Auxin Signaling and Transport Discovery of the First Plant Signaling Molecule – Auxin and its Transport • Embryo development • Organ initiation and positioning • Vascular tissue differentiation • Shoot and root elongation • Growth responses to light and gravity • Apical hook formation AUXIN mediates embryos Auxin biosynthesis Signal Transduction Biochemical Approach to Identify Auxin Receptor Isolation of auxin binding proteins - 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 Molecular Biology Approach to Elucidate Auxin Signaling Does auxin regulate gene expression? - Rapidly upregulated mRNAs (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 ARFs auxin response factors – TF controlling, Aux/IAA expression QVVGWPPVRSYRK S Homo and hetero- dimerisation Protein stability Homo and hetero- dimerisation AuxRE binding DBD MR III IV IVIIIIII ARF Aux/IAA bdl mutation Hardtke and Berleth 1998 Homo and hetero- dimerisation Forward genetics Genetic Approach to Identify Auxin Receptor - Auxin resistant (axr): axr1 – axr6 - Transport inhibitor response (tir): tir1 – tir7 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 Subunits of ubiquitin ligase Auxin resistant (axr): axr1 – axr6 Auxin Transport inhibitor response (tir): SCF TIR1 Mutant Screen at Seedling Level bdl Some ARFs are Activators, and AUX/IAAs Repressors of Auxin Response QVVGWPPVRSYRK S Homo and hetero- dimerisation Protein stability Homo and hetero- dimerisation AuxRE binding DBD MR III IV IVIIIIII ARF Aux/IAA bdl mutation Hardtke and Berleth 1998 bdl mp 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 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. 0 IAA Local Application of Auxin Induces Organ Formation Reinhardt et al. 03 NPA NPA Bern 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 Physiology of Auxin Transport Chemiosmotic hypothesis Auxin Influx aux1 is Resistant to Auxin aux1 phenotype Transport properties of different auxins aux1WT aux1WT 2,4D 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 Membrane topology model The Arabidopsis PIN Gene Family Comparison of Arabidopsis PIN proteins Phylogenetic tree What is Molecular Role of 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 NAA accumulation kinetics 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 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 -Interested in novel players in auxin homeostasis - Search for proteins with PIN like protein organisation - SMART tool from EMBL (finds proteins with topology of interest) PILS1 At1g20925 PILS4 At1g76530 PILS6 At5g01990 PILS7 At5g65980The PILS (PIN-likes) gene family Aim of the project: Initial characterisation of the PILS family members PINs vs PILS: Protein sequences PIN2 PIN1 PIN3 PIN7 PIN4 PIN8 PIN5 PIN6 PILS7 PILS5 PILS1 PILS3 PILS4 PILS2 PILS6 0.05 Limited AA sequence similarities reveal that PINs and PILS form two distinct protein families PIN proteins PILS proteins PINs vs PILS: Topology PIN1 (ClassII PINs): - Long hydrophylic loop PIN8 (ClassI PINs): - Short hydrophylic loop PILS2 - Short hydrophylic loop -PILS proteins share the same predicted protein topology as the PINs -The short central hydrophylic loop make the PILS more related to the Class I PINs Phenotypes of pils mutants ER Localization Yeast: IAA transport PILS genes in Arabidopsis Barbez et al., 2012, unpublished Cellular Polarity of PIN Localization and Directionality of Intercellular Auxin Flow pin2Col-0 PIN2 – Root Gravitropism PIN2 protein 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 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 proteins are rate-limiting factors in auxin efflux from cells and the polarity of their subcellular localization determines direction of intercellular auxin flow Constitutive Cycling of 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 PM-ATPase PIN2/ATPase PIP2 BRI1 NAA /BFA PIN1 2,4-D/BFA BFA in sur2IAA/BFA 2-NAA/BFA Ethylene/BFA Place of Auxin Action in Protein Cycling BFA Auxin + BFA BFA compartment BFA compartment PIN1 PIN1 Paciorek et al., 2005 Auxin Inhibits Endocytosis 0 NAA Uptake of endocytic tracer FM4-64 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 DR5 PIN1 2,4-D/ CHX 2,4-D 2,4-D/MG132 /BFA unpublished 2,4-D/CHX /BFA BFA 2,4-D/ MG1320 Auxin Inhibits PIN Internalization and Stimulates its Efflux PIN2 at PM Auxin efflux Paciorek et al., 2005 BFA 2,4-D/BFAIAA/BFA Novel Pathway of Auxin Action DR5 PIN1 2,4-D/ CHX 2,4-D 2,4-D/MG132 /BFA 2,4-D/CHX /BFA BFA 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 lines intragenic extragenic sequencing cloning cycling polarity important residues novel genes Mutant Screen for Components of PIN Polarity and Cycling EMS mutagenesis. Screening for polarity and cycling defects. 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 PINs at the cell surface thus stimulating auxin efflux. This auxin effect involves novel, genetically tractable auxin pathway Novel Pathway for Auxin Signaling Auxin Inhibits PIN Internalization and Stimulates its Transport PIN2 at PM Auxin efflux Paciorek et al., 2005 BFA 2,4-D/BFANAA/BFA Auxin Signaling for Endocytosis Dhonukshe et al., 2007; Robert et al., 2011; unpublished PIN endocytosis Auxin ABP1 ABP1 Clathrin Auxin effect on PIN endocytosis requires ROP6/RIC1 activity unpublished ROP6/RIC1 acts downstream of ABP1 to regulate endocytosis unpublished ABP1/ROP signaling for endocytosis and polarity ABP1 ROP Clathrin PIN endocytosis Auxin ? ? ? unpublished PIN polarity Gravitropism Vascular tissue formation