Signal Transduction Auxin Signaling and Transport Discovery of the First Plant Signaling Molecule – Auxin and its Transport • Embryo development • Organ initiation and positioning AUXIN mediates embryos • Organ initiation and positioning • Vascular tissue differentiation • Shoot and root elongation • Growth responses to light and gravity • Apical hook formation Signal Transduction Biochemical Approach to Identify Auxin Receptor Isolation of auxin binding proteins - Azidolabeling- Azidolabeling - Affinity chromatography Protein sequencing, cDNA screening, gene identification => Auxin Binding Protein (ABP1) Reverse Genetic – Embryo Lethal abp1 Mutant ABP1 Antisense BY-2 Cells Display Defects in Auxin Dependent Cell Elongation ABP1 – Structure Optimistic Model for ABP1 Action Genetic Approach to Identify Auxin Receptor - Auxin resistant (axr): axr1 – axr6 - Transport inhibitor response (tir): tir1 – tir7 Morphological mutants (monopteros, bodenlos,Morphological mutants (monopteros, bodenlos, etc.) => Role of regulated protein degradation and transcriptional regulation in auxin signaling None of the identified gene looks like a receptor Forward genetics EMS Mutagenesis Auxin resistant (axr): axr1 – axr6 Auxin Transport inhibitor response (tir) Subunits of ubiquitin ligase SCF TIR1 Molecular Biology Approach to Elucidate Auxin Signaling Does auxin regulate gene expression? - Rapidly upregulated mRNAs (GH3, SAUR, AUX/IAA genes)(GH3, SAUR, AUX/IAA genes) - One hybrid screen with Auxin Response Elements => Auxin Response Factors (ARF) - Two hybrid => AUX/IAAs interact with ARFs Molecular Biology Approach to Elucidate Auxin Signaling RT-PCR - IAA + IAA Some ARFs are Activators, whereas Aux/IAA Repressors of Auxin Response Homo and hetero-Protein IVIIIIII Aux/IAA QVVGWPPVRSYRK S Homo and hetero- dimerisation Protein stability Homo and hetero- dimerisation AuxRE binding DBD MR III IV ARF bdl mutation Hardtke and Berleth 1998 Mutant Screen at Seedling Level AUX/IAAs are stabilized in enhancer TIR1 mutant Pull-down GST–IAA7 Genomic Auxin Signaling Summary for Auxin Signaling Biochemical approach – auxin binding protein ABP1 binds auxin, important in embryogenesis, role in endocytosis Genetic approach – role of protein degradationGenetic approach – role of protein degradation (axr1, tir1) Molecular approach – auxin regulates expression ARE in promotors of auxin regulated genes ARF transcription factors binds to ARE AUX/IAA proteins repress ARF and are degraded upon auxin signal Local Auxin Gradients in Plant Development Sabatini et al. 99; Friml et al. 02; Ottenschläger et al. 03; Benková et al. 03; Friml et al. 03 IAA Local Application of Auxin Induces Organ Formation Bern Reinhardt et al. 03 NPA NPA Auxin Transport Proteins involved in auxin transport -PIN proteins (efflux) -AUX1 proteins (influx) Role of GNOM dependent vesicle trafficking PIN proteins cycling and its role • Embryo development • Organ initiation and positioning AUXIN TRANSPORT mediates embryos • Organ initiation and positioning • Vascular tissue differentiation • Shoot and root elongation • Growth responses to light and gravity • Apical hook formation Physiology of Auxin Transport Chemiosmotic hypothesis Auxin Influx aux1 is Resistant to Auxin aux1 phenotype Transport properties of different auxins 2,4D aux1WT aux1WT NAA - NAA + NAA NAA Rescues aux1 Phenotype AUX1 – Expression and Localization AUX1 proteinAUX1::GUS PIN1/AUX1 Auxin Efflux PIN1 – the Auxin Efflux Carrier? pin1 mutant PIN1 protein pin2Col-0 PIN2 – Root Gravitropism PIN2 protein The Arabidopsis PIN Gene Family Comparison of Arabidopsis PIN proteins Phylogenetic tree Membrane topology model What is Molecular Role of PIN Proteinsof PIN Proteins in Auxin Transport? PINs Are Essential Components of Auxin Transport Putative topology of PIN proteins Phylogenetic tree - All defects in pin loss-off-function mutants are in auxin transport-dependent processes and can be phenocopied by auxin transport inhibitors - Local auxin distribution (gradients) are affected in pins - Polar PIN localization determines direction of auxin flow PINs Are Rate-limiting Factors in Auxin Efflux -DEX Inducible PIN1 expression +DEX-DEX +DEX NAA accumulation kinetics Petrasek et al., 2006 PIN-induced Phenotypes in BY-2 Cells - DEX + DEX + DEX/+ NPA - DEX/- auxin unpublished Expression of PINs in HeLa and Yeast Heterologous PIN2 expression auxin efflux activity Petrasek et al., 2006 PIN gene family Predicted PIN Protein Topology PINs in Arabidopsis Root gravitropismApical organogenesis Root merist. ? Embryo Tropisms ? ? Auxin Transport in Yeast ER-based PIN5-dependent auxin transport Mravec et al., 2009 PIN5 regulates auxin metabolism Mravec et al., 2009 PIN5-dependent auxin transport into ER Yeast Mravec et al., 2009 Updated model for auxin transport unpublished Cellular Polarity of PIN Localization and Directionality of Intercellular Auxin Flow PIN2pr::PIN1:GFPPIN2pr::PIN2:HA PIN2pr::PIN1:HA PIN-specific Signals for Polar Targeting PIN1/PIN1:GFP apical basal localization apical basal localization basal localization Wisniewska et al. 2006 PIN Polarity Determines Direction of Auxin Flow pin2 (eir1, agr1) PIN2::PIN2:HA DR5rev::GFP gravitropism PIN2::PIN1:HA PIN2::PIN1:GFP-3 PIN2::PIN1:GFP-2 Wisniewska et al. 2006 PIN proteins are rate-limiting factors in auxin efflux from cells andand the polarity of their subcellular localization determines direction of intercellular auxin flow Constitutive Cycling of PINsof PINs + BFA - BFA PIN1 Subcellular Movement untreated Geldner et al., 2001 Dynamic Movement of PIN Proteins Subcellular Cycling – Means to Modulate Protein Activity? BFA Auxin Inhibits Internalization of Plasma Membrane Proteins NAA /BFA PM-ATPase PIN2/ATPase PIP2 BRI1PIN1 2,4-D/BFA BFA in sur2IAA/BFA 2-NAA/BFA Ethylene/BFA Place of Auxin Action in Protein Cycling BFA Auxin + BFA PIN1 PIN1 BFA compartment BFA compartment PIN1 PIN1 Paciorek et al., 2005 Auxin Inhibits Endocytosis 0 Uptake of endocytic tracer FM4-64 NAA 0‘ 30‘ 60‘ Paciorek et al., 2005 Auxin Increases PIN Levels at Cell Surface and Stimulates its own Efflux PIN2 levels at PM Auxin efflux in tobacco cells Paciorek et al., 2005 Novel Pathway of Auxin Action PIN1 2,4-D/MG132 /BFA 2,4-D/CHX /BFA BFA DR5 2,4-D/ CHX 2,4-D unpublished 2,4-D/ MG1320 Auxin Inhibits PIN Internalization and Stimulates its Efflux PIN2 at PM Auxin effluxBFA Paciorek et al., 2005 2,4-D/BFAIAA/BFA Novel Pathway of Auxin Action PIN1 2,4-D/MG132 /BFA 2,4-D/CHX /BFA BFA DR5 2,4-D/ CHX 2,4-D 2,4-D/ MG1320 Auxin analogues: mapping the binding sites Robert et al., 2010 Auxin-mediated regulation of transcription and endocytosis involve different binding sites Robert et al., 2010 Mechanisms for endocytosis in animals Clathrin is required for PIN internalization in Arabidopsis protoplasts Dhonukshe et al., 2007 Auxin targets clathrin mechanism of endocytosis Robert et al., 2010 ABP1 positively regulates endocytosis Robert et al., 2010 ABP1 mediates auxin effect on endocytosis Robert et al., 2010 ABP1 mediates auxin effect on clathrin Robert et al., 2010 ABP1- and TIR1-dependent Signaling unpublished ABP1 PIN:GFP Mutant Screen for Components of PIN Polarity and Cycling EMS mutagenesis. Screening for polarity and cycling defects. mutant lines intragenic extragenic sequencing cloning cycling polarity important residues novel genes unpublished “Cell Biological” Mutant Screens in Progress: Auxin effect on endocytosis: 3 confirmed mutants wt 30´ NAA 30 µM/90´BFA 50 µM mutant Auxin-resistant BFA patches mutants unpublished Auxin inhibits endocytosis including internalization of PIN proteins This is mechanism by which auxin stabilizes Novel Pathway for Auxin Signaling This is mechanism by which auxin stabilizes PINs at the cell surface thus stimulating auxin efflux. This auxin effect involves novel, genetically tractable auxin pathway