Root gravitropism and root hair development constitute coupled developmental responses regulated by auxin homeostasis in the Arabidopsis root apex

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1 Research Root gravitropism and root hair development constitute coupled developmental responses regulated by auxin homeostasis in the Arabidopsis root apex Stamatis Rigas 1 *, Franck Anicet Ditengou 2 *, Karin Ljung 3, Gerasimos Daras 1, Olaf Tietz 2, Klaus Palme 2,4,5,6 and Polydefkis Hatzopoulos 1 1 Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, Athens, , Greece; 2 Institute of Biology II, Faculty of Biology, Albert-Ludwigs-University of Freiburg, Sch anzlestrasse 1, D-79104, Freiburg, Germany; 3 Department of Forest Genetics and Plant Physiology, Umea Plant Science Centre, Swedish University of Agricultural Sciences, SE , Umea, Sweden; 4 Centre of Biological Systems Analysis, Albert-Ludwigs-University of Freiburg, Habsburgerstr. 49,D-79104, Freiburg, Germany; 5 Freiburg Institute of Advanced Sciences (FRIAS), Albert-Ludwigs-University of Freiburg, Albertstrasse 19,D-79104, Freiburg, Germany; 6 Centre for Biological Signalling Studies (bioss), Albert-Ludwigs-University of Freiburg, Albertstrasse 19,D-79104, Freiburg, Germany Author for correspondence: Polydefkis Hatzopoulos Tel: phat@aua.gr Received: 9 November 2012 Accepted: 10 November 2012 doi: /nph Key words: auxin transport, gravitropism, PIN1, root hair, TRH1. Summary Active polar transport establishes directional auxin flow and the generation of local auxin gradients implicated in plant responses and development. Auxin modulates gravitropism at the root tip and root hair morphogenesis at the differentiation zone. Genetic and biochemical analyses provide evidence for defective basipetal auxin transport in trh1 roots. The trh1, pin2, axr2 and aux1 mutants, and transgenic plants overexpressing PIN1, all showing impaired gravity response and root hair development, revealed ectopic PIN1 localization. The auxin antagonist hypaphorine blocked root hair elongation and caused moderate agravitropic root growth, also leading to PIN1 mislocalization. These results suggest that auxin imbalance leads to proximal and distal developmental defects in Arabidopsis root apex, associated with agravitropic root growth and root hair phenotype, respectively, providing evidence that these two auxin-regulated processes are coupled. Cell-specific subcellular localization of TRH1-YFP in stele and epidermis supports TRH1 engagement in auxin transport, and hence impaired function in trh1 causes dual defects of auxin imbalance. The interplay between intrinsic cues determining root epidermal cell fate through the TTG/ GL2 pathway and environmental cues including abiotic stresses modulates root hair morphogenesis. As a consequence of auxin imbalance in Arabidopsis root apex, ectopic PIN1 mislocalization could be a risk aversion mechanism to trigger root developmental responses ensuring root growth plasticity. Introduction Cellular differentiation and morphogenesis are major developmental processes modulating the growth of multicellular organisms. The interplay of environmental cues and the interdependence of hormones play a major role in these two processes. Auxin, the tryptophan-derived plant hormone, has been implicated in the patterning or growth of virtually every tissue (Teale et al., 2006). During primary growth, auxin is mainly synthesized at the shoot apex and moves towards the root tip through the vascular tissues of stems. Components within the stellar tissues that facilitate polar auxin transport form an auxin maximum at the root tip columella through the acropetal directional auxin flow for the maintenance of root stem cells (Sabatini et al., 1999; *These authors contributed equally to this work. Friml et al., 2002a; Blilou et al., 2005). A stable asymmetric auxin distribution is established around the meristematic zone, where auxin moves in a basipetal orientation to the elongation zone through the root peripheral tissues, including the cortical and epidermal cell layers. AUXIN RESISTANT 1/LIKE AUX1 (AUX1/LAX) auxin influx carriers, PIN-FORMED (PIN) auxin efflux facilitators, P-GLYCOPROTEIN (MDR/PGP/ABCB) efflux transporters and the potassium transporter TRH1 are key components of the network that mediates polar auxin transport and regulates root morphogenesis and growth (Vicente- Agullo et al., 2004; Blilou et al., 2005; Vieten et al., 2005; Wisniewska et al., 2006; Mravec et al., 2008). In roots, auxin modulates major developmental processes, including primary root growth, lateral root formation, gravitropism and root hair morphogenesis. 1130

2 New Phytologist Research 1131 The primary root of most plants exhibits gravitropism, the tendency after germination to grow parallel to the gravity vector downwards into the soil to ensure water and nutrient supply. On the basis of the starch statolith theory of gravity perception, columella cells in the root cap constitute the gravity-sensing site in roots as a result of amyloplast sedimentation (Blancaflor et al., 1998). During gravistimulation, auxin transport components mediate the perception, transmission and response to a gravity stimulus, consistent with the role of auxin as the primary gravitropic signal (Swarup et al., 2005; Abas et al., 2006). The gravity-induced retargeting of the auxin efflux facilitator PIN3 presumably modulates auxin asymmetric redistribution at the root apex (Friml et al., 2002b; Kleine-Vehn et al., 2010). Molecular, genetic and biochemical evidence supports the widely appreciated Cholodny Went theory proposing that auxin asymmetry results in a differential response, causing growth curvature of the root towards the gravitational force. The auxin influx and efflux facilitators AUX1 (Bennett et al., 1996; Swarup et al., 2005) and PIN2 (AGR1/EIR1/WAV6) (Chen et al., 1998; Luschnig et al., 1998; M uller et al., 1998; Abas et al., 2006), respectively, promote differential auxin flux rates in the basipetal direction by channelling auxin from the root cap to the elongation zone, thereby maintaining the lateral auxin gradient initially established by PIN3. In Arabidopsis, root hair structures emerge from highly differentiated epidermal cells, the trichoblasts in the H position, arranged in alternating files with the atrichoblasts in the N position (Dolan et al., 1994; Scheres et al., 1994). Auxin has a pivotal role in root hair development, mainly acting downstream of the TTG/GL2 pathway that determines root epidermal cell fate (Masucci & Schiefelbein, 1996; Pitts et al., 1998). Numerous Arabidopsis mutants defective in auxin response or transport have been identified with the root hair phenotype. Root hairs of the axr1 mutant are shorter than those of the wild-type (Cernac et al., 1997; Pitts et al., 1998). The AXR1 protein has features of the ubiquitin-activating enzyme E1 and is required for a normal hormonal response (Pozo et al., 1998). The sar1 mutant suppresses any phenotypic aspect of the axr1 background, including root hair density (Cernac et al., 1997). The AXR2, AXR3 and SHY2 genes encode for Arabidopsis Aux/IAA proteins, and thereby they are rapidly induced as a primary response to auxin (Nagpal et al., 2000; Knox et al., 2003). Mutations in the domain II destruction box of AXR3/IAA17 and SHY2/IAA3, which confer protein stability and increased auxin resistance by reducing molecular interactions with SCF TIR1, have opposite root hair phenotypes (Knox et al., 2003). Although, axr3 plants have essentially no root hairs, shy2 mutants show increased root hair density. Mutations in the AXR2 gene show a reduction in root hair abundance (Nagpal et al., 2000). In addition to axr2, rhd6 mutant plants show a dramatic reduction in hair density. However, unlike rhd6 plants, the application of either auxin or ethylene does not rescue the axr2 phenotype (Masucci & Schiefelbein, 1996). Auxin transport mutants aux1 (Pitts et al., 1998) and trh1 (Rigas et al., 2001) have shorter root hairs than wild-type seedlings. The AUX1 gene encodes a putative transporter that facilitates auxin influx (Swarup et al., 2001), whereas TRH1 encodes a potassium transporter that is also required for auxin transport in Arabidopsis roots (Vicente-Agullo et al., 2004). In addition to auxin, the gaseous plant hormone ethylene is likely to be the positional signal translocated along the apoplasmic space between underlying cortical cells reaching the trichoblasts (Tanimoto et al., 1995; Masucci & Schiefelbein, 1996; Pitts et al., 1998). Genetic and physiological studies have shed light on the extensive crosstalk between auxin and ethylene in the regulation of root growth. Ethylene stimulates auxin biosynthesis (Ruzicka et al., 2007; Swarup et al., 2007) and auxin transport (Ruzicka et al., 2007), and therefore increases the levels of auxin in epidermal cells. Although the root gravitropic response and root hair formation have been extensively reported to be modulated by auxin, there is no report of the coupling of these two morphogenetic phenomena with auxin homeostasis. Here, we report that Arabidopsis backgrounds with impaired auxin perception or transport show ectopic localization of the PIN1 auxin efflux facilitator in the epidermal cells of the root meristematic zone. The redirection of auxin flow by PIN1 apical (shootward) polar localization in these cells correlates with impaired auxin translocation from the site at which gravity is perceived (root tip) to the growth response region (elongation zone). On the basis of these results, we propose that root gravitropism and root hair elongation constitute coupled developmental responses relying on auxin homeostasis and maxima in the Arabidopsis root apex. Materials and Methods Plant material and growth conditions Arabidopsis thaliana (L.) Heynh. mutants aux1-7 (N3074) and axr2 (N3077) were obtained from Nottingham Arabidopsis Stock Centre, Nottingham, UK; rhd6 seeds were generously provided by Liam Dolan (University of Oxford, Oxford, UK); the 35S:: PIN1 plant line was generated by fusion of the CaMV35S promoter with the Arabidopsis PIN1 gene, as described previously (Benkova et al., 2003). After 48 h of stratification in the dark at 4 C, plants were grown on a near-vertical position on plates containing Murashige Skoog (MS) medium (Duchefa, Haarlem, the Netherlands) supplemented with 0.05% MES-KOH buffer, ph 5.7, 1% sucrose and solidified with 0.4% phytagel (Sigma, St Louis, MO, USA). Seedlings were cultivated for 3 7 d after germination (dag) at 22 C with a cycle of 16 h : 8 h light : dark. For chemical treatment, hypaphorine was prepared as described by Ditengou & Lapeyrie (2000). Immunolocalization PIN1 and PIN2 immunolocalizations in 5-d-old Arabidopsis seedlings were performed using the InsituPro liquid handling robot from Intavis AG (Cologne, Nordrhein-Westfalen, Germany), as described by Friml et al. (2003). The following antibodies and dilutions were used: anti-pin1 (1 : 400) (Paponov et al., 2005), anti-pin2 (1 : 400) (M uller et al., 1998) and Alexa Fluor 488 or Alexa Fluor 546 (1 : 300) secondary antibodies

3 1132 Research New Phytologist (Molecular Probes, Oregon, OR, USA). Fluorescent samples were inspected by a Zeiss Axioplan 2 Imaging confocal laser scanning microscope and Zeiss LSM 510 Image Browser software. Images were processed using Adobe Photoshop. Histological staining Wild-type and trh1 seedlings were initially grown vertically on solidified MS medium for 5 d. These young developing seedlings were subsequently transferred to liquid MS to be treated for several time points and concentrations with naphthalene-1-acetic acid (NAA) (Sigma). The DR5 auxin response transgene was visualized by b-glucuronidase (GUS) staining for 2 h, as described previously (Haralampidis et al., 2002). The specimens were observed under an Olympus BX-50 (Tokyo, Japan) light microscope equipped with a Sony DSC-F707 camera. Endogenous auxin measurements Arabidopsis wild-type Wassilewskija (N1603) and trh1 mutant seedlings were grown on vertical agar plates for 4 and 7 d in longday conditions, as described by Ljung et al. (2005). The most apical (closest to the root tip) 4 mm of the root was collected from each seedling and divided into 1-mm sections. For each sample, 50 sections were pooled, frozen in liquid nitrogen and stored at 80 C; 100 pg 13 C 6 -IAA internal standard was added to each sample before extraction, and the samples were then extracted, purified and analysed by gas chromatography-selected reaction monitoring-mass spectrometry (GC-SRM-MS), as described previously (Ljung et al., 2005). All measurements were performed in triplicate. Morphometric analysis The root hair phenotype of Arabidopsis 5-d-old plants analysed in this study was observed on a Zeiss Stemi SV11 Apo stereomicroscope (Carl Zeiss, Goettingen, Germany), equipped with an Axiocam HR CCD camera (Carl Zeiss, Goettingen, Germany). Morphometric analysis of root shape was performed by scanning 5-d-old seedlings grown vertically on an Epson Perfection 3170 Photo Scanner (Seiko Epson Corp., Nagano-Ken, Japan). The digital images obtained were further analysed using the IMAGEJ software package ( and were processed with Microsoft Office Excel. Vertical growth index calculations were performed as described previously (Grabov et al., 2005). Fluorescent live cell imaging of TRH1 Citrine-YFP was PCR amplified with 5 -TTTGATCAAAGGAG GTGGAGGTGGAGCT-3 forward and 5 -TTGATCAAGG CCCCAGCGGCCGCAGCA-3 reverse primers. The PCR product was cloned into the pgem T-Easy vector (Promega, Madison, WI, USA). The TRH1 gene, including the promoter region, was PCR amplified with 5 -AAGCTTGTCGAC AGGCTGATATCTGGAGTGCTGGTGTGA-3 forward and 5 -TTTGCAACTAAAACTACACAGAGTAGAG-3 reverse primers, and the PCR product was cloned into the pbluescript SK vector. The Citrine-YFP gene was then introduced into the C-terminus of the TRH1 genomic sequence. The resulting TRH1-ΥFP transgene was cloned into the SmaI site of the pgptv-hpt binary vector. The TRH1-ΥFP construct was introduced into trh1 plants by Agrobacterium tumefaciensmediated stable transformation. Transgenic seedlings of the T2 and T3 generations were grown vertically and analysed by confocal microscopy. Results The basipetal route of auxin transport is distorted in trh1 roots Disruption of the TRH1 potassium transporter impairs root hair development (Rigas et al., 2001) and results in agravitropic root growth (Vicente-Agullo et al., 2004). In trh1 mutant plants, the root hair length (Figs 1, 2a) and density (Fig. 2b) were reduced relative to the wild-type Wassilewskija (Ws-0) ecotype background from which the mutant was isolated. In addition, the primary root growth was agravitropic (Fig. 2c). These morphological defects can be restored by exogenous application of auxin, demonstrating a link between TRH1 activity and auxin transport (Vicente-Agullo et al., 2004). These experimental results raised the question of whether auxin transport is defective in trh1 roots, resulting in suboptimal auxin levels at the epidermal tissues spanning the root elongation and differentiation zone. To experimentally address this question, we observed the accumulation of auxin in trh1 plants by monitoring, at cellular resolution, the synthetic auxin-responsive reporter DR5::GUS. The seedlings were grown under the effect of the synthetic auxin NAA. NAA is capable of entering cells by diffusion, but requires the active auxin efflux mechanism to be translocated. In the absence of NAA, the auxin response was restricted to the quiescent centre and to the initials or mature columella cells of the wild-type root tip (Fig. 3a), whereas, in trh1 roots, additional sites of auxin response were detected in protoxylem cell files (Fig. 3b). Exogenous application of auxin for certain time points or under concentrations ranging from 10 to 60 nm revealed a completely different pattern of auxin distribution between the trh1 and wild-type roots. Apart from the quiescent centre and columella initials or mature columella cells, wild-type roots showed enhanced auxin accumulation in cortical, epidermal and lateral root cap cells at the meristematic region. However, at the central cylinder, DR5 expression was observed in a narrow column of stellar cells (Fig. 3a). By contrast, at the root tip of trh1 roots, DR5 expression was marginally detected at the cortical, epidermal and lateral root cap cells only under the application of 60 nm NAA (Fig. 3b). Likewise, in the distal elongation zone, the auxin response gradually expanded from the central cylinder to the root peripheral cell files as the duration of auxin application or the concentration of auxin increased. Together, these data demonstrate that, in wild-type roots, the pattern of auxin response, as recorded by DR5::GUS expression, coincides with the reported basipetal route of auxin (Fig. 3c), whereas, in trh1

4 New Phytologist Research 1133 (a) (b) (c) (d) (e) (f) (g) (h) Fig. 1 The root hair phenotype of 5-d-old Arabidopsis thaliana seedlings. The primary root of wild-type Columbia (Col-0) (a), trh1 (b), pin2 (c), axr2-1 (d), aux1-7 (e), 35S:: PIN1 (f) and Col-0 (g) plants grown under the effect of 100 lm hypaphorine, and rhd6 (h). Bars, 200 lm. roots, this pattern is distorted (Fig. 3d). Furthermore, the increased expression of DR5 in the stele of the thr1 background reveals that auxin is acropetally directed to trh1 root tips without being redistributed further. The malfunction of the basipetal auxin transport route in trh1 roots leads to auxin imbalance which may perturb auxin-dependent developmental processes, such as the root response to gravity and root hair development. tip of 7-d-old trh1 plants was significantly higher than that of the wild-type plants. Elevated auxin accumulation provides evidence to support the acropetal direction of auxin to trh1 root tips, as revealed previously by DR5 expression in the stele of the trh1 background (Fig. 3b), but the active auxin efflux system mediating basipetal auxin distribution displays abnormal function, creating auxin imbalance. Auxin distribution is different in trh1 and wild-type root tips Given the key role of directional auxin flow in plant developmental processes, we performed endogenous IAA measurements to verify experimentally that auxin homeostasis is perturbed in the trh1 root apex. In wild-type root tips, the basipetal IAA concentration gradient is established just before the leaf-mediated IAA pulse reaches the tip, accompanied by a significant increase in IAA concentration in the first millimetre of the root tip (Bhalerao et al., 2002). Hence, the levels of free IAA were measured 4 and 7 d after germination in sections of wild-type and trh1 root tips. IAA levels were slightly higher in the first millimetre and marginally lower above the third millimetre of 4-d-old trh1 root tips relative to the wild-type tips (Fig. 4a). Within the central region of the root tips extending between the first and the third millimeter, the content of auxin in trh1 tips was similar to that of the wildtype. However, the content of auxin was increased in 7-d-old trh1 root tips (Fig. 4b). Auxin concentration over the entire root TRH1 cell-specific localization supports the proximal and distal defects of auxin suboptimal concentration in the trh1 root apex As the auxin accumulation pattern and IAA quantitative analysis revealed auxin imbalance in the trh1 root apex, it is important to delineate its functional role in hormone transport. To track TRH1 localization, the YFP reporter gene was fused at the C-terminus of the TRH1 genomic fragment including the promoter sequence. Confocal microscopy live cell imaging revealed the polar plasma membrane localization of TRH1 at the cells of the epidermis, cortex and endodermis in the meristematic zone (Fig. 5a). In the stele of the root elongation zone, TRH1 was co-localized with the polar auxin efflux carrier PIN1 on the basal (rootward) cell side, consistent with the role of TRH1 in auxin transport (Fig. 5b). The fact that TRH1 is localized in both the central cylinder at the elongation zone and in the peripheral root layers, including the epidermis and cortex, at the meristematic

5 1134 Research New Phytologist (a) (b) plasma membrane revealed that TRH1 is also partially localized at the plasma membrane (Fig. 5c). On brefeldin A (BFA) application, FM4-64 is internalized, becoming a tool for the analysis of endocytosis and vesicle trafficking. This treatment indicated that TRH1 was not completely co-localized with FM4-64 (Fig. 5d). TRH1 localization at intracellular membrane structures is reminiscent of the route of auxin translocation within the cell from the cytoplasm to the nucleus, potentially through the endoplasmic reticulum (ER). Intracellular auxin transport has already been reported for the PIN5-type subclass formed by the Arabidopsis PIN5, PIN6 and PIN8 members of the PIN protein family that is evolutionarily older than the plasma membranelocalized PIN1-type subclass (Mravec et al., 2009). PIN5-mediated auxin uptake into the ER lumen might have a relevant role in reducing auxin availability for plasma membrane-based auxin exporters and increasing the amount of auxin in the ER pool, where enzymes of auxin metabolism are compartmentalized. (c) Fig. 2 Biometric analysis of the primary root in Arabidopsis thaliana backgrounds. Graphs presenting: (a) root hair length (in lm) of the examined plant genotypes; (b) root hair density as the number of hairs per primary root millimetre of the examined plant genotypes; and (c) the primary root shape of the examined plant genotypes quantified by the vertical growth index (VGI). Error bars represent standard deviations of the means. zone suggests that it could participate in both acropetal and basipetal auxin transport. Consequently, in trh1 roots, auxin imbalance is associated with developmental defects proximal and distal to the root apex. The proximal defect is the agravitropic root behaviour that most likely originates from the improper transport of auxin to the quiescent centre and columella region and/or from the hormonal gradient formed between the stele and the quiescent centre and columella region. The root hair phenotype is the distal defect attributed to the distorted basipetal auxin transport. Intriguingly, TRH1 is predominantly localized to internal compartments of the root epidermis (Fig. 5c,d). Apart from the evident internal localization, the FM4-64 dye that labels the TRH1 activity is required for PIN1 localization As trh1 plants showed an impaired auxin homeostasis pattern, we hypothesized that the subcellular topology of the PIN auxin efflux carriers might be modified. To test this hypothesis, we determined the localization of the PIN auxin carriers. Immunolocalization revealed that only PIN1 was ectopically expressed in specific cell types of the trh1 meristematic zone. In the wild-type roots, PIN1 is predominantly localized with a basal polarity to the bottom side of the stele and endodermis cells (Fig. 6a). Furthermore, PIN1 is detected at the quiescent centre and the cortex. At the meristematic zone surrounding the root initial cells, the PIN1 localization signal fades out, being restricted at the initials of the epidermis and lateral root cap, whereas it is marginally identified at the two epidermal cells above the quiescent centre (Fig. 6b). In contrast with wild-type roots, in trh1 roots, PIN1 is detected at the epidermal cells of the entire meristematic zone and at the transition zone, a border between the root meristem and elongation zone (Fig. 6c,d). PIN1 is localized on the apical (shoot apex facing) side of the epidermal cells at the PIN2 position (Blilou et al., 2005; Vieten et al., 2005). The ectopic localization pattern of PIN1 is restored in trh1 plants functionally complemented by the insertion of the TRH1 genomic locus (Supporting Information Fig. S1), generating normally elongated root hairs (Fig. 2a,b) and having primary roots responding to gravity (Fig. 2c). Together, these results indicate that, in trh1 root tips, the ectopic polar arrangement of PIN1 results from auxin imbalance, and hence is most likely a response to attenuate the defect in basipetal hormonal transport. PIN1 is mislocalized in Arabidopsis backgrounds with defects in gravity response and root hair development Auxin imbalance in trh1 root tips caused developmental defects, including agravitropic root growth and root hair phenotype, and ectopic PIN1 mislocalization at the meristematic region. This raised the question of whether it also occurs in other auxin mutants. The PIN2 gene encodes for an auxin efflux carrier that

6 New Phytologist Research 1135 (a) (c) (b) (d) Fig. 3 The basipetal auxin distribution profile is modified in the trh1 auxin transport mutant of Arabidopsis thaliana. Concentration- and time-dependent elevation of auxin accumulation in wild-type (a) and trh1 (b) roots following the expression pattern of the synthetic auxin response transgene DR5::GUS. Schematic representation of enhanced responses in wild-type (c) and trh1 (d) roots that appear at late stages of 1-naphthalene acetic acid (NAA) application. One representative image of three independent experiments, with 6 10 seedlings per experiment, is shown. (a) (b) Fig. 4 Auxin concentration gradient in 1-mm root tip sections of Arabidopsis thaliana wild-type and trh1 genetic background. (a) Auxin content in the first millimetre of the root tip from 4-d-old trh1 (open squares) plants is higher than that in the wild-type (closed squares), whereas it gradually decreases in the fourth millimetre. (b) Over the entire root tip of 7-d-old trh1 plants (open triangles), the IAA concentration is significantly higher than that of the wild-type (closed triangles). Error bars indicate standard deviation (SD). is predominantly found with apical polarity in the root epidermis and lateral root cap and basal polarity in the cortex (Blilou et al., 2005; Vieten et al., 2005). PIN2 is a major component for basipetal auxin transport, being involved in root gravitropism (Abas et al., 2006). In addition to the agravitropic root growth phenotype (Fig. 2c), pin2 mutants also have a root hair phenotype (Fig. 1c), with reduced root hair length and density (Fig. 2a, b). Immunolocalization experiments revealed that PIN1 is localized on the apical side of pin2 epidermal cells at the meristematic zone (Fig. 6e). This pattern of PIN1 ectopic localization in pin2 mutants is consistent with previous observations supporting the hypothesis of functional synergistic interactions within the PIN members for compensation of the redundant auxin distribution network (Vieten et al., 2005; Wisniewska et al., 2006). The localization pattern of PIN1 was also investigated in the root tips of axr2-1 auxin signalling (Nagpal et al., 2000) and aux1-7 auxin transport (Pitts et al., 1998) mutants. Not only were the root hair length and density problematic in both axr2-1 (Figs 1d, 2a,b) and aux1-7 (Figs 1e, 2a,b) backgrounds, but also the growth of the primary root was agravitropic (Fig. 2c). PIN1 was ectopically localized on the apical side of axr2-1 (Fig. 6f) and aux1-7 epidermal cells at the meristematic zone (Fig. 6g,h), as for the other mutant backgrounds. Taken together, the data demonstrate that auxin mutants with ectopic PIN1 localization at the

7 1136 Research New Phytologist (a) (b) (c) (d) Fig. 5 Subcellular localization of TRH1 in Arabidopsis thaliana root apex. (a) Live cell imaging of TRH1-YFP transcriptional fusion in Arabidopsis root tip driven by the TRH1 promoter. (b) Double immunofluorescence labelling shows that TRH1 (green fluorescence) is co-localized with PIN1 (red fluorescence) to the basal side of stellar root cells in the elongation zone. Right panel corresponds to an overlay of the left and middle panel images, revealing that green fluorescence coincides with red. White arrowheads in (a, b) depict TRH1 asymmetric localization. (c) On root epidermis, TRH1 is mainly localized to the intracellular membrane structures and is partially co-localized with FM4-64 at the plasma membrane. (d) Application of 25 lm brefeldin A (BFA) for 80 min internalized FM4 64 without affecting TRH1 topology. (c, d) Green fluorescence corresponds to the signal from the green channel for yellow fluorescent protein (YFP), red fluorescence to the FM4 64 stain and yellow fluorescence shows a merge of the green and red channels. 4,6-Diamidino-2-phenylindole (DAPI) staining was used to visualize the cell nucleus by blue fluorescence. Bars: (a) 20 lm; (b d) 5 lm. meristematic zone have developmental defects including root gravitropism and root hair development. Overexpression of Arabidopsis PIN1 causes root agravitropism and defects in root hair growth To assess whether PIN1 ectopic mislocalization is associated directly with the root developmental defects in Arabidopsis, the PIN1 gene was introduced in wild-type plants expressed under the control of the CaMV35S constitutive promoter. Immunolocalization of PIN1 in the transgenic overexpressor lines revealed that the auxin efflux carrier was ectopically localized at the apical (shootward) side of epidermal cells at the meristematic region (Fig. 6i,j). Furthermore, PIN1 overexpressors showed pronounced defects in root gravitropism (Fig. 2c), as reported previously by Petrasek et al. (2006), but also impaired root hair development (Figs 1f, 2a,b). These results are consistent with the data of auxin-deficient mutants, indicating that, in PIN1 overexpressor lines, the topology of the PIN1 protein at the apical side of epidermal cells at the meristematic region is associated with root agravitropism and the root hair phenotype. Hypaphorine treatment results in PIN1 ectopic mislocalization The fungal hypaphorine, the betaine of tryptophan, is an indole alkaloid that belongs in a class of IAA antagonists. Hypaphorine is a signalling molecule released by the fungus during Pisolithus tinctorius/eucalyptus globulus symbiotic association (Ditengou & Lapeyrie, 2000; Ditengou et al., 2000). Hypaphorine shares structural similarities with IAA and the auxin precursor tryptophan (Fig. 7). Although auxin results in the inhibition of primary root elongation, the application of hypaphorine counteracts the inhibitory effect of auxin restoring the primary root growth and hence acting as a natural auxin inhibitor (Fig. S2). The application of 100 lm hypaphorine on wild-type seedlings led to apical (shootward) polar localization of PIN1 on root epidermal cells at the meristematic zone (Fig. 6k,l). Likewise, hypaphorine had a dramatic effect on root hair morphogenesis (Figs 1g, 2a,b), whereas no effect was recorded in terms of agravitropic root growth (Fig. 2c). Interestingly, the root hair length and density were strongly inhibited as the concentration shifted from 100 to 500 lm. The increased concentration of 500 lm hypaphorine also had a slight effect on agravitropic root growth relative to a concentration of 100 lm. This agravitropic root growth phenotype induced by hypaphorine when applied at relatively high concentration could be attributed to the inability of the compound to be transported within the root tissues. Therefore, the effect is strictly restricted during the uptake on the epidermal and cortical peripheral root cell layers, inhibiting basipetal auxin transport. Thus, perturbation of auxin homeostastis results in ectopic PIN1 localization on the apical side of the epidermal cells at the meristematic zone, causing the defect of root hair development distal to the root apex.

8 New Phytologist Research 1137 (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k) (l) (m) Fig. 6 PIN1 protein immunolocalization in 5-d-old Arabidopsis thaliana roots. (a) Columbia (Col-0); boxed area in (a) is magnified in (b). PIN1 is exclusively localized in the initials of epidermal and lateral root cap cells. (c) trh1; boxed area in (c) is magnified in (d). Arrowheads depict the apical polar PIN1 mislocalization on root epidermal cells at the meristematic zone. (e) pin2, (f) axr2-1, (g) aux1-7; boxed area in (g) is magnified in (h). (i, j) 35S::PIN1. (k) Effect of 100 lμ hypaphorine on Arabidopsis wild-type roots; boxed area in (k) is magnified in (l) showing the lateral root cap and epidermal cells at the meristematic zone. (m) rhd6. Immunolocalization signals are green for PIN1, red for PIN2 and yellow on PIN1 and PIN2 co-localization. Fig. 7 Comparative chemical structures of the natural auxin antagonist/ inhibitor hypaphorine (betaine of tryptophan) and the natural occurring auxin indole-3-acetic acid (IAA), derived from tryptophan, showing their structural similarities. RHD6 is autonomous to the environmental/hormonal signalling pathway Unlike the other Arabidopsis mutants, rhd6 has the most severe root hair phenotype (Fig. 1h). RHD6 is required for the selection of the bulging site to initiate root hair elongation on the trichoblast (H-position) (Masucci & Schiefelbein, 1994) and, therefore, rhd6 is almost hairless (Fig. 2a,b). RHD6 is an important downstream regulator of the TTG/ GL2 pathway, and auxin or ethylene can restore the rhd6 root hair phenotype (Masucci & Schiefelbein, 1994, 1996). Hence, hormones positively regulate root hair development independently of RHD6 (Cho & Cosgrove, 2002; Jang et al., 2011). Interestingly, immunolocalization experiments revealed that PIN1 is normally localized on the cells of the rhd6 root tips (Fig. 6m). Furthermore, the gravity response of rhd6 roots is not disturbed, being similar to that of wild-type roots (Fig. 2c). These results are in agreement with previous genetic and physiological studies reported by Cho & Cosgrove (2002), indicating that there are two independent pathways for root hair initiation (Fig. 8). The first pathway is dependent on the external environmental stimuli that result in alterations in hormonal homeostasis in the Arabidopsis root apex. The second pathway includes RHD6 as a downstream component of the intrinsic developmental TTG/GL2 pathway that is involved in the initiation of root hair development. Consequently, RHD6 belongs to the developmental pathway that is independent of the environmental/hormonal pathway. The results indicating that PIN1 is not ectopically mislocalized in rhd6 root tips and rhd6 roots show no agravitropic growth support this model, suggesting that the environmental/hormonal signalling pathway converges with the developmental intrinsic pathway downstream of RHD6. The position

9 1138 Research New Phytologist Fig. 8 Model showing the distal effect of auxin imbalance in the Arabidopsis root apex. Root hair morphogenesis is a procession of developmental events modulated by the interplay of intrinsic and environmental cues. The intrinsic pathway determines root epidermal cell fate through the TTG/GL2 pathway. The environmental pathway modulates hormonal transport or signalling. The environmental/hormonal signalling pathway converges with the intrinsic developmental pathway downstream of RHD6. of RHD6 in this developmental cascade explains why only the root hair morphogenesis is perturbed in rhd6 roots, but not agravitropic root growth. Discussion In the root, auxin is delivered acropetally to the root apex, where the direction of auxin flux is reversed to basipetal, diverted via cortical and epidermal cells, and, subsequently, an auxin reflux loop is created redirecting auxin to the root tip. This auxin circulation stabilizes the auxin maximum, and hence cell patterning and polarity in the root meristem (Sabatini et al., 1999; Blilou et al., 2005). Auxin has been implicated in root hair development acting downstream of the TTG/GL2 pathway that determines root epidermal cell fate (Masucci & Schiefelbein, 1996; Pitts et al., 1998). Several A. thaliana mutants encoding for genes involved in either the auxin response or transport have been characterized as root hair defective. The TRH1 gene encodes a potassium transporter that is required for auxin transport in Arabidopsis roots (Rigas et al., 2001; Vicente-Agullo et al., 2004). In addition to the root hair phenotype, trh1 seedlings show agravitropic root growth. These phenotypic features raise the issue of whether root gravitropism and root hair morphogenesis constitute coupled auxin-mediated developmental processes relying on auxin homeostasis in the Arabidopsis root apex. In trh1, the auxin-sensitive reporter DR5::GUS revealed an abnormal pattern of auxin distribution on exogenous application of NAA. Wild-type roots showed enhanced auxin accumulation in specific stele cells at the central cylinder and, subsequently, in the epidermis and lateral root cap, indicative of the basipetal auxin translocation route. By contrast, in trh1 roots, no response to auxin application was apparent at epidermal and lateral root cap cells. Furthermore, the pattern of auxin accumulation in the distal elongation zone was radically expanded from the central cylinder to the epidermal tissues. These results strongly suggest defects in both basipetal and acropetal auxin transport. Quantitative analysis of free IAA levels in successive sections of trh1 roots indeed confirmed the auxin imbalance. As the deviation of the auxin optimum in trh1 roots results in the agravitropic response and root hair phenotype, the localization of PIN auxin efflux facilitators was investigated. Unlike the wild-type roots, ectopic PIN1 localization was observed on the apical (shootward) side of the epidermal cells lying in the meristematic zone of trh1 roots. Interestingly, in wild-type plants, ectopic PIN1 localization in root epidermal cells has been reported as a result of the inhibition of auxin polar transport or exogenous auxin application (Vieten et al., 2005). Such experimental approaches, even though they may seem contradictory, support the hypothesis, based on the data obtained from trh1 analysis and exogenous hormone application, that auxin imbalance in the Arabidopsis root apex results in ectopic PIN1 mislocalization. Given the fact that trh1 facilitates auxin transport (Vicente- Agullo et al., 2004), the distribution of TRH1 in the root apex implies that it may participate in both the acropetal and basipetal route. At the meristematic zone, TRH1 is co-localized with PIN1 in the central cylinder, facilitating the acropetal polar transport of auxin. In the central cylinder of hormone-nontreated roots, TRH1 redirects auxin efflux, as evidenced by DR5 expression, for the establishment of auxin maxima in the root tip. In epidermal and cortical cells, TRH1 facilitates the basipetal auxin route, as indicated by polar TRH1 localization, together with the DR5 results in auxin-treated roots. The outcome of auxin distribution and transport in trh1 roots is that auxin imbalance leads to developmental defects proximal and distal to the root apex. The former corresponds to the root agravitropic behaviour, whereas the latter refers to the root hair phenotype. TRH1 at the cellular level is localized in both the plasma membrane and internal compartments. This is in agreement with genetic studies confirming that, in addition to the plasma membrane, TRH1 could be localized in intracellular membrane structures (Desbrosses et al., 2003). The dual localization of TRH1 in both the plasma membrane and the endomembrane system opens up the possibility of dual TRH1-mediated auxin transport processes within the cell. TRH1 could limit the availability of cytosolic auxin and enhance the availability of intracellular auxin for signalling or metabolism. Ectopic PIN1 localization in the root apex caused by auxin imbalance, which, in turn, is associated with proximal and distal developmental defects, is not only restricted to trh1 roots. PIN1 was mislocalized in pin2, axr2 and aux1 auxin-deficient mutants and in transgenic plants overexpressing the PIN1 gene, all characterized by an impaired gravity response and root hair development. Similar observations of PIN1 polar localization on the apical side of epidermal cells have been reported previously for pin2 roots, suggesting functional synergistic interactions within the PIN members for compensation of the redundant auxin distribution network and auxin imbalance (Vieten et al., 2005; Wisniewska et al., 2006). Interestingly, the agravitropic root growth of pin2 roots was restored when PIN1 gene expression was driven by the PIN2 promoter, resulting in localization of the PIN1 transgene on the apical side of root epidermal cells, substituting for PIN2 (Wisniewska et al., 2006). This shows that

10 New Phytologist Research 1139 PIN1, when placed under the PIN2 promoter, could partially compensate for PIN2 loss of function in pin2 mutants. In accordance with this conclusion, pin2 mutants show substantially elevated auxin levels in the root tip and accumulate DR5 activity ectopically in the lateral root cap, showing that the basipetal auxin transport mechanism is defective (Sabatini et al., 1999; Rashotte et al., 2000). In the aux1 mutant, PIN1 ectopic localization could be attributed to the reduced root basipetal auxin transport (Swarup et al., 2005), whereas substantial experimental data on the axr2 mutant indicate that components of the Aux/IAA signal transduction pathway are involved in the regulation of the PIN gene family (Vieten et al., 2005). In line with PIN1 expression under the control of an inducible promoter system (Petrasek et al., 2006), PIN1 overexpression driven by the CaMV35S promoter led to pronounced defects in root gravitropism and to impaired root hair development. The application of hypaphorine reveals that chemical-mediated auxin imbalance, as evidenced by root agravitropism and root hair defects, also leads to ectopic PIN1 localization in the Arabidopsis root apex. Hypaphorine is the major indolic compound isolated from the fungus Pisolithus tinctorius, which establishes ectomycorrhizas with Eucalyptus globulus and inhibits root hair elongation (Ditengou & Lapeyrie, 2000; Ditengou et al., 2000). Hypaphorine had a significant negative effect on root hair morphogenesis, but marginally affected gravitropic root behaviour. The former effect is caused by impaired basipetal auxin transport, whereas the latter most likely results from suboptimal auxin translocation within the root tissues. In line with these results, the defects in root hair development and the gravitropic response of aem1 roots in rice are correlated with auxin imbalance (Debi et al., 2005). On the basis of the experimental data, we designed a model scheme, illustrated in Fig. 9, postulating that auxin homeostasis in root epidermis is essential for root hair development and the root gravitropic response. In the case of impaired auxin response or transport, PIN1 is mislocalized at the apical side of root epidermal cells, probably in an attempt to attenuate auxin imbalance, leading to aberrant root hair growth and root agravitropic response. These findings are in agreement with previously reported results indicating that PIN-dependent auxin (a) translocation is modulated by an elegant autoregulatory molecular mechanism. Auxin itself orchestrates both the transcription of PIN genes and the post-transcriptional protein stability in response to environmental or developmental signals (Vieten et al., 2005; Abas et al., 2006; Wisniewska et al., 2006). The environmental cues may include nutrient deficiency, salinity, water scarcity or mechanical impedance, conveying the abiotic stress signal to the root. The external/environmental pathway, in turn, modulates auxin and ethylene transport or signalling. In several aspects of plant development, such as root hair elongation, auxin signalling acts downstream of ethylene signalling (Strader et al., 2010). Ethylene stimulates auxin biosynthesis (Ruzicka et al., 2007; Swarup et al., 2007) and auxin transport (Ruzicka et al., 2007), increasing the levels of auxin in epidermal cells. In trichoblasts, auxin accumulation is dependent on atrichoblast-localized AUX1, promoting root hair elongation (Pitts et al., 1998; Jones et al., 2009). As auxin or ethylene restores the rhd6 root hair phenotype (Masucci & Schiefelbein, 1994, 1996), and the hormonalregulated development of root hairs is independent of RHD6 (Cho & Cosgrove, 2002; Jang et al., 2011), the intrinsic developmental pathway converges with the environmental/hormonal signalling pathway downstream of RHD6 (Fig. 8). Our results confirm the concept that root gravitropism and root hair morphogenesis are coupled developmental processes synchronized by auxin homeostasis. This, in turn, is modulated by environmental stress conditions to ensure plant adaptation and survival in changing environments. This concept is in agreement with the role of the TRH1 potassium transporter in the sensing of external K + and the regulation of potassium-dependent root development. Under low potassium availability, the root agravitropic behaviour is triggered in wild-type plants and the pattern of auxin distribution becomes similar to that of trh1 roots (Vicente-Agullo et al., 2004). This response to K + deprivation is important to enable the root to avert soil patches with low potassium and to explore soil layers with higher nutrient content. Likewise, TRH1 has been reported to be involved in the ammonium-induced loss of root gravitropism in the Arabidopsis root apex (Zou et al., 2012). Excessive ammonium inhibited Arabidopsis root gravitropism, increasing the angle of the root tip (b) Fig. 9 Schematic representation of the proximal and distal effects of auxin in the root apex. (a) Root gravitropism is the developmental process relying on the proximal effect of auxin homeostasis in the root apex, whereas root hair morphogenesis, which depends on the basipetal route of auxin transport, is the process relying on the distal effect. (b) Defects in auxin transport or signalling result in PIN1 ectopic localization on the apical side of epidermal cells at the meristematic zone. Auxin imbalance affects the two developmental processes, resulting in enhanced root bending and arrest of root hair elongation.

11 1140 Research New Phytologist from the gravity vector. This could be a mechanism of risk aversion by the root system via TRH1-mediated auxin redistribution in the root cap. Ectopic PIN1 mislocalization at the meristematic zone in the Arabidopsis root apex results from auxin imbalance and is associated with certain developmental responses. To what extent environmental cues and hormone crosstalk may induce endogenous auxin redistribution through PIN1-dependent mislocalization to provide the root system with flexible growth, ensuring adaptability and survival, remains an exciting question for future studies. Acknowledgements We gratefully acknowledge support from the EU Research Training Network TIPNET (project no. HPRN-CT ), DFG-SFB 592, the Excellence Initiative of the German Federal and State Governments (EXC 294), Bundesministerium f ur Forschung und Technik (BMBF), Deutsches Zentrum f ur Luft und Raumfahrt (DLR 50WB1022), the Freiburg Initiative for Systems Biology (FRISYS), the European Union Framework 6 Program (AUTOSCREEN, LSHG-CT ), European Molecular Biology Organization and Greek State Scholarships Foundation. We also gratefully acknowledge support by the Freiburg Life Imaging Center and Roland Nitschke for advice. References Abas L, Benjamins R, Malenica N, Paciorek T, Wisniewska J, Moulinier-Anzola JC, Sieberer T, Friml J, Luschnig C Intracellular trafficking and proteolysis of the Arabidopsis auxin-efflux facilitator PIN2 are involved in root gravitropism. Nature Cell Biology 8: Benkova E, Michniewicz M, Sauer M, Teichmann T, Seifertova D, J urgens G, Friml J Local, efflux-dependent auxin gradients as a common module for plant organ formation. Cell 115: Bennett MJ, Marchant A, Green HG, May ST, Ward SP, Millner PA, Walker AR, Schulz B, Feldmann KA Arabidopsis AUX1 gene: a permease-like regulator of root gravitropism. Science 273: Bhalerao RP, Ekl of J, Ljung K, Marchant A, Bennett M, Sandberg G Shoot-derived auxin is essential for early lateral root emergence in Arabidopsis seedlings. Plant Journal 29: Blancaflor EB, Fasano JM, Gilroy S Mapping the functional roles of cap cells in the response of Arabidopsis primary roots to gravity. Plant Physiology 116: Blilou I, Xu J, Wildwater M, Willemsen V, Paponov I, Friml J, Heidstra R, Aida M, Palme K, Scheres B The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots. Nature 433: Cernac A, Lincoln C, Lammer D, Estelle M The SAR1 gene of Arabidopsis acts downstream of the AXR1 gene in auxin response. Development 124: Chen R, Hilson P, Sedbrook J, Rosen E, Caspar T, Masson PH The Arabidopsis thaliana AGRAVITROPIC 1 gene encodes a component of the polar-auxin-transport efflux carrier. Proceedings of the National Academy of Sciences, USA 95: Cho HT, Cosgrove DJ Regulation of root hair initiation and expansin gene expression in Arabidopsis. Plant Cell 14: Debi BR, Chhun T, Taketa S, Tsurumi S, Xia K, Miyao A, Hirochika H, Ichii M Defects in root development and gravity response in the aem1 mutant of rice are associated with reduced auxin efflux. Journal of Plant Physiology 162: Desbrosses G, Josefsson C, Rigas S, Hatzopoulos P, Dolan L AKT1 and TRH1 are required during root hair elongation in Arabidopsis. Journal of Experimental Botany 54: Ditengou FA, Beguiristain T, Lapeyrie F Root hair elongation is inhibited by hypaphorine, the indole alkaloid from the ectomycorrhizal fungus Pisolithus tinctorius, and restored by indole-3-acetic acid. Planta 211: Ditengou FA, Lapeyrie F Hypaphorine from the ectomycorrhizal fungus Pisolithus tinctorius counteracts activities of indole-3-acetic acid and ethylene but not synthetic auxins in eucalypt seedlings. Molecular Plant Microbe Interactions 13: Dolan L, Duckett CM, Grierson C, Linstead P, Schneider K, Lawson E, Dean C, Poethig RS, Roberts K Clonal relationships and cell patterning in the root epidermis of Arabidopsis. Development 120: Friml J, Benkova E, Blilou I, Wisniewska J, Hamann T, Ljung K, Woody S, Sandberg G, Scheres B, J urgens G et al. 2002a. AtPIN4 mediates sink-driven auxin gradients and root patterning in Arabidopsis. Cell 108: Friml J, Benkova E, Mayer U, Palme K, Muster G Automated whole mount localisation techniques for plant seedlings. Plant Journal 34: Friml J, Wisniewska J, Benkova E, Mendgen K, Palme K. 2002b. Lateral relocation of the auxin efflux regulator AtPIN3 mediates tropism in Arabidopsis. Nature 415: Grabov A, Ashley MK, Rigas S, Hatzopoulos P, Dolan L, Vicente-Agullo F Morphometric analysis of root shape. New Phytologist 165: Haralampidis K, Milioni D, Rigas S, Hatzopoulos P Combinatorial interaction of cis elements specifies the expression of the Arabidopsis AtHsp90-1 gene. Plant Physiology 129: Jang G, Yi K, Pires ND, Menand B, Dolan L RSL genes are sufficient for rhizoid system development in early diverging land plants. Development 138: Jones AR, Kramer EM, Knox K, Swarup R, Bennett MJ, Lazarus CM, Leyser HM, Grierson CS Auxin transport through non-hair cells sustains roothair development. Nature Cell Biology 11: Kleine-Vehn J, Ding Z, Jones AR, Tasaka M, Morita MT, Friml J Gravityinduced PIN transcytosis for polarization of auxin fluxes in gravity-sensing root cells. Proceedings of the National Academy of Sciences, USA 107: Knox K, Grierson CS, Leyser O AXR3 and SHY2 interact to regulate root hair development. Development 130: Ljung K, Hull AK, Celenza J, Yamada M, Estelle M, Normanly J, Sandberg G Sites and regulation of auxin biosynthesis in Arabidopsis roots. Plant Cell 17: Luschnig C, Gaxiola R, Grisafi P, Fink G EIR1, a root specific protein involved in auxin transport, is required for gravitropism in Arabidopsis thaliana. Genes & Development 12: Masucci JD, Schiefelbein JW The rhd6 mutation of Arabidopsis thaliana alters root-hair initiation through an auxin- and ethylene-associated process. Plant Physiology 106: Masucci JD, Schiefelbein JW Hormones act downstream of TTG and GL2 to promote root hair outgrowth during epidermis development in the Arabidopsis root. Plant Cell 8: Mravec J, Kubes M, Bielach A, Gaykova V, Petrasek J, Skupa P, Chand S, Benkova E, Zazımalova E, Friml J Interaction of PIN and PGP transport mechanisms in auxin distribution-dependent development. Development 135: Mravec J, Skupa P, Bailly A, Hoyerova K, Krecek P, Bielach A, Petrasek J, Zhang J, Gaykova V, Stierhof YD et al Subcellular homeostasis of phytohormone auxin is mediated by the ER-localized PIN5 transporter. Nature 459: M uller A, Guan C, G alweiler L, T anzler P, Huijser P, Marchant A, Parry G, Bennett M, Wisman E, Palme K AtPIN2 defines a locus of Arabidopsis for root gravitropism control. EMBO Journal 17: Nagpal P, Walker LM, Young JC, Sonawala A, Timpte C, Estelle M, Reed JW AXR2 encodes a member of the Aux/IAA protein family. Plant Physiology 123: Paponov IA, Teale WD, Trebar M, Blilou I, Palme K The PIN auxin efflux facilitators: evolutionary and functional perspectives. Trends in Plant Science 10: Petrasek J, Mravec J, Bouchard R, Blakeslee JJ, Abas M, Seifertova D, Wisniewska J, Tadele Z, Kubes M, Covanova M et al PIN proteins perform a rate-limiting function in cellular auxin efflux. Science 312: Pitts RJ, Cernac A, Estelle M Auxin and ethylene promote root hair elongation in Arabidopsis. Plant Journal 16: