Signal Transduction Mutant screens for ethylene pathway genes Triple response Ethylene signal transduction pathway Ethylene signal transduction pathway Ethylene signal transduction pathway His-kinases in Arabidopsis Response Regulators in Arabidopsis Ethylene signal transduction pathway Genetic interactions CYTOKININ ­ what is important? Synthesis ­ IPT genes Degradation ­ CK-oxidase Signal transduction ­ forward genetics activation tagging CK regulated genes Cytokinins Effect of CK on regeneration Eklef Manipulating of CK levels by overexpression of bacterial IPT Takei Isolation of Arabidopsis IPTs Werner Isolation of CK-oxidase (AtCKX) AtCKXs overexpression in tobacco Effect of AtCKX on tobacco root Morphology of AtCKX tobacco plants Frank Tumor shoot development (tsd) and pasticcino (pas) mutants Use of CyclinB::GUS for cell division monitoring Kakimoto Isolation of CK independent (cki1) mutant Kakimoto Identification of CKI1 gene Verification - 35S::CKI1 transgene Inoue Next strike - CK response mutant (cre1) Piece of genius - complementation Brandstatter CK responsive genes - ARRs Phenotypes of arrs wt Hwang His kinase transduction pathway His kinase pathway components in Arabidopsis Games with protoplasts - Reporters for different pathways ARR 6 RD29A GH3 LUC LUC LUC cytokinin auxin ABA Hwang Games with protoplasts AHPs ­ shuttle to nucleus Opposite effects of two classes of ARRs on CK signalling Games with protoplasts Auxin Signaling and Transport Discovery of the First Plant Signaling Molecule ­ Auxin and its Transport ˇ 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 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 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 Forward genetics , a n d s e v e r a l h u n d r e d l i n e s w i t h i n t e r e s t i n g E x p r e s s i o n p a t u i t a b l e f o r t h e m a i n 3 9 5 n m e x c i t a t i o n a n d 5 0 9 s o b u t y r i c a c i d ( I B A ) f o r i n d u c t i o n o f r o o t s . o f s h o o t f o r m a t i o n . m i d . u r e .EMS Mutagenesis Mutant Screen at Seedling Level 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 Some ARFs are Activators, whereas Aux/IAA 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 Auxin-responsive gene ON 26S auxin SCF OFF Auxin-responsive gene BDL MP 0 20 40 60 80 100 120 140 160 180 200 BDL BDL BDL MP-C BDL - GNOM MP-C MillerUnits Y2H Genomic Auxin Signaling Summary for Auxin Signaling Biochemical approach ­ auxin binding protein ABP1 binds auxin, important in embryogenesis, precise role unclear 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 Auxin Transport Proteins involved in auxin transport -PIN proteins (efflux) -AUX1 proteins (influx) ˇ Embryo development * Organ initiation and positioning * Vascular tissue differentiation * Shoot and root elongation * Growth responses to light and gravity * Apical hook formation AUXIN TRANSPORT mediates embryos 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 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 proteins are rate-limiting factors in auxin efflux from cells and the polarity of their subcellular localization determines direction of intercellular auxin flow Cellular Polarity of PIN Localization 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/PIN2 apical basal localization basal localization Wisniewska et al., 2006 PIN Polarity Determines Direction of Auxin Flow eir1 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 Auxin in Embryonic Apical-Basal Axis Formation Friml et al. 03 DR5 DR5 + NPA PIN7 PIN1 pin1,3,4,7 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 NAA/BFABFA H+ATPase endosoms Auxin Effect on Endosome Trafficking Paciorek et al., 2005 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 0` 30` 60` 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 gravity vector gravity stimulated gravity + NPA in planta Correlation between Cycling and Auxin Flow DR5rev::GFP PIN2:GFP not stimulated PIN2:GFP 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 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 M/90´BFA 50 M 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