Game of thrones among AUXIN RESPONSE FACTORs—over 30 years of MONOPTEROS research

Abstract

For many years, research has been carried out with the aim of understanding the mechanism of auxin action, its biosynthesis, catabolism, perception, and transport. One central interest is the auxin-dependent gene expression regulation mechanism involving AUXIN RESPONSE FACTOR (ARF) transcription factors and their repressors, the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) proteins. Numerous studies have been focused on MONOPTEROS (MP)/ARF5, an activator of auxin-dependent gene expression with a crucial impact on plant development. This review summarizes over 30 years of research on MP/ARF5. We indicate the available analytical tools to study MP/ARF5 and point out the known mechanism of MP/ARF5-dependent regulation of gene expression during various developmental processes, namely embryogenesis, leaf formation, vascularization, and shoot and root meristem formation. However, many questions remain about the auxin dose-dependent regulation of gene transcription by MP/ARF5 and its isoforms in plant cells, the composition of the MP/ARF5 protein complex, and, finally, all the genes under its direct control. In addition, information on post-translational modifications of MP/ARF5 protein is marginal, and knowledge about their consequences on MP/ARF5 function is limited. Moreover, the epigenetic factors and other regulators that act upstream of MP/ARF5 are poorly understood. Their identification will be a challenge in the coming years.

A review of the action of MP/ARF5 in auxin-dependent regulation of gene expression, its role in the plant developmental process, and available tools for molecular analysis.

Introduction

The name auxin comes from the Greek word ‘auxein’, meaning ‘growth’. The first reports of auxin date back to 1880, when Charles and Francis Darwin hypothesized the existence of ‘a growth-regulating stimulus’ controlling the process of phototropism (Darwin and Darwin, 1897). Darwin proposed that the ‘stimulus’, though he could not identify it, moves from the site of stimulus perception to the area of responsibility, the bending site. In 1926, Fritz Went isolated a chemical compound affecting the elongational growth of Avena sativa seedlings lacking a shoot apical meristem (SAM; Went, 1926). A few years later, the Fritz Kögl team isolated and identified indole-3-acetic acid (IAA) from human urine (Kögl et al., 1934). Larry Vanderhoef and his group in 1976 suggested that the initial response to auxin is driven by a rapid process, whereas a second response involves gene expression (Vanderhoef et al., 1976). The first reports of auxin’s potential influence on the transcriptional regulation of many genes appeared in the 1980s (reviewed in Guilfoyle and Key, 1986; Theologis, 1986). Since then, the development of molecular techniques and the use of model plants have led to the identification of many genes involved in auxin metabolism, perception, and transport (Paciorek and Friml, 2006; Quint and Gray, 2006; Calderon-Villalobos et al., 2010).

The process of auxin biosynthesis in plants is not fully understood, and intensive research is still underway to uncover and understand all the enzymes involved. Two pathways have been identified for the synthesis of endogenous auxin IAA, one dependent on and one independent of l-tryptophan (l-Trp). The l-Trp-independent pathway is poorly understood, in contrast to the l-Trp-dependent pathway, for which many enzymes have already been identified. The l-Trp-mediated IAA biosynthesis pathway can be further divided into four routes. In the first route, tryptophan aminotransferases [TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) and TAA1-RELATED 1-2 (TAR1-TAR2)] synthesize indolyl-3-pyruvic acid (IPyA) from l-Trp which is converted into IAA by the flavin monooxygenases YUCCA (YUC1–YUC11) (Stepanova et al., 2008, 2011). This mode of IAA synthesis is common in the plant world (Zhao, 2012). Another auxin biosynthesis pathway requires tryptophan oxidase activity to produce indolyl-3-acetaldoxime (IAAox). IAAox can be converted into indolyl-3-acetonitrile (IAN), indolyl-3-acetamide (IAM), and indolyl-3-methyl glucosinolates. The hydrolysis of IAN, probably under the influence of nitrile aminohydrolases (NITRILASE 1–4, NIT1–NIT4), produces IAA (Lehmann et al., 2017). IAN can transform into IAM catalyzed by NITs (Sugawara et al., 2009). l-Trp is also directly converted to IAM. Lastly, IAM can be hydrolyzed by indolyl-3-acetamide hydrolase (AMI1) into IAA (Pollman et al., 2006; Novák et al., 2012).

Proteins facilitating the transport of IAA between and within cells are well known and well characterized. Among them, plasma membrane-localized AUX1, LIKE-AUX1 (LAX), and NITRATE TRANSPORTER 1.1 (NRT1.1) proteins import IAA into the cell. IAA moves inside the cell, in and out the endoplasmic reticulum, and exits the cell mainly via the activity of PIN-FORMED (PIN) and PILS (PIN-LIKE) auxin exporters. Members of the ARABIDOPSIS THALIANA ATP-BINDING CASSETTE B (ABCB) family can mediate auxin influx and efflux (reviewed by Barbosa et al., 2018).

During more than three decades of intensive research on auxin, four groups of IAA receptors have been identified: membrane- and apoplast-localized AUXIN BINDING PROTEIN 1 (ABP1) together with its plasma membrane-localized partner TRANSMEMBRANE KINASE (TMK1–TMK4), TRANSPORT INHIBITOR RESPONSE 1 (TIR1), and its homologs AUXIN SIGNALING F-boxes (AFB1–AFB5) localized in the nucleus and cytosol, and nuclear-located S-PHASE KINASE-ASSOCIATED PROTEIN 2a (SKP2a), and ETTIN/AUXIN RESPONSE FACTOR 3 (ETT/ARF3) (reviewed by Yu et al., 2022). The central role of the nuclear auxin perception mechanism is transcriptional reprogramming, conducted by AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) and AUXIN RESPONSE FACTOR (ARF) proteins. Aux/IAA repressors are ubiquitinated in the presence of auxin and subsequently degraded, relieving the repression on ARF-targeted loci. In the Arabidopsis genome, 23 ARF genes and 29 Aux/IAA repressors were identified (Ulmasov et al., 1997b; Guilfoyle and Hagen, 2001; Liscum and Reed, 2002). Among ARFs, ARF5, also called MONOPTEROS (MP) or INDOLE-3-ACETIC ACID INDUCIBLE 24 (IAA24), was one of the first identified (Hardtke and Berleth, 1998). MP/ARF5 is a transcription factor (TF) that regulates the expression of targeted genes in an auxin dose-dependent manner (Mattsson et al., 2003; Krogan et al., 2014). The monopteros (mp) mutant was discovered after screening a population of Landsberg erecta (Ler) Arabidopsis thaliana mutants obtained by ethyl methanesulfonate (EMS) chemical mutagenesis (Mayer et al., 1991). The MP/ARF5 locus and sequence in chromosome 1 were characterized in detail by Hardtke and Berleth (1998). The Arabidopsis MP/ARF5 gene is 4736 bp long, consisting of 13 exons (Fig. 1A). MP/ARF5 belongs to class A-ARF (together with ARF6-9 and ARF19), the best-studied ARF group (Guilfoyle and Hagen, 2007; Finet et al., 2013). Although MP/ARF5 in model plants such as Arabidopsis has been well characterized, its identities and potential roles in non-model plants are less studied. With increasing numbers of sequenced plant genomes, the ARF family and the MP/ARF5 gene have been identified at the whole-genome level in Arabidopsis (Okushima et al., 2005) and in many plant species. The knowledge about MP/ARF5 action is being slowly explored in commercially important species (Xu et al., 2019; Yue et al., 2020; Mei et al., 2022). Transferring knowledge about MP/ARF5 function to non-model plants of important agro-botanical and industrial significance is a challenge for the coming years, requires further studies, and may help better understand MP/ARF5 action.

Fig. 1.

Structure of the MP/ARF5 gene. Dark green boxes, the 5ʹ- and 3ʹ-untranslated regions; lines, introns; light green boxes, exons (A). Domains present in canonical MP/ARF5 protein (B) and its isoform MP11ir (C) involved in auxin-dependent and independent regulation of gene expression; DBD, DNA-binding domain; MR, middle region domain; PB1/III-IV, Phox/Bem1/domain III and IV; DD, dimerization domin; B3, B3 DNA-binding domain; AC, ancillary subdomain; Q, glutamine; S, serine; L, leucine; P, phosphoryl group. Possible interactions of MP/ARF5, MP11ir with ARF, and Aux/IAA proteins (D–H). MP/ARF5–MP/ARF5 homodimer (D); heterodimer MP/ARF5–canonical ARFs (E); heterodimer MP/ARF5–truncated ARFs (F); MP/ARF5–Aux/IAA complex (G); homodimer of truncated MP11ir isoform (H). Created with BioRender.com.

Evolution and origin of the nuclear auxin response pathway

A nuclear auxin response is conserved across land plants and requires the presence of the three major auxin signaling proteins, ARFs, Aux/IAAs, and TIR1/AFBs. These three signaling components appeared in and are limited to land plants (Lavy and Estelle, 2016; Leyser, 2018; Mutte et al., 2018). Phylogenomic analyses and genetic approaches revealed that bryophytes, including many model species such as Physcomitrium patens for the mosses, Selaginella moellendorffii for the lycophytes (Floyd and Bowman, 2007; Rensing et al., 2008), and Marchantia polymorpha for the liverworts (Flores-Sandoval et al., 2015; Kato et al., 2015), possess all the canonical elements, with all the conserved domains necessary for the nuclear auxin pathway (Kato et al., 2017), and act similarly to the auxin signaling machinery in angiosperms (Flores-Sandoval et al., 2015, 2016; Kato et al., 2015, 2017, 2018). Fewer gene copies for the canonical components (ARFs, Aux/IAAs, TIR1/AFBs, and TPL) are found in bryophytes compared with angiosperms (Paponov et al., 2009; Flores-Sandoval et al., 2015; Kato et al., 2015; Lavy et al., 2016).

Some algae are sensitive to the application of exogenous IAA (Ohtaka et al., 2017), and auxin-like core elements exist in different algal species (Cooke et al., 2002; Stirk et al., 2002). However, no clear evidence demonstrated that algae have an ARF–Aux/IAA complex mediating the auxin signaling pathway as in land plants (Lau et al., 2009). For example, neither Aux/IAA nor TIR/AFB proteins were detected in red algae. Still, an ARF-like structure was found, with 20–30 conserved amino acids replacing the B3 DNA-binding domain and a C-terminal bromodomain instead of the Phox/Bem1 (PB1) domain (Mutte et al., 2018). Charophyte ARFs have all the characteristic functional subdomains, but the Aux/IAAs are non-canonical, lacking the degron domain (Mutte et al., 2018). In land plants, ARFs are classified into A, B, and C classes (Finet et al., 2013). Many charophytes encode two groups of ARFs, group C and a combined A/B group (Mutte et al., 2018; Martin-Arevalillo et al., 2019). The three ARFs classes were probably derived from a common ancestral proto-ARF protein with class C-like characteristics (Martin-Arevalillo et al., 2019). Taken together, the ancestral genes encoding all the auxin core components required for a functional nuclear auxin response, including auxin biosynthesis, transport, and signaling, were present, established, and conserved in all terrestrial plants (Flores-Sandoval et al., 2015; Bowman et al., 2017, 2019; Mutte et al., 2018) and evolved from an algal auxin-independent pathway ancestor (Martin-Arevalillo et al., 2019).

Structure of MP/ARF5 protein

The canonical MP/ARF5 protein contains 902 amino acids with a mol. wt of 99.65 kDa. In addition to the canonical protein, two isoforms were identified in Arabidopsis: ARF5ʹ with unknown function (891 amino acids, 98.45 kDa) (TAIR; https://www.arabidopsis.org/) and MP11ir (815 amino acids, 89.94 kDa) (Cucinotta et al., 2021). The canonical MP/ARF5 protein is composed of three main domains: the N-terminal DNA-binding domain (DBD), the middle region (MR) domain, and the Phox/Bem1 (PB1) known as a C-terminal domain (previously called domain III and IV) (Cancé et al., 2022) (Fig. 1B). The MP11ir isoform lacks a C-terminal PB1 domain (Fig. 1C).

The DBD consists of three subdomains: the B3 DNA-binding motif flanked by two dimerization domains (DDs) and, at the C-terminus of the DBD domain, an ancillary subdomain (AC), related to the Tudor domain (a five-stranded β-barrel-like structure). The AC subdomain interacts tightly with the DD subdomains. However, its exact function remains unknown (Boer et al., 2014). Furthermore, the DD mediates homodimerization (Fig. 1D) of MP/ARF5 and heterodimerization with other ARF proteins (Fig. 1E, F). MP/ARF5 can act as a monomer and dimer. Homodimerization is necessary for its action and binding to the promoter of its target genes, and may define MP/ARF5–DNA binding specificity in vivo (Boer et al., 2014; Fontana et al., 2023). However, the role of potential heterodimers is still under discussion (Cancé et al., 2022; Caumon and Vernoux, 2023). MP/ARF5 binds to DNA via a single B3 DNA-binding domain, a plant-specific domain of ~110 amino acids. The B3-type TF superfamily contains 118 members in Arabidopsis, having functions mainly related to hormone response, and includes the ARFs, LEC2-ABI3-VAL (LEAFY COTYLEDON 2-ABSCISIC ACID INSENSITIVE 3-VP1-ABI3-LIKE, LAVs), RELATED TO ABI3/VP1 (RAVs), and REPRODUCTIVE MERISTEM (REM) (Yamasaki et al., 2013). The B3 domain is essential for MP/ARF5 binding to auxin-responsive elements (AuxREs) in auxin-induced gene promoters. It has been demonstrated by DNA affinity purification sequencing (DAP-seq) that the hexanucleotide-target motif 5ʹ-TGTCGG-3ʹ rather than the canonical AuxRE 5ʹ-TGTCTC-3ʹ is the preferred binding sequence for MP/ARF5 (O’Malley et al., 2016; Freire-Rios et al., 2020). A ChIP-seq analysis showed an overlap of target genes between three positive ARFs [MP/ARF5, ARF6, and NON-PHOTOTROPHIC HYPOCOTYL 4 (NPH4)/ARF7] and one repressor ARF10 (Xie et al., 2022, Preprint). It suggests that ARFs may compete for binding to AuxREs, depending on the cell types. The DBD of MP/ARF5 can recognize three combinations of two AuxRE motifs when located in different DNA strands, facing each other (inverted repeats, IRs), the same strand, in the same direction, one after the other (direct repeats, DRs), or different DNA strands back-to-back (overturned repeats, ERs). Additionally, MP/ARF5 binds with a strong affinity to two AuxREs separated with specific spacing, namely IR7–8 and IR18; DR4–5, DR14–16, and DR25; and ER3, ER13, and ER23, where the numbers indicate the number of bases between two AuxREs (Stigliani et al., 2019; Freire-Rios et al., 2020).

The sequence of the amino acid residues of the MR domain is specific for each ARF TF and determines whether the TF acts as a transcriptional activator or repressor (Li et al., 2016; Powers and Strader, 2020). In the case of MP/ARF5, ARF6–ARF8, and ARF19 proteins, their MR domain is rich in amino acids such as glutamine, serine, and leucine, implying that they belong to the activators (Tiwari et al., 2003; Guilfoyle and Hagen, 2007). This was confirmed in Arabidopsis (Tiwari et al., 2003), rice (Shen et al., 2010), and tomato (Zouine et al., 2014). The MR domain of the repressor ARFs (ARF1–ARF4, ARF9–ARF18, and ARF20–ARF22) is enriched in serine, proline, leucine, and glycine (Ulmasov et al., 1997a; Guilfoyle and Hagen, 2007). MP/ARF5 can be phosphorylated on Thr163 in the DBD and Ser647 in the MR (Fig. 1B, D) by interacting with a glycogen synthase kinase BIN2-LIKE 1 (BIL1). This phosphorylation attenuates the interaction between MP/ARF5 and Aux/IAA repressors, thus enhancing MP/ARF5 activity (Han et al., 2018).

Lastly, the PB1 domain, at the C-terminal end of the MP/ARF5 protein, provides positive and negative electrostatic interfaces for directional protein interaction (Korasick et al., 2014). MP/ARF5 cooperates with short-lived nuclear repressors, Aux/IAA proteins, through this domain. Computer simulations showed that MP/ARF5 could interact with almost all Aux/IAA proteins (excluding IAA6, IAA9, and IAA26), and in vitro analyses confirmed the interaction between MP/ARF5 and IAA1, IAA3, IAA13–IAA14, IAA17, and IAA19 (Vernoux et al., 2011) (Fig. 1G). The PB1 domain also contributes to the dimerization and condensation of A-type ARF factors (Jing et al., 2022). Indeed, deletion of the PB1 domain lowers ARF–ARF interaction affinities (Nanao et al., 2014; Powers et al., 2019).

The MP/ARF5 isoform, MP11ir, lacking the PB1 domain, is produced by RNA alternative splicing during ovule development. The retention of intron 11 in the transcript results in a stop codon in the preserved intron (Fig. 1A), leading to the translation of a truncated protein (Fig. 1C, H). By polysome profiling using pre-fertilization inflorescences, it was suggested that MP11ir transcripts might escape nonsense-mediated mRNA decay to produce a functional truncated protein. The MP11ir isoform is insensitive to Aux/IAA repression. It was also proposed that MP11ir could work independently of auxin. The ectopic expression of MP11ir partially complements the mp/arf5 mutant phenotypes during reproductive development, suggesting that certain MP/ARF5 functions bypass its interaction with Aux/IAA repressors. Coherently, MP11ir activates its target genes in regions of the ovule that are characterized by low auxin concentrations (Cucinotta et al., 2021).

Summing up, despite the structure of the MP/ARF5 protein being known, details on how MP/ARF5 functions in vivo are still being uncovered. The detailed role of MR and PB1 domains in forming different protein complexes [MP/ARF5–Aux/IAAs; MP/ARF5–ARFs heterodimerization and oligomerization; MP/ARF5–TFs and MP/ARF5–chromatin remodelers (discussed below)] are continually being discovered. What other post-transcriptional modifications does the MP/ARF5 protein undergo, and how does it affect its stability and activity? These questions remain open. Additionally, the role of the MP/ARF5 isoforms in plant developmental processes is puzzling.

MP/ARF5-dependent regulation of gene expression

The AuxRE motif recognized by MP/ARF5 is found in the promoter of 11 092 genes and MP/ARF5 protein directly regulates the expression of 4585 target genes in an auxin-dependent way (Xie et al., 2022, Preprint). However, it is essential to note that the molecular mechanisms carried out by MP/ARF5 vary depending on the stages of plant development. It was postulated that in low cellular auxin levels, MP/ARF5 interacts with Aux/IAA repressors (and negative regulators of auxin signaling), mainly IAA12, known as BODENLOS (BDL). MP/ARF5–BDL/IAA12 interaction occurs via their respective PB1 domains. BDL/IAA12 recruits the transcriptional co-repressors TOPLESS (TPL) and TOPLESS-RELATED (TPR), which interact with the histone deacetylase enzyme HDA19 (Long et al., 2006). HDA19 removes the acetyl group from the histone H3 and H4 tails, resulting in condensed chromatin, and the expression of auxin-responsive genes is repressed (Hollender and Liu, 2008) (Fig. 2A).

Fig. 2.

Mechanism of nuclear auxin signaling pathway and auxin-dependent regulation of gene expression by MP/ARF5. BDL/IAA12 repressor binds to MP/ARF5 transcription factor at low cellular auxin levels. BDL/IAA12 repressor recruits co-repressors: TPL/TPRs. Additionally, HDA19 maintains chromatin in an unlicensed repressive state. This interaction prevents MP/ARF5 from driving gene transcription (A). At high cellular auxin concentrations, the hormone is perceived by the TIR1–BDL/IAA12 co-receptor complex, followed by ubiquitination and proteasomal degradation of the BDL/IAA12 protein. The chromatin-remodeling complex containing BRM, SYD, histone acetylases, and other TFs physically associates with MP/ARF5. It facilitates chromatin opening and further activation of gene transcription in response to auxin (B). At a suboptimal auxin level, MP/ARF5 may oligomerize with MP/ARF5 or other ARFs, which can shut down expression of the target genes (C). Ac, acetyl group; ASK1, ARABIDOPSIS SKP1 HOMOLOG; Aux/IAA, AUXIN/INDOLE-3-ACETIC ACID; BDL/IAA12, BODENLOS/INDOLE-3-ACETIC ACID 12; BRM/SYD, BRAHMA/SPLAYED; CUL1, CULLIN 1; E2, UBIQUITIN-LIGASE; HAT, HISTONE ACETYLASE; HDA19, HISTONE DEACETYLASE 19; MP/ARF5, MONOPTEROS/AUXIN RESPONSE FACTOR 5; RBX1, RING-BOX 1; TF, transcription factor; TIR1/AFB, TRANSPORT INHIBITOR RESISTANT1/AUXIN SIGNALING F-BOX; TPL/TPR, TOPLESS/TOPLESS RELATED; Ub, ubiquitin. Created with BioRender.com.

In the presence of auxin, BDL/IAA12 is recruited by the SCF^TIR1/AFB complex, ubiquitinated, and degraded by the 26S proteasome. In the absence of BDL/IAA12, MP/ARF5 recruits chromatin-remodeling complexes containing the BRAHMA (BRM) or SPLAYED (SYD) SWI/SNF ATPases via its MR domain, and this unlocks chromatin through nucleosome eviction and histone acetylation (Wu et al., 2015). MP/ARF5 can also interact with the bZIP11 TF, which binds to the G-box-related cis-element motif in the vicinity of AuxREs. The bZIP11 TF can recruit the multiprotein SAGA complex (SPT–ADA–GCN5–ACETYLTRANSFERASE), which acetylates nearby histones to open up the chromatin and allow gene transcription by RNA polymerase II (Pol II) (Weiste and Dröge-Laser, 2014; Wu et al., 2015) (Fig. 2B).

Furthermore, it could not be excluded that when cellular auxin levels are at suboptimal concentrations, MP/ARF5 protein might oligomerize with itself or other ARFs, inhibiting the expression of auxin-responsive genes (Fig. 2C). The formation of heterodimers between ARF proteins (i.e. MP/ARF5–ARF1, MP/ARF5–NPH4/ARF7) was experimentally demonstrated (Vernoux et al., 2011). The ARF–ARF oligomerization was observed in vitro (Korasick et al., 2014; Nanao et al., 2014) and in vivo for ARF19 (Powers et al., 2019). This MP/ARF5–ARF oligomerization scenario is plausible. Indeed, plants with MP/ARF5 constitutive overexpression (Hardtke et al., 2004) display a pin-like phenotype—a naked inflorescence—similar to the phenotypes of the mp/arf5 mutant (Przemeck et al., 1996).

Truncated MPΔ proteins

To elucidate the complex action of MP/ARF5 and its MP11ir isoform, lines expressing truncated MPΔ proteins (Table 1) may be a powerful tool. These transgenic lines revealed novel MP/ARF5 functions and may help to dissect the functional relevance of MP/ARF5–ARF or MP/ARF5–Aux/IAA interactions. Lines with a truncated MP/ARF5 protein lack the whole PB1 domain (III and IV) or only domain IV, like MPΔ or mp^abn, respectively (Garrett et al., 2012; Krogan et al., 2012). The truncated MPΔ protein is presumably not repressible by its Aux/IAA partners, mainly BDL/IAA12, which results in an auxin-independent and enhanced expression of the known MP/ARF5 target genes. The truncated proteins MPΔ and MP^abn partially rescue the mp/arf5 phenotypic defects but also cause leaf defects that were not previously associated with either mp/arf5 loss- or gain-of-function mutations (Garrett et al., 2012; Krogan et al., 2012). Additionally, it was shown that the MPΔ protein stimulated the ectopic initiation of new organs, specifically in the peripheral zone of the apical meristem (Ma et al., 2019). Interestingly, during in vitro culture, MPΔ significantly increases the frequencies of de novo shoot formation in Arabidopsis tissue and may be used to overcome organogenic recalcitrance from recalcitrant explants and species (Ckurshumova and Berleth, 2015; Gonzalez et al., 2021). A recent study identified the target genes of MP/ARF5 and truncated MPΔ proteins in 3-day-old seedlings (un)treated with auxin (Xie et al., 2022, Preprint). Only 922 gene promoters out of 4585 were commonly bound by both proteins (Fig. 3A). While MP/ARF5 specifically binds to the promotor of 258 genes, MPΔ interacted with a much higher number of promoters (3405). Interestingly, IAA treatment affected the binding profiles of both proteins differently. In the presence of IAA, 339 target genes were bound by MP/ARF5 (1143 genes in the absence of IAA). For 302 genes out of 339, the interaction was insensitive to the presence of IAA (MP/ARF5 target genes with and without IAA treatment). In contrast, 3404 genes were bound by MPΔ in the presence of IAA (3970 without IAA), among which the interaction was auxin insensitive for 3047 genes (Fig. 3B). This ChIP-seq analysis indicates that the deletion of the PB1 domain modifies the binding profile of MP/ARF5 and renders the protein auxin insensitive. Moreover, differences in the AuxRE motifs preferentially bound by MP/ARF5 and MPΔ suggest that the presence of the PB1 domain may affect homodimer formation and binding to DNA (Xie et al., 2022, Preprint). It raises the possibility that the action of MP/ARF5 and MPΔ may differ at the molecular level and, therefore, differentially impact plant development. An unanswered question is whether the absence of the PB1 domain affects MP/ARF5 interaction with other proteins: ARFs, TFs, and chromatin remodelers. Does the absence of the PB1 domain affect the protein structure and formation of homodimers, heterodimers, and oligomers? In light of research outcomes showing that the PB1 domain is involved in the efficient binding of ARF protein to DNA (Fontana et al., 2022), it would be interesting to investigate how the lack of the PB1 domain in MPΔ affects cis-element recognition in the promoter of controlled genes. Does MP/ARF5, like ETT/ARF3, possess an IAA-binding domain and can non-canonically regulate gene expression? Answering those questions would require further studies.

Table 1.

Genetic material to study MP/ARF5 PB1 domain function.

Line name	Characterization	References
Chemical and insertional mutants
mp abn chemical mutagenesis	A nonsense codon at position 837 corresponds to the mpabn mutation.	Garrett et al. (2012)
mp-14 (SALK_001058) insertional mutagenesis	SALK_001058 has a T-DNA insertion in the 11th exon of MP at the beginning of the sequence coding for the C-terminal domain. Mutant plants have completely penetrant defects only in some developmental processes that depend on MP.	Odat et al. (2014)
Transgenic line
MPΔ MPΔ-2 MPΔ-3	Driven by the endogenous MP promoter (3.3 kb upstream of the start codon). MPΔ encodes amino acids 1–813 of MP, followed by a cloning artifact of 20 extra amino acids (DLEELARISPIVQTFGNKVS). MPΔ-2 and MPΔ-3 encode amino acids 1–794 and 1–813 of MP, respectively, and lack extra residues.	Krogan et al. (2012)
pMP::MPΔ pAS1::MPΔ	pMP::MPΔ contains the endogenous MP promoter (3302 kb upstream of the start codon) and the endogenous MP transcriptional sequence truncated 730 bp downstream of the stop codon. MPΔ encodes amino acids 1–813 of MP without extra residues. In pAS2::MPΔ, MPΔ is controlled by the AS2 promoter containing 3303 bp upstream of the AS2 start codon and 18 bp of the N-terminal AS2 coding region.	Qi et al. (2014)
pUBQ10::MP794-YPet	A 794 amino acid truncated version of MP cDNA was amplified with primer set 648f/675r as a translational fusion to C-terminal YPet to generate UBQ10>> MP794-YPet.	Bhatia et al. (2016)
pMP::MP∆-GR pMP::MP∆-EAR-GR	To construct pMP::MP∆-GR, a 6695 bp MP genomic DNA fragment (containing 3231 bp upstream and 3461 bp downstream of the start codon, respectively) was amplified and fused in-frame to GR. To construct pMP::MP∆-EAR-GR, a 120 bp fragment coding for amino acids 183–222 of AtERF4 was amplified and fused to the C-terminus of pMP::MP∆.	Guan et al. (2017)
pPXY::GR-ARF5ΔIII/IV p35S::ARF5ΔIII/IV	To generate pPXY::GR-ARF5ΔIII/IV (pKB25), the GR-ARF5ΔIII/IV fragment, including a stop codon, was amplified from pKB17 using the MP_for18/MP_rev16 primer pair and inserted in pTOM50 using NcoI/Cfr9I restriction sites. To generate p35S::ARF5ΔIII/IV (pKB40), the ARF5ΔIII/IV CDS was amplified from pKB25 using MP_for17/MP_rev15 and introduced into pGreen0229-35S using XbaI/EcoRI restriction sites.	Brackmann et al. (2018)
pHMG::MP∆	The pHMG promoter corresponds to a 1347 bp fragment upstream of the At1g76110 locus. To generate MPΔ, a fragment of the MP cDNA coding for a truncated protein before domain III (amino acids 1–794) was amplified.	Ma et al. (2019)
XVE::ARF5Δ DR5rev::GFP	The first 2382 nucleotides of MP/ARF5 (encoding the first 794 amino acids and lacking the C-terminal PB1 domain) were cloned into the β-estradiol-inducible, gateway-compatible vector pMDC7. This vector was used to transform plants carrying the DR5rev::GFP reporter.	Gonzalez et al. (2021)
pUBQ10::MPΔ-TagRFP	pMOA34-pUBQ10-loxP-GUS-35S-polyA-loxP-MPΔ-TagRFP construct contains a 2389 bp UBQ10 promoter fragment up to the start codon and a 3461 bp genomic fragment for the MP coding region.	Guan et al. (2022)
Other approaches
GD-ARF5M Protoplast transfection assays	The first and last amino acids from MP/ARF5 in ARF5M, were 349 and 766.	Tiwari et al. (2003)
MPΔC Y3H/BiFC	To generate estradiol-inducible MP, a truncated version of MP was missing the C-terminal PB1 domain (amino acids 795–902) and was amplified from cDNA, cloned into pENTR/D-TOPO (Thermo Fisher). The clone was shuttled into the estradiol-inducible expression vector pMDC7.	Wu et al. (2015)
MP ΔIII,IV GR::MP ΔIII,IV Protoplast transfections Dual-luciferase reporter assay system	Truncated MP protein lacking domains III and IV.	Lau et al. (2011) Herud et al. (2016)
pUAS::MPΔ-GUS p35S::lox-MPΔ-YFP pUBQ10::XVE-amiMP-3AT	pSm43GW-pUAS::MPΔ-GUS-OcsT [OS 30.1] combines pENTR41R-6xUAS2, p221z-cMPdelta, p2R3e-GUS-OcsT into pSm43GW. pBm43GW-p35S::lox-MPΔ-YFP [OS 74.1] combines p1R4z-p35S:lox, p221z-cMPdelta, p2R3a-venYFP-3AT into pBm43GW. pBm43GW-pUBQ10::XVE-amiMP-3AT [RM25.1] combines p1R4a-pUBQ10::XVE, pENTR-AtMIR167a-MP; p2R3a-3AT into pBm43GW	Smetana et al. (2019)

A list of mutants after chemical mutagenesis, insertional mutagenesis, and transgenic lines used to study the truncated MP/ARF5 protein, which acts independently on Aux/IAA repressors.

Fig. 3.

Set of genes directly regulated by canonical MP/ARF5 and truncated MPΔ in vivo. Venn diagram showing the overlap between canonical MP/ARF5 and truncated MPΔ gene targets (A). The number of genes under direct MP/ARF5 or MPΔ regulation in response to auxin (B). Venn diagram created with the jvenn tool (Bardou et al., 2014).

Role of the MP/ARF5 transcription factor during the developmental processes

Molecular genetics and cell biological studies have revealed the involvement of MP/ARF5 in several aspects of plant development in vivo and in vitro, mainly in the model plant Arabidopsis, with the help of numerous tools and transgenic lines (Table 2). This section will illustrate how MP/ARF5 controls genes involved in zygotic embryogenesis, meristem establishment, development of various plant organs, and vascularization patterns through subsequent transcriptional steps.

Table 2.

Mutants after chemical mutagenesis, insertional mutagenesis, and transgenic lines used to study the canonical MP/ARF5 protein.

Tool	Line name	References
Chemical and insertional mutants	arf5-1 SALK_023812	Okushima et al. (2005)
	arf5-2 (mpS319) SALK_021319	Donner et al. (2009)
	mp-11 SAIL_1265_F06	Odat et al. (2014)
	mp-12 SALK_149553	Odat et al. (2014)
	mp-13 WiscDsLoxHs148_11H	Odat et al. (2014)
	WiscDsLox489-492C10	Odat et al. (2014)
	SALK_144183	Odat et al. (2014)
	WiscDsLoxHs148_12G	Odat et al. (2014)
	mpBS1354	Hardtke and Berleth (1998)
	mpG12	Hardtke and Berleth (1998)
	mpG92	Hardtke et al. (2004)
	mpB4149	Weijers et al. (2005)
	mpBS62	Vidaurre et al. (2007)
Constitutively overexpressed line	p35S::MP	Mattsson et al. (2003) Hardtke et al. (2004)
Inducible lines	pMP::MP-GR	Krogan et al. (2014)
	pUBQ10::MP-GR	Donner et al. (2009) Yamaguchi et al. (2015)
Silenced lines asRNA or amiRNA	p35S::MPAS	Hardtke et al. (2004)
	pMP::amiRARFMP	Z. Liu et al. (2018)
	pUBQ10::XVE>>amiMP	Smetana et al. (2019)
Transcriptional reporter lines	pMP::n3×GFP	Schlereth et al. (2010) Rademacher et al. (2012)
	pMP::erYFP	Smetana et al. (2019)
Translational reporter lines	pMP::MP-GUS	Vidaurre et al. (2007) Krogan et al. (2012) Krogan and Berleth (2012) Krogan et al. (2014) Carey and Krogan (2017)
	pMP::MP-GFP	Cole et al. (2009) Schlereth et al. (2010) Krogan et al. (2012)
	pMP::MP-YFP	Bhatia et al. (2016)
	pMP::MP:ECFP	Donner et al. (2009)
Tag lines	pMP::6×HA	Weijers et al. (2006)
	pMP::MP-HA	Krogan et al. (2014)
	pMP::MP-6×HA	Wu et al. (2015)

Involvement of MP/ARF5 in zygotic embryogenesis

MP/ARF5 is essential for development of the female reproductive organ (gynoecium), as shown by the mp/arf5 mutant displaying smaller gynoecia with a reduction of the stigmatic, stylar, and ovary tissues (Okada et al., 1991; Przemeck et al., 1996; Hardtke and Berleth 1998; Benkova et al., 2003; Bencivenga et al., 2012). The phenotype is more dramatic in arf3 arf5 double mutants (Pekker et al., 2005), with a near complete loss of ovary tissues. After sexual reproduction and during zygotic embryogenesis, MP/ARF5 is expressed in the lower tier of the globular embryo (Fig. 4A). MP/ARF5 transcripts and MP/ARF5 proteins are broadly expressed in the vascular and ground tissue embryonic cells. Subsequently, at the heart stage, MP/ARF5 is transcribed in the cells of the adaxial side of the cotyledons, the SAM, the quiescent center, the 5–10 fast-dividing cells above the quiescent center, and in subdomains of the vascular tissue (Fig. 4A) (Cole et al., 2009; Rademacher et al., 2012). The mp/arf5 globular embryos are characterized by the aberrant formation of the hypophysis and the vascular tissue due to defects in the division plan: periclinal rather than anticlinal. It results in a disrupted embryonic body plan (embryos lacking hypocotyl and root, and, in the strong mutant alleles, with a single cotyledon) (Fig. 4B) (Mayer et al., 1991; Möller et al., 2017). The MP/ARF5 protein controls the expression of numerous genes involved in embryonic divisions and cell specification (Fig. 4C). The embryonic root initiation process greatly depends on the antagonistic interaction between MP/ARF5 and its repressor BDL/IAA12 protein (Hamann et al., 2002; Schlereth et al., 2010), and both the loss-of-function mp/arf5 and gain-of-function bdl/iaa12 mutants are defective in hypophysis specification (Hamann et al., 1999). Interestingly, MP/ARF5 and BDL/IAA12 proteins accumulate in embryonic cells (Weijers et al., 2006; Schlereth et al., 2010), but neither accumulates in the hypophysis, implying that MP/ARF5 drives the hypophysis specification non-cell-autonomously (Hamann et al., 2002; Weijers et al., 2006). The embryonic root initiation also depends on auxin production and transport. Auxin synthesis occurs in the upper tier protoderm of the globular embryo, followed by its transport by the auxin efflux carrier PIN-FORMED 1 (PIN1) and auxin influx carriers AUX1 and LAX2 to the ground tissue, where auxin levels increase (Friml et al., 2003; Robert et al., 2013, 2015; Wabnik et al., 2013). Auxin induces the degradation of the BDL/IAA12 protein, hence promoting the MP/ARF5 activity (Weijers et al., 2006; Schlereth et al., 2010; Herud et al., 2016) to induce the transport of auxin, through the controlled PIN1, AUX1, and LAX2, to the hypophysis precursor (Weijers et al., 2006; Schlereth et al., 2010; Robert et al., 2013, 2015). In the provascular tissue, MP/ARF5 transcriptionally controls the activity of the TFs TARGET OF MONOPTEROS 5 (TMO5) and TMO7, which are critical for embryonic root initiation and formation (Schlereth et al., 2010; Möller et al., 2017). TMO5 and TMO7 belong to the basic helix–loop–helix (bHLH) family and are active in the hypophysis-adjacent embryo cells. TMO5 functions cell autonomously and dimerizes with the bHLH TF LONESOME HIGHWAY (LHW). Together, they regulate periclinal cell divisions, vascular initial cell production, vascular cell proliferation, and xylem fate determination (De Rybel et al., 2013). TMO7 was discovered to be mobile, from the embryo to the uppermost suspensor cell (Schlereth et al., 2010). It contributes to root formation by regulating the asymmetric hypophysis cell division to give rise to the quiescent center and columella cells.

Fig. 4.

Molecular mechanism of MP/ARF5 action during the developmental process. MP/ARF5 expression pattern during embryo development in the globular and heart stage (A). The phenotype of the mp/arf5 mutant in comparison with the wild type (B). Set of MP/ARF5-controlled genes during embryogenesis (C). Localization of MP/ARF5 expression during post-embryogenic leaf development (D). Effect of MP/ARF5 mutation on leaf development (E). Genes under MP/ARF5 control during leaf formation (F). MP/ARF5 activity in the post-embryogenic process of vascularization (G). Effect of MP/ARF5 mutation on the structure of vascular tissues (H). MP/ARF5 controlled genes during canalization (I). Expression pattern of MP/ARF5 during SAM (J) and inflorescence (L) development. Structure of the SAM in the mp/arf5 mutant in comparison with the wild type (K). Inflorescence of wild-type plants with developed flowers and naked inflorescence of the mp/arf5 mutant (M). Genes regulated by MP/ARF5 during the formation of meristems. Other processes under MP/ARF5 control (O). Yellow box, genes coding auxin transporters; brown box, gene involved in miR165/166 activity inhibition; blue box, genes coding transcription factors. AB, abaxial domain; Ac, acetyl group; AD, adaxial domain; B3, DNA-binding domain; BRM/SYD, BRAHMA/SPLAYED; CR, chromatin remodeler; CZ, central zone; DD, dimerization domain; HAT, HISTONE ACETYLASES; MD, middle domain; MP/ARF5, MONOPTEROS/AUXIN RESPONSE FACTOR 5; P, phosphoryl group; PB1, Phox/Bem1 domain; P, primordium; PZ, peripheral zone; SAM, shoot apical meristem; TF, transcription factor; V, vascular tissue; WT, wild type. Created with BioRender.com.

AP2 domain-containing TFs PLETHORA 1 (PLT1) and PLT2 are redundantly required for correct quiescent center specification, leading to the maintenance of the stem cells in the root meristem (Aida et al., 2004). Their expression in the lower tier embryo requires MP/ARF5 and NPH4/ARF7. The SHORT ROOT (SHR) gene expressed in the provascular cells encodes a mobile TF promoting asymmetric division within the ground tissue, giving rise to root endodermis and cortex (Moreno-Risueno et al., 2015). SHR also requires the transcriptional activity of MP/ARF5 (Möller et al., 2017). Genes encoding the zinc finger TFs, NO TRANSMITTING TRACT (NTT), and its paralogs WIP DOMAIN PROTEIN 4 (WIP4) and WIP5, are the targets of MP/ARF5 and required for root meristem initiation (Crawford et al., 2015). Likewise, WRKY23 acts downstream of ARF5–BDL/IAA12 during embryogenesis and formation of the hypophysis. Moreover, expression patterns of MP/ARF5, BDL/IAA12, and WRKY23 overlap in the inner cells of young embryos, excluding the protoderm. The pWRKY23::GUS activity in the inner cells of a 16-cell stage embryo was absent in the gain-of-function (auxin-resistant) bdl/iaa12 embryos. Additionally, the morphological defects observed in the wrky23 mutant are very similar to those seen in mp/arf5 and bdl/iaa12 mutants (Grunewald et al., 2013).

Many genes linked to embryogenic root formation were documented to be a direct downstream target of the MP/ARF5. Also, in post-embryonic development, MP/ARF5 is involved in root formation. MP/ARF5 directly regulates the expression of MIR390 in the primary root meristem (Dastidar et al., 2019); miR390 triggers the biogenesis of 21 nucleotide secondary siRNAs called ta-siRNAs targeting ARF2, ETT/ARF3, and ARF4 transcripts (Marin et al., 2010). MP/ARF5 is also expressed during lateral root founder cell specification, nuclear migration, and lateral root initiation processes. ETHYLENE RESPONSE FACTOR 114 (ERF114) and ERF115 activate auxin signaling via induction of MP/ARF5 and promote lateral root development (Canher et al., 2020, 2022). In dividing pericycle cells, the BDL/IAA12–MP/ARF5-mediated auxin response guarantees an organized lateral root patterning downstream of SOLITARY-ROOT (SLR)/IAA14 (de Smet et al., 2010).

Less is known about the role of MP/ARF5 in SAM and cotyledon formation during embryogenesis. MP/ARF5 controls the expression of the DORNRÖSCHEN/ENHANCER OF SHOOT REGENERATION 1 (DRN/ESR1) gene, encoding an AP2 transcription factor involved in embryonic patterning, auxin perception, and transport (Chandler et al., 2008; Cole et al., 2009). Based on ChIP and phenotypic analysis, it was demonstrated that MP/ARF5 positively regulates DRN/ESR1 via binding to two AuxRE motifs in its promoter. This genetic interaction between MP/ARF5 and DRN/ESR1 is also essential for DRN/ESR1 to contribute to the development of embryonic cotyledons (Cole et al., 2009). These results align with studies that showed a lack of pDRN::GFP activity in the cotyledon tips of the mp/arf5 insertional mutant (Okushima et al., 2005). The role of ARF5 in fruit development has been confirmed in tomatoes, where SlARF5 plays a role in fruit initiation and is associated with auxin/gibberellic acid crosstalk (S. Liu et al., 2018), and the members of the LcARF5A/B clade may promote fruit abscission in litchi (Zhang et al., 2019). In apples, MdARF5 directly binds to the promoters of ethylene biosynthetic genes MdACS3a, MdACS1, and MdACO1 to induce their expression, initiating fruit ripening (Yue et al., 2020).

Polar auxin transport, properly organized cell divisions, cell specification, and intercellular communication are required to form the zygotic embryo—MP/ARF5 appears to coordinate all these aspects.

Leaf formation and establishment of its abaxial–adaxial polarity

During embryogenic development, MP/ARF5 governs the formation of embryo apical–basal polarity. MP/ARF5 is also responsible for establishing organ polarity during post-embryonic development. We can distinguish three domains in a leaf: adaxial, abaxial, and, on the borderline, middle domains. The adaxial domain has lower auxin levels than the abaxial, leading to a specific high auxin response in the middle domain (Qi et al., 2014; Guan et al., 2017; Burian et al., 2022). In early leaf developmental stages, MP/ARF5 is expressed in leaf primordia and most leaf regions (Hardtke et al., 2004; Wenzel et al., 2007; Krogan and Berleth, 2012; Schuetz et al., 2019; Burian et al., 2022). The initial broad expression domain of MP/ARF5 is gradually restricted to the leaf veins, and adaxial and middle domains (Pekker et al., 2005; Guan et al., 2017) (Fig. 4D). The Arabidopsis mp/arf5 and tomato slarf5 mutant leaf blades have a reduced surface area (Fig. 4E) (Israeli et al., 2019; Schuetz et al., 2019), and both arf3 arf5 and arf5 arf7 double mutants could not form leaves and displayed enlarged meristems, indicating that MP/ARF5 functions synergistically with ETT/ARF3 and NPH4/ARF7 (Schuetz et al., 2019). The adaxial–abaxial polarity is altered in the leaves of the gain-of-function mutant MPΔ. The mutant displays an irregular and narrow leaf shape due to restricted laminar expansion, disrupted adaxial–abaxial polarity, and vasculature hypertrophy, compared with the hypotrophy observed in mp/arf5 loss-of-function mutants. It implies that MP/ARF5 plays a prominent role in establishing organ polarity (Krogan and Berleth, 2012). MP/ARF5 directly activates the expression of WUSCHEL-RELATED HOMEOBOX 1 (WOX1) and PRESSED FLOWER (PRS) in the middle domain, leading to an outgrowth of the leaf (Guan et al., 2017) (Fig. 4F).

Vascular tissue formation

MP/ARF5 is strongly expressed in pre-vascular, pre-procambial, procambial, and cambial cells (Fig. 4G), but less abundantly in the already differentiated tissues, namely the phloem and xylem (Schuetz et al., 2019; Agustí and Blázquez, 2020). MP/ARF5 activity is crucial for embryonic and post-embryonic vascular tissue development. The mp/arf5 mutant embryos have an incomplete vascular system and are defective in the formation of the root and the embryonic body axis (Hamann et al., 1999; Hobbie et al., 2000). The mutant embryos also have fewer cell files in the vascular tissue (Fig. 4H) (De Rybel et al., 2013). MP/ARF5 is implicated in leaf vein patterning and development (Fig. 4I). MP/ARF5 and PIN1 expression patterns overlapped in pre-procambial veins. In addition, their respective mutants have leaf vascular defects (Wenzel et al., 2007; Bhatia et al., 2016; Krogan et al., 2016). MP/ARF5 also positively and directly regulates the expression of the HD-ZIPIII gene ARABIDOPSIS THALIANA HOMEOBOX GENE 8 (ATHB8) through binding to the AuxREs in its promoter. ATHB8 is involved in leaf pre-procambial cell fate acquisition (Donner et al., 2009). Similarly, in the woody species Populus tomentosa, PtoIAA9 interacts with PtoARF5 to form a PtoIAA9–PtoARF5 module that directly controls the expression of the homeobox PtoHB7 and PtoHB8 genes, encoding HD-ZIPIII TFs responsible for mediating the secondary xylem development (Xu et al., 2019).

During embryogenesis, TMO5 and its closest homolog, TMO5-LIKE1 (T5L1), play a pivotal role in vascular development in roots. Both genes are expressed in an MP/ARF5-dependent manner in the xylem precursor cells of the root and the vasculature of the globular embryo (Schlereth et al., 2010; De Rybel et al., 2013). The mature embryos and seedlings of the tmo5 t5l1 double mutant display severe defects in the vascular tissue, proving that MP/ARF5 mediates vascular tissue establishment during embryogenesis through its two direct target genes, TMO5 and T5L1 (De Rybel et al., 2013). The heterodimer TMO5–LHW induces the expression of LONELY GUY 3 (LOG3) and LOG4, encoding cytokinin biosynthetic enzymes. Cytokinin is required for the periclinal cell divisions within procambial cells. MP/ARF5 can also induce the expression of the Dof5.8 gene in the pre-procambial stage (Konishi et al., 2015). MP/ARF5 is post-transcriptionally modified during the vascularization process by BIN2-LIKE 1 (BIL1), a glycogen synthase kinase. BIL1 phosphorylates MP/ARF5 and enhances its activity in regulating the expression of ARABIDOPSIS RESPONSE REGULATOR (ARR) genes, ARR7 and ARR715. ARR7 and ARR15 are negative regulators of the cytokinin response, leading to inhibition of cambial activity (Han et al., 2018). Additionally, MP/ARF5 predominantly promotes xylem production by directly activating xylem-related genes and repressing WOX4 (Brackmann et al. 2018). However, the conditional knockdown of MP/ARF5 also leads to the down-regulation of WOX4 (Smetana et al., 2019), questioning the molecular mechanisms by which MP/ARF5 regulates WOX4.

Shoot apical meristem and inflorescence development

The MP/ARF5 protein controls embryonic and post-embryonic SAM development. MP/ARF5 accumulates in both differentiated and undifferentiated SAM cells (Hardtke and Berleth, 1998; Schlereth et al., 2010; Zhao et al., 2010), specifically in the peripheral zone and in the developing primordia, but not in the central zone (Fig. 4J) (Burian et al., 2022). The mp/arf5 mutant initiates rosette leaves at a slightly reduced rate (Fig. 4K) (Bennett et al., 1996; Przemeck et al., 1996; Gälweiler et al., 1998). The development of axillary branches necessitates stem cell maintenance, axillary meristem initiation, differentiation, and axillary bud outgrowth. Axillary meristems are formed from a small number of stem cells with meristematic competence located in cauline leaf axils. During embryonic and post-embryonic development, ARGONAUTE 10 (AGO10), spatiotemporally controlled by auxin, brassinosteroids, and light, promotes embryo and axillary meristem development through miR165/166 sequestration, resulting in increased levels of miR165/166 target transcripts: PHABULOSA (PHB), PHAVOLUTA (PHV), and REVOLUTA (REV). In axils of young leaves, MP/ARF5 activates BRASSINAZOLE-RESISTANT 1 (BZR1) and PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), which directly repress AGO10 transcription and prevent axillary meristem initiation. In axils of older leaves, MP/ARF5 up-regulates AGO10 expression to promote axillary meristem initiation (Turchi et al., 2013; Zhang et al., 2020). Interestingly, MP/ARF5 may be a direct target of PHB, indicating a complex interaction mechanism resembling a feedback loop (Qi et al., 2014; Müller et al., 2016; Guan et al., 2017).

During inflorescence formation, MP/ARF5 accumulates in the central zone, peripheral zone, and the developing flower primordia (Fig. 4L) (Bhatia et al., 2016). MP/ARF5 is crucial for inflorescence development. The mp/arf5 mutant and plants with a constitutive MP/ARF5 overexpression give rise to pin-shaped inflorescences (Fig. 4M) (Hardtke et al., 2004; Carey and Krogan, 2017). The MPΔ lines develop flowers, but floral organs, such as petals, are narrow with marked increased vascularization (Krogan and Berleth, 2012). Besides MP/ARF5, loss-of-function mutations in the genes involved in auxin biosynthesis, the auxin efflux carrier PIN1, and the AGC kinase NAKED PINS IN YUC MUTANTS5 (NPY5) abolish floral meristem formation, resulting in the naked inflorescence stem phenotype (Reinhardt et al., 2000; Vernoux et al., 2000; Cheng et al., 2006, 2008). These observations indicate that auxin biosynthesis, transport, and signaling are indispensable in floral meristem initiation and inflorescence organization. On the one hand, MP/ARF5 contributes to flower initiation by directly up-regulating the primary floral development regulator genes (Fig. 4N) such as LEAFY (LFY), AINTEGUMENTA (ANT), AINTEGUMENTA-LIKE 6/PLETHORA 3 (AIL6/PLT3), FILAMENTOUS FLOWER (FIL), TMO3/CYTOKININ RESPONSE FACTOR 2 (CRF2), SCARECROW (SCR), and SHR (Cole et al., 2009; Krizek, 2009; Yamaguchi et al., 2013; Wu et al., 2015; Bahafid et al., 2022, Preprint). On the other hand, MP/ARF5 monitors flower primordium initiation by contributing to reprogramming cell identities from stem cell descendent to primordium founder cell fate in the inflorescence. Auxin-activated MP/ARF5 recruits proteins from the SWI/SNF chromatin-remodeling complex, which opens the chromatin to the repressing TFs binding the promoter of the essential regulators for flower initiation (Wu et al., 2015). SYD and BRM, two related Arabidopsis SWI/SNF ATPases (Bezhani et al., 2007; Shu et al., 2022), are expressed in flower primordia (Wu et al., 2012). In addition, brm-3 syd-5 double mutants exhibit pin-like inflorescence phenotypes, indicating their crucial role in initiating flower primordium. Interestingly, ChIP experiments identified SYD and BRM binding to regulatory sites of MP/ARF5 targets in the proximity of the AuxREs bound by MP/ARF5. It suggests that MP/ARF5 recruits this SWI/SNF chromatin-remodeling complex to its target loci to loosen the compacted chromatin and triggers the transcriptional activation of these loci (Wu et al., 2015). Additionally, the down-regulation of the TF genes SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS (BP), key pluripotency genes stimulating meristematic fate, is crucial for reproductive primordium initiation. MP/ARF5 contributes indirectly to STM repression by up-regulating FIL. In parallel, MP/ARF5 acts with ETT/ARF3 and ARF4 repressors expressed in incipient reproductive primordia and promoter of flower initiation through histone acetylation (Chung et al., 2019). MP/ARF5 also activates the expression of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6) (Besnard et al., 2014), which encodes a negative regulator of the cytokinin signaling pathway (Mähönen et al., 2006). AHP6 has been shown to non-cell-autonomously suppress meristematic cell activity in the tissue surrounding floral primordia and thus to assist in establishing phyllotaxis, the regular pattern of primordia emergence (Besnard et al., 2014). Notably, it down-regulates, together or in parallel with MP/ARF5 (Zhao et al., 2010), the expression of type-A ARR (Chahtane et al., 2013), negatively regulating cytokinin responses. ARR7 and ARR15 are potent regulators of SAM function and negative regulators of cytokinin signaling, and their promoters are targets of MP/ARF5-mediated auxin signaling (Zhao et al., 2010). It was demonstrated that the up-regulation of SHR expression at sites of organ initiation depends on auxin, acting through MP/ARF5. In the central zone, the SHR-target SCR-LIKE 23 (SCL23) physically interacts with WUSCHEL (WUS), a key regulator of stem cell maintenance. SCL23 and WUS expression is subject to negative feedback regulation from stem cells through the CLAVATA (CLV) signaling pathway (Bahafid et al., 2022, Preprint). MP/ARF5 mediates auxin signaling responses in the peripheral zone and, in the central zone, binds directly to the promoter of DRN and represses its activity (Luo et al., 2018). DRN is expressed in the center of the meristem, where it up-regulates CLV3 expression, suggesting a mechanism where MP/ARF5-mediated auxin signaling controls stem cell activity by regulating DRN expression. This provides a scenario where WUS establishes a minimal auxin signaling activity in the central zone. This activity requires MP/ARF5 to fine-tune CLV3 activity, helping to regulate stem cell homeostasis. For organ primordia specification in the peripheral zone, temporal information carried by auxin is essential, as is the repression of STM expression by ETT/ARF3, ARF4, and MP/ARF5 auxin signaling effectors (Luo et al., 2018; Chung et al., 2019; Ma et al., 2019).

The general absence of H3K27me3, a hallmark of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2) activity, at the MP/ARF5 locus indicates that their regulation does not rely on this epigenetic mechanism. Interestingly, the H3K4me3 marks, and accessible regions identified by FANS-ATAC on the MP/ARF5 promoter, were present, which suggests a constitutively active MP/ARF5 locus. These properties suggest that the chromatin configuration of the MP/ARF5 locus allows it to be actively transcribed in different tissues and at different developmental stages. They also imply that the spatial expression pattern specific to the MP/ARF5 gene does not result primarily from alternative chromatin states with contrasting accessibility. The regulatory system of MP/ARF5 expression might be based on the transcriptional activators, repressors, and post-translational modifications that modulate the activity of MP/ARF5. A few proteins can potentially regulate MP/ARF5 expression, namely KNOTTED-LIKE FROM ARABIDOPSIS THALIANA 1 (KNAT1), SCHLAFMUTZE (SMZ), Dof1.8, and LOB DOMAIN-CONTAINING PROTEIN 3 (LBD3). In particular, Dof1.8 may repress MP/ARF5 expression, as MP/ARF5 and DOF1.8 genes show complementary expression patterns (Truskina et al., 2021). It cannot be excluded that MP/ARF5 may also be regulated during the translation process via the GTPase ROP2–TARGET OF RAPAMYCIN (TOR) pathway. The levels of translation reinitiation of MP/ARF5 can be increased several fold without affecting mRNA stability (Schepetilnikov and Ryabov, 2017).

Role of MP/ARF5 in developmental processes in vitro

MP/ARF5 is crucial for indirect de novo shoot organogenesis, and its expression determines the acquisition of shoot progenitor cell identity (Liu et al., 2022, Preprint). Callus formation is inhibited in the mp/arf5 mutant, and explants are impaired in producing shoots (Zhang et al., 2021). In contrast, transgenic lines with a dominantly acting variant of MPΔ showed increased regeneration, even from recalcitrant tissues (Ckurshumova et al., 2014; Gonzalez et al., 2021). Interestingly, the MPΔ variant is associated with a higher WUS expression in spots where the shoot formation occurred. MP/ARF5 can positively regulate downstream cytokinin response factor TMO3/CRF2 (Ckurshumova et al., 2014). The crf2 mutation abolishes the formation of SAMs on calli of MPΔ cotyledon explants and diminishes the WUS and STM expression. In contrast, TMO3/CRF2 overexpression confers higher shoot-forming properties in a wild-type background. Thus, the shoot-promoting influence of MPΔ is likely to be partially conferred by TMO3/CRF2. ARF4 is also involved in shoot regeneration and depends on the presence of MP/ARF5 (Zhang et al., 2021). Additionally, MP/ARF5 is involved in the auxin-dependent induction of somatic embryogenesis. The MP/ARF5 gene is the most highly up-regulated member of the ARF family during somatic embryogenesis induction, and the mp/arf5 mutant rarely regenerates somatic embryos (Wójcikowska and Gaj, 2017).

Stress response

ARF5 is involved in abiotic and biotic stress responses. In Ipomoea batatas, IbARF5 regulates salt and drought tolerance, and the overexpression of IbARF5 up-regulates ABA biosynthetic genes (Kang et al., 2018). In lettuce, the ARF5 gene was studied to understand the molecular mechanism underlying the phenomenon of early bolting caused by high temperatures and reducing the quality and taste of the lettuce leaf. The relative ARF5 expression was significantly down-regulated in lettuce varieties susceptible to bolting under high temperatures, suggesting that ARF5 may be involved in vegetable bolting (Pan et al., 2019). The Osarf5 mutant showed an increased tolerance or enhanced resistance to rice dwarf virus infection, lower disease incidence, and accumulation of rice dwarf virus proteins (Qin et al., 2020). It was demonstrated that auxin inhibits stomatal development through nuclear TIR1/AFB-mediated auxin signaling and that MP/ARF5 binds to the STOMAGEN promoter, inhibiting its expression in mesophyll and stopping stomatal development (Zhang et al., 2014).

Concluding remarks and future perspectives

Among the members of the ARF family, MP/ARF5 is at the center of scientific interest. MP/ARF5 is the key TF regulating almost every aspect of plant development. Research on it contributes knowledge about the regulation of gene expression, particularly in response to auxin. Analytical tools allow the study of the interactions between MP/ARF5 and DNA, as well as MP/ARF5 and other proteins, revealing an extremely complex interaction network. Many genes that may be under the direct control of MP/ARF5 have been recognized. The challenge of the next few years will be to learn how the level of MP/ARF5 as an oligomer, dimer, or monomer affects its function. It will also be essential to discover in detail the activator and repressor regulatory elements acting upstream of MP/ARF5. It is interesting to study epigenetic factors affecting the activity of the MP/ARF5 locus, especially in the context of recent data indicating that this locus is constantly open (Truskina et al., 2021). Are modifications of DNA and histones or the composition of nucleosomes containing various histone variants responsible for this open chromatin state? This question remains unanswered. When single-cell DNA and histone modification profiling are widely adopted in plants, it will help to uncover how MP/ARF5 is epigenetically regulated. Another open question remains: is MP/ARF5 essential and acting as a key player in plant development? Is MP/ARF5 unique among other ARF genes? Or is there a bias in this statement because ARF5 has been and is the focus of so many studies? Many years of detailed research on the process of zygotic embryogenesis have led to the identification of 510 EMBRYO-DEFECTIVE (EMB) genes in Arabidopsis involved in the zygotic embryogenic process, whose mutation induces defects during embryogenesis (Meinke, 2020). It seems likely that most genes encoding TFs will play a key role at some stages of plant development in vivo. However, numerous studies are still needed to identify the function of all TF genes, especially those acting redundantly. Until then, we can conclude that MP/ARF5 reigns supreme over the other members of the ARFs and most of the genes encoding TFs.

Glossary

Abbreviations

ARF

AUXIN RESPONSE FACTOR

ARR

ARABIDOPSIS RESPONSE REGULATOR

Aux/IAA

AUXIN/INDOLE-3-ACETIC ACID

AuxRE

auxin-responsive element

BDL

BODENLOS

BRM

BRAHMA

CRF2

CYTOKININ RESPONSE FACTOR 2

DBD

DNA-binding domain

DD

dimerization domain

DRF

DORNRÖSCHEN

ERF

ETHYLENE RESPONSE FACTOR

ESR1

ENHANCER OF SHOOT REGENERATION 1

ETT

ETTIN

HDA19

HISTONE DEACETYLASE 19

IAA

indole-3-acetic acid

IAM

indolyl-3-acetamide

IAN

indolyl-3-acetonitrile

MP

MONOPTEROS

MR

middle region

NPH4

NON-PHOTOTROPHIC HYPOCOTYL 4

PB1

Phox/Bem1

PIN

PIN-FORMED

SAM

shoot apical meristem

TF

transcription factor

TMO

TARGET OF MONOPTEROS

WUS

WUSCHEL

Author contributions

BW and SB: design, reviewing the referenced articles, and writing; BW: preparing the figures; BW and SB: preparing the tables; HSR: design and revising the manuscript. All authors approved the final version of the text.

Conflict of interest

No conflict of interest was declared.

Funding

This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic with the European Regional Development Fund-Project ‘Centre for Experimental Plant Biology’ (no. CZ.02.1.01/0.0/0.0/16_019/0000738). BW was supported by the Polish National Agency for Academic Exchange (BPN/BEK/2021/1/00278/U/00001). The funding bodies had no role in the study design, collection, analysis, data interpretation, and manuscript writing.