Characterization of a pine multigene family containing elicitor- responsive stilbene synthase genes

Young pine seedlings respond to environmental stress by induced synthesis of pinosylvin, a stilbene phytoalexin. Heartwood of pine trees is characterized by a high content of pinosylvin. The formation of pinosylvin from cinnamoyl-CoA and three molecules malonyl-CoA catalysed by pinosylvin synthase is typical of the genus Pinus. Its enzyme activity not detectable in unstressed seedlings is substantially increased upon application of stimuli like UV-light or infection with the phytopathogenic fungus Botrytis cinerea. A genomic DNA library was screened with pinosylvin synthase cDNA pSP-54 as a probe. Ten clones were isolated and grouped into five subclasses according to the size of their introns. After subcloning into plasmid T7T3, four different members of the five gene subclasses were characterized by sequencing. Emphasis was put on isolating various promoters and analyzing and comparing their responsiveness. The amino acid sequences deduced from genes PST-1, PST-2, PST-3 and PST-5 shared an overall identity of more than 95%. In gene PST-5, the putative translation start site ATG was replaced by CTG. While promoter regions near the TATAA box were almost identical PST-1, PST-2 and PST-3, further upstream sequences differed substantially. Differences in promoter strength were analysed both in transgenic tobacco plants and by transient expression in tobacco protoplasts. Constructs used contained the bacterial -glucuronidase under the control of the promoters of pine genes PST-1, PST-2 and PST-3. Upon treatment with UV light or fungal elicitor, the promoter of PST-1 showed highest responsiveness and led to tissue-specific expression in vascular bundles. The data suggest that in pine the gene product of PST-1 is responsible for both the stress response in seedlings and pinosylvin formation in the heartwood.     

Metabolic engineering of Saccharomyces cerevisiae for the synthesis of the wine-related antioxidant resveratrol

The stilbene resveratrol is a stress metabolite produced by Vitis vinifera grapevines during fungal infection, wounding or UV radiation. Resveratrol is synthesised particularly in the skins of grape berries and only trace amounts are present in the fruit flesh. Red wine contains a much higher resveratrol concentration than white wine, due to skin contact during fermentation. Apart from its antifungal characteristics, resveratrol has also been shown to have cancer chemopreventive activity and to reduce the risk of coronary heart disease. It acts as an antioxidant and anti-mutagen and has the ability to induce specific enzymes that metabolise carcinogenic substances. The objective of this pilot study was to investigate the feasibility of developing wine yeasts with the ability to produce resveratrol during fermentation in both red and white wines, thereby increasing the wholesomeness of the product. To achieve this goal, the phenylpropanoid pathway in Saccharomyces cerevisiae would have to be introduced to produce p-coumaroyl-CoA, one of the substrates required for resveratrol synthesis. The other substrate for resveratrol synthase, malonyl-CoA, is already found in yeast and is involved in de novo fatty-acid biosynthesis. We hypothesised that production of p-coumaroyl-CoA and resveratrol can be achieved by co-expressing the coenzyme-A ligase-encoding gene (4CL216) from a hybrid poplar and the grapevine resveratrol synthase gene (vst1) in laboratory strains of S. cerevisiae. This yeast has the ability to metabolise p-coumaric acid, a substance already present in grape must. This compound was therefore added to the synthetic media used for the growth of laboratory cultures. Transformants expressing both the 4CL216 and vst1 genes were obtained and tested for production of resveratrol. Following beta-glucosidase treatment of organic extracts for removal of glucose moieties that are typically bound to resveratrol, the results showed that the yeast transformants had produced the resveratrol beta-glucoside, piceid. This is the first report of the reconstruction of a biochemical pathway in a heterologous host to produce resveratrol.     

The wheat Lr34 multipathogen resistance gene confers resistance to anthracnose and rust in sorghum

The ability of the wheat Lr34 multipathogen resistance gene (Lr34res) to function across a wide taxonomic boundary was investigated in transgenic Sorghum bicolor. Increased resistance to sorghum rust and anthracnose disease symptoms following infection with the biotrophic pathogen Puccinia purpurea and the hemibiotroph Colletotrichum sublineolum, respectively, occurred in transgenic plants expressing the Lr34res ABC transporter. Transgenic sorghum lines that highly expressed the wheat Lr34res gene exhibited immunity to sorghum rust compared to the low‐expressing single copy Lr34res genotype that conferred partial resistance. Pathogen‐induced pigmentation mediated by flavonoid phytoalexins was evident on transgenic sorghum leaves following P. purpurea infection within 24–72 h, which paralleled Lr34res gene expression. Elevated expression of flavone synthase II, flavanone 4‐reductase and dihydroflavonol reductase genes which control the biosynthesis of flavonoid phytoalexins characterized the highly expressing Lr34res transgenic lines 24‐h post‐inoculation with P. purpurea. Metabolite analysis of mesocotyls infected with C. sublineolum showed increased levels of 3‐deoxyanthocyanidin metabolites were associated with Lr34res expression, concomitant with reduced symptoms of anthracnose.     

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.     

In vitro tolerance to Botrytis cinerea of grapevine 41B rootstock in transgenic plants expressing the stilbene synthase Vst1 gene under the control of a pathogen-inducible PR 10 promoter.

Resveratrol is a major phytoalexin in grapevine but its synthesis in response to phytopathogen attack decreases with grape berry ripening. A chimeric gene combining an alfalfa PR 10 promoter and Vst1 (Vitis stilbene synthase 1) gene was introduced into the genome of 41B rootstock. Transgenic plants were analysed for resveratrol production in leaves infected with Botrytis using an in vitro test. Among the 50 transgenic lines analysed, some exhibited a production lower than the non-transgenic control, but others accumulated resveratrol from 5-100-fold. Moreover, in the latter clones, symptoms were highly reduced in response to infection. These results were a good indication that the combination of a pathogen-inducible promoter and a defence gene may increase tolerance against fungi in grapevine. The efficacy of this approach should be further tested by experiments conducted in the vineyard.     

Expression pattern, genomic structure, and promoter analysis of the gene encoding stilbene synthase from Chinese wild Vitis pseudoreticulata

The gene encoding stilbene synthase (STS) plays a central role in many biochemical and physiological actions, and its metabolite resveratrol possesses broad-spectrum resistance to pathogens, as well as diverse pharmacological properties, notably an anticancer effect. Here, we report the expression analysis of the gene encoding STS and its promoter function from a powdery mildew (PM)-resistant Chinese wild Vitis pseudoreticulata, and compare it with two PM-susceptible cultivated grapevines, Vitis vinifera cvs. Carignane and Thompson Seedless. We show an unusual expression pattern of STS in V. pseudoreticulata, which differs markedly from that of the cultivated species. Sequence comparisons reveal that the genomic DNA sequences encoding STS in the three grapevines are highly conserved, but a novel residue mutation within the key motif of STS is solely present in V. pseudoreticulata. Moreover, the STS promoter in V. pseudoreticulata displays a significantly different structure from that found in the two V. vinifera. The three promoter-driven GUS differential expression patterns in transformed tobacco plants induced with Alternaria alternata, methyl jasmonate, and wounding indicated that the structurally different STS promoter of V. pseudoreticulata is responsible for its specific regulatory function. We also demonstrate that the expression of STS genes from their native promoters are functional in transformed tobacco and retain pathogen inducibility. Importantly, the genomic DNA-2 of V. pseudoreticulata under its native promoter shows good induction and the maximum level of resveratrol content. These findings further our understanding of the regulation of STS expression in a resistant grapevine and provide a new pathogen-inducible promoter system for the genetic improvement of plant disease resistance.     

Extracellular invertase is involved in the regulation of clubroot disease in Arabidopsis thaliana

Clubroot disease of Brassicaceae is caused by an obligate biotrophic protist, Plasmodiophora brassicae. During root gall development, a strong sink for assimilates is developed. Among other genes involved in sucrose and starch synthesis and degradation, the increased expression of invertases has been observed in a microarray experiment, and invertase and invertase inhibitor expression was confirmed using promoter::GUS lines of Arabidopsis thaliana. A functional approach demonstrates that invertases are important for gall development. Different transgenic lines expressing an invertase inhibitor under the control of two root-specific promoters, Pyk10 and CrypticT80, which results in the reduction of invertase activity, showed clearly reduced clubroot symptoms in root tissue with highest promoter expression, whereas hypocotyl galls developed normally. These results present the first evidence that invertases are important factors during gall development, most probably in supplying sugars to the pathogen. In addition, root-specific repression of invertase activity could be used as a tool to reduce clubroot symptoms.     

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.     

Co-expression of VpROMT gene from Chinese wild Vitis pseudoreticulata with VpSTS in tobacco plants and its effects on the accumulation of pterostilbene

Plant secondary metabolites, such as stilbenes, have fungicidal potential and have been found in several plant species. Stilbenes in grapevine, such as resveratrol and pterostilbene, have recently attracted much attention, they are not only helping the plant to fight against pathogen attack, but they are also being widely used as ingredients of fungicide, anti-inflammatory drugs, antioxidant, and anti-infective agents. However, resveratrol O-methyltransferase gene, related with the synthesis of pterostilbene from resveratrol, has not been characterized effectively from Chinese wild Vitis pseudoreticulata. In this study, a candidate of resveratrol O-methyltransferase gene designated as VpROMT was isolated from a powdery mildew-resistant Chinese wild V. pseudoreticulata 'Baihe-35-1', and characterization studies were performed. Expression studies showed that VpROMT was predominantly expressed in developing roots yet not found in the leaves, stems, nor tendrils when the plants are not challenged. Results of qRT-PCR showed that VpROMT was rapidly induced by Erysiphe necator in V. pseudoreticulata and by methyl-jasmonate, UV-irradiation in suspension culture cells of Vitis romanetii. The expression level varies in different tissues of grapevine, which MeJA and UV-C treatment significantly upregulated the expression of VpROMT gene while UV-B treatment failed to. Co-expression of VpROMT and grapevine stilbene synthase (VpSTS) gene leads to the accumulation of pterostilbene in leaves of tobacco (Nicotiana tabacum) indicating that VpROMT was able to catalyze the biosynthesis of pterostilbene from resveratrol in over-expression transgenic tobacco plants.     

Elicitor-induced formation of free and cell-wall-bound stilbenes in cell-suspension cultures of Scots pine (Pinus sylvestris L.)

Treatment of Pinus sylvestris L. cell-suspension cultures with an elicitor preparation from the pine needle pathogen Lophodermium seditiosum, resulted in a severalhundredto thousandfold accumulation of the stilbenes pinosylvin and pinosylvin 3-O-methyl ether in methanolic cell extracts. There was a simultaneous induction of the biosynthetic enzymes phenylalanine ammonia-lyase (E.C. 4.3.1.5.) and stilbene synthase (pinosylvin-forming, E.C. 2.3.1.146). For the first time, an incorporation of stilbenes into the cell wall fraction as well as stilbene excretion into the extracellular space was demonstrated in addition to intracellular accumulation.Abbreviations PAL phenylalanine ammonia-lyase - PS pinosylvin - PSM pinosylvin 3-O-methyl ether - STS stilbene synthase (pinosylvin-forming) This study has been supported by the Bayerisches Staatsministerium für Landesentwicklung und Umweltfragen, Fonds der chemischen Industrie, and EUROSILVA. The authors wish to thank their colleagues L. Gößl for maintaining the Scots pine cell-suspension culture, and Dr. G. Bahnweg for supplying the Lophodermium mycelia.     

PHOSPHATIDYLSERINE SYNTHASE1 is required for microspore development in Arabidopsis thaliana

Phosphatidylserine (PS) has many important biological roles, but little is known about its role in plants, partly because of its low abundance. We show here that PS is enriched in Arabidopsis floral tissues and that genetic disruption of PS biosynthesis decreased heterozygote fertility due to inhibition of pollen maturation. At1g15110, designated PSS1, encodes a base-exchange-type PS synthase. Escherichia coli cells expressing PSS1 accumulated PS in the presence of l-serine at 23°C. Promoter-GUS assays showed PSS1 expression in developing anther pollen and tapetum. A few seeds with pss1-1 and pss1-2 knockout alleles escaped embryonic lethality but developed into sterile dwarf mutant plants. These plants contained no PS, verifying that PSS1 is essential for PS biosynthesis. Reciprocal crossing revealed reduced pss1 transmission via male gametophytes, predicting a rate of 61.6%pss1-1 pollen defects in PSS1/pss1-1 plants. Alexander's staining of inseparable qrt1-1 PSS1/pss1-1 quartets revealed a rate of 42% having three or four dead pollen grains, suggesting sporophytic pss1-1 cell death effects. Analysis with the nuclear stain 4',6-diamidino-2-phenylindole (DAPI) showed that all tetrads from PSS1/pss1-1 anthers retain their nuclei, whereas unicellular microspores were sometimes anucleate. Transgenic Arabidopsis expressing a GFP-LactC2 construct that binds PS revealed vesicular staining in tetrads and bicellular microspores and nuclear membrane staining in unicellular microspores. Hence, distribution and/or transport of PS across membranes were dynamically regulated in pollen microspores. However, among unicellular microspores from PSS1/pss1-2 GFP-LactC2 plants, all anucleate microspores showed little GFP-LactC2 fluorescence, suggesting that pss1-2 microspores are more sensitive to sporophytic defects or show partial gametophytic defects.     

In vitro inhibition of Sphaeropsis sapinea by natural stilbenes

The effects of pinosylvin, pinosylvin monomethyl ether, pinosylvin dimethyl ether, and resveratrol on the fungal shoot blight and canker pathogen of conifers Sphaeropsis sapinea were examined in vitro. Effects of compounds, isolates, and concentrations on both conidial germination and mycelial growth were significant (values of P < 0.001), indicating inhibitory activity of these compounds.     

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is a biotrophic fungal pathogen and the causal agent of corn smut on maize. This disease is responsible for significant yield losses of approximately $1.0 billion annually in the U.S.¹ Several methods including crop rotation, fungicide application and seed treatments are currently used to control corn smut². However, host resistance is the only practical method for managing corn smut. Identification of crop plants including maize, wheat, and rice that are resistant to various biotrophic pathogens has significantly decreased yield losses annually^3-5. Therefore, the use of a pathogen inoculation method that efficiently and reproducibly delivers the pathogen in between the plant leaves, would facilitate the rapid identification of maize lines that are resistant to U. maydis. As, a first step toward indentifying maize lines that are resistant to U. maydis, a needle injection inoculation method and a resistance reaction screening method was utilized to inoculate maize, teosinte, and maize x teosinte introgression lines with a U. maydis strain and to select resistant plants.Maize, teosinte and maize x teosinte introgression lines, consisting of about 700 plants, were planted, inoculated with a strain of U. maydis, and screened for resistance. The inoculation and screening methods successfully identified three teosinte lines resistant to U. maydis. Here a detailed needle injection inoculation and resistance reaction screening protocol for maize, teosinte, and maize x teosinte introgression lines is presented. This study demonstrates that needle injection inoculation is an invaluable tool in agriculture that can efficiently deliver U. maydis in between the plant leaves and has provided plant lines that are resistant to U. maydis that can now be combined and tested in breeding programs for improved disease resistance.     

A stress-inducible resveratrol O-methyltransferase involved in the biosynthesis of pterostilbene in grapevine

Stilbenes are considered the most important phytoalexin group in grapevine (Vitis vinifera) and they are known to contribute to the protection against various pathogens. The main stilbenes in grapevine are resveratrol and its derivatives and, among these, pterostilbene has recently attracted much attention due both to its antifungal and pharmacological properties. Indeed, pterostilbene is 5 to 10 times more fungitoxic than resveratrol in vitro and recent studies have shown that pterostilbene exhibits anticancer, hypolipidemic, and antidiabetic properties. A candidate gene approach was used to identify a grapevine resveratrol O-methyltransferase (ROMT) cDNA and the activity of the corresponding protein was characterized after expression in Escherichia coli. Transient coexpression of ROMT and grapevine stilbene synthase in tobacco (Nicotiana benthamiana) using the agroinfiltration technique resulted in the accumulation of pterostilbene in tobacco tissues. Taken together, these results showed that ROMT was able to catalyze the biosynthesis of pterostilbene from resveratrol both in vitro and in planta. ROMT gene expression in grapevine leaves was induced by different stresses, including downy mildew (Plasmopara viticola) infection, ultraviolet light, and AlCl(3) treatment.     

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     

Genetic transformation of a fruit-specific,highly expressed stilbene synthase gene from Chinese wild Vitis quinquangularis

Planta - The stilbene synthase gene VqSTS6, from Chinese wild type Vitis quinquangularis accession Danfeng-2, increases the resveratrol content and pathogen resistance of transgenic plants of V....     

核基质结合区对转爬山虎芪合酶基因烟草中产生白藜芦醇的作用

采用核基质结合区(MARs)来提高转芪合酶基因(STS)烟草(Nicotianatabacum L.)中白藜芦醇产物的含量.MARs是细胞中能与核基质特异紧密结合的DNA片段,体外结合实验表明克隆自酵母的MARs序列能特异地与烟草核基质结合.芪合酶是白藜芦醇生物合成中的关键酶,用RT-PCR方法从川鄂爬山虎(Parthenocissus henryana(Hemsl.)Diels et Gilg)中克隆了与葡萄芪合酶基因有较高同源性的芪合酶编码区,将其置于CaMV35SΩ强启动子下,分别构建两侧带有MARs及不含MARs序列的表达载体,通过农杆菌介导转化烟草.Northern blot及HPLC等分析表明STS基因已整合至烟草染色体中并正常转录,且表达的外源芪合酶在烟草中可催化其底物合成白藜芦醇产物.与对照相比,MARs的存在使转芪合酶基因烟草中白藜芦醇的含量平均提高了约一倍.MARs在转芪合酶基因植物中的应用也为获得抗病性更强、白藜芦醇含量更高、更保健的转基因果蔬的研究奠定了基础.