植物激素——水杨酸

1、Salicylates: Defense and MoreSalicylates plant hormones and painkillersSalicylic AcidAcetylsalicylic AcidASPIRINPhoto credits: Geaugagrrl; Sten PorseNamed for white willow Salix albaNamed for Spiraea, the former name for meadowsweetHow does aspirin work in people? Image courtesy cytochrome c, artfav

2、orProtection from heart attacks, strokes and maybe cancer! Cyclo-oxygenaseProstaglandinsPainFeverInflammationDecreased platelet adhesionProstaglandins are a family of lipid-derived signaling molecules with diverse effectsIts main effect is to limit prostaglandin productionSalicylic acid is used to t

3、reat viral warts, acne and sun-damaged skinHigh concentrations of SA are used - the effect is mainly due to removing the outer layers of skin Before SAAfter SAHow do salicylates work in plants? SAs effects in plants and animals are unrelated plants do not make prostaglandins and their epidermis is v

4、ery differentExpression of pathogen-induced genesIncreased synthesis of SASignal (e.g. Pathogen, UV light, developmental cues)In plants SAs main effect is to alter patterns of gene expressionDEFENSEDevelopmental responsesStress responsesMicroorganisms are (hemi)biotrophic or necrotrophic Cell death

5、accompanies or precedes colonization by necrotrophsBiotrophs or hemibiotrophs can live within their host tissue without causing (immediate) deathToxinPseudomonas syringae in intercellular spaceBestwick, C.S., Brown, I.R., Bennett, M., and Mansfield, J.W. (1997). Localization of hydrogen peroxide acc

6、umulation during the hypersensitive reaction of lettuce cells to Pseudomonas syringae pv phaseolicola. Plant Cell 9: 209-221.Salicylates participate in plant defenses to biotrophic pathogensTo a first approximation, insects and necrotrophs trigger jasmonate production, and biotrophs trigger salicyla

7、te productionJasmonatesSalicylatesTranscriptional responsesHistory of salicylate researchNational Library of Medicine; Raskin, I. (1992). Salicylate, a new plant hormone. Plant Physiol. 99: 799-803.Hippocrates wrote about the use of willow to relieve pain 2400 years agoIn 1979 White showed that pre-

8、treatment of a leaf with aspirin or SA conferred resistance to tobacco mosaic virus (TMV)In 1987 endogenous SA was shown to be responsible for heat production in Arum lily flowersIn 1990 SA was shown to be an endogenous signal in defense responses Untreated sideSA treatedCao, H., Bowling, S.A., Gord

9、on, A.S., and Dong, X. (1994). Characterization of an Arabidopsis mutant that is nonresponsive to inducers of systemic acquired resistance. Plant Cell 6: 1583-1592. Rusterucci, C., Aviv, D.H., Holt, B.F., III, Dangl, J.L., and Parker, J.E. (2001). The disease resistance signaling components EDS1 and

10、 PAD4 are essential regulators of the cell death pathway controlled by LSD1 in Arabidopsis. Plant Cell 13: 2211-2224. Delaney, T.P., Uknes, S., Vernooij, B., Friedrich, L., Weymann, K., Negrotto, D., Gaffney, T., Gut-Rella, M., Kessmann, H., Ward, E. and Ryals, J. (1994). A central role of salicylic

11、 acid in plant disease resistance. Science. 266: 1247-1250 reprinted with permission of AAAS. Gorlach, J., Volrath, S., Knauf-Beiter, G., Hengy, G., Beckhove, U., Kogel, K.H., Oostendorp, M., Staub, T., Ward, E., Kessmann, H., and Ryals, J. (1996). Benzothiadiazole, a novel class of inducers of syst

12、emic acquired resistance, activates gene expression and disease resistance in wheat. Plant Cell 8: 629-643.Tools to study SA and disease in plantsPathogen growth rateSA analoguesPR-1 expression (days after infection)Trypan blue stains fungal structures within the plant tissue and dead or damaged pla

13、nt cells.SA treatment slows pathogen growthPathogen infection or SA treatment induces PATHOGENESIS-RELATED GENE1 (PR-1)expressionFrom Delaney, T.P., Uknes, S., Vernooij, B., Friedrich, L., Weymann, K., Negrotto, D., Gaffney, T., Gut-Rella, M., Kessmann, H., Ward, E. and Ryals, J. (1994). A central r

14、ole of salicylic acid in plant disease resistance. Science. 266: 1247-1250, reprinted with permission from AAAS.Before SA-deficient plants were identified genetically, NahG expression was used to investigate SAs roles. ControlNahG-expressing plantSalicylate hydroxylase encoded by NahGSAcatecholAltho

15、ugh it is very effective, concerns about its specificity and effects of catechol mean researchers now prefer other methodsThe bacterial enzyme encoded by NahG hydrolyzes SA to catecholLecture outlineSynthesis, conjugation and transportPerception and signalingNPR1, transcriptional and chromatin effec

16、tsSalicylates in whole-plant processesPathogen responsesPerception, local and systemic responsesCross-talk between SA and JAOther functionsAbiotic stressThermogenesisImage credit: Prof. Dr. Otto Wilhelm Thom Flora von Deutschland, sterreich und der Schweiz 1885, Gera, GermanySynthesis, conjugation a

17、nd transportMost SA is synthesized from chorismic acidReprinted from Mtraux, J.-P. (2002). Recent breakthroughs in the study of salicylic acid biosynthesis. Trends in Plant Science 7: 332-334 with permission from Elsevier. Isochorismate synthase (ICS) converts chorismic acid to isochorismic acid. Th

18、e enzyme that converts isochormic acid to salicylic acid hasnt been identified in plants. Flow through the shikimate pathway increases with stress, stimulating SA production. A small amount of SA is derived from cinnamic acid. In many plants ICS1 is upregulated by pathogen exposureICS1PR1Expression

19、of ICS1 increases upon exposure to a pathogenPR1 is an SA-induced pathogen responsive geneReprinted by permission from Macmillan Publishers Ltd. Wildermuth, M.C., Dewdney, J., Wu, G., and Ausubel, F.M. (2001). Isochorismate synthase is required to synthesize salicylic acid for plant defence. Nature

20、414: 562-565 copyright 2001. Col-0sid2Mutants in ICS1 are blocked in SA synthesis and susceptible to diseaseNawrath, C. and Metraux, J.-P. (1999). Salicylic acid inductiondeficient mutants of Arabidopsis express PR-2 and PR-5 and accumulate high levels of camalexin after pathogen inoculation. Plant

21、Cell. 11: 1393-1404.Reduced accumulation of free and conjugated SAIncreased growth of Hyaloperonospora arabidopsidispathogen sid2 and eds16 are ICS1 mutantsSA synthesis is controlled by positive and negative factors Transcription factors that specifically activate or repress ICS1 expression have bee

22、n identifiedPathogen infection, UV light, ozone and abiotic stresses promote SA accumulationSharma, Y.K., Len, J., Raskin, I., and Davis, K.R. (1996). Ozone-induced responses in Arabidopsis thaliana: the role of salicylic acid in the accumulation of defense-related transcripts and induced resistance

23、. Proc. Natl. Acad. Sci. USA 93: 5099-5104, copyright 1996, National Academy of Sciences USA. Ozone treatmentSA is modified and conjugated to active and inactive compoundsUsed with permission from Vlot, A.C., Dempsey, D.M.A., and Klessig, D.F. (2009). Salicylic acid, a multifaceted hormone to combat

24、 disease. Annu. Rev. Phytopath. 47: 177-206, permission conveyed through Copyright Clearance Center, Inc.Salicylic acid O-glucosideSalicyloyl glucose esterMethyl salicylateMethyl salicylate O-glucosideSA is synthesized in the chloroplast, conjugated in the cytoplasm and sequestered in the vacuolepla

25、stidMeSAICSSASAG (Salicylic acid O-glucoside)SASAGSGESGE (Salicyloyl glucose ester)-glucose-glucoseLEAF 3MeSASAMeSA accumulates in response to pathogensSeskar, M., Shulaev, V., and Raskin, I. (1998). Endogenous methyl salicylate in pathogen-inoculated tobacco plants. Plant Physiol. 116: 387-392.; Sh

26、ulaev, V., Leon, J., and Raskin, I. (1995). Is salicylic acid a translocated signal of systemic acquired resistance in tobacco? Plant Cell 7: 1691-1701.SACH3MeSA3Levels of MeSA increase dramatically upon pathogen infectionLeaf 3 was inoculated with tobacco mosaic virus at time 0MeSA may be the form

27、of SA transported systemically SACH3MeSASA and MeSA were detected in an adjacent leaf (leaf 8)83LEAF 3LEAF 8MeSASAMeSASASeskar, M., Shulaev, V., and Raskin, I. (1998). Endogenous methyl salicylate in pathogen-inoculated tobacco plants. Plant Physiol. 116: 387-392.; Shulaev, V., Leon, J., and Raskin,

28、 I. (1995). Is salicylic acid a translocated signal of systemic acquired resistance in tobacco? Plant Cell 7: 1691-1701.Regulation of SA accumulationSAICS1Genetic studies have identified some of the signals that transduce pathogen perception to SA synthesisThe eds1 mutant shows enhanced disease susc

29、eptibilityParker, J.E., Holub, E.B., Frost, L.N., Falk, A., Gunn, N.D., and Daniels, M.J. (1996). Characterization of eds1, a mutation in Arabidopsis suppressing resistance to Peronospora parasitica specified by several different RPP genes. Plant Cell 8: 2033-2046.Wild-typeeds1Wild type: Pathogen gr

30、owth is arrested by plant defense responseeds1:Plant fails to respond and pathogen growth is uncheckedThe eds1 mutant is deficient in PR1 gene expression but rescued by SAFalk, A., Feys, B.J., Frost, L.N., Jones, J.D.G., Daniels, M.J., and Parker, J.E. (1999). EDS1, an essential component of R gene-

31、mediated disease resistance in Arabidopsis has homology to eukaryotic lipases. Proc. Natl. Acad. Sci. USA 96: 3292-3297 copyright 1999 National Academy of Sciences USA. Wild-typeeds1Pathogen recognitionEDS1SADefense responseNo PR1 induction by pathogenSAEDS1 and PAD4 are related lipase-like proteins

32、 that form a complexReprinted by permission from Macmillan Publishers Inc (EMBO) Feys, B.J., Moisan, L.J., Newman, M.-A., and Parker, J.E. (2001). Direct interaction between the Arabidopsis disease resistance signaling proteins, EDS1 and PAD4. EMBO J 20: 5400-5411. Zhou, N., Tootle, T.L., Tsui, F.,

33、Klessig, D.F., and Glazebrook, J. (1998). PAD4 functions upstream from salicylic acid to control defense responses in Arabidopsis. Plant Cell 10: 1021-1030.The pad4 mutant is deficient in SA accumulationPathogen recognitionEDS1 / PAD4 /SAG101SADefense responsePathogen recognitionNDR1Other genes are

34、also needed for pathogen-induced SA accumulationSARegulation of SA synthesisSAICS1Additional mutants with altered SA accumulation continue to be characterizedPAD4 / EDS1/ SAG101NDR1Synthesis, modification and accumulation of SA - summarySA accumulation is correlated with ICS1 expressionTranscription

35、 factors that control ICS1 transcription have been identifiedOther factors necessary for SA synthesis have been identified and their molecular functions are under investigationSA can be conjugated for storage, or converted to MeSA for transportImage credit: Prof. Dr. Otto Wilhelm Thom Flora von Deut

36、schland, sterreich und der Schweiz 1885, Gera, GermanyPerception and signalingSANPR1NPR1NPR1 monomerization and nuclear importRegulation of transcriptionPRReceptor? Pathogens induce an enormous change in transcription. NPR1 is key to this responseWanted SA receptorSASeveral SA-binding proteins have

37、been identified. None seem to be a receptor in the traditional sense. 2 H2O22 H2O + O2 catalaseSASA binds to catalase and ascorbic peroxidase and inhibits their action, leading to a build up of H2O2 and other reactive oxygen species.SABP2 is a MeSA esterase that is feedback inhibited by its product,

38、 SA. SAMeSASABP2SAICS1EDS1PAD4NPR1Several WRKYs (transcription factors)Many genes respond to SA: Early genes amplify the signalEarlyLateAdapted from van den Burg, H.A., and Takken, F.L.W. (2009). Does chromatin remodeling mark systemic acquired resistance? Trends Plant Sci. 14: 286-294.Pathogen reco

39、gnitionEDS1 / PAD4 SATranscription responseICS1NPR1PR-1PR-5BGL2NIMIN1ATNUDT6FRK1Several WRKYs (transcription factors)NPR1 is a major activator of SA-mediated responsesDelaney, T.P., Friedrich, L., and Ryals, J.A. (1995). Arabidopsis signal transduction mutant defective in chemically and biologically

40、 induced disease resistance. Proc. Natl. Acad. Sci. USA 92: 6602-6606 copyright 1995 National Academy of Sciences USA. Wild-typenpr1 (aka nim1)Plants were treated with SA, then three days later challenged with a fungal pathogen (Hyaloperonospora arabidopsidis)NPR1 is necessary for defense responses

41、and at the core of SA signal transductionNPR1 is necessary and sufficient for downstream signaling and defenseReprinted from Cao, H., Glazebrook, J., Clarke, J.D., Volko, S., and Dong, X. (1997). The Arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing

42、ankyrin repeats. Cell 88: 57-63 with permission from Elsevier; Cao, H., Li, X., and Dong, X. (1998). Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene in systemic acquired resistance. Proc. Natl. Acad. Sci. USA 95: 6531-6536 copyright National Academy

43、of Sciences, USA. Wild-typenpr1Pseudomonas syringae infectionBgl2-GUS (PR gene) expressionLoss of function: more susceptibleGain of function: more resistantNPR1PR gene expressionResistanceSANPR1 oligomerizes via redox-sensitive CysteinesHSSHSSReductionOxidizationSA accumulation contributes to a redu

44、cing environment, which causes multimeric NPR1 to monomerizeNPR1NPR1NPR1NPR1NPR1NPR1NPR1Monomeric NPR1 is imported into the nucleus (here it is GFP-labeled)SANPR1SASAKinkema, M., Fan, W., and Dong, X. (2000). Nuclear localization of NPR1 is required for activation of PR gene expression. Plant Cell 1

45、2: 2339-2350.In the nucleus, SA-activated NPR1 promotes transcriptionNPR1NO SA+ SA-TGAs-WRKYs+TGAs+TGAs+WRKYsIn the absence of SA and activated NPR1, negative regulators repress defense genesIn the presence of SA and activated nuclear NPR1, positive regulators activate defense genesNPR1 binds TGA tr

46、anscription factors and promotes DNA bindingZhang, Y., Fan, W., Kinkema, M., Li, X., and Dong, X. (1999). Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene. Proc. Natl. Acad. Sci. USA 96: 6523-6528;

47、Despres, C., DeLong, C., Glaze, S., Liu, E., and Fobert, P.R. (2000). The Arabidopsis NPR1/NIM1 protein enhances the DNA binding activity of a subgroup of the TGA family of bZIP transcription factors. Plant Cell 12: 279-290.Yeast-two hybrid assay showing NPR1/ TGA interactionNPR1+TGAsNPR1+TGAsFree D

48、NABoundDNADNA binding of TGA2 is enhanced by NPR1The Arabidopsis genome encodes 10 TGA factors. Some are positive and some negative regulators of defense genes-TGAs-TGAs+TGAs+TGAsTGA2 is both a repressor and an activator of transcriptionBoyle, P., Le Su, E., Rochon, A., Shearer, H.L., Murmu, J., Chu

49、, J.Y., Fobert, P.R., and Despres, C. (2009). The BTB/POZ domain of the Arabidopsis disease resistance protein NPR1 interacts with the repression domain of TGA2 to negate its function. Plant Cell 21: 3700-3713.SA / NPR1NPR1NPR1-binding to TGA2 masks its repressor domain and activates transcriptionWR

50、KYs are a large family of transcription factors There are 74 Arabidopsis WRKYs and 90 rice WRKYs, many of which are involved in defense signaling. Some promote and some repress transcription -WRKY+WRKYGenes induced by WRKYs include NPR1 and ICS1 signal amplification! SA+WRKYICS1NPR1NPR1WRKYs were ch

51、aracterized in parsley cell culturesInteraction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes. P J Rushton, J T Torres, M Parniske, P Wernert, K Hahlbrock, and I E Somssich EMBO J. 1996 October 15; 15(20): 56905700.WRKY transcription f

52、actors were characterized as activators of PR1 gene expressionWRKY = Trp-Arg-Lys-Tyr The network of WRKY transcription factor interactions is complexReprinted from Eulgem, T., and Somssich, I.E. (2007). Networks of WRKY transcription factors in defense signaling. Curr. Opin. Plant Biol. 10: 366-371

53、with permission from Elsevier.This large family of transcription factors controls expression of many defense genes. Some are activators, some repressors, one is a resistance protein, and some are direct targets of resistance proteinsThe MLA resistance protein inhibits a negative-acting WRKYShen, Q.-

54、H., Saijo, Y., Mauch, S., Biskup, C., Bieri, S., Keller, B., Seki, H., lker, B., Somssich, I.E., and Schulze-Lefert, P. (2007). Nuclear activity of MLA immune receptors links isolate-specific and basal disease-resistance responses. Science 315: 1098-1103. Reprinted with permssion from AAAS.When barl

55、ey perceives the pathogen, the MLA protein moves into the nucleus and inhibits repressive WRKYs to activate PR genesThe complexity of defense gene regulation protects makes it hard for pathogens to defeat and provides flexibility and specificity to the responseThe PR-1 promoter is subject to chromat

56、in modification Mosher, R.A., Durrant, W.E., Wang, D., Song, J., and Dong, X. (2006). A comprehensive structure-function analysis of Arabidopsis SNI1 defines essential regions and transcriptional repressor activity. Plant Cell 18: 1750-1765.SAHistone modifications that activate transcription are enr

57、iched by SA treatment+SA+SAIn the absence of SA:Gene OFFIn the presence of SA: Gene ONHistone deacetylase activity is correlated with pathogen responseKim, K.-C., Lai, Z., Fan, B., and Chen, Z. (2008). Arabidopsis WRKY38 and WRKY62 transcription factors interact with histone deacetylase 19 in basal

58、defense. Plant Cell 20: 2357-2371.HDA19had19-3WTHDA19-OELess resistantMore resistantHistone deacetylation leads to a closed chromatin state and inactive genesHDA19WKRY-WRKYPR-1HDA19 turns off the gene encoding a repressive WRKY, leading to enhanced PR-1 expressionsni1 is a suppressor of npr1Li, X.,

59、Zhang, Y., Clarke, J.D., Li, Y. and Dong, X. (1999). Identification and cloning of a negative regulator of systemic acquired resistance, SNI1, through a screen for suppressors of npr1-1. Cell. 98: 329-339. Wang, S., Durrant, W.E., Song, J., Spivey, N.W., and Dong, X. (2010). Arabidopsis BRCA2 and RA

60、D51 proteins are specifically involved in defense gene transcription during plant immune responses. Proc. Natl. Acad. Sci. USA 107: 22716-22721.npr1 = no PR expressionnpr1sni1 = suppressor of npr1 = constitutive PR expression In the sni1 mutant, PR-1 genes are expressed all the time even in non-indu