Auxin

1. Auxin • Growth • Phototropism and gravitropism • Branching • Embryonic patterning • Stem cell maintenance • Organ initiation Indole-3-acetic acid (IAA), the most abundant natural auxin

2. Auxin controls growth Charles Darwin studied the way seedlings bend towards light, a direct effect of auxin action. Site of signal perception Site of response Coleoptile drawing from Darwin, C., and Darwin, F. (1881) The power of movement in plants. Available online.

3. Darwin concluded that a signal moves through the plant controlling growth “We must therefore conclude that when seedlings are freely exposed to a lateral light some influence is transmitted from the upper to the lower part, causing the latter to bend.” Coleoptile drawing from Darwin, C., and Darwin, F. (1881) The power of movement in plants. Available online. Photograph of Darin statue by Patche99z

4. Differential cell growth is a result of auxin movement to the shaded side Auxin accumulation on shaded side stimulates elongation and bending. Cell length Auxin concentration Esmon, C.A. et al. (2006) A gradient of auxin and auxin-dependent transcription precedes tropic growth responses. Proc. Natl. Acad. Sci. USA 103: 236–241. Friml, J., et al. (2002) Lateral relocation of auxin efflux regulator PIN3 mediates tropism in Arabidopsis. Nature 415: 806-809.

5. Repositioning the tip can induce bending in uniform light Asymmetric tip placement causes bending Tip replaced only on one side Tip removed Tip replaced Control Paal (1919) showed that removing the tip and replacing it on one side of the base is sufficient to cause the bending Before After

6. In the 1930s, auxin was purified and shown to promote growth Angle of curvature is proportional to amount of auxin in block Frits Went collected auxin from shoot tips into agar blocks... ...and showed that the material collected in the agar blocks was the growth-promoting substance. This bending assay for the growth-promoting effect of auxin was used as a basis for its purification. Indole-3-acetic acid, IAA Redrawn from Went, F.W. (1935) Auxin, the plant growth-hormone. Bot. Rev. 1: 162-182.

7. Auxin biosynthesis IAA is produced from tryptophan (Trp) via several semi-independent pathways and one Trp-independent pathway. v Environmental and developmental control of the genes controlling auxin biosynthesis, conjugation and degradation maintain auxin homeostasis. Mano, Y. et al (2010) The AMI1 gene family: indole-3-acetamide hydrolase functions in auxin biosynthesis in plants J Exp Bot 61: 25-32, by permission of Oxford University Press.

8. Auxin moves in part by a chemiosmotic mechanism Cell wall pH 5.5 Cytoplasm pH 7 Auxin is a charged anion (IAA-) in the cytoplasm (pH 7) IAA- IAAH IAA- + H+ In the more acidic cell wall (pH 5.5) some is uncharged (IAAH). The uncharged form crosses the plasma membrane into the cell where it is deprotonated and unable to exit other than through specific transporters IAAH IAA- + H+ Redrawn from Robert, H.S., and Friml, J. (2009) Auxin and other signals on the move in plants. Nat. Chem. Biol. 5: 325-332.

9. Auxin transport is polarized Auxin transport out of cells is controlled by three families of transport proteins that collectively control the directionality of auxin movement. Asymmetric distribution of the transporters controls polar auxintransport Cell wall pH 5.5 Cytoplasm pH 7 IAA- IAAH PIN1 is responsible for auxin flow from shoot apex to root apex IAA- + H+ IAAH IAA- + H+ PIN1 localizes to the lower surface of root cortex cells Net flow of auxin Redrawn from Robert, H.S., and Friml, J. (2009) Auxin and other signals on the move in plants. Nat. Chem. Biol. 5: 325-332.

10. Auxin controls plant development Promotes and specify lateral organ initiation at the shoot apical meristem Inhibits branching in the shoot Controls patterning and vascular development Maintains stem cell fate at the root apical meristem Promotes branching in the root Wolters, H., and Jürgens, G. (2009). Survival of the flexible: Hormonal growth control and adaptation in plant development. Nat. Rev. Genet. 10: 305–317.

11. Many of auxin’s effects are mediated by changes in gene expression Genes controlling cell growth Genes involved in signaling Genes coordinating other hormone response pathways

12. Auxin-induced changes in gene expression Low Auxin ARF Aux/IAA In low-auxin conditions, auxin responsive factors (ARFs) are complexed with Aux/IAA proteins, which repress their transcriptional activity

13. In high auxin, the TIR1 auxin receptor binds Aux/IAA proteins Low Auxin High Auxin TIR1 ARF ARF Aux/IAA Aux/IAA Auxin Auxin TIR1 TIR1

14. TIR1 is an F-box protein, part of the SCF ubiquitin ligase complex F-box/ TIR1 Aux/IAA Auxin Auxin TIR1 TIR1 TIR1 TIR1 ubiquitin F-box/ TIR1 SCF complex

15. Auxin binding to TIR1 initiates proteolysis of Aux/IAA proteins Ub ARF 26S proteasome Aux/IAA Removal of the Aux/IAA repressors allows ARFs to dimerizeand activatetranscription F-box/ TIR1 + Auxin Growth promotion Similar to DELLA degradation induced by binding of GA to its receptor, GID1

16. Cytokinins (CKs) • Cell division • Control of leaf senescence • Control of nutrient allocation • Root nodule development • Stem cell maintenance • Regulate auxin action trans-zeatin, a cytokinin

17. Cytokinins are a family of related adenine-like compounds Isopentenyl adenine trans-zeatin dihydrozeatin cis-zeatin Hirose, N., Takei, K., Kuroha, T., Kamada-Nobusada, T., Hayashi, H., and Sakakibara, H. (2008). Regulation of cytokinin biosynthesis, compartmentalization and translocation. J. Exp. Bot. 59: 75–83.

18. Cytokinin (CK) biosynthesis Regulated by CK and nitrogen Tissue specific; auxin, CK and ABA sensitive Adenosine phosphate-isopentenyltransferase Meristem specific Cytokininriboside 5'-monophosphate phosphoribohydrolase (LOG) = CK activating enzyme Active form Inactive form CKX Upregulated by CK and ABA cytokininoxidase/dehydrogenase = irreversible CK inactivation CK biosynthesis and inactivation are strongly regulated by CK, other hormones and exogenous factors Hirose, N., Takei, K., Kuroha, T., Kamada-Nobusada, T., Hayashi, H., and Sakakibara, H. (2008). Regulation of cytokinin biosynthesis, compartmentalization and translocation. J. Exp. Bot. 59: 75–83.

19. CK act antagonistically to auxins Promotes and specify lateral organ initiation at the shoot apical meristem Inhibits branching in the shoot Auxin Promotes stem cell fate at the shoot apical meristem Promotes branching in the shoot CK Maintains stem cell fate at the root apical meristem Promotes branching in the root Inhibits branching in the shoot Promotes differentiation at the root apical meristem Wolters, H., and Jürgens, G. (2009). Survival of the flexible: Hormonal growth control and adaptation in plant development. Nat. Rev. Genet. 10: 305–317.

20. Auxin and cytokinin regulate each other’s function at the root apex Through effects on each other’s synthesis, transport and response, auxin and cytokinin establish two mutually exclusive domains that coordinate cellular activities at the root apex. Auxin transport Cytokinin Cell differentiation Auxin transport and response Cytokinin biosynthesis Cell division Auxin

21. Auxin, cytokinin and strigolactones control branching Root branches, called lateral roots, are promoted by auxin and inhibited by CK Shoot branches are promoted by CK and strigolactones and inhibited by auxin Branching controls every aspect of plant productivity from nutrient uptake to crop yields. Coleus shoot image by Judy Jernstedt, BSA ; lateral root image from Casimiro, I., et al. (2001) Auxin transport promotes Arabidopsis lateral root initiation. Plant Cell 13: 843-852.

22. Agrobacterium tumefaciens’ T-DNA encode CK T-DNA A lowerdose of CK producesthe typicaltumorcaused by Agrobacteriumtumefaciens

23. Cytokinin signaling is mediated by a two-component system Input domain Transmitter domain Receiver domain Output domain H D Histidine Kinase Response Regulator A two-component system is a short signaling pathway that moves information form an input to an output. In bacteria it usually consists of two proteins, a histidine kinase (HK) with a conserved histidine residue (H) and a response regulator (RR) with a conserved aspartate residue (D).

24. Information and phosphoryl groups are relayed between the components ATP ADP P P H D Histidine Kinase Response Regulator Stimulation of the input domain activates the kinase activity, phosphorylating the transmitter domain. The phosphoryl group is subsequently relayed to the response regulator.

25. Two component signaling in plants D H D Hybrid histidine kinase H Histidine phosphotransferase (HPt) Response regulator Most plant HKs are hybrid histidine kinase, which transfer the phosphoryl group to a histidine phosphotransferase. The HPt transfers it to a response regulator.

26. Cytokinin control of gene expression in Arabidopsis Plasma membrane H H Type B Arabidopsis response regulators (ARRs) are transcription factors Receptor D D H HPt Type A or C ARRs interfere with CK signaling through as yet unknown means. D A-ARR or C-ARR D ? B-ARR Transcription

27. Ethylene • Control of fruit ripening • Control of leaf and petal senescence • Control of cell division and cell elongation • Sex determination in some plants • Control of root growth • Stress responses H H C C H H

28. Ethylene promotes senescence of leaves and petals Air (control) 7 days ethylene In gas-lit houses, plants were harmed by the ethylene produced from burning gas. Aspidistra is ethylene- resistant and so became a popular houseplant! Ethylene promotes leaf and petal senescence. Cotton plants Beyer, Jr., E.M. (1976) A potent inhibitor of ethylene action in plants. Plant Physiol. 58: 268-271.

29. Ethylene shortens the longevity of cut flowers and fruits Ethylene levels can be managed to maintain fruit freshness, commercially and at home. Old strategies to limit ethylene effects: Limit production - high CO2 or low O2 Removal from the air -KMnO4 reaction, zeolite absorption Newer Strategies to limit ethylene effects: Interfere with ethylene binding to receptor – sodium thiosulfate (STS), diazocyclopentadiene (DACP), others Reprinted from Serek, M., Woltering, E.J., Sisler, E.C., Frello, S., and Sriskandarajah, S. (2006) Controlling ethylene responses in flowers at the receptor level. Biotech. Adv. 24: 368-381 with permission from Elsevier.

30. Molecular genetic approaches can limit ethylene synthesis ACC synthase ACC oxidase ACC S-adenosyl methionine Ethylene (1-aminocyclopropane-1-carboxylic acid) Antisense ACC synthase Introduction of antisense constructs to interfere with expression of biosynthesis enzymes is an effective way to control ethylene production. H H C C H H Control Theologis, A., Zarembinski, T.I., Oeller, P.W., Liang, X., and Abel, S. (1992) Modification of fruit ripening by suppressing gene expression. Plant Phys. 100: 549-551.

31. Ethylene-regulated gene expression is negatively regulated Ethylene Air Air In the absence of ethylene, CTR kinases bind the receptor and prevents transcription. Receptor CTR Ethylene binding to the receptor releases CTR, which self-inactivates, permitting transcription. Benavente, L.M., and Alonso, J.M. (2006) Molecular mechanisms of ethylene signaling in Arabidopsis. Mol. BioSyst. 2: 165–173. Reproduced by permission of The Royal Society of Chemistry (RSC) for the European Society for Photobiology, the European Photochemistry Association, and the RSC. Diagram adapted from Cuo, H., and Ecker, J.R. (2004) The ethylene signaling pathway: new insights. Curr. Opin. Plant Biol. 7: 40-49.

32. Ethylene perception mutants interfere with ripening Air or Ethylene Wild type Several mutations that affect ethylene perception and signaling interfere with fruit ripening. Receptor Green-ripe Never-ripe2 CTR Never-ripe Barry, C. S., et al. (2005) Ethylene insensitivity conferred by the Green-ripe and Never-ripe 2 ripening mutants of tomato. Plant Physiol. 138: 267-275.

33. Hormonal responses to biotic stress Bacteria, fungi, viruses – Biotrophic organisms Jasmonic Acid Herbivores – insects, other animals, fungi – Necrotrophic organisms Salicylic Acid Photo credits: A. Collmer, Cornell University; Salzbrot.

34. Salicylic Acid – plant hormone and painkiller • Response to biotrophic pathogens • Induced defense response • Systemic acquired resistance Salicylic Acid Salicylic acid is named for the willow Salix whose analgesic properties were known long before the chemical was isolated. Acetylsalicylic Acid = aspirin

35. Synthesis of SA There are at least two pathways for SA synthesis. The blue-highlighted pathway involves the key enzyme isochorismate synthase (ICS), which is induced by pathogen infection Reprinted from Métraux, J.P. (2002) Recent breakthroughs in the study of salicylic acid biosynthesis. Trends Plant Sci. 7: 332-334, with permission from Elsevier.

36. Plants recognize PAMPS (pathogen-associated molecular patterns) Flagellin is a conserved bacterial protein recognized by plants, also known as a pathogen-associated molecular pattern (PAMP). PAMP-triggered immunity SA Recognition of PAMPs by a plant cell triggers a set of immune responses known as PAMP-triggered Immunity (PTI) mediated by salicylic acid Immune Responses Flagellin monomers Redrawn from Pieterse, C.M.J, Leon-Reyes, A., Van der Ent, S., and Saskia C M Van Wees, S.C.M. (2009) Nat. Chem. Biol. 5: 308 – 316.

37. Some pathogens elicit a stronger defense response Specific bacterial proteins (effectors) delivered by the pathogens inhibit PTI Effector-triggered immunity Plants express resistance genes (R genes) that recognize specific bacterial effectors The interaction of an R protein with an effector protein promotes a stronger immune response, including the Hypersensitive Response (HR), and SA is involved in transducing this signal R SA HR Immune Responses HR (celldeath) Redrawn from Pieterse, C.M.J, Leon-Reyes, A., Van der Ent, S., and Saskia C M Van Wees, S.C.M. (2009) Nat. Chem. Biol. 5: 308 – 316.

38. The hypersensitive response seals the pathogen in a “tomb” of dead cells HR No HR Without a hypersensitive response, the pathogen can multiply The HR kills the infected cells and cells surrounding them and prevents the pathogen from spreading Drawing credit Credit: Nicolle Rager Fuller, National Science Foundation; Photo reprinted by permission of Macmillan Publishers Ltd. Pruitt, R.E., Bowman, J.L., and Grossniklaus, U. (2003) Plant genetics: a decade of integration. Nat. Genet. 33: 294 – 304.

39. Systemic Acquired Resistance (SAR) SAR The infected site sends signals, methyl salicylic acid (MeSA) and azelaic acid (AzA) through the veins and through airborne compounds that prime other tissues, making them resistant to pathogen attack. This effect is called Systemic Acquired Resistance (SAR). SA SA MeSA MeSA MeSA AzA AzA Drawing based on Vlot, A.C., Klessig, D.F., and Park, S.-W. (2008). Systemic acquired resistance: The elusive signal(s). Curr. Opin. Plant Biol. 11: 436–442.

40. Jasmonic Acid • Response to necrotrophic pathogens • Induction of anti-herbivory responses • Production of herbivore-induced volatiles to prime other tissues and attract predatory insects

41. JA biosynthesis occurs in 3cellular compartments Cytoplasm JAR1 conjugation JA-ILE JA conjugated to isoleucine (JA-ILE) is the active compound From Acosta, I., et al. (2009) tasselseed1 is a lipoxygenase affecting jasmonic acid signaling in sex determination of maize. Science 323: 262 – 265. Reprinted with permission from AAAS.

42. Jasmonic Acid signaling contributes to defense against herbivory WTMutant without JA When exposed to hungry fly larvae, plants unable to produce JA have low rates of survival. McConn, M., et al. (1997) Jasmonate is essential for insect defense in Arabidopsis. Proc. Natl. Acad. Sci. USA 94: 5473-5477.

43. JA induces the expression of anti-herbivorous chemicals including protease inhibitors Wound-induced signals Insect oral secretions Protease inhibitors Feeding deterants R.J. Reynolds Tobacco Company Slide Set and R.J. Reynolds Tobacco Company, Bugworld.org

44. JA also stimulates production of volatile signalling compounds Herbivore-induced volatiles prime other tissues (and other plants) for attack making them unpalatable (indicated in red). Reprinted from Matsui, K. (2006) Green leaf volatiles: hydroperoxidelyase pathway of oxylipin metabolism. Curr. Opin. Plant Biol. 9: 274-280, with permission from Elsevier.

45. Herbivore-induced volatiles are recognized by carnivorous and parasitoid insects Tim Haye, Universität Kiel, Germany Bugwood.org; R.J. Reynolds Tobacco Company Slide Set and R.J. Reynolds Tobacco Company, Bugworld.org

46. JA-induced changes in gene expression Low JA-Ile High JA-Ile Defense genes JA-responsive transcription factor JAZ protein JA-Ile No transcription F-box protein receptor (COI1) Transcription Proteolysis

47. Crosstalk between hormone signaling pathways H1 H1 H2 H1 H2 Response Response Crosstalk (or cross-regulation) occurs when two pathways are not independent. It can be positive and additive or synergistic, or negative.

48. Crosstalk between hormone signaling pathways H1 H1 H1 H2 H2 H2 H1 H2 Response Response Response Crosstalk can affect the synthesis, transport or signaling pathway of another hormone. Crosstalk (or cross-regulation) occurs when two pathways are not independent. It can be positive and additive or synergistic, or negative.

49. Synergistic requirement for JA and ET signaling in defense response JA and ET signaling are both required for high-level expression of ERF1, a TF that induces defense gene expression NO JA response NO ET response JA Defense genes ERF1 ERF1 and ET Lorenzo, O., Piqueras, R., Sánchez-Serrano, J.J., and Solano, R. (2003) ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defense. Plant Cell 15: 165-178.

50. Negative interaction between JA and SA in defense responses In defense signaling, the JA and SA pathways are mutually antagonistic (locally), and both are antagonized by ABA. Why does ABA reduce SA and JA signaling? Perhaps a plant that is already stressed and producing high levels of ABA may be better off temporarily restricting its responses to pathogens. Reprinted from Spoel, S.H.,  and Dong, X. (2008) Making sense of hormone crosstalk during plant immune responses. Cell Host Microbe 3: 348-351 with permission from Elsevier.