Patterning in Plant Development Jiří Friml, ZMBP Tübingen Plants and Animals Live Different Lives Arabidopsis Embryogenesis Comparison of embryo development in Angiosperms Chenopodiad Caryophyllad Solanad Asterad Piperad Modified after Johri et al. 1992 How can such a protected system be investigated experimentally? Mutant screen at seedling level Genetic Interference with Auxin Response and Transport Disrupts Embryo Patterning monopteros bodenl gno 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 Auxin Transport Chemiosmotic hypothesis Molecular Genetics of Auxin Efflux PIN1 PIN2 PIN3 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 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 treatmen 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 CycBmargins + IAA + IAA + NPA Wt pin1,3 pin1,3,4 + IAA + IAA Common module for organ formation Aerial organogenesis Underground organogenesis Cotyledons, leaves, flowers, ral organs, ovules, integuments Lateral roots 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 PIN1/PIN1:GFP apical basal localization basal localization Wisniewska et al. 2006 PIN Polarity Determines Direction of Auxin Flow pin2 (eir1, agr1) PIN2::PIN2:HA PIN2::PIN1:HA PIN2::PIN1:GFP-3 DR5rev::GFP gravitropism PIN2::PIN1:GFP-2 Wisniewska et al. 2006 PIN:GFP mutant lines intragenic extragenic sequencing cloning cycling polarity important residues novel genes Mutant Screen for Components of Polarity and Recycling EMS mutagenesis. Screening for polarity and cycling defects. 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 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 Rapid Changes in PIN Polarity for Redirection of Auxin Flow PIN1 in embryogenesis PIN3 for gravitropism Friml et al. 2002Friml et al. 2003 0’ 2’ Subcellular Cycling of PIN Proteins + BFA - BFA Geldner et al., 2003 UV-activated PIN2-EosFP Protoplasts + BFA - BFA Dhonukshe et al., 2007 PIN3 Polarity Switch in Gravitropic Response Nucleus StatolitsAtPIN3 Root turned on its side Friml et al. 02 DNA RNA PID ARF GN BIG Cell-biological Determinants Auxin Gradients Plant Development TROPISMS Phototropism Gravitropism - NPA - NPA+ NPA + NPA Asymmetric Auxin Distribution Underlies Tropisms Col-0 Col-0Atpin3 Atpin3 Hypocotyl phototropism Hypocotyl gravitropism 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 Tropisms Col-0 Atpin3 Col-0 (NPA) light dark DR5 pin3 Hypocotyl and Apical Hook Phenotypes 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 gravity stimulated gravity + NPA DR5rev::GFP Root Gravitropism PIN2 localizationStatoliths - gravity perception Relocation of PIN3 during Gravitropism 0 min PIN3 in vertical root PIN3 in root on its side 2 min 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 PIN-dependent Auxin Gradients in Plant Development