JASMONATES American Society of Plant Biologists

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1 JASMONATES

2 Plants are surrounded by many enemies with a common goal to eat them! Insects Bacteria Nematodes Fungi Oomycetes Photo by Gilbert Ahlstrand courtesy USDA, Photo by Eric Erbe, digital colorization by Christopher Pooley, D'Arcy, C. J., D. M. Eastburn, and G. L. Schumann Illustrated Glossary of Plant Pathology. The Plant Health Instructor

3 Pathogens and pests cause significant crop losses Most plants are resistant to most pests, but a few organisms cause tremendous damage. 25% or more of potential harvests can be lost to insects and disease! Jasmonates and salicylates are hormones that participate in plant defense responses Phytophthora capsici on cucumber (Cucumis sativus) European corn borer Ostrinia nubilalis in its host Zea mays Images courtesy Clemson University - USDA Cooperative Extension Slide Series, Bugwood.org; Charles Averre, North Carolina State University, Bugwood.org

4 Microorganisms are (hemi)biotrophic or necrotrophic Biotrophs or hemibiotrophs can live within their host tissue without causing (immediate) death Cell death accompanies or precedes colonization by necrotrophs Toxin Pseudomonas syringae in intracellular space Bestwick, C.S., Brown, I.R., Bennett, M., and Mansfield, J.W. (1997). Localization of hydrogen peroxide accumulation during the hypersensitive reaction of lettuce cells to Pseudomonas syringae pv phaseolicola. Plant Cell 9:

5 Insects eat leaves, flowers, seeds and roots and spread diseases Leaf beetle (Phratora laticollis) on European aspen (Populus tremula) Boll weevil (Anthonomus grandis grandis) on cotton (Gossypium hirsutum) Image credits: Petr Kapitola, State Phytosanitary Administration, Bugwood.org ; Alton N. Sparks, Jr., University of Georgia, Bugwood.org

6 Jasmonates participate in plant defenses to insects and necrotrophs To a first approximation, insects and necrotrophs trigger jasmonate production, and biotrophs trigger salicylate production Jasmonates Transcriptional responses Salicylates

7 Jasmonates also contribute to developmental and growth controls Flower development Seed development Trichome formation JA also controls: cell cycle, root extension, leaf senescence, stomata closure, and mutualistic interactions... Li, L., Zhao, Y., McCaig, B.C., Wingerd, B.A., Wang, J., Whalon, M.E., Pichersky, E., and Howe, G.A. (2004). The tomato homolog of CORONATINE-INSENSITIVE1 is required for the maternal control of seed maturation, jasmonate-signaled defense responses, and glandular trichome development. Plant Cell 16: ; Reprinted by permission from Macmillan Publishers Ltd. Thines, B., Katsir, L., Melotto, M., Niu, Y., Mandaokar, A., Liu, G., Nomura, K., He, S.Y., Howe, G.A., and Browse, J. (2007). JAZ repressor proteins are targets of the SCFCOI1 complex during jasmonate signalling. Nature 448:

8 Lecture outline Jasmonate biochemistry Jasmonate signaling: COI1- JAZ coreceptor JAZ repressors Transcriptional responses Jasmonates in whole-plant processes: Responses to insects Responses to pathogens and other microorganisms Cross-talk in defense signaling Jasmonates in development and other functions

9 Jasmonates synthesis, conjugation, transport and degradation Like other esters, methyl jasmonate smells good it is the dominant scent from jasmine flowers CH 2 COOCH 3 O Jasmonate de méthyle Methyl jasmonate was purified from Jasminum grandiflorium in 1962 Demole, E. Lederer, E., and Mercier, D. (1962) Isolement et détermination de la structure du jasmonate de méthyle, constituant odorant charactéristique de l èssence de jasmin. Helv. Chim. Acta 45:

10 Jasmonates include jasmonic acid (JA) and derivatives Jasmonic acid (JA) JA-Isoleucine (JA-Ile) OH (3R,7S)-jasmonic acid a.k.a. (+)-7-iso-jasmonic acid JA-Ile Methyl jasmonate (MeJA) OCH 3 Yan, J., Zhang, C., Gu, M., Bai, Z., Zhang, W., Qi, T., Cheng, Z., Peng, W., Luo, H., Nan, F., Wang, Z., and Xie, D. (2009). The Arabidopsis CORONATINE INSENSITIVE1 protein Is a jasmonate receptor. Plant Cell 21:

11 Jasmonates are produced preventively in flowers and induced as a defense response in other tissues Wounding, pathogens or herbivores (or molecules derived from them) trigger very rapid accumulation of jasmonates MeJA JA Untreated control Induction of jasmonate production in cell suspension culture (Rauvolfia canescens, a medicinal plant) by adding yeast cell wall elicitor Rauvolfia canescens Gundlach, H., Müller, M.J., Kutchan, T.M., and Zenk, M.H. (1992). Jasmonic acid is a signal transducer in elicitor-induced plant cell cultures. Proc. Natl. Acad. Sci. 89: (Photo from Wikipedia)

12 JA accumulates in a circadian pattern in phase with herbivory Herbivory Hormone accumulation JA and SA cycle in opposite phasing JA SA Resistance to herbivory decreases when Arabidopsis cycles out-of-phase to the insects Reprinted from Goodspeed, D., Chehab, E.W., Min-Venditti, A., Braam, J., Covington, M.F. (2012). Arabidopsis synchronizes jasmonate-mediated defense with insect circadian behavior. Proc. Natl. Acad. Sci. USA 109:

13 Jasmonate synthesis occurs in the plastid, peroxisome and cytosol Lipase α-linolenic acid (18:3) (in membrane) LOX AOS AOC OPDA OPDA OPR3 β-oxidation ACX1 plastid JA peroxisome JA-Ile MeJA cytosol

14 Jasmonate precursors are derived from membrane lipids Membrane lipid Lipase α-linolenic acid Free fatty acid MEMBRANE Lipolytic enzymes are necessary for JA synthesis and are under tight regulation DAD1, DGL, GLA1 Free fatty acid Ishiguro, S., Kawai-Oda, A., Ueda, J., Nishida, I., and Okada, K. (2001). The DEFECTIVE IN ANTHER DEHISCENCE1 gene encodes a novel phospholipase A1 catalyzing the initial step of jasmonic acid biosynthesis, which synchronizes pollen maturation, anther dehiscence, and flower opening in Arabidopsis. Plant Cell 13:

15 In Arabidopsis DAD1 and DGL lipases are expressed in different tissues DAD1 is expressed in anthers and is necessary for male fertility In Arabidopsis the DGL lipase is expressed in rosette leaves and involved in wound response Ishiguro, S., Kawai-Oda, A., Ueda, J., Nishida, I., and Okada, K. (2001). The DEFECTIVE IN ANTHER DEHISCENCE1 gene encodes a novel phospholipase A1 catalyzing the initial step of jasmonic acid biosynthesis, which synchronizes pollen maturation, anther dehiscence, and flower opening in Arabidopsis. Plant Cell 13: ; Reprinted from Hyun, Y., Choi, S., Hwang, H.-J., Yu, J., Nam, S.-J., Ko, J., Park, J.-Y., Seo, Y.S., Kim, E.Y., Ryu, S.B., Kim, W.T., Lee, Y.-H., Kang, H., and Lee, I. (2008). Cooperation and functional diversification of two closely related galactolipase genes for jasmonate biosynthesis. Developmental Cell 14: with permission from Elsevier.

16 Jasmonates are derived from free fatty acids Linolenic acid has 18 carbons and 3 double bonds so is called an 18:3 octadecanoid Another way to draw linolenic acid

17 LOX is a lipoxygenase Molecular oxygen is added to the C13 position by 13- lipoxygenase (LOX) Acosta I. F., and Farmer E. E. (2008). Jasmonates. In The Arabidopsis Book (The American Society of Plant Biologists), pp

18 Allene oxide synthase (AOS) catalyzes the first committed step Allene oxide synthase (AOS) dehydrates 13- HPOT to form an unstable allene epoxide 13-HPOT is 13(S)- hydroxyperoxyoctadecatrienoic acid Acosta I. F., and Farmer E. E. (2008). Jasmonates. In The Arabidopsis Book (The American Society of Plant Biologists), pp

19 Allene oxide cyclase (AOC) defines the stereochemistry The unstable epoxide spontaneously forms a mixture of cis and trans OPDA BUT, in the presence of AOC it forms only the cis form. Acosta I. F., and Farmer E. E. (2008). Jasmonates. In The Arabidopsis Book (The American Society of Plant Biologists), pp

20 A parallel set of reactions starts with a 16-carbon fatty acid O O COOH COOH cis-(+)-opda dnopda These reactions take place in plastids Reprinted from Schaller, A., and Stintzi, A. (2009). Enzymes in jasmonate biosynthesis - Structure, function, regulation. Phytochemistry 70: with permission from Elsevier.

21 OPDA and dnopda move into the peroxisome through a transporter O O COOH COOH cis-(+)-opda dnopda The subsequent reactions take place in the peroxisome CTS / PXA1 Reprinted from Schaller, A., and Stintzi, A. (2009). Enzymes in jasmonate biosynthesis - Structure, function, regulation. Phytochemistry 70: with permission from Elsevier.

22 In the peroxisome, OPDA is first reduced and then β-oxidized OPDA reductase (OPR3) β-oxidation is a multistep process whose net result is the removal of two carbon groups Three cycles of β- oxidation shorten the 18 carbon OPDA to the 12 carbon jasmonic acid Vick, B.A., and Zimmerman, D.C. (1984). Biosynthesis of jasmonic acid by several plant species. Plant Physiol. 75:

23 OPCL1 OPC-8:CoA ligase 1 β-oxidation occurs by the action of three enzymes Acyl-coenzyme A oxidase (ACX) Multifunctional protein (MFP) 2-trans-enoyl-CoA hydratase Multifunctional protein (MFP) 1-3-hydroxyacyl-CoA dehydrogenase Repeat to shorten chain by two more carbons 3-ketoacyl-CoA thiolase (KAT) Li, C., Schilmiller, A.L., Liu, G., Lee, G.I., Jayanty, S., Sageman, C., Vrebalov, J., Giovannoni, J.J., Yagi, K., Kobayashi, Y., and Howe, G.A. (2005). Role of β-oxidation in jasmonate biosynthesis and systemic wound signaling in tomato. Plant Cell 17:

24 ACX1 acts during β-oxidation of JA biosynthesis in the peroxisome Expression in pollen of ACX1, encoding an enzyme required for jasmonate biosynthesis ACX1 YFP YFP Schilmiller, A.L., Koo, A.J.K. and Howe, G.A. (2007). Functional diversification of acyl-coenzyme A oxidases in jasmonic acid biosynthesis and action. Plant Physiol. 143:

25 Jasmonic acid is exported to the cytosol for further modification Lipase α-linolenic acid (18:3) (in membrane) LOX AOS AOC OPDA OPDA OPR3 β-oxidation ACX1 plastid JA peroxisome JA-Ile MeJA

26 Jasmonic acid can be conjugated to amino acids by JAR1 JA JAR1 JA-Ile OH isoleucine NH2 Active form JAR1 is an amino acid conjugase

27 JAR1 was identified as the jasmonate-insensitive mutant jar1 Arabidopsis jar1 mutants produce jasmonic acid but cannot make JA-Ile jar1 mutants are also called far-red insensitive 219 (fin219) due to their phenotype in seedlings (etiolation under far-red light) jar1 WT Root growth on MeJA is less inhibited in jar1 mutants. Staswick, P.E., Su, W., and Howell, S.H. (1992). Methyl jasmonate inhibition of root growth and induction of a leaf protein are decreased in an Arabidopsis thaliana mutant. Proc. Natl. Acad. Sci. USA 89: ; Staswick, P.E., and Tiryaki, I. (2004). The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine in Arabidopsis. Plant Cell 16:

28 Jasmonate can be reversibly esterified to MeJA O JA-methyl transferase (JMT) O COOH JA Methyl jasmonate esterase (MJE) COOCH 3 MeJA MeJA is volatile and may be produced for transport

29 MeJA production is developmentally controlled and wound-induced JMT expression peaks at anther dehiscence Increased MeJA production confers protection against necrotrophic pathogens JMT is induced by wounding or MeJA Seo, H.S., Song, J.T., Cheong, J.-J., Lee, Y.-H., Lee, Y.-W., Hwang, I., Lee, J.S., and Choi, Y.D. (2001). Jasmonic acid carboxyl methyltransferase: A key enzyme for jasmonate-regulated plant responses. Proc. Natl. Acad. Sci. USA 98:

30 There are other forms of jasmonate but their functions are not yet known JA introduced into cultured cells is incorporated into several compounds possibly for storage or degradation? Swiatek, A., Dongen, W.V., Esmans, E.L., and Onckelen, H.V. (2004). Metabolic Fate of Jasmonates in Tobacco Bright Yellow-2 Cells. Plant Physiol. 135:

31 WIPK and SIPK contribute to rapid activation of JA synthesis enzymes WIPK wound-induced protein kinase? wounding SIPK salicylic-acid induced protein kinase chloroplast? SIPK NPR1 WIPK These protein kinases might activate enzymes for very rapid JA production glycerolipids GLA 1 18:3 LOX 3 13(S)-OOH-18:3 Stroma AOS EOT AOC OPDA OPDA JA peroxisome Kallenbach, M., Alagna, F., Baldwin, I.T., and Bonaventure, G. (2010). Nicotiana attenuata SIPK, WIPK, NPR1, and fatty acid-amino acid conjugates participate in the induction of jasmonic acid biosynthesis by affecting early enzymatic steps in the pathway. Plant Physiol. 152: ; Bonaventure, G. and Baldwin, I.T. (2010) New insights into the early biochemical activation of jasmonic acid biosynthesis in leaves. Plant Signal Behaviour 5:

32 Cytochrome P450 inactivates JA-Ile Precursor Active Inactive Loss-of-function mutant cyp94b3-1 accumulates JA-Ile Cytochrome P450 CYP94B3 mediates catabolism and inactivation of JA-Ile Koo, A.J., Cooke, T.F., Howe, G.A. (2011) Cytochrome P450 CYP94B3 mediates catabolism and inactivation of the plant hormone jasmonoyl-l-isoleucine. Proc. Natl. Acad. Sci. USA 108:

33 Control of active hormone levels Lipases are developmentally and wound regulated AOS and AOC are wound-regulated (via a wound-induced protein kinase) AOS Lipase LOX * Jasmonate synthesis is developmentally regulated as well * OPDA * AOC OPDA Entry into peroxisome may be regulated JA-Ile JA * OPR3 β-oxidation MeJA JAR1 activity and JMT activity are upregulated by wounding Ishiguro, S., Kawai-Oda, A., Ueda, J., Nishida, I., and Okada, K. (2001). The DEFECTIVE IN ANTHER DEHISCENCE1 gene encodes a novel phospholipase A1 catalyzing the initial step of jasmonic acid biosynthesis, which synchronizes pollen maturation, anther dehiscence, and flower opening in Arabidopsis. Plant Cell 13:

34 Synthesis, conjugation, transport and degradation - summary Jasmonate accumulation is developmentally regulated and stimulated by wounding, some insects and some pathogens Jasmonates are oxylipins, derived from the oxidation of free fatty acids Jasmonate synthesis occurs in plastids and peroxisomes. Conjugation reactions occur in the cytosol JA-Ile is the most active compound whereas MeJA may be a transport form JA-Ile is degraded by cytochrome P450 oxidases

35 Perception and signaling JA-Ile binding by the COI1-JAZ coreceptor Ubiquitination and degradation of JAZ Transcriptional activation by MYC2 and others JAZ Z MYC2 BD AD J SCF COI1 JA- Ile MYC2 BD AD Gene expression

36 Coronatine is a bacterial compound and powerful jasmonate mimic MeJA Coronatine is a toxin produced by some pathogenic bacteria that mimics jasmonate action and structurally resembles JA-Ile COR Control MeJA or coronatine (COR) induce defense compounds in cultured cells of California poppy Coronatine (3R,7S)-JA-Ile Reprinted from Weiler, E.W., Kutchan, T.M., Gorba, T., Brodschelm, W., Niesel, U., and Bublitz, F. (1994). The Pseudomonas phytotoxin coronatine mimics octadecanoid signalling molecules of higher plants. FEBS Letters 345: 9-13 with permission from Elsevier. Yan, J., Zhang, C., Gu, M., Bai, Z., Zhang, W., Qi, T., Cheng, Z., Peng, W., Luo, H., Nan, F., Wang, Z., and Xie, D. (2009). The Arabidopsis CORONATINE INSENSITIVE1 protein Is a jasmonate receptor. Plant Cell 21:

37 The Arabidopsis mutant coi1 is insensitive to coronatine and MeJA WT coi1 coi1 MeJA coi1 WT WT Control COR Feys, B., Benedetti, C.E., Penfold, C.N., and Turner, J.G. (1994). Arabidopsis mutants selected for resistance to the phytotoxin coronatine are male sterile, insensitive to methyl jasmonate, and resistant to a bacterial pathogen. Plant Cell 6:

38 Tomato jai1 mutant is also deficient in jasmonate responses MeJA-resistant Deficient in JA-induced transcription JAI1 is the tomato orthologue of COI1 Insect sensitive Li, L., Zhao, Y., McCaig, B.C., Wingerd, B.A., Wang, J., Whalon, M.E., Pichersky, E., and Howe, G.A. (2004). The tomato homolog of CORONATINE-INSENSITIVE1 is required for the maternal control of seed maturation, jasmonate-signaled defense responses, and glandular trichome development. Plant Cell 16:

39 Map-based cloning and a functional assay were used to clone COI1 Functional assay for COI1: COI1 activity is needed for JAor wound-induced activation of PThi2.1-GUS reporter construct. (Thionins are JA-induced defense proteins) From Xie, D.-X., Feys, B.F., James, S., Nieto-Rostro, M., and Turner, J.G. (1998). COI1: An Arabidopsis gene required for jasmonate-regulated defense and fertility. Science 280: Reprinted with permission from AAAS.

40 Genetic mapping led to two candidate genes and the functional assay showed which has COI1 activity YES YES YES NO Genetic complementation of coi1 mutant by COI1 gene From Xie, D.-X., Feys, B.F., James, S., Nieto-Rostro, M., and Turner, J.G. (1998). COI1: An Arabidopsis gene required for jasmonate-regulated defense and fertility. Science 280: Reprinted with permission from AAAS.

41 COI1 is closely related to TIR1 and other auxin receptors (AFB) F-box proteins COI1 protein family (jasmonate coreceptors) This similarity suggests a mode of action! TIR1 / AFB protein family (auxin receptors) Reprinted from Katsir, L., Chung, H.S., Koo, A.J.K., and Howe, G.A. (2008). Jasmonate signaling: a conserved mechanism of hormone sensing. Curr. Opin. Plant Biol. 11: , with permission from Elsevier. Reprinted from Chico, J.M., Chini, A., Fonseca, S., and Solano, R. (2008). JAZ repressors set the rhythm in jasmonate signaling. Curr. Opin. Plant Biol. 11: with permission from Elsevier.

42 The jasmonate receptor consists of COI1 and JAZ co-receptors COI1 JA- Ile The COI1-JAZ co-receptor has > 100 fold greater affinity for the ligand than either COI1 or JAZ alone Reprinted by permission from Macmillan Publishers Ltd. Nature: Sheard, L.B., Tan, X., Mao, H., Withers, J., Ben-Nissan, G., Hinds, T.R., Kobayashi, Y., Hsu, F.-F., Sharon, M., Browse, J., He, S.Y., Rizo, J., Howe, G.A., and Zheng, N. (2010) Jasmonate perception by inositol-phosphate-potentiated COI1-JAZ co-receptor. Nature 468: copyright 2010.

43 COI1 is a component of the SCF ubiquitin ligase complex SCF COI1 COI1 F-box protein SKP1 F-box COI1 protein SCF complex SCF ubiquitin ligase complex (Named for SKP1, CUL1 and F-box proteins) CUL1 (The auxin receptor TIR1 is a component of the SCF TIR1 complex)

44 Ubiquitin ligase complexes ubiquitinate target proteins Ubiquitin The F-box protein recognizes F-box protein Target Target and binds to the target protein. The complex then transfers SKP1 ubiquitin proteins to the target CUL1

45 Proteolytic targets are covalently linked to ubiquitin Target Ubiquitin Ubiquitin is a small (76 aa) protein that targets proteins for proteolytic degradation Ubiquitin by Rogerdodd

46 Ubiquitinated targets are proteolyzed by the 26S proteasome Target The proteasome breaks down target proteins and recycles ubiquitin 26S proteasome

47 Jasmonate (like auxin) signaling requires repressor degradation Aux/IAA ARF Repressor Transcriptional activator JAZ MYC2 TIR1 Auxin F-box protein hormone COI1 JA-Ile Aux/IAA Repressor JAZ + hormone In the absence of hormone, a repressor interferes with a transcriptional activator. The hormone promotes an interaction between the repressor and an F-box protein, leading to repressor degradation and transcriptional activation ARF Transcriptional activator MYC2

48 JAZ proteins are repressors of jasmonate signaling Reprinted from Chung, H.S., Niu, Y., Browse, J., and Howe, G.A. (2009). Top hits in contemporary JAZ: An update on jasmonate signaling. Phytochemistry 70: with permission from Elsevier.

49 JAZ proteins are rapidly degraded in the presence of jasmonates Protein stability was assayed in transgenic plants expressing JAZ-GFP or JAZ-GUS fusions Control JAZ-GFP + jasmonate Reprinted by permission from Macmillan Publishers Ltd. [Nature] Thines, B., Katsir, L., Melotto, M., Niu, Y., Mandaokar, A., Liu, G., Nomura, K., He, S.Y., Howe, G.A., and Browse, J. (2007). JAZ repressor proteins are targets of the SCFCOI1 complex during jasmonate signalling. Nature 448: and Chini, A., Fonseca, S., Fernandez, G., Adie, B., Chico, J.M., Lorenzo, O., Garcia-Casado, G., Lopez-Vidriero, I., Lozano, F.M., Ponce, M.R., Micol, J.L., and Solano, R. (2007). The JAZ family of repressors is the missing link in jasmonate signalling. Nature 448: copyright 2007.

50 JAZ genes are transcriptionally upregulated by jasmonates Many JAZ genes are rapidly induced by JA application Similarly, transcription of the Aux/IAA repressor genes are rapidly upregulated by auxin Reprinted by permission from Macmillan Publishers Ltd. [Nature] Thines, B., Katsir, L., Melotto, M., Niu, Y., Mandaokar, A., Liu, G., Nomura, K., He, S.Y., Howe, G.A., and Browse, J. (2007). JAZ repressor proteins are targets of the SCFCOI1 complex during jasmonate signalling. Nature 448:

51 JAZ proteins have conserved Jas and ZIM/TIFY domains The Jas domains facilitate interactions with COI1 and MYC2 proteins. The ZIM domains (also called TIFY domains) are dimerization domains for interactions with other JAZ proteins and NINJA Chung, H.S., and Howe, G.A. (2009). A critical role for the TIFY motif in repression of jasmonate signaling by a stabilized splice variant of the JASMONATE ZIMdomain protein JAZ10 in Arabidopsis. Plant Cell 21:

52 In the jai3-1 mutant, the JAI3 protein lacks a Jas domain A mutation changes the encoded protein so it lacks the Jas domain and no longer interacts with COI1. SCF COI1 JA- Ile Therefore it is not degraded and continues to repress JA responses including root inhibition by MeJA jasmonateinsensitive3 Reprinted by permission from Macmillan Publishers Ltd. [Nature] Chini, A., Fonseca, S., Fernandez, G., Adie, B., Chico, J.M., Lorenzo, O., Garcia-Casado, G., Lopez-Vidriero, I., Lozano, F.M., Ponce, M.R., Micol, J.L., and Solano, R. (2007). The JAZ family of repressors is the missing link in jasmonate signalling. Nature 448: copyright 2007.

53 Plants expressing stabilized JAZ are deficient in insect defenses Plants with stabilized JAZ proteins are deficient in defense responses. This is clearly seen by the growth rate of insects feeding on them! Chung, H.S., Koo, A.J.K., Gao, X., Jayanty, S., Thines, B., Jones, A.D., and Howe, G.A. (2008). Regulation and function of Arabidopsis JASMONATE ZIM-domain genes in response to wounding and herbivory. Plant Physiol. 146:

54 The JAZ ZIM domain facilitates interactions with NINJA NINJA = Novel Interactor of JAZ JAZ ZIM NINJA Jas And NINJA interacts with TOPLESS... Image by Hector Gomez

55 TOPLESS represses transcription in absence of hormones Aux/IAA ARF BD III/IV III/IV EAR TPL JAZ MYC2 AD BD Z J NINJA EAR TPL TOPLESS binds directly to the EAR domain of the Aux/IAA repressors TOPLESS binds indirectly to JAZ, through NINJA

56 Direct targets of JAZ and downstream regulation of transcription factors Reprinted from Kazan, K., Manners, J.M. (2011). JAZ repressors and the orchestration of phytohormone crosstalk. Trends Plant Sci. 17: 22-31, with permission from Elsevier.

57 The auxin signaling pathway Aux/IAA III/IV ARF III/IV BD EAR TPL SCF TIR1 IAA IAA Aux/IAA III/IV 1. Auxin binds to SCF TIR1 and Aux/IAA EAR

58 The auxin signaling pathway Aux/IAA III/IV ARF III/IV BD EAR TPL SCF TIR1 IAA 2. Aux/IAA ubiquitinated and degraded by 26S proteasome IAA 1. Auxin binds to SCF TIR1 and Aux/IAA

59 The auxin signaling pathway Aux/IAA III/IV ARF III/IV BD EAR TPL SCF TIR1 IAA 2. Aux/IAA ubiquitinated and degraded by 26S proteasome IAA 1. Auxin binds to SCF TIR1 and Aux/IAA ARF BD AD 3. Degradation of repressor permits transcriptional activation by ARF transcription factors

60 The jasmonate signaling pathway JAZ Z NINJA EAR TPL MYC2 BD AD J JA-Ile SCF COI1 JA- Ile 1.JA-Ile binds to SCF COI1 and JAZ protein

61 The jasmonate signaling pathway JAZ MYC2 AD BD Z J NINJA EAR TPL JA-Ile SCF COI1 2. JAZ ubiquitinated and degraded by 26S proteasome JA- Ile 1.JA-Ile binds to SCF COI1 and JAZ protein

62 The jasmonate signaling pathway JAZ MYC2 AD BD Z J NINJA EAR TPL JA-Ile SCF COI1 1.JA-Ile binds to SCF COI1 and JAZ protein MYC2 2. JAZ ubiquitinated and degraded by 26S proteasome JA- Ile BD AD 3. Degradation of repressor permits transcriptional activation by MYC2 transcription factors

63 jin1 is deficient in JA-induced transcription and encodes MYC2 MYC2 upregulates: JAZ genes AtVSP mrna LOX3 (JA synthesis) VSP2 (wound response) MYC2 MYC2 Berger, S., Bell, E., and Mullet, J.E. (1996). Two methyl jasmonate-insensitive mutants show altered expression of AtVsp in response to methyl jasmonate and wounding. Plant Physiol. 111: ; Lorenzo, O., Chico, J.M., Sanchez-Serrano, J.J., and Solano, R. (2004). JASMONATE-INSENSITIVE1 encodes a MYC transcription factor essential to discriminate between different jasmonate-regulated defense responses in Arabidopsis. Plant Cell 16:

64 MYC2 downregulates some pathogen-response genes MYC2 Lorenzo, O., Chico, J.M., Sanchez-Serrano, J.J., and Solano, R. (2004). JASMONATE-INSENSITIVE1 encodes a MYC transcription factor essential to discriminate between different jasmonate-regulated defense responses in Arabidopsis. Plant Cell 16:

65 JA-regulated genes can be positively or negatively regulated by ethylene PDF1.2 requires both JA and ethylene VSP1 is down regulated by ethylene Reprinted from Memelink, J. (2009). Regulation of gene expression by jasmonate hormones. Phytochemistry 70: with permission from Elsevier.See also Zhu, Z., et al., (2011). Derepression of ethylene-stabilized transcription factors (EIN3/EIL1) mediates jasmonate and ethylene signaling synergy in Arabidopsis. Proc. Natl. Acad. Sci. USA 108:

66 The Mediator complex is a key regulator of JA-dependent defense The Mediator complex acts as a bridge between RNA polymerase II and transcription factors and fine-tunes diverse regulatory inputs Kidd, B.N., Edgar, C.I., Kumar, K.K., Aitken, E.A., Schenk, P.M., Manners, J.M. and Kazan, K. (2009). The Mediator Complex subunit PFT1 is a key regulator of jasmonate-dependent defense in Arabidopsis. Plant Cell. 21:

67 MYC2 BD JAZ AD Core jasmonate signaling Z J NINJA There is also extensive cross-talk with other signaling pathways! EAR TPL summary JA-Ile SCF COI1 1.JA-Ile binds to SCF COI1 and JAZ protein MYC2 JA- Ile BD 2. JAZ ubiquitinated and degraded by 26S proteasome AD 3. Degradation of repressor permits transcriptional activation by MYC2 and other transcription factors

68 Jasmonates in whole-plant processes Responses to insects Responses to pathogens and other microorganisms Cross-talk between JA and SA pathways Jasmonates in development and other processes Mithofer, A., and Boland, W. (2008). Recognition of herbivory-associated molecular patterns. Plant Physiol. 146:

69 Plants and insects have shared a long period of co-evolution > 400 mya Image credit: L. Shyamal based on work by Bruce Tiffney

70 Plants are not passive victims Plants have constitutive and induced defenses Constitutive always present Chemical toxins, irritants (e.g. cyanogenic glycosides, alkaloids, tannins) Physical thorns, trichomes Induced responses can be direct or indirect Direct responses production of toxic or anti-nutritive compounds (e.g. proteinase inhibitors) Indirect responses volatile compounds recognized by carnivorous or parasitoid insects Linamarin, a cyanogenic glycoside from cassava Geocoris feeding on Manduca eggs, attracted by plant-emitted volatiles Trichome photo credit: NRC-CNRC (Harry Turner). Geocoris photo by M. Stitz from Allmann, S., and Baldwin, I.T. Insects betray themselves in nature to predators by rapid Isomerization of green leaf volatiles. Science 329: reprinted with permission from AAAS.

71 There are many kinds of arthropods and feeding behaviors Adult green peach aphid (Myzus persicae) feeding on a dill plant Beet armyworm (Spodoptera exigua) damage to sweet pepper (Capsicum annuum) S. littoralis caterpillar feeding on a barrel medic (Medicago truncatula) leaf Tomato leaf showing damage caused by a leaf miner Image credits: Syncroscopy; David Riley; N3v3rl4nd; Mithofer, A., and Boland, W. (2008). Recognition of herbivory-associated molecular patterns. Plant Physiol. 146:

72 Plants respond to oral secretions as well as mechanical damage Proteinase inhibitor gene induction is much faster when insect saliva or regurgitate is present Plants recognize insectderived compounds as well as mechanical damage Korth, K.L., and Dixon, R.A. (1997). Evidence for chewing insect-specific molecular events distinct from a general wound response in leaves. Plant Physiol. 115:

73 Plants produce proteinase inhibitors that deter and weaken herbivores Clarence Ryan ( ) was a pioneer in the study of plant responses to herbivorous insects In 1972, Green & Ryan found that plants produce proteinase inhibitors (PIs) in response to herbivory A four-fold increase in PIs was recorded after 24 hours Control plant Plant infested with herbivore Green, T.R., and Ryan, C.A. (1972). Wound-induced proteinase inhibitor in plant leaves: A possible defense mechanism against insects. Science 175: Photo courtesy of Washington State University and Scott Bauer, USDA

74 Plants produce proteinase inhibitors that interfere with digestion MUNCH MUNCH Normally food that enters the digestive system is broken down by digestive enzymes so the nutrients can be absorbed Green, T.R., and Ryan, C.A. (1972). Wound-induced proteinase inhibitor in plant leaves: A possible defense mechanism against insects. Science 175:

75 Plants produce proteinase inhibitors that interfere with digestion I feel sick Proteinase inhibitors (PIs) make insects sick or even die Green, T.R., and Ryan, C.A. (1972). Wound-induced proteinase inhibitor in plant leaves: A possible defense mechanism against insects. Science 175:

76 Jamonate signaling contributes to defenses against herbivory WT Mutant without JA When exposed to hungry fly larvae, plants unable to produce JA have low rates of survival McConn, M., Creelman, R.A., Bell, E., Mullet, J.E., and Browse, J. (1997). Jasmonate is essential for insect defense in Arabidopsis. Proc. Natl. Acad. Sci. USA 94:

77 Jasmonates accumulate extremely quickly after wounding W (wounded) part of leaf U (unwounded) part of leaf When a leaf is crushed with forceps, the level of JA in the wounded and unwounded part of the leaf increases within a minute Unwounded plant Glauser, G., Dubugnon, L., Mousavi, S.A.R., Rudaz, S., Wolfender, J.-L., and Farmer, E.E. (2009). Velocity estimates for signal propagation leading to systemic jasmonic acid accumulation in wounded Arabidopsis. J. Biol. Chem. 284:

78 The defense responses occurs in unwounded leaves as well Elevated PIs were recorded from unwounded leaves as well suggesting a mobile signal Control plant Plant infested with herbivore extract from distant leaf Green, T.R., and Ryan, C.A. (1972). Wound-induced proteinase inhibitor in plant leaves: A possible defense mechanism against insects. Science 175:

79 PI genes are induced by jasmonate treatment, locally and systemically Proteinase inhibitor gene expression is induced by Wounding or Methyl Jasmonate. MeJA vapor promotes PI accumulation in exposed leaves but also unexposed leaves of the same plant 2 6 No MJ control Farmer, E.E., Johnson, R.R., and Ryan, C.A. (1992). Regulation of expression of proteinase inhibitor genes by methyl jasmonate and jasmonic acid. Plant Physiol. 98:

80 Systemic signaling does not involve movement of JA-Ile but may involve other jasmonates The nature of the mobile signal is still not resolved it may include: Jasmonates Hydraulic signals Ionic signals Wang, L., Allmann, S., Wu, J., and Baldwin, I.T. (2008). Comparisons of LIPOXYGENASE3- and JASMONATE-RESISTANT4/6-silenced plants reveal that jasmonic acid and jasmonic acid-amino acid conjugates play different roles in herbivore resistance of Nicotiana attenuata. Plant Physiol. 146:

81 Tomatoes and related plants produce systemin, a peptide signal Systemin is produced by cleavage of a larger protein prosystemin Ryan, C.A., and Pearce, G. (2003). Systemins: A functionally defined family of peptide signals that regulate defensive genes in Solanaceae species. Proc. Natl. Acad. Sci. USA 100: copyright 2003 National Academy of Sciences USA.

82 Expression of systemin in the rootstock induces PIs in the scion WT 35S: Prosys WT WT Initially these data suggested that systemin itself is the mobile signal, but we now believe systemin s role is only at the wound site McGurl, B., Orozco-Cardenas, M., Pearce, G., and Ryan, C.A. (1994). Overexpression of the prosystemin gene in transgenic tomato plants generates a systemic signal that constitutively induces proteinase inhibitor synthesis. Proc. Natl. Acad. Sci. USA 91:

83 The COI1 receptor is required at the systemic site but JA synthesis is not WT 13 jai1 (COI1 receptor mutant) 0 spr-2 (JA synthesis mutant) 150 WT 35S: Prosys 35S: Prosys McGurl, B., Orozco-Cardenas, M., Pearce, G., and Ryan, C.A. (1994). Overexpression of the prosystemin gene in transgenic tomato plants generates a systemic signal that constitutively induces proteinase inhibitor synthesis. Proc. Natl. Acad. Sci. USA 91:

84 Systemin at the wound site generates or amplifies the systemic signal The mobile signal ( X ) is still being debated Li, L., Li, C., Lee, G.I., and Howe, G.A. (2002). Distinct roles for jasmonate synthesis and action in the systemic wound response of tomato. Proc. Natl. Acad. Sci. USA 99: copyright 2002 National Academy of Sciences USA.

85 Indirect responses include volatile compounds that attract carnivorous and parasitic insects And insects that lay their eggs in the herbivore, which gets eaten when they hatch! Volatiles attract carnivorous insects that eat the herbivore Tim Haye, Universität Kiel, Germany Bugwood.org; R.J. Reynolds Tobacco Company Slide Set and R.J. Reynolds Tobacco Company, Bugworld.org

86 Charles Darwin had thoughts about parasitic insects I cannot persuade myself that a beneficent and omnipotent God would have designedly created the Ichneumonidae with the express intention of their feeding within the living bodies of caterpillars. Charles Darwin, 1860

87 Jasmonate mediates defenses against necrotrophic pathogens Arabidopsis plants infected with Botrytis cinerea Photo credit Gary Loake

88 JA-insensitive plants are more susceptible to pathogens Alternaria brassicicola Botrytis cinerea Thomma, B.P.H.J., Eggermont, K., Penninckx, I.A.M.A., Mauch-Mani, B., Vogelsang, R., Cammue, B.P.A., and Broekaert, W.F. (1998). Separate jasmonate-dependent and salicylate-dependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens. Proc. Natl. Acad. Sci. USA 95: copyright 1998 National Academy of Sciences USA.

89 JA-induces plant defenses against pathogens JA-mediated responses to pathogens include production of phytoalexins, and antimicrobial peptides or proteins Peanut kernel infected by a soil fungus (Aspergillus niger). Yellow-colored phytoalexin is locally produced by the kernel tissues (arrow) Photo credit: USDA

90 Jasmonates protect plants through induced resistance Prior exposure to a pest, or colonization with some nonpathogenic bacteria can prime a plant for enhanced responses. Beneficial bacteria or fungi Pseudomonas fluorescens WCS417r primes the plant to elicit a stronger, faster response to MeJA Adapted from Frost, C.J., Mescher, M.C., Carlson, J.E., and De Moraes, C.M. (2008). Plant defense priming against herbivores: Getting ready for a different battle. Plant Physiol. 146: ; Conrath, U., Beckers, G.J.M., Flors, V., García-Agustín, P., Jakab, G.b., Mauch, F., Newman, M.-A., Pieterse, C.M.J., Poinssot, B., Pozo, M.J., Pugin, A., Schaffrath, U., Ton, J., Wendehenne, D., Zimmerli, L., and Mauch-Mani, B. (2006). Priming: Getting ready for battle. Mol. Plant-Microbe Interact. 19:

91 The mechanisms of priming are not fully understood Perhaps signaling molecules or transcription factors increase in an abundant, inactive state, leading to an enhanced defense response upon subsequent attack Reprinted from Conrath U. (2011). Molecular aspects of defence priming. Trend Plant Sci 16: with permission from Elsevier.

92 JA seems to play a role in establishing mutualistic interactions Arbuscular mycorrhiza Legume-rhizobia symbiosis Reprinted from Hause, B., and Schaarschmidt, S. (2009). The role of jasmonates in mutualistic symbioses between plants and soil-born microorganisms. Phytochemistry 70: with permission from Elsevier.

93 Cross-talk between JA and SAmediated signaling Remarkably, there is considerable crosstalk between JA and SA signaling pathways Jasmonates Salicylates

94 Exogenous application of SA enhances success of an herbivore Trichoplusia ni (cabbage looper) herbivory is enhanced by SA and inhibited by MeJA Cui, J., Bahrami, A.K., Pringle, E.G., Hernandez-Guzman, G., Bender, C.L., Pierce, N.E., and Ausubel, F.M. (2005). Pseudomonas syringae manipulates systemic plant defenses against pathogens and herbivores. Proc. Natl. Acad. Sci. USA 102: copyright 2005 National Academy of Sciences USA.

95 Salicylates repress jasmonate signaling JA SA PDF1.2 PR-1 SA-induced JA-induced JA induces expression of PDF1.2, but in the presence of JA + SA the gene is not activated. SA overrides JA. Koornneef, A., and Pieterse, C.M.J. (2008). Cross talk in defense signaling. Plant Physiol. 146:

96 Some insects induce SA responses and repress JA responses - + Silverleaf whitefly nymphs induce SA defenses which suppress JA defenses, increasing their survival SA-induced JA-induced JA-induced Zarate, S.I., Kempema, L.A., and Walling, L.L. (2007). Silverleaf whitefly induces salicylic acid defenses and suppresses effectual jasmonic acid defenses. Plant Physiol. 143:

97 Salicylates also make plants more susceptible to necrotrophs The necrotrophic fungus Alternaria brassicicola reproduces better on Arabidopsis plants treated with salicylic acid (SA) JA PDF1.2 SA PR-1 SA tends to override JAmediated responses Spoel, S.H., Johnson, J.S., and Dong, X. (2007). Regulation of tradeoffs between plant defenses against pathogens with different lifestyles. Proceedings of the National Academy of Sciences 104: copyright National Academy of Sciences USA.

98 NPR1 is a node through which SA and JA pathways intersect NONEXPRESSER OF PR GENES1 (NPR1) is activated by SA, and interferes with JA signaling The Mediator complex subunit16 (MED16) has been implicated with this signaling pathway (downstream of NPR1) in order to call RNA Pol II and initiate transcription RNA Pol II MED16 Spoel, S.H. et al. (2003). NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathways through a novel function in the cytosol. Plant Cell 15: Zhang, X. et al. (2012). The Arabidopsis Mediator complex subunit16 positively regulates salicylate-mediated systemic acquired resistance and jasmonate/ethylene-induced defense pathways. Plant Cell 24:

99 Coronatine stimulates JA responses, suppressing SA response JA SA Coronatineproducing bacterium Coronatine is an excellent mimic of JA-Ile. Production of coronatine significantly enhances the pathogenicity of bacteria that produce it Defense

100 Different pathogens elicit different signal signatures Plants are able to perceive the type of pathogen or pest threatening them, and respond in different, appropriate ways. We still don t understand much of this recognition process De Vos, M., Van Oosten, V.R., Van Poecke, R.M.P., Van Pelt, J.A., Pozo, M.J., Mueller, M.J., Buchala, A.J., Metraux, J.-P., Van Loon, L.C., Dicke, M., and Pieterse, C.M.J. (2005). Signal signature and transcriptome changes of Arabidopsis during pathogen and insect attack. Mol. Plant-Microbe Interact. 18:

101 Ethylene and jasmonate pathways confer specificity to the response Pathogens induce ethylene and jasmonate production, and activate a different subset of genes as those induced by wounding or herbivory Lorenzo, O., Chico, J.M., Sanchez-Serrano, J.J., and Solano, R. (2004) JASMONATE-INSENSITIVE1 encodes a MYC transcription factor essential to discriminate between different jasmonate-regulated defense responses in Arabidopsis. Plant Cell 16:

102 Defense pathways coordinate with ABA and developmental signals Reprinted from Spoel, S.H., and Dong, X. (2008) Making sense of hormone crosstalk during plant immune responses. Cell Host Microbe 3: with permission from Elsevier.

103 Jasmonates integrate signals in plant growth and development Senescence Root growth inhibition Tuber formation Trichome formation Flower development Wasternack, C., and Hause, B. (2013). Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. An update to the 2007 review in Annals of Botany. Ann. Bot. 111: by permission of Oxford University Press.

104 Jasmonates contribute to developmental and growth controls Flower development Seed development Trichome formation JA also controls: cell cycle, root extension, leaf senescence, stomata closure, and mutualistic interactions... Li, L., Zhao, Y., McCaig, B.C., Wingerd, B.A., Wang, J., Whalon, M.E., Pichersky, E., and Howe, G.A. (2004). The tomato homolog of CORONATINE-INSENSITIVE1 is required for the maternal control of seed maturation, jasmonate-signaled defense responses, and glandular trichome development. Plant Cell 16: ; Reprinted by permission from Macmillan Publishers Ltd. Thines, B., Katsir, L., Melotto, M., Niu, Y., Mandaokar, A., Liu, G., Nomura, K., He, S.Y., Howe, G.A., and Browse, J. (2007). JAZ repressor proteins are targets of the SCFCOI1 complex during jasmonate signalling. Nature 448:

105 JA regulates stamen development GA antagonizes positive effect of JA on stamen development Qi, T., Huang, H., Song, S., and Xie, D. (2015). Regulation of jasmonate-mediated stamen development and seed production by a bhlh-myb complex in Arabidopsis. Plant Cell. 27:

106 Jasmonates and nutrients Jasmonates promote nutrient reallocation away from leaves into roots There is a connection between jasmonates, nutrients and energy reserves that may contribute to defense and survival Jasmonates promote tuber formation in potato Potassium-starved Arabidopsis accumulate jasmonates and are more resistant to insects Armengaud, P., Breitling, R., and Amtmann, A. (2010) Coronatine-Insensitive 1 (COI1) mediates transcriptional responses of Arabidopsis thaliana to external potassium supply. Mol. Plant 3:

107 Control of primary root development involves both auxin and jasmonate Reprinted from Kazan, K., Manners, J.M. (2011). JAZ repressors and the orchestration of phytohormone crosstalk. Trends Plant Sci. 17: 22-31, with permission from Elsevier; Reprinted with permission from Hoffmann, M., Hentrich, M., Pollmann, S. (2011). Auxin-oxylipin crosstalk: Relationship of antagonists. J. Integr. Plant Biol. 53:

108 Auxin and jasmonate act antagonistically on leaf senescence Ultimately, via WRKY57 JA induces leaf senescence Auxin suppresses leaf senescence Reprinted from Jian et al. (2014). Arabidopsis WRKY functions as a node of convergence for jasmonic acid- and auxin-mediated signaling in jasmonic-acid induced leaf senescence. Plant Cell 26:

109 Ethylene and JA act antagonistically on apical hook formation EIN3 is induced by ethylene, and will ultimately induce apical hook formation. On the other hand, JA-induced MYC2 and EBF1 repress EIN3 activity, inhibiting apical hook formation by repressing expression of HLS1 Zhang, X et al. (2014). Jasmonate-activated MYC2 represses ETHYLENE INSENSITIVE3 activity to antagonize ethylene-promoted apical hook formation in Arabidopsis. Plant Cell. 26:

110 Competition between GA and JA Normal situation growth and defense are balanced DELLAs competing with MYCs for JAZs binding while JAZs competing with PIFs for DELLA binding Elevated GA - growth Elevated JA - defense Reprinted from Kazan, K., Manners, J.M. (2011). JAZ repressors and the orchestration of phytohormone crosstalk. Trends Plant Sci. 17: 22-31, with permission from Elsevier. See also Hou, X. et al. (2010) DELLAs modulate jasmonate signaling via competitive binding to JAZ. Dev. Cell 19:

111 Jasmonates integrate with light signals via JAZ and and JAR1 Hsieh, H.L., and Okamoto, H. (2014). Molecular interaction of jasmonate and phytochrome A signalling. J Exp Bot. 65: by permission of Oxford University Press; Robson, F., Okamoto, H., Patrick, E., Harris, S.-R., Wasternack, C., Brearley, C. and Turner, J.G. (2010). Jasmonate and phytochrome A signaling in Arabidopsis wound and shade responses are integrated through JAZ1 stability. Plant Cell. 22:

112 Ongoing questions JA What signals trigger JA synthesis and signaling pathways? How does the plant discriminate between threats? What is the systemic signal? JA-Ile MeJA COI1 JA- Ile What do the different JAZ proteins do? How is the diversity of JA responses controlled in specific organs and cell types?

113 How does the plant prioritize its stress and defense responses? Pathogens (biotrophs) Biotic stress SA ABA SA-response genes Stress-response genes Lorenzo, O., Chico, J.M., Sanchez-Serrano, J.J., and Solano, R. (2004) JASMONATE-INSENSITIVE1 encodes a MYC transcription factor essential to discriminate between different jasmonate-regulated defense responses in Arabidopsis. Plant Cell 16: Robert-Seilaniantz, A., Grant, M., Jones, J.D.G. (2011). Hormone crosstalk in plant disease and defense: More than just JASMONATE-SALICYLATE antagonism. Annu. Rev. Phytopathol. 49:

114 How can plant scientists prevent crop losses to disease and insects? Most plants are resistant to most pests. Nevertheless, pre- and post-harvest crops losses can be devastating. Understanding jasmonates helps plant scientists and breeders enhance plant resistance and optimize development Photo credit: ARS USDA