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Terpenoids are present in all organisms but are especially abundant in plants, with more than 30,000 compounds. Not only do they play an important role in the life of plant, but also have high commercial values. However, the content of... more
Terpenoids are present in all organisms but are especially abundant in plants, with more than 30,000 compounds. Not only do they play an important role in the life of plant, but also have high commercial values. However, the content of many important terpenoids in plant is very low. Therefore, how to improve the inefficient production of terpenoids is an urgent task. Metabolic engineering has been one of the most potential technologies to improve terpenoids production in recent years, following the study of metabolic pathway and regulation mechanism of terpenoids. Although there are some breakthroughs, metabolic engineering of terpenoids is still full of challenges because of the lack of knowledge on metabolic control of most terpenoids. Functional genomics approaches, including transcriptomics, proteomics and metabolomics, are potential tools for exploring of metabolic engineering. Integrating transcriptomics and metabolomics is an effective way to discover new genes involved in me...
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Polycyclic polyprenylated acylphloroglucinols (PPAPs) exhibit a broad range of biological activities, such as antidepressant, antibacterial, antiviral and antitumor properties. The content of PPAPs in the producing plants is often quite... more
Polycyclic polyprenylated acylphloroglucinols (PPAPs) exhibit a broad range of biological activities, such as antidepressant, antibacterial, antiviral and antitumor properties. The content of PPAPs in the producing plants is often quite low, and their complex structures make total chemical synthesis difficult and economically impractical. Progresses in synthetic biology provide an alternative approach for the production of these valuable compounds. Here we present our results on reconstruction of the biosynthetic pathways of PPAP precursors in yeast. Based on our previous works on the biosynthesis of PPAPs, genes involved in the formation of benzophenones and xanthones from Hypericum sp. and other organisms were expressed in yeast either episomally or by integration into the genome. The production of the expected products reached around 0.5 mg/l, which is high enough to be the substrate for enzymes of subsequent biosynthetic steps. The yeast strains will be further engineered by int...
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Plant anthranoids are medicinally used for their purgative properties. Their scaffold was believed to be formed by octaketide synthase (OKS), a member of the superfamily of type III polyketide synthase (PKS) enzymes. Here, a cDNA encoding... more
Plant anthranoids are medicinally used for their purgative properties. Their scaffold was believed to be formed by octaketide synthase (OKS), a member of the superfamily of type III polyketide synthase (PKS) enzymes. Here, a cDNA encoding OKS of Polygonum cuspidatum was isolated using a homology-based cloning strategy. When produced in Escherichia coli, P. cuspidatum octaketide synthase (PcOKS) catalyzed the condensation of eight molecules of malonyl-CoA to yield a mixture of unphysiologically folded aromatic octaketides. However, when the ORF for PcOKS was expressed in Arabidopsis thaliana, the anthranoid emodin was detected in the roots of transgenic lines. No emodin was found in the roots of wild-type A. thaliana. This result indicated that OKS is the key enzyme of plant anthranoids biosynthesis. In addition, the root growth of the transgenic A. thaliana lines was inhibited to an extent that resembled the inhibitory effect of exogenous emodin on the root growth of wild-type A. th...
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Seven hairy root lines with the properties of fast growth and high artemisinin contents were selected from 747 hairy roots induced by transformation of Artemisia annua L. strain 025 with Agrobacterium rhizogenes ATCC15834. The differences... more
Seven hairy root lines with the properties of fast growth and high artemisinin contents were selected from 747 hairy roots induced by transformation of Artemisia annua L. strain 025 with Agrobacterium rhizogenes ATCC15834. The differences of growth rates and artemisinin contents among the 7 selected hairy root lines were extremely significant, of which HR-9 gave the highest yield of artemisinin, reaching 33.25 mg/y.L. The differences of growth rates and artemisinin contents among hairy roots, untransformated roots and callus were also significant. There were no obvious lag phase in batch culture of hairy roots of Artemisia annua L. The exponential growth phase was 7 approximately 15 days after inculation. The growth rate reached the highest on the 11th day, and the cultures reached a stationary phase on the 20th day. The properties of artemisinin content of hairy roots were obviously "related to growth". The artemisinin content decreased slowly during the exponential phase...
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A type III polyketide synthase cDNA and the corresponding gene (PcPKS2) were cloned from Polygonum cuspidatum Sieb. et Zucc. Sequencing results showed that the ORF of PcPKS2 was interrupted by three introns, which was an unexpected... more
A type III polyketide synthase cDNA and the corresponding gene (PcPKS2) were cloned from Polygonum cuspidatum Sieb. et Zucc. Sequencing results showed that the ORF of PcPKS2 was interrupted by three introns, which was an unexpected finding because all type III PKS genes studied so far contained only one intron at a conserved site in flowering plants, except for an Antirrhinum majus chalcone synthase gene. Besides the unusual gene structure, PcPKS2 showed some interesting characteristics: (1) the CHS "gatekeepers" Phe215 and Phe265 are uniquely replaced by Leu and Cys, respectively; (2) recombinant PcPKS2 overexpressed in Escherichia coli efficiently afforded 4-coumaroyltriacetic acid lactone (CTAL) as a major product along with bis-noryangonin (BNY) and p-hydroxybenzalacetone at low pH; however, it effectively yielded p-hydroxybenzalacetone as a dominant product along with CTAL and BNY at high pH. Beside p-hydroxybenzalacetone, CTAL and BNY, a trace amount of naringenin chalcone could be detected in assays at different pH. Furthermore, 4-coumaroyl-CoA and feruloyl-CoA were the only cinnamoyl-CoA derivatives accepted as starter substrates. PcPKS2 did not accept isobutyryl-CoA, isovaleryl-CoA or acetyl-CoA as substrate. DNA gel blot analysis indicated that there are two to four PcPKS2 copies in the P. cuspidatum genome. RNA gel blot analysis revealed that PcPKS2 is highly expressed in the rhizomes and in young leaves, but not in the roots of the plant. PcPKS2 transcripts in leaves were induced by pathogen infection, but not by wounding.
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The flowering promoting factor1 ( fpf1) from Arabidopsis thaliana was transferred into Artemisia annua L. via Agrobacterium tumefaciens. The fpf1 gene was firstly inserted in the binary vector pBI121 under the control of CaMV 35S promoter... more
The flowering promoting factor1 ( fpf1) from Arabidopsis thaliana was transferred into Artemisia annua L. via Agrobacterium tumefaciens. The fpf1 gene was firstly inserted in the binary vector pBI121 under the control of CaMV 35S promoter to construct the plant expression vector pBIfpf1, then leaf explants of A. annua were infected with A. tumefaciens LBA4404 containing pBIfpf1, and induced shoots. Transgenic plants were obtained through the selection with kanamycin. PCR, PCR-Southern and Southern blot analyses confirmed that the foreign fpf1 gene had been integrated into the A. annua genome. RT-PCR and RT-PCR-Southern analyses suggested that the foreign fpf1 gene had expressed at the transcriptional level. Under short-day conditions, the flowering time of fpf1 transgenic plants was about 20 days earlier than the non-transformed plants; however, no significant differences were detected in artemisinin content between the flowering transgenic plants and the non-flowering non-transgenic plants. These results showed that flowering is not a necessary factor for increasing the artemisinin content, furthermore, there may be no direct linkage between flowering and artemisinin biosynthesis.
Research Interests: Complementary and Alternative Medicine, Plant Biology, Biology, Phytotherapy, Humans, and 10 morePolymerase Chain Reaction, Light, Flowers, Sesquiterpenes, Artemisia Annua, Artemisinin, PLANT PROTEINS, Plant Leaves, reverse transcriptase polymerase chain reaction, and Pharmacology and pharmaceutical sciences
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Research Interests: Chemistry, Phytochemistry, Medicine, Cell Culture, Biological Sciences, and 14 moreTemperature, Iron, Terpenes, Solubility, CHEMICAL SCIENCES, Specific Activity, Substrate Specificity, Hypericum, Hydrogen-Ion Concentration, Phloroglucinol, Molecular Structure, Prenylation, Fpp, and Medical and Health Sciences
We report a rapid and simple method for isolating the 5'-end of plant genes from genomic DNA by polymerase chain reaction (PCR) with TATA-box-based degenerate primers (TDPs). The TDPs were specially designed... more
We report a rapid and simple method for isolating the 5'-end of plant genes from genomic DNA by polymerase chain reaction (PCR) with TATA-box-based degenerate primers (TDPs). The TDPs were specially designed according to the TATA box, which is conserved in the promoter region of most plant genes. The unknown 5'-ends of several genes in different plants were isolated by PCR with gene-specific primers of the known core fragment and the TDPs. Our method does not require the arduous RNA manipulations and expensive enzyme treatments of the popular rapid amplification of cDNA ends (RACE) and its variants, and so could be a cheap practical alternative.
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A simple poly(dimethylsiloxane) (PDMS) microchip was fabricated with an integrated contactless conductivity detector. This microchip consists of a PDMS cover sheet with embedded channels and a glass substrate holding the electrodes. The... more
A simple poly(dimethylsiloxane) (PDMS) microchip was fabricated with an integrated contactless conductivity detector. This microchip consists of a PDMS cover sheet with embedded channels and a glass substrate holding the electrodes. The electrodes are insulated from the microchannel by a PDMS membrane. n-Dodecyl β-D-maltoside was utilized to modify the channel surface to improve its hydrophilicity and to reduce the strong
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A contactless conductivity detector integrated into a poly(dimethylsiloxane) microchip for electrophoresis is presented. It adopted the simplest configuration of electrodes commonly used in this detection mode for capillary... more
A contactless conductivity detector integrated into a poly(dimethylsiloxane) microchip for electrophoresis is presented. It adopted the simplest configuration of electrodes commonly used in this detection mode for capillary electrophoresis microchips. Although the chip is based on a simple and effective design, it is able to obtain low detection levels due to the low noise of the detection circuit. A circuit based on a lock-in amplifier was designed on printed circuit boards to read out the signal. The property of the detection cell was studied by applying excitation signals of different frequencies and different amplitudes. It was found that the best detection limit could be achieved with a frequency of 50 kHz and amplitude of 20 V. The performance of the detector was demonstrated by successfully separating and detecting several inorganic ions and also a mixture of heavy metal ions. An average detection limit of 0.4 μM was obtained for inorganic cations. This value is significantly improved compared to similar microchip-based detectors. The presented detector could be promising for mass production due to its properties, such as simple construction, high degree of integration, high performance and low cost.
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The latest progresses of the application of Ri plasmid on the studies of theoretical problems of plant science, the production of plant secondary metabolites, plant species improvement and plant cultivation are reviewed.
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A soil column split-root experiment was designed to investigate the ability of apple replant disease (ARD)-causing agents to spread in soil. ‘M26’ apple rootstocks grew into a top layer of Control soil, followed by a barrier-free... more
A soil column split-root experiment was designed to investigate the ability of apple replant disease (ARD)-causing agents to spread in soil. ‘M26’ apple rootstocks grew into a top layer of Control soil, followed by a barrier-free split-soil layer (Control soil/ARD soil). We observed a severely reduced root growth, concomitant with enhanced gene expression of phytoalexin biosynthetic genes and phytoalexin content in roots from ARD soil, indicating a pronounced local plant defense response. Amplicon sequencing (bacteria, archaea, fungi) revealed local shifts in diversity and composition of microorganisms in the rhizoplane of roots from ARD soil. An enrichment of operational taxonomic units affiliated to potential ARD fungal pathogens (Ilyonectria and Nectria sp.) and bacteria frequently associated with ARD (Streptomyces, Variovorax) was noted. In conclusion, our integrated study supports the idea of ARD being local and not spreading into surrounding soil, as only the roots in ARD soil...
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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... more
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...
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We recently characterized a gene-terpene network that is associated with artemisinin biosynthesis in self-pollinated (SP) Artemisia annua, an effective antimalarial plant. We hypothesize that an alteration of gene expression in the... more
We recently characterized a gene-terpene network that is associated with artemisinin biosynthesis in self-pollinated (SP) Artemisia annua, an effective antimalarial plant. We hypothesize that an alteration of gene expression in the network may improve the production of artemisinin and its precursors. In this study, we cloned an isopentenyl pyrophosphate isomerase (IPPI) cDNA, AaIPPI1, from Artemisia annua (Aa). The full-length cDNA encodes a type I IPPI containing a plastid transit peptide (PTP) at its amino terminus. After removal of the PTP, the recombinant truncated AaIPPI1 isomerized isopentenyl pyrophosphate (IPP) to dimethyl allyl pyrophosphate (DMAPP) and vice versa. The steady-state equilibrium ratio of IPP/DMAPP in the enzymatic reactions was approximately 1:7. The truncated AaIPPI1 was overexpressed in the cytosol of the SP A. annua variety. The leaves of transgenic plants produced approximately 4% arteannuin B (g/g, dry weight, dw) and 0.17-0.25% artemisinin (g/g, dw), th...
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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... more
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 phytoa...
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Plant-specific type III polyketide synthase (PKS) produces a variety of plant secondary metabolites with notable structural diversity and biological activity. So far 14 plant-specific type III PKS have been identified according to their... more
Plant-specific type III polyketide synthase (PKS) produces a variety of plant secondary metabolites with notable structural diversity and biological activity. So far 14 plant-specific type III PKS have been identified according to their enzymatic products, and the corresponding genes have been cloned and characterized. The differences among the various PKS are mainly in their substrate specificities, the number of their condensation reactions, and the type of ring closure of their products. However, numerous studies have revealed the common features among the plant-specific type III PKS, which include sequence homology, similar gene structure, conserved amino acid residues in the reaction center, enzymatic characteristics and reaction mechanism. We briefly reviewed 14 plant-specific type III PKS to better understand genetic and metabolic engineering of plant-specific type III PKS.
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ABSTRACT In response to pathogen attack, the Pyrinae (formerly Maloideae) produce biphenyls and/or structurally related dibenzofurans as phytoalexins. The carbon backbone of these inducible defence compounds is formed by biphenyl synthase... more
ABSTRACT In response to pathogen attack, the Pyrinae (formerly Maloideae) produce biphenyls and/or structurally related dibenzofurans as phytoalexins. The carbon backbone of these inducible defence compounds is formed by biphenyl synthase (BIS). The enzyme catalyzes the sequential condensation of benzoyl-CoA with three molecules of malonyl-CoA to yield 3,5-dihydroxybiphenyl, precursor of the biphenyl and dibenzofuran phytoalexins. BIS belongs to the superfamily of type III polyketide synthases (PKSs), which generate a diverse array of plant secondary metabolites. Formation of biphenyls and dibenzofurans in response to fire blight (Erwinia amylovora) infection was studied in Malus species and Malus domestica cultivars. In response to elicitation, cell cultures of mountain ash (Sorbus aucuparia) accumulated three biphenyls and two dibenzofurans. These compounds were isolated and their structures were determined. Similar phytoalexins were also detected in fire blight-infected M. domestica shoots. Interestingly, co-occurrence of both classes of phytoalexins is rare. Therefore, S. aucuparia cell cultures could serve as a model system for studying the biosynthesis of biphenyl and dibenzofuran phytoalexins. INTRODUCTION Considering the economic importance, little is known of the disease resistance mechanisms in the Pyrinae (formerly Maloideae; Potter et al., 2007). This rosaceous subtribe includes economically important fruit trees, such as apple (Malus domestica) and pear (Pyrus communis). It is known that, in response to pathogen attack, several Rosaceae species synthesize biphenyls and structurally related dibenzofurans as phytoalexins (Fig. 1; Kokubun and Harborne, 1995a). The ability to produce both classes of metabolites as inducible defence compounds is unique to the Pyrinae (Kokubun and Harborne, 1995a). To date, the best-known biphenyl phytoalexin is aucuparin, which was first isolated from mountain ash (Sorbus aucuparia), another member of the subtribe Pyrinae (Erdtman et al., 1963). Since then, biphenyls have been detected as phytoalexins following fungal infection in the sapwood of Pyrinae species (Kemp et al.,
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Biphenyls and dibenzofurans are the phytoalexins of the Malinae involving apple and pear. Biosynthesis of the defence compounds includes two O-methylation reactions. cDNAs encoding the O-methyltransferase (OMT) enzymes were isolated from... more
Biphenyls and dibenzofurans are the phytoalexins of the Malinae involving apple and pear. Biosynthesis of the defence compounds includes two O-methylation reactions. cDNAs encoding the O-methyltransferase (OMT) enzymes were isolated from rowan (Sorbus aucuparia) cell cultures after treatment with an elicitor preparation from the scab-causing fungus, Venturia inaequalis. The preferred substrate for SaOMT1 was 3,5-dihydroxybiphenyl, supplied by the first pathway-specific enzyme, biphenyl synthase (BIS). 3,5-Dihydroxybiphenyl underwent a single methylation reaction in the presence of S-adenosyl-L-methionine (SAM). The second enzyme, SaOMT2, exhibited highest affinity for noraucuparin, however, the turnover rate was greater with 5-hydroxyferulic acid. Both substrates were only methylated at the meta-positioned hydroxyl group. The substrate specificities of the OMTs and the regiospecificities of their reactions were rationalized by homology modeling and substrate docking. Interaction of ...
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Research Interests: Kinetics, Tobacco, Biological Sciences, Phylogeny, Plant Physiology, and 12 morePlant diseases, Sesquiterpenes, Molecular cloning, Malus, Substrate Specificity, Gas Chromatography/mass Spectrometry, Amino Acid Sequence, Subcellular Fractions, PLANT PROTEINS, Protein Transport, Sorbus, and Molecular Sequence Data
Plant-specific type III polyketide synthase (PKS) produces a variety of plant secondary metabolites with notable structural diversity and biological activity. So far 14 plant-specific type III PKS have been identified according to their... more
Plant-specific type III polyketide synthase (PKS) produces a variety of plant secondary metabolites with notable structural diversity and biological activity. So far 14 plant-specific type III PKS have been identified according to their enzymatic products, and the corresponding genes have been cloned and characterized. The differences among the various PKS are mainly in their substrate specificities, the number of their condensation reactions, and the type of ring closure of their products. However, numerous studies have revealed the common features among the plant-specific type III PKS, which include sequence homology, similar gene structure, conserved amino acid residues in the reaction center, enzymatic characteristics and reaction mechanism. We briefly reviewed 14 plant-specific type III PKS to better understand genetic and metabolic engineering of plant-specific type III PKS.
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Artemisinin from Artemisia annua has become one of the most important drugs for malaria therapy. Its biosynthesis proceeds via amorpha-4,11-diene, but it is still unknown whether the isoprenoid precursors units are obtained by the... more
Artemisinin from Artemisia annua has become one of the most important drugs for malaria therapy. Its biosynthesis proceeds via amorpha-4,11-diene, but it is still unknown whether the isoprenoid precursors units are obtained by the mevalonate pathway or the more recently discovered non-mevalonate pathway. In order to address that question, a plant of A. annua was grown in an atmosphere containing 700 ppm of 13CO2 for 100 min. Following a chase period of 10 days, artemisinin was isolated and analyzed by 13C NMR spectroscopy. The isotopologue pattern shows that artemisinin was predominantly biosynthesized from (E,E)-farnesyl diphosphate (FPP) whose central isoprenoid unit had been obtained via the non-mevalonate pathway. The isotopologue data confirm the previously proposed mechanisms for the cyclization of (E,E)-FPP to amorphadiene and its oxidative conversion to artemisinin. They also support deprotonation of a terminal allyl cation intermediate as the final step in the enzymatic con...
Research Interests: Phytochemistry, Carbon Dioxide, Malaria, NMR Spectroscopy, Pharmacognosy, and 13 moreBiological Sciences, Phytotherapy, Carbon Isotopes, Artemisia, Sesquiterpenes, TERPENE, CHEMICAL SCIENCES, Antimalarials, Artemisia Annua, Biosynthetic Pathways, Chemical Structure, Molecular Structure, and Medical and Health Sciences
The effect of the gene dosage on the expression of rRNAs was studied in Hypericum perforatum. The methylation levels of rDNA were analysed using the isoschizomers MspI and HpaII and eleven additional methylation-sensitive enzymes. No... more
The effect of the gene dosage on the expression of rRNAs was studied in Hypericum perforatum. The methylation levels of rDNA were analysed using the isoschizomers MspI and HpaII and eleven additional methylation-sensitive enzymes. No differences in rDNA methylation were observed between diploids and tetraploids at an early ontogenetic stage.
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cDNAs encoding Hypericum sampsonii benzophenone synthase (HsBPS) and chalcone synthase (HsCHS) were isolated and functionally characterized. Differential expressions of HsBPS and HsCHS were monitored using quantitative polymerase chain... more
cDNAs encoding Hypericum sampsonii benzophenone synthase (HsBPS) and chalcone synthase (HsCHS) were isolated and functionally characterized. Differential expressions of HsBPS and HsCHS were monitored using quantitative polymerase chain reaction (PCR). In the vegetative stage, HsBPS was highly expressed in the roots; its transcript level was approx. 100 times higher than that of HsCHS. Relatively high transcript amounts of HsBPS were also detected in older leaves, whereas the youngest leaves contained higher transcript amounts of HsCHS. In the reproductive stage, maximum HsCHS expression was detected in flowers, the transcript level being approx. 5 times higher than that of HsBPS. The inversed situation with a 10-fold difference in the expression levels was observed with fruits. High transcript amounts for both proteins were found in roots.
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Amorpha-4,11-diene synthase (ADS) and Cyt P450 monooxygenase (CYP71AV1) in Artemisia annua L. are two key enzymes involved in the biosynthesis of artemisinin. The promoters of ADS and CYP71AV1 contain E-box elements, which are putative... more
Amorpha-4,11-diene synthase (ADS) and Cyt P450 monooxygenase (CYP71AV1) in Artemisia annua L. are two key enzymes involved in the biosynthesis of artemisinin. The promoters of ADS and CYP71AV1 contain E-box elements, which are putative binding sites for basic helix-loop-helix (bHLH) transcription factors. This study successfully isolated a bHLH transcription factor gene from A. annua, designated as AabHLH1, from a cDNA library of the glandular secretory trichomes (GSTs) in which artemisinin is synthesized and sequestered. AabHLH1 encodes a protein of 650 amino acids containing one putative bHLH domain. AabHLH1 and ADS genes were strongly induced by ABA and the fungal elicitor, chitosan. The transient expression analysis of the AabHLH1-green fluorescent protein (GFP) reporter gene revealed that AabHLH1 was targeted to nuclei. Biochemical analysis demonstrated that the AabHLH1 protein was capable of binding to the E-box cis-elements, present in both ADS and CYP71AV1 promoters, and pos...
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It is well known in the literature that cinnamyl alcohol dehydrogenase (CAD) reduces hydroxycinnamyl aldehydes, such as coumaryl, coniferyl, and sinapyl aldehydes, to their corresponding alcohols in the presence of NADPH, and these... more
It is well known in the literature that cinnamyl alcohol dehydrogenase (CAD) reduces hydroxycinnamyl aldehydes, such as coumaryl, coniferyl, and sinapyl aldehydes, to their corresponding alcohols in the presence of NADPH, and these alcohols act as the precursors of lignin biosynthesis. Here, we report the isolation of a cDNA encoding an NADP(+)-dependent CAD, designated as AaCAD, from the cDNA library using glandular secretory trichomes of Artemisia annua as the source of mRNA. A phylogenetic analysis indicated that AaCAD was clustered with AtCAD4 and AtCAD5, which were involved in monolignol biosynthesis from Arabidopsis. Semi-quantitative RT-PCR showed that the AaCAD transcript was abundant mostly in leaf and root, followed by flower, and lowest in stem. Functional and enzymatic assays showed that the recombinant enzyme was able to reversibly reduce a variety of common CADs substrates, namely geranial, cinnamyl aldehyde, sinapyl aldehyde, coniferyl aldehyde, and a sesquiterpenoid ...
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ABSTRACT Biosynthesis of phytoalexins is a plant defence strategy against pathogens. Shoots of the apple (Malus × domestica) cultivar ‘Holsteiner Cox’ formed biphenyls and dibenzofurans when inoculated with the fire blight bacterium,... more
ABSTRACT Biosynthesis of phytoalexins is a plant defence strategy against pathogens. Shoots of the apple (Malus × domestica) cultivar ‘Holsteiner Cox’ formed biphenyls and dibenzofurans when inoculated with the fire blight bacterium, Erwinia amylovora. The phytoalexins were only present in the transition zone of stems, whereas the leaves were devoid of the defence compounds. The scaffold of the phytoalexins is formed by biphenyl synthase (BIS), a type III polyketide synthase. In apple, BIS is encoded by a gene family, members of which fall into four subfamilies. Representative BIS cDNAs were cloned from fire blight-infected shoots of ‘Holsteiner Cox’ and functionally expressed. The preferred starter substrates were benzoyl-CoA and salicoyl-CoA, leading to the formation of 3,5-dihydroxybiphenyl and 4-hydroxycoumarin, respectively, in the presence of malonyl-CoA as extender molecule. The four subfamilies were differentially regulated after inoculation of shoots with E. amylovora. The BIS3 gene was expressed in stems, with maximum transcript levels in the transition zone. The BIS3 protein was immunochemically localized to the parenchyma of the bark. Dot-shaped immunofluorescence was restricted to the junctions between neighbouring cortical parenchyma cells. Leaves contained transcripts for BIS2 which, however, were not translated into immunodetectable BIS protein. The understanding of phytoalexin metabolism may aid in improving apple resistance to fire blight.
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Biphenyls are unique phytoalexins produced by plants belonging to Pyrinae, a subtribe of the economically important Rosaceae family. The formation of aucuparin, a well-known biphenyl, is induced by yeast extract (YE) in cell cultures of... more
Biphenyls are unique phytoalexins produced by plants belonging to Pyrinae, a subtribe of the economically important Rosaceae family. The formation of aucuparin, a well-known biphenyl, is induced by yeast extract (YE) in cell cultures of Sorbus aucuparia. However, the molecular mechanism underlying YE-induced activation of biphenyl biosynthesis remains unknown. Here we demonstrate that the addition of YE to the cell cultures results in a burst of reactive oxygen species (ROS; H(2)O(2) and O(2) (-)), followed by transcriptional activation of the biphenyl synthase 1 gene (BIS1) encoding the key enzyme of the biphenyl biosynthetic pathway and aucuparin accumulation. Pretreatment of the cell cultures with ROS scavenger dihydrolipoic acid and NADPH oxidase-specific inhibitor diphenylene iodonium abolished all of the above YE-induced biological events. However, when the cell cultures was pretreated with superoxide dismutase specific inhibitor N,N-diethyldithiocarbamic acid, although O(2) (-) continued to be generated, the H(2)O(2) accumulation, BIS1 expression and aucuparin production were blocked. Interestingly, exogenous supply of H(2)O(2) in the range of 0.05-10 mM failed to induce aucuparin accumulation. These results indicate that endogenous generation of H(2)O(2) rather than that of O(2) (-) is a key factor in YE-induced accumulation of biphenyl phytoalexins in cell cultures of S. aucuparia.
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Although a number of plant natural products are derived from benzoic acid, the biosynthesis of this structurally simple precursor is poorly understood. Hypericum calycinum cell cultures accumulate a benzoic acid-derived xanthone... more
Although a number of plant natural products are derived from benzoic acid, the biosynthesis of this structurally simple precursor is poorly understood. Hypericum calycinum cell cultures accumulate a benzoic acid-derived xanthone phytoalexin, hyperxanthone E, in response to elicitor treatment. Using a subtracted complementary DNA (cDNA) library and sequence information about conserved coenzyme A (CoA) ligase motifs, a cDNA encoding cinnamate:CoA ligase (CNL) was isolated. This enzyme channels metabolic flux from the general phenylpropanoid pathway into benzenoid metabolism. HcCNL preferred cinnamic acid as a substrate but failed to activate benzoic acid. Enzyme activity was strictly dependent on the presence of Mg2+ and K+ at optimum concentrations of 2.5 and 100 mm, respectively. Coordinated increases in the Phe ammonia-lyase and HcCNL transcript levels preceded the accumulation of hyperxanthone E in cell cultures of H. calycinum after the addition of the elicitor. HcCNL contained a...