1Balance of Hormones regulate growth and developmentEnvironmental factors regulate hormone levelslight- e.g. phototropismgravity- e.g. gravitropismtemperatureMode of action of each hormone1. Signal perception by a receptor on or in target cells.2. Signal transduction and amplification viaa. R --> -->2 messengers ---> modify/activate proteinb. R -----> ------> gene expression3. Response: changes in protein activities and/or gene expression---Æ -Æ growth/development responseBalance of hormones regulate growth and developmentBalance of IAA: cytokinindetermines root or shoot development.e.g. in pith tissue Hormones are used to regenerate transgenic plants.23-. Shoot and root development depend on ratio of IAA/CK[Auxin] rooting[Cytokinin]shoot21-4 Taiz. Tumour induction by cytokinin.[ Agrobacterium-mediated transformation- introducing new gene into plants]Box 21-1 Taiz. Regenerating whole plants from transgenic cells or tissues.Balance of hormones regulate growth and development1. Shoot and root development depend on ratio of Auxin IAA: cytokinin2. Leaf abscission depends on auxin and ABA3. Seed development and germination depends on relative amount of ABA and GAEffect depends on•Type of hormones•conc•balance of hormones219- 27. Light causes redistribution of auxin to shaded side in phototropism.Environmental signals regulate hormone levels. How?How can one monitor hormone levels in plants?• Monitor auxin levels using a reporter gene GUS or GFP.• A promoter that is activated by auxin is fused to GUS or GFP.a. DR5:: GUS (enzyme gives blue color)b. DR5:: GFP (green fluorescence protein)• Plant is transformed with a. or b. construct.Gene is incorporated into genome and inherited.Progeny plants carry a or b.Figure 1 Auxin response during differential hypocotyl growth. a–d, Expression of the DR5::GUS reporter in hypocotyl of untreated (a, c) or auxin efflux inhibitor (NPA)-treated (b, d) wild-type seedlings upon stimulation by light (a, b) or gravity (c, d). Insets show details of DR5::GUS expression. Scale bars, 400 µm. e, Asymmetric expression of DR5::GUS reporter in the apical hook of an etiolated seedling. Scale bar, 50 µm. Friml J. et al 2002. NatureAuxin level changes in response to light and to gravity are due to auxin transport.Light- inhib + NPAGravity- inhib + NPA InhibFigure 2. pin3 mutants. b, c, pin3 hypocotyls are defective in gravitropic(b) as well as phototropic(c) responses. d, pin3 mutants are defective in root gravitropismFriml J. et al 2002. NatureEvidence auxin transport is critical for phototropismGenetic evidence:Mutants of auxin transportrespond poorly to light and to gravityAuxin levels can be changed by auxin efflux carriersPIN1 Basipetal auxin transport in the stemPIN2 Basipetal auxin transportPIN3 Lateral auxin efflux in gravitropic responsesPIN1-GFP is localized to base of cellPIN3-GFP is localized to lateral side of cellFriml, J. , Wisniewska, J. , Benkova, E. , Mendgen, K. & Palme, K. (2002) Nature 415, 806-809.Geldner N. 2001 Nature19-33 Taiz. Why do roots placed horizontally bend downwards?Gravitropism depends on distribution of hormones3Gravity changes auxin distributionDR5::GFP signal reflect auxin concBeforegravistimulationAfter 3 h19.15 In Arabidopsis, PIN transport proteins direct the flow of auxin (Part 2)19-30. Taiz. Root cap cells are important for sensing gravityamyloplastQ: What is the sensor for gravity?19-30. Taiz. Gravity may be sensed by a root cap cell according to the distribution of starch-storing plastids Mode of Action of Hormones1. Signal perception by a receptor in/on target cell2. Signal transduction and amplification3. Responses: a. molecular-early gene expression-late gene expressionproteins activatedrepressor removedb. Cellular & Physiological responseAuxinStimulate cell elongationHow? Two modes of actiona. Stimulates H+efflux (Membrane protein trafficking )b. Cause changes in gene expressionRemoval of repressors415-21. Taiz. What causes cell elongation?Acid pH causes wall extension.15-22. Wall protein expansins are required for acid growth.19-23. Auxin induces H+extrusion and increases cell extensionHow does auxin stimulate cell elongation?Acid growth theory•Auxin induce H+extrusion•Ion uptake --> osmotic water uptake•Turgor pressure builds up•Wall loosens, allowing cell wall to expandHow does auxin stimulate H+ extrusion?Hypotheses:•Activate the PM H+-pumping ATPase•Increase synthesis of new PM H+pumps19-18. Model of auxin-induced H+ extrusion and cell expansion 19-31. Auxin also induced expression of specific mRNAs519-31 Many SAUR (small auxin upregulated RNA) are transcription factorsAuxin induces expression of early and late genesEarly genes- [primary response genes]expressed in 5-60 min after adding auxininclude transcription factorshave roles in intercellular communicationstress adaptationLate Genes [secondary response genes]target genesAuxin receptor identified in 2005.TIR1 receptorAuxin binds to TIR1 complex.Auxin activates TIR1 (F-box protein)TIR1 complex targets the degradation of IAA repressor proteins.Removal of repressor activates transcription of early genes.19.40 The auxin receptorModel for auxinbinding to TIR auxinreceptorRemoval of repressor protein by degradationAfterBinding of TIR1 to IAA7 depends on auxin conc• Plant (expressing TIR1-myc) extract + GST-IAA7 (repressor)+ 0 - 50 auxin• GST-IAA7 will pull down X if it binds to IAA7.• Bound protein identified by SDS-PAGE & Immunoblotwith anti-myc is TIR1•3H-IAA interacts with TIR1 complex.•Crude extracts (TIR1-myc) + GST-IAA7 + 3H-IAA•pull-down & count cpm (a) •Competed by IAA, 2,4-D, NAA (b)Dharmasiri et al 2005 Nature6Where is TIR1 localized?• GFP-TIR1 DNA is expressed in tobacco leaf cells.Located to nucleus• 4 related F-box proteins in Arabidopsis are auxinreceptors. • So quadraple mutants were made to knock out all 4 proteins.Dharmasiri et al 2005 Dev CellAuxin-responsive gene expression is severely reduced in tir1 mutantSeedling growth affected in tir1 (4 genes) mutantsRoot tip in Wt (col, Ws), and in mutantsTIR1 and AFB genes act collectivelyWt-col Ws 2x mut 3x 4xSpecificity in IAAs and ARFs• 23 ARFs, and 29 Aux/IAA proteins•E.g.• Root & Hypocotyl growth, ARF2, 7, 8, 19• Tropism, ARF 2, 7, 19• Embryogenesis, ARF5•flowerSummary: Auxin action• 1. Increase H+ pumping• 2. Change gene expressionAuxin binding to TIR receptor stimulated