Rapid report. Distinct subclades of Aux/IAA genes are direct targets of ARF5/MP transcriptional regulation. Research

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1 Research Rapid report Distinct subclades of Aux/IAA genes are direct targets of ARF/MP transcriptional regulation Author for correspondence: Thomas Berleth Tel: thomas.berleth@utoronto.ca Received: June 1 Accepted: 3 July 1 Naden T. Krogan 1,, Xiaojun Yin 1, Wenzislava Ckurshumova 1 and Thomas Berleth 1 1 Department of Cell and Systems Biology, University of Toronto, Willcocks Street, Toronto, ON MS 3B, Canada; Department of Biology, American University, Massachusetts Avenue NW, Washington, DC 1, USA New Phytologist (1) : 7 3 doi: /nph.199 Key words: Arabidopsis development, Aux/ IAA genes, AUXIN RESPONSE FACTOR (ARF), regulatory network, transcriptional regulation. Summary The regulatory interactions between AUXIN RESPONSE FACTORS (ARFs) and Aux/IAA repressors play a central role in auxin signal transduction. Yet, the systems properties of this regulatory network are not well established. We generated a steroid-inducible ARF/MONOPTEROS (MP) transgenic background to survey the involvement of this factor in the transcriptional regulation of the entire Aux/IAA family in Arabidopsis thaliana. Target genes of ARF/MP identified by this approach were confirmed by chromatin immunoprecipitation, in vitro gel retardation assays and gene expression analyses. Our study shows that ARF/MP is indispensable for the correct regulation of nearly one-half of all Aux/IAA genes, and that these targets coincide with distinct subclades. Further, genetic analyses demonstrate that the protein products of multiple Aux/IAA targets negatively feed back onto ARF/MP activity. This work indicates that ARF/MP broadly influences the expression of the Aux/IAA gene family, and suggests that such regulation involves the activation of specific subsets of redundantly functioning factors. These groups of factors may then act together to control various processes within the plant through negative feedback on ARF. Similar detailed analyses of other Aux/IAA ARF regulatory modules will be required to fully understand how auxin signal transduction influences virtually every aspect of plant growth and development. Introduction The phytohormone auxin regulates numerous aspects of plant growth, including cell division and elongation, the response to environmental stimuli and tissue patterning (Vanneste & Friml, 9). Many of these processes require coordinated cell-to-cell movement of auxin to form concentration gradients that direct cell behavior and/or fate changes (Vanneste & Friml, 9). Auxin is perceived by members of the TIR1 F-box family of receptors, whose activities affect the composition of transcription factor complexes that control auxin-responsive gene expression (Chapman & Estelle, 9). The AUXIN RESPONSE FACTORS (ARFs), of which there are 3 in Arabidopsis, contain a B3-type DNA-binding domain (DBD) that recognizes cis-acting Auxin Response Elements (AuxREs) of target genes. Downstream of the DBD is a weakly conserved central region (which functions in either transcriptional activation or repression) and two conserved dimerization domains, III and IV, the structural nature of which has recently been determined (Korasick et al., 1; Nanao et al., 1). Gene regulation is best understood for activating ARFs and, in the current model, ARFs are physically bound and antagonized by Aux/ IAA repressor proteins (9 members in Arabidopsis) (Guilfoyle & Hagen, 1). Shared domains III and IV facilitate Aux/IAA ARF dimerization, whereas Aux/IAA-mediated antagonism of ARF activity is conferred by transcriptional repression domain I. Binding of auxin to TIR1 proteins promotes their association with Aux/IAA domain II, resulting in targeted degradation of the repressor and liberation of activator ARFs to promote auxinresponsive transcription. Among these targets appear to be the Aux/ 7 New Phytologist (1) : 7 3 Ó 1 The Authors New Phytologist Ó 1 New Phytologist Trust

2 New Phytologist Rapid report Research 7 IAA genes themselves, the induction of which establishes a negative feedback loop to downregulate the auxin response on dissipation of the hormone signal. Genetic approaches to assign specific roles to individual ARFs and Aux/IAAs have been hindered by apparent functional redundancies among the numerous members of each family (Chapman & Estelle, 9). Details of Aux/IAA roles have mainly come from dominant domain II mutations that stabilize the resulting protein, whereas only a few ARFs have been characterized by lossof-function alleles (Woodward & Bartel, ). This includes the activating ARF MONOPTEROS (MP)/ARF, which regulates organ formation and auxin-inducible responses throughout Arabidopsis development (Hardtke & Berleth, 199). More recently, gain-of-function constructs and searches for transcriptional targets have been used to reveal functions masked by genetic redundancy (Scacchi et al., 1; Krogan et al., 1; De Rybel et al., 13). Although a basic outline of auxin-inducible transcription has been established, the regulatory properties of individual ARF and Aux/IAA proteins are only beginning to emerge. For example, the auxin inducibilities and expression profiles of Aux/IAAs are highly divergent (Abel et al., 199) and the identities of their individual regulators are largely unknown. To simulate the systems properties of the Aux/IAA ARF network, it will be necessary to establish a precise matrix of regulatory interactions between specific members of both families and to demonstrate the directness and in vivo relevance of such interactions. As a first step towards understanding which Aux/IAA genes are controlled by individual ARFs in different parts of the plant, we have used a variety of strategies to determine the suite of Aux/IAA genes directly regulated by MP. Materials and Methods Plant material and growth conditions Unless stated otherwise, Arabidopsis thaliana (L.) Heynh seeds were plated and plants were grown as described previously (Hardtke et al., ). The mutant alleles used were mpg1 (Hardtke & Berleth, 199), nph-1 (Harper et al., ), axr-1 (Yang et al., ) and msg-3 (Tatematsu et al., ). Dexamethasone (DEX) treatment for induction of MP GR (glucocorticoid receptor) was 3 lm DEX and 1% (v/v) Silwet L-77. For mp nph MP::MP-GR quantitative reverse transcriptionpolymerase chain reaction (RT-PCR), dissected 1 d after germination (DAG) vegetative shoot apical meristems (SAMs) or 9 DAG roots (Supporting Information Fig. S1) were incubated for h in liquid media treatments with or without 1% (v/v) Silwet L-77. Transgene construction To make MP::MP-GR, the hormone-binding domain of GR was amplified by PCR and inserted in-frame into an AflII site at the 3 end of the MP open reading frame (ORF) surrounded by noncoding sequence (Fig. 1a). MP::MP-GUS has been described previously (Vidaurre et al., 7). To make Aux/IAA transcriptional reporter genes, 191, 7, 1 and 19 bp upstream of Ó 1 The Authors New Phytologist Ó 1 New Phytologist Trust the translational start codons of IAA1, IAA19, IAA and IAA9, respectively, were fused to the b-glucuronidase (GUS) reporter gene. Quantitative real-time RT-PCR A SuperScript first-strand synthesis system (Invitrogen, Burlington, ON, Canada) was used for reverse transcription of total RNA samples. Quantitative real-time RT-PCR on cdna template employed an MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Data analysis, including threshold cycle (C t ) determination, was performed by accompanying Optical System Software. Thermocycling conditions were 9 C for 9 s, followed by cycles of 9 C for 3 s, C for 3 s and 7 C for 3 s. Absence of non-specific amplification and primer dimers was verified by melting curve analysis and electrophoresis of final reactions. Standard curves were generated for each primer pair using serial template dilutions, and amplification efficiencies (E) were calculated according to the formula E = 1 ( 1/ slope) (Pfaffl, 1). Relative induction ratios (R) ofaux/iaas normalized against ACT7 were calculated using the equation: R ¼ðE Aux=IAA Þ DC t Aux=IAA ðcontrol sampleþ =ðe ACT7 Þ DC t ACT7 ðcontrol sampleþ (Pfaffl, 1). Primer sequences are provided in Supporting Information Table S1. Chromatin immunoprecipitation (ChIP) ChIP experiments on 1 DAG MP::MP-HA (Weijers et al., ) seedling tissue was performed as described previously (Gomez-Mena et al., ) with minor modifications. IP buffer was modified to contain 1 mm HEPES (ph 7.), mm NaCl, 1 lm ZnSO, 1% Triton X-1 and.% sodium deoxycholate. Samples were pre-cleared with protein A-agarose, and 3 ll anti-igg antibody was added to mock samples, whereas 3 ll anti-ha (hemagglutinin) antibody (Roche Diagnostics, Laval, QC, Canada) was added to test IP samples. Protein A- agarose beads (Roche Diagnostics) were added to both samples and, following incubation and centrifugation, were washed twice with Lysis buffer ( mm HEPES (ph 7.), 1 mm NaCl, 1 mm EDTA, 1% Triton X-1 and.1% sodium deoxycholate), twice with Wash buffer (1 mm Tris-HCl (ph ), mm LiCl,.% NP-,.% sodium deoxycholate and 1 mm EDTA) and once with TE (1 mm Tris-HCl (ph ) and 1 mm EDTA). Elution was performed with TE and 1% SDS incubated at C overnight, followed by a 37 C incubation in the presence of lgml 1 proteinase K. Isolated DNA was purified by phenol/chloroform extraction, followed by ethanol precipitation. DNA was resuspended in 1 ll water and ll of each sample were used as template in quantitative PCR. Primer sequences are given in Table S. Electrophoretic mobility shift assays (EMSAs) The purification of His-MP(3) protein and its use in EMSA experiments have been described previously (Scacchi et al., 1). New Phytologist (1) : 7 3

3 7 Research Rapid report New Phytologist (a) (b) (c) (d) (e) Fig. 1 Dexamethasone (DEX)-mediated phenotypic rescue of Arabidopsis mp MP:: MP-GR. (a) MP::MP-GR transgene. Boxes depict open reading frames; lines represent non-coding sequence. (b, c) Seedling and vascular patterning. Absence of hypocotyl and root formation (b, top) and reduced vascular patterning (c, left) under normal growth conditions, and restoration of normal seedling (b, bottom) and vascular (c, right) patterning following DEX application during embryogenesis. (d) DEX application at the seedling stage induces adventitious roots (arrows) from the basal peg (arrowheads). (e) Lateral organ induction. Time course of floral bud initiation from a pin-like inflorescence (bottom) subjected to 1 d of continual DEX treatment. New Phytologist (1) : 7 3 EMSA reactions contained 1 9 Binding Buffer ( mm HEPES/ KOH (ph 7.), 1 mm EDTA, mm MgCl, 1 mm KCl, 1% (v/v) glycerol, 1 mm dithiothreitol (DTT) and. mm phenylmethylsulfonylfluoride (PMSF)), 1 ng poly(didc), endlabeled radioactive ( 3 P) DNA probe,. lg purified protein and variable amounts of competitor DNA. Nonspecific competitors used for IAA1, IAA19 and IAA EMSAs corresponded to 3 to 1 bp (relative to translational start) of IAA1, 19 to Ó 1 The Authors New Phytologist Ó 1 New Phytologist Trust

4 New Phytologist Rapid report Research 77 1 bp of IAA19,and 91 to 1 bp of IAA, respectively. The prokaryotic control protein containing an amino-terminal His-tag was prephenate dehydrogenase (gift from S. Singh, University of Toronto, Canada). Microtechniques and microscopy Analysis of GUS activity was as described by Scarpella et al. () with modifications (Table S3a,b). Results and Discussion Post-translational regulation of MP activity The expression of most Aux/IAA genes is rapidly auxin responsive, and their promoters often contain multiple conserved AuxRE binding sites (Chapman & Estelle, 9). To detect potential direct regulation of Aux/IAA genes by a specific ARF with genetically characterized functions, we fused the hormone-binding domain of the mammalian GR to MP, and expressed it under its natural promoter in mp mutant Arabidopsis plants (mp MP::MP- GR) (Fig. 1). In this context, the MP fusion protein enters the nucleus and regulates transcription only on exposure to a glucocorticoid steroid, such as DEX (Wang et al., 3). As shown in Fig. 1, untreated mp MP::MP-GR plants are indistinguishable from mp mutants. On DEX application at any stage, structures missing in mp mutants are instantly generated, and subsequent development becomes indistinguishable from the wildtype (Fig. 1b e). Importantly, DEX-treated mp MP::MP-GR plants do not show defects associated with MP mis- or overexpression (Hardtke et al., ). We conclude that the mp MP::MP-GR genetic background offers a post-translational switch between the absence of MP activity and approximately wild-type MP dosage. Therefore, as described below, this inducible background was used to determine which Aux/IAA genes are regulated by MP in different tissue types. A matrix of MP-regulated Aux/IAA genes Expression and genetic data indicate that MP acts predominantly in embryos, meristems and organ primordia (Hardtke & Berleth, 199). Further, in these locations, MP acts redundantly with at least one other ARF, NPH/ARF7 (Hardtke et al., ). MP masks NPH function, but, in the absence of MP activity, residual auxin signal transduction through NPH dampens the severity of the mutant phenotype. We confirmed this genetic relationship by completely normalizing the extreme phenotype of the mp nph double mutant through DEX induction of MP-GR (Fig. S1). In order to capture the full extent of MP-mediated gene regulatory input, we used RNA selectively isolated from pooled SAM or root tissue (with associated organ primordia) of mp nph MP::MP-GR plants in a gene-by-gene quantitative real-time RT-PCR analysis. In three replicates, we observed strong and reproducible transcript abundances for all but two (IAA1, IAA1) Aux/IAA genes (Fig. ). Consistent with MP being subject to negative regulation by Aux/ IAA proteins (Tiwari et al., ), gene induction was strongest in Ó 1 The Authors New Phytologist Ó 1 New Phytologist Trust the absence of protein synthesis (Fig. a, A, C, D vs A, D ). Remarkably, of the 7 assessed Aux/IAAs, approximately one-half (IAA1 IAA, IAA11, IAA13, IAA19, IAA, IAA9 IAA31)were directly upregulated 3- to 3-fold on MP activation (Fig. a,b, A, C, D vs A, C ). Further, in each case, this upregulation occurred in both the shoot and root (Fig. a,b). Notably, MP dependence of Aux/IAA expression correlated with gene phylogeny (summarized in Fig. S). For instance, strong MP dependence was displayed by all members of three previously characterized groups: IAA1 IAA; IAA, IAA, IAA19; and IAA, IAA3, IAA31 (Liscum & Reed, ; Remington et al., ; Overvoorde et al., ). In addition, expression of the phylogenetically solitary gene IAA9 was also very strongly dependent on MP. To gain an insight into the function of IAA9, we analyzed the expression profile conferred by its promoter. We observed staining in the vasculature of cotyledons, leaves, hypocotyl and root (including in the columella) (Fig. S3a d), closely resembling MP distribution (Fig. Sa). Together with exhibiting a dependence on MP for appropriate expression (Fig. S3e), IAA9::GUS displayed auxin responsiveness (Fig. S3f, g). In summary, these analyses indicate that MP directly regulates nearly one-half of all Aux/IAA genes, and that this regulation affects identical sets of genes in shoots and roots. Remarkably, these genes belong to specific Aux/IAA clades, each comprising genes with apparently overlapping functions (Overvoorde et al., ). In vivo and in vitro binding of MP to Aux/IAA upstream regulatory regions To assess whether MP directly binds the promoters of presumptive target genes, we performed ChIP on MP::MP- HA seedling tissue using an anti-ha antibody. At least one consensus AuxRE ( -TGTCTC-3 ) was found within 1 bp upstream of the translational start codon of the tested potential targets (Fig. 3a). As shown in Fig. 3(b), specific AuxREs in the promoters of most target genes were enriched in ChIP samples (between - and 13-fold). This was in contrast with AuxREcontaining promoter elements of IAA and IAA (Fig. S), which were not enriched above the levels of other elements used as negative controls. Because our binding site confirmation was restricted to a 1 bp upstream interval, it is not surprising that MP binding sites of a small subset of target genes could not be confirmed. For example, it is possible that the responsiveness of IAA and IAA to MP-GR activation is mediated through non-consensus AuxREs in the immediate upstream regions or through consensus AuxREs more remotely located, including those in introns. The fact that the perfectly conserved AuxREs in the promoters of IAA and IAA were not enriched in MP immunoprecipitated samples stresses the authenticity of the other reproducibly identified binding sites. Such in vivo binding sites are expected to be context dependent and not a mere reflection of the conservation of consensus AuxRE elements. MP truncations lacking dimerization domains III and IV appear to act as irrepressibly active variants of MP in plant development New Phytologist (1) : 7 3

5 7 Research Rapid report New Phytologist (a) 1 IAA1 1 IAA 1 IAA3 7 IAA IAA 1 IAA. IAA7 IAA IAA9 IAA1 IAA11 1 IAA IAA1. IAA IAA17.. IAA1 1 IAA19 IAA 3. IAA. IAA IAA IAA9 1 IAA3 7 IAA IAA IAA IAA3 Fig. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assessment of Aux/IAA expression on MONOPTEROS (MP) activation in Arabidopsis. (a) mp nph MP::MP-GR shoot apical meristems were treated for h with 1 lm indole-3-acetic acid (IAA) ( A ), 1 lm IAA and 3 lm cycloheximide (CHX) ( A, C ), 1 lm IAA and 3 lm dexamethasone (DEX) ( A, D ) or 1 lm IAA, 3 lm CHX and 3 lm DEX ( A, C, D ). (b) mp nph MP:: MP-GR roots were subjected to only three treatments because of the low abundance of tissue: lm 1-naphthalene acetic acid (NAA) and 3 lm CHX ( A, C ), 3 lm CHX and 3 lm DEX ( C, D ) or lm NAA, 3 lm CHX and 3 lm DEX ( A, C, D ). For both (a) and (b), MP-mediated Aux/IAA gene induction was determined by comparing A, C, D with A, C. Therefore, three biological (a) or technical (b) replicates were performed on these two treatments. Values for A, C were set to 1, whereas SD bars are presented on A, C, D data. New Phytologist (1) : 7 3 Ó 1 The Authors New Phytologist Ó 1 New Phytologist Trust

6 New Phytologist Rapid report Research 79 (b) IAA1. IAA IAA3 1 IAA IAA IAA.. IAA7.. IAA 3. IAA9 IAA1 IAA11 1 IAA IAA1 IAA1 1. IAA17 IAA IAA19 3 IAA 3. IAA IAA IAA IAA IAA IAA31 Fig. (Continued) IAA3 IAA IAA3 (Krogan et al., 1). These findings imply that MP can bind DNA in the absence of its domains III and IV, consistent with structural data showing that ARF dimers cooperatively bind target sequences Ó 1 The Authors New Phytologist Ó 1 New Phytologist Trust through their DBDs (Boer et al., 1). We tested the dispensability of MP domains III and IV in DNA binding with EMSAs on AuxREs from three promoters using a recombinant truncated MP New Phytologist (1) : 7 3

7 Research Rapid report New Phytologist (a) (c) (d) (b) Fig. 3 Binding of MONOPTEROS (MP) to Arabidopsis Aux/IAA upstream regulatory regions. (a) Schematics of Aux/IAA promoter regions. Arrows depict open reading frames, drawn to scale relative to the promoter sequence. Flags represent Auxin Response Elements (AuxREs) (TGTCTC or GAGACA). Solid lines delineate promoter regions tested by chromatin immunoprecipitation (ChIP) in (b), whereas dashed lines designate regions used as electrophoretic mobility shift assay (EMSA) probes in (d). (b) Anti-HA ChIP of MP::MP-HA seedlings showing fold enrichment of Aux/IAA genes (relative to Aux/IAA abundance in IgG control ChIP samples). Bars, SE. (c) Schematic of recombinant His-MP(3) protein with DNA-binding domain (DBD) and amino-terminal His-tag (drawn to scale). (d) EMSAs using Aux/IAA DNA probes and recombinant MP protein depicted in (a) and (c), respectively. Arrows indicate recombinant MP complex positions. Lanes 3 and lanes contain increasing amounts (91, 9, 91) of specific unlabeled competitor DNA and nonspecific unlabeled DNA lacking consensus AuxREs, respectively. Lane E contains protein from a purification of bacteria harboring empty expression vector. Lane H contains an unrelated, purified prokaryotic protein with the same amino-terminal His-tag as the MP protein. New Phytologist (1) : 7 3 Ó 1 The Authors New Phytologist Ó 1 New Phytologist Trust

8 New Phytologist Rapid report Research 1 (a) protein (Fig. 3c). As shown in Fig. 3(d), DNA fragments from all three promoters were specifically bound by MP protein. These results are in agreement with MP directly regulating Aux/IAA genes, and confirm that ARF domains III and IV are not required for binding site recognition. (b) (c) In planta expression of MP target genes Because our MP-GR transgene is driven by the genuine MP promoter, and because our expression data were derived from isolated tissues with high MP activity, we believe that the identified Aux/IAA genes are genuine downstream targets of MP in plant development. To gain support for this idea, we compared published Aux/IAA expression patterns with the distribution of a functional MP::MP-GUS reporter, which is expressed predominantly in the vasculature of cotyledons, hypocotyl and root (Fig. Sa) (Vidaurre et al., 7). The expression patterns of many Aux/IAA genes putatively targeted by MP have been studied at tissue-specific resolution, and these show overlap with MP distribution in organ vasculature (Tatematsu et al., ; Weijers et al., ). To further increase the resolution of this analysis, we assessed in detail whether the expression profiles of IAA1, IAA19 and IAA, representing three different Aux/IAA subclades (Liscum & Reed, ; Remington et al., ; Overvoorde et al., ), were consistent with being regulated by MP. As illustrated in Fig. S, the GUS expression profiles of IAA1, IAA19 and IAA matched expectations with regard to their spatio-temporal distribution during development and their auxin inducibility. Fig. Antagonistic relationships between MONOPTEROS (MP) and Aux/ IAA targets in Arabidopsis. (a, b) Mean hypocotyl lengths of seedlings germinated and grown in the dark in the absence (open bars) or presence (closed bars) of the synthetic auxin,-dichlorophenoxyacetic acid (1 nm) for d. Bars, SE. (c) Dark-field view of cleared cotyledons showing extent of vascular patterning. Wild-type (wt) cotyledons produce closed distal vascular loops. Bars,. mm. Ó 1 The Authors New Phytologist Ó 1 New Phytologist Trust MP target genes antagonize MP activity It is possible that MP triggers the expression of specific Aux/IAA target genes because their corresponding protein products negatively feed back on MP function through dimerization. Auxinmediated degradation of Aux/IAAs activates MP, ensuring concomitant transcriptional upregulation of the respective Aux/ IAA genes. This restores the original Aux/IAA protein abundance and returns the system to its ground state after an auxin pulse. This type of antagonistic genetic interaction with MP has been established for only one Aux/IAA, namely BDL/IAA1 (Hardtke et al., ; Weijers et al., ; Lau et al., 11). We therefore analyzed the antagonistic relationships between MP and one Aux/ IAA gene from each clade identified as being MP regulated (IAA1, IAA19 and IAA). Gain-of-function mutations in AXR/IAA1 and MSG/IAA19 which stabilize the corresponding gene products oppose auxin-responsive shortening of hypocotyls on germination in the dark (Figs a,b, S) (Tatematsu et al., ; Yang et al., ), whereas ubiquitously expressed MP mediates hypocotyl shortening in response to auxin (Hardtke et al., ). As shown in Fig., overexpression of MP restores auxin sensitivity in hypocotyls of both Aux/IAA mutants. Further, plants overexpressing IAA show features of mp mutants, such as decreased vascular patterning in lateral organs (Fig. c) (Sato & Yamamoto, ). A protein variant of MP believed to operate independently of Aux/IAA repression (Krogan et al., 1) can overcome the effect of IAA overexpression, resulting in increased lateral organ vascularization New Phytologist (1) : 7 3

9 Research Rapid report New Phytologist (Fig. c). Collectively, our data demonstrate that auxin-responsive processes, such as hypocotyl growth and vascular development, are antagonistically regulated through the relative abundance of an ARF vs the protein abundance of its transcriptional target genes. Thus, our data provide new examples of circuit regulatory modules between specific ARF and Aux/IAA genes, which include Aux/IAA transcriptional regulation. It is unlikely that such regulatory interactions are restricted to the cases described here. Beyond these three genes, others have been shown to interfere with traits that are promoted by MP activity. For example, the target gene IAA13 has been reported to interfere with embryonic root formation, similar to its close relative IAA1 (Weijers et al., ). Further, like IAA, overexpression of the target genes IAA3 and IAA31 results in reduced leaf vasculature (Sato & Yamamoto, ). Together, these findings suggest that it is common for an individual ARF to transcriptionally activate the genes whose protein products repress its activity. Natural developmental target genes The identification of direct target genes of transcription factors using their post-translationally regulated derivatives is well established in plants (Wang et al., 3). However, although many studies have used strong constitutive promoters to drive GR fusion gene expression, the work presented here employed the natural regulatory sequences of MP (Fig. 1a). Phenotypic assays confirmed that MP activity was supplied in approximately natural dosage and distribution, suggesting that affected transcripts represented authentic MP targets. The authenticity of identified target genes was confirmed by in vivo binding assays in many cases. Where tested, auxin-responsive expression patterns overlapped with MP, further supporting their roles as MP target genes. The participation of MP in the regulation of a large fraction of all Aux/IAA genes does not diminish the roles of other ARFs. An Aux/ IAA gene may be regulated by a number of ARFs, as these are expressed in distinct domains and have been shown to act redundantly as well as non-redundantly (Hardtke et al., ; Okushima et al., ; Rademacher et al., 11), and may function in transcriptional regulation as ARF ARF heterodimers. Our saturating survey implicated MP in the regulation of approximately one-half of the Aux/IAAs, which is consistent with diverse Aux/IAA-controlled processes (ranging from organ initiation to the control of differential growth) being dependent on MP (Hardtke & Berleth, 199). The MP targets IAA1 and IAA19 have been implicated in auxin-mediated hypocotyl elongation, a trait that is affected by MP activity (Hardtke et al., ; Tatematsu et al., ; Yang et al., ). Moreover, MP function appears to focus on specific Aux/IAA subclades, potentially representing distinct processes, each controlled by redundantly acting Aux/IAAs (Overvoorde et al., ). From this, we propose that MP constitutes a critical component in a large portion of all Aux/IAAregulating transcriptional complexes. Within these, other proteins, including other ARFs, may confer specificity, distinguishing individual subclades. A role for MP in the regulation of Aux/IAAs is also suggested by the fact that the induction of many Aux/IAAs by cycloheximide (CHX) was far weaker in the absence of MP New Phytologist (1) : 7 3 activity (Fig. a, A, C vs A, C, D ). CHX-mediated activation of auxin-inducible genes has long suggested the existence of labile proteins, presumably the Aux/IAA proteins themselves, repressing activating transcription factors (Guilfoyle & Hagen, 1). Following this interpretation, the dependence on MP for CHXmediated induction of Aux/IAA transcripts implicates MP as a central, normally repressed transcriptional activator in this network. Acknowledgements We thank Jeffrey Long for allowing quantitative real-time RT-PCR analyses to be performed in his laboratory. We also thank Dolf Weijers, Kotaro Yamamoto and Jason Reed for Arabidopsis seeds. Support for this work came from the Center for Analysis of Genome Evolution and Function (CAGEF) and a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant to T.B., and from NSERC and Ontario Graduate Scholarship in Science and Technology (OGSST) postgraduate fellowships to N.T.K. References Abel S, Nguyen MD, Theologis A The PS-IAA/-like family of early auxin-inducible mrnas in Arabidopsis thaliana. Journal of Molecular Biology 1: Boer DR, Freire-Rios A, van den Berg WAM, Saaki T, Manfield IW, Kepinski S, Lopez-Vidrieo I, Franco-Zorrilla JM, de Vries SC, Solano R et al. 1. Structural basis for DNA binding specificity by the auxin-dependent ARF transcription factors. Cell 1: Chapman EJ, Estelle M. 9. Mechanism of auxin-regulated gene expression in plants. Annual Review of Genetics 3:. De Rybel B, Moller B, Yoshida S, Grabowicz I, Barbier de Reuille P, Boeren S, Smith RS, Borst JW, Weijers D. 13. A bhlh complex controls embryonic vascular tissue establishment and indeterminate growth in Arabidopsis. Developmental Cell : 37. Gomez-Mena C, de Folter S, Costa MMR, Angenent GC, Sablowski R.. Transcriptional program controlled by the floral homeotic gene AGAMOUS during early organogenesis. Development 13: 9 3. Guilfoyle TJ, Hagen G. 1. Getting a grasp on domain III/IV responsible for Auxin Response Factor IAA protein interactions. Plant Science 19:. Hardtke CS, Berleth T The Arabidopsis gene MONOPTEROS encodes a transcription factor mediating embryo axis formation and vascular development. EMBO Journal 17: Hardtke CS, Ckurshumova W, Vidaurre DP, Singh SA, Stamatiou G, Tiwari SB, Hagen G, Guilfoyle TJ, Berleth T.. Overlapping and non-redundant functions of the Arabidopsis auxin response factors MONOPTEROS and NONPHOTOTROPIC HYPOCOTYL. Development 131: Harper RM, Stowe-Evans EL, Luesse DR, Muto H, Tatematsu K, Watahiki MK, Yamamoto K, Liscum E.. The NPH locus encodes the Auxin Response Factor ARF7, a conditional regulator of differential growth in aerial Arabidopsis tissue. Plant Cell 1: Korasick DA, Westfall CS, Lee SG, Nanao MH, Dumas R, Hagen G, Guilfoyle TJ, Jez JM, Strader LC. 1. Molecular basis for AUXIN RESPONSE FACTOR protein interaction and the control of auxin response repression. Proceedings of the National Academy of Sciences, USA 111: 7 3. Krogan NT, Ckurshumova W, Marcos D, Caragea AE, Berleth T. 1. Deletion of MP/ARF domains III and IV reveals a requirement for Aux/IAA regulation in Arabidopsis leaf vascular patterning. New Phytologist 19: Lau S, De Smet I, Kolb M, Meinhardt H, Jurgens G. 11. Auxin triggers a genetic switch. Nature Cell Biology 13: Ó 1 The Authors New Phytologist Ó 1 New Phytologist Trust

10 New Phytologist Rapid report Research 3 Liscum E, Reed JW.. Genetics of Aux/IAA and ARF action in plant growth and development. Plant Molecular Biology 9: 37. Nanao MH, Vinos-Poyo T, Brunoud G, Thevenon E, Mazzoleni M, Mast D, Laine S, Wang S, Hagen G, Li H et al. 1. Structural basis for oligomerization of auxin transcriptional regulators. Nature Communications : 317. Okushima Y, Overvoorde PJ, Arima K, Alonso JM, Chan A, Chang C, Ecker JR, Hughes B, Lui A, Nguyen D et al.. Functional genomic analysis of the AUXIN RESPONSE FACTOR gene family members in Arabidopsis thaliana: unique and overlapping functions of ARF7 and ARF19. Plant Cell 17: 3. Overvoorde PJ, Okushima Y, Alonso JM, Chan A, Chang C, Ecker JR, Hughes B, Liu A, Onodera C, Quach H et al.. Functional genomic analysis of the AUXIN/INDOLE-3-ACETIC ACID gene family members in Arabidopsis thaliana. Plant Cell 17: Pfaffl MW. 1. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research 9: e. Rademacher EH, Moller B, Lokerse AS, Llavata-Peris CI, van den Berg W, Weijers D. 11. A cellular expression map of the Arabidopsis AUXIN RESPONSE FACTOR gene family. Plant Journal : 97. Remington DL, Vision TJ, Guilfoyle TJ, Reed JW.. Contrasting modes of diversification in the Aux/IAA and ARF gene families. Plant Physiology 13: Sato A, Yamamoto KT.. Overexpression of the non-canonical Aux/IAA genes causes auxin-related aberrant phenotypes in Arabidopsis. Physiologia Plantarum 133: 397. Scacchi E, Salinas P, Gujas B, Santuari L, Krogan N, Ragni L, Berleth T, Hardtke CS. 1. Spatio-temporal sequence of cross-regulatory events in root meristem growth. Proceedings of the National Academy of Sciences, USA 17: Scarpella E, Francis P, Berleth T.. Stage-specific markers define early steps of procambium development in Arabidopsis leaves and correlate termination of vein formation with mesophyll differentiation. Development 131: 3 3. Tatematsu K, Kumagai S, Muto H, Sato A, Watahiki MK, Harper RM, Liscum E, Yamamoto KT.. MASSUGU encodes Aux/IAA19, an auxin-regulated protein that functions together with the transcriptional activator NPH/ARF7 to regulate differential growth responses of hypocotyl and formation of lateral roots in Arabidopsis thaliana. Plant Cell 1: Tiwari SB, Hagen G, Guilfoyle TJ.. Aux/IAA proteins contain a potent transcriptional repression domain. Plant Cell 1: Vanneste S, Friml J. 9. Auxin: a trigger for change in plant development. Cell 13: Vidaurre DP, Ploense S, Krogan NT, Berleth T. 7. AMP1 and MP antagonistically regulate embryo and meristem development in Arabidopsis. Development 13: 1 7. Wang R, Zhou X, Wang X. 3. Chemically regulated expression systems and their applications in transgenic plants. Transgenic Research 1: 9. Weijers D, Benkova E, Jager KE, Schlereth A, Hamann T, Kientz M, Willmoth JC, Reed JW, Jurgens G.. Developmental specificity of auxin response by pairs of ARF and Aux/IAA transcriptional regulators. EMBO Journal : Weijers D, Schlereth A, Ehrismann JS, Schwank G, Kientz M, Jurgens G.. Auxin triggers transient local signaling for cell specification in Arabidopsis embryogenesis. Developmental Cell 1: 7. Woodward AW, Bartel B.. Auxin: regulation, action, and interaction. Annals of Botany 9: Yang X, Lee S, So J, Dharmasiri S, Dharmasiri N, Ge L, Jensen C, Hangarter R, Hobbie L, Estelle M.. The IAA1 protein is encoded by AXR and is a substrate of SCF TIR1. Plant Journal : Supporting Information Additional supporting information may be found in the online version of this article. Fig. S1 Dexamethasone (DEX)-dependent phenotypic rescue of Arabidopsis mp nph MP::MP-GR individuals. Fig. S General phylogenetic relationships between Arabidopsis Aux/IAA genes. Fig. S3 IAA9 reporter gene expression patterns in Arabidopsis. Fig. S MP and Aux/IAA reporter gene expression patterns in Arabidopsis. Fig. S Schematics of Arabidopsis IAA and IAA promoter regions. Fig. S Auxin-mediated inhibition of Arabidopsis hypocotyl growth. Table S1 Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) primer sequences Table S Chromatin immunoprecipitation (ChIP) primer sequences Table S3 (a) b-glucuronidase (GUS) staining conditions of nonauxin-induced tissues; (b) GUS staining conditions of auxininduced tissues (and corresponding control mock-treated tissues) Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Ó 1 The Authors New Phytologist Ó 1 New Phytologist Trust New Phytologist (1) : 7 3