TY - JOUR AB - Auxin directs plant ontogenesis via differential accumulation within tissues depending largely on the activity of PIN proteins that mediate auxin efflux from cells and its directional cell-to-cell transport. Regardless of the developmental importance of PINs, the structure of these transporters is poorly characterized. Here, we present experimental data concerning protein topology of plasma membrane-localized PINs. Utilizing approaches based on pH-dependent quenching of fluorescent reporters combined with immunolocalization techniques, we mapped the membrane topology of PINs and further cross-validated our results using available topology modeling software. We delineated the topology of PIN1 with two transmembrane (TM) bundles of five α-helices linked by a large intracellular loop and a C-terminus positioned outside the cytoplasm. Using constraints derived from our experimental data, we also provide an updated position of helical regions generating a verisimilitude model of PIN1. Since the canonical long PINs show a high degree of conservation in TM domains and auxin transport capacity has been demonstrated for Arabidopsis representatives of this group, this empirically enhanced topological model of PIN1 will be an important starting point for further studies on PIN structure–function relationships. In addition, we have established protocols that can be used to probe the topology of other plasma membrane proteins in plants. © 2016 The Authors AU - Nodzyński, Tomasz AU - Vanneste, Steffen AU - Zwiewka, Marta AU - Pernisová, Markéta AU - Hejátko, Jan AU - Friml, Jirí ID - 1145 IS - 11 JF - Molecular Plant TI - Enquiry into the topology of plasma membrane localized PIN auxin transport components VL - 9 ER - TY - JOUR AB - Apical dominance is one of the fundamental developmental phenomena in plant biology, which determines the overall architecture of aerial plant parts. Here we show apex decapitation activated competition for dominance in adjacent upper and lower axillary buds. A two-nodal-bud pea (Pisum sativum L.) was used as a model system to monitor and assess auxin flow, auxin transport channels, and dormancy and initiation status of axillary buds. Auxin flow was manipulated by lateral stem wounds or chemically by auxin efflux inhibitors 2,3,5-triiodobenzoic acid (TIBA), 1-N-naphtylphtalamic acid (NPA), or protein synthesis inhibitor cycloheximide (CHX) treatments, which served to interfere with axillary bud competition. Redirecting auxin flow to different points influenced which bud formed the outgrowing and dominant shoot. The obtained results proved that competition between upper and lower axillary buds as secondary auxin sources is based on the same auxin canalization principle that operates between the shoot apex and axillary bud. © The Author(s) 2016. AU - Balla, Jozef AU - Medved'Ová, Zuzana AU - Kalousek, Petr AU - Matiješčuková, Natálie AU - Friml, Jirí AU - Reinöhl, Vilém AU - Procházka, Stanislav ID - 1147 JF - Scientific Reports TI - Auxin flow mediated competition between axillary buds to restore apical dominance VL - 6 ER - TY - JOUR AB - Tissue patterning in multicellular organisms is the output of precise spatio–temporal regulation of gene expression coupled with changes in hormone dynamics. In plants, the hormone auxin regulates growth and development at every stage of a plant’s life cycle. Auxin signaling occurs through binding of the auxin molecule to a TIR1/AFB F-box ubiquitin ligase, allowing interaction with Aux/IAA transcriptional repressor proteins. These are subsequently ubiquitinated and degraded via the 26S proteasome, leading to derepression of auxin response factors (ARFs). How auxin is able to elicit such a diverse range of developmental responses through a single signaling module has not yet been resolved. Here we present an alternative auxin-sensing mechanism in which the ARF ARF3/ETTIN controls gene expression through interactions with process-specific transcription factors. This noncanonical hormonesensing mechanism exhibits strong preference for the naturally occurring auxin indole 3-acetic acid (IAA) and is important for coordinating growth and patterning in diverse developmental contexts such as gynoecium morphogenesis, lateral root emergence, ovule development, and primary branch formation. Disrupting this IAA-sensing ability induces morphological aberrations with consequences for plant fitness. Therefore, our findings introduce a novel transcription factor-based mechanism of hormone perception in plants. © 2016 Simonini et al. AU - Simonini, Sara AU - Deb, Joyita AU - Moubayidin, Laila AU - Stephenson, Pauline AU - Valluru, Manoj AU - Freire Rios, Alejandra AU - Sorefan, Karim AU - Weijers, Dolf AU - Friml, Jirí AU - Östergaard, Lars ID - 1151 IS - 20 JF - Genes and Development TI - A noncanonical auxin sensing mechanism is required for organ morphogenesis in arabidopsis VL - 30 ER - TY - JOUR AB - Differential cell growth enables flexible organ bending in the presence of environmental signals such as light or gravity. A prominent example of the developmental processes based on differential cell growth is the formation of the apical hook that protects the fragile shoot apical meristem when it breaks through the soil during germination. Here, we combined in silico and in vivo approaches to identify a minimal mechanism producing auxin gradient-guided differential growth during the establishment of the apical hook in the model plant Arabidopsis thaliana. Computer simulation models based on experimental data demonstrate that asymmetric expression of the PIN-FORMED auxin efflux carrier at the concave (inner) versus convex (outer) side of the hook suffices to establish an auxin maximum in the epidermis at the concave side of the apical hook. Furthermore, we propose a mechanism that translates this maximum into differential growth, and thus curvature, of the apical hook. Through a combination of experimental and in silico computational approaches, we have identified the individual contributions of differential cell elongation and proliferation to defining the apical hook and reveal the role of auxin-ethylene crosstalk in balancing these two processes. © 2016 American Society of Plant Biologists. All rights reserved. AU - Žádníková, Petra AU - Wabnik, Krzysztof T AU - Abuzeineh, Anas AU - Gallemí, Marçal AU - Van Der Straeten, Dominique AU - Smith, Richard AU - Inze, Dirk AU - Friml, Jirí AU - Prusinkiewicz, Przemysław AU - Benková, Eva ID - 1153 IS - 10 JF - Plant Cell TI - A model of differential growth guided apical hook formation in plants VL - 28 ER - TY - JOUR AB - Plants adjust their growth according to gravity. Gravitropism involves gravity perception, signal transduction, and asymmetric growth response, with organ bending as a consequence [1]. Asymmetric growth results from the asymmetric distribution of the plant-specific signaling molecule auxin [2] that is generated by lateral transport, mediated in the hypocotyl predominantly by the auxin transporter PIN-FORMED3 (PIN3) [3–5]. Gravity stimulation polarizes PIN3 to the bottom sides of endodermal cells, correlating with increased auxin accumulation in adjacent tissues at the lower side of the stimulated organ, where auxin induces cell elongation and, hence, organ bending. A curvature response allows the hypocotyl to resume straight growth at a defined angle [6], implying that at some point auxin symmetry is restored to prevent overbending. Here, we present initial insights into cellular and molecular mechanisms that lead to the termination of the tropic response. We identified an auxin feedback on PIN3 polarization as underlying mechanism that restores symmetry of the PIN3-dependent auxin flow. Thus, two mechanistically distinct PIN3 polarization events redirect auxin fluxes at different time points of the gravity response: first, gravity-mediated redirection of PIN3-mediated auxin flow toward the lower hypocotyl side, where auxin gradually accumulates and promotes growth, and later PIN3 polarization to the opposite cell side, depleting this auxin maximum to end the bending. Accordingly, genetic or pharmacological interference with the late PIN3 polarization prevents termination of the response and leads to hypocotyl overbending. This observation reveals a role of auxin feedback on PIN polarity in the termination of the tropic response. © 2016 Elsevier Ltd AU - Rakusová, Hana AU - Abbas, Mohamad AU - Han, Huibin AU - Song, Siyuan AU - Robert, Hélène AU - Friml, Jirí ID - 1212 IS - 22 JF - Current Biology TI - Termination of shoot gravitropic responses by auxin feedback on PIN3 polarity VL - 26 ER - TY - JOUR AB - The Auxin Binding Protein 1 (ABP1) is one of the most studied proteins in plants. Since decades ago, it has been the prime receptor candidate for the plant hormone auxin with a plethora of described functions in auxin signaling and development. The developmental importance of ABP1 has recently been questioned by identification of Arabidopsis thaliana abp1 knock-out alleles that show no obvious phenotypes under normal growth conditions. In this study, we examined the contradiction between the normal growth and development of the abp1 knock-outs and the strong morphological defects observed in three different ethanol-inducible abp1 knock-down mutants ( abp1-AS, SS12K, SS12S). By analyzing segregating populations of abp1 knock-out vs. abp1 knock-down crosses we show that the strong morphological defects that were believed to be the result of conditional down-regulation of ABP1 can be reproduced also in the absence of the functional ABP1 protein. This data suggests that the phenotypes in abp1 knock-down lines are due to the off-target effects and asks for further reflections on the biological function of ABP1 or alternative explanations for the missing phenotypic defects in the abp1 loss-of-function alleles. AU - Michalko, Jaroslav AU - Glanc, Matous AU - Perrot Rechenmann, Catherine AU - Friml, Jirí ID - 1221 JF - F1000 Research TI - Strong morphological defects in conditional Arabidopsis abp1 knock-down mutants generated in absence of functional ABP1 protein VL - 5 ER - TY - JOUR AB - The dynamic localization of endosomal compartments labeled with targeted fluorescent protein tags is routinely followed by time lapse fluorescence microscopy approaches and single particle tracking algorithms. In this way trajectories of individual endosomes can be mapped and linked to physiological processes as cell growth. However, other aspects of dynamic behavior including endosomal interactions are difficult to follow in this manner. Therefore, we characterized the localization and dynamic properties of early and late endosomes throughout the entire course of root hair formation by means of spinning disc time lapse imaging and post-acquisition automated multitracking and quantitative analysis. Our results show differential motile behavior of early and late endosomes and interactions of late endosomes that may be specified to particular root hair domains. Detailed data analysis revealed a particular transient interaction between late endosomes—termed herein as dancing-endosomes—which is not concluding to vesicular fusion. Endosomes preferentially located in the root hair tip interacted as dancing-endosomes and traveled short distances during this interaction. Finally, sizes of early and late endosomes were addressed by means of super-resolution structured illumination microscopy (SIM) to corroborate measurements on the spinning disc. This is a first study providing quantitative microscopic data on dynamic spatio-temporal interactions of endosomes during root hair tip growth. AU - Von Wangenheim, Daniel AU - Rosero, Amparo AU - Komis, George AU - Šamajová, Olga AU - Ovečka, Miroslav AU - Voigt, Boris AU - Šamaj, Jozef ID - 1238 IS - JAN2016 JF - Frontiers in Plant Science TI - Endosomal interactions during root hair growth VL - 6 ER - TY - JOUR AB - The shaping of organs in plants depends on the intercellular flow of the phytohormone auxin, of which the directional signaling is determined by the polar subcellular localization of PIN-FORMED (PIN) auxin transport proteins. Phosphorylation dynamics of PIN proteins are affected by the protein phosphatase 2A (PP2A) and the PINOID kinase, which act antagonistically to mediate their apical-basal polar delivery. Here, we identified the ROTUNDA3 (RON3) protein as a regulator of the PP2A phosphatase activity in Arabidopsis thaliana. The RON3 gene was map-based cloned starting from the ron3-1 leaf mutant and found to be a unique, plant-specific gene coding for a protein with high and dispersed proline content. The ron3-1 and ron3-2 mutant phenotypes [i.e., reduced apical dominance, primary root length, lateral root emergence, and growth; increased ectopic stages II, IV, and V lateral root primordia; decreased auxin maxima in indole-3-acetic acid (IAA)-treated root apical meristems; hypergravitropic root growth and response; increased IAA levels in shoot apices; and reduced auxin accumulation in root meristems] support a role for RON3 in auxin biology. The affinity-purified PP2A complex with RON3 as bait suggested that RON3 might act in PIN transporter trafficking. Indeed, pharmacological interference with vesicle trafficking processes revealed that single ron3-2 and double ron3-2 rcn1 mutants have altered PIN polarity and endocytosis in specific cells. Our data indicate that RON3 contributes to auxin-mediated development by playing a role in PIN recycling and polarity establishment through regulation of the PP2A complex activity. AU - Karampelias, Michael AU - Neyt, Pia AU - De Groeve, Steven AU - Aesaert, Stijn AU - Coussens, Griet AU - Rolčík, Jakub AU - Bruno, Leonardo AU - De Winne, Nancy AU - Van Minnebruggen, Annemie AU - Van Montagu, Marc AU - Ponce, Maria AU - Micol, José AU - Friml, Jirí AU - De Jaeger, Geert AU - Van Lijsebettens, Mieke ID - 1247 IS - 10 JF - PNAS TI - ROTUNDA3 function in plant development by phosphatase 2A-mediated regulation of auxin transporter recycling VL - 113 ER - TY - JOUR AB - Plant growth and architecture is regulated by the polar distribution of the hormone auxin. Polarity and flexibility of this process is provided by constant cycling of auxin transporter vesicles along actin filaments, coordinated by a positive auxinactin feedback loop. Both polar auxin transport and vesicle cycling are inhibited by synthetic auxin transport inhibitors, such as 1-Nnaphthylphthalamic acid (NPA), counteracting the effect of auxin; however, underlying targets and mechanisms are unclear. Using NMR, we map the NPA binding surface on the Arabidopsis thaliana ABCB chaperone TWISTED DWARF1 (TWD1).We identify ACTIN7 as a relevant, although likely indirect, TWD1 interactor, and show TWD1-dependent regulation of actin filament organization and dynamics and that TWD1 is required for NPA-mediated actin cytoskeleton remodeling. The TWD1-ACTIN7 axis controls plasma membrane presence of efflux transporters, and as a consequence act7 and twd1 share developmental and physiological phenotypes indicative of defects in auxin transport. These can be phenocopied by NPA treatment or by chemical actin (de)stabilization. We provide evidence that TWD1 determines downstreamlocations of auxin efflux transporters by adjusting actin filament debundling and dynamizing processes and mediating NPA action on the latter. This function appears to be evolutionary conserved since TWD1 expression in budding yeast alters actin polarization and cell polarity and provides NPA sensitivity. AU - Zhu, Jinsheng AU - Bailly, Aurélien AU - Zwiewka, Marta AU - Sovero, Valpuri AU - Di Donato, Martin AU - Ge, Pei AU - Oehri, Jacqueline AU - Aryal, Bibek AU - Hao, Pengchao AU - Linnert, Miriam AU - Burgardt, Noelia AU - Lücke, Christian AU - Weiwad, Matthias AU - Michel, Max AU - Weiergräber, Oliver AU - Pollmann, Stephan AU - Azzarello, Elisa AU - Mancuso, Stefano AU - Ferro, Noel AU - Fukao, Yoichiro AU - Hoffmann, Céline AU - Wedlich Söldner, Roland AU - Friml, Jirí AU - Thomas, Clément AU - Geisler, Markus ID - 1251 IS - 4 JF - Plant Cell TI - TWISTED DWARF1 mediates the action of auxin transport inhibitors on actin cytoskeleton dynamics VL - 28 ER - TY - JOUR AB - n contrast with the wealth of recent reports about the function of μ-adaptins and clathrin adaptor protein (AP) complexes, there is very little information about the motifs that determine the sorting of membrane proteins within clathrin-coated vesicles in plants. Here, we investigated putative sorting signals in the large cytosolic loop of the Arabidopsis (Arabidopsis thaliana) PIN-FORMED1 (PIN1) auxin transporter, which are involved in binding μ-adaptins and thus in PIN1 trafficking and localization. We found that Phe-165 and Tyr-280, Tyr-328, and Tyr-394 are involved in the binding of different μ-adaptins in vitro. However, only Phe-165, which binds μA(μ2)- and μD(μ3)-adaptin, was found to be essential for PIN1 trafficking and localization in vivo. The PIN1:GFP-F165A mutant showed reduced endocytosis but also localized to intracellular structures containing several layers of membranes and endoplasmic reticulum (ER) markers, suggesting that they correspond to ER or ER-derived membranes. While PIN1:GFP localized normally in a μA (μ2)-adaptin mutant, it accumulated in big intracellular structures containing LysoTracker in a μD (μ3)-adaptin mutant, consistent with previous results obtained with mutants of other subunits of the AP-3 complex. Our data suggest that Phe-165, through the binding of μA (μ2)- and μD (μ3)-adaptin, is important for PIN1 endocytosis and for PIN1 trafficking along the secretory pathway, respectively. AU - Sancho Andrés, Gloria AU - Soriano Ortega, Esther AU - Gao, Caiji AU - Bernabé Orts, Joan AU - Narasimhan, Madhumitha AU - Müller, Anna AU - Tejos, Ricardo AU - Jiang, Liwen AU - Friml, Jirí AU - Aniento, Fernando AU - Marcote, Maria ID - 1264 IS - 3 JF - Plant Physiology TI - Sorting motifs involved in the trafficking and localization of the PIN1 auxin efflux carrier VL - 171 ER -