Biochemical Plant Responses to Ozone : II. Induction of Stilbene Biosynthesis in Scots Pine (Pinus sylvestris L.) Seedlings

Formation of the stilbenes pinosylvin and pinosylvin 3-methyl ether, as well as the activity of the biosynthetic enzyme stilbene synthase (pinosylvin-forming), were induced several hundred- to thousandfold in primary needles of 6-week-old pine (Pinus sylvestris L.) seedlings upon exposure to a single pulse of ozone of at least 0.15 microliters per liter. The seedlings required 4 hours of exposure as a minimum for the induction of stilbene biosynthesis when exposed to 0.2 microliters per liter ozone. Both stilbene synthase activity and stilbene accumulation increased with the duration of ozone treatment. The activity of phenylalanine ammonia-lyase and the activity of chalcone synthase, a key enzyme of the flavonoid pathway that uses the same substrates as stilbene synthase, were also stimulated about twofold by ozone. Stilbene biosynthesis appears to represent the first example of a dose-dependent biochemical response to ozone in a conifer species and may serve as a useful biomarker to study stress impacts on pine trees.     

Cell-suspension culture of Arachis hypogaea L.: model system of specific enzyme induction in secondary metabolism

A peanut (Arachis hypogaea L.) cell-suspension culture susceptible to selective induction of stilbene formation was established. The principles of defense responses of the whole plant were found to be retained in the artificial system. The suspension culture was characterized by its growth curve and by various biochemical parameters. In the stationary phase, reached 8 d after transfer to a new medium, the formation of stilbenes and stilbene synthase could be induced without altering the levels of other enzymes. Eighteen hours after applying an artificial elicitor (ultraviolet-C light) or 4 h after eliciting with a crude preparation of Phytophthora cambivora cell walls, phenylalanineammonia-lyase activity was increased eightfold and stilbene-synthase activity 20-fold. The activity of phenylalanine ammonia-lyase reached its peak at a slightly different time from that of stilbene synthase. The main products of L-phenylalanine metabolism in the induced cells were resveratrol, 3,3,5-trihydroxy-4-methoxystilbene and isopentenylresveratrol. Likewise, feruloyl-CoA reductase, as a parameter of lignin formation, was enhanced following induction, albeit with a different time course and with a less steep increase than found for phenylalanine ammonia-lyase and stilbene synthase.     

Precursor-directed biosynthesis of stilbene methyl ethers in Escherichia coli

Stilbenes are bioactive compounds that show beneficial effects for humans, such as anti-tumor activity and survival improvement. Resveratrol, a representative of stilbenes and showing various health-improving activities, is rapidly metabolized in humans, and modified resveratrols are therefore desired as anti-cancer drugs and dietary polyphenols. An Escherichia coli system, in which an artificial stilbene biosynthetic pathway, including steps of phenylalanine ammonia-lyase, 4-coumarate:CoA ligase, and stilbene synthase, was reconstructed, produced stilbenes in high yields: resveratrol from tyrosine and pinosylvin from phenylalanine. To incorporate a stilbene methyltransferase gene into this E. coli system, cDNA of Os08g06100 in Oryza sativa was expressed and its O-methylating activity toward stilbenes was confirmed. Incorporation of the pinosylvin methyltransferase (OsPMT) gene into the pathway established in E. coli led to production of mono- and di-methylated stilbenes. Furthermore, the OsPMT gene turned out to be useful in production of unnatural stilbene methyl ethers due to its rather relaxed substrate specificity; various carboxylic acids supplemented as precursors, such as p-fluorocinnamic acid, 3-(2-furyl)acrylic acid, 3-(2-thienyl)acrylic acid, and 3-(3-pyridyl)acrylic acid, to the E. coli system carrying the steps of 4-coumarate:CoA ligase, stilbene synthase, and OsPMT were converted to stilbene dimethyl ethers with the corresponding carboxylic moiety.     

Molecular cloning and functional expression of a stress-induced multifunctional O-methyltransferase with pinosylvin methyltransferase activity from Scots pine (Pinus sylvestris L.)

Formation of pinosylvin (PS) and pinosylvin 3-O-monomethyl ether (PSM), as well as the activities of stilbene synthase (STS) and S-adenosyl-l-methionine (SAM):pinosylvin O-methyltransferase (PMT), were induced strongly in needles of Scots pine seedlings upon ozone treatment, as well as in cell suspension cultures of Scots pine upon fungal elicitation. A SAM-dependent PMT protein was purified and partially characterised. A cDNA encoding PMT was isolated from an ozone-induced Scots pine cDNA library. Southern blot analysis of the genomic DNA suggested the presence of a gene family. The deduced protein sequence showed the typical highly conserved regions of O-methyltransferases (OMTs), and average identities of 20–56% to known OMTs. PMT expressed in Escherichia coli corresponded to that of purified PMT (40 kDa) from pine cell cultures. The recombinant enzyme catalysed the methylation of PS, caffeic acid, caffeoyl-CoA and quercetin. Several other substances, such as astringenin, resveratrol, 5-OH-ferulic acid, catechol and luteolin, were also methylated. Recombinant PMT thus had a relatively broad substrate specificity. Treatment of 7-year old Scots pine trees with ozone markedly increased the PMT mRNA level. Our results show that PMT represents a new SAM-dependent OMT for the methylation of stress-induced pinosylvin in Scots pine needles.     

Induction of stilbene synthase by Botrytis cinerea in cultured grapevine cells

The interaction between Botrytis cinerea Pers. and grapevine (Vitis vinifera L.) was studied in a model system of reduced complexity. Cultured plant cells and fragments of fungal cell wall were used to simulate some of the processes taking place upon infection of grapevine with B. cinerea. A soluble glucan elicitor was prepared from the fungal cell wall by acid hydrolysis. Like the insoluble wall preparation, the soluble fragment derived from the cell wall acted upon plant cells in eliciting stilbene formation. In grapevine cells, the interaction with the fungus led to a dramatic shut-off general protein synthesis and to the selective formation of a small set of proteins involved in induced resistance. The proteins synthesized de novo with highest rates were stilbene synthase (StiSy) and l-phenylalanine ammonia-lyase (PAL). Stilbene synthase was purified to apparent homogeneity and its molecular properties were characterized. The enzyme is a homodimer with subunit M_r 43 000 and pl = 5.4. Although there were indications of the presence of isoenzymes, these were not distinguished by charge differences. In size, the grapevine StiSy shows microheterogeneity and differs from the appreciably larger enzyme prepared from peanut. Prior to induction by fungal attack, virtually no stilbenes are formed in the plant cell. Upon induction of the pathway leading to the stilbene resveratrol, StiSy activity determines the ratelimiting step in the metabolic sequence. The highly induced grapevine cells produce and secrete resveratrol and derivatives which are known to be fungistatic.Abbreviations PAL l-phenylalanine ammonia-lyase - SDS-PAGE sodium dodecyl sulfate-polyacrylamine gel electrophoresis - StiSy stilbene synthase (resveratrol forming) The authors thank Dr. Blaich, Bundesforschungsanstalt Geilweilerhof, Siebeldingen, F.R.G., for provision of callus culture. This paper is based on research supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.     

Molecular analysis of chalcone and dihydropinosylvin synthase from Scots pine (Pinus sylvestris), and differential regulation of these and related enzyme activities in stressed plants

Chalcone synthase (CHS) and stilbene synthase (STS) are closely related polyketide synthases which are key enzymes in the biosynthesis of flavonoids and stilbenes. Scots pine (Pinus sylvestris) is an interesting plant for a direct comparison of the enzymes. It not only contains the usual flavonoids, but also an unusual chalcone derivative (pinocembrin), and it synthesizes stilbenes of the pinosylvin type. We analysed a CHS and a STS by molecular cloning and functional expression in Escherichia coli. The CHS was active not only with 4-coumaroyl-CoA (to naringenin chalcone), but also with cinnamoyl-CoA (leading to pinocembrin). The STS was identified as dihydropinosylvin synthase, because it preferred dihydrocinnamoyl-CoA to cinnamoyl-CoA. The protein deviated in 47 positions from the CHS consensus. It had 73.2% identity with the CHS from P. sylvestris and only 65.3% with a STS from peanut (Arachis hypogaea). We also investigated the regulation of both enzyme types in P. sylvestris plantlets exposed to stress. CHS was present in non-stressed plantlets, and induction led to a transient increase with a peak after 16 h. STS¹ type activities were regulated differently and were absent in non-stressed plantlets. Increases were observed after a lag period of at least 6 h, and highest activities were obtained after 30 h. The analysis of the reactions in the plant extracts and the substrate specificity of the cloned STS indicate that the plants contain at least two different types of STS: the cloned dihydropinosylvin synthase and a pinosylvin synthase which preferentially utilizes cinnamoyl-CoA as substrate.     

The effect of explant origin and collection season on stilbene biosynthesis in cell cultures of Vitis amurensis Rupr.

Plant cell and tissue cultures are considered as a source of valuable secondary metabolites but usually produce insufficient level of the compounds, which is the limiting factor for their application in biotechnology. We obtained 18 callus cell cultures from different organs of wild grape Vitis amurensis Rupr. collected at different seasons and analyzed stilbene accumulation in combination with calli growth parameters. This analysis showed that temporal and tissue origin of the calli affected the rate of stilbene biosynthesis. Stem-derived calli accumulated higher stilbene levels and exhibited a higher expression of phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS) genes than calli derived from the leaves and petioles. The highest content of stilbenes was detected in the calli initiated from grapevine stems collected in the autumn. In general, all “autumn” cell cultures contained more than 2 mg g^??1 dry wt (up to 11 mg g^??1 dry wt) and exhibited high PAL and STS genes expression in comparison with the calli initiated in the summer. The content of stilbenes in the “autumn” cell cultures were comparable to the highest stilbene contents detected in other plant sources described in the literature. Thus, selecting the most optimal explant source for cell culture establishment could be an effective approach towards developing plant cell cultures producing high stilbene levels.

     

Antifungal activity of stilbenes in in vitro bioassays and in transgenic<Emphasis Type="Italic"> Populus</Emphasis> expressing a gene encoding pinosylvin synthase

The effect of two stilbene compounds, pinosylvin and resveratrol, on the growth of several fungi was evaluated in plate tests. Wood decay tests were carried out with birch and aspen samples impregnated with the two stilbenes. In plate experiments, resveratrol had an enhancing effect on growth at concentrations where pinosylvin was already enough to prevent the growth of most fungi studied. Pinosylvin impregnated at 0.2% (w/w) concentration significantly reduced the decay caused by all fungi except Phellinus tremulae. In contrast, a resveratrol content of 0.8%, did not protect the wood from decay. A pinosylvin-synthase-encoding gene from Pinus sylvestris was transferred into aspen (Populus tremula) and two hybrid aspen clones (Populus tremula×tremuloides) by Agrobacterium tumefaciens-mediated transformation. Transgenic plants accumulated pinosylvin synthase-specific mRNA and showed stilbene synthase enzyme activity in vitro. Transgenic aspen line H4 showed increased resistance to Phellinus tremulae, while two hybrid aspen transformants decayed faster than the control trees. However, we were unable to detect the accumulation of stilbenes in the transgenic plantlets.Communicated by P. Debergh     

A novel process for obtaining pinosylvin using combinatorial bioengineering in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Pinosylvin as a bioactive stilbene is of great interest for food supplements and pharmaceuticals development. In comparison to conventional extraction of pinosylvin from plant sources, biosynthesis engineering of microbial cell factories is a sustainable and flexible alternative method. Current synthetic strategies often require expensive phenylpropanoic precursor and inducer, which are not available for large-scale fermentation process. In this study, three bioengineering strategies were described to the development of a simple and economical process for pinosylvin biosynthesis in Escherichia coli. Firstly, we evaluated different construct environments to give a highly efficient constitutive system for enzymes of pinosylvin pathway expression: 4-coumarate: coenzyme A ligase (4CL) and stilbene synthase (STS). Secondly, malonyl coenzyme A (malonyl-CoA) is a key precursor of pinosylvin bioproduction and at low level in E. coli cell. Thus clustered regularly interspaced short palindromic repeats interference (CRISPRi) was explored to inactivate malonyl-CoA consumption pathway to increase its availability. The resulting pinosylvin content in engineered E. coli was obtained a 1.9-fold increase depending on the repression of fabD (encoding malonyl-CoA-ACP transacylase) gene. Eventually, a phenylalanine over-producing E. coli consisting phenylalanine ammonia lyase was introduced to produce the precursor of pinosylvin, trans-cinnamic acid, the crude extraction of cultural medium was used as supplementation for pinosylvin bioproduction. Using these combinatorial processes, 47.49 mg/L pinosylvin was produced from glycerol.     

Vanadate mimics effects of fungal cell wall in eliciting gene activation in plant cell cultures

Cell-suspension cultures of peanut (Arachis hypogaea L.) can be used as a very sensitive and rapidly responding physiological system for monitoring extracellular signals. Elicitors effect the activation of the genes that code for a set of enzymes synthesizing stilbenes. Within 2–6 h after administering micromolar, concentrations of orthovanadate to the suspended cells, the enzyme activities of phenylalanine ammonia-lyase, stilbene synthase, and cinnamate 4-hydroxylase increased 10-to 100-fold. The transient time course of induction, and the quality and quantity of gene expression found with vanadate as artificial elicitor were very similar to those observed after biotic stress generated by fungal cell walls. The dose-response of vanadate as an elicitor of gene expression in intact cells matched precisely its inhibitory effect on the ATPase activity of isolated plasma membrane. By concentrating, on the profiles of cinnamate 4-hydroxylase activity, we observed differences between the effects elicited by fungal cell wall or vanadate when different stages of cell development were analyzed. Unlike the fungal elicitor, vanadate did not induce the hydroxylase activity when cells at the stationary phase of the cell cycle were used. This lack of response was not the result of a decrease in membrane biosynthesis. The finding, that the effects of vanadate and fungal elicitor are additive indicates that vanadate does not interfere negatively with the perception of the biotic signal but rather addresses the same intracellular intermediate of the signalling process. We hypothesize that membrane potentials created or modulated by ATPases may be intermediates in the signal chain, starting with the recognition process at the plasma membrane and eventually leading to the production of stilbenes as low-molecular-weight plant-defence products.Abbreviations ER endoplasmic reticulum - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol deceased     

Intracellular location of NADP+-linked malic enzyme in C3 plants

Ultraviolet light induces anthocyanin biosynthesis in cell cultures of an Afghan cultivar of Daucus carota (Daucus carota L. ssp. sativus). Simultaneous treatment with a fungal elicitor from Pythium aphanidermatum results in an inhibition of the catalytic activity of chalcone synthase (CHS), which in turn correlates with an inhibition of anthocyanin biosynthesis. On immunoblots, one isoenzyme (40 kDa) of CHS disappears upon elicitor treatment. On an mRNA level, only the mRNA for the 40-kDa-CHS is active after treatment with ultraviolet light. After inhibition of anthocyanin biosynthesis by the elicitor the enzyme protein disappears and the CHS mRNA is strongly diminished. This inhibition depends on the concentration of the elicitor. In addition, elicitor treatment leads to an induction of the general phenylpropanoid pathway as well as to the accumulation of 4-hydroxybenzoic acid which is covalently bound to wall polysaccharides of the carrot cells. The possible function of phenylalanine ammonia-lyase in providing precursors for 4-hydroxybenzoic acid is discussed.Abbreviations CHI chalcone isomerase - CHS chalcone synthase - PAL phenylalanine ammonia-lyase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We are grateful to Professor K. Hahlbrock (Max-Planck-Institut für Züchtungsforschung, Köln, FRG) for providing us with antisera to CHS and PAL, respectively. This work was supported by a grant from the Deutsche Forschungsgemeinschaft and scholarships from the Friedrich-Ebert-Stiftung (J. G.), the Landesgraduierten-förderungsgesetz Baden-Württemberg (J.-P. S) and the Gerhard-Rösch-Stiftung (D. S.). We thank R. Hofmann for her excellent technical assistance.     

Coordinate induction by UV light of stilbene synthase,phenylalanine ammonia-lyase and cinnamate 4-hydroxylase in leaves of vitaceae

A stilbene synthase catalyzing the formation of resveratrol from 4-hydroxycinnamoyl-CoA and malonyl-CoA was found in the leaves of several Vitaceae. This stilbene synthase and two other enzymes functioning on the route from phenylalanine to stilbenes were induced concurrently upon irradiation of the leaves with UV light. With leaves of Cissus antarctica, an increase of stilbene synthase activity, more than hundred-fold, was observed with a maximum appearing 15 h after the induction with UV light.Abbreviations EDTA Na₂-ethylenediaminotetraacetate - Hepes 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid - Mes morpholinoethanesulfonic acid     

Antibacterial effects of knotwood extractives on paper mill bacteria

Hydrophilic knotwood extracts from 18 wood species were assessed in disc diffusion and liquid culture tests for antibacterial effects against three species of paper mill bacteria. The Pinus sylvestris, P. resinosa, P. contorta, and P. banksiana extracts decreased or inhibited bacterial growth. The susceptibility order was P. sylvestris > P. resinosa > P. contorta > P. banksiana, correlating with the concentrations of pinosylvin and pinosylvin monomethyl ether in these wood species. Also, Pseudotsuga menziesii and Thuja occidentalis extracts had a small inhibitory effect. The Gram-positive Bacillus coagulans was more susceptible to the extracts than the Gram-negative Burkholderia multivorans and Alcaligenes xylosoxydans. The main components in the Pinus knotwood extracts were pinosylvin monomethyl ether and pinosylvin, suggesting these to be the active components. Therefore, pure pinosylvin, pinosylvin monomethyl ether, and dihydro-pinosylvin monomethyl ether were also tested. All compounds showed antibacterial effects. However, higher concentrations were needed for these pure compounds than for the knotwood extracts. Pinosylvin had stronger antibacterial effects than pinosylvin monomethyl ether. This work shows that knotwood extracts, especially from Pinus species, have a potential for use as natural biocides in papermaking.     

Synthesis of phenylalanine ammonia-lyase in anthocyanin-containing and anthocyanin-free callus cells of Daucus carota L.

When callus cells of Daucus carota are grown on a medium containing gibberellic acid (GA₃) in a physiological concentration of 3x10^-6 M the cells cease to accumulate anthocyanins. This anthocyanin-free cell line has a very low activity of phenylalanine ammonia-lyase. After density labelling with D₂O an intensive de novo synthesis of the phenylalanine ammonia-lyase (E.C. 4.3.1.5; PAL) in the anthocyanin-containing cells does occur. 58% of the C-bound H-atoms are replaced by deuterium. The anthocyanin-free cells show only a very low enzyme synthesis which is difficult to detect with density labelling experiments. To ascertain that de novo synthesis occurs in the anthocyanin-free cells, the incorporation of ¹⁴C-labelled amino acids into the partially purified enzyme protein was measured after separation of the protein a) in CsCl gradients and b) on polyacrylamide gels. In both cases the enzyme bears ¹⁴C-label. These results suggest that in the anthocyanin-free cells de novo synthesis of PAL is still occuring but the synthesis is reduced in comparison to the anthocyanin-containing cells.Abbreviations GA₃ gibberellic acid - PAL phenylalanine ammonia-lyase (E.C.4.3.1.5) - DCb anthocyanin-containing cells - DCw anthocyanin-free cells     

Rapid induction of phenylalanine ammonia-lyase and chalcone synthase mRNAs during fungus infection of soybean (Glycine max L.) roots or elicitor treatment of soybean cell cultures at the onset of phytoalexin synthesis

The differential regulation of the activities and amounts of mRNAs for two enzymes involved in isoflavonoid phytoalexin biosynthesis in soybean was studied during the early stages after inoculation of primary roots with zoospores from either race 1 (incompatible, host resistant) or race 3 (compatible, host susceptible) of Phytophthora megasperma f.sp. glycinea, the causal fungus of root rot disease. In the incompatible interaction, cloned cDNAs were used to demonstrate that the amounts of phenylalanine ammonia-lyase and chalcone synthase mRNAs increased rapidly at the time of penetration of fungal germ tubes into epidermal cell layers (1–2 h after inoculation) concomitant with the onset of phytoalxxin accumulation; highest levels were reached after about 7 h. In the compatible interaction, only a slight early enhancement of mRNA levels was found and no further increase occurred until about 9 h after inoculation. The time course for changes in the activity of chalcone synthase mRNA also showed major differences between the incompatible and compatible interaction. The observed kinetics for the stimulation of mRNA expression related to phytoalexin synthesis in soybean roots lends further support to the hypothesis that phytoalexin production is an early defense response in the incompatible plant-fungus interaction. The kinetics for the enhancement of mRNA expression after treatment of soybean cell suspension cultures with a glucan elicitor derived from P. megasperma cell walls was similar to that measured during the early stages of the resistant response of soybean roots.Abbreviations cDNA copy DNA - CHS chalcone synthase - PAL phenylalanine ammonia-lyase     

Pisatin metabolism in pea (Pisum sativum L.) cell suspension cultures

Cell suspension cultures were established from germinating pea (Pisum sativum L.) seeds. This cell culture, which accumulated pisatin, consisted mostly of single cells containing a few cell aggregates. The cells responded to treatment with a yeast glucan preparation with transient accumulation of pisatin in both cells and culture media. Addition of pisatin to cell cultures resulted in increased synthesis of pisatin. Phenylalanine ammonia-lyase, chalcone synthase and isoflavone reductase activities were present in untreated cells. Upon treatment with an elicitor preparation the activities of the first two enzymes showed a rapid, transient increase up to 20 hours after treatment. Isoflavone reductase showed a major and minor peak at 16 and 36 h, respectively, after elicitor treatment. The time course of the enzyme activity and pisatin accumulation is consistent with an elicitor-mediated response.Abbreviations CHS chalcone synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - IFR isoflavone reductase - 2iP 6-(dimethylallylamino)-purine - MS Murashige & Skoog basal salt medium - PAL phenylalanine ammonia-lyase - PMSF phenylmethylsulfonyl fluoride - POPOP 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene - PPO 2,5-diphenyloxazole     

Involvement of phenylalanine ammonia-lyase in the development of pollen in broccoli (Brassica oleracea L.)

Summary To determine whether phenylalanine ammonia-lyase (EC 4.3.1.5) is involved in the maturation of microspores to fertile pollen, anthers of a fertile strain of broccoli (Brassica oleracea L.) were studied in a comparison with anthers of a cytoplasmic male sterile strain. In the normal fertile strain, immature anthers of about 2 mm in length exhibited higher phenylalanine ammonia-lyase activity than mature anthers or those shorter than 2 mm. The 2-mm-long anthers corresponded to the mononucleate stage, just after release of the microspores during pollen development. Immunohistochemical localization of phenylalanine ammonia-lyase in the anthers indicated that the protein was present predominantly in the tapetal cells. The immature anthers of cytoplasmic male sterile broccoli had a lower phenylalanine ammonia-lyase activity than those of the normal fertile strain. The level of phenylalanine ammonia-lyase activity in the immature anthers was positively correlated with the number of fertile pollen grains at the flowering stage in both strains. It seems possible, therefore, that phenylpropanoid metabolism, which involves phenylalanine ammonia-lyase, may play an important role in the maturation of microspores in flowering plants.Abbreviations CHS chalcone synthase - CMS cytoplasmic male sterility - DAPI 4, 6-diamidmo-2-phenylindole dihydrochloride - PAL L-phenylalanine ammonia-lyase     

Regulation of isoflavonoid metabolism in alfalfa

Isoflavonoids are believed to play important roles in plant-microbe interactions. During infection of alfalfa (Medicago sativa) leaves with the fungal pathogen Phoma medicaginis, rapid increases in mRNA levels and enzyme activities of isoflavone reductase, phenylalanine ammonia-lyase, chalcone synthase and other defense genes are observed within 1 to 2 hours. The phytoalexin medicarpin and its antifungal metabolite sativan increase beginning at 4 and 8 hours, respectively, along with other isoflavonoids. In contrast, during colonization of alfalfa roots by the symbiotic mycorrhizal fungus Glomus versiforme, expression of the general phenylpropanoid and flavonoid genes phenylalanine ammonia-lyase and chalcone synthase increases while mRNA levels for the phytoalexin-specific isoflavone reductase decrease. The total isoflavonoid content of colonized roots increases with time and is higher than that of uninoculated roots, but the accumulation of the antifungal medicarpin is somehow suppressed.An isoflavone reductase genomic clone has been isolated, promoter regions have been fused to the reporter gene -glucuronidase, and the promoter-reporter fusions have been transformed into tobacco and alfalfa. Using histological staining, we have studied the developmental and stress-induced expression of this phytoalexin-specific gene in whole plants at a more detailed level than other methods allow. The isoflavone reductase promoter is functional in tobacco, a plant which does not synthesize isoflavonoids. Infection of transgenic alfalfa plants by Phoma causes an increase in -glucuronidase staining, as does elicitation of transgenic alfalfa cell cultures, indicating that this promoter fusion is a good indicator of phytoalexin biosynthesis in alfalfa.Abbreviations CA4H cinnamic acid 4-hydroxylase - CHI chalcone isomerase - CHOMT chalcone O-methyltransferase - CHS chalcone synthase - 4CL 4-coumarate:CoA ligase - COMT caffeic acid O-methyltransferase - FGM malonylated glucoside of formononetin - GUS -glucuronidase - IFOH isoflavone 2-hydroxylase - IFR isoflavone reductase - IFS isoflavone synthase - IOMT isoflavone 4-O-methyltransferase - MGM medicarpin 3-O-glucoside-6-O-malonate - PAL L-phenylalanine ammonia-lyase - PTS pterocarpan synthase - VAM vesicular arbuscular mycorrhizal - X-gluc 5-bromo-4-chloro-3-indolyl--D-glucuronide     

Elicitor induction of furanocoumarin biosynthetic pathway in cell cultures ofRuta graveolens

Treatment with an autoclaved culture homogenate of the yeastRhodotorula rubra induces rapid accumulation of acridone epoxides, furoquinolines and furanocoumarins in cell cultures ofRuta graveolens (L). The increased accumulation is preceeded by an induction of enzymes of the biosynthetic pathways. In the case of furanocoumarins induction was shown for phenylalanine ammonia-lyase (PAL), 4-coumarate: CoA ligase (4-CL) and S-adenosyl-l-methionine: xanthotoxol O-methyltransferase (XOMT). For PAL and 4-CL time courses of induced activity showed an early maximum, 8–12 h after treatment, whereas XOMT was found to reach its maximum later, about 36–42 h after treatment. The elicitor dose-response curve showed saturation at an elicitor concentration of 1%. At any time during the whole culturing period cells responded to elicitiation but the maximum enzyme activities induced were lower at the late stages. Experiments with different suspension culture strains, a shoot teratoma culture and hydroponically grown sterile photomixotrophic plants were performed to assess the influence of differentiation on constitutive activities of these enzymes and their inducibility by elicitation. Constitutive furanocoumarin accumulation was positively correlated with the level of differentiation. Although induction of PAL, 4-CL and XOMT activity always accompanied induced furanocoumarin accumulation no absolute correlation existed between induced enzyme activities and the induced product level or relative product increase.Abbreviations 4-CL 4-coumarate:CoA ligase - COMT S-adenosyl-l-methionine:caffeic acid 3-O-methyltransferase - PAL phenylalanine:ammonia-lyase - XOMT S-adenosyl-l-methionine:xanthotoxol O-methyltransferase     

Expression of transgenic stilbene synthases in wheat causes the accumulation of unknown stilbene derivatives with antifungal activity

The expression of foreign phytoalexins in a new host is thought to increase fungal resistance, since host-specific pathogens have not experienced selection for detoxifying or metabolising the novel antifungal compounds. Two resveratrol synthase genes vst1 and vst2 from grapevine (Vitis vinifera L.) and the pinosylvin synthase gene pss from pine (Pinus sylvestris L.) were stably transformed into bread wheat. The expression of the target genes is regulated by stress-inducible grapevine promoters. The vst1 and vst2 promoters were functional in wheat and retained their expression profiles described for grapevine. ALL vst and pss transgenic lines accumulated stilbene derivatives upon induction by UV light. The detected stilbenes showed a remarkable similarity to resveratrol and pinosylvin, however were found to be more hydrophilic than resveratrol and pinosylvin. Upon inoculation with the biotrophic pathogen Puccinia recondita f.sp. tritici several vst expressing wheat lines showed a significant reduction of disease symptoms (19 +/- 9% to 27 +/- 8%) compared to wild-type plants. The reduction of disease symptoms was even more obvious after inoculation with the facultative biotrophic pathogen Septoria nodorum Berk. and ranged from 42 +/- 13% to 71 +/- 4%. None of the four tested pss expressing lines showed a reduction in disease incidence.