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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 GENE ENCODING 4-COUMARATE:COA-LIGASE By Mary Ellard B.Sc. (ions.) National University of Ireland, 1987 M.Sc. National University of Ireland, 1988  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF BOTANY  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA  March 1994  ©  Mary Ellard, 1994  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department  of  The University of British Columbia Vancouver, Canada Date  DE.6 (2/88)  c’  /  /  ‘  -  Abstract  Many of the diverse end-products of the phenyipropanoid pathway play an important role in 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 of the parsley genes encoding this enzyme is increased in response to wounding or challenge with a fungal elicitor from Phytophthora megasperma. Using a number different systems I have investigated the stress response of parsley  4 CL. Using transgenic tobacco as a heterologous system to study the wound response, I showed that the same fragment of the  4 CL-i promoter mediates wound-responsiveness  and a strong response to exogenously applied methyl jasmonate. The  4 CL-i promoter  also mediates responsiveness to linolenic acid, from which jasmonates are synthesised via a lipoxygenase mediated step. In parsley cells in suspension culture, linolenic acid also activates 4CL expression, and expression of a number of phenyipropanoid and other defense-related genes is induced by jasmonates in this system. A similar response to jasmonates is observed in whole parsley plants. Jasmonate treatment of parsley cells activates the expression of genes of the furanocoumarin specific branch pathway of phenylpropanoid metabolism and results in secretion of furanocoumarins (which are known phytoalexins in parsley) by the cell cultures. In the presence of an inhibitor of lipoxygenase activity, n-propylgallate, the response of 4CL to stress was decreased in both systems suggesting that de novo synthesis of jasmonates may be required for these stress responses. This suggests that jasmonates may mediate the stress responses of 4CL and other defense-related genes 11  In transgenic Arabidopsis, the parsley 4 CL-i promoter is responsive to endogenously generated wound signals and directed strong localised expression of the reporter gene GUS at wound-sites. This may provide the basis for a genetic screen to identify genes whose products are necessary for the wound response of 4 CL-i.  111  Table of Contents  Abstract  ii  List of Figures  viii  Abbreviations  xi  Acknowledgements  xii  1  General introduction  2  Experimental procedures  13  2.1  4C1-1-GUS fusions  13  2.2  Generation of transgenic tobacco plants  13  2.3  Plant treatments  13  2.3.1  Wounding experiments  14  2.3.2  Ethephon and ABA treatment  14  2.3.3  1  Treatment of plants with methyl jasmonate, jasmonic acid and linolenic acid  2.4  2.3.4  Remote treatment of tobacco with MJ  2.3.5  Treatment of tobacco plants with n-propylgallate  15  2.3.6  Treatment of tobacco with elicitor  15  Parsley cell culture growth and treatments  16  2.4.1  Growth of cell cultures  16  2.4.2  Treatment of cell cultures with elicitor  16  iv  2.4.3  Isolation and analysis of total coumarins from cell culture medium  2.4.4  Treatment of cell cultures with methyl jasmonate, jasmonic acid,  2.4.5 2.5  3  linolenic acid and ABA  17  Treatment of cell cultures with inhibitors  17  RNA isolation and analysis  17  2.5.1  RNA and poiy A RNA isolation  17  2.5.2  Northern and slot blots  18  2.6  Histochemical localisation of GUS activity  19  2.7  Mutagenesis and screening of Arabidopsis  20  Expression of 4 CL-i in response to wounding in transgenic tobacco  21  3.1  Introduction  21  3.2  Results  30  3.2.1  Expression of 4 CL-i in transgenic tobacco  3.2.2  Expression of 4 CL-i-GUS gene fusions in response to wounding  3.2.3  The effect of ethylene on the expression of 4 CL-i in tobacco  3.2.4  The effect of ABA treatment on  4 CL-i promoter activity  35  3.2.5  Expression of 4 CL-i-GUS gene fusions in response to MJ  37  3.2.6  Effect of methyl jasmonate, jasmonic acid, and linolenic acid on the parsley  3.2.7 3.3 4  16  4 CL-i promoter in transgenic tobacco  30  .  .  .  .  31 33  39  The effect of a potent inhibitor of lipoxygenase on the wound response 41  Discussion  43  The role of jasmonates in the elicitor response in parsley cell cultures 52 4.1  Introduction  52  4.2  Results  58  V  4.2.1  The effect of methyl jasmonate, jasmonic acid, and linolenic acid on  4.2.2  4 CL expression in parsley cells  The effect of methyl jasmonate versus elicitor on expression of genes in the phenyipropanoid pathway  4.2.3  5  59  Accumulation of furanocoumarins in response to methyl jasmonate and elicitor treatment  62  4.2.4  The response of parsley cell cultures to ABA  62  4.2.5  The response to MJ of a group of non-phenylpropanoid elicitoractivated genes: ELI’s  65  4.2.6  The MJ response in whole parsley plants  66  4.2.7  The effect of lipoxygenase inhibitors on elicitor-inducible gene ex  4.2.8 4.3  58  pression  68  4 CL-i elicitor-responsiveness in transgenic tobacco  71  Discussion  Expression of parsley  74  4 CL-i in Arabidopsis thaliana  84  5.1  Introduction  84  5.2  Results  87  5.2.1  Wound-inducible expression of a parsley 4CL-i promoter-GUS fu sion in transgenic Arabidopsis  87  5.2.2  The effect of ethylene on 4CL expression in Arabidopsis  88  5.2.3  Response of 4CL to methyl jasmonate in Arabidopsis  92  5.2.4  A genetic screen for identification of genes involved in mediating the wound response  5.3  92  Discussion  93  vi  6  Conclusions and future directions  98  6.1  Conclusions  98  6.2  Future directions  100  Bibliography  104  VII  List of Figures  1.1  The phenyipropanoid pathway  3.2  Model proposed by Farmer and Ryan (1992) for wound inducible expres  .  sion of proteinase inhibitor genes  2  27  3.3  Expression of 4 CL-i in response to wounding in transgenic tobacco  3.4  Expression of GUS in response to wounding in transgenic tobacco line 801-8 34  3.5  Expression of GUS in response to wounding in plants of transgenic tobacco  .  .  line 810  35  3.6  Expression of 4 CL-i in response to ethephon in transgenic tobacco.  3.7  Expression of GUS in response to ABA in transgenic tobacco line 80 1-8  3.8  Expression of GUS in response to methyl jasmonate in tobacco plants of  .  .  transgenic line 801-8 3.9  32  36 36  38  The effect of methyl jasmonate vapour on GUS expression in plants of transgenic line 801-8  39  3.10 The effect of methyl jasmonate on GUS and tobacco 4CL expression in plants of transgenic line 810  40  3.11 Expression of GUS and tobacco 4CL in response to treatment with methyl jasmonate, jasmonic acid and linolenic acid in plants of line 801-8  .  .  .  42  3.12 The effect of the lipoxygenase inhibitor npropylgallate (nPG) on the wound response in plants of line 801-8  44  4.13 Response of parsley cell suspension culture cells to exogenously applied methyl jasmonate, jasmonic acid, and linolenic acid viii  60  4.14 The effect of methyl jasmonate, and elicitor treatments on the expression of genes in the phenyipropanoid pathway  61  4.15 Levels of furanocoumarins secreted by methyl jasmonate or elicitor treated parsley cell suspension cultures  63  4.16 Thin layer chromatography of furanocoumarins from culture media of MJ and elicitor treated cells  64  4.17 Expression of PAL and 4CL in response to treatment with ABA  65  4.18 The effect of methyl jasmonate, or elicitor treatments on the expression of defense-related genes in parsley cells  67  4.19 Response to methyl jasmonate and wounding of phenylpropanoid and other defense-related genes in parsley plants  69  4.20 The effect of ibuprofen (IP) and phenylbutazone (PB) on elicitor- inducible expression of defense-related genes  70  4.21 The effect of the lipoxygenase inhibitor, n-propylgallate on elicitor-induc ible expression of defense-related genes in parsley  72  4.22 The effect of n-propylgallate (nPG) treatment on furanocoumarin levels in culture fluids of parsley cell suspension cultures  73  4.23 The effect of Pmg elicitor treatment on 4 CL-i promoter activity in a plant of transgenic line 801-8  74  5.24 Accumulation of GUS activity in wounded leaf discs of transgenic Ara bidopsis line 204-1-3  89  5.25 Accumulation of GUS activity at wound sites in leave of Arabidopsis line 204-1-3  90  5.26 Accumulation of GUS and Arabidopsis 4CL transcripts in wounded leaves of Arabidopsis line 204-1-3  91  ix  5.27 Accumulation of GUS and Arabidopsis 4CL transcripts in ethephon treated leaves of Arabidopsis line 204-1-3  91  5.28 The effect of methyl jasmonate treatment on .4CL expression in transgenic Arabidopsis  93  x  Abbreviations  4CL  4-coumarate:CoA ligase  BMT  S-adenosyl-L-methionine:bergaptol 0-methyltransferase  CR5  chalcone synthase  ELI  elicitor induced  GUS  ,8-glucuronidase  HRGP  hydroxyproline-rich glycoprotein  IP  ibuprofen  JA  jasmonic acid  LA  linolenic acid  MJ  methyl jasmonate  uP G  n-propylgallate  PAL  phenylalanine ammonia lyase  PB  phenylbutazone  PT  proteinase inhibitor  Pmg  Phytophthora megasperma  PR  pathogenesis-related  TyrDC  tyrosine decarboxylase  Ubi4  polyubiquitin 4  Ubiq  ubiquitin  xi  Acknowledgements  I gratefully acknowledge the support and help of Dr. Carl Douglas under whose super vision this project was conducted. His generosity with his patience and time has been most sincerely appreciated. I am also grateful for the input and interest of my supervisory committee, 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: Arabidopsis were grown with a major contribution from the green fingers of Sheldon Marcuvitz. Cheerful research assistance was provided by Karma Carrier and technical advice, en couragement and all important perspective was provided by Gopal. Assembly of the thesis and figures was rendered painless thanks to very generous help with I4TEX from Dr. Stewart Schultz, useful computing tips from Dr. Stephen Lee and the occasional use of EM Lab computing facilities provided by Michael Weis. I am grateful to BC Research for the use of their scanning densitometry equipment and I thank Dr. Bob Gawley for taking the time to train me in its use. Thanks also to the staff of the botany office, in particular, Tami and Judy for endless favours and interest in my progress. Finally, a special thank you for the support I have received from the Vancouver branch of the Ellard clan, Fionnuala, Gerry, Conor and Fiona.  xl’  Chapter 1  General introduction  Unique to higher plants, the pathways of phenyipropanoid metabolism have long held the interest of biologists. This thesis deals with regulation of the expression of a gene encod ing 4-coumarate CoA:ligase, a pivotal enzyme of general phenyipropanoid metabolism. Among the compounds synthesised from this pathway are pigments, antimicrobial com pounds (phytoalexins) and structural components of the cells wall, e.g. lignin, suberin and wall bound phenolics. The carbon skeleton for all these compounds is derived from phenylalanine. Phenylalanine is synthesised via the shikimic acid pathway using phos phoenol pyruvate from the tricarboxylic acid cycle and erythrose-4-phosphate derived from the pentose phosphate shunt (Davies et al., 1964). Phenylalanine made from this pathway can then enter into phenyipropanoid metabolism. The phenyipropanoid path way can be divided into the core reactions of general phenyipropanoid metabolism and a 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 the activity of phenylalanine ammonia lyase (PAL) to yield cinnamic acid. Cinnamic acid is hydroxylated to 4-coumaric acid by cinnamate 4-hydroxylase. The activities of hydroxy lases and 0-methyl transferases upon 4-coumaric acid can yield derivatives of 4-coumaric acid e.g. ferulic acid (3’ methoxylated) and sinapic acid, (3’, 5’ methoxylated). The for mation of CoA esters of these compounds is believed to be an important branch point  1  Chapter 1. General introduction  2  LAVONOIDS [pFLAVANOlDS  COUMAR1NS  I GENERAL COOK  1 [  SOLUBLE STERS1  /  PHENYLPROPANOID COOH  METABOLISM  COOH  COSCoA  NH2 4CL  04H  RXR PhenylaIan-e  Cinnaniic Acid  OH  OH  4-coumarc Acid  CoA(R=RH)  4-coumaroyl-  / [LIGNIN  1 L SUBERIN  1LOTHER WALL-BOUND PHENOLlC]1 STILBENES  Figure 1.1: Schematic showing core reactions of general phenyipropanoid metabolism as well as some of the major branch pathways  Chapter 1. General introduction  3  between the general phenylpropanoid pathway and the branch pathways since these es ters 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 de velopment, in specific tissues, and in times of stress, must be met in plant cells. For exam ple, developing tracheary elements in xylem tissue require production of lignin monomers. Flavonoid pigments of fruits and flowers are required in the differentiated cell types in these organs, and production of phytoalexins and structural components of the cell wall (lignin, suberin and other wall bound phenolics) is an important adaptation to environ mental stresses. The activation of phenylpropanoid metabolism in response to pathogen challenge has been demonstrated in a number of systems including potato, bean, al falfa, and soybean (reviewed by Hahibrock and Scheel, 1989). In the legumes, pathogen attack leads to the accumulation of isoflavonoid phytoalexins. This accumulation is pre ceded by an increase in mRNAs and proteins for enzymes of the general phenyipropanoid pathway and the flavonoid specific branch pathway (chalcone synthase and chalcone iso merase) (Dixon et at., 1992; Hahlbrock and Scheel, 1989; Dixon and Lamb, 1990). In suspension-cultured bean cells treated with elicitor, defense genes involved in biosynthe sis of phytoalexins are activated within two-three minutes of elicitor treatment (Lamb et at., 1989). This represents one of the most rapid gene activation systems in plant cells in response to an exogenous sigiial. In the bean system, cinnamyl-alcohol dehydrogenase (CAD), an enzyme involved in lignin biosynthesis is also rapidly activated upon elicitor treatment (Walter et at., 1988). In hybrid poplar suspension cultures treated with an elicitor there are coordinate and transient increases in PAL and JCL mRNA, followed by a 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 cell wall-bound phenolic metabolites. Wounding and pathogen attack drastically change the  Chapter 1. General introduction  4  composition of cell walls in potato tubers. There is a slow wound-induced deposition of suberin phenolics (reaching maximum levels after 5-10 days), whereas suberisation caused by a pathogen attack occurs rapidly, within one day (Hammerschmidt, 1984). Direct ev idence for the role of inducible defense responses in the expression of disease resistance is provided by the observation that aminooxyphenyipropionic acid (a specific inhibitor of PAL) renders soybean seedlings susceptible to normally avirulent races of Phytophthora megasperma f. sp. glycinea (Pmg) (Dixon and Lamb, 1990). My interests lie in under standing how the requirements for phenyipropanoid metabolites subsequent to stress are met at the level of gene regulation. Because of its pivotal role in the phenylpropanoid pathway 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 of the flavonoid specific branch pathway of phenyipropanoid metabolism in cells is a re sponse that is specifically associated with UV light treatment and not elicitor treatment (Douglas, 1992). Parsley cells respond to UV irradiation with vacuolar accumulation of UV absorbing ilavonoids (Hahibrock et al., 1981; Matern et al., 1983). The key en zyme for entry into flavonoid synthesis is chalcone synthase (CHS) which catalyses the formation of naringenin chalcone by condensation of 3 molecules of malonyl-CoA with 4-coumaroyl-CoA, the major product of 4CL (Hahibrock and Scheel, 1989). The parsley  CHS gene is present in a single copy, and has been studied extensively because of its pivotal metabolic role and its strong light-induced transcriptional activation (Chappell and Hahlbrock, 1984). Treatment with a fungal elicitor preparation from Pmg causes parsley cells in sus pension culture to secrete a complex mixture of coumarin derivatives with antifungal activity into the culture media (Tietjen et al., 1993; Hauffe et al., 1986). The linear fura nocoumarins, marmesin and psoralen, their coumarin precursor, umbelliferone, and the methoxylated psoralen derivatives, xanthotoxin, bergapten, and isopimpinellin, have been  Chapter 1. General introduction  5  identified in the coumarin derivatives from cultured parsley cells. Essentially this same mixture of compounds accumulates in infection droplets of parsley leaves inoculated with spores of the Pmg fungus, and all of the furanocoumarins mentioned above are antibioti cally active and considered to be potent phytoalexins in parsley (reviewed by Hahibrock and Scheel 1989). The biosynthetic pathway to the furanocoumarins is not as well char acterised as the pathway to flavonoid synthesis. Nevertheless, antisera raised against SAM:xanthotoxol 0-methyltransferase (XMT) and SAM:bergaptol 0-methyltransferase (BMT) which catalyse the final methoxylation of xanthotoxin and bergapten, were used to isolate the parsley genes encoding these enzymes (Hauffe et al. 1988). Activated CoA esters which act as substrates for both of these pathways are thought to be formed by the catalytic activity of 4CL (Douglas et al., 1992). The 4CL enzyme was first partially purified from UV irradiated cultures of parsley (Petroselinum hortense), by Knobloch and Hahlbrock (1977). They identified only one 4CL species in the final enzyme preparation. It had a molecular weight of 67,000, exhibited an absolute require ment for ATP and was largely specific for 4-coumarate and other derivatives of cinnamic acid. Isolation of genomic clones and cDNA clones for 4CL from parsley (Petroselinum crispum) 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 highly  homologous; the gene sequences are over 95% identical in coding regions and introns, and they display a high degree of sequence identity for several hundred basepairs upstream of the transcription start site. Using a gene-specific probe from an intron fragment specific to 4CL-2 and a fragment common to both genes as hybridisation probes against run-off transcripts from elicitor or light-treated cells, it was shown that there is no differential expression of these two genes in parsley cells (Douglas et al., 1987).  Comparison of  the deduced amino acid sequences of the genes shows there are only three differences in amino acid sequence between the two isoenzymes, and only one of these (asparagine  Chapter 1. General introduction  6  versus aspartate) causes a charge difference. This small difference in primary structure enables the efficient separation of the isoenzymes on two-dimensional gels and on ionexchange columns, but was apparently insufficient to allow separation of the two forms using techniques available in 1977. By expression of 4 CL-i and 4CL-2 in E. coli it was shown that the substrate affinities of the two enzymes are apparently identical (Lozoya et al., 1988). The properties of 4CL and the genes which encode it have been studied in a number of other organisms. In potato, similar to the situation in parsley, 4CL is encoded by two structurally similar genes, St4CL-1 and 2 (Becker-André et al., 1991). The observed nucleotide differences in the coding regions result in three neutral amino acid differences and one charge difference suggesting that the two encoded 4CL isoenzymes probably have similar properties. These authors showed that mRNAs from both of the potato  4 CL genes accumulate to equal levels in suspension-cultured cells and whole plant tissue independent of stress treatment or organ analysed. In other organisms in which the enzymatic properties of partially purified 4CL have been studied, the situation is quite different. In soybean, petunia, pea, poplar and maize for example, there appear to be different 4CL isoenzymes which display different substrate affinities for differently substituted hydroxycinnamic acids (Knobloch and Hahlbrock, 1975; Wallis and Rhodes, 1977; Grand et al., 1983; Vincent and Nicholson, 1987). Presumably, controlling the level of the different isoenzymes is a way in which the flow of hydroxycinnamic acids to the various branch pathways of phenylpropanoid metabolism can be regulated. In support of this, three classes of cDNAs which may encode three 4CL isoenzymes in soybean have been isolated (Uhlmann and Ebel, 1993). Members of this gene family are differentially expressed in soybean cells treated with /3-glucan elicitors of Phytophthora megasperma or in soybean roots infected with either an incompatible or compatible race of the fungus. Recently, a number of different 4CL cDNAs have been isolated from a  Chapter 1. General introduction  7  poplar cDNA library suggesting the existence of different gene family members in this organism (S. Allina and C. Douglas, unpublished results). In contrast, Arabidopsis (like parsley) appears to contain only a single gene encoding 4CL (Lee, Ellard and Douglas, unpublished results). For the purposes of dissecting components of the transduction pathway involved in stress-induced expression of genes like 4CL, the essentially single gene system reduces the complexity of the task since each parsley gene responds to a variety of stresses and developmental signals. Parsley PAL on the other hand, is encoded by a family of four genes and three of these are responsive to different stress stimuli and are over 90% similar to one another at the nucleotide level (Lois et aL, 1989). In bean, PAL is also encoded by a family of genes and the three PAL genes encode distinct polypeptide isoforms (Liang et al., 1989). The transcripts from these genes exhibit markedly different patterns of accumulation leading to 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 preferentially induced thus setting a priority for synthesis of phenyipropanoid products under these conditions. Another feature of particular interest in the bean system is the apparent regulation of PAL transcript accumulation by pathway intermediates (Mavandad, et al., 1990). Accumulation of PAL transcripts in elicitor-treated bean cells is repressed by relatively high concentrations of trans-cinnamic acid, the immediate product of the PAL reaction, whereas low concentrations increase PAL. As might be predicted based on our knowledge of the end products of the phenyl propanoid pathway with their diverse functions in development and their response to numerous environmental stresses, regulation of the expression of 4CL and other phenyl propanoid genes is tightly controlled. Developmentally regulated patterns of expression must be integrated with tissue-specific requirements for phenyipropanoid products and to add to the complexity, there is a rapid and transient accumulation of phenyipropanoid  Chapter 1. General introduction  8  products subsequent to stress. Using in vitro run-off transcription, it was shown that the accumulation of PAL,  4 CL, CHS and BMT in response to elicitor and UV light is  controlled largely at the level of transcription (Chappell and Hahibrock, 1984; Douglas et al., 1987; Lois et al., 1989; Hahlbrock and Scheel, 1989). This is followed by mRNA accu mulation and increased enzymes levels (Hahlbrock and Scheel, 1989) with PAL and 4CL regulation occurring in a coordinate manner at all levels, and activation of the branch pathway enzymes e.g. BMT and CHS occuring later. Schmelzer et al. (1989) used in situ hybridisation to look at the temporal and spatial patterns of expression of some elicitor inducible genes, including PAL, 4CL and BMT in a whole plant system, parsley seedlings. In uninfected tissue, BMT expression is confined to oil-duct epithelial cells. (Parsley plants constitutively secrete furanocoumarins into the lumen of oil ducts: the function of this is not known). PAL and  4 CL are expressed  constitutively 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 expression is induced to high levels at the site of infection. Wounding causes accumulation of 4CL and PAL mRNA, however, the wound response is more diffuse than that seen after Pmg challenge i.e., there is not a sharp border between the unaffected area and the area where transcripts are induced, as is the case with pathogen infection. This work was extended by Wu and Hahibrock (1992) who showed activation of CHS gene expression in the epidermis and also showed that the expression of all genes in developing parsley seedling was dependent on light. Thus, in cell cultures and in plants, these genes must respond to a complex array of signals to ensure that phenyipropanoid compounds are synthesised when required. Regulation of gene expression can occur at a number of levels including initiation of transcripts, mRNA stability, mRNA processing or translation. However, most gene reg ulation occurs at the transcriptional level (Alberts et al. 1989). How genes are regulated  Chapter 1. General introduction  9  transcriptionally has been reviewed by a number of authors (Ptashne et al., 1988; Berk and Schmidt, 1990; Clover, 1989). Regulation of transcription is thought to occur by the binding of specific proteins factors within the nucleus (trans-acting factors) to DNA bind ing regions (cis-acting elements). These DNA sequence elements are 6-20 bp long and are situated within several kilobases of transcription initiation sites. DNA/protein interac tions 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 is the C-Box (C/A-CACGTGGC) present in the photoregulated rbcS-1A (small subunit  of 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 et al., 1989; Staiger et al., 1989). Another important cis-acting element identified within a chs promoter is the H-Box element C 7 [CCTACC(N T]) which has been suggested to be of importance in the response of this gene to stress and developmentally regulated expression of the bean chsi5 promoter (Yu et al., 1993). A H-box and G-Box element act in combination to control the expression of one of the members of the bean chsi5 gene family in response to the phenyipropanoid intermediate para-coumaric acid (Loake  et al., 1992). Although CHS is the best characterised of the phenylpropanoid gene in terms of its cis-acting sequences, a number of elements within other phenylpropanoid genes have been identified. Transcriptional activation of PAL-i is associated with the appearance of three inducible in vivo footprints. Two of these occur in response to both UV light and elicitor and the third is seen only in response to elicitor (Lois et al., 1989). Expression of  4 CL-i in response to elicitor, UV light and tissue-specific signals revealed a separation of cis-acting elements mediating stress and tissue-specific regulation. Exonic sequences are required in conjunction with promoter sequences for expression in response to UV light  Chapter 1. General introduction  10  and elicitor (Douglas et al., 1991). In contrast a 210 bp fragment of the same promoter mediates tissue-specific and developmentally regulated expression of this gene (Hauffe et al., 1991) and a number of putative elements (both positive and negative) within the  4 CL-i promoter have been identified which control spatial patterns of expression of this gene 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 conserved between the two genes. Trans-acting factors which control the initiation of transcription by RNA polymerase II can be divided into two groups, general factors and activators (Holdsworth et aL, 1992). General transcription factors are responsible for the assembly of the preinitia tion complex at the “TATA” box and control constitutive expression. Activators are a heterogeneous class of sequence-specific DNA-binding proteins that interact with the preinitiation complex to bring about high regulated levels of expression. A number of classes of transcription factors or trans-acting factors have been identified in eukaryotes (Glover et al., 1989) and these trans-acting factors have been characterised according to their characteristic binding motif e.g., 11TH (helix-turn-helix) characterised by two opposed c-he1ices joined by a turn of the helix, zinc finger containing a number of cys teine or histidine residues (or a combination of both) for binding of a zinc ligand and the leucine zipper motif characterised by the presence of two amphipathic a-helices which adhere to each other to make a dimer forming region—hence the zipper analogy. Until very recently, our knowledge of transcription factors came solely from animal and fungal systems. In the last few years, more that 40 cDNA clones encoding putative transcrip tion factors have been isolated from plants (reviewed by IKatagiri and Chua, 1992). For example, 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 the DNA occurs, and a leucine zipper element. A plant leucine zipper protein specifically  Chapter 1. General introduction  11  recognises 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 pro tein is the TGA1a tobacco bZIP transcription activator, which binds to the activation sequence element of the cauliflower mosaic virus 35S promoter (Katagiri et al., 1992). A human in vitro transcription system (based on HeLa cell nuclear extracts) was used to demonstate the ability of this binding factor to increase the number of initiation com plexes (Katagiri et al., 1990). Another less well characterised example is the recently identified bZIP protein OHP1 from Maize which interacts with opaque 2, a regulatory gene controlling zein storage protein deposition (Pysh et al., 1993). Transcription factors of 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 correlated with the onset of CHS transcription (Schulze-Lefert et al., 1989a and b). One of these footprints, Box II, contains a 0-box sequence and three parsley cDNAs encoding bZIP proteins which interact in a sequence specific with this element have been isolated. These proteins were named CPRF’s (Common Plant Regulatory Factors) and in support of a functional role for these factors, the massive light mediated increase in CHS mRNA is preceded by induced expression of one of these genes, CPRF-i. A parsley DNA binding protein (BPF-1) that is involved in stress induced phenyl propanoid 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 pro moter. This protein specifically binds a cis-acting element called Box P. This P box is present in all known PAL and 4CL promoters analysed to date and it may play a role in the coordinate induction of expression of these two genes. There is also a very precise correlation 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 strong evidence thatBPF-i may play a key role in regulation of expression of PAL and 4CL in  Chapter 1. General introduction  12  parsley. This is the first report of a trans-acting factor that may be involved in inducible defense 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 the detection of the stress signal to the genetic regulatory apparatus. I used two heterologous systems 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 the stress response of 4 CL. The chapters are divided accordingly.  Chapter 2  Experimental procedures  2.1  401-i-GUS fusions  The 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 the  GUS gene in pRT99-GUSJD/Kozak, a derivative of pRT99-GUS-JD (Schulze-Lefert et al., 1989) that contains a consensus eukaryotic (“Kozak”) translation start site (Kozak, 1981).  2.2  Generation of transgenic tobacco plants  I cloned  4 Cl-i-GUS fusions as EcoRi-Hindill fragments into BIN19 (Bevan, 1984), and  introduced them into Agrobacterium t’amefaciens by triparental matings with Escherichia coli strains (Ditta et al., 1980). The structure of all BIN 19 constructions in Agrobacterium was confirmed using the screening method of Ebert et al. (1987). I transformed tobacco leaf discs, and generated plants, by standard methods (Horsch et al., 1985). 2.3  Plant treatments  Plant treatments described were performed in a minimum of two independent experi ments and the data presented represent typical results obtained.  13  Chapter 2. Experimental procedures  2.3.1  14  Wounding experiments  I wounded excised leaves by slicing tissue into 1-2mm strips, and incubated the wounded tissue on filter paper moistened with MS (Murashige and Skoog) media (Gibco labo ratories) in the absence of hormones. For this and all other Arabidopsis and parsley experiments, 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 necessary to control for the effects of developmentally regulated 4CL expression by distributing leaves of different developmental ages among sample points. For the 0 hour time point, I excised leaves and immediately froze them in liquid nitrogen without further wounding. 2.3.2  Ethephon and ABA treatment  I sprayed to run off plants that were fully grown and non-flowering with a 10 mg/ml solution 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 sprayed control plants with 0.01% ethanol alone and harvested tissue after incubation at 22°C for the periods indicated. 2.3.3  Treatment of plants with methyl jasmonate, jasmonic acid and linole nic acid  Stock solutions of methyl jasmonate (Bedoukian Research Inc., Danbury, CT) and jas monic 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 or a 1% Triton solution (controls). Due to its volatility, methyl jasmonate-treated plants were enclosed in a bell jar (other treatments were left uncovered) and treatments were  Chapter 2. Experimental procedures  15  conducted in constant light at 28°C until harvest of tissue. 2.3.4  Remote treatment of tobacco with MJ  I placed young tobacco plants in an airtight chamber together with 9 cotton tipped sticks/plant. Each stick had been dipped in 500 pls of a 1/1,000 dilution of methyl jasmonate, diluted in 95% ethanol to speed vapourisation. I treated the control plants with 95% ethanol alone. 2.3.5  Treatment of tobacco plants with n-propylgallate  I placed petioles of excised tobacco leaves in n-propylgallate (3,4,5-trihydroxybenzoic acid n-propyl ester) (Sigma) dissolved in 0.2 mM potassium phosphate, p11 7, for 12 hours (described by Staswick et al., 1991). Control leaves were incubated in buffer. I then either wounded leaves and incubated them on MS media for 24 hours as described above (section 2.3.1), or immediately froze them in liquid nitrogen. In contrast to all other plant treatments the results presented for this experiment represent data from a single experiment. 2.3.6  Treatment of tobacco with elicitor  I treated plants with Fmg elicitor as described by Douglas et. al., 1991. Briefly, I placed petioles of groups of excised leaves in a solution of 500 ig/ml Pmg to allow uptake, and harvested them after 2 hours. Control leaves were placed in water alone.  Chapter 2. Experimental procedures  2.4 2.4.1  16  Parsley cell culture growth and treatments Growth of cell cultures  Parsley cell suspension cultures were grown in the dark in modified B5 media as described by Ragg et. al., (1981). Cells were subcultured weekly by transferring 28mls of cells into 200 ml of media in a 1 L Erlenmeyer flask. I performed all treatments 5 days after transfer to new media. 2.4.2  Treatment of cell cultures with elicitor  All elicitor treatments were carried out using 1O-2Oml aliquots of cells which had been aseptically transfered to a 250 ml Erlenmyer flask. I treated cells with aliquots of Pmg elicitor (made as described by Ayers et al. 1976). Elicitor was kindly provided by Klaus Hahlbrock and Shona Ellis. The elicitor was dissolved in sterile water and added to the cells to a final concentration of 50 g/ml. I incubated cells in the dark at 28°C with continuous shaking (110 rpm) and harvested them, at the time points specified, by filtration through a Buchner funnel. Cells were used for RNA analysis and media from treated cells was used for extraction of total coumarins. 2.4.3  Isolation and analysis of total coumarins from cell culture medium  I isolated coumarins from culture medium and performed TLC, as described by Kom brink and Hahlbrock (1986), with the following modifications. For quantification, after extracting coumarins into chloroform, I immediately read the OD onm without dilution 32 or further rotary evaporation. For thin layer chromatography, extracts were evaporated to dryness and coumarins resuspended in lml chloroform.  Chapter 2. Experimental procedures  2.4.4  17  Treatment of cell cultures with methyl jasmonate, jasmonic acid, lin olenic acid and ABA  I prepared solutions as in Section 2.3.3, and conducted treatments as follows: I aseptically transferred 10-20m1 aliquots of cells to 125 or 250m1 Erhlenmeyer flasks to which the treatment compound had been added. I treated control cells with solvent alone. Cells were 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 were enclosed in plastic bags for the treatment period. 2.4.5  Treatment of cell cultures with inhibitors  I used the following lipoxygenase inhibitors in the parsley cells: ibuprofen (a-methyl-44[2-methylpropyl] benzeneacetic acid), n-phenylbutazone (4-butyl- 1 ,2-diphenyl-3,5-pyrolidinedione), n-propylgallate (3,4,5-trihydroxybenzoic acid n-propyl ester), salicylhy droxamic acid (n ,2-dihydroxybenzamide) and antipyrene (2,3-dimethyl- 1-phenyl-3-pyrazolin-5-one). I prepared stock solutions of inhibitors dissolved overnight in sterile 0.2M potassium phosphate, p11 7.0 as described by Staswick et a!. (1991) and added them to 10-20 ml aliquots of cell cultures at a final concentration of 50 pM 16-l8hrs before elicitor experiments and returned cells to the dark with constant shaking. Control cells were treated identically but with buffer alone. All inhibitors were from Sigma. 2.5 2.5.1  RNA isolation and analysis RNA and poly A RNA isolation  RNA was isolated from frozen tissue ground to a fine powder in a mortar and pestle. I extracted 1-5 g ground tissue in 5-10 ml guanidiniuin-HCL extraction buffer consisting of 8 M Guanidinium HCL, 20 mM MES (2[N-Morpholino]ethanesulfonic Acid) buffer  Chapter 2. Experimental procedures  18  and 20 mM EDTA, pH 7.0. After thawing the mixture it was extracted once in an equal volume of phenol-chloroform (1:1) and once in an equal volume of chloroform. I precipitated nucleic acids in the aqueous phase by adding 0.05 volumes 1 M acetic acid and 0.7 volumes 95% ethanol and I selectively precipitated RNA from this mixture of nucleic 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 (Sambrook et al., 1989). 2.5.2  Northern and slot blots  I separated RNA (10 jtg/lane) on 1.2% agarose gels in 1 X MOPS (3-[N-Morpholino]propane-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 distilled water. I stained gels in 2 ig/m1 ethidium bromide and destained in distilled water to en sure 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 Minifold II apparatus (Schleicher and Schuell). A random primer labelling kit from Boehringer was used to prepare radioactive hybridisation probes. Hybridization probes were prepared using inserts purified from cDNA clones after digestion with EcoRI, unless otherwise noted. Parsley CL probes were made using the 2kb insert of the  4 CL-i cDNA (Douglas et al., 1987; Lozoya et al., 1988). I detected  endogenous tobacco 4CL RNA using probes made from the 2kb insert of a potato 4CL cDNA (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 probes  Chapter 2. Experimental procedures  19  were made using inserts from cDNA clones described by Somssich et al. (1989). The orig inal 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 a PAL cDNA clone (Lois et al., 1989); BMT probes were made using a 2kb HindIII-EcoRI insert from a BMT cDNA (Hahlbrock and Scheel, 1989). The insert of a tomato cDNA clone (a Sal I, EcoRl fragment) encoding ubiquitin (a gift of Luca Comai, University of 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 diges tion of pRT99-GUSJD (Schulze-Lefert et al., 1989). I carried out hybridisations in a solution containing 6 X SSC, 0.5% SDS, 5 X Denhardts solution and 0.01 M EDTA at 68°C for 14-16 hours (Sambrook et al., 1989). I carried out high stringency washes at 68°C in 0.2 X SSC and low stringency washes at 68 °C in 2 X SSC. Except as noted below, washes after hybridisations with homologous probes were at high stringency and with non-homologous probes at low stringency. After hybridisation with the tomato ubiquitin probe, washes were carried out at high stringency and after hybridisation with GUS probes, washes were performed at low stringency. 2.6  Histochemical localisation of GUS activity  GUS 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 mM sodium 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-bromo-4-chloro-3-indoleglucuronide (X- GLUC, Clontech, Palo Alto, CA). After staining the tissue was cleared in 95% ethanol.  Chapter 2. Experimental procedures  2.7  20  Mutagenesis and screening of Arabidopsis  Arabidopsis seeds for mutagenesis were obtained by selfing of the line 204-1-3 (homozy gous 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 100 mls of 0.3% ethyl methane sulfonate (EMS) (Sigma). After incubation (with occasional mixing) for 12 hours, I washed the seeds 15 times over the course of 3 hours with distilled water. I then sowed the seeds at a density of one per cm 2 in 12 cm square pots and harvested seeds in bulks of 50 plants. I sowed 1,000 seeds from each bulk and screened them when they were four weeks old. The screening was conducted as follows: Wounded leaf segments were obtained by excising leaf fragments with a scissors and then pinching the tissue with a blunt forceps. I incubated wounded leaf segments for 24 hours in MS media (no hormones) in 4 X 6 well microtitre plates. Tissue was stained with X-Gluc and cleared in the microtitre plate (without fixing) as described in Section 2.6.  Chapter 3  Expression of .4 CL-i in response to wounding in transgenic tobacco  3.1  Introduction  In nature, plants are frequently subjected to wound stress from factors such as wind, herbivores, insects, fungi and other pathogens. Normal developmental processes within the plant, e.g., abscission and growth cracks as well as human activities, such as pruning and ringing, also create wounds. The resulting loss of compartmentalisation and increased susceptibility to pathogen attack and water loss pose a great threat to the health of the plant. Unable to flee such threats, plants have evolved a complex set of physiological and biochemical responses which occur in the neighbouring stressed but unbroken cells. The responses of plants to wounding can for convenience be divided into those that are immediate, occurring in a matter of minutes after wounding, and those which are slow, occurring in a matter of hours. The immediate responses include changes in mem brane structure and function. Fatty acids are released from the membrane and can be oxidised to ethylene, ethane and a variety of other compounds (Bostock 1989; Davies et al., 1987). The action of lipoxygenase on the polyunsaturated octadecanoid fatty acids  linolenic and linoleic acid released from damaged cell membranes, results in the forma tion 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 the characteristic odours of freshly cut or damaged plant tissue (Siedow, 1991) and Croft et al., (1993) suggest that these volatile products play an antipathogenic role. Changes  21  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  Ca influx and C1 + in membrane ion fluxes result in a net increase in 2  22  and K+ efflux,  suggesting a collapse in the electrogenic H+/K+ATPase in the plasmalemma (Davies et al., 1987). There is a rapid activation of callose synthase, resulting in the deposition of callose at the wound site (Davies et al., 1987), and an activation of wall associated polysaccharidases that lead to the release of oligosaccharins (Albersheim and Darvell, 1985). Wounding also leads to an increase in the biosynthesis of ethylene, “the wound hormone”, the evolution of which has long been associated with stress in plants (Bostock and Sterner, 1989). The second group of responses (the slow responses) for the most part are controlled at the level of gene expression. Changes in plant gene expression associated with wounding generally fall into two categories in terms of spatial patterns of induction. Genes whose expression is induced in a localised manner in the plant are involved in strengthening of the cell wall surrounding the wound site or preventing opportunistic pathogen attack, or both. There have been reports of wound-inducible expression of genes encoding at least two classes of proteins that play a structural role in the cell wall. The best characterised class are the hydroxyproline-rich glycoproteins (HRGPs), represented by extensin (Sauer et al., 1990).  Another class of wound-inducible proteins are the proline- or hydrox  yproline-rich proteins (PRPs), and genes encoding these are expressed in response to wounding in carrot roots and soybean hypocotyls (Sheng et al., 1991). In light of the vital role played by compounds derived from the phenyipropane skeleton as structural components of the cell wall, it is hardly surprising that the genes of the phenyipropanoid pathway are also induced upon wounding, resulting in the production of suberin and lignin monomers to protect against mechanical damage. In addition, genes encoding anionic peroxidase, the enzyme which may catalyse polymerisation of lignin monomers from the phenylpropanoid pathway, are inducible by wounding (Mohan et al., 1993). Other genes whose expression is induced in a localised manner in response to wounding  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  23  include 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 sys temic and implies the existence of a transmissible signal to communicate to tissues distal from the wound site. Genes whose expression is induced systemically in response to wounding include the win (wound-induced) genes from poplar (Davis et al., 1991) and the win and wun gene families identified in potato (Stanford et al., 1989; Logemann et al., 1989). The functions of these gene products are unknown, although the win-S poplar gene shows sequence similarity to trypsin and Kunitz-Type proteinase inhibitor genes (Bradshaw et al., 1990). Despite uncertainty about the function of these genes, their expression patterns have been described and promoter elements mediating the response to wounding identified (Siebertz et at., 1989). By far the best characterised change in gene expression in response to wounding involves the systemic expression of proteinase inhibitor (P1) genes identified in the Solan aceae. Fl genes encode potent inactivators of proteolytic enzymes. There are two classes of PT proteins that have been isolated and characterised in potato tuber (reviewed by Ryan, 1992). They are encoded by two nonhomologous gene families; the PT-I class have a molecular mass of 8,000kDa and inhibit chymotrypsin while the second class (PT-il) have a reactive site for both trypsin and chymotrypsin and are larger, with a molecular mass of 12,000kDa. Several lines of evidence show that these compounds are a chemical defense against Lepidopteran insect attack. Firstly, P1 proteins are not normally present in leaves, yet, upon wounding they accumulate to extraordinarily high levels. Upon multiple wound treatments, levels of PT-I and -II mRNA in the the Bonny Best variety of potato can account for 1% of the total poly A mRNA population (Ryan, 1992). Secondly, these inhibitors trigger physiological feedback mechanisms in insects which results in them feeling prematurely satiated and starvation of the animal results. The most convincing  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  24  evidence, however, for a role for these compounds as a defense against herbivory lies in the observation that transforming plants with P1 genes under the control of a constitutive promoter 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 gene that 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, PT proteins prevent small mammals, birds, and insects from eating the fruit before the seeds are fully developed. Later, the level of inhibitors decreases and the fruit is more likely to be eaten and thus dispersed (Pearce et al., 1988). Many genes involved in defense responses are encoded by multigene families, and the different 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 differen tially 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 that differ 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 dis tinct 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 pro moter 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 example of 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-Il promoter (Lorbeth et al., 1992). Two elements direct wound-inducible expression: an upstream quantitative element conferring maximal levels of expression upon wounding, and a wound regulatory element downstream from this quantitative enhancer region. A  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  25  separate regulatory element directs constitutive promoter activity in flowers. A region of the P-IlK gene that both binds a protein factor present specifically in nuclei of wounded tissue and is necessary for wound inducible gene expression has been identified (Palm et al., 1990). Identifying the cis-acting elements of wound-inducible genes, however, represents but one approach to understanding the mechanism of wound-inducible gene expression. An other aspect of the process that needs to be elucidated and integrated with this informa tion is the nature of the wound signal within the plant cell, and in the case of systemically  induced genes, the nature of the transportable wound signal. The earliest wound signal identified in plants, “Riccas factor,” was described by Ricca in 1916 (Davies, 1987). This factor is made in response to wounding in injured tissue and travels in the transpiration stream. The chemical nature of this compound has never been elucidated despite its obvious importance. Another potential wound signal, research into which is now apparently out of vogue, is traumatin, the wound hormone originally  identified as trans-2-dodecenedioic acid by English et al. (1939). More recently, trau matin has been identified as 12-oxo-trans-10-dodecenedioic acid which could potentially be generated from membrane lipids broken down as a result of wounding (Zimmerman and Coudron, 1979). Although there is no longer mention of traumatin in the literature, the importance  of lipid-based signalling is apparent as a result of the work of Ryan’s group on the wound inducible expression of proteinase inhibitor genes.  The lipid-derived, volatile  plant compound methyl jasmonate (MJ) is capable of inducing synthesis of proteinase inhibitors in plant tissue (Farmer and Ryan, 1990). Moreover, incubating tomato plants in the same chamber as Artemesia tridentata (sagebrush), which produces large amounts of MJ, was sufficient to cause systemic transcriptional activation of these genes. MJ and the free acid, jasmonic acid (JA), are present in most organs of most plant species  Chapter 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, followed by additional modifications (Vick and Zimmerman, 1987a and b). Intermediates in the pathway to JA biosynthesis, as well as linolenic acid (LA), similarly activate the synthesis of proteinase inhibitors (Farmer and Ryan, 1992).  Oligogalacturonides isolated from  tomato leaf cell walls applied through cut petioles of excised plants, can induce the expression 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 a systemic wound signal, however, this hypothesis was refuted with the demonstration that radioactively labelled oligogalactouronides are not transported throughout tomato plants when placed on wounds in leaves  (  Baydoun and Fry, 1985). Then followed the isolation  and 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 in a manner similar to wounding. It is 10,000 times more potent than oligouronides, active at concentrations in the fmole range (Ryan, 1992).  Previously thought of as simple  molecules, lacking the complexity of animal peptide signalling molecules, the isolation of systemin has changed the way we view plant hormones. It has been suggested that abscisic acid (ABA) plays a role in the stress inducible expression of P1 genes. Potato mutants deficient in the synthesis of ABA fail to accu mulate pin2 mRNA in wounded leaves and this correlates with the absence of the rise in endogenous leaf ABA concentration, which normally occurs in wild-type plants upon wounding (Peña-Cortés et al., 1989). While this work supports some role for ABA in the 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 consensus ABA responsive element (ABRE) within the promoter region responsible for activity in  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  Herbivores  Patbogens  (wounding) Systemic I ystenin Signals  Localized  OtQowo  Signals  .  I captor  27  Plasma Membrane  Receptor  Unolenlc Acid  LOX Dehydrase 6-Oxidation  Jasmotc Acid I (Methyl Jasmonate)  JAReceptor  Ji  p’  Gene Activation Protelnastlnhllbltors  Figure 3.2: A model proposed by Farmer and Ryan (1992) for wound inducible expression of 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 the endogenous ABA concentration apparent after drought (Peña-Cortés et al., 1989), and finally, it has been pointed out that although ABA is an inducer of pin2 mRNA in leaves of normal potato plants, it does not similarly affect tomato Fl mRNA accumulation despite the fact that both are similarly affected by wounding (Ryan, 1992). The role of ABA in this system therefore remains unclear. The work on the response of Fl genes and the discovery of systemin has been synthe sised into a model (see Figure 3.2) for stress inducible gene expression, which I have used as a paradigm for this work on wound-inducible expression of 4 CL-i. Briefly, the model suggests that oligouronides are a signal in localised wound responses, while systemin is the signal in systemic wound responses. The release of these signals somehow (possibly  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  28  via a lipase) causes release of LA from the membrane, which gives rise via lipoxyge nase to de novo synthesis of JA, which mediates gene activation by an as yet unknown mechanism. Since the discovery that MJ mediates wound-inducible Fl gene expression, it has been 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 result of release of linolenic acid from the cell membrane after wounding. In support of this hypothesis, inhibitors of the JA biosynthetic pathway decreased vsp mRNA induction in response to wounding but not in response to MJ (Staswick et al., 1991). Two novel wound-inducible genes whose expression is also induced in response to jasmonates 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 the first example of the involvement of a proteinase in plant defense mechanisms. It has been hypothesised that the wound response of this gene plays a role in intracellular protein turnover to provide a flow of aminoacids from existing into newly synthesised proteins. The second gene identified appears to encode a biosynthetic threonine deaminase which may be involved in biosynthesis of amino acids of the aspartate family. This gene is highly expressed in floral tissue and also in response to wounding and jasmonates. A tomato gene encoding threonine deaminase has been isolated (Samach et al., 1991) and its expression is similarly high in floral tissue and is induced in leaf and floral tissue in response to jasmonates (Samach, 1993). The role of threonine deaminase in plant defense is unclear. It has been pointed out that it is difficult to see how overproduction of isoleucine can defend plants against pathogens and therefore threonine deaminase may participate in another metabolic pathway leading to products participating in the plant’s defense response (Samach, 1993).  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  29  In contrast, it is apparent how the production of phenyipropanoid products around wound sites could be of adaptive value, providing structural barriers and antimicrobial  compounds to ward against potential pathogen attack. Parsley 4 CL transcripts accu mulate 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. MJ has been implicated in wound-inducible expression of a soybean CHS gene and there is a correlation between the accumulation of jasmonates in soybean hypocotyls and induc tion of CHS gene expression (Creelman et al., 1992). Treatment of parsley cells with the jasmonate precursor, 12-oxo-phytodienoic acid, causes increases in the accumulation of mRNA for 4CL, CHS, and PAL (Dittrich et aL, 1992). Whether these data reflect a  functional 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 ethylene biosynthesis as a result of the activity of the biosynthetic enzyme for ethylene, ACC syn thase (S-adenosyl-L-methionine methylthioadenosine-lyase) (Bostock and Sterner, 1989).  An interesting approach to assessing the role of ethylene in wound-inducible gene ex pression in tomato pericarp has been described (ilenstrand and ilanda, 1989). Using competitive inhibitors of ethylene action, the level of translatable products from poly A RNA from wounded tissue treated with the inhibitor was compared with that of poiy A RNA from wounded tissue which had not been treated with the inhibitors. It was estimated that less that 15% of mRNA species induced in wounded tissue is affected by the presence of inhibitors. Such an approach, however, does not include genes whose expression may be regulated by levels of ethylene that are low enough to be available despite the presence of the inhibitors, so it may be an underestimate of the effect of ethylene on wound-inducible gene expression. The expression of a number of the wound inducible genes introduced in this chapter is also induced by ethylene. They are: bean  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  30  chitinase (Brogue et al., 1986), /3-glucanase (Simmons et al., 1992), HRGFs (Ecker and Davis 1987; Lawton and Lamb 1987; Ryder et al., 1987), and in carrot roots PAL, CHS and 4CL (Ecker and Davis, 1987). Raz and Fluhr (1993) used the plant pathogenesis re sponse as a paradigm to investigate ethylene-dependent signal transduction in the plant cell and showed that a transient rise in protein phosphorylation inducible by ethylene treatment is required for the pathogenesis response. Induction of such an increase in phosphorylation by treatment with inhibitors of phosphatases elicited the response in the absence of ethylene. Another component of the pathway of ethylene inducible gene expression requires calcium (Raz and Fluhr, 1992), since blocking calcium fluxes with chelators inhibited ethylene-dependent induction of chitinase accumulation. Artificially increasing cytosolic calcium levels by treatments with a calcium ionophore or a calcium pump blocker stimulated chitinase accumulation. This chapter is concerned with the wound-inducible expression of the parsley 4 CL-i gene. I used a transgenic tobacco system to investigate what promoter region is necessary for wound-inducible expression, and also investigated the potential role in this system of some compounds shown to be involved in wound-inducible expression of other genes. I present data which provides evidence for the involvement of MJ in a signalling pathway leading to the wound activation of 4 CL-i which is similar to that proposed for wound inducible proteinase inhibitor gene expression. 3.2  3.2.1  Results Expression of 4CL-i in transgenic tobacco  The ease with which tobacco can be transformed and regenerated coupled with its large leaf size and relatively short regeneration time makes it an ideal choice for study of wound-inducible gene expression (Schell, 1987). The use of this heterologous system to  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  investigate the wound-induced expression of  4 CL-i  31  required, however, that I initially  establish that the wound response of the parsley gene is reproduced in transgenic to bacco. Six independent lines of tobacco plants (TI plants) were available which were transformed with a large genomic clone for  4 CL-i  (containing l500bp of upstream pro  moter sequences). Using a tobacco plant from each of these lines, I wounded excised leaves, as described in Experimental Procedures, pooled the tissue and analysed total RNA 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 the tobacco 4CL but does not cross hybridise with parsley  4 CL-i  4 CL-i. Conversely, the parsley  cDNA does not cross-hybridise to mRNA from the tobacco 4CL genes. This al  lowed me to monitor the level of mRNA accumulation RNA from of both the endogenous and the introduced gene in the same experiment, providing an internal control. Figure 3.3 shows that wounding strongly induced the accumulation of both the introduced 4CLi 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 still present after 24 hours. A control was performed where an excised leaf was removed and left in water for 3 hours (3 C). Leaf excision caused a small systemic accumulation of both 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 endogenous  tobacco 4CL genes. 3.2.2  Expression of 4 CL-i-GUS gene fusions in response to wounding  In order to establish if the observed wound-inducible increase in the mRNA level for 4CLi was being mediated by the transformation, tobacco  4 CL-i promoter, I generated, by Agrobacterium mediated plants transgenic for a -597bp of the parsley 4 CL-i promoter  fused to the reporter gene GUS and used these plants in wounding experiments. The  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  o  3  6  32  24 3C  St4CL  Pc4CL  Figure 3.3: Wound-induced accumulation of tobacco 4CL and parsley 4 CL-i transcripts in tobacco plants transgenic for an intact 4 CL-i gene. Duplicate northern blots were hybridised to a potato 4CL cDNA probe (St4CL) for detection of endogenous tobacco 4CL transcripts or to a parsley 4CL cDNA probe (Fc4CL) for detection of transcripts from the introduced 4 CL-i gene. 10tg RNA isolated from leaves 0, 3, 6, and 24 hours after wounding was loaded per lane. Control leaves (3C) were detached and placed in water for 3 hours without further wounding. results of a northern analysis of RNA accumulation in plants of the transgenic line, 801-8 is shown in Figure 3.4. GUS RNA accumulated strongly and rapidly, in response to wounding and the increased levels of GUS RNA closely paralleled the accumulation of endogenous tobacco 4CL RNA, detected by the St4CL probe. Thus the -597 bp  4 CL-i  promoter fragment alone confers wound-inducibility upon the GUS reporter gene. A 210bp  4 CL-i promoter fragment confers full developmentally regulated expression  upon 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 wound response, 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 tenfold drop 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 necessary  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  33  to isolate poiy A RNA rather than total RNA from wounded tissue to detect GUS RNA. Figure 3.5 shows the results of the hybridisation of poiy A mRNA from wounded tissue from plants of the transformed line 810-11, with a GUS probe. For comparison, a similar experiment using line 801-6, (with -597 bp of promoter sequences controlling GUS expression) 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 by the larger promoter fragment in the 801-6 plant. In order to control for different amounts of 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 expression is not affected by wounding. Thus, sequences mediating wound-inducible expression of the  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 tobacco  In 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 ex pression of  4 CL-i.  Treatment with ethephon has been used in other systems to achieve  ethylene treatment (Broglie et al., 1986; Brederode et al., 1991). To determine if ethylene could modulate changes in parsley 4 CL-i expression, I treated plants from two indepen dent lines of tobacco transgenic for a genomic copy of parsley 4 CL-i (containing l500bp of promoter sequences) with ethephon. I pooled tissue from each plant and isolated total RNA 6 and 24 hours after treatment. The results of a northern blot analysis of this RNA are shown in Figure 3.6. A northern blot hybridised with a probe for St4CL demonstrated that the tobacco genes are ethephon-responsive: Increased levels of tobacco mRNA were present at 6 and 24 hours after treatment. Similarly, hybridisation with a probe for parsley  4 CL  showed that the expression of the introduced gene is responsive  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  3C  0  3  6  24  3C  0  3  24  Hours  *2kb  0S St4CL  6  34  GUS  Probe  Figure 3.4: Wound-induced accumulation of tobacco CL and GUS transcripts in a tobacco plant transgenic for a 597-hp parsley 4CL-1 promoter-GUS fusion. Leaves of plant 801-8 were detached, then wounded for the times shown, total RNA prepared, and 10 ,ug RNA loaded per lane. Northern blots were hybridized with an StCL cDNA probe to detect endogenous tobacco 4CL RNA, and a GUS probe to detect GUS RNA. The 3C time point is control in which unwounded excised leaves were incubated 3 hours in water.  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  UBIQ  GUS  St4CL  GUS  35  0  6  —  24 Plant:  810-11  801-6  Figure 3.5: Accumulation of GUS transcripts in response to wounding of leaves from a tobacco plant of transgenic line 810. Poly A RNA was isolated from leaves of plant 810-11, transgenic for a 210-hp 4CL-i promoter-GUS fusion, which had been wounded for the times given (in hours). A slot blot loaded with 500 ng 810-11 RNA per slot was hybridized 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 similarly wounded leaves of plant 801-6 (597-bp promoter-GUS fusion) was loaded on a slot blots and hybridized to GUS and St.4CL probes. to ethephon application. I performed similar experiments using a tobacco line trans genic for a 597bp parsley 4CL-i promoter-GUS fusion. However, northern blot analysis with a GUS hybridisation probe failed to show definitively whether the .4CL-i promoter mediated ethylene-responsiveness of the parsley .4CL-i gene. 3.2.4  The effect of ABA treatment on  .4 CL-i  promoter activity  Pefla-Cortés et al. (1989) showed that treatment of transgenic tobacco plants with ABA induced accumulation of P1-Il mRNA. To determine if the parsley .4 CL-i promoter may be similarly responsive I treated 801-8 tobacco plants with 100pM ABA.  Northern  analysis (Figure 3.7) showed no accumulation of GUS mRNA above control levels after 6 hours (the high level of expression at the 0 hour time point overloading of the gel as determined by ethidium-bromide staining) and a small accumulation of transcripts after 24 hours. A similar pattern of hybridisation was seen on a northern blot hybridised with  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  0  Hours  24  6  —4  2kb  Pc4CL  St4CL  Probe:  36  Figure 3.6: Accumulation of tobacco 4CL and parsley 4 CL-i RNA in response to ethep hon. lOmg/ml 2-chloroethephon was applied to leaves of tobacco plants transgenic for an intact parsley 4 CL-i gene. 10 tg total RNA from leaves, harvested at the times indi cated after ethephon application, was loaded on duplicate RNA gels, and northern blots hybridized with St4CL and Pc4CL cDNA probes to detect endogenous tobacco 4CL and parsley 4CL transcripts, respectively.  Time (h) 2Kb  *‘-.  0 lIjpPr  Control 6  24  1 0OM ABA 24 6  Probe: GUS  Figure 3.7: The effect of ABA on GUS expression in plants transgenic for a -597bp parsley 4 CL-i promoter fragment directing expression of GUS (801 plants). Leaf tissue from 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.  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  37  a probe for St4CL (data not shown), suggesting the absence of a rapid response of either the endogenous genes or the introduced 4 CL-i-GUS to exogenously applied ABA in this experiment. Experiments are described in Chapter 4, in which the effect of a range of ABA concentrations on parsley  4 CL-i expression in parsley cells was tested. Those  experiments suggested that the gene is not ABA-responsive in cell cultures. 3.2.5  Expression of 4CL-i-GUS gene fusions in response to MJ  The work reviewed by Ryan et al. (1992) and Staswick et al. (1992) suggests that there is a role for MJ as a second messenger in wound-inducible gene expression. I used the transgenic tobacco system to address whether MJ may have a role in the wound-inducible expression of 4 CL-i. Since the parsley 4 CL-i promoter mediates the wound response of the parsley gene in the tobacco system, it follows that if MJ is a mediator of this response then the  4 CL-i promoter should be similarly MJ- responsive. I treated tobacco plants transgenic for the -597bp parsley 4 CL-i promoter GUS fusion (line 801-8) with 1mM MJ and used northern blots to assay for the induced accumulation of tobacco 4CL and  GUS mRNA. Figure 3.8 shows that MJ treatment led to a massive increase in GUS RNA levels by 6 hours and that GUS mRNA levels remained high for at least 24 hours after treatment. Endogenous tobacco 4CL levels as assayed by hybridisation to the St4CL probe, 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 in  transgenic tobacco Farmer and Ryan (1990) showed that MJ originating from Artemesia tridentata was capable of inducing proteinase inhibitor accumulation in neighboring tomato plants. In light of the high level of responsiveness of the 597bp promoter fragment, I considered the possibility that direct application of MJ was not necessary for promoter activation. I  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  0  Probe:  6  6C  24  GUS  0  6  6C  24  24C  38  hours  St4CL  Figure 3.8: Expression of GUS in response to methyl jasmonate in tobacco plants of transgenic 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 pro moter-GUS fusion (801-8), and RNA isolated at 0, 6, and 24 hours. 10 g total RNA was loaded on duplicate RNA gels, and northern blots hybridized to a GUS probe or to SL4CL to detect endogenous tobacco 4CL RNA. tested the ability of MJ to act remotely in this system by exposing plants from transgenic tobacco line 80 1-8, to MJ vapour without direct application of the compound. The results in Figure 3.9 show that levels of GUS mRNA were unaffected by MJ applied in this manner. I hybridised a duplicate northern blot with a probe for St4CL to determine if endogenous tobacco 4CL mRNA accumulation was similarly unaffected by MJ applied in this manner. Only a very weak signal was detected on this blot, despite a long exposure and 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 levels after indirect application of MJ. Thus, under the conditions I used, activation of parsley  4 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 fusions  To determine if the same 2lObp promoter fragment that mediated the wound response of the  4 CL-i promoter was also capable of mediating the response to MJ, I treated plants  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  OC  2kb  6MJ  6C  39  24MJ  -  GUS  probe  Figure 3.9: The effect of methyl jasmonate vapour on GUS expression in plants of trans genic 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 jasmon ate in 95% ethanol was applied to 9 cotton dipped dowels surrounding but not in direct contact with the plants. Control plants (C) were treated with 95% ethanol alone. Tissue was harvested at the times shown in hours. Accumulation of transcripts for GUS was determined 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 8107), with MJ and estimated the level of expression on slot blots. Figure 3.10 shows that in both these transformants, GUS mRNA (under the direction of a 2lObp promoter fragment) had accumulated to high levels in response to MJ after 6 hours. These high levels of GUS mRNA were still present 24 hours after treatment. 3.2.6  Effect of methyl jasmonate, jasmonic acid, and linolenic acid on the parsley  4 CL-i promoter in transgenic tobacco  I used plants of the transgenic line 801-8 to determine the effect on GUS expression of different concentrations of MJ, JA, and o-1ino1enic acid (a-LA). Plants were sprayed with solutions containing 0, 0.1, 1, and 5 mM concentrations of each compound, RNA isolated 6 hours after treatment, and northern blots performed. Hybridisation with a  GUS probe showed GUS mRNA accumulated to approximately 60% of maximum levels  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  40  Time: hours  0 6C  III  6MJ 6C 24MJ  Plant:  810-11  810-7  Figure 3.10: The effect of methyl jasmonate on GUS and tobacco 4CL expression in plants of transgenic line 810. Methyl jasmonate-induced accumulation of GUS and endogenous tobacco 4CL (St4CL) transcripts in tobbaco plants transgenic for a 210-bp parsley 4 CL-i promoter- GUS fusion. Plants from two independent transgenic tobacco lines (810-11 and 810-7) 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 duplicate blots and hybridized to GUS or St4CL probes. Blots were subsequently stripped and hybridized to a ubiquitin (UBIQ) probe.  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  41  after treatment with 0.1 mM MJ and a-LA and approximately 30% maximum levels after JA treatment. In all cases the maximum response as measured by mRNA accumulation was seen after treatment with a solution containing 1mM of each compound. Increasing the concentration of the applied compounds to 5 mM resulted in a marked decrease in  GUS mRNA accumulation. Hybridisation with a probe for StCL showed that the endogenous tobacco 4CL gene responded in a similar manner to the introduced GUS gene. Plants were also treated with 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 and SL4CL probes were weak and did not increase above constitutive levels after treatment with LA (data not shown). This suggests that the induction of gene expression observed after treatment with a-LA may be as a consequence of its entry into the biosynthetic pathway for JA. 3.2.7  The effect of a potent inhibitor of lipoxygenase on the wound response  According to the model proposed by Farmer and Ryan (1992), the wound response is mediated by endogenous JA synthesised de novo via a lipoxygenase. Inhibition of lipoxy genase activity would therefore be predicted to prevent the wound response. The inhibitor n-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 expression of the  4 CL-1-G US fusion in transgenic tobacco line 801-8. Plants were treated for 10  hours with buffer alone or with 1 t5 M or 50MM nPG and subsequently wounded or har vested without wounding. The accumulation of GUS RNA was assayed using northern blots hybridised to a GUS and the accumulation of RNA for the endogenous tobacco  CL gene was assayed using a homologous probe for tobacco 4CL (NqCL) (Diana Lee,  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  42  75 C 0  E  50  CC  z C) >  25  CC  C)  0 0  0.1  10  mM methyl jasmonate E 0 E  100  x  CC  E  75  C 0 0  50  E  CC  z  25  C) > CC  C)  0 0  0.1  10  mM jasmonic acid E 0 E  100  CC  E 75 C 0 0  E  50  CC  z C) > CC  C)  25 0 0  0.1  1  10  Figure 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 801-8 were treated for 24 hours with 0, 0.1, 1, and 5 mM of each compound and expression of GUS (0) and tobacco jCL (.) determined on northern blots. Plants corresponding to the 0 mM concentration were treated with 1% Triton alone. Blots were scanned with a densitometer and the band intensities plotted as a function of concentration and normalised to hybridisation with a probe for ubiquitin.  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  43  unpublished results). In the plants pre-treated with buffer alone, there was an accumu lation 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 decrease in the response to wounding of the GUS reporter gene directed by the  4 CL-i promoter  which was most apparent at 5OzM nPG where the wound response appeared to be ab sent. Hybridisation with a tobacco 4CL probe revealed a similar marked response to the inhibitor. The filter hybridised with the Nt4CL probe was subsequently rehybridised to a probe for tomato ubiquitin (Figure 3.12). The presence of a discrete band which hybridised to the ubiquitin probe in all lanes demonstrated that nPG treatment did not lead to any significant degredation of RNA. Since nPG is known to be a potent inhibitor of tobacco lipoxygenase, these results suggest that a lipoxygenase activity may be re quired for the wound-induced expression of tobacco 4CL and the wound-responsiveness of the parsley 4 CL-i promoter in tobacco.  3.3  Discussion  In transgenic tobacco, the parsley  4 CL-i gene is responsive to wounding in a manner  similar to the wound response of the endogenous tobacco 4CL genes. The sequences mediating the response of the  4 CL-i gene to wounding are contained within a 2lObp  promoter region. The parsley 4 CL-i promoter and the endogenous tobacco 4CL genes are also 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 of the 4 CL-i promoter and a similar marked decrease in the wound response of the tobacco  4 CL genes. This may be a reflection of the requirement of lipoxygenase for synthesis of JA to mediate the wound response as was proposed in the model suggested by Farmer and Ryan (1992) (Figure 3.2).  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  44  Buffer 5iM nPG 5OiM nPG 0 24 0 24 0 24  GUS Nt4CL Ubiq  I  Figure 3.12: The effect of the lipoxygenase inhibitor n-propylgallate (nPG) on the wound response in plants of line 801-8. 801-8 plants were treated with 0 (buffer), 5 or 50 tM nPG and subsequently either wounded and incubated for 24 hours (24), or harvested im mediately without wounding (0). The accumulation of GUS and tobacco 4CL transcripts was estimated on a northern blot. The filter hybridised with Nt4CL was then stripped and rehybridised with a probe for ubiquitin. The ease with which it can be transformed coupled with its large leaf size, makes tobacco an ideal choice as a heterologous system in which to study the wound response of the parsley 4CL-i gene (Schell, 1987). The use of a heterologous system made it nec essary to initially determine if the parsley 4 CL-i gene is wound-responsive in transgenic tobacco. Northern analysis of tobacco plants harbouring a genomic clone for parsley  4 CL-i showed that, similar to its expression in parsley (Schmelzer et al., 1989), mRNA accumulated in a localised manner in response to wounding. Wound-inducible expres sion of the parsley gene was paralleled by accumulation of mRNA for tobacco  4 CL. This  means that the tobacco wound signal, in whatever form it takes within the cells, is also recognised by the parsley  4 CL-i gene, suggesting that the wound response is conserved  between the two species. In parsley leaves,  4 CL-i is also responsive to attempted in  fection by Fhytophthora megasperma (Fmg) and irradiation with UV containing white  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  45  light (Schmelzer et al.,, 1989). The response to these stresses is also conserved between tobacco and parsley, since it was shown that in tobacco transgenic for a parsley  4 CL-i  gene, mRNA for both the introduced gene and the endogenous gene accumulates in re sponse to Pmg elicitor treatment and UV light treatment (Douglas et al., 1991). In situ hybridisations were used in tobacco to examine cell type and tissue-specific expression patterns of parsley  4 CL-i, and it was demonstrated that these patterns of expression  are closely correlated to those of the endogenous 4CL genes (Reinold et al., 1993). This provides 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 study regulation of  4 CL-i gene expression. In contrast, expression of the wound-inducible  potato gene wine is not conserved between tobacco and its homologous host. The reg ulatory sequences of the win2 gene direct dramatic wound-inducible expression of GUS in the leaves of potato; however, no induction of win2-GUS gene expression is observed in 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 tobacco  4CL in response to MJ, LA and JA (Figure 3.11), since this again indicates a high level of conservation of signalling systems between parsley and tobacco. Uknes et al. (1993) recently highlighted the importance of monitoring endogenous gene activity as a control in transgenic systems. This group used GUS as a reporter of tobacco PR ia (pathogenesis related) promoter activity and documented two artifactual patterns of  GUS expression which were not seen with the endogenous tobacco PR-ia gene or with the PR-ia promoter directing expression of other genes. One was ectopic expression of GUS in anthers and pollen, the other anomaly was GUS RNA accumulation in response to cycloheximide treatment. These workers suggest that the GUS coding sequence contains  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  46  a promoter element that responds to cycloheximide treatment. Monitoring endogenous gene 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 genes are highly conserved and  4 CL-i and 2 are not differentially regulated in response to UV  light and elicitor (Douglas, 1987). Recent work, however, suggests that these genes may be differentially regulated in response to wounding in roots (Lois et al., 1992). Differential expression in leaves was not studied. In contrast to the wound-inducible expression of  4 CL-i in roots, parsley 4CL-2 expression was shown to be either repressed or not induced in wounded roots. It seems highly unlikely that there is differential expression in leaves at the level of transcription since the two parsley 4CL genes differ in only one position within 210 bps upstream of the transcription start site (Douglas et al., 1987) and a 210 bp  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 expression  of  4 CL-i in response to UV light and Pmg elicitor treatment since promoter sequences  alone do not confer responsiveness to these stimuli upon a GUS reporter gene (Douglas et al., 1991). The fact that the 4 CL-i promoter sequences alone confer a strong response to wounding upon GUS, suggests that the pathways mediating responsiveness to wounding diverge, at least at the level of DNA/promoter interactions, from intracellular pathways mediating elicitor- and UV light-inducible expression. The sequences responsible for a myriad of cell type specific-expression modes of 4 CL-i in transgenic tobacco are located within 2lObp upstream of the transcription start site (Hauffe et al., 1991), and distinct promoter domains which specify tissue and cell type specific patterns of expression by combinatorial interactions have been identified (Hauffe et at., 1993). Since the same 2lObp fragment can also specify wound- and MJ-inducible mRNA accumulation, it is possible 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  47  expression. In vivo DNA/protein interactions within this region have been investigated by in vivo footprinting and an approximately 200bp region 5’ of the transcription start site showed strong in vivo protection (ilauffe et at., 1991). Although these in vivo foot prints are constitutive, (they do not appear to be modified by fungal elicitor), they may still play an important role in stress-regulated (CL-1 expression. They may represent re gions where modification of constitutively bound factors produced by an environmental, developmental, or tissue-specific signal may cause changes in gene expression. Identifica tion of the elements within the CL-1 promoter responsible for MJ mediated expression will determine more precisely the correlation between developmental and tissue-specific patterns of expression, and jasmonate-induced expression. Meanwhile, there is an inter esting correlation between levels of jasmonates in tissue, and CL expression; both are highest 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, where it is involved in MJ-mediated responses (Kim et at., 1992), and it is also present within a parsley CHS gene (Schulze-Lefert et at., 1989). It remains to be seen if this element mediating the response of soybean vsp to MJ, is the same element mediating the wound response 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 environmental or internal stimuli, it may do so by a mechanism that is distinct from that controlling wound responsive vsp gene expression. MJ was first implicated as a signal molecule in wound inducible gene expression when Farmer and Ryan (1990) showed that airborne MJ emanating from Artemesia tridentata could induce the expression of proteinase inhibitor genes in nearby tomato plants. This experiment conclusively demonstrated that a signal from one plant can cause defense gene  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  48  activation in another plant. This means that, theoretically, plants could communicate via volatile signals. I investigated the possibility that such remote application of MJ could affect parsley promoter activity in tobacco plants. Figure 3.9 demonstrates that airborne MJ does not activate the  4 CL-i promoter, and the tobacco 4CL genes were similarly  unaffected (data not shown). Similarly, Andresan et al. (1992) demonstrated that JA induced accumulation of leaf proteins is not sensitive to volatile MJ in intact barley seedlings. Thus, interplant communication via MJ may not be a general mechanism activating the expression of MJ-responsive genes. It is possible, however, that 4CL genes are less sensitive to MJ than Fl genes and very high concentrations of airborne MJ may mediate 4CL expression. Similarities in structure, physical properties and the activity of ABA and jasmonate have been pointed out (Staswick 1992). Both induce the accumulation of proteinase inhibitors 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 and  the endogenous tobacco 4CL genes were unresponsive to ABA after 6 hours, suggesting ABA does not activate the 4 CL-i promoter within the time frame which would indicate a potential role for this molecule as a component of the signal transduction pathway mediating the rapid response to wounding. This suggests there is not a direct role for this compound in the wound-activated expression of 4CL in this system. In this respect  4CL regulation is similar to that of soybean vsp, which is MJ inducible but not ABA responsive (Anderson, 1989). Ethephon, on the other hand, appeared to mimic wound-inducible expression of the entire  4 CL-i gene. Unfortunately, due to difficulties in demonstrating accumulation of  GUS mRNA in response to ethephon, I was unable to determine if the 4 CL-i promoter is ethephon-responsive. Hybridisation of northern blots loaded with RNA from ethephon treated plants gave irreproducible results and it appeared as if the GUS transcript was  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  49  unstable in the presence of ethephon. Although there is precedence in the literature for using 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 that upon breakdown of ethephon to release ethylene, phosphonic acid is released which by itself may be a stress upon the plant. Direct application of ethylene gas would avoid this potential problem and may help elucidate the role (if any) of ethylene in the wound response. Another potentially useful approach involves looking at the wound response of plants deficient in ethylene biosynthesis. Ethylene biosynthesis can be blocked by antisense 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 components of signal transduction pathways. One of the goals of the work in this chapter was to determine if endogenous JA or MJ may play a role in the wound-activated expression of the  4 CL-i  promoter in  transgenic tobacco, using as a working hypothesis the model developed by Farmer and Ryan (1992) (Figure 3.2). (In chapter 4, I have used the parsley cell suspension culture system to investigate the possibility of a role for jasmonates in the response of 4CL1 to Pmg elicitor using the cell suspension culture system). The data presented here support this model. Firstly, both MJ and JA strongly induce the expression of the  GUS reporter gene under the control of 4 CL-i promoter and expression of the tobacco 4CL genes was similarly activated by jasmonates (Figure 3.11).  The parsley 4CL-i  promoter is also wound-inducible, and the same 2lObp promoter confers responsiveness to jasmonates (Figure 3.10) and wounding (Figure 3.5). Comparison of Figures 3.4 and 3.5 with Figures 3.8 and Figure 3.11 shows that jasmonates are more powerful in this regard than wounding. Farmer and Ryan (1992) similarly reported more powerful induction of proteinase inhibitor genes by JA than by wounding. In this case they hypothesised a receptor system which is not saturable by wounding but is saturable by direct application  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  50  of 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 Zim merman, 1984; Vick and Zimmerman, 1987a and b) and a-LA specifically activates the -LA had no effect on transcript accumulation). Since not 7 4 CL-i promoter in tobacco ( only JA but its biosynthetic precursor can activate 4 CL-i expression, these data are con sistent with the lipid-based signalling system proposed by Farmer and Ryan (1992), and the observed activity of LA may be a result of its conversion to JA in vivo. Moreover, my results suggest that lipoxygenase activity sufficient for conversion of LA to biologically active amounts of JA is present constitutively in tobacco leaf cells. This would mean that 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 proteinase inhibitor gene expression (Farmer and Ryan, 1992). Thirdly, in the presence of nPC, the wound response of both the tobacco 4CL gene and the  4 CL-i promoter was decreased in a dose dependent manner. Inhibitors of tobacco  lipoxygenase 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 ability of LA to induce 4CL expression is a reflection of its conversion into jasmonates with a role in wound signalling, lipoxygenase inhibition would diminish the wound-response of 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 directly demonstrate inhibition of tobacco lipoxygenase and even if such data were available a non-specific mode of action of the inhibitor cannot be excluded. nPG is a free radical scavenger (Vick and Zimmerman, 1987b) and may well interfere with any number of other cellular processes within the tobacco cells. In Chapter 4, however, I present data  Chapter 3. Expression of 4CL-1 in response to wounding in transgenic tobacco  51  which suggests that in parsley cells at least, cellular signalling processes important in mediating 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 MJ responsiveness 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 transgenic  parsley and tobacco, indicated that this promoter by itself is not sufficient to confer elicitor- and UV light-responsiveness upon GUS. Thus, although my results point to a possible role for JA or MJ as endogenous signalling compounds mediating woundinducible expression of  4 CL-i,  they do not by themselves support a role for JA or MJ  in the elicitor or light response. The fact that different regulatory sequences associated with the  .4 CL-i  are required for MJ/JA and wound-responsiveness, suggests signalling  pathways mediating these responses are distinct from each other. In Chapter 4, using the cell culture system, I investigate further whether there may be a role for jasmonates in signalling of phenyipropanoid gene expression in response to elicitor.  Chapter 4  The role of jasmonates in the elicitor response in parsley cell cultures  4.1  Introduction  The study of stress-induced phenyipropanoid metabolism in parsley plants has been greatly aided by the availability of the parsley cell suspension culture system. Parsley cells are easy to propagate in simple, synthetic media and the cells respond synchronously and uniformly to environmental stress (Hahibrock and Scheel, 1989). Much of the work on elucidating the enzymology of the pathway and its induction was done using this system. Parsley cell cultures treated with UV light accumulate flavonoids thought to have a UV protective function, and after treatment with a glycoprotein elicitor from Phytophthora megasperma (Fmg elicitor), which simulates an infection, secrete fura nocoumarins with antifungal activity (reviewed by Hahibrock and Scheel, 1989). The accumulation of these products is preceded by the rapid transcriptional activation of genes for phenylalanine ammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL). This transcriptional induction is followed by increases in levels of the respective mRNAs and proteins (Hahibrock et al., 1981; Ragg et al., 1981). Activation of genes of the general phenylpropanoid pathway therefore occurs in response to both stimuli. Activation of the branch pathways however is signal specific. UV light specifically activates transcription of chalcone synthase (CHS), (a large number of other enzymes involved in biosynthe sis of fiavonoids have been shown to be activated at the catalytic level at least), and  52  Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures  53  elicitor activates transcription of genes encoding enzymes involved in biosynthesis of fu ranocoumarins 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 signal transduction leading from perception of stress to gene activation. Important questions include, how stress signals are perceived by the cell, how they are communicated via second messengers to the nucleus, and ultimately how changes in gene expression are brought about. Recent work has begun unravelling what may be involved in the initial recognition of the elicitor by the plant cell. Renelt et al., (1993) demonstrated the presence of competable binding sites on the parsley cell membrane which recognise the protein moiety of the Pmg elicitor. It is known also that two of the initial events leading to gene activation are; a transient change in the in vivo phosphorylation patterns of proteins, and a change in permeability of the plasma cell membrane to Ca +, IP, K+ and C1 2 (Dietrich et al., 1990; Scheel et. al., 1990). Plasma membranes of soybean similarly carry 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, there are fundamental differences in elicitor recognition between the two species; firstly, it is a proteinaceous constituent, (not carbohydrate), of the mycelial cell wall that is recognised by the parsley cells; and secondly, there is no evidence that GTP binding proteins, which participate in elicitation in soybean cells (Legendre et al., 1992) are involved in the parsley cell response (Renelt et. al., 1993). A number of phenyipropanoid and other defense related genes have been isolated from parsley, including PAL, jCL, CHS, HRGP and genes encoding two pathogenesis related proteins, PR1 and PR2. These genes are transcriptionally activated upon elicitor treatment and mRNA for these genes accumulates in elicitor-treated cells (Hahlbrock  Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures  54  and Scheel, 1989; Chappell and Hahibrock, 1984; Sommsich et al., 1986). In a systematic search for other parsley genes that are transcriptionally activated upon elicitor treatment of cell cultures, Sommsich et al., (1989) isolated clones for a large group of defense related genes called ELI genes. These clones fall into four classes based on their transcription kinetics. For example, the most abundant ELI class has a maximum of transcriptional activation at two hours after elicitor treatment. This class accounts for 30% of the isolated clones and these genes are identical in kinetics of activation with FRi (pathogenesis related) genes. Since their original isolation, a function has been assigned to the encoded products of some of the ELI genes. ELI 5, a member of the class which is activated latest (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 in defense has been supported by a strong association between its activation in Arabidopsis and the presence of a specific resistance gene (Kiedrowski et al., 1992). Using in situ hybridisation, it has been demonstrated that mRNAs for many of these defense-related genes isolated from cell cultures accumulate at infection sites in intact parsley plants with a temporal pattern of accumulation very similar to that seen in the suspension culture system (Schmelzer et al., 1989). Along with molecular probes, another tool required to understand how the expres sion of defense-related genes is regulated is a transformation system. Plant protoplasts are very amenable to direct DNA transfer and an important step in development of the parsley cell culture system was the demonstration that parsley protoplasts retain differ ential responsiveness to UV light and elicitor  (  Dangl et al., 1987). The use of parsley  protoplasts for transient expression studies, after PEG-mediated DNA transfer, enabled  Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures  55  functional identification of light-responsive elements within the CHS promoter (Schuize Lefert et al., 1989) and an elicitor-responsive region within the promoter of the gene encoding 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 UV  light and elicitor. In stably transformed parsley cells, it was shown that deleting the pro moter 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 expres sion. It was found, however, that promoter fragments fused to the GUS reporter gene were not able to confer stress-inducible expression upon that gene in either stably or tran siently transformed parsley cells. This led to the suggestion that downstream sequences were required for inducible expression. Since addition or removal of introns did not affect expression of the gene, the coding sequences were implicated as harbouring the required control elements (transcriptional or post-transcriptional). Furthermore, since a CaMV 85S 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 exonic sequences for correctly regulated expression. Further support for this hypothesis came from the observation that transformation of  4 CL-i-GUS fusions into tobacco, resulted  in little or no accumulation of histochemically detectable GUS activity or GUS mRNA in response to the stress stimuli, despite the responsiveness of the endogenous tobacco  4 CL gene  in the same experiment.  The nature of the second messengers involved in signal transduction of the elicitor response to the nucleus is unclear. There is some evidence that the intracellular transduc tion chains are Ca 2 dependent (Renalt et al., 1993). Another candidate for intracellular transmission of elicitor-generated signals are jasmonates, which have been suggested to be 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 defence responses, there is not yet sufficient evidence to prove such a role. They showed that treatment of Rauvolfia canescens and Eschscholtzia californica cultures with a yeast elic itor led to rapid and transient accumulation of endogenous JA. They also show in this system that jasmonate treatment induces expression of a number of genes involved in defense, including PAL. In rice, hydroperoxides and hydroxides of linoleic and linolenic acids, 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 powdery mildew Erysiphe graminis f. sp. hordei has been suggested (Schweizer et al., 1993). JA directly inhibits appressoria differentiation of the fungus, and appears not to be involved in signal transduction leading to induction of pathogenesis-related proteins. They used an inhibitor of transcription, cordycephin, to show that repression of transcription does not 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 or linoleic acid which is oxidised to 12-oxo-phytodecanoic acid (12-oxo-PDA). 12-oxo-PDA is converted 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 the incorporation of molecular oxygen into polyunsaturated fatty acids that possess a cis, cis 1,4-pentadiene structure. It is believed that lipoxygenase activity is ubiquitous in the plant 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 studied more extensively than any other plant lipoxygenase, perhaps due to the fact that soybean seeds and hypocotyls contain particularly high levels of lipoxygenase (reviewed by Siedow, 1991). Lipoxygenases have, however, been isolated and characterised from other species  Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures  57  including tobacco (Fournier et al., 1993), rice, soybean, cotton and sunflower (reviewed by 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 between the Fe(II) and Fe(III) states during catalysis giving rise to formation of large amounts of hydroperoxide. cDNAs encoding lipoxygenases from a number of species have recently been isolated. Included are clones for genes encoding lipoxygenases from soybean (Siedow et 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 soybean and one from Arabidopsis, accumulates in response to MJ (Grimes at at., 1992; Bell and Mullet, 1993), suggesting that MJ may regulate its own biosynthesis and that induction of lipoxygenase expression by MJ may be important for increasing jasmonate levels under certain 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 was accompanied by a two-fold increase in 4GL and CHS mRNA and a five-fold increase in PAL mRNA accumulation (Dittrich et at., 1992). The significance of these data to the role of JA in elicitor-mediated signalling is unclear since the accumulation of flavonoids is a specific response to light, not elicitor, in parsley cells (Hahibrock and Scheel, 1989), and the reported induction of mRNA accumulation is much lower than that observed after elicitor treatment (Douglas et al.,1987; Lois et al., 1989; Hahibrock and Scheel, 1989). In other work, Kauss et al. (1992) suggested that MJ enhances the effect of elicitor in parsley cells. They looked at the effect of 5pM MJ on furanocoumarin accumulation and report minimal accumulation associated with treatment with MJ alone at this concentration. Treatment with 2OpM MJ increased furanocoumarin accumulation but levels were still 100-fold less than those observed after elicitor treatment. However, the major effect of MJ reported in this paper is that pretreatment of parsley cells with MJ prior to elicitor  Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures  58  treatment, greatly enhances the elicitor response. Whether jasmonates act directly in parsley cells to induce furanocoumarin phytoalexin accumulation and defense gene activation, and whether the elicitor-induced expression of phenylpropanoid and other defense-related genes may be mediated by endogenous JA has not been addressed. In this chapter, I investigated the role played by jasmonates in the response of parsley cells to elicitor, and present data on the elicitor response in tobacco that is relevant to this question.  4.2  Results  4.2.1  The effect of methyl jasmonate, jasmonic acid, and linolenic acid on 4CL expression in parsley cells  Treatment of parsley cell cultures with the JA precursor 12-oxo-phytodienoic acid leads to moderate increases in PAL, 4CL and CHS mRNA levels, which could be due to con version of 12-oxo-phytodienoic acid to JA (Dittrich et al., 1992). I wished to directly test the activity of jasmonates on phenyipropanoid gene expression in parsley cells, and chose  4 CL  as a representative gene for initial dose-response experiments. I tested the ability  of range of concentrations of JA, its methyl ester MJ, and a-linolenic acid (os-LA), the ubiquitous membrane fatty acid from which JA is synthesised (Vick and Zimmerman, 1984) to induce accumulation of 4CL transcripts in parsley suspension cultured cells. I measured transcript accumulation by hybridisation of northern blots of total RNA from treated cells, to a parsley 4 CL-i cDNA (Douglas et al., 1987) and estimated band intensi ties by scanning densitometry. The signal from the 4CL probe was normalised to a signal obtained from a duplicate blot hybridised with a probe for Ubi4, a parsley polyubiquitin gene 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 accumulation  Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures  59  was seen at 100 M. At a concentration higher than this, (300 tiM), the accumulation of  transcripts decreased. The free acid (JA) was more active at a lower concentrations than the methyl-ester. Of the concentrations tested, the maximum activity of LA in inducing  4CL transcript accumulation was at 1.0 1 tM and its activity declined quite rapidly as con centration increased. The first step in JA biosynthesis involves the oxygenation of cr-LA via lipoxygenase to 13(S)-Hydroperoxylinolenic acid (13(S) HPOTrE) (Vick and Zimmer man, 1984). The effect of cr-LA was maximal at 3OpM, and increasing the concentration to lOOiiM only slightly decreased the relative 4CL expression. A high concentration of cr-LA (300tM) had little effect on transcript accumulation. To examine the specificity of a-LA in inducing 4CL mRNA accumulation, I also treated the cells with a closely related isomer of a-LA, -7 LA, which does not enter the biosynthetic pathway. -7 LA had no 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 genes in the phenyipropanoid pathway  Pmg elicitor treatment of parsley cells activates the transcription of genes encoding en  zymes of the general phenyipropanoid pathway and the furanocoumarin-specific branch pathway including L-methionine-bergaptol 0-methyltransferase (BMT), an enzyme cat alysing the final methylation of the phytoalexin bergaptol (Hauffe et al., 1988). The result of this activation of gene expression is the secretion of furanocoumarins into the media. If the ability of MJ to induce 4CL transcriptional activation is a reflection of its  role as a signalling intermediate in the response of cells to elicitor, I would expect MJ to induce patterns of gene expression similar in timing and quantity to those induced by elicitor treatment. To test this, I compared the MJ-induced expression of PAL, 4CL and BMT to their expression in response to elicitor. I used slot blots, coupled with scanning  Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures  60  100 S 5  o  S  75  50  Ct  z  25  0 > Ct  5  0 0  S  1 100 10 iM methyl jasmonate  1000  100 10 iM jasmonic acid  1000  100  1000  100  x  75 C 0  o  SCt  50  z 0)  25  > Ct  0  S  0  1  0  1  100  75  50  c  25  0)  > Ct  0 10 iM linolenic acid  Figure 4.13: Response of parsley cell suspension culture cells to exogenously applied methyl 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 point were treated with 0.1% Triton alone. They were incubated for 6 hours in the dark with constant shaking. The level of mRNA for parsley 4CL was determined by northern hybridisation coupled with scanning densitometry. Duplicate blots (loaded with lOjigs of RNA per lane) were hybridised with a probe for parsley 4CL and Ubi, a parsley ubiquitin gene whose expression is unaffected by elicitor. The signal from the (CL probe was standardised to the Ubi( signal and relative mRNA amount (as a percentage of maximum) plotted as a function of the log of concentration.  Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures Methyl Jasmonate  Elicitor Time (h)  0  2  6  9  2  o ,-  Control (-  .i  11  4  2  6  9  Probe:  PAL  I I  j  I  I  •1•  61  4CL  BMT  Iii  Figure 4.14: The effect of methyl jasmonate, and elicitor treatments on the expression of genes in the phenylpropanoid pathway. Parsley cells were treated either with 3OpM methyl jasmonate or 5Opg/ml Pmg elicitor. Control cells were treated with 0.1% Triton alone. Cells were harvested at 2, 6 and 9 hours after treatments, total RNA extracted and 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 both treatments. 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 occurred more slowly. These results are similar to previous results obtained in this system (Lois et al., 1989; Hahibrock and Scheel, 1989). The accumulation of CL and PAL transcripts was also rapidly induced in response to treatment with 30 pM MJ but was lower than the elicitor-induced accumulation. The kinetics of PAL and 4CL RNA accumulation ap peared similar in MJ and elicitor-treated cells. Activation of BMT expression in response to MJ appeared to occur more rapidly than to elicitor (compare 6 hour time points) and  Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures  62  reached a level about twofold more than that seen after elicitor treatment by 24 hours. 4.2.3  Accumulation of furanocoumarins in response to methyl jasmonate and elicitor treatment  To further test the relative responses of parsley cells to MJ and elicitor, I compared the accumulation of furanocoumarins in cultures 24 hours after treatment with either MJ or elicitor. Furanocoumarins were extracted from the culture filtrate with chloroform and quantified spectrophotometrically (Kombrink and 1{ahlbrock, 1986). In contrast to previously published data by Kauss et al. (1992) who reported a minimal accumulation of furanocoumarins subsequent to treatment with MJ alone, the level of furanocoumarins in this experiment was tenfold above that in control cells, and one third the level seen in elicitor-treated cells (Figure 4.15). To determine if the same furanocoumarin deriva tives were produced after MJ treatment as after elicitor treatment, I analysed extracts of furanocoumarins 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 furanocoumarins in the extracts (data not shown). A qualitatively identical pattern was induced by MJ treatment and elicitor treatment. Because equal amounts of the extracts were chro matographed, the amount of each product was lower in the MJ sample (Figure 4.16).  4.2.4  The response of parsley cell cultures to ABA  ABA has been proposed to activate the expression of defense-related genes and shows some 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 the expression of phenyipropanoid genes in parsley cell suspension cultures, I treated cells  Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures  .2)  0 C  63  2  1.5  I  E  D 0  o  1.  0 0 0  O.5  o conhi  MJ  eIkitor  Figure 4.15: Levels of furanocoumarins secreted by methyl jasmonate or elicitor treated parsley cell suspension cultures. Cells were treated with methyl jasmonate (3OiiM) or Pmg elicitor (5Ozgs/ml), incubated for 24 hours and furanocoumarins extracted in chloro form. The 0D 320 of the extracts was read and the amount of furanocoumarins estimated from the extinction coefficient.  Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures  2Ol  -  C  MJ  64  50 iii Eli  C  MJ  Eli Solvent front  -•4-  Origin  Figure 4.16: Thin layer chromatography of furanocoumarins from culture media of MJ and elicitor treated cells. Parsley cells were treated with either 50 gs/ml Fmg elicitor or 30 tM methyl jasmonate. Furanocoumarin derivatives were extracted from the culture fluids and 20 and 50 ls aliquots of extract were separated by thin layer chromatography.  Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures  1 jiM ABA 1 OjiM ABA 1 00 jiM ABA  Controi Time (h)  0  6  65  24  6  24  6  24  6  24  probe: PAL 4CL  Figure 4.17: Expression of PAL and CL in response to treatment with ABA. Parsley cells were treated with 1, 10 and 100 zM ABA which was initially dissolved in a minimal volume of 95% ethanol, total RNA extracted after 6 and 24 hours and transcripts for Pc4CL detected by northern blotting. Control cells were treated with distilled water containing 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 accumula tion on a northern blot. After 6 hours, no increase in 4CL or PAL mRNA accumulation above that in control cells was observed over the range of concentrations used in this ex periment (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 there was an accumulation of mRNA for both genes in the control cells this induction may have masked an ABA response at this time point. However it appeared that there was no rapid ABA induced accumulation of transcripts suggesting that it does not induce expression of these genes within the time frame required to support a role for ABA in the rapid elicitor response. 4.2.5  The response to MJ of a group of non-phenyipropanoid elicitor-act ivated genes: ELI’s  The range of elicitor-mediated responses in parsley cells includes the transcriptional ac tivation of a large set of putative defense-related genes (Sommsich et at., 1989) which fall  Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures  66  into four classes based on the kinetics of their transcriptional activation. I reasoned that if the MJ induced expression of phenylpropanoid genes is a reflection of a general role for jasmonates in the response of parsley cells to elicitor, then these genes would be similarly elicitor-responsive. I chose to examine the expression of ELI 7’, HRGP, TyrDC and ELI 3 which represent 3 of the 4 kinetic classes. (The 4th class is represented by PAL and  4 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 simi larities to the response seen after elicitor treatment. Figure 4.18 shows that, consistent with previously published results (Sommsich et al., 1989), expression of all four of these genes was induced by Pmg elicitor treatment. ELI 7 and TyrDC mRNA accumulation was highest at the 6 hour time point, and accumulation of mRNA for ELI 3 and HRGP was 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. In terms of timing, the response of HRGP and ELI 3 to MJ was quite similar to their responses to elicitor and the level of induction was also similar. The expression of ELI ‘7 was unaffected by treatment with 3OtM MJ. 4.2.6  The MJ response in whole parsley plants  In order to determine if the response of this suite of elicitor-inducible genes to MJ is a phenomenon unique to cell cultures, I tested the responses of the general phenylpropa noid genes and other elicitor-inducible genes to 1mM MJ in intact parsley plants. This concentration was chosen because it was the concentration at which optimal activation of the 4 CL-i promoter occured in transgenic tobacco (Figure 3.11). I also wished to estab lish if expression of the non-phenylpropanoid elicitor-inducible genes, shown above to be MJ-inducible, is also wound-inducible. Figure 4.19 shows a northern blot of total RNA from wounded and MJ treated parsley plants, hybridised to cDNAs for parsley PAL, 4CL  Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures  Time 0 (h)  1  Methyl jasmonate  Elicitor 2  6  9  2  6  9  Control 9  6  IIHR  1,1  III  I i  I S I  1,1 I I  I I  Probe:  2  ELI3  •  Ii I  67  TyrDC  EL17  I I I  I I a  Figure 4.18: The effect of methyl jasmonate, or elicitor treatments on the expression of defense-related genes in parsley cells. Cells were treated as described for Figure 4.14. Slot blots loaded with 2 1 ug 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  68  and other elicitor-inducible genes. Expression of HRGP and ELI 3 was strongly induced by MJ treatment, consistent with results in cell cultures. The expression of Eli 7 which was not induced by MJ in cell cultures, was similarly unaffected in whole plants. Expres sion of the non-phenylpropanoid genes did not appear to be activated by wounding, in fact expression of HRGP and Eli 3 appeared to be repressed by wounding. Despite its high level of induced expression in cell cultures (see Figure 4.18), expression of TyrDC was not detectable on northern blots using total RNA, even with probes labelled to very high specific activity (data not shown). In whole plants, the parsley 4CL and PAL genes were expressed, after treatment with MJ, in a manner similar to their expression in cell cultures. 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. Wounding rapidly induced mRNA levels for parsley CL and PAL genes and increased mRNA levels were still present after 24 hours. 4.2.7  The effect of lipoxygenase inhibitors on elicitor-inducible gene expres sion  In the model proposed by Farmer and Ryan (Figure 3.2), JA is synthesised de novo in response to stress via lipoxygenase activity. Correlative evidence presented in this Chapter and by others (Gundlach et al., 1992) suggests that endogenously synthesised JA could play a role in the intracellular transmission of an elicitor-generated signal to the nucleus. I used an approach similar to that described in Chapter 3, to further test the hypothesis that MJ is a mediator of the elicitor response in parsley cells. A specific prediction of the model of Farmer and Ryan, if it holds true for this system, is that lipoxygenase inhibitors would prevent elicitor-inducible gene expression by preventing the elicitor-stimulated biosynthesis of endogenous JA. I tested the effect of a number of lipoxygenase inhibitors on the elicitor-responsiveness of several of these genes. The  Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures  Methyl jasmonate  Wounduig  0  —  3  69  6  24  0  6  6C  24  24C  —  4CL  —  PAL ELI 3 ELI 7 HRGP  Figure 4.19: Response to methyl jasmonate and wounding of phenylpropanoid and other defense-related genes in parsley plants. Excised leaves were wounded as described in Figure 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 light until harvest of tissue. Accumulation of transcripts for CL, PAL, ELI 3, 7, and HRGP was 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  PAL  4CL  TyrDC  ELI3  70  Ubi4  PB IP+EIi PB  +  Eli Eli  Figure 4.20: The effect of ibuprofen (IP) and phenylbutazone (PB) on elicitor inducible expression of defense-related genes in parsley cell cultures. Cultures were pretreated for 16 hours with the inhibitors and then treated with elicitor. mRNA accumulation from these cells was compared on slot blots (loaded with 2 jg total RNA) with mRNA from cells treated with elicitor without prior inhibitor treatment. Blots were hybridised with cDNAs for PAL, 4CL, TyrDG, ELI 3 and Ubi4. Control cells were treated with a comparable volume of phosphate buffer. inhibitors ibuprofen and phenylbutazone which were effective in inhibition of wound inducible 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 (Figure 4.20). Similarly, the inhibitors salicylhydroxamic acid and antipyrene, had no effect on the 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 inducible expression 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, PAL and 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  71  In contrast, HRGP and ELI 3, were still fully responsive to elicitor (Figure 4.21). The apparent response of E1i3 to treatment with nPG alone was not present in a duplicate experiment (data not shown) and possibly reflects an abberation in loading. The appar ent elicitor responsiveness of some of the genes despite nPG treatment suggested that inhibition of elicitor-induced expression of CL, PAL and HRGP transcription was not simply 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, I tested the effect of nPG pretreatment on elicitor-induced furanocoumarin accumulation. Figure 4.22 shows that, in this experiment, Pmg elicitor stimulated the accumulation of approximately 1.0 tmoles furanocoumarins per gram fresh weight, and that MJ induced the accumulation was about one third this amount, as previously observed (Figure 4.15). Incubation of cells with nPG alone did not induce furanocoumarin accumulation. How ever, incubation with nPG prior to elicitor treatment resulted in a 10-fold decrease in elicitor-induced accumulation of furanocoumarins. Addition of MJ to nPG-treated cells resulted in furanocoumarin accumulation similar to that observed with Mi alone. Thus, nPG treatment specifically blocked the responsiveness of the cells to elicitor, but not to MJ. 4.2.8  CL-1 elicitor-responsiveness in transgenic tobacco  The above data are consistent with the hypothesis that MJ plays a role in the elicitor response, including activation of 4CL expression. However, previous work suggested that the  4 CL-i promoter alone is not sufficient for elicitor responsiveness in transgenic  tobacco or stably transformed parsley cell cultures (Douglas et al., 1991).  Since the  4 CL-i promoter is MJ responsive in tobacco, as shown in Chapter 3 (Figure 3.8), it is difficult to reconcile those observations with a role for MJ-mediated signalling in the elicitor response in tobacco. As part of my work, I looked at the effect of MJ and elicitor  Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures  PAL  4CL  TyrDC  ELI3  HPRG  Ubi4  —  —  72  nPG nPG  +  Eli  —  Eli  C MJ  ..  :. •. .  .•  Figure 4.21: The effect of the lipoxygenase inhibitor, n-propylgallate (nPG) on elici tor-inducible expression of defense-related genes in parsley. Treatment with the inhibitor nPG, 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 line 801-8, derived from the selfing of the primary transformant 801-8. The 801-8 primary transformant was used in the original work on elicitor-induced carries a GUS gene under the direction of -597bp of the  4 CL-i expression and  4 CL-i promoter. Surprisingly,  the Fl individual of line 801-8 examined showed a marked increase in GUS expression in response to Pmg elicitor (Figure 4.23), showing that, in this individual at least, the  4 CL-i promoter was elicitor responsive. An explanation for this anomalous result may lie in the fact that the plants used in these experiments had been through at least one meiosis, giving the potential for a change in the introduced construct, rendering it now capable of elicitor responsiveness. Thus, in tobacco, the  4 CL-i promoter appears to be  capable of responding to elicitor as well as wounding and MJ application.  Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures  73  1.0  CD  0.8  —  Co  CD  0.6  0.4  I  0.2  -  -  -  0  control  nPG  nPG  +  MJ  MJ  nPG  +  Eli  Eli  Figure 4.22: The effect of n-propylgallate treatment on furanocoumarin levels in culture fluids of parsley cell suspension cultures. Cultures were pretreated for 16 hours with nPG and 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 30 M MJ (MJ) or 50 tM Pmg elicitor (Eli), untreated (C). After 24 hours furanocoumarins were isolated and quantified as described in Figure 4.15.  Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures  74  Elicitor 0  2C  2  0  2C  2  -.2kb  Probe:  St4CL  GUS  Figure 4.23: The effect of Pmg elicitor treatment on 4 CL-i promoter activity in a plant of transgenic line 801-8. Leaves from the selfed offspring of the original transformed line 801-8 (transgenic for a -597bp 4CL-i promoter-GUS fusion), were incubated in 500 izg/ml Pmg elicitor for 3 hours. Control leaves were incubated in water. Duplicate blots were hybridised to probes for St4CL (to detect tobacco 4CL transcripts) and GUS. 4.3  Discussion  The rapid and transient change in gene expression caused by the addition of Pmg elicitor to the parsley cells in suspension culture make this an ideal system in which to study regulation of stress-induced gene expression. Using this greatly simplified system I have investigated signalling involved in stress responses, with particular attention paid to the role of jasmonates. Although direct evidence is lacking, a number of workers have suggested MJ may play a role in signalling (Reviewed by Staswick, 1992). In support of this notion, MJ is distributed in many plant species (Meyer et. al., 1984), and bears great similarity in structure and biosynthesis to eicosanoids, a group of important mammalian regulatory compounds which function as stress related second messengers (Anderson, 1989). In Chapter 3, I presented evidence consistent with the hypothesis that the wound response, mediated by the  4 CL-i promoter in tobacco, is mediated by jasmonates. In  this Chapter, I used parsley cells in suspension culture to further test the hypothesis that  Chapter 4.  The role of jasmonates in the elicitor response in parsley cell cultures  75  jasmonates 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 case  different 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 role as a signalling intermediate, it is probably not acting alone. Other signalling molecules may be required as well as JA. There is evidence suggesting that changes in protein Ca levels may play a role (Renalt et al., + phosphorylation patterns, and intracellular 2  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 gene expression seen after elicitor treatment. ABA can probably be ruled out as a signalling molecule with a direct role altering  4 CL  or other phenyipropanoid gene expression in  reponse to stress, since a range of concentrations of ABA had no effect on the expression of 4CL or PAL within the time frame necessary to suggest it may have a role as a signalling molecule in the rapid response to elicitor  .  We cannot, however, rule out the  possibility that it may be acting (as it does in other systems) as a translational regulator of gene expression (reviewed by Gallie, 1993), or in combination with other signalling molecules to bring about changes in phenyipropanoid gene expression. In support of a model where other components to the transduction pathway besides MJ are involved, is the observation that the level of furanocoumarins accumulated in MJ treated cells, although significantly higher than control cells (10 fold higher), was one third less than the level seen in elicitor treated cells. Lozoya et al., (1991) report that activation of both the flavonoid- and the elicitor- specific branch pathways of the phenyipropanoid pathway by simultaneous treatment with both Pmg elicitor and UV light results in an overall decrease in the level of furanocoumarin secretion, as compared  Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures  76  to elicitor treatment alone. This decrease, however, appears to occur at the posttran scriptional level since transcription rates and mRNA accumulation are unaffected. Since treatment of cells with MJ activates both the flavonoid pathway (Dittrich et al., 1992) and the furanocoumarin pathway perhaps the low coumarin accumulation is a reflection of a similar phenomenon. It could be hypothesised that activation of both pathways by MJ decreases the carbon flow to furanocoumarin biosynthesis relative to the carbon flow occurring subsequent to specific activation of furanocoumarin biosynthesis after elicitor treatment. If this is the case I would assume that the observed MJ activation of gene ex pression 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 stimula tion of both pathways does not occur. Although quantaatively the MJ-induced coumarin accumulation differed from elicitor-induced coumarin accumulation, qualitatively both profiles of coumarin accumulation were similar. Methyl j asmonate therefore stimulates the 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 in reduced 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 to plant tissue (Anderson, 1989). To avoid the possibility of JA in treated cells reaching toxic levels, I conducted all methyl jasmonate experiments in the parsley cells using a concentration of 30 pM, even though a slightly higher .4CL expression was observed at 100 pM. Kauss et al., (1992) similarly measured the effect of a range of concentrations of MJ on furanocoumarin secretion in parsley cells. In contrast to the data presented here, they reported an accumulation of furanocoumarin derivatives after treatment with 100 pM MJ of approximately 1/50th the level measured in elicitor treated cells. They do not report absolute furanocoumarin levels. This discrepancy may be explainable. Staswick (1992) pointed out that results of MJ treatment are difficult to compare among  Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures  77  laboratories. The reason is that different stereoisomers of jasmonates are possible.  (-  )Jasmonate is easily epimerised to the (+)isomer, which apparently has lower biological activity. Commercial preparations may differ in the ratio of the different isomers present and the MJ preparation used in my work was obtained from a different source than that used by Kauss et al. (1992). Another possible explanation is the fact that these workers dissolved MJ in ethanol, rather than Triton (as reported here). MJ is not readily soluble, and potentially Triton X-100 is a better choice of solvent than ethanol. At lower concentrations JA was more active than the methyl ester in inducing increases in 4CL mRNA accumulation. This is to be expected since JA is the biologically active form of  j 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 were originally isolated from parsley cells based on their rapid transcriptional activation after elicitor treatment. Based on DNA sequence comparisons, functions have been assigned to some of these genes. The fact that expression of these genes is induced by elicitor treatment suggests they play a role in defense (Sommsich et al., 1989). ELI 3, HRGP and TyrDC expression was induced by MJ treatment, however ELI 7 expression was unaffected under the same conditions. The fact that MJ did not affect expression of all of these genes implies the presence of multiple elicitor-induced signalling pathways for induction of elicitor-responsive genes. This hypothesis is consistent with the apparent lack of conserved promoter elements among the elicitor responsive genes, 4CL, PR2 and ELI 7 (I. Sommsich, personal communication). Furthermore, of the non-phenyipropanoid defense-related genes tested, the elicitor reponse of TyrDC was uniquely affected by nPG treatment, which implies further branching of signalling pathways for the elicitor response. This gene may therefore share signal transduction pathway components with the phenyipropanoid genes. The other genes which were induced by MJ, but unaffected  Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures  78  by nPG, may be induced by other lipoxygenase independent pathways. Both 4CL and PAL are inducible by both wounding and elicitor (Hahlbrock and Scheel, 1989). Endogenous JA levels may therefore play a role in the response to either or 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 is an endogenous signal involved in their regulation, it is more likely to play a role in the elicitor 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 was unaffected showing that the MJ response is present in whole plants as well as cells in culture. a-LA activated CL expression in the cell cultures and this response was maximum at 30 M. At 300 t1 M expression decreased markedly. The most likely explanation for this is that linolenic acid is toxic to the cells at higher concentrations. The ability of a-LA to induce CL-1 mRNA accumulation in the cells, may reflect its conversion to JA. This is supported by the observation that -7 LA, which does not enter the biosynthetic pathway to JA (Hamberg and Gardner, 1992) had no effect on 4CL expression (data not shown). If this is the case it means that lipoxygenase activity in the cells is present constitutively at sufficiently high levels to convert LA to biologically active levels of JA. Thus, induction of lipoxygenase activity may not be a prerequisite for the elicitor response.  Elicitor  perception at the cell membrane may result in release via lipase activity of LA from cell membrane as proposed by Farmer and Ryan, (1992).  Induction of lipoxygenase  activity may however be important in stress responses in other systems since its activity is induced in tobacco cells treated with elicitors from Phytophthora parasitica (Fournier et. al. 1993), oat infected with Puccinia coronata avenac (Yamamoto and Tani, 1986), and in rice infected with Magnaporthe grisea (Ohta et.  al.  1991).  Also, increased  lipoxygenase gene expression is associated with pathogen attack in Arabidopsis (Melan  Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures  79  et. al., 1993). The parsley lipoxygenase and its inhibitors have not been characterised. I therefore tested the ability of a large number of known lipoxygenase inhibitors to affect the elici tor response. Five of these, ibuprofen, phenylbutazone, antipyrene, n-propylgallate and salicylhydroxamic acid, which are known inhibitors of soybean lipoxygenase, inhibited wound-inducible expression of vsp genes in soybean hypocotyls (Staswick et al., 1991). In parsley cells, only n-propylgallate, an inhibitor of tobacco lipoxygenase which inhib ited wound-induced activation of the 4CL promoter in transgenic tobacco (Figure 3.12), affected the elicitor-responsiveness of 4CL. Elicitor-responsiveness of PAL, and TyrDC was also affected by treatment with nPG. Furthermore, elicitor-induced furanocoumarin synthesis was dramatically reduced by treatment with nPG (Figure 4.22). The ability of nPG to markedly decrease the stress responses in both systems suggests that endoge nous JA could play a general role in the stress-induced signalling which culminates in the transcriptional activation of 4CL and other parsley defense-related genes. nPG may be inhibiting de novo synthesis of endogenous JA released from the cell membrane after perception of the elicitor response, and JA may be required as an intracellular signal mediating changes in gene expression. In the parsley system, nPG does not appear to be non-specifically inhibiting cellular processes since some of the elicitor-inducible genes tested retained elicitor-responsiveness after nPG treatment, and expression of the constitutive 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 cells after inhibition restored activation of the phenylpropanoid pathway, as measured by ac cumulation of furanocoumarins (Figure 4.22). One interpretation of these results is that addition of MJ bypassed the block in lipoxygenase activity caused by nPG, restoring the signalling pathway within the cells. Regardless, it cannot be assumed that the effect of nPG are not attributable to  Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures  80  something other than an inhibition of lipoxygenase activity. Lipoxygenase activities in nPG treated cells were not measured, and furthermore even if such assays were to reveal an inhibition of lipoxygenase activity after nPG treatment it would not necessarily mean that this was the sole effect of the inhibitor. nPG may well affect other processes important in the elicitor response. nPG is an antioxidant that functions by reacting with free-radical intermediates of the reaction (Vick and Zimmerman, 1987b). It has recently been suggested that reactive oxygen species can induce pathogen-related gene expression (Jones, 1994). A rapid (within 1-5 minutes) burst of hydrogen peroxide is released from cultured soybean cells treated with PGA (polygalacturonic acid) elicitor and may stimulate subsequent defense pathways (Legendre ci al., 1992). A similar rapid oxidative burst occurs in parsley cells treated with Pmg elicitor (Dierck Scheel, personal communication). nPG may interfere with this hypothetical signalling mechanism. Despite the potential for non-specific activity of the inhibitor, there is good evi dence that JA plays a role in intracellular signalling of the elicitor response. Much of the complete elicitor response is induced by jasmonates, i.e. activation of genes of the furanocoumarin specific branchpathway of phenyipropanoid metabolism, secretion of fu ranocoumarins from MJ treated cell, and MJ inducible expression of other defense genes and the ability of LA to induce 4CL gene expression at least, also supports the existence of 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 that this response is mediated by promoter elements. A promoter fragment also directed MJ and JA inducible GUS expression, and nPG inhibited wound-induced activation of the  4 CL-i promoter. This suggests that JA could be an endogenous signal in transmitting the wound signal to the nucleus to induce 4CL expression.  Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures  81  The work described in this chapter shows that endogenous JA may activate 4CL ex pression in parsley cells, and that JA may be an intracellular signal involved in activating  4 CL expression following elicitor perception. If so, these results must be considered in view of the ability of the  4 CL-i promoter to respond to elicitor and MJ treatment. If  MJ/JA is an intracellular signal synthesized in response to elicitor, one would predict that the  4 CL-i promoter responds similarly to JA/MJ and elicitor. However, previous  work showed that the 4 CL-i promoter alone is not sufficient for elicitor-responsivenesss in transgenic tobacco or parsley cells (Douglas et al., 1991), while the  4 CL-i promoter  clearly responds to MJ in the same transgenic tobacco lines (Figure 3.11). However, the results presented in Figure 4.23 show that the Fl progeny of at least one primary tobacco transformant (801-8) in which the  4 CL-i promoter was elicitor-unresponsive (Douglas  et al, 1991) has acquired the potential for elicitor-responsiveness. One possible explanation is an epigenetic modification of the construct in the original transformant that was reversed after meiosis. Methylation of cytosines is frequently correlated with inactivation of gene expression. Multiple unlinked copies of a homologous gene have been shown to lead to reversible inactivation of the genes involved (Reviewed by Matzke and Matzke, 1993). In this case however, the 801-8 primary transformant segregated 3:1 for Kanamycin resistance upon selfing indicating a single insertion or several tightly linked insertions.  Several studies have shown that multiple copies of  closely linked or tandemly arranged transgenes in plants can also lead to inactivation that is correlated with reduced activity.  Such methylation-associated inactivation is  reversible through one or more generations. It is conceivable that an inactivation of a promoter element required for elicitor inducibility could result in the recovered elicitor inducibility in offspring of primary transformants. A second explanation may lie in the fact that the original work looked at the elicitor response at the level of GUS enzymatic  Chapter 4. The role of jasmonates in the elicitor response in parsley cell cultures  82  activity rather than RNA accumulation as described here. The observation that the 4CL1 promoter is capable of elicitor-responsiveness as well as wound and MJ-responsiveness is consistent with a potential role for JA as an intracellular signal involved in mediating responsiveness 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 MJ responsiveness of the 4 CL-i gene in parsley cells cannot yet be made. I therefore cannot say if the known lack of promoter-responsiveness to elicitor in parsley cells is associated with a presence or absence of promoter-responsiveness to MJ in the same system. Parsley cells can readily be transiently transformed and this system has been successfully used to 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 in this system has proved difficult (Klaus Hahibrock personal communication). For these reasons, definitive results regarding the elements required for MJ-induced expression in parsley cells are best obtained using stably transformed cells. Parsley cells are difficult to transform stably and I was unable to generate stable transformants. In spite of the un certainties regarding the promoter sequences required for MJ and elicitor-responsiveness in parsley, the accumulated data presented in this chapter argue strongly for a potential role 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 observations in Chapter 3 which suggest a role for MJ in the stress response of  4 CL-i. I have also  presented data which suggests not only a role for MJ in the response of 4CL, but also in the response of genes involved in furanocoumarin biosynthesis, and other elicitor inducible genes. It is clear that a single transduction pathway does not mediate the  Chapter 4.  The role of jasmonates in the elicitor response in parsley cell cultures  83  elicitor response but more likely there exists a diversity of signalling pathways controlling expression of defense-related genes.  Chapter 5  Expression of parsley 4CL-1 in Arabidopsis thaliana  5.1  Introduction  Genetic approaches are potentially powerful for identifying second messengers involved in regulating gene expression. By identifying mutants which no longer respond to exter nal stimuli in the normal way, genes whose products are required for signal transduction can be identified genetically. In plants, a useful organism for such studies is Arabidopsis thaliana. The usefulness of Arabidopsis for laboratory work in classical and molecular genetics is well known and has been extensively reviewed (Meyerowitz 1989; Meyerowitz and Pruitt, 1985; Estelle and Somerville 1986). It is small, very prolific, and has a gener ation 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. The existence of RFLP maps and the low amount of repetitive DNA has made possible the isolation 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, Ara bidopsis is readily transformable. A leaf disc transformation assay using Agrobacterium tumefaciens has been described (Lloyd et at., 1986). Other transformation protocols have been reported including a root explant transformation method, and transformation via rooty tumours (Van Sluys et al., 1987; Valvekens et at., 1988). It is not surprising that a great number of research programs take advantage of these attributes.  84  Chapter 5. Expression of parsley 4CL-1 in Arabidopsis thaliana  85  A disadvantage of Arabidopsis is that, in contrast to systems like the parsley cell suspension culture system, there is less information regarding the responses of this plant to challenge with potential pathogens. Due to the interest in developing Arabidopsis as a model system, there have been a number of recent reports in the literature dealing with the response of Arabidopsis to pathogen attack. For example, pathogen-induced activation of the Arabidopsis acidic chitinase promoter has been analysed, and elements mediating induced expression identified (Samac et aL, 1991).  The induction of Ara  bidopsis defense genes by virulent and avirulent Pseudomonas syringae strains has been studied and a putative avirulence gene involved in the interaction has been cloned (Dong et al., 1991). A plant defense gene, ELI3, has been identified in Arabidopsis which ap pears to play a functional role in establishing a resistant phenotype in the interaction between Arabidopsis and phytopathogenic Pseudomonas syringae strains. Furthermore, a suspension culture system has been developed in which putative defense responses are induced by treatment with two different elicitors, a bacterial pectin-degrading en zyme a-1,4-endopolygalacturonic acid lyase (PGA lyase), and Pmg elicitor (Davis and Ausubel, 1989). In this system, an increase in PAL activity and increases in PAL and CL mRNAs were reported. Elicitor stimulated accumulation of two mRNAs associated with lignin deposition, caffeic acid O-methyltransferase and peroxidase but no increase in chalcone synthase mRNA was observed, suggesting that flavonoid derivatives are not produced as defense compounds ii Arabidopsis. There has also been a report of induction of genes encoding 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHP), the first enzyme in the shikimate pathway, in response to wounding and pathogen attack (Keith et al., 1991). The phenomenon of systemic acquired resistance (SAR) in Ara bidopsis has recently been studied (Uknes et aL, 1992). SAR is the phenomenon whereby a pathogen infection results in a hypersensitive response at the site of infection, and leads to subsequent resistance to attack by a range of pathogens. This response has been  Chapter 5. Expression of parsley 4CL-1 in Arabidopsis thaliana  86  well characterised in tobacco and is correlated with the expression of a group of genes called SAR (systemic acquired resistance) genes (Ward et al., 1991). By treatment with a chemical, INA (2,6-dichioroisonicotinic acid), these workers were able to mimic the hypersensitive response in Arabidopsis, with subsequent acquired resistance to a number of pathogens and expression of SAR genes. Some genes of phenyipropanoid metabolism have been cloned from Arabidopsis. A clone for the single chalcone synthase gene has been isolated (Feinbaum and Ausubel, 1988) and promoter sequences required for its light regulated expression identified (Fein baum 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 single copy 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 gene family of four to five members, comparable in size to the gene family in parsley and bean (Lois et al., 1989; Ohi et al., 1990). Expression of one of these Arabidopsis PAL genes, PAL-i, has been partially characterised. The PAL-i promoter is activated early in seedling development and in adult plants it is highly expressed in vascular tissue and in flowers, a pattern similar to expression of parsley CCL-i in transgenic tobacco (Reinold et al.,1993). The Arabidopsis PAL-i promoter contains elements responsible for conferring accumulation of GUS mRNA in response to wounding, heavy-metal stress, and light. The genetics of Arabidopsis could be exploited to identify genes encoding components of the signalling pathways which mediate responses such as these. The first requirement for the identification by mutagenesis of genes involved in signal transduction is a genetic screen for phenotypes by which mutants can be distinguished from wild type plants. In this chapter, I discuss stress-inducible expression of .4CL in Arabidopsis. I show that wound-inducible GUS expression, directed by the  .4 CL-i pro  moter in transgenic Arabidopsis, provides a screen which could enable the identification  Chapter 5. Expression of parsley 4CL-1 in Arabidopsis thaliana  87  of mutants in transduction pathways leading to wound-activation gene expression. 5.2  Results  5.2.1  Wound-inducible expression of a parsley 4CL-1 promoter-GUS fusion in transgenic Arabidopsis  To determine if the  4 CL-i promoter is wound-responsive in Arabidopsis, I used a line transgenic for a 1.5kb parsley 4 CL-i promoter fused to GUS (204-1-3), which had been generated by Agrobacterium-mediated transformation of roots of ecotype C24 (R. Moselei and C. Douglas, unpublished results). The primary transformant which gave rise to 2041-3 appeared to contain the T-DNA at a single locus, since, after selfing, FT progeny segregated 3:1 for kanamycin resistance carried on the T-DNA. After confirming that line 204-1-3 was homozygous for the T-DNA, I used Southern blots to attempt to estimate the number of T-DNA inserts at a single locus. The pattern of hybridisation was not reconcilable with a single T-DNA insertion, but also was not consistent with simple tandem insertions, suggesting some rearrangements of the T-DNA (data not shown). I used a histochemical assay to detect wound-inducible GUS expression in leaf discs cut from these plants. This assay is based in the cleavage of a substrate, X-gluc, by the enzymatic activity of GUS, to yield an indigo dye. Figure 5.24 shows the accumulation of GUS activity around the margins of the disc and also at a tear which resulted from forceps handling. A control leaf disc which was fixed and stained immediately after wounding, 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 leaf material from the plant immediately prior to wounding, I wounded the leaf while it was still attached to the plant, thus mimicking herbivory. Figure 5.25 shows how this induced accumulation of GUS activity localised at the wound site of the attached leaf. The control  Chapter 5. Expression of parsley 4CL-1 in Arabidopsis thaliana  88  leaves which were removed, and stained immediately after wounding, showed only some GUS 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 the parsley  4 CL-i promoter) and Arabidopsis 4CL mRNAs in wounded excised leaves. Ara  bidopsis 4CL transcripts were detected using a cDNA clone for Arabidopsis 4CL. The northern blot shown in Figure 5.26 shows that the Arabidopsis 4CL ene was responsive to wounding. Transcript accumulation was seen by 2 hours after wounding and a high level of transcripts was still present 24 hours after wounding. Similar wound-inducible expression was seen for GUS driven by the  4 CL-i promoter. The 4 CL-i promoter is  therefore sensitive to the wound signal in Arabidopsis. In both cases maximum tran script levels were observed by 2 hours after wounding. This peak, however, was more pronounced for accumulation of GUS transcripts, and the level of GUS transcripts was lower at the later time points. 5.2.2  The effect of ethylene on 4CL expression in Arabidopsis  In 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 ethylene responsive and to determine if the sequences mediating this response are contained within the promoter region of the parsley  4 CL-i gene, I treated 204-1-3 plants with 10 mg/ml  ethephon and analysed Arabidopsis 4CL and GUS mRNA levels on northern blots. The  4 CL gene responded strongly to treatment with ethephon, and by 24 hours a large in crease in transcript levels was observed. The parsley  4 CL-i promoter also appears to  direct 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 with the GUS probe detected a second band which migrated faster than the 2 kb GUS mRNA suggesting the presence of a degradation product of GUS mRNA.  Chapter 5. Expression of parsley 4CL-1 in Arabidopsis thaliana  89  C  w  Figure 5.24: Accumulation of GUS activity in wounded leaf discs of transgenic Ara bidopsis line 204-1-3. Leaf discs were punched from Arabidopsis plants transgenic for a l500bp parsley 4CL-1 promoter GUS-fusion (204-1-3). These discs were either immedi ately stained with X-Gluc (C) to detect GUS activity, or incubated for 24 hours on filter paper moistened with MS media without hormones (W) prior to staining.  Chapter 5. Expression of parsley 4CL-1 in Arabidopsis thaliana  90  4*  C w  n Figure 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 either removed and stained immediately with X-Gluc to detect GUS activity (C), or removed and stained 24 hours after wounding (W).  Chapter 5. Expression of parsley 4CL-1 in Arabidopsis thaliana  Time  6  2  0  Trne  24  6  2  0  91  24  2kb  I  GUS  Probe:  2kb  4CL  Probe:  Figure 5.26: Accumulation of GUS and Arabidopsis 4CL transcripts in wounded leaves of Arabidopsis line 204-1-3. Leaves were detached from Arabidopsis plants transgenic for a l500bp parsley 4 CL-i promoter GUS fusion (line 204-1-3) and they were wounded by slicing into 1-2 mm strips. Tissue was then either frozen immediately (0 hour) or incubated on MS media (without hormones) for the times indicated. 10 pg of RNA was isolated and separated on a formaldehyde gel. Duplicate blots were hybridised to cDNA probes for Arabidopsis 4CL or GUS.  Time  0  6  24  Time hrs  0  6  2kb  Probe:  GUS  24 kb  Probe:  4CL  Figure 5.27: Accumulation of GUS and Arabidopsis 4CL transcripts in Arabidopsis ethephon treated leaves of transgenic line 204-1-3. Arabidopsis plants transgenic for a l500bp parsley 4 CL-i promoter GUS fusion (line 204-1-3) were sprayed with 10 mg/ml 2-chioroethanephosphonic acid and tissues harvested at the 0, 6, and 24 hours. 10 pg of total RNA was loaded on duplicate northern blots which were hybridised with probes for Arabidopsis CL to detect endogenous Arabidopsis 4CL transcripts, or GUS to detect the activity of the introduced gene construct.  Chapter 5. Expression of parsley 4CL-1 in Arabidopsis thaliana  5.2.3  92  Response of 4CL to methyl jasmonate in Arabidopsis  I showed that the parsley 4 CL-i promoter, as well as being wound-responsive, is strongly methyl jasmonate-responsive in transgenic tobacco (Figure 3.8). In analogous experi ments in transgenic Arabidopsis, 204-1-3 plants were treated with 1 mM and 10 mM methyl jasmonate and accumulation of GUS and 4CL transcripts measured by northern blotting. Figure 5.28 shows there was an accumulation, above the level seen in control plants, of transcripts for Arabidopsis 4CL in response to both concentrations of methyl jasmonate. There was also, directed by the parsley 4 CL-i promoter, an accumulation of  GUS transcripts. Again, a second smaller band was detected with the GUS hybridisation probe suggesting the presence of a smaller mRNA product made either in vivo or in vitro during the extraction process. The control plants were treated with 1% Triton X-100 (in which methyl jasmonate was dissolved) alone and this treatment, appeared to result in some transcript accumulation. Thus, the effect of the Triton may be masking a larger MJ response in these plants indicating that a more effective way of applying MJ should be devised. 5.2.4  A genetic screen for identification of genes involved in mediating the wound response  The accumulation of histochemically detectable GUS activity around wound sites in plants of the transgenic line 204-1-3 provided a phenotype which could be readily assayed in a screen for mutants defective in wound-induced signal transduction leading to the activation of the parsley 4 CL-i promoter. Thus, I mutagenised seeds from this line with EMS, (Ml generation), and allowed them to self to produce the M2 generation in which recessive mutations which might affect the wound-inducible expression of the introduced  GUS fusion would be expected to segregate. In order to try to avoid analysis of cis  Chapter 5. Expression of parsley 4CL- 1 in Arabidopsis thaliana  OC  1mMM.1 24 6  S  1OmMMJ 24 6  93  24C  GUS  4CL  Figure 5.28: The effect of methyl jasmonate treatment on 4CL expression in transgenic Arabidopsis. 204-1-3 Arabidopsis plants were sprayed with 1 mM and 10 mM solutions of methyl jasmonate dissolved in 1.0% Triton X-100. Control plants were sprayed with 1.0% Triton X-100 alone. Plants were harvested at the times shown in hours (h), 10 pg of RNA was separated on a formaldehyde gel and duplicate blots hybridised with probes for Arabidopsis 4CL to detect endogenous if CL transcripts, or GUS to detect the activity of the introduced gene. mutations, in which the parsley J CL-i promoter or the GUS structural gene rather than genes 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 but retained tissue-specific expression of GUS. Thus, any potential mutant would contain a functional transgene, although cis-mutations in promoter elements required for wound inducible expression would be possible. I individually screened 5,000 M2 plants and in this number there was no mutant which lacked the wound response and retained tissue specific expression.  5.3  Discussion  With a view to using Arabidopsis as a model genetic system for studying regulation of phenylpropanoid gene expression, I have studied the expression of both the parsley  Chapter 5. Expression of parsley 4CL- 1 in Arabidopsis thaliana  94  4 CL-i and the Arabidopsis 4CL gene in response to wounding in Arabidopsis. The Arabidopsis 4CL gene responded strongly to wounding by accumulation of mRNA, and the parsley 4 CL-i promoter responded to wound signals produced in Arabidopsis, since it directed wound inducible accumulation of GUS mRNA. The accumulation of GUS transcripts 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 that the 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 accumulation of GUS activity provided the basis for a genetic screen which, it was hoped, would allow genetic identification of loci for genes important in signal transduction mediating woundinduced 4CL gene expression. An important requirement in a screen based like this one on introduction of a trans gene, is a way to distinguish between cis-mutations, in which the introduced gene con struct has been mutated, and trans-mutations, where expression of the introduced con struct has been affected by mutation of another locus which acts upon it. One approach to overcoming this problem is the use of a transformed line in which there is more than one transgene (e.g. promoter- GUS fusion). A trans mutation would therefore affect all the introduced constructs and the probability of inactivating all the introduced GUS genes with cis mutations would be small. Southern analysis of 204-1-3, the homozygous transformed line used in this study, suggested more than one insertion but was ambigu ous in defining the number and structure of inserted T-DNAs. Since I was not able to establish with certainty the manner in which the introduced construct was arranged within the plant, I used an alternative approach and screened only for mutations which lacked wound inducible expression of GUS but retained tissue-specific expression. In a  Chapter 5. Expression of parsley 4CL-l in Arabidopsis thaliana  95  preliminary screen of 5,000 M2 plants, I failed to identify any such mutants. I would ex pect non wound-responsive mutations to be rare, and screening for mutations which still retain tissue-specific expression could further reduce the number of target genes being screened for. Results in Chapter 3 showed that in transgenic tobacco, the 2lObp pro moter fragment that directs tissue-specific expression of parsley 4CL-1 is also sufficient to activate gene expression in response to wounding. This indicates that the signalling pathways involved in directing tissue-specific and wound-inducible expression of  CL-  1 could converge on the same promoter elements and thus could share components in common. If the situation is similar in Arabidopsis, the number of target genes affecting wounding and not tissue-specific expression may be small. There has been a report in the literature of a successful genetic screen for signal transduction 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 screened to obtain a small number of mutants affected in chloroplast-induced activation of the promoter of a nuclear gene encoding chlorophyll a/b binding protein (cab). Their strategy differed in that they generated a transgenic line of Arabidopsis which carried two different reporter genes (GUS and NP Ti!), each driven by the CABS promoter. They screened M2 plants for mutants that, in the absence of chloroplast development, expressed both reporter genes driven by the CAB2 promoter, and discarded potential mutants in which expression of only one of the transgenes was affected. From a screen of 100,000 M2 plants they isolated 9 plants which had heritable mutant phenotypes in which CAB2GUS expression was uncoupled from chioroplast development. The Arabidopsis gene responded to ethylene, applied as ethephon and the  4 CL-i  promoter appears to direct responsiveness to ethephon upon a GUS reporter gene. How ever, induced accumulation of GUS transcripts appeared to be lower than the response of the endogenous Arabidopsis gene. This would make analysis of promoter elements  Chapter 5. Expression of parsley 4CL-1 in Arabidopsis thaliana  96  mediating this response difficult. Other limitations of using ethephon to study ethylene mediated changes in gene expression were discussed in Chapter 3. A genetic approach may prove more successful in elucidating the role, if any, for ethylene in phenylpropanoid gene expression. Mutants in Arabidopsis that are insensitive to ethylene have been de scribed (Bleecker et al., 1988; Pickett et al., 1990). The mutation etrl for example is a dominant mutation that inhibits a diverse number of processes affected by ethylene e.g., cell elongation, promotion of seed germination, enhancement of peroxidase activity, and feedback suppression of ethylene synthesis by ethylene. It is thought that the ETRJ gene product 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 is similar to the prokaryotic two-component regulators (Chang et al., 1993). In bacteria these systems consist of two proteins, a sensor and a regulator which function together to regulate adaptive responses to environmental stimuli. The ETRJ gene product encodes a single protein with similarities to both components. It is thought that ETR1 may be a sensor of ethylene with its amino terminus acting as the sensor and the signal being transduced 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 is a candidate for perception of the signal. Potentially therefore, analysis of wound-induced CL expression in etri mutant lines could provide evidence to support a function for ethylene in the wound response. In this work, the massive response to MJ present in tobacco plants and parsley cells did not appear to be present in Arabidopsis. The 4CL1-GUS transgene showed some response 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, it may be that a larger MJ response was masked by the response of the cells to the Triton X-100 in which the MJ was dissolved. Applications of MJ in an alternative solvent should  Chapter 5. Expression of parsley 4CL-1 in Arabidopsis thaliana  97  clarify this. It is possible therefore that the proposed signalling pathways involving MJ are present in Arabidopsis and in support of this there has been a report in the literature of 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 3 hours. Identification of mutants in Arabidopsis which are lacking the MJ response should prove a valuable approach to elucidating the functional significance of the MJ response.  Chapter 6  Conclusions and future directions  6.1  Conclusions  Using 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, mechanical wounding and pathogen attack (simulated by elicitor treatment). My work has focused mainly on the phenyipropanoid gene CL, using it as a model gene to gain a under standing of the intracellular events which occur subsequent to stress, and which lead to changes in expression of defense-related genes. It is perhaps a measure of the primary importance of the wound response that induc tion of 4CL expression in response to to this stress is conserved among three different families, Apiaceae (parsley), Solanaceae (tobacco) and Brassicaceae (Arabidopsis). The conservation of the wound response enabled me to use a transgenic tobacco system to demonstrate that the same 2lObp promoter fragment that mediates tissue-specific ex pression (HaufFe et al., 1991) is responsive to wounding and MJ. Potentially, the same trans-acting factors may be mediating all three responses, and the signal transduction pathways triggered by these stimuli may share common components. I have identified factors which may be involved in signalling pathways leading to the expression 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 and ethylene responses may not be independent of each other since, for example, MJ induces  98  Chapter 6. Conclusions and future directions  99  ethylene 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 in the signal transduction pathway. This is supported by the observation that although the same qualitative patterns of antimicrobial furanocoumarins accumulate in MJ-treated and elicitor-treated cells, MJ treatment alone does not induce the same level of fura nocoumarin accumulation in the cell culture system as is induced by elicitor treatment. Furthermore, in the parsley cell culture system the elicitor-induced changes in patterns of expression of a number of genes, often differed in timing or level of inducibility from patterns of expression seen after MJ treatment. ABA does not appear to play a direct role as a signal molecule in the activation of phenyipropanoid gene expression in tobacco or cultured parsley cells since the 4CL gene was not rapidly ABA responsive in these systems. This does not rule out the possibility that in a complex interplay of signalling molecules, ABA may play a role. It may activate gene expression in combination with other 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 gene in the parsley cell culture system, the endogenous  4 CL  genes in tobacco and the ac  tivation of the parsley 4CL promoter in transgertic tobacco. In the presence of an in hibitor 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 activation  was greatly reduced. In parsley cells, the same inhibitor reduced the elicitor responsive ness of a number of defense-related genes  (4 CL, PAL,  and TyrDC. This suggests that  the LA-inducible expression of 4GL in parsley cells and the LA-induced activation of the  4 CL-i  promoter in tobacco could reflect an important role for the conversion of LA to  JA in intracellular signalling following perception of these stresses. The similarity between eicosanoids in animals and jasmonates has been pointed out  Chapter 6. Conclusions and future directions  (Staswick et al., 1992; Anderson, 1989).  100  Both compounds are produced via similar  lipoxygenase mediated pathways. In mammals, eicosanoids are a diverse class of potent metabolic regulators. The main classes of eicosanoids are the prostaglandins, prosta cyclin, leukotrienes, thromboxanes, and lipoxins (Reviewed by Anderson, 1989). These molecules are not required for growth and development of animal tissues and are consid ered secondary hormones. It is interesting to note that one of the main classes of stimuli that elicit eicosanoid synthesis are tissues stresses such as trauma, disease, and allergy. Plant lipoxygenases are inhibited by nonsteroidal antiinflamatory drugs (Vick and Zim merman, 1987b). Perhaps a parallel exists between plant responses to pathogen attack and mechanical stress, and animal responses to equivalent traumas. Plants are not the only organisms that respond to MJ. It is, for example, an active component of the female attracting pheromone released by male oriental fruit moths (Baker et al., 1981) and it is found in at least one pathogenic fungus (Staswick, 1992). In view of its volatile nature, it is possible that other organisms may influence changes in gene expression in plants and that this could play a role in the interaction between plants and potential pathogens. 6.2  Future directions  An extension of the molecular biological studies I have started here will be valuable in gaining an understanding of the signal transduction pathways mediating expression of  4 CL.  For example, the marked response to MJ conferred on a GUS reporter gene by  the 210 bp  4 CL-i  promoter fragment should enable the identification of a MJ-responsive  element or elements within the promoter. By comparing this element (or elements) to those known to be required for tissue-specific expression (Hauffe et al., 1993), it will be possible to determine if the signal transduction pathways controlling spatial patterns of expression, share a common end point with those conferring the response to methyl  Chapter 6. Conclusions and future directions  101  j asmonate. The data presented here suggests that lipoxygenase activity is required for the stress response 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 reports of characterisation of parsley lipoxygenses. Consequently, in this work I relied upon information known about inhibition of soybean and tobacco lipoxygenases.  Only n  propylgallate, a known inhibitor of tobacco lipoxygenase, affected the stress-inducible expression of the genes in the parsley system. Characterisation of the lipoxygenases in parsley and the genes that encode them would provide a useful tool for continuation of these studies. As well, it must be demonstrated that n-propylgallate, which I used as an inhibitor of lipoxygenases, actually inhibits lipoxygenase activity and reduces the biosynthesis 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 trans duction pathways will be genetic studies. To initiate such an approach I characterised the wound-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 re sponse. Such mutations are rare, requiring long and laborious screening. Such large scale screening was beyond the scope of this work. Recently, however, a screening approach which will avoid arduous large scale screening for rare signal transduction mutants in localised induction of defense genes, has been described a(de Maagd et al., 1993). A rice basic chitinase promoter which was deleted to l6Obp 5’ of the transcription start, was fused to the selectable alcohol dehydrogenase (ADB) from Avabidopsis and a similar pro moter fragment was also fused to GUS. This construct was introduced into Arabidopsis line ROO2, an ADH (alcohol dehydrogenase) null mutant. Truncating the promoter in this manner abolished most of the developmental expression while retaining inducibility  Chapter 6.  Conclusions and future directions  102  by wounding. This group are selecting mutants based on their ability to survive allyl alcohol treatment. In plants in which the chitinase promoter fragment is activated nor mally, the ADH produced will convert allyl alcohol into the toxic acrolein, killing the plant. A potential problem with this approach in the case of 4CL gene expression is the fact that the same promoter fragment that is required for the wound response mediates high levels of tissue specific and developmentally regulated expression. It may however prove useful for other defense-related genes. The isolation of mutants in Arabidopsis that are deficient in the biosynthesis of or response to jasmonates will be of great value in determining the functional relevance of gene activation in response to these compounds. Such mutants would allow definitive testing of the hypothesis that jasmonates are part of an intracellular signalling network required for gene activation in response to external stimuli. Regardless of the approach, an increased understanding of plant responses to stress will be valuable. Our understanding of plant signalling has for a long time lagged behind that which exists in animal systems. The rapid response of a gene such as 4CL to stresses is useful as a paradigm for plant signalling in general. From a practical point of view, the advantages of increased understanding of plant defense are obvious. One advantage being the potential for engineering of lines with increased resistance to pathogen attack and the mechanical stresses inflicted by the environment and horticultural practices. 4CL is also of major practical importance because of its role in the production of the activated substrates to the lignin branch pathway of phenyipropanoid metabolism. For example, there is considerable interest in modifying the type of lignin in wood. Decreased lignin content would make herbaceous plants such as alfalfa more digestible and better quality animal forage and the pulp industry would save considerable resources if there was less lignin (or one of a composition that is more readily removed) in trees. A prerequisite  Chapter 6. Conclusions and future directions  103  to all these applications is an understanding of how expression of 4CL, as a model phe nylpropanoid gene, is regulated. 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