Accumulation of furanocoumarins in Ruta graveolens L. shoot culture

Ruta graveolens L. shoots cultured in stationary liquid phase produced furanocoumarins: psoralen, bergapten, xanthotoxin, isopimpinellin and imperatorin at the amount totalling almost 1 g/100 g dry wt of the shoots. The dominating metabolites were therapeutically important compounds: xanthotoxin – 0.33 g/100 g dry wt and bergapten – 0.32 g/100 g dry wt. Maximum contents of the majority of the compounds were observed on 28th day of culture.     

Elicitor induced secondary metabolism in Ruta graveolens L.

In vitro cultures of Ruta graveolens L. respond with rapid accumulation of acridone epoxides, furoquinolines and furanocoumarins, when challenged with autoclaved homogenate of the yeast Rhodotorula rubra. A transient increase of several enzymes of the respective biosynthetic pathways was measured but we still look for the key regulatory enzymes. We investigated whether the branch point enzymes of the shikimic acid pathway anthranilate synthase (AS) and chorismate mutase (CM) possibly play such a role. The two enzymes compete for chorismate. AS forms anthranilate, the precursor amino acid of acridone and furoquinoline alkaloids. CM channels chorismate into phenylalanine, tyrosine and phenylpropanoid biosynthesis. Elicitation resulted in a transient increase of the activity of both enzymes. Relative induction rates were 2–4 fold for AS and about 1.5 fold for CM. Constitutive CM activity, however, is about 1000 fold higher than AS activity. As in other plants 2 isoforms of CM are expected to be present in R. graveolens. A differential determination of the activity of the isoforms via the tryptophan activation rate proved to be ambiguous. Some evidence for the specific induction of a plastidic form of CM was obtained by inhibition of translation. The time courses of CM induction show CM not to be a key enzyme in elicitor induction of furanocoumarin accumulation. In comparison to other enzyme activities induction of anthranilate synthase activity corresponds closest to inducible acridone epoxide accumulation indicating a key role in its regulation. Induction of AS and CM was inhibited by actinomycin D and chloramphenicol while cycloheximid inhibited AS induction only.Abbreviations ACT actinomycin D - AS anthranilate synthase - CAP chloramphenicol - CHX cycloheximid - 4-CL 4-coumarate CoA ligase - CM chorismate mutase - DTT dithiothreitol - NMT S-adenosyl-L-methionine:anthranilic acid N-methyltransferase - PAL phenylalanine ammonia lyase - XOMT S-adenosylmethionine: xanthotoxol-O-methyltransferase     

Potential allelochemicals from the essential oil of Ruta graveolens

The essential oil of aerial parts of Ruta graveolens was obtained by hydrodistillation with a 0.74% yield on a dry weight basis. Thirty-eight components were identified by GC and GC-MS analyses. 2-Ketones predominated in the essential oil, with undecan-2-one (46.8%) and nonan-2-one (18.8%) as the main constituents. The essential oil and some of its constituents were tested for their allelopathic activity in vitro on radish germination and radicle growth in light and darkness. The essential oil and some of its minor constituents were effective and dose-dependent inhibitors of both the germination and radicle growth; 2-ketones are not active. The possible allelopathic activity of rue essential oil and some its isolated constituents is reported.     

Unusually divergent 4-coumarate:CoA-ligases from <Emphasis Type="Italic">Ruta graveolens</Emphasis> L.

Most angiosperms encode a small family of 4-coumarate:CoA-ligases (4CLs) activating hydroxycinnamic acids for lignin and flavonoid pathways. The common rue, Ruta graveolens L., additionally produces coumarins by cyclization of the 4-coumaroyl moiety, possibly involving the CoA-ester, as well as acridone and furoquinoline alkaloids relying on (N-methyl)anthraniloyl-CoA as the starter substrate for polyketide synthase condensation. The accumulation of alkaloids and coumarins, but not flavonoids, was enhanced in Ruta graveolens suspension cultures upon the addition of fungal elicitor. Total RNA of elicitor-treated Ruta cells was used as template for RT-PCR amplification with degenerate oligonucleotide primers inferred from conserved motifs in AMP-binding proteins, and two full-size cDNAs were generated through RACE and identified as 4-coumarate:CoA-ligases, Rg4CL1 and Rg4CL2, by functional expression in yeast cells. The recombinant enzymes differed considerably in their preferential affinities to cinnamate (Rg4CL1) or ferulate (RgCL2) besides 4-coumarate, but did not activate hydroxybenzoic or (N-methyl)anthranilic acid. Most notably, the Rg4CL1 polypeptide included an N-terminal extension suggesting a chloroplast transit peptide. The genes were cloned and revealed four exons, separated by 1056, 94 and 54 bp introns for RgCL1, while Rg4CL2 was composed of five exons interupted by four introns from 113 to 350 bp, and the divergent heritage of these genes was substantiated by phylogenetic analysis. Both genes were expressed in shoot, leaf and flower tissues of adult Ruta plants with preference in shoot and flower, whereas negligible expression occurred in the root. However, Rg4CL1 was expressed much stronger in the flower, while Rg4CL2 was expressed mostly in the shoot. Furthermore, considerable transient induction of only Rg4CL1 was observed upon elicitation of Ruta cells, which seems to support a role of Rg4CL1 in coumarin biosynthesis. Alexander Endler and Stefan Martens contributed equally to the work.     

Unusual pseudosubstrate specificity of a novel 3,5-dimethoxyphenol O-methyltransferase cloned from Ruta graveolens L

A cDNA was cloned from Ruta graveolens cells encoding a novel O-methyltransferase (OMT) with high similarity to orcinol or chavicol/eugenol OMTs, but containing a serine-rich N-terminus and a 13 amino acid insertion between motifs IV and V. Expression in Escherichia coli revealed S-adenosyl-l-methionine-dependent OMT activity with methoxylated phenols only with an apparent Km of 20.4 for the prime substrate 3,5-dimethoxyphenol. The enzyme forms a homodimer of 84 kDa, and the activity was insignificantly affected by 2.0 mM Ca2+ or Mg2+, whereas Fe2+, Co2+, Zn2+, Cu2+ or Hg2+ were inhibitory (78-100%). Dithiothreitol (DTT) suppressed the OMT activity. This effect was examined further, and, in the presence of Zn2+ as a potential thiol methyltransferase (TMT) cofactor, the recombinant OMT methylated DTT to DTT-monomethylthioether. Sets of kinetic OMT experiments with 3,5-dimethoxyphenol at various Zn2+/DTT concentrations revealed the competitive binding of DTT with an apparent Ki of 52.0 microM. Thus, the OMT exhibited TMT activity with almost equivalent affinity to the thiol pseudosubstrate which is structurally unrelated to methoxyphenols.     

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     

Specific accumulation and revised structures of acridone alkaloid glucosides in the tips of transformed roots of Ruta graveolens

The root tips of Ruta graveolens (common rue) show strong autofluorescence of acridone alkaloids, which are characteristic secondary metabolites of this plant. To study the specific distribution and accumulation of acridone alkaloids in various root segments of Ruta graveolens, root material was harvested from genetically transformed root cultures and extracts were investigated by chromatographic techniques and HPLC-(1)H NMR spectroscopy. The cells of the elongation and differentiation zones contained acridone glucosides and large amounts of acridone alkaloids, mainly rutacridone. Gravacridondiol glucoside was identified as the dominant secondary compound of the root tips and its structure revised by means of spectroscopic methods. In addition, minor acridones, including the structurally revised gravacridontriol glucoside and unknown natural products, were found in the root tip.     

Effective biotic elicitation of <Emphasis Type="Italic">Ruta graveolens</Emphasis> L. shoot cultures by lysates from <Emphasis Type="Italic">Pectobacterium atrosepticum</Emphasis> and <Emphasis Type="Italic">Bacillus</Emphasis> sp.

Growth of Ruta graveolens shoots was induced when Bacillus sp. cell lysates were added to the culture medium. Elicitation of coumarin by this lysate was also very effective; the concentrations of isopimpinelin, xanthotoxin and bergapten increased to 610, 2120 and 1460 μg g⁻¹ dry wt, respectively. It also had a significant effect on the production of psoralen and rutamarin (680 and 380 μg g⁻¹ dry wt) and induced the biosynthesis of chalepin, which was not detected in the control sample, up to 47 μg g⁻¹ dry wt With lysates of the Pectobacterium atrosepticum, their effect on growth was not so significant and had no effect on the induction of coumarin accumulation. But elicitation with this lysate was much more effective for inducing the production of furoquinolone alkaloids; the concentrations of γ-fagarine, skimmianine, dictamnine and kokusaginine rose to 99, 680, 172 and 480 μg g⁻¹ dry wt, respectively.     

Molecular cloning and expression profiling of a chalcone synthase gene from hairy root cultures of Scutellaria viscidula Bunge

A cDNA encoding chalcone synthase (CHS), the key enzyme in flavonoid biosynthesis, was isolated from hairy root cultures of Scutellaria viscidula Bunge by rapid amplification of cDNA ends (RACE). The full-length cDNA of S. viscidula CHS, designated as Svchs (GenBank accession no. EU386767), was 1649 bp with a 1170 bp open reading frame (ORF) that corresponded to a deduced protein of 390 amino acid residues, a calculated molecular mass of 42.56 kDa and a theoretical isoelectric point (pI) of 5.79. Multiple sequence alignments showed that SvCHS shared high homology with CHS from other plants. Functional analysis in silico indicated that SvCHS was a hydrophilic protein most likely associated with intermediate metabolism. The active sites of the malonyl-CoA binding motif, coumaroyl pocket and cyclization pocket in CHS of Medicago sativa were also found in SvCHS. Molecular modeling indicated that the secondary structure of SvCHS contained mainly α-helixes and random coils. Phylogenetic analysis showed that SvCHS was most closely related to CHS from Scutellaria baicalensis. In agreement with its function as an elicitor-responsive gene, the expression of Svchs was induced and coordinated by methyl jasmonate. To our knowledge, this is the first report to describe the isolation and expression of a gene from S. viscidula.     

Formation of edulinine and furoquinoline alkaloids from quinoline derivatives by cell suspension cultures of Ruta graveolens

The formation of furoquinoline alkaloids and of edulinine, elaborated by cell suspension cultures of Ruta graveolens, was found to occur by way of 4-hydroxy-2-quinolone. Other substrates transformed to furoquinolines included 4-hydroxy- and 4-methoxy-3-(3-methyl-2-butenyl)-2-quinolone, known earlier as natural precursors in studies with whole plants. Involvement of dictamnine as a natural precursor of 8-methoxydictamnine (γ-fagarine) and skimmianine was proved using ¹⁴C-labelled compounds. Edulinine in the cell suspensions was formed from such substrates as 4-hydroxy-N-methyl-2-quinolone, 4-hydroxy-3-(3-methyl-2-butenyl)-N-methyl-2-quinolone and its 4- methyl ether; this is probably the natural biosynthetic sequence. Changes in alkaloid yields were noted upon prolonged subculturing.     

Characterization and heterologous expression of hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/benzoyltransferase from elicited cell cultures of carnation, Dianthus caryophyllus L.

Benzoyl-CoA:anthranilate N-benzoyltransferase catalyzes the first committed reaction of phytoalexin biosynthesis in carnation (Dianthus caryophyllus L.), and the product N-benzoylanthranilate is the precursor of several sets of dianthramides. The transferase activity is constitutively expressed in suspension-cultured carnation cells and can be rapidly induced by the addition of yeast extract. The enzyme was purified to homogeneity from yeast-induced carnation cells and shown to consist of a single polypeptide chain of 53 kDa. Roughly 20% of the sequence was identified by micro-sequencing of tryptic peptides, and some of these sequences differed in a few amino acid residues only suggesting the presence of isoenzymes. A specific 0.8 kb cDNA probe was generated by RT-PCR, employing degenerated oligonucleotide primers complementary to two of the tryptic peptides and using poly(A)⁺ RNA from elicited carnation cells. Five distinct benzoyltransferase clones were isolated from a cDNA library, and three cDNAs, pchcbt1–3, were sequenced and shown to encode full-size N-benzoyltransferases. The translated peptide sequences revealed more than 95% identity among these three clones. The additional two clones harbored insert sequences mostly homologous with pchcbt1 but differing in the 3-flanking regions due to variable usage of poly(A) addition sites. The identity of the clones was confirmed by matching the translated polypeptides with the tryptic enzyme sequences as well as by the activity of the benzoyltransferase expressed in Escherichia coli. Therefore, carnation encodes a small family of anthranilate N-benzoyltransferase genes. In vitro, the benzoyltransferases exhibited narrow substrate specificity for anthranilate but accepted a variety of aromatic acyl-CoAs. Catalytic rates with cinnamoyl- or 4-coumaroyl-CoA exceeded those observed with benzoyl-CoA, although the corresponding dianthramides did not accumulate in vivo. Thus the cDNAs described represent also the first hydroxycinnamoyltransferases cloned from plants, which classifies the enzymes as hydroxycinnamoyl/benzoyltransferases.     

Molecular cloning,sequence determination and heterologous expression of nucleoside diphosphate kinase from Pisum sativum

Protein sequence data derived from the N-terminal region of a 17 kDa polypeptide associated with the microsomal membrane fraction from Pisum sativum was used to design degenerate oligonucleotides which were used to amplify P. sativum cDNA via the polymerase chain reaction (PCR). Amplified cDNA was used as a probe to screen a P. sativum cDNA library and a cDNA clone, NDK-P1 was isolated and sequenced. The protein encoded by NDK-P1 had a calculated molecular mass of 16485 Da and possessed substantial homology with nucleoside diphosphate kinases (NDKs) isolated and cloned from other sources. High levels of expression of NDK-P1 protein were achieved in Escherichia coli using a T7-driven expression system. Recombinant NDK-P1 protein was shown to possess NDK activity and had similar biochemical characteristics to NDKs isolated from other sources. The Michaelis constants for a variety of nucleoside diphosphate (NDP) substrates were found to be broadly similar to those reported for other NDKs, with thymidine nucleotides being the sustrates of greatest affinity.     

Molecular cloning and expression analysis of a monosaccharide transporter gene OsMST4 from rice (Oryza sativa L.)

Two putative glycosyltransferases in Arabidopsis thaliana, designated reduced residual arabinose-1 and -2 (RRA1 and RRA2), are characterized at the molecular level. Both genes are classified in CAZy GT-family-77 and are phylogenetically related to putative glycosyltranferases of Chlamydomonas reinhardtii. The expression pattern of the two genes was analyzed by semi-quantitative RT-PCR using mRNA extracted from various organs of bolting Arabidopsis thaliana plants. In addition, promoter::gusA analysis of transgenic Arabidopsis thaliana containing a fusion between either the RRA-1 or -2 promoter fragment and the gusA reporter gene showed that whereas the RRA1 promoter was primarily active in the apical meristem, the expression pattern of the RRA2 promoter was more diverse but also highly active in the meristematic region. In addition, T-DNA mutant insertion lines of both RRA-1 and -2, were identified and characterized at the molecular and biochemical level. Monosaccharide compositional analyses of cell wall material isolated from the meristematic region showed a ca. 20% reduction in the arabinose content in the insoluble/undigested cell wall residue after enzymatic removal of xyloglucan and pectic polysaccharides. These data indicate that both RRA-1 and -2 play a role in the arabinosylation of cell wall component(s).     

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.     

Inhibition of flavonoid biosynthesis by gibberellic acid in cell suspension cultures of Daucus carota L.

In carrot cells (Daucus carota L.), cultured in the presence of gibberellic acid, anthocyanin synthesis is blocked at the level of chalcone synthase. By feeding suitable precursors for anthocyanins (naringenin, eriodictyol, dihydroquercetin) biosynthesis of cyanidin glycosides can be restored. After addition of these substrates to the culture medium in the presence of gibberellic acid, the activity of chalcone synthase remained as low as in the control without precursors. The highest increase in anthocyanin content was achieved using dihydroquercetin as the added precursor. The time course of this supplementation showed a rapid response; within 4 h a substantial increase in anthocyanin could be observed. In contranst, the flavonol quercetin is not a precursor for cyanidin. The fact that naringenin was also accepted for cyanidin synthesis leads to the conclusion that hydroxylation in 3-position of ring B in Daucus carota takes place at the flavonoid stage.Abbreviations CHI Chalcone isomerase - CHS chalcone synthase - DMSO dimethylsulfoxide - GA₃ gibberellic acid - PAL phenylalanine ammonia-lyase