containing Transport Inhibitor Response 1 (TIR1) and its homologous Auxin-signaling F Box Proteins (AFBs) proteins, the transcriptional co-repressors AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA), and the ARF transcription factors (Wright and Nemhauser, 2015; Kong et al., 2016). Activation of gene expression as a result of IAA-mediated assembly of TIR1/AFB proteins with AUX/IAA transcriptional regulators has been accepted as the canonical auxin signalling pathway (Figure 1). During auxin limitation, Aux/IAA protein binds to the C-terminal domain of ARFs and its co-repressor TOPLESS (TPL) to repress transcription. TPL recruits chromatin remodeling enzymes such as Histone Deacetylase 19 (HDA19) (Figure 1A) and also interacts with Mediator multiprotein complex (Figure 1B) to prevent ARF transcriptional output. For one case, the HDA19 acts as a physical impediment to maintain chromatin closure at the promoters of ARF-regulated auxin responsive genes (Szemenyei et al., 2008; Qiao et al., 2018) (Figure 1A). For another, ARFs interacts with the Mediator complex via its MR region and Aux/IAA via their PB1domains. The recruited TPL by the domain I of Aux/IAA inturn interacts with the CDK8 of the Mediator complex. Under high auxin concentration, TIR1/AFB forms SCF^TIR1/AFBs ubiquitin complex and triggers Aux/IAAs polyubiquitylation and degradation via the 26S proteasome, resulting in the dissociation of ARFs to TPL-HDA19 and Mediator complex. The eviction of TPL facilitates a permissive chromatin conformation and an increase in the accessibility of transcription factors on the promoters of auxin responsive genes (Wang and Estelle, 2014; Jing et al., 2015) (Figure 1A), and permits the ARFs-Mediator complex to recruit RNA polymerase II and leading to the initiation of gene expression (Figure 1B). Comparison of TIR1, AUX/IAA and ARF orthologues across land plants and charophycean algae indicate that the assembly of the canonical auxin transcriptional response pathway is a land plant innovation.

The auxin-related developmental defects of ett mutants suggested that ETT/ARF3 could regulate auxin signaling independently of the canonical pathway. A fundamental difference between the ETT/ARF3-mediated and the canonical models of auxin signaling is that the former does not primarily require protein degradation to activate gene expression. It was suggested that ETT/ARF3 translates local auxin concentrations to developmental outputs in the gynoecium, although the molecular mechanisms governing this occurrence had not yet been discovered (Simonini et al., 2016). ETT/ARF3 has been reported to participate in auxin dependent protein-protein interactions with several transcription factors belonging to different families, and that these interactions are relevant for auxin responsiveness of specific tissues or cell types during development (Simonini et al., 2016). In the absence of auxin, ETT/ARF3 recruits TPL to its target loci via its ES domain. TPL, in turn, recruits HDA19 to promote deacetylation of histones and repress target gene expression (Figure 2A). In the presence of auxin, ETT/ARF3 can directly interact with the auxin molecule via the ES domain, suggesting that binding of auxin disrupts the interaction between ETT/ARF3 and its corepressor TPL (Kuhn et al., 2020) (Figure 2A), which permits the regulation of auxin-responsive genes.

Another non-canonical auxin-dependent signaling mechanism involves the Trans-Membrane Protein Kinases (TMPKs) pathway. The TMPK subfamily was first linked to auxin signal transduction when the phenotypes of double, triple, and quadruple tmpk mutants showed cell expansion and proliferation defects, miniaturized organs, infertility, and a reduced sensitivity to exogenously applied auxin (Dai et al., 2013). Kinase cascades are rapid and could be involved in rapid, non-canonical signalling. At high auxin concentration, TMPK phosphorylates AUX/IAA at its domain II, which interferes with the poly-ubiquitination and degradation of the AUX/IAA-TPL repressor complex by the SCF^TIR1/AFBs ubiquitin complex, thus inhibiting the transcriptional regulation of auxin-responsive genes (Figure 2B).

Plant root system plays crucial role in regulating and optimizing plant growth and development. They are important plant organs that absorb water and nutrients from soils and translocate them to the shoot (Stone et al., 2001; Sainju et al., 2005), as well as providing a means to monitor the soil for a range of environmental conditions (Overvoorde et al., 2010). Moreover, roots provide mechanical support to plants and distribute hormones that regulate numerous physiological and biochemical processes associated with growth and development of plants. Seed plants have evolved a complex root system consisting of at least three root types, i.e., the primary root, lateral roots, and adventitious roots. Since the discovery of auxins, they have been characterized to be closely related to root development. Root phenotypes associated with auxin signaling are dosage dependent, and include the length of epidermal-derived root hairs, primary root length, number and length of lateral roots and response to gravity (Ishida et al., 2008; Peret et al., 2009). ARFs have been reported to regulate various aspects of root morphogenesis and architecture in several plant species (Table 1).

Photosynthesis is crucial for the existence of the vast majority of life on earth. Plants are primary producers that form the base of every ecosystem and fuel the next tropic level by utilizing photosynthesis to transform water, sunlight and carbon dioxide into oxygen and simple energy for utilization. The photosynthetic process is the principal energy source for all organisms on earth. Leaf anatomy, such as mesophyll thickness and chloroplast abundance and distribution, influences the photosynthetic capacity of plants (Oguchi et al., 2003). Moreover, the shape, size, and chlorophyll content of plant leave influence its photosynthetic capability and efficiency (Guan et al., 2017). Auxin has been proven to play central roles in leaf developmental processes such as leaf initiation, blade formation, compound leaf patterning and leaf inclination (Xiong and Jiao, 2019), with active participation of ARFs in numerous plant species (Schuetz et al., 2019), as outlined in Table 1.

The flattening of leaves to form broad blades with wider surface area is a pronounced adaptation by plants to maximize photosynthetic ability and efficiency. Adaxial-expressed ARF5/MP directly binds to the promoters of WOX1 and Pressed Flower (PRS) and activate their expression in the leaf marginal domain to enable leaf flattening, while redundant abaxial-enriched ARF2/ARF3/ARF4 repressors suppress WOX1 and PRS expression to maintain the abaxial identity (Guan et al., 2017). While arf3, arf5 and arf7 single mutants formed normal leaves in Arabidopsis, mp/arf3 or mp/arf7 displayed a breakdown in leaf formation with novel leaf structure not present in any of the single mutants, suggesting that ARF3 and ARF7 regulates rosette leaf formation and that their functions overlap and act parallel with those of ARF5/MP (Schuetz et al., 2019). ARF6 and ARF8 activate the expression of DWARF4 (DWF4), a pivotal enzyme in brassinosteroids (BR) synthesis. BRs, in turn, facilitate the demethylation of cell wall pectin, resulting in isotropic in-plane cell wall loosening, which ultimately gives rise to leaves with diverse shapes and overseeing the proximal-distal growth of leaf reproductive organs (Xiong et al., 2021). ARF2 and ARF7, with the help of IAA14, suppressed the expression of chlorophyll biosynthesis gene Protochlorophyllide Oxidoreductase A (PORA) and Genomes Uncoupled 5 (GUN5) in matured leaves, resulting in reduced chloroplast number and structure in mesophyll cells and eventual reduction in photosynthetic efficiency (Luo et al., 2023).

Leaf inclination/angle is a component of crop architecture and fundamental property of plant canopy structure, which is required for light interception, canopy photosynthesis, and energy balance. Leaf inclination of rice results mainly from the asymmetric cell division and elongation of adaxial and abaxial cells at the lamina joint (Zhou et al., 2017), which is regulated by the biosynthesis or signaling of auxin. In rice, OsARF4 participates in leaf inclination regulation via auxin and brassinosteroid (BR) signaling pathways: osarf4 mutants displayed increase in cell differentiation on the adaxial side, resulting in increased leaf inclination; however, OsARF4-overexpressing lines manifested a decrease in leaf inclination, resulting in erect leaves (Qiao et al., 2022). In another experiment, OsIAA6 interacts with OsARF1 to suppress auxin signaling and regulates leaf inclination, with rice brassinazole resistant (OsBZR1), the key transcription factor in BR signaling, binding directly to the promoter of OsIAA6 to stimulate its transcription (Xing et al., 2022), suggesting that OsIAA6–OsARF1 module regulates rice leaf inclination through synergistic action of auxin and BR. The mutant ds1 showed reduced BR sensitivity and leaf angle through a mechanism involving DS1’s interaction with OsARF11 to regulate OsBRI1 expression (Liu X. et al., 2018). Loss-of-function mutant of OsARF11, osarf11-1, displayed phenotypes with reduced plant height and leaf angle of flag leaves compared to WT in rice (Sakamoto et al., 2013). OsARF19 controls rice leaf angles by positively regulating OsGH3-5 and OsBRI1. OsARF19-overexpression rice lines showed an enlarged lamina inclination compared to WT due to its increased adaxial cell division in an auxin and brassinosteroid-dependent manner, resulting from direct activation of the early auxin responsive gene OsGH3-1 and Brassinosteroid Insensitive 1 (OsBRI1) (Zhang et al., 2015). Auxin induces OsARF6 and OsARF17 to independently and synergistically bind directly to the Increased Leaf Angle1 (ILA1) promoter and activate its expression to control secondary cell wall composition of the lamina joint to determine flag leaf angle (Zhang et al., 2015).

Mutation in maize leafbladeless1 (lbl1), that disrupt ta-siRNA biogenesis, give rise to plants with thread-like leaves that have lost top/bottom polarity. Misregulation of tasiR-ARFs target, ETT/ARF3, has emerged as the basis for the lbl1 leaf polarity defects, with plants expressing arf3a transcripts displaying insensitivity to tasiR-ARF-directed cleavage and recapitulating the phenotypes observed in lbl1 (Dotto et al., 2014). Auxin plays important roles in regulating both age-dependent and dark-induced senescence through the actions of several auxin-related genes, such as YUCCA6, Small Auxin Upregulated RNA36 (SAUR36), and Indole-3-acetic Acid Inducible 29 (IAA29) (Kim et al., 2011; Hou et al., 2013; Jiang et al., 2014). ZmbHLH112 can repress the expression of Aux/IAA related genes, and promote the binding of ARF to AUXRE in the promoter of their target genes to regulate the elongation of leaf angle cells (Zhang et al., 2022).

Flowers constitute the reproductive structures in plants and lead to formation of fruit and seed after fertilization. Unlike leaves and roots that appear as single organs, flowers have evolved into a stable plant reproductive composite structure, composed of multiple organs arranged in an orderly pattern (Endress, 2010). ARFs have been reported to modulate auxin-dependent regulation of floral organ organization mostly in Arabidopsis (Table 1). The ett/arf3mutant displayed phenotypes with abnormal floral meristem patterning and gynoecium development in Arabidopsis (Sessions et al., 1997), whiles arf1 and arf2 loss-of-function mutants illustrated abnormal abscission of floral organs (Ellis et al., 2005). Mutation analyses revealed that ARF1 and ARF2 regulated plant leaf senescence and floral organ exfoliation, and the ETT/ARF3 gene influenced defect in pistil and flower meristem formation in Arabidopsis thaliana (Nishimura et al., 2005; Quint and Gray, 2006). ARF3 has been functionally characterized to participate in regulatory pathway that modulate gynoecium morphogenesis, self-incompatibility, de novo organ-regeneration, and organ polarity (Tantikanjana and Nasrallah, 2012). ARF6 and ARF8 regulated JA biosynthesis and floral organ development via suppression of class I KNOX genes KNAT2 and KNAT6, with arf6arf8 plants displaying defective phenotypes such as aberrant vascular patterning and lack of epidermal cell differentiation in petals, which were partially suppressed by mutations in KNAT2 or KNAT6 (Tabata et al., 2010).

Floral organ development significantly influences plant reproduction and seed quality, yet its underlying regulatory mechanisms are still largely unknown, especially in crop plants. Disruption of OsARF19 regulates floral organ development and plant architecture in rice. ARF6, ARF12, ARF17, and ARF25, manifested overlapping functions in flower opening and stigma size: Single mutant, arf12, showed a reduced plant height and aborted apical spikelets, while mutation in ARF12 together with mutation in either ARF6, ARF17, or ARF25 led to the same defective phenotypes including the failed elongation of stamen filaments, increased stigma size, and morphological alteration of lodicule (Zhao et al., 2022). AUX/IAA-ZmARF complexes have been reported to predominantly affect maize reproductive growth (Ori, 2019). ZmIAA29 can influence maize florescence by interacting with ZmARF2, ZmARF7, and ZmARF25 (Ma et al., 2023). AUX/IAA proteins Barren Inflorescence 1 and Barren Inflorescence4 and ARFs forms multiple BIF1/BIF4-ARFs transcriptional repression modules involved in the regulation of the boundary basic helix-loop-helix transcription factor Barren Stalk1 (BA1), during the initial stages of reproductive organogenesis in maize and influence its inflorescence architecture (Galli et al., 2015).

Most of the gains made towards functional characterization of ARF family proteins have focused largely on their role in plant growth and development. On the contrary, the role of auxin in regulating stress responses in plants has not received much attention. However, recent molecular approaches such as expression profiling have hinted that auxin might exert some regulatory role on plant responses to environmental stress conditions (Ha et al., 2013). It is suggested that auxin might either be acting alone or together with other key phytohormones in regulating plant response to abiotic stresses such as drought, cold, temperature extremities and salinity (Zahir et al., 2010; Lee et al., 2012). These abiotic stresses affect plant viability and development, which may result in changes in plant growth and crop yield, as well as, disturbance of physiological processes such as photosynthetic or mineral uptake rates (Kou et al., 2022). Genomic studies and expression analysis revealed that, numerous ARF family proteins were differentially expressed in various species in response to key abiotic stress such as drought, salinity or cold (Jain and Khurana, 2009), suggesting that these ARFs are active participants in abiotic stress response in plant species (Table 1).

Biotic stresses are those adverse conditions that normally affectplant growth due to their interaction with deleterious microorganisms such as fungi, bacteria, viruses, viroids, phytoplasmas and nematodes. These microorganisms mainly growth either on or inside plant tissues and inflict varied damages leading to symptoms like chlorosis, stunting, rotting, or local lesions formation. Compared to the role of ARF TFs in regulating responses to abiotic stresses, the role of theses TFs in biotic stress response regulation has not received much research attention. The role of auxin and its signaling pathway on plan- pathogen association has long been reported (Bari and Jones, 2009).

In Arabidopsis, the transcript of ARF1 and ARF2 were repressed by F. oxysporum, whiles arf2, arf1 and arf2/arf1 displayed phenotypes with increased resistance to F. oxysporum relative to WT, these outcomes suggest that ARF1 and ARF2 promote susceptibility to F. oxysporum infestation (Lyons et al., 2015). Misregulation of ARF8 results in developmental abnormalities manifested by viral suppressors of RNA (VSR) transgenic plants and also for the phenotypes displayed during normal viral infection caused by the HcPro-encoding Turnip mosaic virus (TuMV) (Jay et al., 2011). Some OsARFs also play crucial roles in host antiviral immune defense. OsARF12 and OsARF16 interacted with OsIAA10 to positively regulate rice antiviral defense against rice dwarf virus (RDV) through a mechanism involving binding of OsARF12 to the AuxRE in promoter of OsWRK13 to activate its transcription (Qin et al., 2020). Overexpression of OsARF17 reduced accumulation of the black-streaked dwarf virus (BSDV) and rice black-streaked dwarf virus (RBSDV), whiles the accumulation of these virus and severity of their symptoms increased in osarf17 knockout mutant rice lines (Zhang et al., 2020). In maize, expression of ZmARF6 and ZmARF18 genes increased significantly in response to Colletotrichum graminicola and F. verticillioides (Saidi and Hajibarat, 2020), suggesting that these ARFs could act as positive regulators to stresses induced by Colletotrichum graminicola and F. verticillioides.

Histone acetylation is a key histone modification mechanism that appears to be a dynamic reversible switch for inter-conversion between permissive and repressive transcriptional states of chromatin domains (Zhou and Hu, 2010). The co-repressor TOPLESS (TPL) recruits HDA19 to the auxin signaling repressor, AUX/IAA, in an EAR motif-dependent manner, and that the function of GCN5/HAG1 histone acetylase is directly opposed to the function of IAA12/BDL-TPL-HDA19 repressor complex in the ARF-dependent expression of auxin responsive genes (Long et al., 2006; Szemenyei et al., 2008). ARF18-HISTONEDEACETYLASE6 (HDA6) module regulates floral organ identity in Rosa hybrid: Silencing of RhHAD6 increases H3K9/K14 acetylation levels at the site adjacent to the RhARF18-binding site in the promoter of its downstream target, RhAG, and reduces petal number (Chen J. et al., 2021), which indicates that RhARF18 probably recruit RhHDA6 to the RhAG promoter to repress RhAG transcription.

DNA methylation is one of the prominent epigenetic modifications that occur extensively in living organisms (Wang et al., 2009). DNA methylation causes changes in chromatin state in plant cells undergoing dedifferentiation (Koukalova et al., 2005), and can also help to establish or maintain the undifferentiated cell state in plants (Berdasco et al., 2008). In plants, DNA demethylation depends on four bifunctional 5-methylcytosine glycosylases: Repressor of silencing 1 (ROS1), Demeter (DME), DME-like 2 (DML2), and DML3, which remove methylated bases and cleave the DNA backbone at abasic sites. The increased expression of AUXIN RESPONSE FACTOR3 (ARF3) in met1 indeed was due to DNA demethylation, suggesting that DNA methylation regulates de novo shoot regeneration by modulating auxin signaling (Li et al., 2011). BOBBERY1 (BOB1), an Arabidopsis orthologue of eukaryotic NudC domain proteins, and ELONGATA3 (ELO3), the catalytic subunit of the hioghly conserved elongator complex in Arabidopsis, has been revealed through genetic analysis to repress expression of ARF3 and ARF4, along with AS1-AS2 (Takahashi et al., 2013). BIN2 has been reported to phosphorylate ARF7 and ARF19, and in contrast to reducing activity of ARF2, ARF7, and ARF19 phosphorylation enhanced their transactivation activity, which is attributed to reduced ARF7 and ARF19 interactions with the Aux/IAA repressors.

Protein phosphorylation is the most prominent PTM that acts as a crucial cellular regulatory mechanism to either activate or deactivate enzymes and receptors by phosphorylation or dephosphorylation events, which are respectively catalyzed by kinases and phosphatases at serine, threonine, or tyrosine residues (Ardito et al., 2017). The mitogen-activated protein kinases (MAPK) cascades are conserved signaling mechanism comprising reversible phosphorylation through a cascade of ATP-dependent protein kinases, which regulates multiple aspects of plant growth and development. It has been proposed that auxin signal transduction is mediated by the conserved MAPK signaling cascade (Mizoguchi et al., 1994). Auxin-induced MPK14 phosphorylated and stabilized non-canonical IAA33 and enhanced its competitiveness over canonical repressor IAA5 for binding site on promoters of ARF10 and ARF16, which mitigated inhibition of ARF10 and ARF16 by IAA5 and promoted the identity of root distal stem cell (DSC) and negatively regulated auxin signaling (Lv et al., 2020). In the canonical NAP, drought-induced MPK3/MPK6 phosphorylates and stabilizes IAA15 by inhibiting TIR1-mediated ubiquitination of IAA15, which inturn represses the transcriptional activation of LBD genes by ARF7 and ARF19, leading to limited lateral root development under drought stress in Arabidopsis (Kim et al., 2022). In the non-canonical NAP, some TMK1 family members function as PM-resident receptors or part of a receptor complex, perceiving extracellular auxin and transducing these signals into various phosphorylation events (Tan et al., 2021). The cytosolic and nucleus-translocated C terminus of TMK1 specifically interacts with and phosphorylates non-canonical IAA32 and IAA34 repressors of auxin signaling, thereby regulating ARF transcription factors to dictate differential growth of the apical hook (Cao et al., 2019). Other phosphorylation events have been reported to regulate ARF protein functions. For example, ARF2 mostly represses the expression of the HAK5 potassium transporter gene, meanwhile ARF2 is phosphorylated under low potassium stress to abolish its ability to bind to the promoter of HAK5 and diminishes its repressive effects on HAK5 (Zhao et al., 2016). The BRASSINOSTEROID-INSENSITIVE 2 (BIN2) has been implicated to target and phosphorylate ARF7 and ARF19: Phosphorylation of ARF7 and ARF19 suppresses their interaction the AUX/IAA repressor, which eventually enhances the transcription activity of ARF7 and ARF19 to regulate their downstream target gene LBD16 and LBD29 to promote lateral root organogenesis (Cho et al., 2014).

Previous studies on the role of proteolytic regulation in auxin signaling have focused on degradation of their interacting partner, the Aux/IAA proteins, as described above. Although ARF proteins have been shown to be degraded through the 26S mediated ubiquitination, and the degradation process occurs independent of IAAs (Salmon et al., 2008), not much data has been generated regarding degradation of ARFs. Degradation analysis in ARFs show that 37°C treatment increased the protein levels of HA-ARF5/MP, HA-ARF6, and HA-ARF10. On the contrary, there was a pronounced reduction in protein levels of these HA-ARFs by ABA, 4°C and salt treatments, whiles MG132 inhibited the reduction of HA-ARF6 level by ABA and 4°C treatments, suggesting that the ARF protein levels are regulated by multiple factors and that these treatments decrease HA-ARF6 level through 26S proteasome-mediated protein degradation (Li K. et al., 2020). MG132 suppressed the ethylene-dependent decrease in ARF2 protein levels during apical hook development, which strongly suggests that the ethylene-mediated degradation of ARF2 protein is via 26S proteasome degradation pathway (Li et al., 2004). Functional characterization of F-box protein AUXIN RESPONSE FACTOR F-BOX1 (ARF1) SCF^ARF1) revealed that this E3 ubiquitin ligase directly interacts with ARF7 and ARF19 to promote their degradation, and regulate their accumulation, condensation, and nucleo-cytoplasmic partitioning, which triggers downstream auxin responses (Jing et al., 2022).

Small ubiquitin-like modifier (SUMO) is emerging as an important posttranslational modification that regulates plant development and defense pathways (Orosa-Puente et al., 2018; Niu et al., 2019). SUMO is covalently attached to the lysine residues of target proteins, which could modulate protein activity, stability, localization, and protein-protein interactions of target proteins (Vierstra, 2009), converse to protein degradation as witnessed during ubiquitination. SUMOylation is a crucial PTM that has significantly affected various plant responses to stress and environmental changes (Benlloch and Lois, 2018). MdARF8 is directly SUMOylated by apple SUMO E3 ligase MdSIZ1, which enhances protein stability of MdARF8, and facilitates LR formation in apple (Zhang C. L. et al., 2021). The uneven distribution of water in the soil has a direct influence on plant growth and root architecture, which are regulated by the SUMOylation of ARF7 (Bao et al., 2014; Orosa-Puente et al., 2018). SUMOylated ARF7 enhances its binding capacity to IAA3 and negatively regulates ARF7 activity, thereby inhibiting the expression of LBD16 (Orosa-Puente et al., 2018). Conversely, nonSUMOylated ARF7 cannot recruit IAA3 on the moisture side, which leads to an increased expression of LBD16 and promoted LR development (Orosa-Puente et al., 2018).