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Stress-induced expression of a parsley gene encoding 4-coumarate:CoA-ligase Ellard, Mary 1994

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STRESS-INDUCED EXPRESSION OF A PARSLEY GENEENCODING 4-COUMARATE:COA-LIGASEByMary EllardB.Sc. (ions.) National University of Ireland, 1987M.Sc. National University of Ireland, 1988A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF BOTANYWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAMarch 1994© Mary Ellard, 1994In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of_________________The University of British ColumbiaVancouver, CanadaDate c’ / / ‘ -DE.6 (2/88)AbstractMany of the diverse end-products of the phenyipropanoid pathway play an important rolein the response of plants to environmental stresses such as wounding and pathogen attack.A key enzyme in the general phenylpropanoid pathway is 4-coumarate:CoA ligase (4CL)which catalyses the formation of activated esters of hydroxycinnamic acids. Expression ofthe parsley genes encoding this enzyme is increased in response to wounding or challengewith a fungal elicitor from Phytophthora megasperma.Using a number different systems I have investigated the stress response of parsley4 CL. Using transgenic tobacco as a heterologous system to study the wound response, Ishowed that the same fragment of the 4 CL-i promoter mediates wound-responsivenessand a strong response to exogenously applied methyl jasmonate. The 4 CL-i promoteralso mediates responsiveness to linolenic acid, from which jasmonates are synthesised viaa lipoxygenase mediated step.In parsley cells in suspension culture, linolenic acid also activates 4CL expression, andexpression of a number of phenyipropanoid and other defense-related genes is inducedby jasmonates in this system. A similar response to jasmonates is observed in wholeparsley plants. Jasmonate treatment of parsley cells activates the expression of genes ofthe furanocoumarin specific branch pathway of phenylpropanoid metabolism and resultsin secretion of furanocoumarins (which are known phytoalexins in parsley) by the cellcultures. In the presence of an inhibitor of lipoxygenase activity, n-propylgallate, theresponse of 4CL to stress was decreased in both systems suggesting that de novo synthesisof jasmonates may be required for these stress responses. This suggests that jasmonatesmay mediate the stress responses of 4CL and other defense-related genes11In transgenic Arabidopsis, the parsley 4 CL-i promoter is responsive to endogenouslygenerated wound signals and directed strong localised expression of the reporter geneGUS at wound-sites. This may provide the basis for a genetic screen to identify geneswhose products are necessary for the wound response of 4 CL-i.111Table of ContentsAbstractList of FiguresAbbreviationsAcknowledgements1 General introductioniiviiixixii1131313131414141515151616162 Experimental procedures2.1 4C1-1-GUS fusions2.2 Generation of transgenic tobacco plants2.3 Plant treatments2.3.1 Wounding experiments2.3.2 Ethephon and ABA treatment2.3.3 Treatment of plants with methyl jasmonate, jasmonic acid andlinolenic acid2.3.4 Remote treatment of tobacco with MJ2.3.5 Treatment of tobacco plants with n-propylgallate2.3.6 Treatment of tobacco with elicitor2.4 Parsley cell culture growth and treatments2.4.1 Growth of cell cultures2.4.2 Treatment of cell cultures with elicitoriv2.4.3 Isolation and analysis of total coumarins from cell culture medium 162.4.4 Treatment of cell cultures with methyl jasmonate, jasmonic acid,linolenic acid and ABA 172.4.5 Treatment of cell cultures with inhibitors 172.5 RNA isolation and analysis 172.5.1 RNA and poiy A RNA isolation 172.5.2 Northern and slot blots 182.6 Histochemical localisation of GUS activity 192.7 Mutagenesis and screening of Arabidopsis 203 Expression of 4 CL-i in response to wounding in transgenic tobacco 213.1 Introduction 213.2 Results 303.2.1 Expression of 4 CL-i in transgenic tobacco 303.2.2 Expression of 4 CL-i-GUS gene fusions in response to wounding . 313.2.3 The effect of ethylene on the expression of 4 CL-i in tobacco . . . 333.2.4 The effect of ABA treatment on 4 CL-i promoter activity 353.2.5 Expression of 4 CL-i-GUS gene fusions in response to MJ 373.2.6 Effect of methyl jasmonate, jasmonic acid, and linolenic acid onthe parsley 4 CL-i promoter in transgenic tobacco 393.2.7 The effect of a potent inhibitor of lipoxygenase on the wound response 413.3 Discussion 434 The role of jasmonates in the elicitor response in parsley cell cultures 524.1 Introduction 524.2 Results 58V4.2.1 The effect of methyl jasmonate, jasmonic acid, and linolenic acidon 4 CL expression in parsley cells 584.2.2 The effect of methyl jasmonate versus elicitor on expression of genesin the phenyipropanoid pathway 594.2.3 Accumulation of furanocoumarins in response to methyl jasmonateand elicitor treatment 624.2.4 The response of parsley cell cultures to ABA 624.2.5 The response to MJ of a group of non-phenylpropanoid elicitor-activated genes: ELI’s 654.2.6 The MJ response in whole parsley plants 664.2.7 The effect of lipoxygenase inhibitors on elicitor-inducible gene expression 684.2.8 4 CL-i elicitor-responsiveness in transgenic tobacco 714.3 Discussion 745 Expression of parsley 4 CL-i in Arabidopsis thaliana 845.1 Introduction 845.2 Results 875.2.1 Wound-inducible expression of a parsley 4CL-i promoter-GUS fusion in transgenic Arabidopsis 875.2.2 The effect of ethylene on 4CL expression in Arabidopsis 885.2.3 Response of 4CL to methyl jasmonate in Arabidopsis 925.2.4 A genetic screen for identification of genes involved in mediatingthe wound response 925.3 Discussion 93vi6 Conclusions and future directions 986.1 Conclusions 986.2 Future directions 100Bibliography 104VIIList of Figures1.1 The phenyipropanoid pathway. 23.2 Model proposed by Farmer and Ryan (1992) for wound inducible expression of proteinase inhibitor genes 273.3 Expression of 4 CL-i in response to wounding in transgenic tobacco . . 323.4 Expression of GUS in response to wounding in transgenic tobacco line 801-8 343.5 Expression of GUS in response to wounding in plants of transgenic tobaccoline 810 353.6 Expression of 4 CL-i in response to ethephon in transgenic tobacco. . . 363.7 Expression of GUS in response to ABA in transgenic tobacco line 80 1-8 363.8 Expression of GUS in response to methyl jasmonate in tobacco plants oftransgenic line 801-8 383.9 The effect of methyl jasmonate vapour on GUS expression in plants oftransgenic line 801-8 393.10 The effect of methyl jasmonate on GUS and tobacco 4CL expression inplants of transgenic line 810 403.11 Expression of GUS and tobacco 4CL in response to treatment with methyljasmonate, jasmonic acid and linolenic acid in plants of line 801-8 . . . 423.12 The effect of the lipoxygenase inhibitor npropylgallate (nPG) on the woundresponse in plants of line 801-8 444.13 Response of parsley cell suspension culture cells to exogenously appliedmethyl jasmonate, jasmonic acid, and linolenic acid 60viii4.14 The effect of methyl jasmonate, and elicitor treatments on the expressionof genes in the phenyipropanoid pathway 614.15 Levels of furanocoumarins secreted by methyl jasmonate or elicitor treatedparsley cell suspension cultures 634.16 Thin layer chromatography of furanocoumarins from culture media of MJand elicitor treated cells 644.17 Expression of PAL and 4CL in response to treatment with ABA 654.18 The effect of methyl jasmonate, or elicitor treatments on the expression ofdefense-related genes in parsley cells 674.19 Response to methyl jasmonate and wounding of phenylpropanoid andother defense-related genes in parsley plants 694.20 The effect of ibuprofen (IP) and phenylbutazone (PB) on elicitor- inducibleexpression of defense-related genes 704.21 The effect of the lipoxygenase inhibitor, n-propylgallate on elicitor-inducible expression of defense-related genes in parsley 724.22 The effect of n-propylgallate (nPG) treatment on furanocoumarin levelsin culture fluids of parsley cell suspension cultures 734.23 The effect of Pmg elicitor treatment on 4 CL-i promoter activity in a plantof transgenic line 801-8 745.24 Accumulation of GUS activity in wounded leaf discs of transgenic Arabidopsis line 204-1-3 895.25 Accumulation of GUS activity at wound sites in leave of Arabidopsis line204-1-3 905.26 Accumulation of GUS and Arabidopsis 4CL transcripts in wounded leavesof Arabidopsis line 204-1-3 91ix5.27 Accumulation of GUS and Arabidopsis 4CL transcripts in ethephon treatedleaves of Arabidopsis line 204-1-3 915.28 The effect of methyl jasmonate treatment on .4CL expression in transgenicArabidopsis 93xAbbreviations4CL 4-coumarate:CoA ligaseBMT S-adenosyl-L-methionine:bergaptol 0-methyltransferaseCR5 chalcone synthaseELI elicitor inducedGUS ,8-glucuronidaseHRGP hydroxyproline-rich glycoproteinIP ibuprofenJA jasmonic acidLA linolenic acidMJ methyl jasmonateuP G n-propylgallatePAL phenylalanine ammonia lyasePB phenylbutazonePT proteinase inhibitorPmg Phytophthora megaspermaPR pathogenesis-relatedTyrDC tyrosine decarboxylaseUbi4 polyubiquitin 4Ubiq ubiquitinxiAcknowledgementsI gratefully acknowledge the support and help of Dr. Carl Douglas under whose supervision this project was conducted. His generosity with his patience and time has beenmost sincerely appreciated. I am also grateful for the input and interest of my supervisorycommittee, Drs. Edith Camm, N. Louise Glass and George Haughn.My thanks to colleagues past and present in the Douglas lab and Botany Department.In particular I thank Sandra Allina for helping with Arabidopsis screening: Arabidopsiswere grown with a major contribution from the green fingers of Sheldon Marcuvitz.Cheerful research assistance was provided by Karma Carrier and technical advice, encouragement and all important perspective was provided by Gopal.Assembly of the thesis and figures was rendered painless thanks to very generous helpwith I4TEX from Dr. Stewart Schultz, useful computing tips from Dr. Stephen Lee andthe occasional use of EM Lab computing facilities provided by Michael Weis.I am grateful to BC Research for the use of their scanning densitometry equipmentand I thank Dr. Bob Gawley for taking the time to train me in its use. Thanks alsoto the staff of the botany office, in particular, Tami and Judy for endless favours andinterest in my progress.Finally, a special thank you for the support I have received from the Vancouver branchof the Ellard clan, Fionnuala, Gerry, Conor and Fiona.xl’Chapter 1General introductionUnique to higher plants, the pathways of phenyipropanoid metabolism have long held theinterest of biologists. This thesis deals with regulation of the expression of a gene encoding 4-coumarate CoA:ligase, a pivotal enzyme of general phenyipropanoid metabolism.Among the compounds synthesised from this pathway are pigments, antimicrobial compounds (phytoalexins) and structural components of the cells wall, e.g. lignin, suberinand wall bound phenolics. The carbon skeleton for all these compounds is derived fromphenylalanine. Phenylalanine is synthesised via the shikimic acid pathway using phosphoenol pyruvate from the tricarboxylic acid cycle and erythrose-4-phosphate derivedfrom the pentose phosphate shunt (Davies et al., 1964). Phenylalanine made from thispathway can then enter into phenyipropanoid metabolism. The phenyipropanoid pathway can be divided into the core reactions of general phenyipropanoid metabolism anda variety of specific branch pathways leading to the production of specific end products(Figure 1.1).The first step in the general pathway is the deamination of L-phenylalanine by theactivity of phenylalanine ammonia lyase (PAL) to yield cinnamic acid. Cinnamic acid ishydroxylated to 4-coumaric acid by cinnamate 4-hydroxylase. The activities of hydroxylases and 0-methyl transferases upon 4-coumaric acid can yield derivatives of 4-coumaricacid e.g. ferulic acid (3’ methoxylated) and sinapic acid, (3’, 5’ methoxylated). The formation of CoA esters of these compounds is believed to be an important branch point1Chapter 1. General introduction 2LAVONOIDS [pFLAVANOlDS COUMAR1NS 1 [ SOLUBLE STERS1I /GENERAL PHENYLPROPANOID METABOLISMCOOK COOH COOH COSCoANH204H 4CLRXROH OHPhenyl- Cinnaniic 4-coumarc 4-coumaroyl-aIan-e Acid Acid CoA(R=RH)/_________________________________________[LIGNIN 1 L SUBERIN 1LOTHER WALL-BOUND PHENOLlC]1 STILBENESFigure 1.1: Schematic showing core reactions of general phenyipropanoid metabolism aswell as some of the major branch pathwaysChapter 1. General introduction 3between the general phenylpropanoid pathway and the branch pathways since these esters are used as substrates for the biosynthesis of products such as flavonoids and lignin.This step is catalysed by 4-Coumarate: CoA ligase (4CL) (Douglas et al., 1992).Complex requirements for phenyipropanoid metabolites during specific stages of development, in specific tissues, and in times of stress, must be met in plant cells. For example, developing tracheary elements in xylem tissue require production of lignin monomers.Flavonoid pigments of fruits and flowers are required in the differentiated cell types inthese organs, and production of phytoalexins and structural components of the cell wall(lignin, suberin and other wall bound phenolics) is an important adaptation to environmental stresses. The activation of phenylpropanoid metabolism in response to pathogenchallenge has been demonstrated in a number of systems including potato, bean, alfalfa, and soybean (reviewed by Hahibrock and Scheel, 1989). In the legumes, pathogenattack leads to the accumulation of isoflavonoid phytoalexins. This accumulation is preceded by an increase in mRNAs and proteins for enzymes of the general phenyipropanoidpathway and the flavonoid specific branch pathway (chalcone synthase and chalcone isomerase) (Dixon et at., 1992; Hahlbrock and Scheel, 1989; Dixon and Lamb, 1990). Insuspension-cultured bean cells treated with elicitor, defense genes involved in biosynthesis of phytoalexins are activated within two-three minutes of elicitor treatment (Lamb etat., 1989). This represents one of the most rapid gene activation systems in plant cellsin response to an exogenous sigiial. In the bean system, cinnamyl-alcohol dehydrogenase(CAD), an enzyme involved in lignin biosynthesis is also rapidly activated upon elicitortreatment (Walter et at., 1988). In hybrid poplar suspension cultures treated with anelicitor there are coordinate and transient increases in PAL and JCL mRNA, followed bya 10 to 20 fold increase in extractable PAL and 4CL enzyme activities (Moniz de S et at.,1992). The increase in enzyme activation is associated with an increase in extractable cellwall-bound phenolic metabolites. Wounding and pathogen attack drastically change theChapter 1. General introduction 4composition of cell walls in potato tubers. There is a slow wound-induced deposition ofsuberin phenolics (reaching maximum levels after 5-10 days), whereas suberisation causedby a pathogen attack occurs rapidly, within one day (Hammerschmidt, 1984). Direct evidence for the role of inducible defense responses in the expression of disease resistanceis provided by the observation that aminooxyphenyipropionic acid (a specific inhibitor ofPAL) renders soybean seedlings susceptible to normally avirulent races of Phytophthoramegasperma f. sp. glycinea (Pmg) (Dixon and Lamb, 1990). My interests lie in understanding how the requirements for phenyipropanoid metabolites subsequent to stress aremet at the level of gene regulation. Because of its pivotal role in the phenylpropanoidpathway I have chosen 4CL from parsley as a model gene for these studies.In parsley, in contrast to the above mentioned legume systems, the activation ofthe flavonoid specific branch pathway of phenyipropanoid metabolism in cells is a response that is specifically associated with UV light treatment and not elicitor treatment(Douglas, 1992). Parsley cells respond to UV irradiation with vacuolar accumulationof UV absorbing ilavonoids (Hahibrock et al., 1981; Matern et al., 1983). The key enzyme for entry into flavonoid synthesis is chalcone synthase (CHS) which catalyses theformation of naringenin chalcone by condensation of 3 molecules of malonyl-CoA with4-coumaroyl-CoA, the major product of 4CL (Hahibrock and Scheel, 1989). The parsleyCHS gene is present in a single copy, and has been studied extensively because of itspivotal metabolic role and its strong light-induced transcriptional activation (Chappelland Hahlbrock, 1984).Treatment with a fungal elicitor preparation from Pmg causes parsley cells in suspension culture to secrete a complex mixture of coumarin derivatives with antifungalactivity into the culture media (Tietjen et al., 1993; Hauffe et al., 1986). The linear furanocoumarins, marmesin and psoralen, their coumarin precursor, umbelliferone, and themethoxylated psoralen derivatives, xanthotoxin, bergapten, and isopimpinellin, have beenChapter 1. General introduction 5identified in the coumarin derivatives from cultured parsley cells. Essentially this samemixture of compounds accumulates in infection droplets of parsley leaves inoculated withspores of the Pmg fungus, and all of the furanocoumarins mentioned above are antibiotically active and considered to be potent phytoalexins in parsley (reviewed by Hahibrockand Scheel 1989). The biosynthetic pathway to the furanocoumarins is not as well characterised as the pathway to flavonoid synthesis. Nevertheless, antisera raised againstSAM:xanthotoxol 0-methyltransferase (XMT) and SAM:bergaptol 0-methyltransferase(BMT) which catalyse the final methoxylation of xanthotoxin and bergapten, were usedto isolate the parsley genes encoding these enzymes (Hauffe et al. 1988).Activated CoA esters which act as substrates for both of these pathways are thoughtto be formed by the catalytic activity of 4CL (Douglas et al., 1992). The 4CL enzyme wasfirst partially purified from UV irradiated cultures of parsley (Petroselinum hortense),by Knobloch and Hahlbrock (1977). They identified only one 4CL species in the finalenzyme preparation. It had a molecular weight of 67,000, exhibited an absolute requirement for ATP and was largely specific for 4-coumarate and other derivatives of cinnamicacid. Isolation of genomic clones and cDNA clones for 4CL from parsley (Petroselinumcrispum) in 1987 by Douglas et al. showed that 4CL is encoded by two distinct genes,4 CL-i and 2. Sequence analysis of these genomic clones showed that they are highlyhomologous; the gene sequences are over 95% identical in coding regions and introns, andthey display a high degree of sequence identity for several hundred basepairs upstream ofthe transcription start site. Using a gene-specific probe from an intron fragment specificto 4CL-2 and a fragment common to both genes as hybridisation probes against run-offtranscripts from elicitor or light-treated cells, it was shown that there is no differentialexpression of these two genes in parsley cells (Douglas et al., 1987). Comparison ofthe deduced amino acid sequences of the genes shows there are only three differencesin amino acid sequence between the two isoenzymes, and only one of these (asparagineChapter 1. General introduction 6versus aspartate) causes a charge difference. This small difference in primary structureenables the efficient separation of the isoenzymes on two-dimensional gels and on ion-exchange columns, but was apparently insufficient to allow separation of the two formsusing techniques available in 1977. By expression of 4 CL-i and 4CL-2 in E. coli it wasshown that the substrate affinities of the two enzymes are apparently identical (Lozoyaet al., 1988).The properties of 4CL and the genes which encode it have been studied in a numberof other organisms. In potato, similar to the situation in parsley, 4CL is encoded bytwo structurally similar genes, St4CL-1 and 2 (Becker-André et al., 1991). The observednucleotide differences in the coding regions result in three neutral amino acid differencesand one charge difference suggesting that the two encoded 4CL isoenzymes probablyhave similar properties. These authors showed that mRNAs from both of the potato4 CL genes accumulate to equal levels in suspension-cultured cells and whole plant tissueindependent of stress treatment or organ analysed. In other organisms in which theenzymatic properties of partially purified 4CL have been studied, the situation is quitedifferent. In soybean, petunia, pea, poplar and maize for example, there appear tobe different 4CL isoenzymes which display different substrate affinities for differentlysubstituted hydroxycinnamic acids (Knobloch and Hahlbrock, 1975; Wallis and Rhodes,1977; Grand et al., 1983; Vincent and Nicholson, 1987). Presumably, controlling thelevel of the different isoenzymes is a way in which the flow of hydroxycinnamic acidsto the various branch pathways of phenylpropanoid metabolism can be regulated. Insupport of this, three classes of cDNAs which may encode three 4CL isoenzymes insoybean have been isolated (Uhlmann and Ebel, 1993). Members of this gene family aredifferentially expressed in soybean cells treated with /3-glucan elicitors of Phytophthoramegasperma or in soybean roots infected with either an incompatible or compatible raceof the fungus. Recently, a number of different 4CL cDNAs have been isolated from aChapter 1. General introduction 7poplar cDNA library suggesting the existence of different gene family members in thisorganism (S. Allina and C. Douglas, unpublished results). In contrast, Arabidopsis (likeparsley) appears to contain only a single gene encoding 4CL (Lee, Ellard and Douglas,unpublished results). For the purposes of dissecting components of the transductionpathway involved in stress-induced expression of genes like 4CL, the essentially singlegene system reduces the complexity of the task since each parsley gene responds to avariety of stresses and developmental signals.Parsley PAL on the other hand, is encoded by a family of four genes and three ofthese are responsive to different stress stimuli and are over 90% similar to one another atthe nucleotide level (Lois et aL, 1989). In bean, PAL is also encoded by a family of genesand the three PAL genes encode distinct polypeptide isoforms (Liang et al., 1989). Thetranscripts from these genes exhibit markedly different patterns of accumulation leadingto the selective synthesis of functional variants of the enzymes in different situations.For example, under stress a form with a higher affinity for its substrate is preferentiallyinduced thus setting a priority for synthesis of phenyipropanoid products under theseconditions. Another feature of particular interest in the bean system is the apparentregulation of PAL transcript accumulation by pathway intermediates (Mavandad, et al.,1990). Accumulation of PAL transcripts in elicitor-treated bean cells is repressed byrelatively high concentrations of trans-cinnamic acid, the immediate product of the PALreaction, whereas low concentrations increase PAL.As might be predicted based on our knowledge of the end products of the phenylpropanoid pathway with their diverse functions in development and their response tonumerous environmental stresses, regulation of the expression of 4CL and other phenylpropanoid genes is tightly controlled. Developmentally regulated patterns of expressionmust be integrated with tissue-specific requirements for phenyipropanoid products andto add to the complexity, there is a rapid and transient accumulation of phenyipropanoidChapter 1. General introduction 8products subsequent to stress. Using in vitro run-off transcription, it was shown thatthe accumulation of PAL, 4 CL, CHS and BMT in response to elicitor and UV light iscontrolled largely at the level of transcription (Chappell and Hahibrock, 1984; Douglas etal., 1987; Lois et al., 1989; Hahlbrock and Scheel, 1989). This is followed by mRNA accumulation and increased enzymes levels (Hahlbrock and Scheel, 1989) with PAL and 4CLregulation occurring in a coordinate manner at all levels, and activation of the branchpathway enzymes e.g. BMT and CHS occuring later.Schmelzer et al. (1989) used in situ hybridisation to look at the temporal and spatialpatterns of expression of some elicitor inducible genes, including PAL, 4CL and BMT in awhole plant system, parsley seedlings. In uninfected tissue, BMT expression is confinedto oil-duct epithelial cells. (Parsley plants constitutively secrete furanocoumarins intothe lumen of oil ducts: the function of this is not known). PAL and 4 CL are expressedconstitutively in epidermal tissue (where there is accumulation of flavonoids), in oil ducts,and in developing xylem. In response to Pmg infection PAL, CL and BMT expressionis induced to high levels at the site of infection. Wounding causes accumulation of 4CLand PAL mRNA, however, the wound response is more diffuse than that seen after Pmgchallenge i.e., there is not a sharp border between the unaffected area and the area wheretranscripts are induced, as is the case with pathogen infection. This work was extendedby Wu and Hahibrock (1992) who showed activation of CHS gene expression in theepidermis and also showed that the expression of all genes in developing parsley seedlingwas dependent on light. Thus, in cell cultures and in plants, these genes must respondto a complex array of signals to ensure that phenyipropanoid compounds are synthesisedwhen required.Regulation of gene expression can occur at a number of levels including initiation oftranscripts, mRNA stability, mRNA processing or translation. However, most gene regulation occurs at the transcriptional level (Alberts et al. 1989). How genes are regulatedChapter 1. General introduction 9transcriptionally has been reviewed by a number of authors (Ptashne et al., 1988; Berkand Schmidt, 1990; Clover, 1989). Regulation of transcription is thought to occur by thebinding of specific proteins factors within the nucleus (trans-acting factors) to DNA binding regions (cis-acting elements). These DNA sequence elements are 6-20 bp long and aresituated within several kilobases of transcription initiation sites. DNA/protein interactions at these elements lead mostly to activation of transcription rather than repression.For example, a cis-acting element found within the promoters of many plant genes isthe C-Box (C/A-CACGTGGC) present in the photoregulated rbcS-1A (small subunitof ribulo.se-i, 5-bisphosphate carboxylase/oxygenase) promoter and the root-specific adh(alcohol dehydrogenase) promoter (Chang and Meyerowitz, 1986; Donald and Cashmore,1990). This sequence is also present in light regulated chs promoters (Schulze-Lefert etal., 1989; Staiger et al., 1989). Another important cis-acting element identified withina chs promoter is the H-Box element [CCTACC(N)7] which has been suggested tobe of importance in the response of this gene to stress and developmentally regulatedexpression of the bean chsi5 promoter (Yu et al., 1993). A H-box and G-Box elementact in combination to control the expression of one of the members of the bean chsi5gene family in response to the phenyipropanoid intermediate para-coumaric acid (Loakeet al., 1992).Although CHS is the best characterised of the phenylpropanoid gene in terms of itscis-acting sequences, a number of elements within other phenylpropanoid genes havebeen identified. Transcriptional activation of PAL-i is associated with the appearance ofthree inducible in vivo footprints. Two of these occur in response to both UV light andelicitor and the third is seen only in response to elicitor (Lois et al., 1989). Expression of4 CL-i in response to elicitor, UV light and tissue-specific signals revealed a separation ofcis-acting elements mediating stress and tissue-specific regulation. Exonic sequences arerequired in conjunction with promoter sequences for expression in response to UV lightChapter 1. General introduction 10and elicitor (Douglas et al., 1991). In contrast a 210 bp fragment of the same promotermediates tissue-specific and developmentally regulated expression of this gene (Hauffe etal., 1991) and a number of putative elements (both positive and negative) within the4 CL-i promoter have been identified which control spatial patterns of expression of thisgene in transgenic tobacco. Consistent with the co-ordinate regulation of PAL and 4CL,some of the elements thought to control expression of these genes appear to be conservedbetween the two genes.Trans-acting factors which control the initiation of transcription by RNA polymeraseII can be divided into two groups, general factors and activators (Holdsworth et aL,1992). General transcription factors are responsible for the assembly of the preinitiation complex at the “TATA” box and control constitutive expression. Activators area heterogeneous class of sequence-specific DNA-binding proteins that interact with thepreinitiation complex to bring about high regulated levels of expression. A number ofclasses of transcription factors or trans-acting factors have been identified in eukaryotes(Glover et al., 1989) and these trans-acting factors have been characterised accordingto their characteristic binding motif e.g., 11TH (helix-turn-helix) characterised by twoopposed c-he1ices joined by a turn of the helix, zinc finger containing a number of cysteine or histidine residues (or a combination of both) for binding of a zinc ligand and theleucine zipper motif characterised by the presence of two amphipathic a-helices whichadhere to each other to make a dimer forming region—hence the zipper analogy. Untilvery recently, our knowledge of transcription factors came solely from animal and fungalsystems. In the last few years, more that 40 cDNA clones encoding putative transcription factors have been isolated from plants (reviewed by IKatagiri and Chua, 1992). Forexample, a number of these proteins belong to the bZIP (basic domain/leucine zipper)class of transcription factors which contain a basic region, where the interaction with theDNA occurs, and a leucine zipper element. A plant leucine zipper protein specificallyChapter 1. General introduction 11recognises an ABA response element within a wheat Lea gene (late embryo abundant)(Guiltinan et al., 1990). One of the best characterised examples of a leucine zipper protein is the TGA1a tobacco bZIP transcription activator, which binds to the activationsequence element of the cauliflower mosaic virus 35S promoter (Katagiri et al., 1992). Ahuman in vitro transcription system (based on HeLa cell nuclear extracts) was used todemonstate the ability of this binding factor to increase the number of initiation complexes (Katagiri et al., 1990). Another less well characterised example is the recentlyidentified bZIP protein OHP1 from Maize which interacts with opaque 2, a regulatorygene controlling zein storage protein deposition (Pysh et al., 1993). Transcription factorsof the bZIP class may play a role in light-induced expression of the parsley CHS gene.The appearance of three light dependent in vivo footprints on this promoter is correlatedwith the onset of CHS transcription (Schulze-Lefert et al., 1989a and b). One of thesefootprints, Box II, contains a 0-box sequence and three parsley cDNAs encoding bZIPproteins which interact in a sequence specific with this element have been isolated. Theseproteins were named CPRF’s (Common Plant Regulatory Factors) and in support of afunctional role for these factors, the massive light mediated increase in CHS mRNA ispreceded by induced expression of one of these genes, CPRF-i.A parsley DNA binding protein (BPF-1) that is involved in stress induced phenylpropanoid gene expression has recently been identified (da Costa e Silva et al., 1993).This protein is expressed in response to pathogen attack and binds to the PAL-i promoter. This protein specifically binds a cis-acting element called Box P. This P box ispresent in all known PAL and 4CL promoters analysed to date and it may play a role inthe coordinate induction of expression of these two genes. There is also a very precisecorrelation between the pattern of expression of BPF-1 and expression of PAL and 4 CL.BPF-1 mRNA is induced by elicitor prior to induction of PAL expression. This is strongevidence thatBPF-i may play a key role in regulation of expression of PAL and 4CL inChapter 1. General introduction 12parsley. This is the first report of a trans-acting factor that may be involved in inducibledefense responses.This thesis is concerned with the regulation of CL expression in response to stress.My interests lie in elucidating at least some aspects of the signalling pathway from thedetection of the stress signal to the genetic regulatory apparatus. I used two heterologoussystems in this work, a transgenic tobacco system and a transgenic Arabidopsis systems.I also used parsley cells in suspension culture to investigate transduction signals in thestress response of 4 CL. The chapters are divided accordingly.Chapter 2Experimental procedures2.1 401-i-GUS fusionsThe 4 Cl-i-GUS fusions used in this study were described by Hauffe et al. (1991). Briefly,they consist of 4 Cl-i promoter fragments as transcriptional fusions upstream of theGUS gene in pRT99-GUSJD/Kozak, a derivative of pRT99-GUS-JD (Schulze-Lefert etal., 1989) that contains a consensus eukaryotic (“Kozak”) translation start site (Kozak,1981).2.2 Generation of transgenic tobacco plantsI cloned 4 Cl-i-GUS fusions as EcoRi-Hindill fragments into BIN19 (Bevan, 1984), andintroduced them into Agrobacterium t’amefaciens by triparental matings with Escherichiacoli strains (Ditta et al., 1980). The structure of all BIN 19 constructions in Agrobacteriumwas confirmed using the screening method of Ebert et al. (1987). I transformed tobaccoleaf discs, and generated plants, by standard methods (Horsch et al., 1985).2.3 Plant treatmentsPlant treatments described were performed in a minimum of two independent experiments and the data presented represent typical results obtained.13Chapter 2. Experimental procedures 142.3.1 Wounding experimentsI wounded excised leaves by slicing tissue into 1-2mm strips, and incubated the woundedtissue on filter paper moistened with MS (Murashige and Skoog) media (Gibco laboratories) in the absence of hormones. For this and all other Arabidopsis and parsleyexperiments, I harvested at least one whole plant for each time point used. In tobacco,however, I used a small number of leaves for each time point. This made it necessaryto control for the effects of developmentally regulated 4CL expression by distributingleaves of different developmental ages among sample points. For the 0 hour time point, Iexcised leaves and immediately froze them in liquid nitrogen without further wounding.2.3.2 Ethephon and ABA treatmentI sprayed to run off plants that were fully grown and non-flowering with a 10 mg/mlsolution of 2-chlorethylphosphonic acid (Sigma), and enclosed them in plastic bags. ABA(Sigma) was dissolved in 0.01% ethanol and sprayed on leaves until run-off; I sprayedcontrol plants with 0.01% ethanol alone and harvested tissue after incubation at 22°Cfor the periods indicated.2.3.3 Treatment of plants with methyl jasmonate, jasmonic acid and linolenic acidStock solutions of methyl jasmonate (Bedoukian Research Inc., Danbury, CT) and jasmonic acid (Apex Organics, UK) were dissolved in 1% Triton, and linolenic acid (Sigma)was emulsified by sonication in 1% Triton immediately prior to use (Farmer and Ryan,1992). I treated plants by spraying until run off with a solution of the test compound ora 1% Triton solution (controls). Due to its volatility, methyl jasmonate-treated plantswere enclosed in a bell jar (other treatments were left uncovered) and treatments wereChapter 2. Experimental procedures 15conducted in constant light at 28°C until harvest of tissue.2.3.4 Remote treatment of tobacco with MJI placed young tobacco plants in an airtight chamber together with 9 cotton tippedsticks/plant. Each stick had been dipped in 500 pls of a 1/1,000 dilution of methyljasmonate, diluted in 95% ethanol to speed vapourisation. I treated the control plantswith 95% ethanol alone.2.3.5 Treatment of tobacco plants with n-propylgallateI placed petioles of excised tobacco leaves in n-propylgallate (3,4,5-trihydroxybenzoicacid n-propyl ester) (Sigma) dissolved in 0.2 mM potassium phosphate, p11 7, for 12hours (described by Staswick et al., 1991). Control leaves were incubated in buffer. Ithen either wounded leaves and incubated them on MS media for 24 hours as describedabove (section 2.3.1), or immediately froze them in liquid nitrogen. In contrast to allother plant treatments the results presented for this experiment represent data from asingle experiment.2.3.6 Treatment of tobacco with elicitorI treated plants with Fmg elicitor as described by Douglas et. al., 1991. Briefly, I placedpetioles of groups of excised leaves in a solution of 500 ig/ml Pmg to allow uptake, andharvested them after 2 hours. Control leaves were placed in water alone.Chapter 2. Experimental procedures 162.4 Parsley cell culture growth and treatments2.4.1 Growth of cell culturesParsley cell suspension cultures were grown in the dark in modified B5 media as describedby Ragg et. al., (1981). Cells were subcultured weekly by transferring 28mls of cells into200 ml of media in a 1 L Erlenmeyer flask. I performed all treatments 5 days aftertransfer to new media.2.4.2 Treatment of cell cultures with elicitorAll elicitor treatments were carried out using 1O-2Oml aliquots of cells which had beenaseptically transfered to a 250 ml Erlenmyer flask. I treated cells with aliquots of Pmgelicitor (made as described by Ayers et al. 1976). Elicitor was kindly provided by KlausHahlbrock and Shona Ellis. The elicitor was dissolved in sterile water and added tothe cells to a final concentration of 50 g/ml. I incubated cells in the dark at 28°Cwith continuous shaking (110 rpm) and harvested them, at the time points specified, byfiltration through a Buchner funnel. Cells were used for RNA analysis and media fromtreated cells was used for extraction of total coumarins.2.4.3 Isolation and analysis of total coumarins from cell culture mediumI isolated coumarins from culture medium and performed TLC, as described by Kombrink and Hahlbrock (1986), with the following modifications. For quantification, afterextracting coumarins into chloroform, I immediately read the OD32onm without dilutionor further rotary evaporation. For thin layer chromatography, extracts were evaporatedto dryness and coumarins resuspended in lml chloroform.Chapter 2. Experimental procedures 172.4.4 Treatment of cell cultures with methyl jasmonate, jasmonic acid, linolenic acid and ABAI prepared solutions as in Section 2.3.3, and conducted treatments as follows: I asepticallytransferred 10-20m1 aliquots of cells to 125 or 250m1 Erhlenmeyer flasks to which thetreatment compound had been added. I treated control cells with solvent alone. Cellswere returned to the dark at 28°C with constant shaking for the incubation period.Methyl jasmonate treated flasks (because of its volatile nature) and their controls wereenclosed in plastic bags for the treatment period.2.4.5 Treatment of cell cultures with inhibitorsI used the following lipoxygenase inhibitors in the parsley cells: ibuprofen (a-methyl-4-4[2-methylpropyl] benzeneacetic acid), n-phenylbutazone (4-butyl- 1 ,2-diphenyl-3,5-pyr-olidinedione), n-propylgallate (3,4,5-trihydroxybenzoic acid n-propyl ester), salicylhydroxamic acid (n ,2-dihydroxybenzamide) and antipyrene (2,3-dimethyl-1-phenyl-3-pyra-zolin-5-one). I prepared stock solutions of inhibitors dissolved overnight in sterile 0.2Mpotassium phosphate, p11 7.0 as described by Staswick et a!. (1991) and added themto 10-20 ml aliquots of cell cultures at a final concentration of 50 pM 16-l8hrs beforeelicitor experiments and returned cells to the dark with constant shaking. Control cellswere treated identically but with buffer alone. All inhibitors were from Sigma.2.5 RNA isolation and analysis2.5.1 RNA and poly A RNA isolationRNA was isolated from frozen tissue ground to a fine powder in a mortar and pestle. Iextracted 1-5 g ground tissue in 5-10 ml guanidiniuin-HCL extraction buffer consistingof 8 M Guanidinium HCL, 20 mM MES (2[N-Morpholino]ethanesulfonic Acid) bufferChapter 2. Experimental procedures 18and 20 mM EDTA, pH 7.0. After thawing the mixture it was extracted once in anequal volume of phenol-chloroform (1:1) and once in an equal volume of chloroform. Iprecipitated nucleic acids in the aqueous phase by adding 0.05 volumes 1 M acetic acidand 0.7 volumes 95% ethanol and I selectively precipitated RNA from this mixture ofnucleic acids using 2 M lithium chloride, 10 mM sodium acetate (Logemann et al., 1987).Poly A RNA was prepared by oligo(dT)-cellulose affinity chromatography (Sambrooket al., 1989).2.5.2 Northern and slot blotsI separated RNA (10 jtg/lane) on 1.2% agarose gels in 1 X MOPS (3-[N-Morpholino]pro-pane-sulfonic acid) buffer (20 mM MOPS, 5 mM NaOAc, 1 mM EDTA) and 7.4%formaldehyde. Gels were run in 1 X MOPS buffer and subsequently thoroughly washed(to remove formaldehyde) by shaking for 40-60 minutes in several changes of distilledwater. I stained gels in 2 ig/m1 ethidium bromide and destained in distilled water to ensure equal loading of lanes. Blotting and hybridisation to either Hybond N (Amersham)or Genescreen plus (Du Pont) was done according to the manufacturer’s specifications.When slot blots were used, these were loaded onto Hybond N (Amersham) using a Mini-fold II apparatus (Schleicher and Schuell). A random primer labelling kit from Boehringerwas used to prepare radioactive hybridisation probes.Hybridization probes were prepared using inserts purified from cDNA clones afterdigestion with EcoRI, unless otherwise noted. Parsley CL probes were made using the2kb insert of the 4 CL-i cDNA (Douglas et al., 1987; Lozoya et al., 1988). I detectedendogenous tobacco 4CL RNA using probes made from the 2kb insert of a potato 4CLcDNA (Becker-André et al., 1991) or a mixture of 2 cDNAs for tobacco 4CL (Nt4CL)(Diana Lee, unpublished results). Parsley HRGP, TyrDC E1i3, E1i9, and Ubi4 probesChapter 2. Experimental procedures 19were made using inserts from cDNA clones described by Somssich et al. (1989). The original designations of TyrDC, HRGP, and Ubi4 were Eli 5, ELi9, and CON2 respectively(Somssich et al., 1989). Parsley PAL probes were made using the 1.4kb insert from aPAL cDNA clone (Lois et al., 1989); BMT probes were made using a 2kb HindIII-EcoRIinsert from a BMT cDNA (Hahlbrock and Scheel, 1989). The insert of a tomato cDNAclone (a Sal I, EcoRl fragment) encoding ubiquitin (a gift of Luca Comai, Universityof Washington) was used to prepare probes for the detection of tobacco ubiquitin RNA.GUS probes were made using a 2kb GUS fragment isolated after HindIII-EcoRI digestion of pRT99-GUSJD (Schulze-Lefert et al., 1989). I carried out hybridisations in asolution containing 6 X SSC, 0.5% SDS, 5 X Denhardts solution and 0.01 M EDTA at68°C for 14-16 hours (Sambrook et al., 1989). I carried out high stringency washes at68°C in 0.2 X SSC and low stringency washes at 68 °C in 2 X SSC. Except as notedbelow, washes after hybridisations with homologous probes were at high stringency andwith non-homologous probes at low stringency. After hybridisation with the tomatoubiquitin probe, washes were carried out at high stringency and after hybridisation withGUS probes, washes were performed at low stringency.2.6 Histochemical localisation of GUS activityGUS activity in wounded tissue was localised by a histochemical assay for GUS activity(Jefferson, 1987). I vacuum infiltrated the tissue in 0.5% paraformaldehyde, 100 mMsodium phosphate (pH 7.0), washed thoroughly in 100 mM sodium phosphate (pH 7.0)and subsequently stained Arabidopsis leaf pieces for 3-10 hours in 0.125 mg/mi 5-br-omo-4-chloro-3-indoleglucuronide (X- GLUC, Clontech, Palo Alto, CA). After stainingthe tissue was cleared in 95% ethanol.Chapter 2. Experimental procedures 202.7 Mutagenesis and screening of ArabidopsisArabidopsis seeds for mutagenesis were obtained by selfing of the line 204-1-3 (homozygous for a 1500 bp parsley 4 CL-i promoter- GUS fusion R. Moselei and C. Douglas,unpublished results) and mutagenesis was carried out by placing 20,000 dry seeds in 100mls of 0.3% ethyl methane sulfonate (EMS) (Sigma). After incubation (with occasionalmixing) for 12 hours, I washed the seeds 15 times over the course of 3 hours with distilledwater. I then sowed the seeds at a density of one per cm2 in 12 cm square pots andharvested seeds in bulks of 50 plants. I sowed 1,000 seeds from each bulk and screenedthem when they were four weeks old. The screening was conducted as follows: Woundedleaf segments were obtained by excising leaf fragments with a scissors and then pinchingthe tissue with a blunt forceps. I incubated wounded leaf segments for 24 hours in MSmedia (no hormones) in 4 X 6 well microtitre plates. Tissue was stained with X-Glucand cleared in the microtitre plate (without fixing) as described in Section 2.6.Chapter 3Expression of.4 CL-i in response to wounding in transgenic tobacco3.1 IntroductionIn nature, plants are frequently subjected to wound stress from factors such as wind,herbivores, insects, fungi and other pathogens. Normal developmental processes withinthe plant, e.g., abscission and growth cracks as well as human activities, such as pruningand ringing, also create wounds. The resulting loss of compartmentalisation and increasedsusceptibility to pathogen attack and water loss pose a great threat to the health of theplant. Unable to flee such threats, plants have evolved a complex set of physiological andbiochemical responses which occur in the neighbouring stressed but unbroken cells.The responses of plants to wounding can for convenience be divided into those thatare immediate, occurring in a matter of minutes after wounding, and those which areslow, occurring in a matter of hours. The immediate responses include changes in membrane structure and function. Fatty acids are released from the membrane and can beoxidised to ethylene, ethane and a variety of other compounds (Bostock 1989; Davies etal., 1987). The action of lipoxygenase on the polyunsaturated octadecanoid fatty acidslinolenic and linoleic acid released from damaged cell membranes, results in the formation of a host of lipid catabolites, including C6 volatiles (Hamberg and Gardner, 1992;Vick and Zimmerman, 1987a and b). Some of these compounds are responsible for thecharacteristic odours of freshly cut or damaged plant tissue (Siedow, 1991) and Croftet al., (1993) suggest that these volatile products play an antipathogenic role. Changes21Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 22in membrane ion fluxes result in a net increase in Ca2+ influx and C1 and K+ efflux,suggesting a collapse in the electrogenic H+/K+ATPase in the plasmalemma (Davieset al., 1987). There is a rapid activation of callose synthase, resulting in the depositionof callose at the wound site (Davies et al., 1987), and an activation of wall associatedpolysaccharidases that lead to the release of oligosaccharins (Albersheim and Darvell,1985). Wounding also leads to an increase in the biosynthesis of ethylene, “the woundhormone”, the evolution of which has long been associated with stress in plants (Bostockand Sterner, 1989).The second group of responses (the slow responses) for the most part are controlled atthe level of gene expression. Changes in plant gene expression associated with woundinggenerally fall into two categories in terms of spatial patterns of induction. Genes whoseexpression is induced in a localised manner in the plant are involved in strengthening ofthe cell wall surrounding the wound site or preventing opportunistic pathogen attack, orboth. There have been reports of wound-inducible expression of genes encoding at leasttwo classes of proteins that play a structural role in the cell wall. The best characterisedclass are the hydroxyproline-rich glycoproteins (HRGPs), represented by extensin (Saueret al., 1990). Another class of wound-inducible proteins are the proline- or hydroxyproline-rich proteins (PRPs), and genes encoding these are expressed in response towounding in carrot roots and soybean hypocotyls (Sheng et al., 1991). In light of thevital role played by compounds derived from the phenyipropane skeleton as structuralcomponents of the cell wall, it is hardly surprising that the genes of the phenyipropanoidpathway are also induced upon wounding, resulting in the production of suberin andlignin monomers to protect against mechanical damage. In addition, genes encodinganionic peroxidase, the enzyme which may catalyse polymerisation of lignin monomersfrom the phenylpropanoid pathway, are inducible by wounding (Mohan et al., 1993).Other genes whose expression is induced in a localised manner in response to woundingChapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 23include those encoding lytic enzymes capable of degrading fungal cell wall material, e.g.,chitinase and /3-1,3-glucanase (Simmons et al., 1992).Wound-inducible gene expression that occurs throughout the plant is said to be systemic and implies the existence of a transmissible signal to communicate to tissues distalfrom the wound site. Genes whose expression is induced systemically in response towounding include the win (wound-induced) genes from poplar (Davis et al., 1991) andthe win and wun gene families identified in potato (Stanford et al., 1989; Logemann etal., 1989). The functions of these gene products are unknown, although the win-S poplargene shows sequence similarity to trypsin and Kunitz-Type proteinase inhibitor genes(Bradshaw et al., 1990). Despite uncertainty about the function of these genes, theirexpression patterns have been described and promoter elements mediating the responseto wounding identified (Siebertz et at., 1989).By far the best characterised change in gene expression in response to woundinginvolves the systemic expression of proteinase inhibitor (P1) genes identified in the Solanaceae. Fl genes encode potent inactivators of proteolytic enzymes. There are two classesof PT proteins that have been isolated and characterised in potato tuber (reviewed byRyan, 1992). They are encoded by two nonhomologous gene families; the PT-I class have amolecular mass of 8,000kDa and inhibit chymotrypsin while the second class (PT-il) havea reactive site for both trypsin and chymotrypsin and are larger, with a molecular mass of12,000kDa. Several lines of evidence show that these compounds are a chemical defenseagainst Lepidopteran insect attack. Firstly, P1 proteins are not normally present in leaves,yet, upon wounding they accumulate to extraordinarily high levels. Upon multiple woundtreatments, levels of PT-I and -II mRNA in the the Bonny Best variety of potato canaccount for 1% of the total poly A mRNA population (Ryan, 1992). Secondly, theseinhibitors trigger physiological feedback mechanisms in insects which results in themfeeling prematurely satiated and starvation of the animal results. The most convincingChapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 24evidence, however, for a role for these compounds as a defense against herbivory lies inthe observation that transforming plants with P1 genes under the control of a constitutivepromoter results in increased resistance to attack by lepidopteran insects (Hilder et al.,1987). Proteinase inhibitors are present constitutively in tubers and a unique P1-I genethat is expressed in unripe developing fruit has been identified (Wingate et al., 1991).It has been suggested that in tomato species with vines growing near the ground, PTproteins prevent small mammals, birds, and insects from eating the fruit before the seedsare fully developed. Later, the level of inhibitors decreases and the fruit is more likely tobe eaten and thus dispersed (Pearce et al., 1988).Many genes involved in defense responses are encoded by multigene families, and thedifferent transcripts encoded by these genes accumulate in response to different stresses,e.g., HRGP (Sauer et al., 1990) and wun genes (Logemann, 1988). PAL genes are differentially regulated in response to different environmental cues (Liang et al., 1989). Recently,patterns of expression after wounding for two pro-rich protein genes were described thatdiffer distinctly in timing and location of transcript accumulation (Suzuki et al., 1993).In bean hypocotyl, three HRGP transcripts are induced by wounding, each having a distinct pattern of mRNA accumulation (Corbin et al., 1987). Differential regulation is not,however, always the rule. The activity of a single potato proteinase inhibitor pin2 promoter closely mirrors the pattern of expression of the whole gene family (Keil et al., 1989;Peña-Cortés et al., 1991). The analysis of this pin2 promoter represents the best exampleof functional analysis of a wound inducible gene. Even though tobacco has no pin2 gene,wound-inducible expression of potato pine is faithfully reproduced in transgenic tobacco,enabling identification of elements required for wound-inducible expression of the P1-Ilpromoter (Lorbeth et al., 1992). Two elements direct wound-inducible expression: anupstream quantitative element conferring maximal levels of expression upon wounding,and a wound regulatory element downstream from this quantitative enhancer region. AChapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 25separate regulatory element directs constitutive promoter activity in flowers. A region ofthe P-IlK gene that both binds a protein factor present specifically in nuclei of woundedtissue and is necessary for wound inducible gene expression has been identified (Palm etal., 1990).Identifying the cis-acting elements of wound-inducible genes, however, represents butone approach to understanding the mechanism of wound-inducible gene expression. Another aspect of the process that needs to be elucidated and integrated with this information is the nature of the wound signal within the plant cell, and in the case of systemicallyinduced genes, the nature of the transportable wound signal.The earliest wound signal identified in plants, “Riccas factor,” was described by Riccain 1916 (Davies, 1987). This factor is made in response to wounding in injured tissueand travels in the transpiration stream. The chemical nature of this compound has neverbeen elucidated despite its obvious importance. Another potential wound signal, researchinto which is now apparently out of vogue, is traumatin, the wound hormone originallyidentified as trans-2-dodecenedioic acid by English et al. (1939). More recently, traumatin has been identified as 12-oxo-trans-10-dodecenedioic acid which could potentiallybe generated from membrane lipids broken down as a result of wounding (Zimmermanand Coudron, 1979).Although there is no longer mention of traumatin in the literature, the importanceof lipid-based signalling is apparent as a result of the work of Ryan’s group on thewound inducible expression of proteinase inhibitor genes. The lipid-derived, volatileplant compound methyl jasmonate (MJ) is capable of inducing synthesis of proteinaseinhibitors in plant tissue (Farmer and Ryan, 1990). Moreover, incubating tomato plantsin the same chamber as Artemesia tridentata (sagebrush), which produces large amountsof MJ, was sufficient to cause systemic transcriptional activation of these genes. MJand the free acid, jasmonic acid (JA), are present in most organs of most plant speciesChapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 26(Meyer et, al., 1984). In plants, JA [3 ,oxo-2- (2- cis-pentenyl)-cyclopentane- 1-acetic acid]is synthesised from -lino1enic acid by a lipoxygenase-mediated oxygenation, followedby additional modifications (Vick and Zimmerman, 1987a and b). Intermediates in thepathway to JA biosynthesis, as well as linolenic acid (LA), similarly activate the synthesisof proteinase inhibitors (Farmer and Ryan, 1992). Oligogalacturonides isolated fromtomato leaf cell walls applied through cut petioles of excised plants, can induce theexpression of the proteinase inhibitor genes in leaves in the absence of severe wounding(Bishop et al., 1984). These compounds were originally thought to be candidates for asystemic wound signal, however, this hypothesis was refuted with the demonstration thatradioactively labelled oligogalactouronides are not transported throughout tomato plantswhen placed on wounds in leaves ( Baydoun and Fry, 1985). Then followed the isolationand characterisation of systemin, the first polypeptide hormone from plants (Pearce et al.(1991). This 18 amino acid molecule has all the characteristics of a systemic wound signal.When applied through cut stems this polypeptide induces systemic PT accumulation ina manner similar to wounding. It is 10,000 times more potent than oligouronides, activeat concentrations in the fmole range (Ryan, 1992). Previously thought of as simplemolecules, lacking the complexity of animal peptide signalling molecules, the isolation ofsystemin has changed the way we view plant hormones.It has been suggested that abscisic acid (ABA) plays a role in the stress inducibleexpression of P1 genes. Potato mutants deficient in the synthesis of ABA fail to accumulate pin2 mRNA in wounded leaves and this correlates with the absence of the risein endogenous leaf ABA concentration, which normally occurs in wild-type plants uponwounding (Peña-Cortés et al., 1989). While this work supports some role for ABA inthe wound response, it has been suggested that it may be acting in an indirect manner(Ryan, 1992). Three observations support this hypothesis: firstly, deletion of a consensusABA responsive element (ABRE) within the promoter region responsible for activity inChapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 27Herbivores Patbogens(wounding) Localized__Systemic I Signals OtQowoSignals ysteninI .captor Plasma Membrane ReceptorUnolenlc AcidLOXDehydrase6-OxidationJasmotc Acid I(Methyl Jasmonate)JAReceptor Ji p’Gene ActivationProtelnastlnhllbltorsFigure 3.2: A model proposed by Farmer and Ryan (1992) for wound inducible expressionof proteinase inhibitor genes. See text for details.wounded leaves failed to affect pin2 inducible promoter activity (Lorberth et al., 1992),secondly, pin2 transcription is not affected by water deficit despite the high rise in theendogenous ABA concentration apparent after drought (Peña-Cortés et al., 1989), andfinally, it has been pointed out that although ABA is an inducer of pin2 mRNA in leavesof normal potato plants, it does not similarly affect tomato Fl mRNA accumulationdespite the fact that both are similarly affected by wounding (Ryan, 1992). The role ofABA in this system therefore remains unclear.The work on the response of Fl genes and the discovery of systemin has been synthesised into a model (see Figure 3.2) for stress inducible gene expression, which I have usedas a paradigm for this work on wound-inducible expression of 4 CL-i. Briefly, the modelsuggests that oligouronides are a signal in localised wound responses, while systemin isthe signal in systemic wound responses. The release of these signals somehow (possiblyChapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 28via a lipase) causes release of LA from the membrane, which gives rise via lipoxygenase to de novo synthesis of JA, which mediates gene activation by an as yet unknownmechanism.Since the discovery that MJ mediates wound-inducible Fl gene expression, it hasbeen implicated in the wound-inducible expression of other genes. Mason and Mullet(1990) demonstrated MJ-induced accumulation of soybean vegetative storage proteins(vsp’s) and suggest that this response is mediated by de novo synthesis of JA as a resultof release of linolenic acid from the cell membrane after wounding. In support of thishypothesis, inhibitors of the JA biosynthetic pathway decreased vsp mRNA induction inresponse to wounding but not in response to MJ (Staswick et al., 1991).Two novel wound-inducible genes whose expression is also induced in response tojasmonates and ABA have been recently identified in potato (Hildmann, et al., 1992).One of these genes has a high degree of identity to bacterial aminopeptidase and is thefirst example of the involvement of a proteinase in plant defense mechanisms. It has beenhypothesised that the wound response of this gene plays a role in intracellular proteinturnover to provide a flow of aminoacids from existing into newly synthesised proteins.The second gene identified appears to encode a biosynthetic threonine deaminase whichmay be involved in biosynthesis of amino acids of the aspartate family. This gene ishighly expressed in floral tissue and also in response to wounding and jasmonates. Atomato gene encoding threonine deaminase has been isolated (Samach et al., 1991) andits expression is similarly high in floral tissue and is induced in leaf and floral tissuein response to jasmonates (Samach, 1993). The role of threonine deaminase in plantdefense is unclear. It has been pointed out that it is difficult to see how overproductionof isoleucine can defend plants against pathogens and therefore threonine deaminase mayparticipate in another metabolic pathway leading to products participating in the plant’sdefense response (Samach, 1993).Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 29In contrast, it is apparent how the production of phenyipropanoid products aroundwound sites could be of adaptive value, providing structural barriers and antimicrobialcompounds to ward against potential pathogen attack. Parsley 4 CL transcripts accumulate in a localised manner around wound sites in parsley plants (Schmelzer et al.,1989). What kinds of compounds mediate this response has not been addressed. MJhas been implicated in wound-inducible expression of a soybean CHS gene and there isa correlation between the accumulation of jasmonates in soybean hypocotyls and induction of CHS gene expression (Creelman et al., 1992). Treatment of parsley cells withthe jasmonate precursor, 12-oxo-phytodienoic acid, causes increases in the accumulationof mRNA for 4CL, CHS, and PAL (Dittrich et aL, 1992). Whether these data reflect afunctional role for jasmonates in the wound response has not been investigated.The other example in the literature of a gaseous wound signal or hormone is ethylene.One of the earliest events detectable during plant stress is a rapid increase in ethylenebiosynthesis as a result of the activity of the biosynthetic enzyme for ethylene, ACC synthase (S-adenosyl-L-methionine methylthioadenosine-lyase) (Bostock and Sterner, 1989).An interesting approach to assessing the role of ethylene in wound-inducible gene expression in tomato pericarp has been described (ilenstrand and ilanda, 1989). Usingcompetitive inhibitors of ethylene action, the level of translatable products from poly ARNA from wounded tissue treated with the inhibitor was compared with that of poiyA RNA from wounded tissue which had not been treated with the inhibitors. It wasestimated that less that 15% of mRNA species induced in wounded tissue is affected bythe presence of inhibitors. Such an approach, however, does not include genes whoseexpression may be regulated by levels of ethylene that are low enough to be availabledespite the presence of the inhibitors, so it may be an underestimate of the effect ofethylene on wound-inducible gene expression. The expression of a number of the woundinducible genes introduced in this chapter is also induced by ethylene. They are: beanChapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 30chitinase (Brogue et al., 1986), /3-glucanase (Simmons et al., 1992), HRGFs (Ecker andDavis 1987; Lawton and Lamb 1987; Ryder et al., 1987), and in carrot roots PAL, CHSand 4CL (Ecker and Davis, 1987). Raz and Fluhr (1993) used the plant pathogenesis response as a paradigm to investigate ethylene-dependent signal transduction in the plantcell and showed that a transient rise in protein phosphorylation inducible by ethylenetreatment is required for the pathogenesis response. Induction of such an increase inphosphorylation by treatment with inhibitors of phosphatases elicited the response inthe absence of ethylene. Another component of the pathway of ethylene inducible geneexpression requires calcium (Raz and Fluhr, 1992), since blocking calcium fluxes withchelators inhibited ethylene-dependent induction of chitinase accumulation. Artificiallyincreasing cytosolic calcium levels by treatments with a calcium ionophore or a calciumpump blocker stimulated chitinase accumulation.This chapter is concerned with the wound-inducible expression of the parsley 4 CL-igene. I used a transgenic tobacco system to investigate what promoter region is necessaryfor wound-inducible expression, and also investigated the potential role in this system ofsome compounds shown to be involved in wound-inducible expression of other genes. Ipresent data which provides evidence for the involvement of MJ in a signalling pathwayleading to the wound activation of 4 CL-i which is similar to that proposed for woundinducible proteinase inhibitor gene expression.3.2 Results3.2.1 Expression of 4CL-i in transgenic tobaccoThe ease with which tobacco can be transformed and regenerated coupled with its largeleaf size and relatively short regeneration time makes it an ideal choice for study ofwound-inducible gene expression (Schell, 1987). The use of this heterologous system toChapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 31investigate the wound-induced expression of 4 CL-i required, however, that I initiallyestablish that the wound response of the parsley gene is reproduced in transgenic tobacco. Six independent lines of tobacco plants (TI plants) were available which weretransformed with a large genomic clone for 4 CL-i (containing l500bp of upstream promoter sequences). Using a tobacco plant from each of these lines, I wounded excisedleaves, as described in Experimental Procedures, pooled the tissue and analysed totalRNA by northern hybridisation for the expression of 4 CL-i transcripts. Douglas et al.,(1991) have shown that a potato 4CL (St4CL) clone hybridises at low stringency with thetobacco 4CL but does not cross hybridise with parsley 4 CL-i. Conversely, the parsley4 CL-i cDNA does not cross-hybridise to mRNA from the tobacco 4CL genes. This allowed me to monitor the level of mRNA accumulation RNA from of both the endogenousand the introduced gene in the same experiment, providing an internal control. Figure3.3 shows that wounding strongly induced the accumulation of both the introduced 4CL-i clone and the endogenous tobacco 4CL. Expression of both genes was rapidly induced.High levels of mRNA for both genes were present at 3 hours after wounding and stillpresent after 24 hours. A control was performed where an excised leaf was removed andleft in water for 3 hours (3 C). Leaf excision caused a small systemic accumulation ofboth 4CL transcripts, however, the strongest effect of wounding was a localised response.Thus, the introduced 4 CL-i gene responds to wounding similarly to the endogenoustobacco 4CL genes.3.2.2 Expression of 4 CL-i-GUS gene fusions in response to woundingIn order to establish if the observed wound-inducible increase in the mRNA level for 4CL-i was being mediated by the 4 CL-i promoter, I generated, by Agrobacterium mediatedtransformation, tobacco plants transgenic for a -597bp of the parsley 4 CL-i promoterfused to the reporter gene GUS and used these plants in wounding experiments. TheChapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 32o 3 6 24 3CFigure 3.3: Wound-induced accumulation of tobacco 4CL and parsley 4 CL-i transcriptsin tobacco plants transgenic for an intact 4 CL-i gene. Duplicate northern blots werehybridised to a potato 4CL cDNA probe (St4CL) for detection of endogenous tobacco4CL transcripts or to a parsley 4CL cDNA probe (Fc4CL) for detection of transcriptsfrom the introduced 4 CL-i gene. 10tg RNA isolated from leaves 0, 3, 6, and 24 hoursafter wounding was loaded per lane. Control leaves (3C) were detached and placed inwater for 3 hours without further wounding.results of a northern analysis of RNA accumulation in plants of the transgenic line, 801-8is shown in Figure 3.4. GUS RNA accumulated strongly and rapidly, in response towounding and the increased levels of GUS RNA closely paralleled the accumulation ofendogenous tobacco 4CL RNA, detected by the St4CL probe. Thus the -597 bp 4 CL-ipromoter fragment alone confers wound-inducibility upon the GUS reporter gene.A 210bp 4 CL-i promoter fragment confers full developmentally regulated expressionupon GUS, and is the site of several protein-DNA interactions (Hauffe et al., 1991).To determine if this same 2lObp promoter fragment was sufficient to mediate the woundresponse, I wounded plants harbouring 2lObp of 4 CL-i promoter sequences fused to GUS(810 plants). Deletion of the promoter region from -597bp to -2lObp results in a tenfolddrop in the constitutive level of expression of 4 CL-i-GUS fusions (HaufFe et al., 1991).As a consequence of this low constitutive level of expression of GUS, it was necessarySt4CL Pc4CLChapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 33to isolate poiy A RNA rather than total RNA from wounded tissue to detect GUSRNA. Figure 3.5 shows the results of the hybridisation of poiy A mRNA from woundedtissue from plants of the transformed line 810-11, with a GUS probe. For comparison, asimilar experiment using line 801-6, (with -597 bp of promoter sequences controlling GUSexpression) is shown. There was a clear induction of GUS mRNA in the 810-11 plant,similar to the wound-inducible accumulation of GUS and tobacco 4CL mRNA directed bythe larger promoter fragment in the 801-6 plant. In order to control for different amountsof RNA loaded or different efficiencies of poly A RNA isolation from different samples,the stripped 810-11 filter was re-hybridised with a probe for ubiquitin, whose expressionis not affected by wounding. Thus, sequences mediating wound-inducible expression ofthe 4 CL-i gene are contained within the 2lObp promoter fragment.3.2.3 The effect of ethylene on the expression of 4 CL-i in tobaccoIn light of the role played by ethylene in plant defense responses (Henstrand et al., 1989;Ecker and Davis, 1987), it is a candidate as a second messenger in stress regulated expression of 4 CL-i. Treatment with ethephon has been used in other systems to achieveethylene treatment (Broglie et al., 1986; Brederode et al., 1991). To determine if ethylenecould modulate changes in parsley 4 CL-i expression, I treated plants from two independent lines of tobacco transgenic for a genomic copy of parsley 4 CL-i (containing l500bpof promoter sequences) with ethephon. I pooled tissue from each plant and isolatedtotal RNA 6 and 24 hours after treatment. The results of a northern blot analysis ofthis RNA are shown in Figure 3.6. A northern blot hybridised with a probe for St4CLdemonstrated that the tobacco genes are ethephon-responsive: Increased levels of tobaccomRNA were present at 6 and 24 hours after treatment. Similarly, hybridisation with aprobe for parsley 4 CL showed that the expression of the introduced gene is responsiveChapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 343C 0 3 6 240S3C 0 3 6 24 Hours*2kbFigure 3.4: Wound-induced accumulation of tobacco CL and GUS transcripts in atobacco plant transgenic for a 597-hp parsley 4CL-1 promoter-GUS fusion. Leaves ofplant 801-8 were detached, then wounded for the times shown, total RNA prepared, and10 ,ug RNA loaded per lane. Northern blots were hybridized with an StCL cDNA probeto detect endogenous tobacco 4CL RNA, and a GUS probe to detect GUS RNA. The3C time point is control in which unwounded excised leaves were incubated 3 hours inwater.St4CL GUS ProbeChapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 35GUS St4CL GUS UBIQ06 —24Plant: 801-6 810-11Figure 3.5: Accumulation of GUS transcripts in response to wounding of leaves from atobacco plant of transgenic line 810. Poly A RNA was isolated from leaves of plant810-11, transgenic for a 210-hp 4CL-i promoter-GUS fusion, which had been woundedfor the times given (in hours). A slot blot loaded with 500 ng 810-11 RNA per slot washybridized to a GUS probe, subsequently stripped and hybridized to a ubiquitirt probe(UBIQ) to control for RNA loading. For comparison, 2 ig total RNA from similarlywounded leaves of plant 801-6 (597-bp promoter-GUS fusion) was loaded on a slot blotsand hybridized to GUS and St.4CL probes.to ethephon application. I performed similar experiments using a tobacco line transgenic for a 597bp parsley 4CL-i promoter-GUS fusion. However, northern blot analysiswith a GUS hybridisation probe failed to show definitively whether the .4CL-i promotermediated ethylene-responsiveness of the parsley .4CL-i gene.3.2.4 The effect of ABA treatment on.4 CL-i promoter activityPefla-Cortés et al. (1989) showed that treatment of transgenic tobacco plants with ABAinduced accumulation of P1-Il mRNA. To determine if the parsley .4 CL-i promoter maybe similarly responsive I treated 801-8 tobacco plants with 100pM ABA. Northernanalysis (Figure 3.7) showed no accumulation of GUS mRNA above control levels after6 hours (the high level of expression at the 0 hour time point overloading of the gel asdetermined by ethidium-bromide staining) and a small accumulation of transcripts after24 hours. A similar pattern of hybridisation was seen on a northern blot hybridised withChapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 36Probe: St4CLFigure 3.6: Accumulation of tobacco 4CL and parsley 4 CL-i RNA in response to ethephon. lOmg/ml 2-chloroethephon was applied to leaves of tobacco plants transgenic foran intact parsley 4 CL-i gene. 10 tg total RNA from leaves, harvested at the times indicated after ethephon application, was loaded on duplicate RNA gels, and northern blotshybridized with St4CL and Pc4CL cDNA probes to detect endogenous tobacco 4CL andparsley 4CL transcripts, respectively.Time (h)Control0 6 241 0OM ABA6 24 Probe:2Kb *‘-. lIjpPr GUSFigure 3.7: The effect of ABA on GUS expression in plants transgenic for a -597bpparsley 4 CL-i promoter fragment directing expression of GUS (801 plants). Leaf tissuefrom a plant that had been sprayed with 100 zM ABA or 0.01% ethanol alone (controls),was harvested after 0, 6, and 24 hours. The blot was hybridised to a GUS cDNA probe.0 6 24 Hours—4 2kbPc4CLChapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 37a probe for St4CL (data not shown), suggesting the absence of a rapid response of eitherthe endogenous genes or the introduced 4 CL-i-GUS to exogenously applied ABA in thisexperiment. Experiments are described in Chapter 4, in which the effect of a rangeof ABA concentrations on parsley 4 CL-i expression in parsley cells was tested. Thoseexperiments suggested that the gene is not ABA-responsive in cell cultures.3.2.5 Expression of 4CL-i-GUS gene fusions in response to MJThe work reviewed by Ryan et al. (1992) and Staswick et al. (1992) suggests that thereis a role for MJ as a second messenger in wound-inducible gene expression. I used thetransgenic tobacco system to address whether MJ may have a role in the wound-inducibleexpression of 4 CL-i. Since the parsley 4 CL-i promoter mediates the wound response ofthe parsley gene in the tobacco system, it follows that if MJ is a mediator of this responsethen the 4 CL-i promoter should be similarly MJ- responsive. I treated tobacco plantstransgenic for the -597bp parsley 4 CL-i promoter GUS fusion (line 801-8) with 1mMMJ and used northern blots to assay for the induced accumulation of tobacco 4CL andGUS mRNA. Figure 3.8 shows that MJ treatment led to a massive increase in GUS RNAlevels by 6 hours and that GUS mRNA levels remained high for at least 24 hours aftertreatment. Endogenous tobacco 4CL levels as assayed by hybridisation to the St4CLprobe, increased in a very similar manner in response to MJ treatment.Effect of remote application of MJ on the -597 bp of 4 CL-i promoter intransgenic tobaccoFarmer and Ryan (1990) showed that MJ originating from Artemesia tridentata wascapable of inducing proteinase inhibitor accumulation in neighboring tomato plants. Inlight of the high level of responsiveness of the 597bp promoter fragment, I consideredthe possibility that direct application of MJ was not necessary for promoter activation. IChapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 380 6 6C 24 0 6 6C 24 24C hoursProbe: GUSFigure 3.8: Expression of GUS in response to methyl jasmonate in tobacco plants oftransgenic line 801-8. 1 mM methyl jasmonate (MJ) or 1% Triton X-100 alone (C)was directly applied to leaves of a tobacco plant transgenic for a 597-hp Pc4 CL-i promoter-GUS fusion (801-8), and RNA isolated at 0, 6, and 24 hours. 10 g total RNAwas loaded on duplicate RNA gels, and northern blots hybridized to a GUS probe or toSL4CL to detect endogenous tobacco 4CL RNA.tested the ability of MJ to act remotely in this system by exposing plants from transgenictobacco line 80 1-8, to MJ vapour without direct application of the compound. The resultsin Figure 3.9 show that levels of GUS mRNA were unaffected by MJ applied in thismanner. I hybridised a duplicate northern blot with a probe for St4CL to determine ifendogenous tobacco 4CL mRNA accumulation was similarly unaffected by MJ applied inthis manner. Only a very weak signal was detected on this blot, despite a long exposureand the use of a St4CL probe labelled to very high specific activity (data not shown).This suggests that tobacco 4CL mRNA levels did not increase above constitutive levelsafter indirect application of MJ. Thus, under the conditions I used, activation of parsley4 CL-i and tobacco 4GL appears to require direct application of MJ to the leaves.MJ directed expression of -2lObp 4 CL-i -promoter GUS fusionsTo determine if the same 2lObp promoter fragment that mediated the wound response ofthe 4 CL-i promoter was also capable of mediating the response to MJ, I treated plantsSt4CLChapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 392kb -probeFigure 3.9: The effect of methyl jasmonate vapour on GUS expression in plants of transgenic line 801-8. Plants transgenic for a 597-hp parsley 4 CL-i promoter- GUS fusion(801-8) were exposed to methyl jasmonate vapour. A total of 500 nis of methyl jasmonate in 95% ethanol was applied to 9 cotton dipped dowels surrounding but not in directcontact with the plants. Control plants (C) were treated with 95% ethanol alone. Tissuewas harvested at the times shown in hours. Accumulation of transcripts for GUS wasdetermined on Northern blots loaded with 10 pg total RNA per lane.from two independent lines transgenic for this promoter fused to GUS (810-11 and 810-7), with MJ and estimated the level of expression on slot blots. Figure 3.10 shows thatin both these transformants, GUS mRNA (under the direction of a 2lObp promoterfragment) had accumulated to high levels in response to MJ after 6 hours. These highlevels of GUS mRNA were still present 24 hours after treatment.3.2.6 Effect of methyl jasmonate, jasmonic acid, and linolenic acid on theparsley 4 CL-i promoter in transgenic tobaccoI used plants of the transgenic line 801-8 to determine the effect on GUS expressionof different concentrations of MJ, JA, and o-1ino1enic acid (a-LA). Plants were sprayedwith solutions containing 0, 0.1, 1, and 5 mM concentrations of each compound, RNAisolated 6 hours after treatment, and northern blots performed. Hybridisation with aGUS probe showed GUS mRNA accumulated to approximately 60% of maximum levelsOC 6MJ 6C 24MJGUSChapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 40Time:hours06C6MJ III6C24MJPlant: 810-11 810-7Figure 3.10: The effect of methyl jasmonate on GUS and tobacco 4CL expression in plantsof transgenic line 810. Methyl jasmonate-induced accumulation of GUS and endogenoustobacco 4CL (St4CL) transcripts in tobbaco plants transgenic for a 210-bp parsley 4 CL-ipromoter- GUS fusion. Plants from two independent transgenic tobacco lines (810-11 and810-7) were sprayed with 1 mM methyl jasmonate (MJ) or 1% Triton X-100 alone (C),and RNA isolated after 0, 6, and 24 hours. 2 ,ug total RNA was loaded on duplicateblots and hybridized to GUS or St4CL probes. Blots were subsequently stripped andhybridized to a ubiquitin (UBIQ) probe.Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 41after treatment with 0.1 mM MJ and a-LA and approximately 30% maximum levels afterJA treatment. In all cases the maximum response as measured by mRNA accumulationwas seen after treatment with a solution containing 1mM of each compound. Increasingthe concentration of the applied compounds to 5 mM resulted in a marked decrease inGUS mRNA accumulation.Hybridisation with a probe for StCL showed that the endogenous tobacco 4CL generesponded in a similar manner to the introduced GUS gene. Plants were also treatedwith 7-LA, a stereoisomer of LA which does not enter the biosynthetic pathway to JA(Hamberg and Gardner, 1992). On these blots, signals detected with both GUS andSL4CL probes were weak and did not increase above constitutive levels after treatmentwith LA (data not shown). This suggests that the induction of gene expression observedafter treatment with a-LA may be as a consequence of its entry into the biosyntheticpathway for JA.3.2.7 The effect of a potent inhibitor of lipoxygenase on the wound responseAccording to the model proposed by Farmer and Ryan (1992), the wound response ismediated by endogenous JA synthesised de novo via a lipoxygenase. Inhibition of lipoxygenase activity would therefore be predicted to prevent the wound response. The inhibitorn-propylgallate (nPG) has been shown to be a potent inhibitor of tobacco lipoxygenase(Fournier et al., 1993). I therefore tested the effect of nPG on wound-inducible expressionof the 4 CL-1-G US fusion in transgenic tobacco line 801-8. Plants were treated for 10hours with buffer alone or with 51tM or 50MM nPG and subsequently wounded or harvested without wounding. The accumulation of GUS RNA was assayed using northernblots hybridised to a GUS and the accumulation of RNA for the endogenous tobaccoCL gene was assayed using a homologous probe for tobacco 4CL (NqCL) (Diana Lee,C0ECCzC)>CCC)E0ExCCEC00ECCzC)>CCC)E0ECCEC00ECCzC)>CCC)Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 4275502500 0.1mM methyl jasmonate100 0.1 10mM jasmonic acid100755025010075502501 10Figure 3.11: Expression of GUS and tobacco 4CL to treatment with methyl jasmonate,jasmonic acid and linolenic acid in plants of line 801-8. Tobacco plants of line 801-8were treated for 24 hours with 0, 0.1, 1, and 5 mM of each compound and expressionof GUS (0) and tobacco jCL (.) determined on northern blots. Plants correspondingto the 0 mM concentration were treated with 1% Triton alone. Blots were scannedwith a densitometer and the band intensities plotted as a function of concentration andnormalised to hybridisation with a probe for ubiquitin.0 0.1Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 43unpublished results). In the plants pre-treated with buffer alone, there was an accumulation of RNA for both transcripts, GUS and Nt4CL, present 24 hours after wounding(Figure 3.12). In plants treated with nPG before wounding there was a large decreasein the response to wounding of the GUS reporter gene directed by the 4 CL-i promoterwhich was most apparent at 5OzM nPG where the wound response appeared to be absent. Hybridisation with a tobacco 4CL probe revealed a similar marked response tothe inhibitor. The filter hybridised with the Nt4CL probe was subsequently rehybridisedto a probe for tomato ubiquitin (Figure 3.12). The presence of a discrete band whichhybridised to the ubiquitin probe in all lanes demonstrated that nPG treatment did notlead to any significant degredation of RNA. Since nPG is known to be a potent inhibitorof tobacco lipoxygenase, these results suggest that a lipoxygenase activity may be required for the wound-induced expression of tobacco 4CL and the wound-responsivenessof the parsley 4 CL-i promoter in tobacco.3.3 DiscussionIn transgenic tobacco, the parsley 4 CL-i gene is responsive to wounding in a mannersimilar to the wound response of the endogenous tobacco 4CL genes. The sequencesmediating the response of the 4 CL-i gene to wounding are contained within a 2lObppromoter region. The parsley 4 CL-i promoter and the endogenous tobacco 4CL genes arealso responsive to JA, MJ and LA. In the presence of an inhibitor of tobacco lipoxygenase,n-propylgallate (Fournier et al., 1993), there is a marked decrease in wound activation ofthe 4 CL-i promoter and a similar marked decrease in the wound response of the tobacco4 CL genes. This may be a reflection of the requirement of lipoxygenase for synthesis ofJA to mediate the wound response as was proposed in the model suggested by Farmerand Ryan (1992) (Figure 3.2).Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 44Buffer 5iM nPG 5OiM nPG0 24 0 24 0 24GUSNt4CLUbiqIFigure 3.12: The effect of the lipoxygenase inhibitor n-propylgallate (nPG) on the woundresponse in plants of line 801-8. 801-8 plants were treated with 0 (buffer), 5 or 50 tMnPG and subsequently either wounded and incubated for 24 hours (24), or harvested immediately without wounding (0). The accumulation of GUS and tobacco 4CL transcriptswas estimated on a northern blot. The filter hybridised with Nt4CL was then strippedand rehybridised with a probe for ubiquitin.The ease with which it can be transformed coupled with its large leaf size, makestobacco an ideal choice as a heterologous system in which to study the wound responseof the parsley 4CL-i gene (Schell, 1987). The use of a heterologous system made it necessary to initially determine if the parsley 4 CL-i gene is wound-responsive in transgenictobacco. Northern analysis of tobacco plants harbouring a genomic clone for parsley4 CL-i showed that, similar to its expression in parsley (Schmelzer et al., 1989), mRNAaccumulated in a localised manner in response to wounding. Wound-inducible expression of the parsley gene was paralleled by accumulation of mRNA for tobacco 4 CL. Thismeans that the tobacco wound signal, in whatever form it takes within the cells, is alsorecognised by the parsley 4 CL-i gene, suggesting that the wound response is conservedbetween the two species. In parsley leaves, 4 CL-i is also responsive to attempted infection by Fhytophthora megasperma (Fmg) and irradiation with UV containing whiteChapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 45light (Schmelzer et al.,, 1989). The response to these stresses is also conserved betweentobacco and parsley, since it was shown that in tobacco transgenic for a parsley 4 CL-igene, mRNA for both the introduced gene and the endogenous gene accumulates in response to Pmg elicitor treatment and UV light treatment (Douglas et al., 1991). In situhybridisations were used in tobacco to examine cell type and tissue-specific expressionpatterns of parsley 4 CL-i, and it was demonstrated that these patterns of expressionare closely correlated to those of the endogenous 4CL genes (Reinold et al., 1993). Thisprovides strong evidence to show that the 4 CL-i gene responds to the same cell type,tissue-specific and stress signals that regulate 4CL expression in tobacco. Taken together,these data indicate that a transgenic tobacco system is an ideal one in which to studyregulation of 4 CL-i gene expression. In contrast, expression of the wound-induciblepotato gene wine is not conserved between tobacco and its homologous host. The regulatory sequences of the win2 gene direct dramatic wound-inducible expression of GUSin the leaves of potato; however, no induction of win2-GUS gene expression is observedin transgenic tobacco, despite the presence of win2 homologues in tobacco (Stanford et,al., 1990; Stanford et al. 1989).It is interesting how precisely GUS expression correlates with expression of tobacco4CL in response to MJ, LA and JA (Figure 3.11), since this again indicates a highlevel of conservation of signalling systems between parsley and tobacco. Uknes et al.(1993) recently highlighted the importance of monitoring endogenous gene activity asa control in transgenic systems. This group used GUS as a reporter of tobacco PRia (pathogenesis related) promoter activity and documented two artifactual patterns ofGUS expression which were not seen with the endogenous tobacco PR-ia gene or with thePR-ia promoter directing expression of other genes. One was ectopic expression of GUSin anthers and pollen, the other anomaly was GUS RNA accumulation in response tocycloheximide treatment. These workers suggest that the GUS coding sequence containsChapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 46a promoter element that responds to cycloheximide treatment. Monitoring endogenousgene activity can help bring to light such aberrations.In parsley, 4 CL has been considered an essentially single gene system (Douglas et al.,1992) since the sequences of the coding regions, introns, and flanking DNA of both genesare highly conserved and 4 CL-i and 2 are not differentially regulated in response to UVlight and elicitor (Douglas, 1987). Recent work, however, suggests that these genes maybe differentially regulated in response to wounding in roots (Lois et al., 1992). Differentialexpression in leaves was not studied. In contrast to the wound-inducible expression of4 CL-i in roots, parsley 4CL-2 expression was shown to be either repressed or not inducedin wounded roots. It seems highly unlikely that there is differential expression in leavesat the level of transcription since the two parsley 4CL genes differ in only one positionwithin 210 bps upstream of the transcription start site (Douglas et al., 1987) and a 210bp 4 CL-i promoter fragment is capable of mediating the wound response (Figure 3.5).It has been suggested that coding sequences are involved in regulating expressionof 4 CL-i in response to UV light and Pmg elicitor treatment since promoter sequencesalone do not confer responsiveness to these stimuli upon a GUS reporter gene (Douglas etal., 1991). The fact that the 4 CL-i promoter sequences alone confer a strong response towounding upon GUS, suggests that the pathways mediating responsiveness to woundingdiverge, at least at the level of DNA/promoter interactions, from intracellular pathwaysmediating elicitor- and UV light-inducible expression. The sequences responsible for amyriad of cell type specific-expression modes of 4 CL-i in transgenic tobacco are locatedwithin 2lObp upstream of the transcription start site (Hauffe et al., 1991), and distinctpromoter domains which specify tissue and cell type specific patterns of expression bycombinatorial interactions have been identified (Hauffe et at., 1993). Since the same2lObp fragment can also specify wound- and MJ-inducible mRNA accumulation, it ispossible that MJ could be a common signal in mediating wounding and cell type specific-Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 47expression. In vivo DNA/protein interactions within this region have been investigatedby in vivo footprinting and an approximately 200bp region 5’ of the transcription startsite showed strong in vivo protection (ilauffe et at., 1991). Although these in vivo footprints are constitutive, (they do not appear to be modified by fungal elicitor), they maystill play an important role in stress-regulated (CL-1 expression. They may represent regions where modification of constitutively bound factors produced by an environmental,developmental, or tissue-specific signal may cause changes in gene expression. Identification of the elements within the CL-1 promoter responsible for MJ mediated expressionwill determine more precisely the correlation between developmental and tissue-specificpatterns of expression, and jasmonate-induced expression. Meanwhile, there is an interesting correlation between levels of jasmonates in tissue, and CL expression; both arehighest in young tissue (Groenewald and Visser, 1978).A 50-bp MJ-responsive domain, containing a hexameric G-box motif (CACGTG)and a C-rich sequence, has been identified within a soybean vsp promoter (Mason et al.,1993). This G-box motif has also been identified in the potato P1-Il promoter, whereit is involved in MJ-mediated responses (Kim et at., 1992), and it is also present withina parsley CHS gene (Schulze-Lefert et at., 1989). It remains to be seen if this elementmediating the response of soybean vsp to MJ, is the same element mediating the woundresponse of the vsp gene. This element appears to be absent from the CCL-1 promoter,which means that if MJ is mediating CL-1 expression in response to environmentalor internal stimuli, it may do so by a mechanism that is distinct from that controllingwound responsive vsp gene expression.MJ was first implicated as a signal molecule in wound inducible gene expression whenFarmer and Ryan (1990) showed that airborne MJ emanating from Artemesia tridentatacould induce the expression of proteinase inhibitor genes in nearby tomato plants. Thisexperiment conclusively demonstrated that a signal from one plant can cause defense geneChapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 48activation in another plant. This means that, theoretically, plants could communicate viavolatile signals. I investigated the possibility that such remote application of MJ couldaffect parsley promoter activity in tobacco plants. Figure 3.9 demonstrates that airborneMJ does not activate the 4 CL-i promoter, and the tobacco 4CL genes were similarlyunaffected (data not shown). Similarly, Andresan et al. (1992) demonstrated that JAinduced accumulation of leaf proteins is not sensitive to volatile MJ in intact barleyseedlings. Thus, interplant communication via MJ may not be a general mechanismactivating the expression of MJ-responsive genes. It is possible, however, that 4CL genesare less sensitive to MJ than Fl genes and very high concentrations of airborne MJ maymediate 4CL expression.Similarities in structure, physical properties and the activity of ABA and jasmonatehave been pointed out (Staswick 1992). Both induce the accumulation of proteinaseinhibitors and Brassica seed storage proteins (Mason and Mullet, 1990; Wilen et al.,1991; Wilen et al., 1990). I found that, in transgenic tobacco, the 4 CL-i promoter andthe endogenous tobacco 4CL genes were unresponsive to ABA after 6 hours, suggestingABA does not activate the 4 CL-i promoter within the time frame which would indicatea potential role for this molecule as a component of the signal transduction pathwaymediating the rapid response to wounding. This suggests there is not a direct role forthis compound in the wound-activated expression of 4CL in this system. In this respect4CL regulation is similar to that of soybean vsp, which is MJ inducible but not ABAresponsive (Anderson, 1989).Ethephon, on the other hand, appeared to mimic wound-inducible expression of theentire 4 CL-i gene. Unfortunately, due to difficulties in demonstrating accumulation ofGUS mRNA in response to ethephon, I was unable to determine if the 4 CL-i promoter isethephon-responsive. Hybridisation of northern blots loaded with RNA from ethephontreated plants gave irreproducible results and it appeared as if the GUS transcript wasChapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 49unstable in the presence of ethephon. Although there is precedence in the literature forusing ethephon for ethylene treatment (Henstrand et al., 1989; Ecker and Davis, 1987)there are potential problems associated with its use. The biggest concern is the fact thatupon breakdown of ethephon to release ethylene, phosphonic acid is released which byitself may be a stress upon the plant. Direct application of ethylene gas would avoidthis potential problem and may help elucidate the role (if any) of ethylene in the woundresponse. Another potentially useful approach involves looking at the wound responseof plants deficient in ethylene biosynthesis. Ethylene biosynthesis can be blocked byantisense genes of ACC .synthase or ACC oxidase (Hamilton, 1990; Oeller et al., 1991).In Chapter 5, I will discuss in more detail the use of mutants in identifying componentsof signal transduction pathways.One of the goals of the work in this chapter was to determine if endogenous JAor MJ may play a role in the wound-activated expression of the 4 CL-i promoter intransgenic tobacco, using as a working hypothesis the model developed by Farmer andRyan (1992) (Figure 3.2). (In chapter 4, I have used the parsley cell suspension culturesystem to investigate the possibility of a role for jasmonates in the response of 4CL-1 to Pmg elicitor using the cell suspension culture system). The data presented heresupport this model. Firstly, both MJ and JA strongly induce the expression of theGUS reporter gene under the control of 4 CL-i promoter and expression of the tobacco4CL genes was similarly activated by jasmonates (Figure 3.11). The parsley 4CL-ipromoter is also wound-inducible, and the same 2lObp promoter confers responsiveness tojasmonates (Figure 3.10) and wounding (Figure 3.5). Comparison of Figures 3.4 and 3.5with Figures 3.8 and Figure 3.11 shows that jasmonates are more powerful in this regardthan wounding. Farmer and Ryan (1992) similarly reported more powerful inductionof proteinase inhibitor genes by JA than by wounding. In this case they hypothesised areceptor system which is not saturable by wounding but is saturable by direct applicationChapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 50of JA. Wound-induced expression of 4 CL-i could be regulated by a similar receptor.Secondly, JA is synthesised via a lipoxygenase activity from a-LA (Vick and Zimmerman, 1984; Vick and Zimmerman, 1987a and b) and a-LA specifically activates the4 CL-i promoter in tobacco (7-LA had no effect on transcript accumulation). Since notonly JA but its biosynthetic precursor can activate 4 CL-i expression, these data are consistent with the lipid-based signalling system proposed by Farmer and Ryan (1992), andthe observed activity of LA may be a result of its conversion to JA in vivo. Moreover, myresults suggest that lipoxygenase activity sufficient for conversion of LA to biologicallyactive amounts of JA is present constitutively in tobacco leaf cells. This would meanthat activation of lipoxygenase is not a prerequisite for wound activation in this case.Such appears to be the situation in mammalian eicosanoid signalling (Needleman et al.1986), and it has been suggested that it is the release of linolenic acid from the membrane(via a lipase activity) that is the key event in signalling of wound -inducible proteinaseinhibitor gene expression (Farmer and Ryan, 1992).Thirdly, in the presence of nPC, the wound response of both the tobacco 4CL gene andthe 4 CL-i promoter was decreased in a dose dependent manner. Inhibitors of tobaccolipoxygenase were recently characterised (Fournier et al., 1993), and among those tested,nPG was the strongest inhibitor of in vitro activity. One would predict that, if the abilityof LA to induce 4CL expression is a reflection of its conversion into jasmonates with arole in wound signalling, lipoxygenase inhibition would diminish the wound-responseof 4CL in this system. In the presence of nPO, the wound response was diminished,suggesting that the endogenous lipoxygenase may have been inhibited. I did not directlydemonstrate inhibition of tobacco lipoxygenase and even if such data were available anon-specific mode of action of the inhibitor cannot be excluded. nPG is a free radicalscavenger (Vick and Zimmerman, 1987b) and may well interfere with any number ofother cellular processes within the tobacco cells. In Chapter 4, however, I present dataChapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco 51which suggests that in parsley cells at least, cellular signalling processes important inmediating changes in gene expression are functional subsequent to treatment with nPG.My results show that the 4 CL-i promoter is sufficient to confer both wound- and MJresponsiveness on the GUS reporter gene. In contrast, earlier results (Douglas et al.,,1991) based on the expression of 4 CL-i and 4 CL-i promoter- GUS fusions in transgenicparsley and tobacco, indicated that this promoter by itself is not sufficient to conferelicitor- and UV light-responsiveness upon GUS. Thus, although my results point toa possible role for JA or MJ as endogenous signalling compounds mediating wound-inducible expression of 4 CL-i, they do not by themselves support a role for JA or MJin the elicitor or light response. The fact that different regulatory sequences associatedwith the.4 CL-i are required for MJ/JA and wound-responsiveness, suggests signallingpathways mediating these responses are distinct from each other. In Chapter 4, usingthe cell culture system, I investigate further whether there may be a role for jasmonatesin signalling of phenyipropanoid gene expression in response to elicitor.Chapter 4The role of jasmonates in the elicitor response in parsley cell cultures4.1 IntroductionThe study of stress-induced phenyipropanoid metabolism in parsley plants has beengreatly aided by the availability of the parsley cell suspension culture system. Parsleycells are easy to propagate in simple, synthetic media and the cells respond synchronouslyand uniformly to environmental stress (Hahibrock and Scheel, 1989). Much of the workon elucidating the enzymology of the pathway and its induction was done using thissystem. Parsley cell cultures treated with UV light accumulate flavonoids thought tohave a UV protective function, and after treatment with a glycoprotein elicitor fromPhytophthora megasperma (Fmg elicitor), which simulates an infection, secrete furanocoumarins with antifungal activity (reviewed by Hahibrock and Scheel, 1989). Theaccumulation of these products is preceded by the rapid transcriptional activation ofgenes for phenylalanine ammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL). Thistranscriptional induction is followed by increases in levels of the respective mRNAs andproteins (Hahibrock et al., 1981; Ragg et al., 1981). Activation of genes of the generalphenylpropanoid pathway therefore occurs in response to both stimuli. Activation of thebranch pathways however is signal specific. UV light specifically activates transcriptionof chalcone synthase (CHS), (a large number of other enzymes involved in biosynthesis of fiavonoids have been shown to be activated at the catalytic level at least), and52Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 53elicitor activates transcription of genes encoding enzymes involved in biosynthesis of furanocoumarins such as, S-adenosyl-L-methionirie:bergaptol 0-methyltransferase (BMT)and S-adenosyl-L-methionine:xanthotoxol 0-methyltransferase (XMT) (Hauffe, 1988).This and similar systems can be used to ask questions regarding the nature of signaltransduction leading from perception of stress to gene activation. Important questionsinclude, how stress signals are perceived by the cell, how they are communicated viasecond messengers to the nucleus, and ultimately how changes in gene expression arebrought about.Recent work has begun unravelling what may be involved in the initial recognitionof the elicitor by the plant cell. Renelt et al., (1993) demonstrated the presence ofcompetable binding sites on the parsley cell membrane which recognise the protein moietyof the Pmg elicitor. It is known also that two of the initial events leading to geneactivation are; a transient change in the in vivo phosphorylation patterns of proteins,and a change in permeability of the plasma cell membrane to Ca2+, IP, K+ and C1(Dietrich et al., 1990; Scheel et. al., 1990). Plasma membranes of soybean similarlycarry high affinity binding sites which recognise elicitors derived from the cell wall of P.megasperma f. sp. glycinea (Schmidt and Ebel, 1987; Cosio et al., 1992). However, thereare fundamental differences in elicitor recognition between the two species; firstly, it is aproteinaceous constituent, (not carbohydrate), of the mycelial cell wall that is recognisedby the parsley cells; and secondly, there is no evidence that GTP binding proteins, whichparticipate in elicitation in soybean cells (Legendre et al., 1992) are involved in the parsleycell response (Renelt et. al., 1993).A number of phenyipropanoid and other defense related genes have been isolatedfrom parsley, including PAL, jCL, CHS, HRGP and genes encoding two pathogenesisrelated proteins, PR1 and PR2. These genes are transcriptionally activated upon elicitortreatment and mRNA for these genes accumulates in elicitor-treated cells (HahlbrockChapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 54and Scheel, 1989; Chappell and Hahibrock, 1984; Sommsich et al., 1986). In a systematicsearch for other parsley genes that are transcriptionally activated upon elicitor treatmentof cell cultures, Sommsich et al., (1989) isolated clones for a large group of defense relatedgenes called ELI genes. These clones fall into four classes based on their transcriptionkinetics. For example, the most abundant ELI class has a maximum of transcriptionalactivation at two hours after elicitor treatment. This class accounts for 30% of the isolatedclones and these genes are identical in kinetics of activation with FRi (pathogenesisrelated) genes. Since their original isolation, a function has been assigned to the encodedproducts of some of the ELI genes. ELI 5, a member of the class which is activatedlatest (maximum at 5 hours), was shown to encode tyrosine decarboxylase (TyrDC)which is a key enzyme in the biosynthesis of diverse alkaloids and auxins (Kawalleck et aL,1993). ELI 9 encodes a hydroxypropline-rich glycoprotein (HRGP) (Trezzini et al., 1993).Although a definitive function has not yet been assigned to ELI 3, its importance indefense has been supported by a strong association between its activation in Arabidopsisand the presence of a specific resistance gene (Kiedrowski et al., 1992). Using in situhybridisation, it has been demonstrated that mRNAs for many of these defense-relatedgenes isolated from cell cultures accumulate at infection sites in intact parsley plants witha temporal pattern of accumulation very similar to that seen in the suspension culturesystem (Schmelzer et al., 1989).Along with molecular probes, another tool required to understand how the expression of defense-related genes is regulated is a transformation system. Plant protoplastsare very amenable to direct DNA transfer and an important step in development of theparsley cell culture system was the demonstration that parsley protoplasts retain differential responsiveness to UV light and elicitor ( Dangl et al., 1987). The use of parsleyprotoplasts for transient expression studies, after PEG-mediated DNA transfer, enabledChapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 55functional identification of light-responsive elements within the CHS promoter (SchuizeLefert et al., 1989) and an elicitor-responsive region within the promoter of the geneencoding pathogenesis-related protein 2 (PRy) (van de Löcht et al., 1990).Douglas et al. (1991) investigated the control of 4 CL-i expression iii response to UVlight and elicitor. In stably transformed parsley cells, it was shown that deleting the promoter region to within 174 bp 5’ of the transcription start site of the intact 4 CL-i gene,affects only constitutive expression of the gene and not elicitor or light-induced expression. It was found, however, that promoter fragments fused to the GUS reporter genewere not able to confer stress-inducible expression upon that gene in either stably or transiently transformed parsley cells. This led to the suggestion that downstream sequenceswere required for inducible expression. Since addition or removal of introns did not affectexpression of the gene, the coding sequences were implicated as harbouring the requiredcontrol elements (transcriptional or post-transcriptional). Furthermore, since a CaMV85S promoter or a minimal 90 bp 95S promoter (both unresponsive to light and elicitor)were unresponsive to either stress stimulus when fused to the 4 CL-i cDNA (Douglas,et al., 1991), this meant that promoter elements are required in conjunction with exonicsequences for correctly regulated expression. Further support for this hypothesis camefrom the observation that transformation of 4 CL-i-GUS fusions into tobacco, resultedin little or no accumulation of histochemically detectable GUS activity or GUS mRNAin response to the stress stimuli, despite the responsiveness of the endogenous tobacco4 CL gene in the same experiment.The nature of the second messengers involved in signal transduction of the elicitorresponse to the nucleus is unclear. There is some evidence that the intracellular transduction chains are Ca2 dependent (Renalt et al., 1993). Another candidate for intracellulartransmission of elicitor-generated signals are jasmonates, which have been suggested tobe involved in inducible defense responses by other workers in the field. Gundilach et al.Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 56(1992) state that “Jasmonic acid is a signal transducer in elicitor-induced cell cultures”.Although their work suggests there may be a role for jasmonates in inducible defenceresponses, there is not yet sufficient evidence to prove such a role. They showed thattreatment of Rauvolfia canescens and Eschscholtzia californica cultures with a yeast elicitor led to rapid and transient accumulation of endogenous JA. They also show in thissystem that jasmonate treatment induces expression of a number of genes involved indefense, including PAL. In rice, hydroperoxides and hydroxides of linoleic and linolenicacids, which are intermediates in JA biosynthesis, show strong elicitor activity (Li et al.,1991). A more direct role for JA in defense in the interaction of barley with the powderymildew Erysiphe graminis f. sp. hordei has been suggested (Schweizer et al., 1993). JAdirectly inhibits appressoria differentiation of the fungus, and appears not to be involvedin signal transduction leading to induction of pathogenesis-related proteins. They usedan inhibitor of transcription, cordycephin, to show that repression of transcription doesnot affect inhibition of appressoria differentiation.The biosynthetic pathway for JA has been well established in many plant species(Vick and Zimmerman, 1987a and b). The metabolic cascade begins with cr-linolenic orlinoleic acid which is oxidised to 12-oxo-phytodecanoic acid (12-oxo-PDA). 12-oxo-PDA isconverted via a reduction and a series of /3-oxidations to JA (Vick and Zimmerman, 1984).The first step in the biosynthetic pathway is mediated by lipoxygenase which catalyses theincorporation of molecular oxygen into polyunsaturated fatty acids that possess a cis, cis1,4-pentadiene structure. It is believed that lipoxygenase activity is ubiquitous in theplant kingdom, and is present in a wide variety of plant organs (Vick and Zimmerman,1987a and b; Vick and Zimmerman, 1984). Soybean lipoxygenases have been studiedmore extensively than any other plant lipoxygenase, perhaps due to the fact that soybeanseeds and hypocotyls contain particularly high levels of lipoxygenase (reviewed by Siedow,1991). Lipoxygenases have, however, been isolated and characterised from other speciesChapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 57including tobacco (Fournier et al., 1993), rice, soybean, cotton and sunflower (reviewedby Siedow et al., 1991). The enzyme has been well characterised (Vick and Zimmerman,1987b). It contains one atom of iron essential to its catalytic role. It alternates betweenthe Fe(II) and Fe(III) states during catalysis giving rise to formation of large amounts ofhydroperoxide. cDNAs encoding lipoxygenases from a number of species have recentlybeen isolated. Included are clones for genes encoding lipoxygenases from soybean (Siedowet at., 1991), French bean (Eiben and Slusarenko, 1994) and Arabidopsis (Melan et al.,1993). Interestingly, mRNA for at least two of these lipoxygenase genes, one from soybeanand one from Arabidopsis, accumulates in response to MJ (Grimes at at., 1992; Bell andMullet, 1993), suggesting that MJ may regulate its own biosynthesis and that inductionof lipoxygenase expression by MJ may be important for increasing jasmonate levels undercertain physiological conditions.Treatment of parsley cells with the jasmonate precursor, 12-oxo-phytodienoic acid,led to the accumulation of the flavonoid apiin, over a period of several days, and wasaccompanied by a two-fold increase in 4GL and CHS mRNA and a five-fold increase inPAL mRNA accumulation (Dittrich et at., 1992). The significance of these data to therole of JA in elicitor-mediated signalling is unclear since the accumulation of flavonoids isa specific response to light, not elicitor, in parsley cells (Hahibrock and Scheel, 1989), andthe reported induction of mRNA accumulation is much lower than that observed afterelicitor treatment (Douglas et al.,1987; Lois et al., 1989; Hahibrock and Scheel, 1989). Inother work, Kauss et al. (1992) suggested that MJ enhances the effect of elicitor in parsleycells. They looked at the effect of 5pM MJ on furanocoumarin accumulation and reportminimal accumulation associated with treatment with MJ alone at this concentration.Treatment with 2OpM MJ increased furanocoumarin accumulation but levels were still100-fold less than those observed after elicitor treatment. However, the major effect ofMJ reported in this paper is that pretreatment of parsley cells with MJ prior to elicitorChapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 58treatment, greatly enhances the elicitor response.Whether jasmonates act directly in parsley cells to induce furanocoumarin phytoalexinaccumulation and defense gene activation, and whether the elicitor-induced expressionof phenylpropanoid and other defense-related genes may be mediated by endogenous JAhas not been addressed. In this chapter, I investigated the role played by jasmonatesin the response of parsley cells to elicitor, and present data on the elicitor response intobacco that is relevant to this question.4.2 Results4.2.1 The effect of methyl jasmonate, jasmonic acid, and linolenic acid on4CL expression in parsley cellsTreatment of parsley cell cultures with the JA precursor 12-oxo-phytodienoic acid leadsto moderate increases in PAL, 4CL and CHS mRNA levels, which could be due to conversion of 12-oxo-phytodienoic acid to JA (Dittrich et al., 1992). I wished to directly testthe activity of jasmonates on phenyipropanoid gene expression in parsley cells, and chose4 CL as a representative gene for initial dose-response experiments. I tested the abilityof range of concentrations of JA, its methyl ester MJ, and a-linolenic acid (os-LA), theubiquitous membrane fatty acid from which JA is synthesised (Vick and Zimmerman,1984) to induce accumulation of 4CL transcripts in parsley suspension cultured cells. Imeasured transcript accumulation by hybridisation of northern blots of total RNA fromtreated cells, to a parsley 4 CL-i cDNA (Douglas et al., 1987) and estimated band intensities by scanning densitometry. The signal from the 4CL probe was normalised to a signalobtained from a duplicate blot hybridised with a probe for Ubi4, a parsley polyubiquitingene whose expression is unaffected by elicitor (Kawalleck ci al., 1993a) (Figure 4.13).MJ induced expression at a concentration of 1.0 M and maximum RNA accumulationChapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 59was seen at 100 M. At a concentration higher than this, (300 tiM), the accumulation oftranscripts decreased. The free acid (JA) was more active at a lower concentrations thanthe methyl-ester. Of the concentrations tested, the maximum activity of LA in inducing4CL transcript accumulation was at 1.0 1tM and its activity declined quite rapidly as concentration increased. The first step in JA biosynthesis involves the oxygenation of cr-LAvia lipoxygenase to 13(S)-Hydroperoxylinolenic acid (13(S) HPOTrE) (Vick and Zimmerman, 1984). The effect of cr-LA was maximal at 3OpM, and increasing the concentrationto lOOiiM only slightly decreased the relative 4CL expression. A high concentration ofcr-LA (300tM) had little effect on transcript accumulation. To examine the specificityof a-LA in inducing 4CL mRNA accumulation, I also treated the cells with a closelyrelated isomer of a-LA, 7-LA, which does not enter the biosynthetic pathway. 7-LA hadno effect on 4 CL expression over the range of concentrations used in this experiment(data not shown).4.2.2 The effect of methyl jasmonate versus elicitor on expression of genesin the phenyipropanoid pathwayPmg elicitor treatment of parsley cells activates the transcription of genes encoding enzymes of the general phenyipropanoid pathway and the furanocoumarin-specific branchpathway including L-methionine-bergaptol 0-methyltransferase (BMT), an enzyme catalysing the final methylation of the phytoalexin bergaptol (Hauffe et al., 1988). Theresult of this activation of gene expression is the secretion of furanocoumarins into themedia. If the ability of MJ to induce 4CL transcriptional activation is a reflection of itsrole as a signalling intermediate in the response of cells to elicitor, I would expect MJto induce patterns of gene expression similar in timing and quantity to those induced byelicitor treatment. To test this, I compared the MJ-induced expression of PAL, 4CL andBMT to their expression in response to elicitor. I used slot blots, coupled with scanningChapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 60100S5 75o 50SCtz 250>Ct5 0100Sx75C0o 50SCtz250)>Ct0S1007550c 250)>Ct0Figure 4.13: Response of parsley cell suspension culture cells to exogenously appliedmethyl jasmonate, jasmonic acid and linolenic acid. Cells were treated with 0, 1, 30,and 100 tiM concentrations of each compound. Cells corresponding to the 0 tiM pointwere treated with 0.1% Triton alone. They were incubated for 6 hours in the dark withconstant shaking. The level of mRNA for parsley 4CL was determined by northernhybridisation coupled with scanning densitometry. Duplicate blots (loaded with lOjigsof RNA per lane) were hybridised with a probe for parsley 4CL and Ubi, a parsleyubiquitin gene whose expression is unaffected by elicitor. The signal from the (CL probewas standardised to the Ubi( signal and relative mRNA amount (as a percentage ofmaximum) plotted as a function of the log of concentration.0 1 10 100 1000iM methyl jasmonate0 1 10 100iM jasmonic acid10000 1 10 100 1000iM linolenic acidChapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 61MethylElicitor Jasmonate ControlTime,- (- 2 6 9 Probe:(h) 0 2 6 9 2 o .i11 PALI I j 4CLBMTI •1• I4 IiiFigure 4.14: The effect of methyl jasmonate, and elicitor treatments on the expressionof genes in the phenylpropanoid pathway. Parsley cells were treated either with 3OpMmethyl jasmonate or 5Opg/ml Pmg elicitor. Control cells were treated with 0.1% Tritonalone. Cells were harvested at 2, 6 and 9 hours after treatments, total RNA extractedand 2 pg hybridised on slot blots with probes for CL, PAL, BMT and Ubi.densitometry to quantify accumulation of transcripts for these genes subsequent to bothtreatments.Figure 4.14 shows that elicitor treatment led to the rapid accumulation (by 2 hours)of 4CL and PAL mRNAs, while the induction of BMT RNA accumulation occurredmore slowly. These results are similar to previous results obtained in this system (Lois etal., 1989; Hahibrock and Scheel, 1989). The accumulation of CL and PAL transcriptswas also rapidly induced in response to treatment with 30 pM MJ but was lower thanthe elicitor-induced accumulation. The kinetics of PAL and 4CL RNA accumulation appeared similar in MJ and elicitor-treated cells. Activation of BMT expression in responseto MJ appeared to occur more rapidly than to elicitor (compare 6 hour time points) andChapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 62reached a level about twofold more than that seen after elicitor treatment by 24 hours.4.2.3 Accumulation of furanocoumarins in response to methyl jasmonateand elicitor treatmentTo further test the relative responses of parsley cells to MJ and elicitor, I compared theaccumulation of furanocoumarins in cultures 24 hours after treatment with either MJor elicitor. Furanocoumarins were extracted from the culture filtrate with chloroformand quantified spectrophotometrically (Kombrink and 1{ahlbrock, 1986). In contrast topreviously published data by Kauss et al. (1992) who reported a minimal accumulationof furanocoumarins subsequent to treatment with MJ alone, the level of furanocoumarinsin this experiment was tenfold above that in control cells, and one third the level seenin elicitor-treated cells (Figure 4.15). To determine if the same furanocoumarin derivatives were produced after MJ treatment as after elicitor treatment, I analysed extracts offuranocoumarins by TLC along with standards for the furanocoumarins isopimpinellin,psoralen, xanthotoxin, bergaptol. At least 4 prominent fluorescing spots were identified,some of which comigrated with the standards confirming the presence of furanocoumarinsin the extracts (data not shown). A qualitatively identical pattern was induced by MJtreatment and elicitor treatment. Because equal amounts of the extracts were chromatographed, the amount of each product was lower in the MJ sample (Figure 4.16).4.2.4 The response of parsley cell cultures to ABAABA has been proposed to activate the expression of defense-related genes and showssome structural similarities to JA (Peña-Cortés et al., 1991; PefIa-Cortés et al., 1989;Hamberg and Gardner, 1992). To determine what, if any, effect ABA may have on theexpression of phenyipropanoid genes in parsley cell suspension cultures, I treated cellsChapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 63.2) 21.50CIED0o 1.000O.5oconhiFigure 4.15: Levels of furanocoumarins secreted by methyl jasmonate or elicitor treatedparsley cell suspension cultures. Cells were treated with methyl jasmonate (3OiiM) orPmg elicitor (5Ozgs/ml), incubated for 24 hours and furanocoumarins extracted in chloroform. The 0D320 of the extracts was read and the amount of furanocoumarins estimatedfrom the extinction coefficient.MJ eIkitorChapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 64-2Ol 50 iiiSolventfront-•4- OriginFigure 4.16: Thin layer chromatography of furanocoumarins from culture media of MJand elicitor treated cells. Parsley cells were treated with either 50 gs/ml Fmg elicitor or30 tM methyl jasmonate. Furanocoumarin derivatives were extracted from the culturefluids and 20 and 50 ls aliquots of extract were separated by thin layer chromatography.C MJ Eli C MJ EliChapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 65Controi 1 jiM ABA 1 OjiM ABA 1 00 jiM ABATime (h) 0 6 24 6 24 6 24 6 24 probe:PAL4CLFigure 4.17: Expression of PAL and CL in response to treatment with ABA. Parsleycells were treated with 1, 10 and 100 zM ABA which was initially dissolved in a minimalvolume of 95% ethanol, total RNA extracted after 6 and 24 hours and transcripts forPc4CL detected by northern blotting. Control cells were treated with distilled watercontaining a minimal volume of ethanol and similarly harvested at 6 and 24 hours.with a range of concentrations of ABA and analysed 4CL and PAL transcript accumulation on a northern blot. After 6 hours, no increase in 4CL or PAL mRNA accumulationabove that in control cells was observed over the range of concentrations used in this experiment (Figure 4.17). After 24 hours of treatment there was no mRNA accumulation,above the level in control cells, observed in the ABA treated cells. However, since therewas an accumulation of mRNA for both genes in the control cells this induction mayhave masked an ABA response at this time point. However it appeared that there wasno rapid ABA induced accumulation of transcripts suggesting that it does not induceexpression of these genes within the time frame required to support a role for ABA inthe rapid elicitor response.4.2.5 The response to MJ of a group of non-phenyipropanoid elicitor-activated genes: ELI’sThe range of elicitor-mediated responses in parsley cells includes the transcriptional activation of a large set of putative defense-related genes (Sommsich et at., 1989) which fallChapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 66into four classes based on the kinetics of their transcriptional activation. I reasoned thatif the MJ induced expression of phenylpropanoid genes is a reflection of a general role forjasmonates in the response of parsley cells to elicitor, then these genes would be similarlyelicitor-responsive. I chose to examine the expression of ELI 7’, HRGP, TyrDC and ELI3 which represent 3 of the 4 kinetic classes. (The 4th class is represented by PAL and4 CL). I used slot blots loaded with total RNA from MJ-treated cells to determine what,if any, was the response of these genes to MJ, and to determine if there were any similarities to the response seen after elicitor treatment. Figure 4.18 shows that, consistentwith previously published results (Sommsich et al., 1989), expression of all four of thesegenes was induced by Pmg elicitor treatment. ELI 7 and TyrDC mRNA accumulationwas highest at the 6 hour time point, and accumulation of mRNA for ELI 3 and HRGPwas highest at the later time point of 9 hours, consistent with the published kinetics.Expression of HRGP, ELI 3 and TyrDC was induced by treatment with 3OM MJ. Interms of timing, the response of HRGP and ELI 3 to MJ was quite similar to theirresponses to elicitor and the level of induction was also similar. The expression of ELI ‘7was unaffected by treatment with 3OtM MJ.4.2.6 The MJ response in whole parsley plantsIn order to determine if the response of this suite of elicitor-inducible genes to MJ is aphenomenon unique to cell cultures, I tested the responses of the general phenylpropanoid genes and other elicitor-inducible genes to 1mM MJ in intact parsley plants. Thisconcentration was chosen because it was the concentration at which optimal activation ofthe 4 CL-i promoter occured in transgenic tobacco (Figure 3.11). I also wished to establish if expression of the non-phenylpropanoid elicitor-inducible genes, shown above to beMJ-inducible, is also wound-inducible. Figure 4.19 shows a northern blot of total RNAfrom wounded and MJ treated parsley plants, hybridised to cDNAs for parsley PAL, 4CLChapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 67MethylElicitor jasmonate ControlTime(h) 0 2 6 9 2 6 9 2 6 9 Probe:1 1,1 III I IIHR1,1 i ELI3I S I I I • TyrDCIi EL17I I I I I I I I aFigure 4.18: The effect of methyl jasmonate, or elicitor treatments on the expression ofdefense-related genes in parsley cells. Cells were treated as described for Figure 4.14.Slot blots loaded with 2 1ug of total RNA were hybridised with probes for ELI 3, ELI 7,TyrDC, HRGP and Ubi.Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 68and other elicitor-inducible genes. Expression of HRGP and ELI 3 was strongly inducedby MJ treatment, consistent with results in cell cultures. The expression of Eli 7 whichwas not induced by MJ in cell cultures, was similarly unaffected in whole plants. Expression of the non-phenylpropanoid genes did not appear to be activated by wounding, infact expression of HRGP and Eli 3 appeared to be repressed by wounding. Despite itshigh level of induced expression in cell cultures (see Figure 4.18), expression of TyrDCwas not detectable on northern blots using total RNA, even with probes labelled to veryhigh specific activity (data not shown). In whole plants, the parsley 4CL and PAL geneswere expressed, after treatment with MJ, in a manner similar to their expression in cellcultures. Their expression was induced above levels seen in control plants after 6 hours,and increased mRNA levels were still present at 24 hours after MJ treatment. Woundingrapidly induced mRNA levels for parsley CL and PAL genes and increased mRNA levelswere still present after 24 hours.4.2.7 The effect of lipoxygenase inhibitors on elicitor-inducible gene expressionIn the model proposed by Farmer and Ryan (Figure 3.2), JA is synthesised de novoin response to stress via lipoxygenase activity. Correlative evidence presented in thisChapter and by others (Gundlach et al., 1992) suggests that endogenously synthesisedJA could play a role in the intracellular transmission of an elicitor-generated signal tothe nucleus. I used an approach similar to that described in Chapter 3, to further testthe hypothesis that MJ is a mediator of the elicitor response in parsley cells. A specificprediction of the model of Farmer and Ryan, if it holds true for this system, is thatlipoxygenase inhibitors would prevent elicitor-inducible gene expression by preventingthe elicitor-stimulated biosynthesis of endogenous JA. I tested the effect of a numberof lipoxygenase inhibitors on the elicitor-responsiveness of several of these genes. TheChapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 69Wounduig Methyl jasmonate0 3 6 24 0 6 6C 24 24C— 4CL— —PALELI 3ELI 7HRGPFigure 4.19: Response to methyl jasmonate and wounding of phenylpropanoid and otherdefense-related genes in parsley plants. Excised leaves were wounded as described inFigure 3.3. Whole parsley plants were sprayed with 1 mM methyl jasmonate in 1.0%Triton or with 1.0% Triton alone (controls) and enclosed in a bell jar in constant lightuntil harvest of tissue. Accumulation of transcripts for CL, PAL, ELI 3, 7, and HRGPwas measured by northern blotting. 10gs of RNA was loaded in each lane.Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 70PAL 4CL TyrDC ELI3 Ubi4PBIP+EIiPB + EliEliFigure 4.20: The effect of ibuprofen (IP) and phenylbutazone (PB) on elicitor inducibleexpression of defense-related genes in parsley cell cultures. Cultures were pretreatedfor 16 hours with the inhibitors and then treated with elicitor. mRNA accumulationfrom these cells was compared on slot blots (loaded with 2 jg total RNA) with mRNAfrom cells treated with elicitor without prior inhibitor treatment. Blots were hybridisedwith cDNAs for PAL, 4CL, TyrDG, ELI 3 and Ubi4. Control cells were treated with acomparable volume of phosphate buffer.inhibitors ibuprofen and phenylbutazone which were effective in inhibition of woundinducible vsp (vegetative storage protein) gene expression in soybean (Staswick et al.,1991) had no effect on the elicitor-induced expression of the parsley genes tested (Figure4.20). Similarly, the inhibitors salicylhydroxamic acid and antipyrene, had no effect onthe elicitor-inducible expression of PAL or 4CL (data not shown).The compound n-propylgallate (nPG), is a potent inhibitor of tobacco lipoxygenase(Fournier et al., 1993), and pretreatment of leaves with nPG decreased wound inducibleexpression of a CL-1 promoter-GUS construction in transgenic tobacco (Figure 3.12).Figure 4.21 shows that nPG decreased the elicitor-induced accumulation of CL, PALand TyrDC transcripts by more than 3 fold (as determined by scanning densitometry).Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 71In contrast, HRGP and ELI 3, were still fully responsive to elicitor (Figure 4.21). Theapparent response of E1i3 to treatment with nPG alone was not present in a duplicateexperiment (data not shown) and possibly reflects an abberation in loading. The apparent elicitor responsiveness of some of the genes despite nPG treatment suggested thatinhibition of elicitor-induced expression of CL, PAL and HRGP transcription was notsimply a reflection of the non-specific loss of ability to effect changes in gene expression.As a further test of the ability of nPG to inhibit the elicitor response in parsley cells, Itested the effect of nPG pretreatment on elicitor-induced furanocoumarin accumulation.Figure 4.22 shows that, in this experiment, Pmg elicitor stimulated the accumulation ofapproximately 1.0 tmoles furanocoumarins per gram fresh weight, and that MJ inducedthe accumulation was about one third this amount, as previously observed (Figure 4.15).Incubation of cells with nPG alone did not induce furanocoumarin accumulation. However, incubation with nPG prior to elicitor treatment resulted in a 10-fold decrease inelicitor-induced accumulation of furanocoumarins. Addition of MJ to nPG-treated cellsresulted in furanocoumarin accumulation similar to that observed with Mi alone. Thus,nPG treatment specifically blocked the responsiveness of the cells to elicitor, but not toMJ.4.2.8 CL-1 elicitor-responsiveness in transgenic tobaccoThe above data are consistent with the hypothesis that MJ plays a role in the elicitorresponse, including activation of 4CL expression. However, previous work suggestedthat the 4 CL-i promoter alone is not sufficient for elicitor responsiveness in transgenictobacco or stably transformed parsley cell cultures (Douglas et al., 1991). Since the4 CL-i promoter is MJ responsive in tobacco, as shown in Chapter 3 (Figure 3.8), itis difficult to reconcile those observations with a role for MJ-mediated signalling in theelicitor response in tobacco. As part of my work, I looked at the effect of MJ and elicitorChapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 72PAL 4CL TyrDC ELI3 HPRG Ubi4nPGnPG + Eli—_____EliC— —MJ .. :.•. . .•Figure 4.21: The effect of the lipoxygenase inhibitor, n-propylgallate (nPG) on elicitor-inducible expression of defense-related genes in parsley. Treatment with the inhibitornPG, and loading and hybridisation of slot blots, was as described in Figure 4.20.on GUS expression at the RNA level in the Fl generation of transgenic tobacco line801-8, derived from the selfing of the primary transformant 801-8. The 801-8 primarytransformant was used in the original work on elicitor-induced 4 CL-i expression andcarries a GUS gene under the direction of -597bp of the 4 CL-i promoter. Surprisingly,the Fl individual of line 801-8 examined showed a marked increase in GUS expressionin response to Pmg elicitor (Figure 4.23), showing that, in this individual at least, the4 CL-i promoter was elicitor responsive. An explanation for this anomalous result maylie in the fact that the plants used in these experiments had been through at least onemeiosis, giving the potential for a change in the introduced construct, rendering it nowcapable of elicitor responsiveness. Thus, in tobacco, the 4 CL-i promoter appears to becapable of responding to elicitor as well as wounding and MJ application.Chapter 4. The role of jasmonates in the elicitor response in parsley cell culturesCDCoCDI731.00.8 —0.6 -0.4-0.2 -0__________Figure 4.22: The effect of n-propylgallate treatment on furanocoumarin levels in culturefluids of parsley cell suspension cultures. Cultures were pretreated for 16 hours with nPGand then treated with either 50 M Pmg elicitor (nPG + Eli), 30 tM MJ (nPG + MJ),left untreated (nPG). Controls were pretreated with buffer alone and then treated with 30M MJ (MJ) or 50 tM Pmg elicitor (Eli), untreated (C). After 24 hours furanocoumarinswere isolated and quantified as described in Figure 4.15.control nPG nPG + MJ MJ nPG + Eli EliChapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 74Elicitor0 2C 2 0 2C 2-.2kbProbe: St4CL GUSFigure 4.23: The effect of Pmg elicitor treatment on 4 CL-i promoter activity in a plantof transgenic line 801-8. Leaves from the selfed offspring of the original transformedline 801-8 (transgenic for a -597bp 4CL-i promoter-GUS fusion), were incubated in 500izg/ml Pmg elicitor for 3 hours. Control leaves were incubated in water. Duplicate blotswere hybridised to probes for St4CL (to detect tobacco 4CL transcripts) and GUS.4.3 DiscussionThe rapid and transient change in gene expression caused by the addition of Pmg elicitorto the parsley cells in suspension culture make this an ideal system in which to studyregulation of stress-induced gene expression. Using this greatly simplified system I haveinvestigated signalling involved in stress responses, with particular attention paid tothe role of jasmonates. Although direct evidence is lacking, a number of workers havesuggested MJ may play a role in signalling (Reviewed by Staswick, 1992). In support ofthis notion, MJ is distributed in many plant species (Meyer et. al., 1984), and bears greatsimilarity in structure and biosynthesis to eicosanoids, a group of important mammalianregulatory compounds which function as stress related second messengers (Anderson,1989). In Chapter 3, I presented evidence consistent with the hypothesis that the woundresponse, mediated by the 4 CL-i promoter in tobacco, is mediated by jasmonates. Inthis Chapter, I used parsley cells in suspension culture to further test the hypothesis thatChapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 75jasmonates are involved in mediating changes in gene expression in this system.In parsley cell cultures, expression of the genes of the phenyipropanoid pathway PAL,4 CL and BMT was activated by MJ. However, the response to MJ was in each casedifferent in either the timing of transcript accumulation, or the level of the response.This means that if the ability of MJ to induce expression of these genes reflects its roleas a signalling intermediate, it is probably not acting alone. Other signalling moleculesmay be required as well as JA. There is evidence suggesting that changes in proteinphosphorylation patterns, and intracellular Ca2+ levels may play a role (Renalt et al.,1993) and potentially these may interact with JA to mediate the elicitor response. Indeed,it may be the interaction of a number of second messengers that gives the pattern of geneexpression seen after elicitor treatment. ABA can probably be ruled out as a signallingmolecule with a direct role altering 4 CL or other phenyipropanoid gene expression inreponse to stress, since a range of concentrations of ABA had no effect on the expressionof 4CL or PAL within the time frame necessary to suggest it may have a role as asignalling molecule in the rapid response to elicitor . We cannot, however, rule out thepossibility that it may be acting (as it does in other systems) as a translational regulatorof gene expression (reviewed by Gallie, 1993), or in combination with other signallingmolecules to bring about changes in phenyipropanoid gene expression.In support of a model where other components to the transduction pathway besidesMJ are involved, is the observation that the level of furanocoumarins accumulated inMJ treated cells, although significantly higher than control cells (10 fold higher), wasone third less than the level seen in elicitor treated cells. Lozoya et al., (1991) reportthat activation of both the flavonoid- and the elicitor- specific branch pathways of thephenyipropanoid pathway by simultaneous treatment with both Pmg elicitor and UVlight results in an overall decrease in the level of furanocoumarin secretion, as comparedChapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 76to elicitor treatment alone. This decrease, however, appears to occur at the posttranscriptional level since transcription rates and mRNA accumulation are unaffected. Sincetreatment of cells with MJ activates both the flavonoid pathway (Dittrich et al., 1992)and the furanocoumarin pathway perhaps the low coumarin accumulation is a reflectionof a similar phenomenon. It could be hypothesised that activation of both pathways byMJ decreases the carbon flow to furanocoumarin biosynthesis relative to the carbon flowoccurring subsequent to specific activation of furanocoumarin biosynthesis after elicitortreatment. If this is the case I would assume that the observed MJ activation of gene expression is not analogous to the natural situation within the cell after elicitor treatment,or that the levels of JA produced in vivo in response to this stress, are such that stimulation of both pathways does not occur. Although quantaatively the MJ-induced coumarinaccumulation differed from elicitor-induced coumarin accumulation, qualitatively bothprofiles of coumarin accumulation were similar. Methyl j asmonate therefore stimulatesthe same pattern of accumulation of antimicrobial compounds as elicitor treatment.A high level of MJ (greater than 100 pM) or JA (greater than 1.0 pM) resulted inreduced biological activity in both the parsley (Figure 4.13) and the tobacco system(Figure 3.11). It has been suggested that levels of JA greater than 50 pM are toxic toplant tissue (Anderson, 1989). To avoid the possibility of JA in treated cells reachingtoxic levels, I conducted all methyl jasmonate experiments in the parsley cells using aconcentration of 30 pM, even though a slightly higher .4CL expression was observed at100 pM. Kauss et al., (1992) similarly measured the effect of a range of concentrationsof MJ on furanocoumarin secretion in parsley cells. In contrast to the data presentedhere, they reported an accumulation of furanocoumarin derivatives after treatment with100 pM MJ of approximately 1/50th the level measured in elicitor treated cells. Theydo not report absolute furanocoumarin levels. This discrepancy may be explainable.Staswick (1992) pointed out that results of MJ treatment are difficult to compare amongChapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 77laboratories. The reason is that different stereoisomers of jasmonates are possible. (-)Jasmonate is easily epimerised to the (+)isomer, which apparently has lower biologicalactivity. Commercial preparations may differ in the ratio of the different isomers presentand the MJ preparation used in my work was obtained from a different source thanthat used by Kauss et al. (1992). Another possible explanation is the fact that theseworkers dissolved MJ in ethanol, rather than Triton (as reported here). MJ is not readilysoluble, and potentially Triton X-100 is a better choice of solvent than ethanol. At lowerconcentrations JA was more active than the methyl ester in inducing increases in 4CLmRNA accumulation. This is to be expected since JA is the biologically active form ofj asmonate.I have shown that MJ, in addition to activating phenyipropanoid gene expression,activates the expression of a subset of elicitor-responsive parsley genes. These genes wereoriginally isolated from parsley cells based on their rapid transcriptional activation afterelicitor treatment. Based on DNA sequence comparisons, functions have been assignedto some of these genes. The fact that expression of these genes is induced by elicitortreatment suggests they play a role in defense (Sommsich et al., 1989). ELI 3, HRGPand TyrDC expression was induced by MJ treatment, however ELI 7 expression wasunaffected under the same conditions. The fact that MJ did not affect expression of allof these genes implies the presence of multiple elicitor-induced signalling pathways forinduction of elicitor-responsive genes. This hypothesis is consistent with the apparent lackof conserved promoter elements among the elicitor responsive genes, 4CL, PR2 and ELI7 (I. Sommsich, personal communication). Furthermore, of the non-phenyipropanoiddefense-related genes tested, the elicitor reponse of TyrDC was uniquely affected bynPG treatment, which implies further branching of signalling pathways for the elicitorresponse. This gene may therefore share signal transduction pathway components withthe phenyipropanoid genes. The other genes which were induced by MJ, but unaffectedChapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 78by nPG, may be induced by other lipoxygenase independent pathways.Both 4CL and PAL are inducible by both wounding and elicitor (Hahlbrock andScheel, 1989). Endogenous JA levels may therefore play a role in the response to eitheror both stresses. Whole plant experiments showed that expression of ELI 3 and HRGP,which is strongly induced by MJ, is not locally induced by wounding. Thus, if JA isan endogenous signal involved in their regulation, it is more likely to play a role in theelicitor response rather than the wound response. In whole plants expression of ELI 3,TyrDC, and HRGP was induced by methyl jasmonate, but the expression of ELI 7 wasunaffected showing that the MJ response is present in whole plants as well as cells inculture.a-LA activated CL expression in the cell cultures and this response was maximum at30 M. At 300 1tM expression decreased markedly. The most likely explanation for thisis that linolenic acid is toxic to the cells at higher concentrations. The ability of a-LA toinduce CL-1 mRNA accumulation in the cells, may reflect its conversion to JA. This issupported by the observation that 7-LA, which does not enter the biosynthetic pathwayto JA (Hamberg and Gardner, 1992) had no effect on 4CL expression (data not shown). Ifthis is the case it means that lipoxygenase activity in the cells is present constitutively atsufficiently high levels to convert LA to biologically active levels of JA. Thus, inductionof lipoxygenase activity may not be a prerequisite for the elicitor response. Elicitorperception at the cell membrane may result in release via lipase activity of LA fromcell membrane as proposed by Farmer and Ryan, (1992). Induction of lipoxygenaseactivity may however be important in stress responses in other systems since its activityis induced in tobacco cells treated with elicitors from Phytophthora parasitica (Fournieret. al. 1993), oat infected with Puccinia coronata avenac (Yamamoto and Tani, 1986),and in rice infected with Magnaporthe grisea (Ohta et. al. 1991). Also, increasedlipoxygenase gene expression is associated with pathogen attack in Arabidopsis (MelanChapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 79et. al., 1993).The parsley lipoxygenase and its inhibitors have not been characterised. I thereforetested the ability of a large number of known lipoxygenase inhibitors to affect the elicitor response. Five of these, ibuprofen, phenylbutazone, antipyrene, n-propylgallate andsalicylhydroxamic acid, which are known inhibitors of soybean lipoxygenase, inhibitedwound-inducible expression of vsp genes in soybean hypocotyls (Staswick et al., 1991).In parsley cells, only n-propylgallate, an inhibitor of tobacco lipoxygenase which inhibited wound-induced activation of the 4CL promoter in transgenic tobacco (Figure 3.12),affected the elicitor-responsiveness of 4CL. Elicitor-responsiveness of PAL, and TyrDCwas also affected by treatment with nPG. Furthermore, elicitor-induced furanocoumarinsynthesis was dramatically reduced by treatment with nPG (Figure 4.22). The abilityof nPG to markedly decrease the stress responses in both systems suggests that endogenous JA could play a general role in the stress-induced signalling which culminates inthe transcriptional activation of 4CL and other parsley defense-related genes. nPG maybe inhibiting de novo synthesis of endogenous JA released from the cell membrane afterperception of the elicitor response, and JA may be required as an intracellular signalmediating changes in gene expression. In the parsley system, nPG does not appearto be non-specifically inhibiting cellular processes since some of the elicitor-induciblegenes tested retained elicitor-responsiveness after nPG treatment, and expression of theconstitutive control gene, ubi4, was also unaffected by nPG treatment. Furthermore,nPG-treated cells retained the ability to respond to MJ since addition of MJ to cellsafter inhibition restored activation of the phenylpropanoid pathway, as measured by accumulation of furanocoumarins (Figure 4.22). One interpretation of these results is thataddition of MJ bypassed the block in lipoxygenase activity caused by nPG, restoring thesignalling pathway within the cells.Regardless, it cannot be assumed that the effect of nPG are not attributable toChapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 80something other than an inhibition of lipoxygenase activity. Lipoxygenase activitiesin nPG treated cells were not measured, and furthermore even if such assays were toreveal an inhibition of lipoxygenase activity after nPG treatment it would not necessarilymean that this was the sole effect of the inhibitor. nPG may well affect other processesimportant in the elicitor response. nPG is an antioxidant that functions by reactingwith free-radical intermediates of the reaction (Vick and Zimmerman, 1987b). It hasrecently been suggested that reactive oxygen species can induce pathogen-related geneexpression (Jones, 1994). A rapid (within 1-5 minutes) burst of hydrogen peroxide isreleased from cultured soybean cells treated with PGA (polygalacturonic acid) elicitorand may stimulate subsequent defense pathways (Legendre ci al., 1992). A similar rapidoxidative burst occurs in parsley cells treated with Pmg elicitor (Dierck Scheel, personalcommunication). nPG may interfere with this hypothetical signalling mechanism.Despite the potential for non-specific activity of the inhibitor, there is good evidence that JA plays a role in intracellular signalling of the elicitor response. Much ofthe complete elicitor response is induced by jasmonates, i.e. activation of genes of thefuranocoumarin specific branchpathway of phenyipropanoid metabolism, secretion of furanocoumarins from MJ treated cell, and MJ inducible expression of other defense genesand the ability of LA to induce 4CL gene expression at least, also supports the existenceof a role for endogenous JA in signalling.In this thesis, I have used 4 CL-i as a model gene to study stress induced expression.In Chapter 3, I showed that this gene is wound inducible in transgenic tobacco and thatthis response is mediated by promoter elements. A promoter fragment also directed MJand JA inducible GUS expression, and nPG inhibited wound-induced activation of the4 CL-i promoter. This suggests that JA could be an endogenous signal in transmittingthe wound signal to the nucleus to induce 4CL expression.Chapter 4. The role ofjasmonates in the elicitor response in parsley cell cultures 81The work described in this chapter shows that endogenous JA may activate 4CL expression in parsley cells, and that JA may be an intracellular signal involved in activating4 CL expression following elicitor perception. If so, these results must be considered inview of the ability of the 4 CL-i promoter to respond to elicitor and MJ treatment. IfMJ/JA is an intracellular signal synthesized in response to elicitor, one would predictthat the 4 CL-i promoter responds similarly to JA/MJ and elicitor. However, previouswork showed that the 4 CL-i promoter alone is not sufficient for elicitor-responsivenesssin transgenic tobacco or parsley cells (Douglas et al., 1991), while the 4 CL-i promoterclearly responds to MJ in the same transgenic tobacco lines (Figure 3.11). However, theresults presented in Figure 4.23 show that the Fl progeny of at least one primary tobaccotransformant (801-8) in which the 4 CL-i promoter was elicitor-unresponsive (Douglaset al, 1991) has acquired the potential for elicitor-responsiveness.One possible explanation is an epigenetic modification of the construct in the originaltransformant that was reversed after meiosis. Methylation of cytosines is frequentlycorrelated with inactivation of gene expression. Multiple unlinked copies of a homologousgene have been shown to lead to reversible inactivation of the genes involved (Reviewedby Matzke and Matzke, 1993). In this case however, the 801-8 primary transformantsegregated 3:1 for Kanamycin resistance upon selfing indicating a single insertion orseveral tightly linked insertions. Several studies have shown that multiple copies ofclosely linked or tandemly arranged transgenes in plants can also lead to inactivationthat is correlated with reduced activity. Such methylation-associated inactivation isreversible through one or more generations. It is conceivable that an inactivation of apromoter element required for elicitor inducibility could result in the recovered elicitorinducibility in offspring of primary transformants. A second explanation may lie in thefact that the original work looked at the elicitor response at the level of GUS enzymaticChapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 82activity rather than RNA accumulation as described here. The observation that the 4CL-1 promoter is capable of elicitor-responsiveness as well as wound and MJ-responsivenessis consistent with a potential role for JA as an intracellular signal involved in mediatingresponsiveness to both of these stimuli.The question of whether the 4 CL-i promoter is MJ activated in parsley cell cultures,remains open, and a correlation between regulatory sequences mediating elicitor and MJresponsiveness of the 4 CL-i gene in parsley cells cannot yet be made. I therefore cannotsay if the known lack of promoter-responsiveness to elicitor in parsley cells is associatedwith a presence or absence of promoter-responsiveness to MJ in the same system. Parsleycells can readily be transiently transformed and this system has been successfully usedto analyse the promoters of other elicitor-inducible parsley genes (Schulze-Lefert et al.,1989; van de Löcht et al., 1990). It is not however without problems. For example,transient expression of plasmid DNA is strongly influenced by bacterial strain genotype(Tovar et al., 1993), and analysis of elicitor-induced expression of the PAL promoter inthis system has proved difficult (Klaus Hahibrock personal communication). For thesereasons, definitive results regarding the elements required for MJ-induced expression inparsley cells are best obtained using stably transformed cells. Parsley cells are difficult totransform stably and I was unable to generate stable transformants. In spite of the uncertainties regarding the promoter sequences required for MJ and elicitor-responsivenessin parsley, the accumulated data presented in this chapter argue strongly for a potentialrole of endogenous JA in the stress-activated expression of 4 CL-i in parsley cells.In conclusion, the data presented in this chapter support and extend the observationsin Chapter 3 which suggest a role for MJ in the stress response of 4 CL-i. I have alsopresented data which suggests not only a role for MJ in the response of 4CL, but alsoin the response of genes involved in furanocoumarin biosynthesis, and other elicitorinducible genes. It is clear that a single transduction pathway does not mediate theChapter 4. The role of jasmonates in the elicitor response in parsley cell cultures 83elicitor response but more likely there exists a diversity of signalling pathways controllingexpression of defense-related genes.Chapter 5Expression of parsley 4CL-1 in Arabidopsis thaliana5.1 IntroductionGenetic approaches are potentially powerful for identifying second messengers involvedin regulating gene expression. By identifying mutants which no longer respond to external stimuli in the normal way, genes whose products are required for signal transductioncan be identified genetically. In plants, a useful organism for such studies is Arabidopsisthaliana. The usefulness of Arabidopsis for laboratory work in classical and moleculargenetics is well known and has been extensively reviewed (Meyerowitz 1989; Meyerowitzand Pruitt, 1985; Estelle and Somerville 1986). It is small, very prolific, and has a generation time of as short as 5 weeks. Its five chromosomes contain only 7 X 1O bp of DNA,the smallest genome known in angiosperms. These features allow large scale mutagenesis,and the existence of multiple genetic markers facilitates mapping of new mutations. Theexistence of RFLP maps and the low amount of repetitive DNA has made possible theisolation by chromosome walking of a number of genes identified by mutant phenotypes(Chang et at., 1993; Arondel et al., 1992; Giraudat et at., 1992). Furthermore, Arabidopsis is readily transformable. A leaf disc transformation assay using Agrobacteriumtumefaciens has been described (Lloyd et at., 1986). Other transformation protocols havebeen reported including a root explant transformation method, and transformation viarooty tumours (Van Sluys et al., 1987; Valvekens et at., 1988). It is not surprising thata great number of research programs take advantage of these attributes.84Chapter 5. Expression of parsley 4CL-1 in Arabidopsis thaliana 85A disadvantage of Arabidopsis is that, in contrast to systems like the parsley cellsuspension culture system, there is less information regarding the responses of this plantto challenge with potential pathogens. Due to the interest in developing Arabidopsis asa model system, there have been a number of recent reports in the literature dealingwith the response of Arabidopsis to pathogen attack. For example, pathogen-inducedactivation of the Arabidopsis acidic chitinase promoter has been analysed, and elementsmediating induced expression identified (Samac et aL, 1991). The induction of Arabidopsis defense genes by virulent and avirulent Pseudomonas syringae strains has beenstudied and a putative avirulence gene involved in the interaction has been cloned (Donget al., 1991). A plant defense gene, ELI3, has been identified in Arabidopsis which appears to play a functional role in establishing a resistant phenotype in the interactionbetween Arabidopsis and phytopathogenic Pseudomonas syringae strains. Furthermore,a suspension culture system has been developed in which putative defense responsesare induced by treatment with two different elicitors, a bacterial pectin-degrading enzyme a-1,4-endopolygalacturonic acid lyase (PGA lyase), and Pmg elicitor (Davis andAusubel, 1989). In this system, an increase in PAL activity and increases in PAL andCL mRNAs were reported. Elicitor stimulated accumulation of two mRNAs associatedwith lignin deposition, caffeic acid O-methyltransferase and peroxidase but no increasein chalcone synthase mRNA was observed, suggesting that flavonoid derivatives are notproduced as defense compounds ii Arabidopsis. There has also been a report of inductionof genes encoding 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHP), thefirst enzyme in the shikimate pathway, in response to wounding and pathogen attack(Keith et al., 1991). The phenomenon of systemic acquired resistance (SAR) in Arabidopsis has recently been studied (Uknes et aL, 1992). SAR is the phenomenon wherebya pathogen infection results in a hypersensitive response at the site of infection, andleads to subsequent resistance to attack by a range of pathogens. This response has beenChapter 5. Expression of parsley 4CL-1 in Arabidopsis thaliana 86well characterised in tobacco and is correlated with the expression of a group of genescalled SAR (systemic acquired resistance) genes (Ward et al., 1991). By treatment witha chemical, INA (2,6-dichioroisonicotinic acid), these workers were able to mimic thehypersensitive response in Arabidopsis, with subsequent acquired resistance to a numberof pathogens and expression of SAR genes.Some genes of phenyipropanoid metabolism have been cloned from Arabidopsis. Aclone for the single chalcone synthase gene has been isolated (Feinbaum and Ausubel,1988) and promoter sequences required for its light regulated expression identified (Feinbaum et al., 1991). Recently, a cDNA clone for Arabidopsis 4CL has been isolated (Lee,Eflard and Douglas, in preparation). Like chalcone synthase, it appears to be a singlecopy gene. Many gene families are smaller in Arabidopsis than in other plant species(Meyerowitz 1987). PAL is an exception to this generalisation, existing as a small genefamily of four to five members, comparable in size to the gene family in parsley andbean (Lois et al., 1989; Ohi et al., 1990). Expression of one of these Arabidopsis PALgenes, PAL-i, has been partially characterised. The PAL-i promoter is activated earlyin seedling development and in adult plants it is highly expressed in vascular tissue and inflowers, a pattern similar to expression of parsley CCL-i in transgenic tobacco (Reinold etal.,1993). The Arabidopsis PAL-i promoter contains elements responsible for conferringaccumulation of GUS mRNA in response to wounding, heavy-metal stress, and light.The genetics of Arabidopsis could be exploited to identify genes encoding components ofthe signalling pathways which mediate responses such as these.The first requirement for the identification by mutagenesis of genes involved in signaltransduction is a genetic screen for phenotypes by which mutants can be distinguishedfrom wild type plants. In this chapter, I discuss stress-inducible expression of .4CL inArabidopsis. I show that wound-inducible GUS expression, directed by the.4 CL-i promoter in transgenic Arabidopsis, provides a screen which could enable the identificationChapter 5. Expression of parsley 4CL-1 in Arabidopsis thaliana 87of mutants in transduction pathways leading to wound-activation gene expression.5.2 Results5.2.1 Wound-inducible expression of a parsley 4CL-1 promoter-GUS fusionin transgenic ArabidopsisTo determine if the 4 CL-i promoter is wound-responsive in Arabidopsis, I used a linetransgenic for a 1.5kb parsley 4 CL-i promoter fused to GUS (204-1-3), which had beengenerated by Agrobacterium-mediated transformation of roots of ecotype C24 (R. Moseleiand C. Douglas, unpublished results). The primary transformant which gave rise to 204-1-3 appeared to contain the T-DNA at a single locus, since, after selfing, FT progenysegregated 3:1 for kanamycin resistance carried on the T-DNA. After confirming that line204-1-3 was homozygous for the T-DNA, I used Southern blots to attempt to estimatethe number of T-DNA inserts at a single locus. The pattern of hybridisation was notreconcilable with a single T-DNA insertion, but also was not consistent with simpletandem insertions, suggesting some rearrangements of the T-DNA (data not shown).I used a histochemical assay to detect wound-inducible GUS expression in leaf discscut from these plants. This assay is based in the cleavage of a substrate, X-gluc, by theenzymatic activity of GUS, to yield an indigo dye. Figure 5.24 shows the accumulationof GUS activity around the margins of the disc and also at a tear which resulted fromforceps handling. A control leaf disc which was fixed and stained immediately afterwounding, showed only GUS activity in vascular tissue, and none at the site of wounding.I performed a second type of wound assay in these plants where, rather than remove leafmaterial from the plant immediately prior to wounding, I wounded the leaf while it wasstill attached to the plant, thus mimicking herbivory. Figure 5.25 shows how this inducedaccumulation of GUS activity localised at the wound site of the attached leaf. The controlChapter 5. Expression of parsley 4CL-1 in Arabidopsis thaliana 88leaves which were removed, and stained immediately after wounding, showed only someGUS activity in vascular tissue and none at the wound site.I used the same transgenic line to look at the accumulation of GUS (driven by theparsley 4 CL-i promoter) and Arabidopsis 4CL mRNAs in wounded excised leaves. Arabidopsis 4CL transcripts were detected using a cDNA clone for Arabidopsis 4CL. Thenorthern blot shown in Figure 5.26 shows that the Arabidopsis 4CL ene was responsiveto wounding. Transcript accumulation was seen by 2 hours after wounding and a highlevel of transcripts was still present 24 hours after wounding. Similar wound-inducibleexpression was seen for GUS driven by the 4 CL-i promoter. The 4 CL-i promoter istherefore sensitive to the wound signal in Arabidopsis. In both cases maximum transcript levels were observed by 2 hours after wounding. This peak, however, was morepronounced for accumulation of GUS transcripts, and the level of GUS transcripts waslower at the later time points.5.2.2 The effect of ethylene on 4CL expression in ArabidopsisIn Chapter 3, I showed that an intact copy of the parsley 4 CL-i gene is ethylene-responsive in transgenic tobacco. To determine if the Arabidopsis 4CL gene is ethyleneresponsive and to determine if the sequences mediating this response are contained withinthe promoter region of the parsley 4 CL-i gene, I treated 204-1-3 plants with 10 mg/mlethephon and analysed Arabidopsis 4CL and GUS mRNA levels on northern blots. The4 CL gene responded strongly to treatment with ethephon, and by 24 hours a large increase in transcript levels was observed. The parsley 4 CL-i promoter also appears todirect a small level of GUS mRNA accumulation in response to ethephon (Figure 5.27)but this response is less than the response of the endogenous gene. Hybridisation withthe GUS probe detected a second band which migrated faster than the 2 kb GUS mRNAsuggesting the presence of a degradation product of GUS mRNA.Chapter 5. Expression of parsley 4CL-1 in Arabidopsis thaliana 89wFigure 5.24: Accumulation of GUS activity in wounded leaf discs of transgenic Arabidopsis line 204-1-3. Leaf discs were punched from Arabidopsis plants transgenic for al500bp parsley 4CL-1 promoter GUS-fusion (204-1-3). These discs were either immediately stained with X-Gluc (C) to detect GUS activity, or incubated for 24 hours on filterpaper moistened with MS media without hormones (W) prior to staining.CFigure 5.25: Accumulation of GUS at wound sites in leaves of Arabidopsis line 204-1-3.Leaves of Arabidopsis transgenic for a l500bp parsley 4 CL-i promoter GUS-fusion(204-1-3), were wounded while still attached to the plant. Wounded tissue was eitherremoved and stained immediately with X-Gluc to detect GUS activity (C), or removedand stained 24 hours after wounding (W).Chapter 5. Expression of parsley 4CL-1 in Arabidopsis thaliana 904*wCnChapter 5. Expression of parsley 4CL-1 in Arabidopsis thaliana 91Time 0 2 6 24 Trne 0 2 6 242kb2kbProbe: GUS IProbe: 4CLFigure 5.26: Accumulation of GUS and Arabidopsis 4CL transcripts in wounded leavesof Arabidopsis line 204-1-3. Leaves were detached from Arabidopsis plants transgenicfor a l500bp parsley 4 CL-i promoter GUS fusion (line 204-1-3) and they were woundedby slicing into 1-2 mm strips. Tissue was then either frozen immediately (0 hour) orincubated on MS media (without hormones) for the times indicated. 10 pg of RNA wasisolated and separated on a formaldehyde gel. Duplicate blots were hybridised to cDNAprobes for Arabidopsis 4CL or GUS.Time 0 6 24 Time 0 6 24hrs2kb kbProbe: 4CLProbe: GUSFigure 5.27: Accumulation of GUS and Arabidopsis 4CL transcripts in Arabidopsisethephon treated leaves of transgenic line 204-1-3. Arabidopsis plants transgenic fora l500bp parsley 4 CL-i promoter GUS fusion (line 204-1-3) were sprayed with 10 mg/ml2-chioroethanephosphonic acid and tissues harvested at the 0, 6, and 24 hours. 10 pgof total RNA was loaded on duplicate northern blots which were hybridised with probesfor Arabidopsis CL to detect endogenous Arabidopsis 4CL transcripts, or GUS to detectthe activity of the introduced gene construct.Chapter 5. Expression of parsley 4CL-1 in Arabidopsis thaliana 925.2.3 Response of 4CL to methyl jasmonate in ArabidopsisI showed that the parsley 4 CL-i promoter, as well as being wound-responsive, is stronglymethyl jasmonate-responsive in transgenic tobacco (Figure 3.8). In analogous experiments in transgenic Arabidopsis, 204-1-3 plants were treated with 1 mM and 10 mMmethyl jasmonate and accumulation of GUS and 4CL transcripts measured by northernblotting. Figure 5.28 shows there was an accumulation, above the level seen in controlplants, of transcripts for Arabidopsis 4CL in response to both concentrations of methyljasmonate. There was also, directed by the parsley 4 CL-i promoter, an accumulation ofGUS transcripts. Again, a second smaller band was detected with the GUS hybridisationprobe suggesting the presence of a smaller mRNA product made either in vivo or in vitroduring the extraction process. The control plants were treated with 1% Triton X-100 (inwhich methyl jasmonate was dissolved) alone and this treatment, appeared to result insome transcript accumulation. Thus, the effect of the Triton may be masking a largerMJ response in these plants indicating that a more effective way of applying MJ shouldbe devised.5.2.4 A genetic screen for identification of genes involved in mediating thewound responseThe accumulation of histochemically detectable GUS activity around wound sites inplants of the transgenic line 204-1-3 provided a phenotype which could be readily assayedin a screen for mutants defective in wound-induced signal transduction leading to theactivation of the parsley 4 CL-i promoter. Thus, I mutagenised seeds from this line withEMS, (Ml generation), and allowed them to self to produce the M2 generation in whichrecessive mutations which might affect the wound-inducible expression of the introducedGUS fusion would be expected to segregate. In order to try to avoid analysis of cisChapter 5. Expression of parsley 4CL- 1 in Arabidopsis thaliana 931mMM.1 1OmMMJOC 6 24 6 24 24CS GUS4CLFigure 5.28: The effect of methyl jasmonate treatment on 4CL expression in transgenicArabidopsis. 204-1-3 Arabidopsis plants were sprayed with 1 mM and 10 mM solutionsof methyl jasmonate dissolved in 1.0% Triton X-100. Control plants were sprayed with1.0% Triton X-100 alone. Plants were harvested at the times shown in hours (h), 10 pgof RNA was separated on a formaldehyde gel and duplicate blots hybridised with probesfor Arabidopsis 4CL to detect endogenous if CL transcripts, or GUS to detect the activityof the introduced gene.mutations, in which the parsley J CL-i promoter or the GUS structural gene rather thangenes whose products are required for the wound-induced expression of the transgene,has been mutated, I screened for mutants which showed an altered wound response butretained tissue-specific expression of GUS. Thus, any potential mutant would contain afunctional transgene, although cis-mutations in promoter elements required for woundinducible expression would be possible. I individually screened 5,000 M2 plants and inthis number there was no mutant which lacked the wound response and retained tissuespecific expression.5.3 DiscussionWith a view to using Arabidopsis as a model genetic system for studying regulationof phenylpropanoid gene expression, I have studied the expression of both the parsleyChapter 5. Expression of parsley 4CL- 1 in Arabidopsis thaliana 944 CL-i and the Arabidopsis 4CL gene in response to wounding in Arabidopsis. TheArabidopsis 4CL gene responded strongly to wounding by accumulation of mRNA, andthe parsley 4 CL-i promoter responded to wound signals produced in Arabidopsis, sinceit directed wound inducible accumulation of GUS mRNA. The accumulation of GUStranscripts was less at the later time points than the accumulation of 4CL transcripts.One explanation for this is different stabilities of the two mRNAs. This result shows thatthe signalling pathways in the two species are conserved. When studied histochemically,the accumulation of GUS activity around wound sites (driven by the 4 CL-i promoter),was very sharply localised to the margins of the wound. This pronounced accumulationof GUS activity provided the basis for a genetic screen which, it was hoped, would allowgenetic identification of loci for genes important in signal transduction mediating wound-induced 4CL gene expression.An important requirement in a screen based like this one on introduction of a transgene, is a way to distinguish between cis-mutations, in which the introduced gene construct has been mutated, and trans-mutations, where expression of the introduced construct has been affected by mutation of another locus which acts upon it. One approachto overcoming this problem is the use of a transformed line in which there is more thanone transgene (e.g. promoter- GUS fusion). A trans mutation would therefore affect allthe introduced constructs and the probability of inactivating all the introduced GUSgenes with cis mutations would be small. Southern analysis of 204-1-3, the homozygoustransformed line used in this study, suggested more than one insertion but was ambiguous in defining the number and structure of inserted T-DNAs. Since I was not ableto establish with certainty the manner in which the introduced construct was arrangedwithin the plant, I used an alternative approach and screened only for mutations whichlacked wound inducible expression of GUS but retained tissue-specific expression. In aChapter 5. Expression of parsley 4CL-l in Arabidopsis thaliana 95preliminary screen of 5,000 M2 plants, I failed to identify any such mutants. I would expect non wound-responsive mutations to be rare, and screening for mutations which stillretain tissue-specific expression could further reduce the number of target genes beingscreened for. Results in Chapter 3 showed that in transgenic tobacco, the 2lObp promoter fragment that directs tissue-specific expression of parsley 4CL-1 is also sufficientto activate gene expression in response to wounding. This indicates that the signallingpathways involved in directing tissue-specific and wound-inducible expression of CL-1 could converge on the same promoter elements and thus could share components incommon. If the situation is similar in Arabidopsis, the number of target genes affectingwounding and not tissue-specific expression may be small.There has been a report in the literature of a successful genetic screen for signaltransduction mutants in Arabidopsis, which was also based on expression of a promoter-GUS transgene (Susek et al., 1993). In that study, 100,000 M2 plants were screenedto obtain a small number of mutants affected in chloroplast-induced activation of thepromoter of a nuclear gene encoding chlorophyll a/b binding protein (cab). Their strategydiffered in that they generated a transgenic line of Arabidopsis which carried two differentreporter genes (GUS and NPTi!), each driven by the CABS promoter. They screenedM2 plants for mutants that, in the absence of chloroplast development, expressed bothreporter genes driven by the CAB2 promoter, and discarded potential mutants in whichexpression of only one of the transgenes was affected. From a screen of 100,000 M2plants they isolated 9 plants which had heritable mutant phenotypes in which CAB2-GUS expression was uncoupled from chioroplast development.The Arabidopsis gene responded to ethylene, applied as ethephon and the 4 CL-ipromoter appears to direct responsiveness to ethephon upon a GUS reporter gene. However, induced accumulation of GUS transcripts appeared to be lower than the responseof the endogenous Arabidopsis gene. This would make analysis of promoter elementsChapter 5. Expression of parsley 4CL-1 in Arabidopsis thaliana 96mediating this response difficult. Other limitations of using ethephon to study ethylenemediated changes in gene expression were discussed in Chapter 3. A genetic approachmay prove more successful in elucidating the role, if any, for ethylene in phenylpropanoidgene expression. Mutants in Arabidopsis that are insensitive to ethylene have been described (Bleecker et al., 1988; Pickett et al., 1990). The mutation etrl for example is adominant mutation that inhibits a diverse number of processes affected by ethylene e.g.,cell elongation, promotion of seed germination, enhancement of peroxidase activity, andfeedback suppression of ethylene synthesis by ethylene. It is thought that the ETRJ geneproduct acts early in the ethylene signal transduction pathway, possibly as a receptor.Cloning of this gene by chromosome walking revealed that it encodes a product that issimilar to the prokaryotic two-component regulators (Chang et al., 1993). In bacteriathese systems consist of two proteins, a sensor and a regulator which function together toregulate adaptive responses to environmental stimuli. The ETRJ gene product encodesa single protein with similarities to both components. It is thought that ETR1 may bea sensor of ethylene with its amino terminus acting as the sensor and the signal beingtransduced through phosphate transfer reactions by the two carboxyl-terminal domains.If ethylene is involved in mediating the wound response of the 4CL1 gene, this protein isa candidate for perception of the signal. Potentially therefore, analysis of wound-inducedCL expression in etri mutant lines could provide evidence to support a function forethylene in the wound response.In this work, the massive response to MJ present in tobacco plants and parsley cellsdid not appear to be present in Arabidopsis. The 4CL1-GUS transgene showed someresponse to MJ and the Arabidopsis 4GL gene responded similarly to MJ treatment,however this response is weak relative to that seen in tobacco (Figure 3.8). However, itmay be that a larger MJ response was masked by the response of the cells to the TritonX-100 in which the MJ was dissolved. Applications of MJ in an alternative solvent shouldChapter 5. Expression of parsley 4CL-1 in Arabidopsis thaliana 97clarify this. It is possible therefore that the proposed signalling pathways involving MJare present in Arabidopsis and in support of this there has been a report in the literatureof an Arabidopsis lipoxygenase gene whose expression is induced by treatment with MJ(Bell and Mullet, 1993). These workers also cite a personal communication (R. Creelman)that wounding in Arabidopsis leads to an increase in JA/MJ levels in the plant within 3hours. Identification of mutants in Arabidopsis which are lacking the MJ response shouldprove a valuable approach to elucidating the functional significance of the MJ response.Chapter 6Conclusions and future directions6.1 ConclusionsUsing transgenic tobacco and Arabidopsis, as well as parsley plants and cells in culture,I have studied changes in gene expression in response to two specific stresses, mechanicalwounding and pathogen attack (simulated by elicitor treatment). My work has focusedmainly on the phenyipropanoid gene CL, using it as a model gene to gain a understanding of the intracellular events which occur subsequent to stress, and which lead tochanges in expression of defense-related genes.It is perhaps a measure of the primary importance of the wound response that induction of 4CL expression in response to to this stress is conserved among three differentfamilies, Apiaceae (parsley), Solanaceae (tobacco) and Brassicaceae (Arabidopsis). Theconservation of the wound response enabled me to use a transgenic tobacco system todemonstrate that the same 2lObp promoter fragment that mediates tissue-specific expression (HaufFe et al., 1991) is responsive to wounding and MJ. Potentially, the sametrans-acting factors may be mediating all three responses, and the signal transductionpathways triggered by these stimuli may share common components.I have identified factors which may be involved in signalling pathways leading to theexpression of 4CL. In Arabidopsis and tobacco, ethephon induced the expression of CL,and in all the systems tested, jasmonate activated of 4CL expression. The jasmonate andethylene responses may not be independent of each other since, for example, MJ induces98Chapter 6. Conclusions and future directions 99ethylene production in green and red fruits of tomato (Saniewski and Czapski, 1987).I believe that the integration of a number of signalling compounds may be involved inthe signal transduction pathway. This is supported by the observation that although thesame qualitative patterns of antimicrobial furanocoumarins accumulate in MJ-treatedand elicitor-treated cells, MJ treatment alone does not induce the same level of furanocoumarin accumulation in the cell culture system as is induced by elicitor treatment.Furthermore, in the parsley cell culture system the elicitor-induced changes in patternsof expression of a number of genes, often differed in timing or level of inducibility frompatterns of expression seen after MJ treatment. ABA does not appear to play a directrole as a signal molecule in the activation of phenyipropanoid gene expression in tobaccoor cultured parsley cells since the 4CL gene was not rapidly ABA responsive in thesesystems. This does not rule out the possibility that in a complex interplay of signallingmolecules, ABA may play a role. It may activate gene expression in combination withother signalling molecules.The data I have gathered support the model proposed by Farmer and Ryan (1992)(Figure 3.2). Linolenic acid strongly induces the expression of the parsley 4CL genein the parsley cell culture system, the endogenous 4 CL genes in tobacco and the activation of the parsley 4CL promoter in transgertic tobacco. In the presence of an inhibitor of lipoxygenase enzyme activity, which is necessary for conversion of LA to JA,wound-inducible accumulation of tobacco 4CL mRNA and 4 CL-i promoter activationwas greatly reduced. In parsley cells, the same inhibitor reduced the elicitor responsiveness of a number of defense-related genes (4 CL, PAL, and TyrDC. This suggests thatthe LA-inducible expression of 4GL in parsley cells and the LA-induced activation of the4 CL-i promoter in tobacco could reflect an important role for the conversion of LA toJA in intracellular signalling following perception of these stresses.The similarity between eicosanoids in animals and jasmonates has been pointed outChapter 6. Conclusions and future directions 100(Staswick et al., 1992; Anderson, 1989). Both compounds are produced via similarlipoxygenase mediated pathways. In mammals, eicosanoids are a diverse class of potentmetabolic regulators. The main classes of eicosanoids are the prostaglandins, prostacyclin, leukotrienes, thromboxanes, and lipoxins (Reviewed by Anderson, 1989). Thesemolecules are not required for growth and development of animal tissues and are considered secondary hormones. It is interesting to note that one of the main classes of stimulithat elicit eicosanoid synthesis are tissues stresses such as trauma, disease, and allergy.Plant lipoxygenases are inhibited by nonsteroidal antiinflamatory drugs (Vick and Zimmerman, 1987b). Perhaps a parallel exists between plant responses to pathogen attackand mechanical stress, and animal responses to equivalent traumas. Plants are not theonly organisms that respond to MJ. It is, for example, an active component of the femaleattracting pheromone released by male oriental fruit moths (Baker et al., 1981) and it isfound in at least one pathogenic fungus (Staswick, 1992). In view of its volatile nature, itis possible that other organisms may influence changes in gene expression in plants andthat this could play a role in the interaction between plants and potential pathogens.6.2 Future directionsAn extension of the molecular biological studies I have started here will be valuable ingaining an understanding of the signal transduction pathways mediating expression of4 CL. For example, the marked response to MJ conferred on a GUS reporter gene bythe 210 bp 4 CL-i promoter fragment should enable the identification of a MJ-responsiveelement or elements within the promoter. By comparing this element (or elements) tothose known to be required for tissue-specific expression (Hauffe et al., 1993), it willbe possible to determine if the signal transduction pathways controlling spatial patternsof expression, share a common end point with those conferring the response to methylChapter 6. Conclusions and future directions 101j asmonate.The data presented here suggests that lipoxygenase activity is required for the stressresponse of ,4 CL. Animal lipoxygenases and their inhibitors have been well characterised,probably due to the pharmacological applications of such research (Needleman et al.,1986). Plant lipoxygenases have not received the same attention and there are no reportsof characterisation of parsley lipoxygenses. Consequently, in this work I relied uponinformation known about inhibition of soybean and tobacco lipoxygenases. Only npropylgallate, a known inhibitor of tobacco lipoxygenase, affected the stress-inducibleexpression of the genes in the parsley system. Characterisation of the lipoxygenases inparsley and the genes that encode them would provide a useful tool for continuationof these studies. As well, it must be demonstrated that n-propylgallate, which I usedas an inhibitor of lipoxygenases, actually inhibits lipoxygenase activity and reduces thebiosynthesis of JA under the conditions I used in parsley cells and tobacco plants.A valuable addition to the biochemical and molecular biological studies of signal transduction pathways will be genetic studies. To initiate such an approach I characterised thewound-inducible expression in Arabidopsis of both the parsley and the endogenous 4CL,and I have set up a system which could facilitate isolation of mutants in the wound response. Such mutations are rare, requiring long and laborious screening. Such large scalescreening was beyond the scope of this work. Recently, however, a screening approachwhich will avoid arduous large scale screening for rare signal transduction mutants inlocalised induction of defense genes, has been described a(de Maagd et al., 1993). A ricebasic chitinase promoter which was deleted to l6Obp 5’ of the transcription start, wasfused to the selectable alcohol dehydrogenase (ADB) from Avabidopsis and a similar promoter fragment was also fused to GUS. This construct was introduced into Arabidopsisline ROO2, an ADH (alcohol dehydrogenase) null mutant. Truncating the promoter inthis manner abolished most of the developmental expression while retaining inducibilityChapter 6. Conclusions and future directions 102by wounding. This group are selecting mutants based on their ability to survive allylalcohol treatment. In plants in which the chitinase promoter fragment is activated normally, the ADH produced will convert allyl alcohol into the toxic acrolein, killing theplant. A potential problem with this approach in the case of 4CL gene expression is thefact that the same promoter fragment that is required for the wound response mediateshigh levels of tissue specific and developmentally regulated expression. It may howeverprove useful for other defense-related genes. The isolation of mutants in Arabidopsis thatare deficient in the biosynthesis of or response to jasmonates will be of great value indetermining the functional relevance of gene activation in response to these compounds.Such mutants would allow definitive testing of the hypothesis that jasmonates are partof an intracellular signalling network required for gene activation in response to externalstimuli.Regardless of the approach, an increased understanding of plant responses to stresswill be valuable. Our understanding of plant signalling has for a long time lagged behindthat which exists in animal systems. The rapid response of a gene such as 4CL to stressesis useful as a paradigm for plant signalling in general. From a practical point of view, theadvantages of increased understanding of plant defense are obvious. One advantage beingthe potential for engineering of lines with increased resistance to pathogen attack andthe mechanical stresses inflicted by the environment and horticultural practices. 4CL isalso of major practical importance because of its role in the production of the activatedsubstrates to the lignin branch pathway of phenyipropanoid metabolism. For example,there is considerable interest in modifying the type of lignin in wood. Decreased lignincontent would make herbaceous plants such as alfalfa more digestible and better qualityanimal forage and the pulp industry would save considerable resources if there was lesslignin (or one of a composition that is more readily removed) in trees. A prerequisiteChapter 6. Conclusions and future directions 103to all these applications is an understanding of how expression of 4CL, as a model phenylpropanoid gene, is regulated. 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