Save your TIRs – more to auxin than meets the eye

Summary Auxin has long been known as an important regulator of plant growth and development. Classical studies in auxin biology have uncovered a ‘canonical’ transcriptional auxin‐signalling pathway involving the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F‐BOX (TIR1/AFB) receptors. TIR1/AFB perception of auxin triggers the degradation of repressors and the derepression of auxin‐responsive genes. Nevertheless, the canonical pathway cannot account for all aspects of auxin biology, such as physiological responses that are too rapid for transcriptional regulation. This Tansley insight will explore several ‘non‐canonical’ pathways that have been described in recent years mediating fast auxin responses. We focus on the interplay between a nontranscriptional branch of TIR1/AFB signalling and a TRANSMEMBRANE KINASE1 (TMK1)‐mediated pathway in root acid growth. Other developmental aspects involving the TMKs and their association with the controversial AUXIN‐BINDING PROTEIN 1 (ABP1) will be discussed. Finally, we provide an updated overview of the ETTIN (ETT)‐mediated pathway in contexts outside of gynoecium development.


I. Introduction
The plant hormone auxin and its omnipresent effects on plant growth and development have been the focus of intense studies for more than a century (reviewed in Enders & Strader, 2015).In the last decades, modern molecular genetics and biophysical studies have led to the elucidation of molecular signalling pathways by which auxin exerts its effect in response to both developmental, mechanical and environmental cues (McLaughlin et al., 2021;Ramos B aez & Nemhauser, 2021;Yu et al., 2022).
Auxin functions primarily by regulating gene expression through a pathway that we will refer to as the 'canonical' auxin signalling pathway (Weijers & Wagner, 2016;Leyser, 2018).The core components of this mechanism include members of the AUXIN RESPONSE FACTOR (ARF) family of transcription factors, which bind to specific DNA cis-elements of the genes that they regulate.In the absence of auxin, ARF proteins interact with AUX/ IAA transcriptional repressors via a C-terminal Phox/Bem1p (PB1) domain.When auxin binds F-box proteins of the TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX (TIR1/AFB) family, the affinity of TIR1/AFB towards AUX/IAA increases resulting in the degradation of AUX/IAAs and subsequent derepression of ARF-regulated, auxin-responsive gene expression (Dharmasiri et al., 2005;Kepinski & Leyser, 2005).
Whilst the canonical pathway has the most predominant effect on auxin-mediated gene expression and is highly conserved in plant evolution at least since the emergence of land plants (Mutte et al., 2018;Kato et al., 2020), an increasing amount of data show that auxin also functions via alternative mechanisms.This is perhaps not surprising given the wide range of processes that are affected by auxin.The molecular details of how some of these mechanisms function are beginning to be elucidated, and a common trend appears to be that they often facilitate fast responses; some so fast indeed that they are inconsistent with a pathway that relies on transcription-translation to reach a certain state such as the canonical pathway.Here, we will provide an overview of the latest discoveries in noncanonical effects of auxin on plant growth and development and point out some of the key questions that must be answered to elucidate the role of such alternative auxin signalling pathways.

II. Acid growth
Plant cell size is controlled by a balance between turgor pressure and mechanical properties of the cell wall.For cells to expand, increased cell wall extensibility must therefore accompany an increase in turgor pressure (Lockhart, 1965).The classic Acid Growth Theory suggests that apoplast acidification activates expansins leading to cell wall alterations that facilitate turgor pressure-mediated cell expansion (Kutschera, 1994;Hager, 2003;Takahashi et al., 2012;Arsuffi & Braybrook, 2018).Auxin regulates apoplastic pH through H + -efflux by controlling the activity of plasma membrane proton-pumping ATPases (PM H + -ATPase) in both shoots and roots; however, the mechanism by which auxin mediates its effect differs between these tissues (Li et al., 2022).Whilst auxin facilitates acidification of the apoplast in the shoot through PM H + -ATPase activation, auxin promotes alkalinisation of the apoplast in roots via rapid activation of H + influx.These two modes of action have differing effects on cell growth with apoplast acidification promoting and apoplast alkalinisation inhibiting cell expansion (Spartz et al., 2014;Fendrych et al., 2016;Barbez et al., 2017;Li et al., 2021).
PM H + -ATPases are activated through phosphorylation in response to auxin via at least two distinct mechanisms.First, auxin induces rapid PM H + -ATPase phosphorylation directly by cell surface-localised TRANSMEMBRANE KINASE1 (TMK1) (Li et al., 2021).Second, an indirect effect on PM H + -ATPase phosphorylation by auxin occurs via a canonical nuclear auxin response whereby TIR1/AFB-mediated auxin-induced gene expression leads to inhibition of PP2C phosphatase activity on H + -ATPases (Spartz et al., 2014;Ren et al., 2018;Fig. 1).
Members of the TIR1/AFB family of auxin receptors are also required for rapid auxin responses in the root with reduced apoplast alkalinisation, membrane polarisation and root growth inhibition observed in tir1 and afb mutants (Fendrych et al., 2018).However, the speed of the response precludes a mechanism based on gene expression as in canonical auxin signalling and must instead represent a transcription-independent auxin response.Indeed, such a nontranscriptional branch of TIR1/AFB function has been proposed for stimulating H + influx (Li et al., 2022).Although the molecular mechanism by which this parallel TIR1/AFB activity mediates its effect is currently unknown, the TIR1/AFB protein, AFB1 from Arabidopsis thaliana (Arabidopsis) may be involved as it was recently found to be localised in the cytoplasm of root cells and afb1 mutants exhibit reduced auxin sensitivity in terms of root growth inhibition (Prigge et al., 2020;Li et al., 2021;Serre et al., 2021;Fig. 1).

III. The TRANSMEMBRANE KINASE -AUXIN-BINDING PROTEIN 1 pathway
As previously mentioned, auxin induces rapid apoplast acidification in shoots for cell elongation while the opposite is true in roots where auxin triggers apoplast alkalinisation and growth inhibition.Recent work has uncovered roles for the TMKs in this contrasting auxin effect.The TMKs exist as a family of four receptor-like kinases in Arabidopsis and were proposed as docking proteins for AUXIN-BINDING PROTEIN 1 (ABP1) on the extracellular surface mediating intracellular responses via their cytoplasmic domain (Xu et al., 2014).While the role of ABP1 in this pathway was controversial until very recently, the function of the TMKs, especially that of TMK1, in mediating auxin responses in various aspects of plant development has been made more strongly.
Perception of extracellular auxin changes the biochemical properties of the cytosolic C-terminal kinase domain of TMKs to mediate intracellular signalling cascades.In hypocotyls, TMK1/4 were shown to phosphorylate and activate PM H + -ATPases leading to apoplast acidification and subsequent cell elongation (Lin et al., 2021).Although the TMKs were also demonstrated to activate PM H + -ATPases in root cells, the effect is opposite to that in hypocotyl cells leading to apoplast alkalinisation and growth inhibition (Li et al., 2021).Nonetheless, this discrepancy was resolved when it was demonstrated that the TIR1/AFBs were sufficient to induce auxin-dependent apoplast alkalinisation and that both TIR1/AFB-and TMK-mediated pathways converge to antagonistically regulate apoplast pH for fine-tuning of root growth rates (Li et al., 2021).
Another facet of TMK-mediated auxin signalling involves the activation of Rho-like GTPases (ROPs).ROP2 and ROP6 from Arabidopsis promote auxin-dependent leaf pavement cell interdigitation by remodelling the cytoskeleton and regulating endocytosis of PIN auxin efflux carriers (Xu et al., 2010;Nagawa et al., 2012).Active ROP2/6 accumulation was compromised, and cytoskeletal aberrations were observed in the tmk1/2/3/4 loss-of-function mutant (Xu et al., 2014).In root gravitropism, ROP6 controls the asymmetric redistribution of PIN2 in response to a shift in the gravity vector.An unstructured protein, MEMBRANE-ASSOCIATED KINASE REGULATOR2 (MAKR2), was shown to antagonise ROP6 signalling by interacting with and inhibiting TMK1 activity at the plasma membrane (Marques-Bueno et al., 2021).Auxin perception through the TMK1-mediated pathway triggers the internalisation of MAKR2 into the cytosol to release the inhibition of ROP6 signalling and PIN2 relocalisation (Fig. 1).
In the context of apical hook development, the TMKs were demonstrated to interact with the noncanonical AUX/IAAs, IAA32 and IAA34 through their C-terminal domain (Cao et al., 2019).

New Phytologist
Apical hook bending requires an asymmetric auxin gradient with higher auxin levels on the concave side (Zadnikova et al., 2010(Zadnikova et al., , 2016)).Exogenous auxin was unable to rescue the tmk1 impaired hook bending phenotype, hence implicating a role for TMK1 in downstream signalling.Indeed, it was shown that higher auxin levels on the concave side promoted cleavage of the TMK1 Cterminus by the DA1 family of peptidases (Cao et al., 2019;Gu et al., 2022).While auxin promotes the degradation of canonical  (Dharmasiri et al., 2005;Kepinski & Leyser, 2005).In acid growth, the TIR1/AFB-mediated pathway induces the expression of SMALL AUXIN UP RNA19 (SAUR19) which inhibits the PP2C-D protein phosphatases, allowing H + efflux through H + ATPases and cell wall acidification (Spartz et al., 2014;Ren et al., 2018).The cytosolic AFB1 has also been shown to mediate apoplast alkalinisation but the mechanism is yet to be elucidated (Prigge et al., 2020;Li et al., 2021;Serre et al., 2021).Apoplast acidification is also induced by the phosphorylation of H + ATPases by the TMKs (Li et al., 2021;Lin et al., 2021).ABP1 is secreted from the endoplasmic reticulum (ER) to the plasma membrane (PM) to function as the auxin receptor in complex with the TMKs (Friml et al., 2022).The TMK-ABP1 complex activate ROPs and promotes the internalisation of MAKR2, an antagonist of ROP signalling (Xu et al., 2014;Marques-Bueno et al., 2021).Other TMK-ABP1 activated pathways include the MAPK pathway through the MKK4/5-MPK3/6 module and abscisic acid signalling through ABA INSENSITIVE 1 and 2 (ABI1/2) (Huang et al., 2019;Yang et al., 2021).(b) Auxin perception also promotes DA1-dependent cleavage of the TMK C-terminus domain, which enters the nucleus and phosphorylates IAA32/34 to prevent their degradation (Cao et al., 2019;Gu et al., 2022).IAA32/34 then promotes growth repression on the concave side of the apical hook through their antagonism of ARF activity.(c) ETT interacts with TPL and other transcription factors (TFs) through its ES domain in an auxin-sensitive manner (Simonini et al., 2016(Simonini et al., , 2018;;Kuhn et al., 2020).Direct binding of auxin to ETT disrupts these protein-protein interactions and possibly promotes interactions with partners associated with the activation of gene expression.Auxin is shown as Indole-3-Acetic Acid.
AUX/IAAs, IAA32/34 lack the degron motif and instead accumulate upon auxin treatment.This stabilisation of IAA32/ 34 is mediated by their phosphorylation facilitated by the cleaved TMK1 C-terminal kinase domain, thus allowing them to regulate downstream gene expression for hook development (Cao et al., 2019).
As the TMKs become implicated in many more downstream signalling cascades, such as the Mitogen-Activated Protein Kinase (MAPK) or abscisic acid signalling pathways, it is clear that their kinase activity is integral throughout plant development (Huang et al., 2019;Yang et al., 2021).Nonetheless, the mechanism as to how the TMKs perceive auxin was enigmatic.It was proposed that the TMKs function downstream of ABP1 (Xu et al., 2014).Upon its discovery, ABP1 was considered an important auxin-binding protein mediating rapid nontranscriptional responses based on biochemical and genetical analyses (Hertel et al., 1972;Hesse et al., 1989).Later, it was revealed that the reported phenotypes of the original abp1 mutants were due to the disruption of a neighbouring gene (Gao et al., 2015;Michalko et al., 2015;reviewed in Napier, 2021).However, recent work by Friml et al. (2022) dissipates the ambiguity surrounding the physiological and developmental functions of ABP1.
First, it was shown that ABP1 can bind auxin at apoplastic pH, and this was strengthened by the discovery that ABP1 is partly secreted to the apoplast.Furthermore, analyses of the phosphoproteomes of confirmed null mutants of tmk1 and abp1 revealed hypo-phosphorylation of thousands of targets that are rapidly phosphorylated upon auxin treatment in wild-type plants.Auxininduced cytoplasmic streaming and vasculature regeneration were shown to be disrupted in abp1 and tmk mutants, and the ABP1 (M2X) allele that is defective in auxin-binding failed to complement these phenotypes (Friml et al., 2022).Hence, at this point in time, ABP1 has emerged from its controversial past as a legitimate auxin-binding protein that functions as a receptor with its TMK partners in a subset of fast auxin responses and regenerative development (Fig. 1).

IV. The ETTIN (ETT)-mediated pathway
ETTIN, also known as ARF3, belongs to the B class of ARFs and lacks the PB1 domain necessary for interaction with the AUX/ IAAs, thus excluding its participation in the canonical pathway (Sessions et al., 1997;Finet et al., 2010).Instead, ETT possesses a unique C-terminal domain termed the ETT-Specific (ES) domain that is poorly conserved in overall primary structure between seed plant orthologues but contains phylogenetically well-defined motifs.
The B class ARFs are generally considered to be transcriptional repressors based on biochemical data in transfected carrot suspension protoplasts (Tiwari et al., 2003).Although ETT falls into the B class category based on sequence comparison, it has been demonstrated that ETT has both repressive and activating activities and regulates a subset of its target genes in an auxin-dependent manner (Simonini et al., 2017).Protein-protein interaction between ETT and transcription factors from a wide range of different families is disrupted by auxin (Simonini et al., 2016).The ES domain is indispensable for the auxin-sensitivity of the ETT protein, and mutations in conserved motifs in the ES domain abolish the ability of ETT to sense auxin (Simonini et al., 2016(Simonini et al., , 2018;;Kuhn et al., 2020).
A study by Kuhn et al. (2020) sheds light on the mechanism as to how auxin modulates ETT activity to regulate target genes in a bidirectional manner.Independent biochemical techniques demonstrated the ability of the ES domain to directly bind auxin, and a conserved tryptophan (W505) residue in the ES domain was shown to be important for auxin binding.When auxin levels are low, ETT was shown to interact directly with members of the TOPLESS/TOPLESS-RELATED (TPL/TPR) family through its ES domain, causing the depletion of histone H3K27 acetylation in the promoters of target genes keeping them transcriptionally repressed.When auxin levels increase, ETT binds auxin, disrupting the interaction between ETT and TPL and H3K27 acetylation at the promoters of ETT-regulated genes is restored, resulting in gene expression (Fig. 1).
While a role for the ETT-mediated pathway has been described for gynoecium development, it is still unknown whether this pathway function in other developmental contexts.Given the pleiotropic phenotypes of the auxin-insensitive ETT 2CS mutant, it is likely that ETT-mediated auxin signalling also functions outside the gynoecium (Simonini et al., 2016).ETTIN has been characterised as an abaxial tissue polarity determinant in leaf development and plays important roles in the shoot apical meristem for phyllotaxis and organogenesis (Pekker et al., 2005;Galvan-Ampudia et al., 2020;Burian et al., 2022;Zhang et al., 2022).It was observed that both time of exposure to auxin and auxin concentration affected the competence of cells in the shoot apical meristem to respond to auxin through the downstream output of the DR5 reporter (Galvan-Ampudia et al., 2020).Intriguingly, this DR5 readout of auxin signalling is abolished in the ett-22 mutant and given the defective phyllotaxis of ett mutants, it is possible that ETT-mediated auxin signalling is important for timely organogenesis.
Classical studies in leaf development have revealed that the development of a flat leaf necessitates the juxtaposition of abaxial and adaxial tissue domains (Waites & Hudson, 1995).ETTIN is known to interact with other abaxial domain transcription factors, some in an auxin-sensitive manner, to mediate flat leaf development (Pekker et al., 2005;Simonini et al., 2016).Recently, Burian et al. (2022) demonstrated that the delineation of the adaxial and abaxial boundary is driven by an asymmetric auxin transcriptional output from a uniform auxin field.Interestingly, it was found that ETT acts as an adaxial determinant in the very early stages of leaf primordia development and that its expression pattern matched that of the DR5v2 auxin response reporter.It is tempting to speculate that ETT-mediated auxin signalling might contribute and regulate ETT's role in adaxial and abaxial polarity determination, but more investigation into the downstream auxin-sensitive proteome and transcriptome of both domains is necessary.

V. Conclusions and perspectives
The canonical auxin signalling pathway based on the TIR1/AFB-AUX/IAA-ARF module plays a pivotal role in transducing the

Review
Tansley insight New Phytologist response to auxin in numerous processes.However, it is interesting how long-standing aspects of auxin biology such as the Acid Growth Theory, rapid growth responses and changes in polarity are based on auxin signalling mechanisms that are different from the canonical pathway.Particularly, it is exciting how these mechanisms are now being uncovered and characterised at the molecular level.One common factor between these noncanonical pathways is that they do not involve degradation of a repressor and may therefore immediately switch between states depending on the auxin status.New aspects of the canonical signalling are also still being uncovered.It was recently demonstrated that a protein domain in TIR1/AFB proteins possesses adenylate cyclase activity and thus generate cAMP upon auxin perception (Qi et al., 2022).cAMP production was necessary for TIR1/AFB function in auxin-induced transcriptional regulation, long-term root growth inhibition and gravitropism, but not for rapid responses.This example highlights how the different auxin-response pathways may interact to increase versatility and specificity of auxin responses, and such studies will no doubt form an important direction for future studies into the role of auxin.

Fig. 1
Fig. 1 Auxin signalling pathways in plants.(a) The 'canonical' pathway involves the perception of auxin by the TIR1/AFB-SCF complex which facilitates the degradation of AUX/IAA repressors by the 26 S proteasome, freeing the Class A ARFs to mediate gene expression(Dharmasiri et al., 2005;Kepinski & Leyser, 2005).In acid growth, the TIR1/AFB-mediated pathway induces the expression of SMALL AUXIN UP RNA19 (SAUR19) which inhibits the PP2C-D protein phosphatases, allowing H + efflux through H + ATPases and cell wall acidification(Spartz et al., 2014;Ren et al., 2018).The cytosolic AFB1 has also been shown to mediate apoplast alkalinisation but the mechanism is yet to be elucidated(Prigge et al., 2020;Li et al., 2021;Serre et al., 2021).Apoplast acidification is also induced by the phosphorylation of H + ATPases by the TMKs(Li et al., 2021;Lin et al., 2021).ABP1 is secreted from the endoplasmic reticulum (ER) to the plasma membrane (PM) to function as the auxin receptor in complex with the TMKs(Friml et al., 2022).The TMK-ABP1 complex activate ROPs and promotes the internalisation of MAKR2, an antagonist of ROP signalling(Xu et al., 2014;Marques-Bueno et al., 2021).Other TMK-ABP1 activated pathways include the MAPK pathway through the MKK4/5-MPK3/6 module and abscisic acid signalling through ABA INSENSITIVE 1 and 2 (ABI1/2)(Huang et al., 2019;Yang et al., 2021).(b) Auxin perception also promotes DA1-dependent cleavage of the TMK C-terminus domain, which enters the nucleus and phosphorylates IAA32/34 to prevent their degradation(Cao et al., 2019;Gu et al., 2022).IAA32/34 then promotes growth repression on the concave side of the apical hook through their antagonism of ARF activity.(c) ETT interacts with TPL and other transcription factors (TFs) through its ES domain in an auxin-sensitive manner(Simonini et al., 2016(Simonini et al., , 2018;;Kuhn et al., 2020).Direct binding of auxin to ETT disrupts these protein-protein interactions and possibly promotes interactions with partners associated with the activation of gene expression.Auxin is shown as Indole-3-Acetic Acid.