Title: SCFTIR1/AFB-auxin signalling regulates PIN vacuolar trafficking and auxin fluxes during root gravitropism
Abstract: Article4 December 2012free access SCFTIR1/AFB-auxin signalling regulates PIN vacuolar trafficking and auxin fluxes during root gravitropism Paweł Baster Paweł Baster Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Genetics, Ghent University, Gent, Belgium Search for more papers by this author Stéphanie Robert Stéphanie Robert Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Genetics, Ghent University, Gent, Belgium Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences/Umeå Plant Science Center, Umeå, Sweden Search for more papers by this author Jürgen Kleine-Vehn Jürgen Kleine-Vehn Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Genetics, Ghent University, Gent, Belgium Department of Applied Genetics and Cell Biology, University of Applied Life Sciences and Natural Resources (BOKU), Vienna, Austria Search for more papers by this author Steffen Vanneste Steffen Vanneste Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Genetics, Ghent University, Gent, Belgium Search for more papers by this author Urszula Kania Urszula Kania Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Genetics, Ghent University, Gent, Belgium Search for more papers by this author Wim Grunewald Wim Grunewald Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Genetics, Ghent University, Gent, Belgium Search for more papers by this author Bert De Rybel Bert De Rybel Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Genetics, Ghent University, Gent, Belgium Laboratory of Biochemistry, University of Wageningen, Wageningen, The Netherlands Search for more papers by this author Tom Beeckman Tom Beeckman Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Genetics, Ghent University, Gent, Belgium Search for more papers by this author Jiří Friml Corresponding Author Jiří Friml Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Genetics, Ghent University, Gent, Belgium Institute of Science and Technology Austria (IST Austria), Klosterneuburg, Austria Search for more papers by this author Paweł Baster Paweł Baster Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Genetics, Ghent University, Gent, Belgium Search for more papers by this author Stéphanie Robert Stéphanie Robert Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Genetics, Ghent University, Gent, Belgium Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences/Umeå Plant Science Center, Umeå, Sweden Search for more papers by this author Jürgen Kleine-Vehn Jürgen Kleine-Vehn Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Genetics, Ghent University, Gent, Belgium Department of Applied Genetics and Cell Biology, University of Applied Life Sciences and Natural Resources (BOKU), Vienna, Austria Search for more papers by this author Steffen Vanneste Steffen Vanneste Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Genetics, Ghent University, Gent, Belgium Search for more papers by this author Urszula Kania Urszula Kania Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Genetics, Ghent University, Gent, Belgium Search for more papers by this author Wim Grunewald Wim Grunewald Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Genetics, Ghent University, Gent, Belgium Search for more papers by this author Bert De Rybel Bert De Rybel Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Genetics, Ghent University, Gent, Belgium Laboratory of Biochemistry, University of Wageningen, Wageningen, The Netherlands Search for more papers by this author Tom Beeckman Tom Beeckman Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Genetics, Ghent University, Gent, Belgium Search for more papers by this author Jiří Friml Corresponding Author Jiří Friml Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Genetics, Ghent University, Gent, Belgium Institute of Science and Technology Austria (IST Austria), Klosterneuburg, Austria Search for more papers by this author Author Information Paweł Baster1, Stéphanie Robert1,2, Jürgen Kleine-Vehn1,3, Steffen Vanneste1, Urszula Kania1, Wim Grunewald1, Bert De Rybel1,4, Tom Beeckman1 and Jiří Friml 1,5 1Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Genetics, Ghent University, Gent, Belgium 2Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences/Umeå Plant Science Center, Umeå, Sweden 3Department of Applied Genetics and Cell Biology, University of Applied Life Sciences and Natural Resources (BOKU), Vienna, Austria 4Laboratory of Biochemistry, University of Wageningen, Wageningen, The Netherlands 5Institute of Science and Technology Austria (IST Austria), Klosterneuburg, Austria *Corresponding author. Department of Plant Systems Biology, VIB-Ghent University, Technologiepark 927, 9052 Gent, Belgium. Tel.:+32 9 3313913; Fax:+32 9 3313809; E-mail: [email protected] author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (http://www.embojournal.org) is Jiří Friml (jiri.friml-psb.vib-ugent.be) The EMBO Journal (2013)32:260-274https://doi.org/10.1038/emboj.2012.310 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The distribution of the phytohormone auxin regulates many aspects of plant development including growth response to gravity. Gravitropic root curvature involves coordinated and asymmetric cell elongation between the lower and upper side of the root, mediated by differential cellular auxin levels. The asymmetry in the auxin distribution is established and maintained by a spatio-temporal regulation of the PIN-FORMED (PIN) auxin transporter activity. We provide novel insights into the complex regulation of PIN abundance and activity during root gravitropism. We show that PIN2 turnover is differentially regulated on the upper and lower side of gravistimulated roots by distinct but partially overlapping auxin feedback mechanisms. In addition to regulating transcription and clathrin-mediated internalization, auxin also controls PIN abundance at the plasma membrane by promoting their vacuolar targeting and degradation. This effect of elevated auxin levels requires the activity of SKP-Cullin-F-boxTIR1/AFB (SCFTIR1/AFB)-dependent pathway. Importantly, also suboptimal auxin levels mediate PIN degradation utilizing the same signalling pathway. These feedback mechanisms are functionally important during gravitropic response and ensure fine-tuning of auxin fluxes for maintaining as well as terminating asymmetric growth. Introduction The phytohormone auxin is an important regulator of cell morphogenesis shaping and directing growth of organs within different developmental contexts and in response to environmental signals (Vanneste and Friml, 2009). To ensure optimal growth and development, plants have acquired elaborate mechanisms to control the local auxin homeostasis, including control of auxin metabolism (Cheng et al, 2006, 2007; Stepanova et al, 2008; Tao et al, 2008), subcellular compartmentalization (Mravec et al, 2009; Barbez et al, 2012; Ding et al, 2012) and directional auxin transport mediated by plasma membrane-resident transporters, such as ABCB, PIN-FORMED (PIN) and AUXIN-RESISTANT 1 (AUX1) (Bennett et al, 1996; Geisler et al, 2005; Petrášek et al, 2006; Cho et al, 2007; Swarup et al, 2008; Jones et al, 2009). One of the prominent growth responses mediated by auxin transport is root gravitropism. Changes of the orientation relative to the gravity vector are perceived in the root tip, by the sedimentation of statoliths, defined as gravity-sensing organelles (Harrison and Masson, 2008; Leitz et al, 2009; Morita, 2010). This process appears to induce the relocation of the auxin efflux carriers (Petrášek et al, 2006) PIN3 and PIN7 to the lower side of the gravity-sensing cells, which presumably aligns auxin flux with gravity vector towards the lower side of the root tip (Friml et al, 2002; Harrison and Masson, 2008; Kleine-Vehn et al, 2010). From there, another auxin efflux carrier, PIN2, which is apically (shootward, upper cell side) localized in the lateral root cap and epidermal cells, mediates the directional auxin flow from the root tip to the elongation zone where control of elongation occurs (Luschnig et al, 1998; Müller et al, 1998; Abas et al, 2006; Wiśniewska et al, 2006). Hence, the PIN-mediated establishment of the asymmetric auxin distribution leads to a differential growth between the lower and the upper side of the root. As a consequence, root bends and re-orients in respect to the gravity vector, allowing the efficient exploration of the soil (Firn et al, 2000; Swarup et al, 2005). The mechanisms underlying the PIN3 and PIN7 polarization in gravity-sensing columella cells and control of the PIN2 abundance at the plasma membrane for defined gravitropic response remain largely elusive. Nevertheless, some of the molecular processes controlling the subcellular localization of PIN proteins have been characterized (Grunewald and Friml, 2010). PIN proteins internalize continuously via a clathrin-mediated endocytotic pathway (Dhonukshe et al, 2007; Kitakura et al, 2011) and cycle back to the plasma membrane as shown by pharmacological approaches with a vesicle-budding inhibitor, Brefeldin A (BFA) (Geldner et al, 2001). This permanent cycling leads to a dynamic control of their polar localization and abundance at the plasma membrane (Kleine-Vehn et al, 2008a), which in turn, determines the rate and direction of the auxin flow (Paciorek et al, 2005; Wiśniewska et al, 2006). The constitutive endocytic recycling enables also rapid switches in PIN polarity and, consequently, directionality of auxin fluxes in response to environmental signals, including light and gravity (Friml et al, 2002; Kleine-Vehn et al, 2010; Ding et al, 2011; Rakusová et al, 2011). Besides the control of the polar localization, PIN protein activity can be also regulated by degradation. Numerous studies reported the occurrence of PIN degradation in the vacuoles (Abas et al, 2006; Laxmi et al, 2008; Kleine-Vehn et al, 2008b; Shirakawa et al, 2009; Leitner et al, 2012; Marhavý et al, 2011), to which they are targeted via a BFA-sensitive canonical retrograde trafficking pathway, involving the retromer complex (Kleine-Vehn et al, 2008b). Moreover, PIN2 turnover depends on the proteasomal activity (Sieberer et al, 2000; Abas et al, 2006) and sorting for vacuolar delivery was recently associated with the formation of the polyubiquitin chains linked to the specific lysine residues at the PIN2 hydrophilic loop (Leitner et al, 2012). Together, this data highlights the importance of post-transcriptional regulations in auxin flux determination. Notably, auxin itself modulates its own distribution by providing feedback on PIN biosynthesis and trafficking (Benjamins and Scheres, 2008). Short auxin treatments (⩽2 h) activate the transcription of different PIN genes (Peer et al, 2004; Heisler et al, 2005; Vieten et al, 2005; Scarpella et al, 2006) and can stabilize PIN at the plasma membrane by inhibiting clathrin-mediated internalization (Paciorek et al, 2005; Robert et al, 2010). Recently, it was found that AUXIN-BINDING PROTEIN 1 (ABP1) is a positive regulator of clathrin-mediated endocytosis, which is inhibited upon auxin binding (Robert et al, 2010; Chen et al, 2012). In contrast, prolonged application of auxin also promotes the turnover of PIN proteins via an unknown mechanism (Sieberer et al, 2000; Vieten et al, 2005; Abas et al, 2006). How this duality of auxin action on endocytosis versus degradation is regulated is unknown. The BFA fungal toxin is known to inhibit the activity of specific ADP-ribosylation factor GTP-exchange factors (ARF-GEFs) (Peyroche et al, 1999; Sata et al, 1999; Geldner et al, 2003). In plants, the secretory pathway is readily inhibited by BFA, resulting in the intracellular accumulation of endocytosed plasma membrane proteins such as PIN proteins (Geldner et al, 2001). Upon inhibition of endocytosis (at ∼25 μM of BFA), PIN proteins no longer end up in such a BFA compartments (Paciorek et al, 2005; Men et al, 2008; Kitakura et al, 2011). Interestingly, it has recently been discovered that at higher concentrations (∼50 μM), BFA also inhibits vacuolar targeting and degradation of PIN proteins (Kleine-Vehn et al, 2008b; Kleine-Vehn and Friml, 2008; Robert et al, 2010). Thus, different concentrations of BFA allow discriminating between effects on endocytosis for recycling and targeting for degradation (Robert et al, 2010). Notably, the aforementioned BFA concentration cutoff should not be taken precisely as most likely specificity of the BFA towards specific ARF-GEF's changes gradually. Here, we show that PIN2 protein abundance is dynamically and differentially controlled at the upper and lower sides of a gravistimulated root. Both increased and decreased auxin levels change PIN2 stability by a post-transcriptional regulation of its vacuolar targeting. Moreover, we provide additional data to clarify the involvement of SCFTIR1/AFB-based signalling in auxin-mediated PIN turnover. These findings link auxin-mediated regulation of vesicle transport and asymmetric growth control during gravitropic response. Results Dynamic changes of auxin response and PIN2 abundance in gravistimulated roots To better understand the regulation of auxin transport activity in response to gravity, we have investigated the dynamics of root bending, auxin redistribution and abundance of PIN2 in gravistimulated roots of Arabidopsis thaliana. We have indirectly visualized the auxin redistribution by monitoring the activity of the synthetic auxin-responsive promoter DR5rev (Ulmasov et al, 1997) driving expression of a nuclearly localized VENUS protein (DR5rev::3xVENUS-N7; Heisler et al, 2005). Consistent with previous observations, after 2 h of gravistimulation, Arabidopsis root bent visibly (Figure 1A–F) and an asymmetric increase in DR5, expression was observed at the less elongated (lower) root side (Ottenschläger et al, 2003; Paciorek et al, 2005), whereas at the upper side of the bending root, the DR5 response was reduced (Figure 1G–M). This asymmetry in auxin response was maintained throughout gravity-induced root bending (Figure 1A–M). In time, the growth angle of the root became progressively parallel to the gravity vector (Figure 1F) and with a delay, a balanced DR5 expression was re-establishing (Figure 1L and M). We have confirmed the formation of auxin lateral gradient in roots responding to the gravity with use of highly dynamic DII-VENUS reporter system (see Supplementary Figure 1; Brunoud et al, 2012). This system was previously used to precisely place the timing of auxin accumulation during root gravitropic response (Band et al, 2012). It is important to note that the timing of onset and disappearance of the DR5rev::3XVENUS-N7 signal lags behind the real kinetics of the auxin distribution due to the time needed for VENUS maturation and turnover. Nonetheless, in spite of the inherent shortcomings of this reporter, we were able to demonstrate a clear spatio-temporal regulation of the auxin distribution during gravitropic bending. Figure 1.Localization of PIN2-GFP protein and auxin maxima during root gravitropic response. (A–E) Kinetics of the root bending in seedlings at 0 h (A), 2 h (B), 4 h (C), 8 h (D) and 12 h (E) after gravistimulation. (F) Angle of the root curvature in relation to horizon after gravistimulation. n=3 independent experiments with at least six roots analysed for each assay. (G–L) Activity of DR5rev::3XVENUS-N7 promoter in seedlings at 0 h (G), 2 h (H), 4 h (I), 8 h (J), 12 h (K) and 24 h (L) after gravistimulation. Pictures represent maximum intensity projection of median root sections (10 Z-sections spaced ∼4.5 μm). (M) Quantification of the DR5rev::3XVENUS-N7 expressing nuclei in the epidermal cells of the gravistimulated root. n=3 independent experiments with at least six roots analysed for each assay. Note a minimum of DR5rev::3XVENUS-N7 expression on the upper side as well as maximum on the lower side of the root 8 h after gravistimulation marked on (J) and graph (M) by red and green discontinuous lines, respectively. (N–S) PIN2-GFP protein localization in epidermal and cortical cells at 0 h (N), 2 h (O), 4 h (P), 8 h (Q) and 12 h (R) after gravistimulation. Pictures represent maximum intensity projection of median root sections (10 Z-sections spaced ∼1 μm apart). (S) PIN2-GFP signal intensity in gravistimulated roots. n=3 independent experiments with at least six roots analysed for each assay. Note a decrease in the GFP signal intensity at the upper side of the root between 0 and 4 h after gravistimulation (discontinuous red line on N, P and graph S) as well as, at the lower side of the root, between 2 and 8 h after gravistimulation (discontinuous green line on O, Q and graph S). Error bars represent standard error of the mean (s.e.m.), P-value calculated according to Student's t-test. Signal intensities are coded white to black and blue to yellow corresponding to increasing intensity levels (see colour scale). cor, cortex; epi, epidermis; lower, lower side of gravistimulated root; upper, upper side of gravistimulated root. Scale bar=10 μm. Download figure Download PowerPoint To further characterize the regulation of the efflux carrier activity in response to gravity, we have investigated PIN2 abundance at the plasma membrane of gravistimulated roots. As previously suggested, following gravistimulation, PIN2 distribution became asymmetric between the upper and lower sides of the root, in concordance with an asymmetrical auxin distribution (Paciorek et al, 2005; Abas et al, 2006; Kleine-Vehn et al, 2008b) (Figure 1N–T). We have quantified the plasma membrane-localized PIN2 abundance at the lower and upper sides of horizontally placed roots at different time points after gravistimulation. Within 2 h of gravistimulation, an increase in PIN2 at the plasma membrane of cells on the lower root side was detected, which spatially correlated with an increase in auxin response (Figure 1H, M). Following this temporal stabilization, the PIN2 level at the lower side of the root started to decrease to supposedly re-establish the pre-stimulation levels after 12 h of gravistimulation (Figure 1O–S). Thus, at the lower root side, the PIN2 levels transiently increased before gradually decreasing to the pre-stimulation values. In parallel, at the upper side of the bending root, where auxin response initially decreases (Figure 1G–M), PIN2 protein levels at the plasma membrane steadily decreased in time (Figure 1N–P and S), probably because of higher rates of protein degradation due to an increased targeting to the vacuole (Kleine-Vehn et al, 2008b; Figure 4A and B). Notably, after 4 h of gravistimulation, PIN2 started to accumulate again at the plasma membrane, reaching levels close to the initial pre-stimulation levels at ∼12 h after gravistimulation (Figure 1P–S). Thus, at the upper root side, the PIN2 levels initially decrease, which is followed by an increase leading to re-establishment of the pre-stimulation values. The observed changes in signal intensity infer ∼12 and 14% change in PIN2 abundance on the lower and upper sides of gravistimulated root, respectively. Notably, the recovery of symmetry in PIN2 protein levels at the plasma membrane after 12 h of gravistimulation at both lower and upper sides of the root presumably reflects a re-established symmetric auxin flow, resulting in vertical root growth (Figure 1). Overall, our data shows that a spatio-temporal regulation of the auxin distribution after gravistimulation correlates with complex and differential regulation of the PIN2 abundance at the lower and upper side of gravistimulated roots. Specifically, the increase in auxin response at the lower side of the root is accompanied with the initial increase in PIN2 abundance followed by its gradual decrease. On the other hand, at the upper side of the root, we have detected a decrease in auxin response that is accompanied with initial decrease in PIN2 abundance followed by its gradual increase. Importantly, the differential auxin accumulation in all observed cases preceeded changes in PIN2 abundance at the plasma membrane. The above findings also complement the observation of Luschnig et al (1998) that a missense pin2 allele fails to establish gravity-induced lateral auxin gradient in the root. Auxin promotes PIN2 degradation in the vacuoles at the lower side of the root First, we have addressed the mechanisms underlying the regulation of PIN2 abundance at the lower side of the gravistimulated root. The initial, transient stabilization of PIN2 at the plasma membrane is presumably a result of a documented transient (⩽2 h) inhibitory effect of higher auxin levels on PIN internalization (Paciorek et al, 2005; Robert et al, 2010; Chen et al, 2012; Lin et al, 2012). On the other hand, the following decrease in PIN2 levels that still coincides with a DR5-visualized local increase in auxin response (Figure 1) might be result of the long-term effect of auxin on PIN stability (Sieberer et al, 2000; Vieten et al, 2005). Therefore, we have tested the effect of prolonged (⩾3 h) exogenous auxin application on PIN2 abundance at the plasma membrane. Following NAA treatment, we have observed a reduction of PIN2-GFP levels (in PIN2::PIN2-GFP (eir1-1) transgenic seedlings) at the plasma membrane concomitantly with an increase in a diffused vacuolar GFP signal (Figure 2A–C; see Supplementary Figure 2). This observation was confirmed by a significant reduction of PIN2 abundance in membrane protein extracts from NAA-treated seedlings as detected by western blots (Figure 2D). Figure 2.Auxin effect on PIN protein degradation. (A, B) Intracellular localization of PIN2-GFP (eir1-1) protein in seedlings incubated with DMSO (A) or with 10 μM NAA (B). (C) Relative PIN2-GFP abundance at the plasma membrane versus the intracellular signal in PIN2::PIN2:GFP (eir1-1) expressing line. n=3 independent experiments with at least six roots analysed for each assay and 60 cells counted in total. (D) Total membrane protein fractions probed with anti-PIN2 antibody. PIN2 protein level decreased when seedlings were treated 3 h with 20 μM NAA. PIN2-specific band at ∼70 kDa is marked with the cross. (E, F) Intracellular localization of 35S::PIN2-EosFP (eir1-1) protein in seedlings incubated with DMSO (E) or with auxin (10 μM/14 h) (F). The effect of auxin on 35S::PIN2-EosFP targeting to the vacuole was observed after an extended auxin treatment probably due to the stabilized expression under 35S promoter, similarly to what was observed with 35S::PIP2-GFP (see Supplementary Figure 4G–I). (G) Relative PIN2-EosFP abundance at the plasma membrane versus intracellular signal in 35S::PIN2-EosFP (eir1-1) expressing line. n=3 independent experiments with at least six roots analysed for each assay and ten cells counted for each root. (H, I) Intracellular localization of PIN2::PIN1-GFP protein in seedlings incubated with DMSO (H) or with 10 μM NAA (I). (J) Relative PIN1-GFP abundance at the plasma membrane versus intracellular signal in PIN2::PIN1-GFP expressing line. n=3 independent experiments with at least six roots analysed for each assay and ten cells counted for each root. (K, L) Intracellular localization of PIN2-GFP in eir1-1 background (F1 generation after cross with Col-0) (K) compared to RPS5»iaaM background (F1 generation after cross with PIN2::PIN2-GFP (eir1-1)) (L). (M) Relative PIN2-GFP abundance at the plasma membrane versus intracellular signal. n=1 with 60 cells analysed. Error bars represent standard error of the mean (s.e.m.), P-value calculated according to Student's t-test. Arrowheads highlight differences in PIN protein retention at the plasma membrane and accumulation in the vacuoles. Signal intensities are coded blue to yellow corresponding to increasing intensity levels (see colour scale). Scale bar=10 μm. Download figure Download PowerPoint We have then addressed the cellular mechanism of the auxin effect on PIN2 abundance. In general, protein abundance at the plasma membrane is expected to reflect a sum of transcription, translation, targeting and proteolysis. It has been shown previously that PIN2 transcription does not change dramatically in response to auxin (Sieberer et al, 2000; Shin et al, 2005). Consistently, PIN2 mRNA levels were shown to be induced by auxin with low amplitude and much slower kinetics than other PIN genes or other auxin inducible genes (Vieten et al, 2005; Lee et al, 2009). In agreement with those findings, in our experimental conditions, auxin treatment only mildly affected PIN2 transcription (see Supplementary Figure 3), suggesting that auxin regulates PIN2 levels via a post-transcriptional mechanism. Moreover, auxin-mediated decrease in PIN2 from the plasma membrane occurred regardless of whether PIN2 was expressed under its endogenous (Figure 2A–C) or constitutive, heterologous 35S promoter (Figure 2E–G), suggesting that the increased downregulation of PIN2 is not an indirect effect of an excess of PIN2 protein in the cell's endomembrane system. It is proposed that PIN proteins are degraded in the vacuoles (Laxmi et al, 2008; Kleine-Vehn et al, 2008b; Shirakawa et al, 2009; Marhavý et al, 2011), where GFP-tagged proteins can be visualized after an incubation in the dark (Tamura et al, 2003). In these conditions, we have found a decrease in PIN2-GFP at the plasma membrane and concomitant increase in fluorescence signal in the vacuoles in response to auxin treatment (Figure 2A–C; see Supplementary Figure 2K–M). This strongly suggests that auxin downregulates PIN2 abundance at the plasma membrane by enhancing PIN trafficking to the vacuole. Moreover, the auxin application destabilized both apical and basal PIN1 and PIN2 cargos from the plasma membrane (Figure 2H–J; see Supplementary Figures 2A–C and 4A–C) and to lesser extent also non-polar integral plasma membrane proteins such as BRASSINOSTEROID INSENSITIVE1 (BRI1)-GFP and PLASMA MEMBRANE INTRINSIC PROTEIN2 (PIP2)-GFP (see Supplementary Figure 4D–I). In addition, we could demonstrate that auxin reduces PIN2 protein levels (Figure 2D), thereby strongly suggesting that the observed vacuolar targeting of PINs is associated with protein degradation. To further confirm the auxin effect on the degradation of plasma membrane proteins, we have genetically manipulated the endogenous auxin concentrations in Arabidopsis seedlings. We have constitutively overexpressed the Agrobacterium tumefaciens indoleacetic acid-tryptophan monooxygenase (iaaM) under the strong ribosomal promoter RPS5. The iaaM enzyme converts tryptophan into indole-3-acetamide, which is then hydrolysed to indole-3-acetic acid (IAA) in plant cells (Klee et al, 1987; Romano et al, 1995; Weijers et al, 2001, 2005). The transcription of PIN2 was not altered in the RPS5»iaaM transactivated line (see Supplementary Figure 5). We have then analysed the abundance and intracellular distribution of PIN2-GFP marker crossed into the iaaM background. Similarly to exogenously applied, endogenously produced auxin promoted an increased PIN2 degradation as manifested by higher vacuolar GFP signal (Figure 2K–M). The iaaM expression was shown to elevate cellular auxin concentration 2- to 10-fold (Klee et al, 1987; Romano et al, 1995), Therefore, considering that we have not used additional media supplementation, neither with tryptophan nor with auxin, it can be expected that the physiological threshold of auxin effect on increased PIN degradation is placed in the aforementioned range of auxin concentration change above normal/physiological level. Taken together, this data shows that exogenously applied or endogenously produced auxin mediates the PIN targeting to the vacuole and promotes PIN2 degradation. This auxin effect presumably accounts for the decrease in PIN2 level at the lower side of the gravistimulated root after 4 h. Auxin promotes PIN2 degradation by SCFTIR1/AFB-mediated signalling Next, we have assessed by which signalling pathway auxin promotes PIN2 degradation. We have previously shown that the inhibitory effect of auxin on PIN endocytosis is mediated by an ABP1-dependent signalling. Whereas auxin inhibits endocytosis instantaneously without de novo protein biosynthesis and nuclear auxin signalling (Paciorek et al, 2005; Robert et al, 2010; Chen et al, 2012; Lin et al, 2012), the auxin-induced PIN2 translocation to the vacuole for degradation required prolonged (⩾3 h) auxin treatments (Figure 2A–C, see Supplementary Figure 4A–C). Given the fact that the earliest auxin-induced response proteins are detectable after ∼10–15 min of auxin application (Badescu and Napier, 2006), the auxin effect on the vacuolar targeting might require transcriptional regulation and de novo protein synthesis mediated by the SCFTIR1/AFB pathway (Kepinski