Patterning in Plant Development Role of Auxin Plants and Animals Live Different Lives Arabidopsis Embryogenesis How can such a protected system be investigated experimentally? Genetic Interference with Auxin Response and Transport Disrupts Embryo Patterning monopteros bodenlos gnom Auxin in Early Embryogenesis DR5::GFP IAA localisation DR5::GFP IAA localisation Auxin in Embryogenesis Control NAA 2,4D NPA or BFA Long time treatments DR5::GFP – in vitro Culturing Arabidopsis PIN Protein Family Homology of PIN proteins Phylogenetic tree GUS mRNA Protein PIN7 in Embryogenesis PIN1 Analysis of DR5 activity in pin7 Col-0 pin7 NPA DR5 Embryo Phenotype of pin7 Mutants Col-0 pin7 PIN1 in Early Embryogenesis GUS mRNA Protein pin1 30,4% Enk 6,1% QC markerDR5 basal defects PIN4 protein pin4 Col-0 PIN4 in Embryogenesis Phenotypes of pin Multiple Mutants 4x7 1x3x4 1x3x4x7 1x3x4x7 gnom gnom 1x3x4x7 1x3x4x7 1x3x4x7 1x3x4x7 Auxin and Embryogenesis GlobularTwo-Cell GNOM PIN1 Apical pole specification Root pole specification MP BDL PIN1 MP BDL PIN4 PIN7 PIN7 Canalization hypothesis of auxin flow Feed-back on transport capacity and directionality Vascular tissue regeneration Leave venation Modeling of Embryo Patterning 3D template Cellular parameters Wabnik et al., 2013 2D model ^ ^ ^ ^ ^ ^ ^ Feed-back regulation of PIN polarization by local auxin biosynthesis Robert et al., 2013 Organogenesis Auxin in Cotyledon Formation PIN1 MP BDL GNOM DR5 BFA pins gnom IAA pin1 PIN1 Polarity in Cotyledon Formation Outer layer Globular Heart HeartHeart Inner layers BFA treatment Auxin in Flower and Leave Formation PIN1 localisation pin1 DR5rev::GFP + NPA + NPA DR5 in Floral Organ Formation pin mutants DR5rev::GFP + NPA PIN1 in Floral Organ Formation Ovule defects in pin1 Ovule primordium DR5 in Ovule Formation Ovule with Integuments primordia Ovule primordium PIN1 in Ovule Formation Ovule with Integuments primordia Gynoecium with ovule primordia Lateral Root Development Lateral Root Development in Time DR5 in Lateral Root Formation DR5rev::GUS IAA PIN1:GFP PIN1+ NPA Relocation > Gradients > Primordia PIN1 primordiaDR5 CycB margins + IAA + IAA + NPA Wt pin1,3 pin1,3,4 + IAA + IAA Common module for organ formation Aerial organogenesis Underground organogenesis Cotyledons, leaves, flowers, floral organs, ovules, integuments Lateral roots PIN-dependent Auxin Gradients in Plant Development TROPISMS role for auxin Tropisms: „Movements“ in Plants Phototropism Gravitropism Phototropism Gravitropism - NPA - NPA+ NPA + NPA Asymmetric Auxin Distribution Underlies Tropisms Col-0 Col-0Atpin3 Atpin3 Hypocotyl phototropism Hypocotyl gravitropism pin3 is Defective in Tropisms PIN3 protein in situ RNA hybridization PIN3 in Hypocotyl PIN3 protein in situ RNA hybridization PIN3 in Inflorescence Axis Col-0 Atpin3 PIN3 – Lateral Auxin Transport pin3 phototropism PIN3 protein DR5 - phototropism PIN3 in Phototropic Response Ding et al., 2011 Light-dependent PIN3 relocation 0‘ 2 hours 6 hours Auxin response Col-0 Shoot gravitropic response Bending PIN3 polarizationDR5 response unpublished gravity stimulated gravity + NPA DR5rev::GFP Root Gravitropism PIN2 localizationStatoliths - gravity perception PIN3 0 - 9 09 0 1 8 0 A t p i n 3 0 - 9 09 0 1 8 0 C o l - 0 Root gravitr. pin3 is Defective in gravitropisms Rapid Relocation of PIN3 during Gravitropism 0 min PIN3 in vertical root PIN3 in root on its side 2 min FRAP of PIN3-GFP Gravity-induced PIN3 transcytosis Kleine-Vehn et al., 2010 Nucleus Statolits Vascular tissue Root cap Vertical root Root turned on its side AtPIN3 Model for Root Gravitropism Differential distribution of auxin underlies tropisms PIN-dependent auxin transport acts upstream of auxin distribution External signals can be translated into redirection of auxin flow by rapid changes of PIN polarity Downstream auxin signaling decides about stimulation or inhibition of growth and thus about positive or negative tropism Role of Auxin Distribution in Tropisms Cellular Polarity of PIN Localisation and Directionality of Intercellular Auxin Flow PIN2pr::PIN1:GFP apical basal localization PIN2pr::PIN2:HA PIN2pr::PIN1:HA PIN-specific Signals for Polar Targeting apical basal localization basal localization Wisniewska et al. 2006 Molecular Components of PIN Polar Targeting Ser/Thr protein kinase PINOID (PID) Christensen et al., 2000; Benjamins et al., 2001; Friml et al., 2004 pinoidCol-0 Role of PINOID Kinase in PIN Polar Targeting PIN4 PIN1 Col-0 35S::PID inducible PIDCol-0 Col-0 pinoid PINOID kinase loss-of-function > basal PIN targeting PP2A Phosphatase and PIN Apical-Basal Targeting Michniewicz et al., 2007 PID Phosphorylates PINs Phosphorylation assays in protoplast in vitro phosphorylation Michniewicz et al., 2007 Role of PID in Controlling PIN Polarity > Auxin Flow > Patterning Col-0 RPS5::PID Col-0 RPS5::PID RPS5::PID seedlings PIN1 PIN1 DR5 DR5 Friml et al., 2004 DNA RNA PID ARF GN ABP1 Cell-biological Determinants Auxin GradientsSignals Gravity Light Tissue context Feed-back Friml et al. 2002 Kleine-Vehn et al., 2010 Rakusova et al., 2011 Friml et al., 2002 Ding et al., 2011 Develop. context Benková et al. 2003 Friml et al. 2003 Reinhardt et al. 2003 Wisniewska et al., 2006 Sauer et al., 2006 Paciorek et al., 2005 Robert et al., 2010 Wabnik et al., 2010 Polar Delivery in Plants Animal Plant “Plant Epithelium”: Root-Soil Interface Endodermis PINs IBA Cd BPEN3 Langowski et al., 2010 Niko Geldner, Lausanne Mechanistic Insight into Polar Targeting in Plants PIN2-GFP Lateral Diffusion and PIN Clusters PIN2-GFP 10 min 10 s/frame in press Internalisation „Hot Spots“ unpublished Super Polar Delivery PIN2-GFP unpublished Cell Polarity Maintenance Genetic approaches: Forward Genetic Screens Tanaka et al., 2009, Feraru et al., 2010; Feraru et al., 2011, unpublished Endocytosis ben ...5 Exocytosis bex ...8 Vacuolar Function deg ...3 Apical/Basal Targeting dpt ...4 Outer Polar Targeting dol ...2 Auxin – Endocytosis eon ...6 Chemical Genetic Screens Endocytosis Polar Targeting Reverse Genetics Marker:FP mutant lines novel genes Deep sequencing EMS mutagenesis. Epifluorescence Screening So far mapped in the lab: 11 mutants Beauty of forward genetics gravitropic mutant - epid cells agravitropicgravitropic WT - epid cells P2::P1:HA in pin2 - epid cells Polarity screen - design PIN1 PIN1 PIN2 PIN2 PIN1 PIN1 EMS Feraru et al., 2011 Elena P2P1HAWT-COL A good hit!! Polarity screen Elena Feraru et al., 2011 P2::P1:HA repp1 repp2 repp3 repp4 repp5 regulator of PIN polarity (repp) Feraru et al., 2011 REPP3 Encodes Cellulose Synthase 3 Feraru et al., 2011 Mapping Wild type repp3 Gravitropic growth Polarity change repp3PIN1:HA repp3 Extracellular matrix: Cell wall Degradation of cell wall results in loss of polarity 10’ protoplasting 15’ protoplasting PIN2::PIN2-GFP Feraru et al., 2011 Before After 30 min After 1.5 hours PIN2::PIN2-GFP After 1.5 hours PIN proteins are attached to cell wall Partial degradation of cell wall Feraru et al., 2011 Partial degradation of cell wall Polar cargos are attached to cell wall LTI6b Before After 2 hoursAfter 2 hours PIN1 PIP2a Feraru et al., 2011 Cell Polarity Maintenance unpublished DNA RNA PID ARF GN ABP1 Cell-biological Determinants Auxin GradientsSignals Gravity Light Tissue context Feed-back Friml et al. 2002 Kleine-Vehn et al., 2010 Rakusova et al., 2011 Friml et al., 2002 Ding et al., 2011 Develop. context Benková et al. 2003 Friml et al. 2003 Reinhardt et al. 2003 Wisniewska et al., 2006 Sauer et al., 2006 Paciorek et al., 2005 Robert et al., 2010 Wabnik et al., 2010