Comparative Bioremediation of Tetradecane, Cyclohexanone and Cyclohexane by Filamentous Fungi from Polluted Habitats in Kazakhstan.

Studying the fates of oil components and their interactions with ecological systems is essential for developing comprehensive management strategies and enhancing restoration following oil spill incidents. The potential expansion of Kazakhstan's role in the global oil market necessitates the existence of land-specific studies that contribute to the field of bioremediation. In this study, a set of experiments was designed to assess the growth and biodegradation capacities of eight fungal strains sourced from Kazakhstan soil when exposed to the hydrocarbon substrates from which they were initially isolated. The strains were identified as Aspergillus sp. SBUG-M1743, Penicillium javanicum SBUG-M1744, SBUG-M1770, Trichoderma harzianum SBUG-M1750 and Fusarium oxysporum SBUG-1746, SBUG-M1748, SBUG-M1768 and SBUG-M1769 using the internal transcribed spacer (ITS) region. Furthermore, microscopic and macroscopic evaluations agreed with the sequence-based identification. Aspergillus sp. SBUG-M1743 and P. javanicum SBUG-M1744 displayed remarkable biodegradation capabilities in the presence of tetradecane with up to a 9-fold biomass increase in the static cultures. T. harzianum SBUG-M1750 exhibited poor growth, which was a consequence of its low efficiency of tetradecane degradation. Monocarboxylic acids were the main degradation products by SBUG-M1743, SBUG-M1744, SBUG-M1750, and SBUG-M1770 indicating the monoterminal degradation pathway through ß-oxidation, while the additional detection of dicarboxylic acid in SBUG-M1768 and SBUG-M1769 cultures was indicative of the fungus' ability to undertake both monoterminal and diterminal degradation pathways. F. oxysporum SBUG-M1746 and SBUG-M1748 in the presence of cyclohexanone showed a doubling of the biomass with the ability to degrade the substrate almost completely in shake cultures. F. oxysporum SBUG-M1746 was also able to degrade cyclohexane completely and excreted all possible metabolites of the degradation pathway. Understanding the degradation potential of these fungal isolates to different hydrocarbon substrates will help in developing effective bioremediation strategies tailored to local conditions.

Characterization of the Mycoremediation of n-Alkanes and Branched-Chain Alkanes by Filamentous Fungi from Oil-Polluted Soil Samples in Kazakhstan.

For decades, researchers have focused on containing terrestrial oil pollution. The heterogeneity of soils, with immense microbial diversity, inspires them to transform pollutants and find cost-effective bioremediation methods. In this study, the mycoremediation potentials of five filamentous fungi isolated from polluted soils in Kazakhstan were investigated for their degradability of n-alkanes and branched-chain alkanes as sole carbon and energy sources. Dry weight estimation and gas chromatography-mass spectrometry (GC-MS) monitored the growth and the changes in the metabolic profile during degradation, respectively. Penicillium javanicum SBUG-M1741 and SBUG-M1742 oxidized medium-chain alkanes almost completely through mono- and di-terminal degradation. Pristane degradation by P. javanicum SBUG-M1741 was >95%, while its degradation with Purpureocillium lilacinum SBUG-M1751 was >90%. P. lilacinum SBUG-M1751 also exhibited the visible degradation potential of tetradecane and phytane, whereby in the transformation of phytane, both the mono- and di-terminal degradation pathways as well as α- and ß-oxidation steps could be described. Scedosporium boydii SBUG-M1749 used both mono- and di-terminal degradation pathways for n-alkanes, but with poor growth. Degradation of pristane by Fusarium oxysporum SBUG-M1747 followed the di-terminal oxidation mechanism, resulting in one dicarboxylic acid. These findings highlight the role of filamentous fungi in containing oil pollution and suggest possible degradation pathways.

Novel Drugs Obtained via Biotransformation-In Memory of the Late Scientists Frieder Schauer and Peter Grunwald.

The development of novel drugs is a complex process that requires cost-effective and sustainable techniques [...].

Cytosolic aromatic aldehyde dehydrogenase provides benzoic acid for xanthone biosynthesis in Hypericum.

Benzoic acid is a building block of a multitude of well-known plant natural products, such as paclitaxel and cocaine. Its simple chemical structure contrasts with its complex biosynthesis. Hypericum species are rich in polyprenylated benzoic acid-derived xanthones, which have received attention due to their biological impact on human health. The upstream biosynthetic sequence leading to xanthones is still incomplete. To supply benzoic acid for xanthone biosynthesis, Hypericum calycinum cell cultures use the CoA-dependent non-ß-oxidative pathway, which starts with peroxisomal cinnamate CoA-ligase (HcCNL). Here, we use the xanthone-producing cell cultures to identify the transcript for benzaldehyde dehydrogenase (HcBD), a pivotal player in the non-ß-oxidative pathways. In addition to benzaldehyde, the enzyme efficiently catalyzes the oxidation of trans-cinnamaldehyde in vitro. The enzymatic activity is strictly dependent on the presence of NAD+ as co-factor. HcBD is localized to the cytosol upon ectopic expression of reporter fusion constructs. HcBD oxidizes benzaldehyde, which moves across the peroxisome membrane, to form benzoic acid. Increases in the HcCNL and HcBD transcript levels precede the elicitor-induced xanthone accumulation. The current work addresses a crucial step in the yet incompletely understood CoA-dependent non-ß-oxidative route of benzoic acid biosynthesis. Addressing this step may offer a new biotechnological tool to enhance product formation in biofactories.

A promiscuous coenzyme A ligase provides benzoyl-coenzyme A for xanthone biosynthesis in Hypericum.

Benzoic acid-derived compounds, such as polyprenylated benzophenones and xanthones, attract the interest of scientists due to challenging chemical structures and diverse biological activities. The genus Hypericum is of high medicinal value, as exemplified by H. perforatum. It is rich in benzophenone and xanthone derivatives, the biosynthesis of which requires the catalytic activity of benzoate-coenzyme A (benzoate-CoA) ligase (BZL), which activates benzoic acid to benzoyl-CoA. Despite remarkable research so far done on benzoic acid biosynthesis in planta, all previous structural studies of BZL genes and proteins are exclusively related to benzoate-degrading microorganisms. Here, a transcript for a plant acyl-activating enzyme (AAE) was cloned from xanthone-producing Hypericum calycinum cell cultures using transcriptomic resources. An increase in the HcAAE1 transcript level preceded xanthone accumulation after elicitor treatment, as previously observed with other pathway-related genes. Subcellular localization of reporter fusions revealed the dual localization of HcAAE1 to cytosol and peroxisomes owing to a type 2 peroxisomal targeting signal. This result suggests the generation of benzoyl-CoA in Hypericum by the CoA-dependent non-ß-oxidative route. A luciferase-based substrate specificity assay and the kinetic characterization indicated that HcAAE1 exhibits promiscuous substrate preference, with benzoic acid being the sole aromatic substrate accepted. Unlike 4-coumarate-CoA ligase and cinnamate-CoA ligase enzymes, HcAAE1 did not accept 4-coumaric and cinnamic acids, respectively. The substrate preference was corroborated by in silico modeling, which indicated valid docking of both benzoic acid and its adenosine monophosphate intermediate in the HcAAE1/BZL active site cavity.

Benzoate-CoA ligase contributes to the biosynthesis of biphenyl phytoalexins in elicitor-treated pear cell cultures.

KEY MESSAGE: Benzoate-Coenzyme A ligase enzyme activity catalyzing the conversion of free benzoic acid to benzoyl-CoA was detected and biochemically characterized in the elicitor-treated pear cell cultures. Asian pear (Pyrus pyrifolia) is an economically and nutritionally important fruit-bearing tree of the subtribe Malinae. Upon pathogen attack, pears produce unique benzoate-derived biphenyl phytoalexins. The upstream biosynthesis of the biphenyl in Malinae is still incomplete. Previously, protein preparations from yeast extract-treated pear cultures were able to convert L-phenylalanine to cinnamic acid catalyzed by the activity of the phenylalanine ammonia lyase. The same extract was able to perform a C2 side-chain cleavage of cinnamic acid to benzaldehyde followed by oxidation of the latter to benzoic acid owing to the molecularly-undefined benzaldehyde synthase and benzaldehyde dehydrogenase activities, respectively. The biosynthesis of biphenyls starts with benzoate-Coenzyme A ligase (BZL), which converts benzoic acid to benzoyl-CoA. Subsequently, the previously-defined biphenyl synthase uses benzoyl-CoA to form the biphenyls. The current study reports the first time detection and characterization of BZL activity in elicitor-treated pear cell cultures. The preferred substrate was benzoic acid (Km = 62 ± 4 µM). Magnesium or manganese was prerequisite for the activity, which was enhanced by ~ 70% in the presence of potassium. Maximum BZL activity was observed 18 h post elicitation, which is in agreement with the coordinate induction reported for the enzymes in the same pathway. The induced BZL activity preceded the accumulation of biphenyls supporting its involvement in their biosynthesis.

Cinnamate-CoA ligase is involved in biosynthesis of benzoate-derived biphenyl phytoalexin in Malus × domestica 'Golden Delicious' cell cultures.

Apple (Malus sp.) and other genera belonging to the sub-tribe Malinae of the Rosaceae family produce unique benzoic acid-derived biphenyl phytoalexins. Cell cultures of Malus domestica cv. 'Golden Delicious' accumulate two biphenyl phytoalexins, aucuparin and noraucuparin, in response to the addition of a Venturia inaequalis elicitor (VIE). In this study, we isolated and expressed a cinnamate-CoA ligase (CNL)-encoding sequence from VIE-treated cell cultures of cv. 'Golden Delicious' (M. domestica CNL; MdCNL). MdCNL catalyses the conversion of cinnamic acid into cinnamoyl-CoA, which is subsequently converted to biphenyls. MdCNL failed to accept benzoic acid as a substrate. When scab-resistant (cv. 'Shireen') and moderately scab-susceptible (cv. 'Golden Delicious') apple cultivars were challenged with the V. inaequalis scab fungus, an increase in MdCNL transcript levels was observed in internodal regions. The increase in MdCNL transcript levels could conceivably correlate with the pattern of accumulation of biphenyls. The C-terminal signal in the MdCNL protein directed its N-terminal reporter fusion to peroxisomes in Nicotiana benthamiana leaves. Thus, this report records the cloning and characterisation of a cinnamoyl-CoA-forming enzyme from apple via a series of in vivo and in vitro studies. Defining the key step of phytoalexin formation in apple provides a biotechnological tool for engineering elite cultivars with improved resistance.

Toward enhanced hyperforin production in St. John's wort root cultures.

During the past decades, several trials targeted a stable, sustainable and economic production of St. John's wort (Hypericum perforatum) extract. The value of this extract stems from its use to treat depression and skin irritation due to its hyperforin content. Previously, hyperforin-forming in vitro root cultures were established. Here, detailed growth and production kinetics have been analyzed over 40 days of cultivation. In the first 10 days, sucrose was completely hydrolyzed to glucose and fructose. The ammonium consumption supported the increase in the biomass and hyperforin production. When sucrose was replaced with glucose/fructose, the linear growth phase started 6 days earlier and resulted in a higher space-time-yield. The maximum hyperforin production was 0.82 mg L-1 day-1, which was 67 % higher than in the sucrose-supplemented standard cultivation. Buffering the sucrose-supplemented medium with phosphate caused a 2.7-fold increase in the product to biomass yield coefficient. However, the combination of monosaccharides and buffering conditions did not cause an appreciable improvements in the production performance of the shake flask approaches. A potential scalability from flask to lab-scale stirred bioreactors has been demonstrated. The results obtained offer a basis for a scalable production of hyperforin and a sustainable source for a tissue culture-based phytomedicine.

Sequential regiospecific gem-diprenylation of tetrahydroxyxanthone by prenyltransferases from Hypericum sp.

Polyprenylated acylphloroglucinol derivatives, such as xanthones, are natural plant products with interesting pharmacological properties. They are difficult to synthesize chemically. Biotechnological production is desirable but it requires an understanding of the biosynthetic pathways. cDNAs encoding membrane-bound aromatic prenyltransferase (aPT) enzymes from Hypericum sampsonii seedlings (HsPT8px and HsPTpat) and Hypericum calycinum cell cultures (HcPT8px and HcPTpat) were cloned and expressed in Saccharomyces cerevisiae and Nicotiana benthamiana, respectively. Microsomes and chloroplasts were used for functional analysis. The enzymes catalyzed the prenylation of 1,3,6,7-tetrahydroxyxanthone (1367THX) and/or 1,3,6,7-tetrahydroxy-8-prenylxanthone (8PX) and discriminated nine additionally tested acylphloroglucinol derivatives. The transient expression of the two aPT genes preceded the accumulation of the products in elicitor-treated H. calycinum cell cultures. C-terminal yellow fluorescent protein fusions of the two enzymes were localized to the envelope of chloroplasts in N. benthamiana leaves. Based on the kinetic properties of HsPT8px and HsPTpat, the enzymes catalyze sequential rather than parallel addition of two prenyl groups to the carbon atom 8 of 1367THX, yielding gem-diprenylated patulone under loss of aromaticity of the gem-dialkylated ring. Coexpression in yeast significantly increased product formation. The patulone biosynthetic pathway involves multiple subcellular compartments. The aPTs studied here and related enzymes may be promising tools for plant/microbe metabolic pathway engineering.

A new enzymatic activity from elicitor-treated pear cell cultures converting trans-cinnamic acid to benzaldehyde.

Cell cultures of Asian pear (Pyrus pyrifolia) are known to produce benzoate-derived biphenyl phytoalexins upon elicitor treatment. Although the downstream pathway for biphenyl phytoalexin biosynthesis is almost known, the upstream route of benzoic acid biosynthesis in pear has not been completely elucidated. In the present work, we report benzaldehyde synthase (BS) activity from yeast extract-treated cell suspension cultures of P. pyrifolia. BS catalyzes the in vitro conversion of trans-cinnamic acid to benzaldehyde using a non-oxidative C2 -side chain cleavage mechanism. The enzyme activity was strictly dependent on the presence of a reducing agent, dithiothreitol being preferred. C2 -side chain shortening of the cinnamic acid backbone resembled the mechanisms catalyzed by 4-hydroxybenzaldehyde synthase (HBS) activity in Vanilla planifolia and salicylaldehyde synthase (SAS) activity in tobacco and apple cell cultures. A basal BS activity was also observed in the non-elicited cell cultures. Upon yeast extract-treatment, a 13-fold increase in BS activity was observed when compared to the non-treated control cells. Moreover, feeding of the cell cultures with trans-cinnamic acid, the substrate for BS, resulted in an enhanced level of noraucuparin, a biphenyl phytoalexin. Comparable accumulation of noraucuparin was observed upon feeding of benzaldehyde, the BS product. The preferred substrate for BS was found to be trans-cinnamic acid, for which the apparent Km and Vmax values were 0.5 mM and 50.7 pkat mg-1 protein, respectively. Our observations indicate the contribution of BS to benzoic acid biosynthesis in Asian pear via the CoA-independent and non-ß-oxidative route.

The Petroleum Ether Extract from Hypericum perforatum Root Cultures Exhibits Potent Antiproliferative Activity in Human Keratinocytes and Fibroblasts.

The hyperforin content of Hypericum perforatum herb was repeatedly reported to be responsible for a multitude of pharmacological activities. Our recent report about the hyperforin accumulation in in vitro root cultures of H. perforatum provides an alternative perspective to achieve constant product quality and to serve the rapidly growing market. In this study, the antiproliferative effect of a petroleum ether extract from the in vitro root cultures was investigated. When normalized to 1 µM hyperforin content, the extract reduced the viability of human keratinocytes (HaCaT) and human dermal fibroblast monolayers to 33 and 36%, respectively, after 72 h of incubation. A cytotoxicity assay and live-dead cell staining confirmed that the extract lacked a cytotoxic effect and that the reduction in cell viability was mainly due to the antiproliferative activity. Application of the 1 µM hyperforin-normalized extract to a 3D artificial skin construct significantly reduced the proliferation of HaCaT in the presence of fibroblasts. This effect was proved by the reduction in thickness of the epidermal construct from 100 µm (control) to 17 µm (treated). Notably, 1 µM pure hyperforin lacked effectiveness in both monolayer cultures and 3D artificial skin constructs. Nor were fractions of the extract containing colupulone and xanthones active. The combination of these constituents also failed to reassemble the antiproliferative activity, which indicates a synergistic role of yet unidentified components present in the extract. Our findings may introduce H. perforatum root cultures as a novel lead system for the treatment of hypertrophic scars.

Molecular cloning and functional analysis of a biphenyl phytoalexin-specific O-methyltransferase from apple cell suspension cultures.

MAIN CONCLUSION: This manuscript describes the cloning and functional characterization of a biphenyl phytoalexin biosynthetic gene, 3,5-dihydroxybiphenyl O-methyltransferase from elicitor-treated cell cultures of scab resistant apple cultivar 'Florina'. Apples belong to the subtribe Malinae of the Rosaceae family. Biphenyls and dibenzofurans are the specialized phytoalexins of Malinae, of which aucuparin is the most widely distributed biphenyl. The precursor of aucuparin, 3,5-dihydroxybiphenyl, is a benzoate-derived polyketide, which is formed by the sequential condensation of three molecules of malonyl-CoA and one molecule of benzoyl-CoA in a reaction catalyzed by biphenyl synthase (BIS). This 3,5-dihydroxybiphenyl then undergoes sequential 5-O-methylation, 4-hydroxylation, and finally 3-O-methylation to form aucuparin. A cDNA encoding O-methyltransferase (OMT) was isolated and functionally characterized from the cell cultures of scab-resistant apple cultivar 'Florina' (Malus domestica cultivar 'Florina'; MdOMT) after treatment with elicitor prepared from the apple scab causing fungus Venturia inaequalis. MdOMT catalyzed the regiospecific O-methylation of 3,5-dihydroxybiphenyl at the 5-position to form 3-hydroxy-5-methoxybiphenyl. The enzyme showed absolute substrate preference for 3,5-dihydroxybiphenyl. The elicitor-treated apple cell cultures showed transient increases in the MdOMT (GenBank ID MF740747) and MdBIS3 (GenBank ID JQ390523) transcript levels followed by the accumulation of biphenyls (aucuparin and noraucuparin) and dibenzofuran (eriobofuran) phytoalexins. MdOMT fused with N- and C-terminal yellow fluorescent protein showed cytoplasmic localization in the epidermis of Nicotiana benthamiana leaves. In scab inoculated greenhouse-grown 'Florina' plants, the expression of MdOMT was transiently induced in the stem followed by the accumulation of biphenyl phytoalexins.

Comparative metabolomics of scab-resistant and susceptible apple cell cultures in response to scab fungus elicitor treatment.

Apple scab disease caused by the fungus Venturia inaequalis is a devastating disease that seriously affects quality and yield of apples. In order to understand the mechanisms involved in scab resistance, we performed gas chromatography-mass spectrometry based metabolomics analysis of the cell culture of scab resistant cultivar 'Florina' and scab susceptible cultivar 'Vista Bella' both prior -to and -following treatment with V. inaequalis elicitor (VIE). A total 21 metabolites were identified to be altered significantly in 'Florina' cell cultures upon VIE-treatment. Among 21 metabolites, formation of three new specialized metabolites aucuparin, noraucuparin and eriobofuran were observed only in resistant cultivar 'Florina' after the elicitor treatment. The score plots of principal component analysis (PCA) exhibited clear discrimination between untreated and VIE-treated samples. The alteration in metabolite levels correlated well with the changes in the transcript levels of selected secondary metabolite biosynthesis genes. Aucuparin, noraucuparin and eriobofuran isolated from the 'Florina' cultures showed significant inhibitory effect on the conidial germination of V. inaequalis. The results expand our understanding of the metabolic basis of scab-resistance in apple and therefore are of interest in apple breeding programs to fortify scab resistance potential of commercially grown apple cultivars.

Biotechnological production of hyperforin for pharmaceutical formulation.

Hyperforin is a major active constituent of Hypericum perforatum (St. John's wort). It has amazing pharmacological activities, such as antidepressant properties, but it is labile and difficult to synthesize. Its sensitivity and lipophilicity are challenges for processing and formulation. Its chemical complexity provokes approaches of biotechnological production and modification. Dedifferentiated H. perforatum cell cultures lack appropriate storage sites and hence appreciable hyperforin levels. Shoot cultures are capable of forming hyperforin but less suitable for biomass up-scaling in bioreactors. Roots commonly lack hyperforin but a recently established adventitious root line has been demonstrated to produce hyperforin and derivatives at promising levels. The roots also contained lupulones, the typical constituents of hop (Humulus lupulus). Although shear-sensitive, these root cultures provide a potential production platform for both individual compounds and extracts with novel combinations of constituents and pharmacological activities. Besides in vitro cultivation techniques, the reconstruction of hyperforin biosynthesis in microorganisms is a promising alternative for biotechnological production. The biosynthetic pathway is under study, with omics-technologies being increasingly implemented. These biotechnological approaches may not only yield hyperforin at reasonable productivity but also allow for modifications of its chemical structure and pharmacological profile.

Exodermis and endodermis are the sites of xanthone biosynthesis in Hypericum perforatum roots.

Xanthones are specialized metabolites with antimicrobial properties, which accumulate in roots of Hypericum perforatum. This medicinal plant provides widely taken remedies for depressive episodes and skin disorders. Owing to the array of pharmacological activities, xanthone derivatives attract attention for drug design. Little is known about the sites of biosynthesis and accumulation of xanthones in roots. Xanthone biosynthesis is localized at the transcript, protein, and product levels using in situ mRNA hybridization, indirect immunofluorescence detection, and high lateral and mass resolution mass spectrometry imaging (AP-SMALDI-FT-Orbitrap MSI), respectively. The carbon skeleton of xanthones is formed by benzophenone synthase (BPS), for which a cDNA was cloned from root cultures of H. perforatum var. angustifolium. Both the BPS protein and the BPS transcripts are localized to the exodermis and the endodermis of roots. The xanthone compounds as the BPS products are detected in the same tissues. The exodermis and the endodermis, which are the outermost and innermost cell layers of the root cortex, respectively, are not only highly specialized barriers for controlling the passage of water and solutes but also preformed lines of defence against soilborne pathogens and predators.

Downstream processing of hyperforin from Hypericum perforatum root cultures.

Hyperforin is a major metabolite of the medicinal plant Hypericum perforatum (St. John's Wort) and has recently been found in hormone induced root cultures. The objective of this study is to identify a downstream process for the production of a hyperforin-rich extract with maximum extraction efficiency and minimal decomposition. The maximum extraction time was found to be 60min. The comparison of two equipment concepts for the extraction and solvent evaporation was performed employing two different solvents. While the rotary mixer showed better results for the extraction efficiency than a stirred vessel, the latter set-up was able to handle larger volumes but did not meet all process requirements. For the evaporation the prompt evaporation of the extraction agent using nitrogen stripping led to minor decomposition. In a 5L stirred vessel, the highest specific extraction of hyperforin was 4.3mg hyperforin/g dry weight bio material. Parameters for the equipment design for extraction and solvent evaporation were determined based on the experimental data.

Benzaldehyde dehydrogenase-driven phytoalexin biosynthesis in elicitor-treated Pyrus pyrifolia cell cultures.

Pyrus pyrifolia (Asian pear) cell cultures respond to yeast extract (YE) treatment by accumulating benzoate-derived biphenyl phytoalexins, namely, noraucuparin and aucuparin. Biphenyl phytoalexins are defense-marker metabolites of the sub-tribe Malinae of the family Rosaceae. The substrates for biphenyl biosynthesis are benzoyl-CoA and malonyl-CoA, which combine in the presence of biphenyl synthase (BIS) to produce 3,5-dihydroxybiphneyl. In the non-ß-oxidative pathway, benzoyl-CoA is directly derived from benzoic acid in a reaction catalyzed by benzoate-CoA ligase (BZL). Although the core ß-oxidative pathway of benzoic acid biosynthesis is well-understood, the complete cascade of enzymes and genes involved in the non-ß-oxidative pathway at the molecular level is poorly understood. In this study, we report the detection of benzaldehyde dehydrogenase (BD) activity in YE-treated cell cultures of P. pyrifolia. BD catalyzes the conversion of benzaldehyde to benzoic acid. BD and BIS activities were coordinately induced by elicitor treatment, suggesting their involvement in biphenyl metabolism. Changes in phenylalanine ammonia-lyase (PAL) activity preceded the increases in BD and BIS activities. Benzaldehyde was the preferred substrate for BD (Km=52.0µM), with NAD+ being the preferred co-factor (Km=64µM). Our observations indicate the contribution of BD towards biphenyl phytoalexin biosynthesis in the Asian pear.

Expression of Biphenyl Synthase Genes and Formation of Phytoalexin Compounds in Three Fire Blight-Infected Pyrus communis Cultivars.

Pear (Pyrus communis) is an economically important fruit crop. Drops in yield and even losses of whole plantations are caused by diseases, most importantly fire blight which is triggered by the bacterial pathogen Erwinia amylovora. In response to the infection, biphenyls and dibenzofurans are formed as phytoalexins, biosynthesis of which is initiated by biphenyl synthase (BIS). Two PcBIS transcripts were cloned from fire blight-infected leaves and the encoded enzymes were characterized regarding substrate specificities and kinetic parameters. Expression of PcBIS1 and PcBIS2 was studied in three pear cultivars after inoculation with E. amylovora. Both PcBIS1 and PcBIS2 were expressed in 'Harrow Sweet', while only PcBIS2 transcripts were detected in 'Alexander Lucas' and 'Conference'. Expression of the PcBIS genes was observed in both leaves and the transition zone of the stem; however, biphenyls and dibenzofurans were only detected in stems. The maximum phytoalexin level (~110 µg/g dry weight) was observed in the transition zone of 'Harrow Sweet', whereas the concentrations were ten times lower in 'Conference' and not even detectable in 'Alexander Lucas'. In 'Harrow Sweet', the accumulation of the maximum phytoalexin level correlated with the halt of migration of the transition zone, whereby the residual part of the shoot survived. In contrast, the transition zones of 'Alexander Lucas' and 'Conference' advanced down to the rootstock, resulting in necrosis of the entire shoots.

Benzophenone Synthase and Chalcone Synthase Accumulate in the Mesophyll of Hypericum perforatum Leaves at Different Developmental Stages.

The active medicinal constituents in Hypericum perforatum, used to treat depression and skin irritation, include flavonoids and xanthones. The carbon skeletons of these compounds are formed by chalcone synthase (CHS) and benzophenone synthase (BPS), respectively. Polyclonal antisera were raised against the polyketide synthases from Hypericum androsaemum and their IgG fractions were isolated. Immunoblotting and immunotitration were used to test the IgGs for crossreactivity and monospecificity in H. perforatum leaf protein extract. Immunofluorescence localization revealed that both CHS and BPS are located in the mesophyll. The maximum fluorescence levels were observed in approx. 0.5 and 1 cm long leaves, respectively. The fluorescence intensity observed for CHS significantly exceeded that for BPS. Using histochemical staining, flavonoids were detected in the mesophyll, indicating that the sites of biosynthesis and accumulation coincide. Our results help understand the biosynthesis and underlying regulation of active H. perforatum constituents.