Auxin-regulated Gene Expression Research Articles

Quantitative imaging reveals the role of MpARF proteasomal degradation during gemma germination

The auxin signaling molecule controls a variety of growth and developmental processes in land plants. Auxin regulates gene expression through a nuclear auxin signaling pathway (NAP) consisting of the ubiquitin ligase auxin receptor TIR1/AFB, its Aux/IAA degradation substrate, and DNA-binding ARF transcription factors. Although extensive qualitative understanding of the pathway and its interactions has been obtained, mostly by studying the flowering plant Arabidopsis thaliana, it remains unknown how these translate to quantitative system behavior in vivo, a problem that is confounded by the large NAP gene families in most species. Here, we used the minimal NAP of the liverwort Marchantia polymorpha to quantitatively map NAP protein accumulation and dynamics in vivo through the use of knockin fluorescent fusion proteins. Beyond revealing the dynamic native accumulation profile of the entire NAP protein network, we discovered that the two central ARFs, MpARF1 and MpARF2, are proteasomally degraded. This auxin-independent degradation tunes ARF protein stoichiometry to favor gene activation, thereby reprogramming auxin response during the developmental progression. Thus, quantitative analysis of the entire NAP has enabled us to identify ARF degradation and the stoichiometries of activator and repressor ARFs as a potential mechanism for control of gemma germination.

A non-canonical Aux/IAA gene MsIAA32 regulates peltate glandular trichome development in spearmint.

Phytohormone auxin controls various aspects of plant growth and development. The typical auxin signalling involves the degradation of canonical Aux/IAA proteins upon auxin perception releasing the auxin response factors (ARF) to activate auxin-regulated gene expression. Extensive research has been pursued in deciphering the role of canonical Aux/IAAs, however, the function of non-canonical Aux/IAA genes remains elusive. Here we identified a non-canonical Aux/IAA gene, MsIAA32 from spearmint (Mentha spicata), which lacks the TIR1-binding domain and shows its involvement in the development of peltate glandular trichomes (PGT), which are the sites for production and storage of commercially important essential oils. Using yeast two-hybrid studies, two canonical Aux/IAAs, MsIAA3, MsIAA4 and an ARF, MsARF3 were identified as the preferred binding partners of MsIAA32. Expression of a R2R3-MYB gene MsMYB36 and a cyclin gene MsCycB2-4 was altered in MsIAA32 suppressed plants indicating that these genes are possible downstream targets of MsIAA32 mediated signalling. Ectopic expression of MsIAA32 in Arabidopsis affected non-glandular trichome formation along with other auxin related developmental traits. Our findings establish the role of non-canonical Aux/IAA mediated auxin signalling in PGT development and reveal species-specific functionalization of Aux/IAAs.

CIN-like TCP13 is essential for plant growth regulation under dehydration stress

Key messageA dehydration-inducible Arabidopsis CIN-like TCP gene, TCP13, acts as a key regulator of plant growth in leaves and roots under dehydration stress conditions.Plants modulate their shape and growth in response to environmental stress. However, regulatory mechanisms underlying the changes in shape and growth under environmental stress remain elusive. The CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) family of transcription factors (TFs) are key regulators for limiting the growth of leaves through negative effect of auxin response. Here, we report that stress-inducible CIN-like TCP13 plays a key role in inducing morphological changes in leaves and growth regulation in leaves and roots that confer dehydration stress tolerance in Arabidopsis thaliana. Transgenic Arabidopsis plants overexpressing TCP13 (35Spro::TCP13OX) exhibited leaf rolling, and reduced leaf growth under osmotic stress. The 35Spro::TCP13OX transgenic leaves showed decreased water loss from leaves, and enhanced dehydration tolerance compared with their control counterparts. Plants overexpressing a chimeric repressor domain SRDX-fused TCP13 (TCP13pro::TCP13SRDX) showed severely serrated leaves and enhanced root growth. Transcriptome analysis of TCP13pro::TCP13SRDX transgenic plants revealed that TCP13 affects the expression of dehydration- and abscisic acid (ABA)-regulated genes. TCP13 is also required for the expression of dehydration-inducible auxin-regulated genes, INDOLE-3-ACETIC ACID5 (IAA5) and LATERAL ORGAN BOUNDARIES (LOB) DOMAIN 1 (LBD1). Furthermore, tcp13 knockout mutant plants showed ABA-insensitive root growth and reduced dehydration-inducible gene expression. Our findings provide new insight into the molecular mechanism of CIN-like TCP that is involved in both auxin and ABA response under dehydration stress.

Multi-Trait Biochemical Features of Metarhizium Species and Their Activities That Stimulate the Growth of Tomato Plants

The interaction between Metarhizium spp., a genus of entomopathogenic fungi, and plant roots may provide a resourceful association to induce host plant growth and to control below- and above-ground pests and diseases. We conducted in vivo and in vitro bioassays using native Brazilian isolates of Metarhizium robertsii (ESALQ 1635), M. humberi (ESALQ 1638), and M. anisopliae (ESALQ 1669) to better understand the possible mechanisms related to plant growth promotion traits and colonization of the root system in the model pot-grown tomato (Solanum lycopersicum L.) miniature cultivar ‘Micro-Tom’. In vivo trials revealed that M. robertsii (ESALQ 1635) or M. humberi (ESALQ 1638) inoculated in tomato seedlings improved vegetative and reproductive traits. Inoculated plants with M. robertsii yielded taller plants, longer roots, and more shoot and root dry matters than M. humberi. The number of flowers, along with fresh and dry weight of fruits were significantly increased by M. robertsii in relation to M. humberi-inoculated or uninoculated control plants. M. robertsii and M. humberi were able to endophytically colonize all tomato tissues by up to 30 days after inoculation. Plants inoculated with either M. robertsii or M. humberi increased auxin-induced GUS expression in the roots at 10 and 30 days after inoculation, confirming that Metarhizium induces auxin-regulated gene expression. We also explored their production of key compounds including enzymes, hormones, and metabolites involved in plant growth promotion. The three fungal species grown with or without exogenous tryptophan were able to produce indole-3-acetic acid (IAA) at different titers. All Metarhizium isolates produced phosphatases, phytases, siderophores and chitinases. Of particular importance, the M. robertsii and M. humberi isolates exhibited similar in vitro biochemical profiles, whereas M. anisopliae and Trichoderma harzianum isolates, demonstrated distinct traits from the others. Taken together, we argue that the M. robertsii isolate is more prone than the M. humberi isolate to endophytically colonize tomato plants resulting in improved growth. However, M. humberi (ESALQ 1638) yielded slightly better production of some metabolites in vitro. Thus, we propose that both isolates of M. robertsii and M. humberi could be explored together, as they possess complimentary properties, for use as plant growth promoters.

UV-B photoreceptor UVR8 interacts with MYB73/MYB77 to regulate auxin responses and lateral root development.

The UV-B photoreceptor UVR8 mediates multiple UV-B responses in plants, but the function of UVR8 in regulating root development has not previously been investigated. Here, we show that UV-B light inhibits Arabidopsis lateral root growth in a UVR8-dependent manner. Monomeric UVR8 inhibits auxin responses in a tissue-autonomous manner and thereby regulates lateral root growth. Genome-wide gene expression analysis demonstrated that auxin and UV-B irradiation antagonistically regulate auxin-regulated gene expression. We further show that UVR8 physically interacts with MYB73/MYB77 (MYB DOMAIN PROTEIN 73/77) in a UV-B-dependent manner. UVR8 inhibits lateral root development via regulation of MYB73/MYB77. When activated by UV-B light, UVR8 localizes to the nucleus and inhibits the DNA-binding activities of MYB73/MYB77 and directly represses the transcription of their target auxin-responsive genes. Our results demonstrate that UV-B light and UVR8 are critical for both shoot morphogenesis and root development. The UV-B-dependent interaction of UVR8 and MYB73/MYB77 serves as an important module that integrates light and auxin signaling and represents a new UVR8 signaling mechanism in plants.

Nucleo-cytoplasmic Partitioning of ARF Proteins Controls Auxin Responses in Arabidopsis thaliana.

The phytohormone auxin plays crucial roles in nearly every aspect of plant growth and development. The auxin response factor (ARF) transcription factor family regulates auxin-responsive gene expression and exhibits nuclear localization in regions of high auxin responsiveness. Here we show that the ARF7 and ARF19 proteins accumulate in micron-sized assemblies within the cytoplasm of tissues with attenuated auxin responsiveness. We found that the intrinsically disordered middle region and the folded PB1 interaction domain of ARFs drive protein assembly formation. Mutation of a single lysine within the PB1 domain abrogates cytoplasmic assemblies, promotes ARF nuclear localization, and results in an altered transcriptome and morphological defects. Our data suggest a model in which ARF nucleo-cytoplasmic partitioning regulates auxin responsiveness, providing a mechanism for cellular competence for auxin signaling.

A novel Ca2+-binding protein that can rapidly transduce auxin responses during root growth.

Signaling cross talks between auxin, a regulator of plant development, and Ca2+, a universal second messenger, have been proposed to modulate developmental plasticity in plants. However, the underlying molecular mechanisms are largely unknown. Here, we report that in Arabidopsis roots, auxin elicits specific Ca2+ signaling patterns that spatially coincide with the expression pattern of auxin-regulated genes. We have identified the single EF-hand Ca2+-binding protein Ca2+-dependent modulator of ICR1 (CMI1) as an interactor of the Rho of plants (ROP) effector interactor of constitutively active ROP (ICR1). CMI1 expression is directly up-regulated by auxin, whereas the loss of function of CMI1 associates with the repression of auxin-induced Ca2+ increases in the lateral root cap and vasculature, indicating that CMI1 represses early auxin responses. In agreement, cmi1 mutants display an increased auxin response including shorter primary roots, longer root hairs, longer hypocotyls, and altered lateral root formation. Binding to ICR1 affects subcellular localization of CMI1 and its function. The interaction between CMI1 and ICR1 is Ca2+-dependent and involves a conserved hydrophobic pocket in CMI1 and calmodulin binding-like domain in ICR1. Remarkably, CMI1 is monomeric in solution and in vitro changes its secondary structure at cellular resting Ca2+ concentrations ranging between 10−9 and 10−8 M. Hence, CMI1 is a Ca2+-dependent transducer of auxin-regulated gene expression, which can function in a cell-specific fashion at steady-state as well as at elevated cellular Ca2+ levels to regulate auxin responses.

Identification and expression profiling of the Aux/IAA gene family in Chinese hickory (Carya cathayensis Sarg.) during the grafting process

Auxin is an essential regulator in various aspects of organism growth and development. Members of the Aux/IAA family of genes encode short-lived nuclear proteins and mediate the responses of auxin-regulated gene expression. Here, the first identification and characterization of 22 cDNAs encoding the open reading frame of the Aux/IAA family in Chinese hickory (named as CcIAA) has been performed. The proteins encoded by these genes contain four whole or partially conserved domains of the Aux/IAA family. Phylogenetic analysis indicated that CcIAAs were unevenly distributed among eight different subgroups. The spatio-specific expression profiles showed that most of the CcIAAs preferentially expressed in specific tissues. Three CcIAA genes, including CcIAA11, CcIAA27a2 and CcIAAx, were predominantly expressed in stem. The predominant expression of CcIAA genes in stems might play important roles in vascular reconnection during the graft process. Furthermore, expression profiles of Aux/IAA genes during the grafting process of Chinese hickory have been analysed. Our data suggested that 19 CcIAAs were down-regulated and 3 CcIAAs (including CcIAA28, CcIAA8a and CcIAA27b) were induced, indicating their specializations during the grafting process. The involvement of CcIAA genes at the early stage after grafting gives us an opportunity to understand the role of auxin signalling in the grafting process.

SELF-PRUNING Acts Synergistically with DIAGEOTROPICA to Guide Auxin Responses and Proper Growth Form.

The SELF PRUNING (SP) gene is a key regulator of growth habit in tomato (Solanum lycopersicum). It is an ortholog of TERMINAL FLOWER1, a phosphatidylethanolamine-binding protein with antiflorigenic activity in Arabidopsis (Arabidopsis thaliana). A spontaneous loss-of-function mutation (sp) has been bred into several industrial tomato cultivars, as it produces a suite of pleiotropic effects that are favorable for mechanical harvesting, including determinate growth habit, short plant stature, and simultaneous fruit ripening. However, the physiological basis for these phenotypic differences has not been thoroughly explained. Here, we show that the sp mutation alters polar auxin transport as well as auxin responses, such as gravitropic curvature and elongation of excised hypocotyl segments. We also demonstrate that free auxin levels and auxin-regulated gene expression patterns are altered in sp mutants. Furthermore, diageotropica, a mutation in a gene encoding a cyclophilin A protein, appears to confer epistatic effects with sp Our results indicate that SP affects the tomato growth habit at least in part by influencing auxin transport and responsiveness. These findings suggest potential novel targets that could be manipulated for controlling plant growth habit and improving productivity.

Plant-plant interactions influence developmental phase transitions, grain productivity and root system architecture in Arabidopsis via auxin and PFT1/MED25 signalling.

Transcriptional regulation of gene expression influences plant growth, environmental interactions and plant-plant communication. Here, we report that population density is a key factor for plant productivity and a major root architectural determinant in Arabidopsis thaliana. When grown in soil at varied densities from 1 to 32 plants, high number of individuals decreased stem growth and accelerated senescence, which negatively correlated with total plant biomass and seed production at the completion of the life cycle. Root morphogenesis was also a major trait modulated by plant density, because an increasing number of individuals grown in vitro showed repression of primary root growth, lateral root formation and root hair development while affecting auxin-regulated gene expression and the levels of auxin transporters PIN1 and PIN2. We also found that mutation of the Mediator complex subunit PFT1/MED25 renders plants insensitive to high density-modulated root traits. Our results suggest that plant density is critical for phase transitions, productivity and root system architecture and reveal a role of Mediator in self-plant recognition.

N,N-dimethyl hexadecylamine and related amines regulate root morphogenesis via jasmonic acid signaling in Arabidopsis thaliana

Plant growth-promoting rhizobacteria are natural inhabitants of roots, colonize diverse monocot and dicot species, and affect several functional traits such as root architecture, adaptation to adverse environments, and protect plants from pathogens. N,N-dimethyl-hexadecylamine (C16-DMA) is a rhizobacterial amino lipid that modulates the postembryonic development of several plants, likely as part of volatile blends. In this work, we evaluated the bioactivity of C16-DMA and other related N,N-dimethyl-amines with varied length and found that inhibition of primary root growth was related to the length of the acyl chain. C16-DMA inhibited primary root growth affecting cell division and elongation, while promoting lateral root formation and root hair growth and density in Arabidopsis thaliana (Arabidopsis) wild-type (WT) seedlings. Interestingly, C16-DMA induced the expression of the jasmonic acid (JA)-responsive gene marker pLOX2:uidA, while JA-related mutants jar1, coi1-1, and myc2 affected on JA biosynthesis and perception, respectively, are compromised in C16-DMA responses. Comparison of auxin-regulated gene expression, root architectural changes in WT, and auxin-related mutants aux1-7, tir1/afb2/afb3, and arf7-1/arf19-1 to C16-DMA shows that the C16-DMA effects occur independently of auxin signaling. Together, these results reveal a novel class of aminolipids modulating root organogenesis via crosstalk with the JA signaling pathway.

Auxin Transport Inhibitor 2,3,5 Triiodobenzoic Acid Induced Direct Shoot Morphogenesis in Millingtonia hortensis L.f.

Tissue culture studies have shown that in vitro response of explants is largely dependent on media composition, auxincytokinin ratio and the choice of explants. Pierik defines the phenomenon wherein the position of explants in the mother plant influences its subsequent in vitro growth and development as topophysis. The plant hormone auxin is assumed to provide positional information for patterning during development. It is precise determination of auxins distributed across tissues and how it is sensed in space and time are uncertain. Interaction between several auxin response factors (ARFs), transcriptional activators and repressors in a complex network, control auxin-regulated gene expression.

Constitutive auxin response in Physcomitrella reveals complex interactions between Aux/IAA and ARF proteins.

The coordinated action of the auxin-sensitive Aux/IAA transcriptional repressors and ARF transcription factors produces complex gene-regulatory networks in plants. Despite their importance, our knowledge of these two protein families is largely based on analysis of stabilized forms of the Aux/IAAs, and studies of a subgroup of ARFs that function as transcriptional activators. To understand how auxin regulates gene expression we generated a Physcomitrella patens line that completely lacks Aux/IAAs. Loss of the repressors causes massive changes in transcription with misregulation of over a third of the annotated genes. Further, we find that the aux/iaa mutant is blind to auxin indicating that auxin regulation of transcription occurs exclusively through Aux/IAA function. We used the aux/iaa mutant as a simplified platform for studies of ARF function and demonstrate that repressing ARFs regulate auxin-induced genes and fine-tune their expression. Further the repressing ARFs coordinate gene induction jointly with activating ARFs and the Aux/IAAs.

Tobacco mosaic virus-directed reprogramming of auxin/indole acetic acid protein transcriptional responses enhances virus phloem loading

Vascular phloem loading has long been recognized as an essential step in the establishment of a systemic virus infection. In this study, an interaction between the replication protein of tobacco mosaic virus (TMV) and phloem-specific auxin/indole acetic acid (Aux/IAA) transcriptional regulators was found to modulate virus phloem loading in an age-dependent manner. Promoter expression studies show that in mature tissues TMV 126/183-kDa-interacting Aux/IAAs predominantly express and accumulate within the nuclei of phloem companion cells (CCs). Furthermore, CC Aux/IAA nuclear localization is disrupted upon infection with an interacting virus. In situ analysis of virus spread shows that the inability to disrupt Aux/IAA CC nuclear localization correlates with a reduced ability to load into the vascular tissue. Subsequent systemic movement assays also demonstrate that a virus capable of disrupting Aux/IAA localization is significantly more competitive at moving out of older plant tissues than a noninteracting virus. Similarly, CC expression and overaccumulation of a degradation-resistant Aux/IAA-interacting protein was found to inhibit TMV accumulation and phloem loading selectively in flowering plants. Transcriptional expression studies demonstrate a role for Aux/IAA-interacting proteins in the regulation of salicylic and jasmonic acid host defense responses as well as virus-specific movement factors, including pectin methylesterase, that are involved in regulating plasmodesmata size-exclusion limits and promoting virus cell-to-cell movement. Combined, these findings indicate that TMV directs the reprogramming of auxin-regulated gene expression within the vascular phloem of mature tissues as a means to enhance phloem loading and systemic spread.

De Novo Assembly of Bitter Gourd Transcriptomes: Gene Expression and Sequence Variations in Gynoecious and Monoecious Lines.

Bitter gourd (Momordica charantia L.) is a nutritious vegetable crop of Asian origin, used as a medicinal herb in Indian and Chinese traditional medicine. Molecular breeding in bitter gourd is in its infancy, due to limited molecular resources, particularly on functional markers for traits such as gynoecy. We performed de novo transcriptome sequencing of bitter gourd using Illumina next-generation sequencer, from root, flower buds, stem and leaf samples of gynoecious line (Gy323) and a monoecious line (DRAR1). A total of 65,540 transcripts for Gy323 and 61,490 for DRAR1 were obtained. Comparisons revealed SNP and SSR variations between these lines and, identification of gene classes. Based on available transcripts we identified 80 WRKY transcription factors, several reported in responses to biotic and abiotic stresses; 56 ARF genes which play a pivotal role in auxin-regulated gene expression and development. The data presented will be useful in both functions studies and breeding programs in bitter gourd.

Rootin, a compound that inhibits root development through modulating PIN-mediated auxin distribution

Plant roots anchor the plant to the soil and absorb water and nutrients for growth. Understanding the molecular mechanisms regulating root development is essential for improving plant survival and agricultural productivity. Extensive molecular genetic studies have provided important information on crucial components for the root development control over the last few decades. However, it is becoming difficult to identify new regulatory components in root development due to the functional redundancy and lethality of genes involved in root development. In this study, we performed a chemical genetic screen to identify novel synthetic compounds that regulate root development in Arabidopsis seedlings. The screen yielded a root growth inhibitor designated as ‘rootin’, which inhibited Arabidopsis root development by modulating cell division and elongation, but did not significantly affect shoot development. Transcript analysis of phytohormone marker genes revealed that rootin preferentially altered the expression of auxin-regulated genes. Furthermore, rootin reduced the accumulation of PIN1, PIN3, and PIN7 proteins, and affected the auxin distribution in roots, which consequently may lead to the observed defects in root development. Our results suggest that rootin could be utilized to unravel the mechanisms underlying root development and to investigate dynamic changes in PIN-mediated auxin distribution.

The PB1 domain in auxin response factor and Aux/IAA proteins: a versatile protein interaction module in the auxin response.

An integral part of auxin-regulated gene expression involves the interplay of two types of transcription factors, the DNA binding auxin response factor (ARF) activators and the interacting auxin/indole acetic acid (Aux/IAA) repressors. Insight into the mechanism of how these transcription factors interact with one another has recently been revealed from crystallographic information on ARF5 and ARF7 C-terminal domains (i.e., a protein-protein interaction domain referred to as domain III/IV that is related to domain III/IV in Aux/IAA proteins). Three-dimensional structures showed that this domain in ARF5 and ARF7 conforms to a well-known PB1 (Phox and Bem1) domain that confers protein-protein interactions with other PB1 domain proteins through electrostatic contacts. Experiments verifying the importance of charged amino acids in conferring ARF and Aux/IAA interactions have confirmed the PB1 domain structure. Some in planta experiments designed to test the validity of PB1 interactions in the auxin response have led to updated models for auxin-regulated gene expression and raised many questions that will require further investigation. In addition to the PB1 domain, a second protein interaction module that functions in ARF-ARF dimerization and facilitates DNA binding has recently been revealed from crystallography studies on the ARF1 and ARF5 DNA binding domains.

Root-specific expression of early auxin-regulated

At the variable environmental conditions plant growth and development are under the control of different factors triggered shifts in hormonal balance and followed changes in intensity of hormone-induced gene expression. Root-specific expression of early auxin-regulated genes, belonged to Aux/IAAs, SAURs, ARFs и GH3s gene families, was tested with specially designed chip. An auxin treatment (15, 30 and 60 min) led to increase of IAA1, IAA3, IAA5, IAA11, IAA19 and GH3-1, GH3-3, GH3-5 genes. Intensification of SAUR9 and SAUR10 genes expression was less significant and appeared only at 30 min.

Cyclic GMP is involved in auxin signalling during Arabidopsis root growth and development

The second messenger cyclic guanosine 3',5'-monophosphate (cGMP) plays an important role in plant development and responses to stress. Recent studies indicated that cGMP is a secondary signal generated in response to auxin stimulation. cGMP also mediates auxin-induced adventitious root formation in mung bean and gravitropic bending in soybean. Nonetheless, the mechanism of the participation of cGMP in auxin signalling to affect these growth and developmental processes is largely unknown. In this report we provide evidence that indole-3-acetic acid (IAA) induces cGMP accumulation in Arabidopsis roots through modulation of the guanylate cyclase activity. Application of 8-bromo-cGMP (a cell-permeable cGMP derivative) increases auxin-dependent lateral root formation, root hair development, primary root growth, and gene expression. In contrast, inhibitors of endogenous cGMP synthesis block these processes induced by auxin. Data also showed that 8-bromo-cGMP enhances auxin-induced degradation of Aux/IAA protein modulated by the SCF(TIR1) ubiquitin-proteasome pathway. Furthermore, it was found that 8-bromo-cGMP is unable to directly influence the auxin-dependent TIR1-Aux/IAA interaction as evidenced by pull-down and yeast two-hybrid assays. In addition, we provide evidence for cGMP-mediated modulation of auxin signalling through cGMP-dependent protein kinase (PKG). Our results suggest that cGMP acts as a mediator to participate in auxin signalling and may govern this process by PKG activity via its influence on auxin-regulated gene expression and auxin/IAA degradation.

ARF-Aux/IAA interactions through domain III/IV are not strictly required for auxin-responsive gene expression

Auxin response factors (ARFs), together with auxin/indole acetic acid proteins (Aux/IAAs), are transcription factors that play key roles in regulating auxin-responsive transcription in plants. Current models for auxin signaling predict that auxin response is dependent on ARF-Aux/IAA interactions mediated by the related protein-protein interaction domain (i.e., referred to as the CTD) found in the ARF and Aux/IAA C-terminal regions. When auxin concentrations in a cell are low, ARF activators residing on the promoters of auxin response genes are thought to be inactive because of the association with dominant Aux/IAA repressors. When auxin concentrations are elevated, the Aux/IAA repressors are recruited to auxin receptors and degraded via the ubiquitin-proteasome pathway. Destruction of the Aux/IAA repressors allows the ARF activators to function in derepressing/activating auxin response genes. While this auxin signaling pathway is simple and attractive, it is unclear whether auxin-regulated gene expression is solely dependent on ARF-Aux/IAA interactions. Here we show that auxin can affect the expression of auxin response genes in a manner that is independent of the ARF activator CTD.