Deletion of MP ARF5 domains III and IV reveals a requirement for Aux IAA regulation in Arabidopsis leaf vascular patterning

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1 Research Deletion of MP ARF5 domains III and IV reveals a requirement for Aux IAA regulation in Arabidopsis leaf vascular patterning Naden T. Krogan, Wenzislava Ckurshumova, Danielle Marcos, Adriana E. Caragea and Thomas Berleth Department of Cell and Systems Biology, University of Toronto, 25 Willcocks Street, Toronto, ON, Canada, M5S 3B2 Author for correspondence: Thomas Berleth Tel: thomas.berleth@utoronto.ca Received: 2 November 2011 Accepted: 24 December 2011 doi: /j x Key words: Arabidopsis, Aux IAA genes, AUXIN RESPONSE FACTOR, gene regulation, leaf development, redundant gene function, transcriptional repression, vascular patterning. Summary Combinatorial interactions of AUXIN RESPONSE FACTORs (ARFs) and auxin indole acetic acid (Aux IAA) proteins through their common domains III and IV regulate auxin responses, but insight into the functions of individual proteins is still limited. As a new tool to explore this regulatory network, we generated a gain-of-function ARF genotype by eliminating domains III and IV from the functionally well-characterized ARF MONOPTEROS(MP) ARF5. This truncated version of MP, termed MPD, conferred complementing MP activity, but also displayed a number of semi-dominant traits affecting auxin signaling and organ patterning. In MPD, the expression levels of many auxin-inducible genes, as well as rooting properties and vascular tissue abundance, were enhanced. Lateral organs were narrow, pointed and filled with parallel veins. This effect was epistatic over the vascular hypotrophy imposed by certain Aux IAA mutations. Further, in MPD leaves, failure to turn off the procambium-selecting gene PIN1 led to the early establishment of oversized central procambial domains and very limited subsequent lateral growth of the leaf lamina. We conclude that MPD can selectively uncouple a single ARF from regulation by Aux IAA proteins and can be used as a genetic tool to probe auxin pathways and explore leaf development. Introduction Auxin has been implicated in a stunning array of processes in plants, ranging from the relay of external signals, such as light and gravity, to patterning and organogenesis (Aloni, 1995; Davies, 2004; Kepinski & Leyser, 2005; Jenik et al., 2007; Petrasek & Friml, 2009). Many auxin responses rely on the modulation of gene expression, and the diversity of auxin functions may be reflected, in part, by the complexity of two families of nuclear proteins involved in auxin-dependent gene regulation (Weijers & Jurgens, 2004; Guilfoyle & Hagen, 2007). One family, the AUXIN RESPONSE FACTORs (ARFs), comprise 23 transcription factors that bind to auxin response elements (AuxREs) in the regulatory regions of their target genes (Ulmasov et al., 1997a). A typical ARF consists of a conserved amino-terminal B3-type DNA-binding domain, a weakly conserved central region (which functions in either transcriptional activation or repression) and two conserved carboxy-terminal dimerization domains (III and IV) (Tiwari et al., 2003). Domains III and IV are shared with members of a second family of 29 transcriptional co-regulators, termed Aux IAAs, thereby facilitating ARF Aux IAA physical interaction (Kim et al., 1997; Ulmasov et al., 1999; Tiwari et al., 2001). Aux IAAs also contain two other conserved domains, I and II. Domain I confers transcriptional repression that counteracts the activation properties of ARFs to which the corresponding Aux IAA binds (Ulmasov et al., 1997b; Tiwari et al., 2001, 2004). Domain II contains a conserved degron sequence that is critical for recognition by the SCF TIR1 complex and for subsequent Aux IAA ubiquitination and degradation. Auxin promotes this regulation by binding directly TIR1 and stabilizing its association with Aux IAAs (reviewed in Kepinski, 2007). In this way, auxin induces ARF-mediated gene expression by destabilizing Aux IAAs that bind and repress ARFs (reviewed in Paciorek & Friml, 2006). Knowledge of the roles of individual proteins in both families is still fragmentary, but there is evidence for widespread functional redundancy that obscures many traits associated with lossof-function mutations (reviewed in Chapman & Estelle, 2009). One of the few ARFs that have been characterized by loss-offunction mutations, MONOPTEROS ARF5 (MP in the following), mediates organ and vascular tissue formation throughout the Arabidopsis life cycle (Berleth & Jurgens, 1993; Przemeck et al., 1996; Hardtke & Berleth, 1998; Mattsson et al., 2003; Hardtke et al., 2004). In mp mutants, the vascular tissue is reduced and its development is delayed. Expression of MP becomes gradually restricted to the vasculature as organs mature, and its expression is maintained in these tissues (Hardtke & Berleth, 1998; Hardtke et al., 2004; Wenzel et al., 2007). Finally, MP is a direct 391

2 392 Research New Phytologist regulator of the pre-procambial transcription factor ATHB8 and is also required for the expression of the auxin efflux facilitator PIN1 (Wenzel et al., 2007; Donner et al., 2009). Members of the PIN family, together with other membrane proteins in Arabidopsis, have been implicated in auxin transport on the basis of their mutant phenotypes, expression patterns and subcellular localizations (reviewed in Vieten et al., 2007). In the leaf vascular system, PIN1 seems to be the most important auxin efflux facilitator (Scarpella et al., 2006). PIN1 expression domains are initially very wide, but become gradually confined to apparent routes of auxin flow, the polarity of which is reflected by asymmetric subcellular PIN1 localization. These narrow domains precisely presage sites of procambium formation in the leaf (Scarpella et al., 2006; Wenzel et al., 2007). The expression of PIN1, dependent on auxin and ARF activity (Sauer et al., 2006), and the narrowing of PIN1 expression domains, dependent on auxin transport (Scarpella et al., 2006), are key elements in a partially self-organizing process of leaf venation patterning, in which the regulation of ARF activity must be assumed to be a critical component (reviewed in Scarpella & Helariutta, 2010). The dynamic expression pattern of PIN1 and the positions of leaf procambial strands suggest that sustained PIN1 expression selects procambial cells from ground meristem cells, and that the axis of PIN1 polarity predisposes the main cell axis of the elongated procambial cells (reviewed in Rolland-Lagan, 2008 and Scarpella & Helariutta, 2010). To elucidate the contributions of individual members of both ARF and AUX IAA families to specific auxin responses, phenotypes associated with multiple loss-of-function mutant backgrounds or dominant gain-of-function activities have been analyzed in combination with molecular interaction studies (Sato & Yamamoto, 2008; reviewed in Chapman & Estelle, 2009). Insight into the roles of individual Aux IAAs, for example, is typically derived from phenotypes associated with gain-of-function missense mutations in domain II, which lead to stabilized Aux IAA proteins (reviewed in Woodward & Bartel, 2005; Ploense et al., 2009). In this work, we present a gain-of-function variant of ARF5 MP as a new type of genetic tool to characterize ARF function and interrogate downstream developmental processes. The new genotype, MPD, allows the analysis of MP activity in the absence of Aux IAA interaction domains and provides new ways to address the relationship between vascular pattern and the formation of leaf shape. Materials and Methods Plant material and growth conditions All genotypes are in the Columbia (Col-0) background of Arabidopsis thaliana (L.) Heynh. The marker lines are ATHB8::- GUS (Baima et al., 1995), MP::MP:GUS (Vidaurre et al., 2007), MP::MP:GFP (Schlereth et al., 2010), DR5rev::GFP (Friml et al., 2003) and PIN1::PIN1:GFP (Benkova et al., 2003). The mutants used in this study are mp G12 (Hardtke & Berleth, 1998), iaa12 bdl (Hamann et al., 1999), crane-2 iaa18 (Uechara et al., 2008) and msg2 iaa19 (Tatematsu et al., 2004). Seeds were sterilized and germinated, and plants were grown as described in Hardtke et al. (2004). We refer to days after germination (DAG) as days after exposure of imbibed seeds to light. For germination in the dark, seeds were plated and exposed to fluorescent white light (80 lm m )2 s )1 ) for 2 h to synchronize germination. Subsequently, plates were wrapped in aluminum foil and incubated at 24 C. Cotyledon rooting assay was performed exactly as described in Mattsson et al. (2003). Hypocotyl elongation assays were performed as described in Hardtke et al. (2004). Plasmid construction and plant transformation All three MPD transgenes are driven by the endogenous MP promoter (3.3 kb upstream of the start codon). MPD-2 and MPD-3 also contain the endogenous MP transcriptional termination sequence (730 bp downstream of the stop codon). A previously published MP reporter construct under the control of these noncoding regulatory sequences matches endogenous MP expression patterns and fully rescues mp phenotypic defects (Vidaurre et al., 2007). MPD encodes amino acids of MP, followed by a cloning artifact of 20 extra amino acids (DLEELARISPIV- QTFGNKVS). MPD-2 and MPD-3 encode amino acids and of MP, respectively, and lack any extra residues. All three transgenes were cloned into the binary vector pegad (Basta resistance; Cutler et al., 2000) and transformed into plants by floral dip (Clough & Bent, 1998). Primary transformants were selected on half-strength Murashige and Skoog (1 2 MS) plates containing 10 lg ml )1 Basta. Genotyping MPD (single-copy transgene) was crossed into various lines, and F2 genotypes were determined on the basis of PCR diagnostic products using the following primers: for mp G12, BS1354-f (5 -GAGATGGCCTGGTTCTAAGTGGC-3 ) and BS1354-r (5 -GCCAGTTCAACATCTCGGTTATCG-3 ); for bdl, BDL- F2 (5 -GCTCAAATCTTGTGATGTGAGTG-3 ) and BDL-1 (5 -AGTCCACTAGCTTCTGAGGTTCCC-3 ), followed by BsuRI (HaeIII) (Fermentas, Canada Inc., Burlington, ON, Canada) digestion. The crane-2 iaa18 genotype was determined by direct sequencing. Microscopy Histochemical detection of b-glucuronidase (GUS) activity was performed as described by Scarpella et al. (2004). For dark field images, lateral organs were fixed in ethanol : acetic acid (3 : 1, v v) for 1 h (or overnight) at room temperature. Samples were then washed twice with 95% (v v) ethanol for 1 h and stored in 70% (v v) ethanol at room temperature. Samples were dissected under water, mounted in chloral hydrate : glycerol : water (8 : 3 : 1, w v v), and left at room temperature overnight to allow for complete tissue clearing. When leaves were mounted, they were placed adaxial side down in an attempt to prevent trichomes from interfering with visualization. Samples were viewed with a Leica MZFLIII stereomicroscope (Leica Microsystems,

3 New Phytologist Research 393 Wetzlar, Germany) equipped with a Canon EOS D60 digital camera (Canon, Inc., Tokyo, Japan). For confocal laser scanning microscopy, dissected leaf primordia were mounted in water, adaxial side up, and observed with a Zeiss Axiovert 100M microscope equipped with a Zeiss LSM510 laser module confocal unit (Carl Zeiss). Confocal settings and band pass filters were as described previously (Scarpella et al., 2006). Run-on transcription Transcriptionally active nuclei were prepared from 2 g of 14-dold light-grown Arabidopsis seedlings with a Percoll gradient (Sigma) as described by Folta & Kaufman (2000). The run-on assay utilized c isolated nuclei (in 100 ll) per genotype. The transcription reaction mixture contained 10 ll of10 transcription buffer (Roche Applied Science), 5 ll of each 100 mm CTP, GTP and ATP (Fermentas) and 20 lci 32 P-CTP (3000 Ci mmol )1 ) (Perkin-Elmer, Woodbridge, ON, Canada). Transcription was allowed to proceed for 30 min. The reaction was terminated with the addition of 10 units DNaseI (Fermentas) and subsequent incubation at 37 C for 20 min. RNA was extracted using Trizol reagent (Bioshop, Burlington, ON, Canada). Probes were generated by PCR, purified and quantified. Five hundred nanograms of probe were heat denatured for 5 min at 98 C and dot blotted onto Hybond filter membranes. Hybridization was carried out for 18 h at 42 C. Following hybridization, filters were washed three times for 20 min each, in 0.1% sodium dodecyl sulfate (SDS) and 0.2% saline-sodium citrate (SSC). Transcription rates were determined by assessing the amount of de novo synthesized RNA bound to dot-spotted probes by scintillation counting in 5 ml of scintillation liquid. Results MPD acts as a gain-of-function MP allele Stop codons near the carboxy-terminal border of the MP activation domain (AD) lead to partial loss of MP activity (Berleth & Jurgens, 1993; Hardtke & Berleth, 1998). To eliminate regulation through domains III and IV without interfering with AD function, three different constructs (MPD, MPD-2, MPD-3, Supporting Information Fig. S1) terminating the MP coding sequence downstream of the AD were generated and introduced into wild-type and mp plants. All constructs (Fig. 1a) led to similar phenotypes in stable transgenic lines, including narrow and pointed lateral organs and smaller, irregular rosettes with possibly shorter plastochrons in some lines (Figs 1f m,o,p, S2). As the constructs differ slightly with regard to the position of the truncation, we conclude that the observed phenotype is the consequence of their common feature: the deletion of domains III and IV. Because of the shared defects associated with each of the three transgenes, subsequent descriptions will usually be limited to MPD. MPD defects were inherited as semi-dominant traits in a dosage-dependent manner. Although, in hemizygous MPD, LPD-2 and MPD-3 plants, lateral organ laminae were still partially expanded and remnants of vascular lobes could be observed (Figs 1f i, S2a e,k o), leaves of homozygous plants were narrow and internally consisted mainly of parallel veins (Figs 1j m, S1f j,p t). Both hemizygous and homozygous MPD plants produced abundant flowers, but only hemizygous plants could be propagated. Homozygous MPD flowers were more severely affected and invariably sterile, producing lateral organs with striking increases in vascularization (Fig. 1r,t,v,x). Despite these gain-of-function effects, MPD conferred sufficient natural MP activity to rescue mp loss-of-function defects. For instance, MPD restored embryonic root formation, flower production and even fertility (Fig. 2), features never observed in mp mutants (Berleth & Jurgens, 1993; Przemeck et al., 1996; Hardtke & Berleth, 1998). Further, MPD increased vascular tissue formation in mp lateral organs, far beyond the amounts present in wild-type organs (compare Fig. 2c,d with Fig. 1c). MPD in auxin signal transduction We next asked whether auxin signal transduction was heightened in MPD plants by examining developmental auxin responses, the expression of auxin-dependent reporter genes and the transcription levels of auxin-inducible Aux IAAs. As shown in Fig. 3, roots did not form at the bases of dissected wild-type or mp cotyledons, but, on rare occasions, did initiate from wild-type cotyledons in the presence of exogenously applied auxin (Fig. 3a,b). By contrast, dissected MPD cotyledons occasionally produced roots even in the absence of auxin (Fig. 3a,b), demonstrating the constitutive expression of traits normally requiring hormone treatment. Moreover, the application of auxin resulted in a striking quantitative and qualitative increase in the rooting capacities of MPD cotyledons (Fig. 3a,b), suggesting that other ARFs also contribute to cotyledon rooting. An enhanced auxin response in MPD was also reflected in the strong constitutive expression of the auxin-response reporter DR5rev::GFP. The synthetic DR5 promoter comprising tandemly repeated ARF binding sites is probably targeted by MP. In early MPD leaf primordia, DR5rev::GFP was expressed much more strongly and ubiquitously than in the wild-type, and was maintained until later stages of leaf development, when expression normally subsides (Fig. 3c,f,i,l). Similar expression patterns were observed for functional MP::MP:GFP and MP::MP:GUS translational fusion constructs (Fig. 3d,g,j,m), supporting the notion of self-regulatory auxin-dependent MP expression (Lau et al., 2011), and for the auxin-responsive pre-procambial gene ATHB8, a confirmed direct target of MP (Donner et al., 2009). ATHB8::GUS remained confined to the procambium, but was expressed more strongly at all stages in MPD compared with the wild-type (Fig. 3e,h,k,n). Finally, the auxin-efflux gene PIN1 was expressed more strongly and in a more widespread manner in young MPD leaf primordia (Fig. 4i,o). Auxin-inducible PIN1 expression narrows down prior to vascular cell fate acquisition, as auxin is drained away (Scarpella et al., 2006; Wenzel et al., 2007). MPD leads to a wider PIN1 domain with stronger expression, and the resulting enhanced recruitment of ground meristem cells into the vascular lineage

4 394 Research New Phytologist (a) (b) (f) (c) (g) (d) (h) (e) (i) Fig. 1 MPD transgenes and phenotypes. (a) Schematics of protein domains encoded by MPD transgenes. The DNA-binding domain (DBD), middle activation domain (AD) and dimerization domains III and IV. Striped box of MPD represents 20 unrelated amino acids resulting from a cloning artifact. MPD and MPD-3 each contain a part of domain III but lack domain IV, whereas MPD-2 lacks both domains III and IV altogether. Cotyledons (4 d after germination, DAG) (b, c, f, g, j, k) and first leaves (7 DAG) (d, e, h, i, l, m) of wild-type (wt), hemizygous MPD (MPDhemi) and homozygous MPD (MPDhomo) Arabidopsis thaliana plants. (n p) Rosettes at 14 DAG: (n) wt; (o) MPD hemizygous; (p) MPD homozygous. (q x) Vascular pattern in flower organs: (q, r) sepals; (s, t) petals; (u, v) anthers; (w, x) carpels; wt (q, s, u, w) and MPD homozygous (r, t, v, x) plants. Note that there is overabundant vasculature in all floral organs (arrowheads). Vascular patterning (xylem) of cleared tissue is shown by dark field images (c, e, g, i, k, m, q x). Bars, (b, f, j) 1 mm; (c, d, g, h, k, l) 500 lm; (e, i, m, q t, w, x) 200 lm; (n p) 5 mm; (u, v) 50 lm. (j) (k) (l) (m) Aux IAAs have been shown to be rapidly responsive to auxin, and each contains multiple ARF binding sites in their regulatory regions (Abel et al., 1994; Sato & Yamamoto, 2008). As shown in Fig. 3(o), seven of the nine tested Aux IAAs displayed significantly enhanced expression levels in MPD compared with the wild-type. We conclude that there is constitutively elevated expression of auxin-induced genes in MPD, consistent with the presumed inaccessibility of MPD to negative regulation by Aux IAA proteins. (n) (o) (p) (q) (u) (r) (v) could affect the lateral growth of the leaf lamina (see Discussion section). In summary, auxin-inducible genes in leaf development that are barely expressed in mp mutants, but abundantly expressed in wild-type plants exposed to auxin, are intensely and constitutively expressed in MPD leaves. In order to address whether transcription from potential MPdependent promoters is constitutively enhanced in MPD, we measured the transcription initiation frequencies in a large number of Aux IAA genes in wild-type and MPD nuclei. The selected (s) (w) (t) (x) Impact of MPD on leaf architecture The most conspicuous morphological change caused by MP deregulation occurs in the architecture of shoot lateral organs. In MPD, all leaf-like lateral organs are narrower, more pointed and contain excess midvein vasculature at the expense of the typical reticulate venation pattern of the leaf lamina (Fig. 1k,m,r,t,v,x). In order to explore the developmental basis of this effect, we analyzed the early ontogeny of MPD vegetative leaves and the expression of a functional PIN1 : GFP fusion product (Benkova et al., 2003), as PIN1 has been found to be instrumental in the selection of procambial cells from leaf ground meristem (Scarpella et al., 2006). As PIN1 expression depends on both auxin and MP activity (Scarpella et al., 2006; Wenzel et al., 2007), we also visualized the MP expression domains at the respective stages of leaf development. As shown in Fig. 4, MP is expressed in the central and adaxial regions of leaves from plants at 2 DAG. In MPD, PIN1 distribution essentially coincides with MP expression, being both central and adaxial (Fig. 4a,b,m,n). This is in sharp contrast with that seen in wild-type leaf primordia at the same stage, where PIN1 is restricted to the leaf center (marking the position of the incipient midvein) and absent from the adaxial domain (Fig. 4g,h). Generally, PIN1 expression domains become wider and narrow down more slowly with increasing MPD activity, and this strict dosage dependence suggests a fairly direct regulatory influence of MPD. This effect is easy to follow in MPD hemizygotes, where the formation of midvein and secondary vein lobes is clearly recognizable, but all PIN1 expression domains are generally wider and delayed in their narrowing (Fig. 4c e). The same trend is seen in MPD

5 New Phytologist Research 395 (a) (b) (c) (d) Fig. 2 Complementation of Arabidopsis thaliana mp mutants by MPD. (a) Restoration of primary root formation by MPD. Seedling genotypes from left to right: mp G12, wild-type, MPDhomo mp G12. (b d) Vascular pattern in cotyledons: (b) mp G12 ; (c) MPDhemi mp G12 ; (d) MPDhomo mp G12. (e h) Inflorescences of 3-wk-old plants: (e) mp G12 inflorescences devoid of lateral organs; (f, g) MPDhemi mp G12 inflorescences; restoration of flower formation and partial restoration of fertility; arrow in (g) indicates a long seed-bearing silique; (h) MPDhomo mp G12 inflorescences. (b d) Dark field images of cleared tissues. Bars, (a) 0.1 mm; (b d) 0.5 mm. (e) (f) (g) (h) (a) (c) (d) (e) (f) (g) (h) (b) (i) (j) (k) (o) (l) (m) (n) Fig. 3 Auxin responses in MPD Arabidopsis thaliana. (a, b) Auxin-induced rooting: (a) proportion of root-forming cotyledons, 5 d after germination (DAG), grown in the presence (black bars) or absence (grey bars) of indole-3-butyric acid (IBA); (b) depiction of typical rooting abundance at rooting sites (arrowheads) after 8 d in culture. (c n) Expression of auxin-inducible vascular genes in wild-type (wt) (c e, i k) and MPDhomo (f h, l n). (c, f, i, l) DR5rev::GFP (arrowheads in c, d indicate narrowing expression domains); (d, g) MP::MP:GFP; (j, m) MP::MP:GUS (arrow in j points to residual MP expression); (e, h, k, n) ATHB8::GUS. (o) Transcription of Aux IAA genes in wt (open bars) and MPDhomo (closed bars) in nuclei from 14-DAG seedlings. Nuclear run-on assays quantifying transcription initiation rates. cpm, counts min )1 ; bg, background. Plotted data represent the mean of three independent experiments ± SD. *, P = 0.05; significance of genotype-based difference as determined by Student t-test analysis. (c, d, f, g, i, l) Confocal laser scanning images; (e, h, j, k, m, n) bright field images. Bars, (b) 2 mm; (c, d, f, g) 0.02 mm; (e, h) 0.05 mm; (i k, m, n) 0.2 mm; (l) 0.5 mm.

6 396 Research New Phytologist (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k) (l) (m) (n) (o) (p) (q) (r) Fig. 4 Expression of MP::MP:GFP and PIN1::PIN1:GFP in first-pair leaves of Arabidopsis thaliana. (a, b) MP expression (boxed in blue): (a) at 1 d after germination (DAG); note strong expression in central-adaxial ground meristem and weak expression in epidermis (arrowheads); (b) at 2 DAG; strong expression restricted to adaxial and central domain (arrow). (c e, g k, m q) PIN1 expression: (g, m) at 2 DAG; (h, n) at 3 DAG; note that expression is adaxially expanded in MPDhomo (arrows in m, n) and is congruent with the MP expression domain in (b). (c e, i k, o q) Narrowing of PIN1 expression domains under different MPD dosage. Note wider PIN1 expression domains in MPDhemi (c e) relative to the wild-type (i k), and nearly universal ground meristem PIN1 expression in MPDhomo (o q). The overall polarity of PIN1 subcellular localization is towards the leaf base, even in the extremely wide central domain of MPDhomo primordia (arrowhead in o). (f, l, r) 14-DAG leaves with mostly differentiated veins. Note that leaf shape and size are not affected, even in MPDhomo before 5 DAG. Days after germination, top right; genotype, bottom left; reproducibility fraction in parentheses (number of samples showing the illustrated features total number of inspected samples). (a, b, g, h, m, n) Leaf margin faces viewer. (c f, i l, o r) Adaxial side faces viewer. Note that optical settings used in low magnification images do not allow for clear recognition of PIN1 subcellular polarities. Bars, (a e, g k, m q) 20 lm; (f, l, r) 0.5 mm. homozygotes, but is so extreme that there is only a single central domain occupying nearly the entire ground meristem in leaf primordia up to 3 DAG (Fig. 4o q). As the procambial cells mature, PIN1 expression is reduced in the central and distal areas of the leaf primordium, but, nonetheless, nearly all subepidermal cells express PIN1 at 4 DAG (Fig. 4p). Notably, at this stage, leaf primordia, regardless of MPD dosage, are still relatively normal in size and shape. Subcellular polarities of PIN1 are essentially normal, although they may be less pronounced with increasing MPD dosage, which may reflect the widening of PIN1 expression. The dosage-dependent features support the notion that there is no genuine distortion of polar auxin transport in MPD, but rather a reduced stringency in polarization of PIN1 that is also observed in wide domains of PIN1 expression of wild-type plants (Scarpella et al., 2006). The preservation of typical PIN1 subcellular polarity, the initially normal and ultimately narrow leaf shape and the resulting vascular pattern argue against a primary defect in auxin transport in MPD, although delayed narrowing of PIN1 distribution and some irregularities in leaf initiation, such as occasional leaf fusions, may have suggested this (see the Discussion section). Normally, the basic pattern of PIN1 expression in 4-DAG leaf primordia presages the first- and second-order vein pattern in mature leaves (Scarpella et al., 2004, 2006; Fig. 4j). In MPD hemizygotes, vein patterning shows alterations with regard to the number of secondary vein lobes (Fig. 4d), but the basic architecture and leaf shape are not nearly as altered as in MPD homozygotes (Fig. 4p). In the latter, domains of PIN1 expression develop into a central field of parallel vascular strands or into two major veins running along the entire length of the leaf. These major veins ultimately frame a central region of parallel cell files aligned with the longitudinal axis of the leaf that may not have differentiated into vascular strands (Fig. 4r). The extreme narrowness of MPD homozygous leaves appears to be derived from the loss of lateral lamina growth beyond 5 DAG. Up to this point, leaf size and shape are normal, but

7 New Phytologist Research 397 (a) (b) (c) (d) (e) (f) Fig. 5 Double mutant analysis of MPD and gain-of-function aux iaa alleles. (a e) Dark field images of cleared Arabidopsis thaliana cotyledons 8 d after germination (DAG): (a) iaa12 bdl; (b) MPDhemi iaa12 bdl; (c) MPDhomo iaa12 bdl; (d) iaa18 crane2; (e) MPDhemi iaa18 crane2. (f) Hypocotyl lengths of 6 DAG dark-grown wild-type (wt) and iaa19 msg2 seedlings with (grey bars) or without (black bars) 20 lm indole-3-acetic acid (IAA). Sample sizes are seedlings. Columns represent the mean ± SD. Significance of genotype-based difference as determined by Student s t-test analysis: between the uninduced (mock-treated) values of MPD relative to the other three genotypes, P = 0.01; of the three genotypes inter se, no significant difference P < Bars, (a e) 500 lm. nearly the entire ground meristem expresses PIN1 (Fig. 4q). These cells may thus acquire vascular identity and polarity similar to midvein cells visible as parallel venation in a broad middle domain at 14 DAG (Fig. 4r). After 5 DAG, MPD homozygous leaves exhibit some lateral lamina growth. This appears to occur directly beneath hydathodes, resulting in a series of bulges, each with a short central secondary vein connected to a main longitudinal vein (Fig. 4r). This is in sharp contrast with the situation in MPD hemizygotes, where an only moderately wider midvein leaves enough cells for the initiation of a second order vein with a polarity towards the flanks of the primordium. PIN1 expression domains continue to narrow, albeit more slowly than in the wild-type, and ground meristem cells not expressing PIN1 are abundant and promote isotropic growth and widening of the leaf lamina (Fig. 4c e). Uncoupling of MP function from counteracting Aux IAA activity If MPD confers MP activity devoid of regulatory influence from Aux IAA proteins, phenotypic traits resulting from the interaction of MP with overly stable mutant Aux IAA products should no longer be visible. Instead, one might expect MPD to be epistatic over gain-of-function alleles in the respective Aux IAA genes. Auxin-response traits exclusively regulated through interaction with MP should no longer be affected by gain-of-function mutations in an Aux IAA regulator. However, independent pathways, through another ARF or an unrelated protein, acting on the same regulatory trait, may still limit the auxin response or even be epistatic over MPD. The gain-of-function iaa12 bodenlos (bdl ) mutation is associated with strongly reduced vasculature in cotyledons and other lateral shoot organs (Fig. 5a). Thus, bdl resembles mp loss-offunction alleles, suggesting that MP and IAA12 form a regulatory unit whereby IAA12 inhibits MP activity (Weijers et al., 2005). As shown in Fig. 5(a c), MPD does indeed uncouple MP activity from its upstream regulator BDL, as MPD is epistatic over bdl with regard to vascular patterning. Similar epistasis of MPD can be established over the gain-of-function allele of IAA18 (Fig. 5d,e). By contrast, in the control of hypocotyl elongation through auxin-mediated degradation of IAA19 (Tatematsu et al., 2004), the auxin resistant gain-of-function iaa19 massugu2 allele is epistatic over MPD (Fig. 5f). Collectively, these findings may reflect the regulation of PIN1 by MP or redundantly acting ARFs (Wenzel et al., 2007), as opposed to the control of hypocotyol elongation through a separate pathway involving ARF7 (Tatematsu et al., 2004). In this process, ARF7 has been shown to act nonredundantly to MP (Hardtke et al., 2004).

8 398 Research New Phytologist Discussion The functions of ARFs are largely unknown, presumably because of widespread genetic redundancy within the gene family. Despite this difficulty, the functions of some ARFs have been explored by means of single and multiple loss-of-function mutant genotypes and by ARF mis- or overexpression. Further, phenotypic similarities between ARF loss-of-function and Aux IAA gain-of-function alleles, in combination with molecular data, have revealed individual cases of ARF interactions with Aux IAA proteins (reviewed in Chapman & Estelle, 2009), but, in most cases, the relevance of these interactions in natural processes has remained unclear. In this study, we have introduced a new tool to explore ARF function: an artificial gain-of-function allele of ARF5 via deletion of conserved protein domains III and IV. We presume that the same strategy should be applicable to other ARFs, including those that do not display loss-of-function phenotypes, to shed some light on their functions. This strategy could complement the exploration of ARF functions through mis- and overexpression. Overexpression from strong, ubiquitous promoters can lead to expression in inappropriate tissues or stages, and to binding of artificial targets. By contrast, the deletion of domains III and IV is predicted to simply release the ARF from Aux IAA control, as would normally occur in response to auxin (reviewed in Leyser, 2006). Therefore, the consequences of auxin regulation on the activity of an individual ARF may be probed with such constructs, as opposed to systemic activation by external application of auxin. This interpretation, however, may be too naïve, as there may be other types of regulation (Shin et al., 2007) or unknown functions associated with domains III and IV. Nevertheless, MPD exerts genuine MP activity and, if further unknown protein functions reside within these terminal domains, the investigation of the effects of ARF truncations is a promising way to identify them. Numerous traits of MPD are consistent with loss of Aux IAAmediated repression, including its dominant nature, its activation of auxin-inducible genes, the prolonged expression of auxininducible marker genes in its presence and its epistasis over certain Aux IAA gain-of-function alleles. These observations suggest that MP can affect plant development without the ability to form dimers through domains III and IV. This is interesting, because premature stop codons only a few hundred nucleotides upstream of the truncation site in MPD result in loss-of-function alleles (Hardtke & Berleth, 1998). These genetic properties are plausible if most of the middle domain is required for MP activation, whereas domains III and IV are required to suppress this activation in the presence of a binding Aux IAA protein. As a consequence, only stop codons very close to the junction between the middle domain and domain III would lead to the conspicuous MPD phenotype. This might explain why the highly vascularized, narrow leaf phenotype of MPD has not been picked up in forward genetic screens for leaf mutants (reviewed in Scarpella & Meijer, 2004). Although MPD may represent a constitutively active MP variant for certain target genes, this may not be true for others. Some target genes could be regulated by specific ARF dimers or by still larger transcriptional complexes. MPD could either be excluded from these transcriptional complexes or overshadowed by other types of control. Finally, it is possible that MP functions completely independently of any auxin input in various regulatory processes. In such instances, MPD could still complement the mp mutant, but no gain-of-function properties would be conferred. Because of the likely heterogeneity of transcriptional complexes involving MP, it will require full transcriptome analysis of its target genes to understand the impact of Aux IAA regulation of MP on a gene-by-gene basis. Beyond the investigation of gene regulatory mechanisms, MPD can serve as a powerful tool to better explore patterning mechanisms that rely on MP. Loss-of-function alleles have identified MP as a key regulator of vein formation and as a direct or indirect regulator of PIN1, the PIN family member that is instrumental in leaf vascularization (Scarpella et al., 2006; Wenzel et al., 2007). Whereas loss of MP function results in a dramatic reduction in the leaf venation pattern, MPD confers an overabundance of vascular tissue (Fig. 1). This overabundance, however, is associated with a dramatic change in leaf growth and morphology, raising the possibility that the vascular hypertrophy could be a secondary effect of altered leaf shape. Our analysis of leaf ontogeny in MPD, however, indicates that the pattern of PIN1 expression is dramatically altered from the earliest stages of leaf development, in a way that profoundly delays the narrowing of PIN1 expression domains, long before there is any alteration in leaf shape or size. This delay seems to be under stringent MP control, as it is directly correlated to the dosage of MPD. In wild-type development, sustained expression of PIN1 is considered to be the key event in selecting cells for the vascular lineage (Scarpella et al., 2006). Initially, a subepidermal PIN1 expression domain in the center of the emerging primordium narrows down to presage the position of the leaf midvein. In MPD, this midvein PIN1 expression remains wider and, in the MPD homozygote, fails to narrow at all. In the latter case, there is no sharp demarcation between PIN1-expressing and nonexpressing cells, even at 5 DAG, and nearly all cells may therefore be recruited to the vascular lineage within an enlarged midvein region. The later appearance of these cells as parallel bundles indicates that they have been polarized along the leaf longitudinal axis and have been partitioned into separate strands after 5 DAG. Such partitioning of a broad domain of PIN1 expression later in leaf development has been described previously (Scarpella et al., 2006). As judged by PIN1 expression, very few cells remain at 5 DAG that are not polarized along the leaf longitudinal axis. Thus, there is limited capacity for growth that would increase the width of the lamina (see Fig. 6). When such growth is observed, it appears to occur late in leaf development beneath the position of hydathodes, resulting in local outgrowth on otherwise very narrow leaves (Fig. 4r). Vascular hypertrophy is also observed in auxin transportinhibited plants, but here the vascular system becomes shifted towards the leaf margin, leaves become wider and flower formation is invariably obstructed (Okada et al., 1991; Mattsson et al., 1999; Sieburth, 1999). By contrast, in MPD, leaves are narrower, with reduced peripheral veins, and abundant flowers are

9 New Phytologist Research 399 Fig. 6 Scheme of wild-type and MPDhomo leaf development. White cells: isotropically dividing ground meristem cells that can be turned into procambial cells by sustained PIN1 expression. Grey cells: PIN1-expressing incipient procambial cells that will divide and expand along a main axis predisposed by the orientation of PIN1 polarity. Overlaid arrows show the main axes of PIN1 subcellular polarities. Data from analyses shown in Fig. 4. It is assumed that sustained PIN1 expression selects procambial cells from the ground meristem, and that the polarities of the procambial cells are then maintained throughout leaf growth (Scarpella et al., 2006). The remaining ground meristem cells are still accessible to new directional cues and will otherwise grow isotropically. Two days after germination (DAG), wild-type (wt): narrow central PIN1 expression domain recruits only a few cells to become midvein procambium. The remaining white cells remain accessible to lateral cues (lateral epidermal convergence points; Scarpella et al., 2006) and form second-order PIN1 expression domains (grey side branches) with new lateral polarities at 3 DAG. By contrast, few cells are left responsive to lateral cues in MPDhomo, and the alignment of polarity along the leaf longitudinal axis in the broad central procambial domain limits lateral leaf growth. 14 DAG: where lateral cues can act on sufficient numbers of ground meristem cells later in development, they may induce short second-order veins with limited lateral growth. This leads to stochastic local lateral lamina growth and second-order vein formation, but the basic architecture of the leaf, which is established early, is dominated by a wide central procambial domain in MPDhomo. generated. Therefore, neither the MPD phenotype nor the cellular function of MP suggests a direct negative effect of MPD on auxin transport. For these reasons, we postulate that the primary defect in MPD is the failure to turn off PIN1, required for the narrowing of PIN1 expression domains, because of the inability of Aux IAA proteins to downregulate MP activity (Fig. 4). Restriction of PIN1 expression to only a part of the MP expression domain can most clearly be seen in very early leaf primordia. In the wild-type, the MP expression domain is far larger than that of PIN1, indicating that there is some spatially restricted negative regulation preventing MP from triggering PIN1 expression in the adaxial domain. By contrast, in MPD, the expression domains of MP and PIN1 are virtually congruent, indicating that releasing MP from Aux IAA control translates into the promotion of PIN1 expression in the entire MP expression domain. Theoretical considerations (reviewed in Berleth et al., 2007), as well as observations in leaves with high auxin content (Scarpella et al., 2006), suggest that all subepidermal cells participate through some incremental level of PIN1 expression in the selection of procambial cells from the ground meristem. Restricting PIN1 expression to sharply defined domains should then require gene regulatory mechanisms, the failure of which would result in the phenotype seen in MPD. A number of Aux IAAs, including IAA12, 18, 20, 30 and 31, exhibit gain-of-function or overexpression phenotypes with reduced leaf vascularization (Hamann et al., 1999; Sato & Yamamoto, 2008; Ploense et al., 2009). These would therefore be promising candidates for genes acting redundantly in narrowing down PIN1 expression domains in leaves. MPD is epistatic over the gain-of-function iaa12 bdl and iaa18 crane2 mutations. This demonstrates that, in the regulation of the leaf venation pattern, the release of MP activity from Aux IAA control overrides nearly all other influences, such as those from other ARFs. This is consistent with the fact that ARFs known to influence vascularization act redundantly with MP (Hardtke et al., 2004). By contrast, ARF7 has a clear nonredundant function in regulating hypocotyl length in response to auxin and is thought to do so in a regulatory feedback with IAA19 (Tatematsu et al., 2004). That MPD proves not to be epistatic to the effects of IAA19 gainof-function on hypocotyl expansion could reflect the sharp separation of the pathways through which MP and ARF7 influence hypocotyl length. If so, MPD or other gain-of-function ARF derivatives might be useful to disentangle the assignment of individual ARFs and Aux IAAs to separate auxin response pathways. Acknowledgements We would like to acknowledge project support from a Natural Sciences and Engineering Research Council of Canada (NSERC) discovery grant to T.B., NSERC long-term postgraduate fellowship and a Government of Ontario Scholarship in Science and Technology (OGSST) to N.T.K., NSERC CGSM to A.C., and support from the Center for Analysis of Genome Evolution and Function (CAGEF). We would like to thank G. Morelli, J. Friml, D. Weijers, J. Reed, H. Fukaki, T. Hamann and R. Z. Sung for seeds. References Abel S, Oeller PW, Theologis A Early auxin-induced genes encode shortlived nuclear proteins. Proceedings of the National Academy of Sciences, USA 91: Aloni R The induction of vascular tissues by auxin and cytokinin. In: Davies PJ, ed. Plant hormones: physiology, biochemistry and molecular biology. 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