l-Phenylalanine ammonia-lyase in suspension culture cells of spruce (Picea abies)

The activity of l-phenylalanine ammonia-lyase (PAL) (EC 4.3.1.5) was determined in seedlings, callus cells, cell suspension cultures and in young needles of spruce (Picea abies) (L.) (Karst). PAL activity increased up to 10 fold in response to transferring suspension cultured cells into new cultivation medium. PAL was also induced about 10 fold when callus cells were transferrd into liquid medium. The increase was transient and it required the presence of a carbohydrate.In cell suspension cultures, grown in the dark (white cells), but not in light-grown cultures (green cells), PAL activity was induced up to 30 fold by UV-light.With a cell wall preparation of Rhizosphaera kalkhoffii, a forest pathogenic fungus, used as elicitor, the activity of PAL could be induced more than 10 fold. The degree of induction depended on the elicitor concentration. Induction was prevented by cycloheximide but not by actinomycin D.     

Fungal elicitor-mediated responses in pine cell cultures

A tissue culture system has been developed to examine phenylpropanoid metabolism induced in pine tissues by an ectomycorrhizal symbiont. An elicitor preparation from the ectomycorrhizal fungus Thelephora terrestris Fr. induced enhanced phenolic metabolism in suspension cultured cells of Pinus banksiana Lamb., as indicated by tissue lignification and accumulation of specific methanol-extractable compounds in the cells. Induction of lignification was observed as early as 12 h after elicitation. The activity of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), the entry-point enzyme into phenylpropanoid metabolism, also increased within the same time-frame in elicited cells. Significant increases in PAL activity were evident by 6 h after elicitation, and, by 12 h after elicitation, PAL activity in elicited cells was ten times greater than that in the corresponding controls. Lignification of the elicited tissue was also accompanied by an increase in the activity of other enzymes associated with lignin synthesis, including caffeic acid O-methyl transferase (EC 2.1.1.46), hydroxycinnamate:CoA ligase (EC 6.2.1.12), cinnamyl alcohol dehydrogenase (EC 1.1.1.-), coniferin glucosidase (EC 3.2.1.21) and peroxidase (EC 1.11.1.7). The increase in total peroxidase activity was associated with a change in the pattern of soluble peroxidase isoforms. The pine cell culture-ectomycorrhizal elicitor system provides a good model for molecular analysis of the process of lignification in an economically important softwood species.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 4CL hydroxycinnamate:Coenzyme A ligase (EC 6.2.1.12) - CAD cinnamyl alcohol dehydrogenase (EC 1.1.1.-) - COMT S-adenosyl-l-methionine:caffeate O-methyl transferase (EC 2.1.1.46) - HPLC high-pressure liquid chromatography - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - TGA thioglycolic acid To whom correspondence should be addressedFinancial assistance for this work was provided by the Natural Sciences and Engineering Research Council of Canada.     

Differential accumulation of monolignol-derived compounds in elicited flax (Linum usitatissimum) cell suspension cultures

Lignin and lignans share monolignols as common precursors and are both potentially involved in plant defence against pathogens. In this study, we investigated the effects of fungal elicitors on lignin and lignan metabolism in flax (Linum usitatissimum) cell suspensions. Cell suspension cultures of flax were treated with elicitor preparations made from mycelium extracts of Botrytis cinerea, Phoma exigua and Fusarium oxysporum F ssp lini. Elicitors induced a rapid stimulation of the monolignol pathway, as confirmed by the increase in PAL (phenylalanine ammonia-lyase, EC 4.1.3.5), CCR (cinnamoyl-CoA reductase EC 1.2.1.44) and CAD (cinnamyl alcohol dehydrogenase EC 1.1.1.195) gene expression and PAL activity. At the same time, CCR activity only increased significantly in F. oxysporum-treated cells 24 h post elicitation. On the other hand, CAD activity measured for coniferyl alcohol formation was transiently decreased but a substrate-specific activation of CAD activity was observed in F. oxysporum-treated cells when using sinapyl alcohol as substrate. The accumulation of monolignol-derived products varied according to the elicitor used. B. cinerea or P. exigua-elicited cell cultures were characterised by a reinforcement of the cell wall by a deposit of 8-O-4′-linked non-condensed lignin structures and phenolic monomers, while at the same time no stimulation of 8-8′-linked lignan or 8-5′-linked phenylcoumaran lignan accumulation was observed. Additionally, elicitation of cell cultures with F. oxysporum extracts even triggered a strong incorporation of monolignols in the non condensed labile ether-linked lignin fraction concomitantly with a decrease in lignan and phenylcoumaran lignan accumulation. Several hypotheses are proposed to explain the putative role of these compounds in the defence response of flax cells against pathogens. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. C. Hano and M. Addi contributed equally to this work.     

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     

Biosynthesis of psoralens. Psoralen 5-monooxygenase activity from elicitor-treated Ammi majus cells

Microsomes prepared from cultured Ammi majus cells that had been challenged for 14 h with an elicitor derived from the cell walls of Phytophthora megasperma f.sp. glycinea (Pmg) converted psoralen to bergaptol (5-hydroxypsoralen) in the presence of NADPH and oxygen. The enzymatic activity was characterized as an inducible cytochrome-P-450-dependent monooxygenase associated with the endoplasmic reticulum. All of the steps involved in bergapten (5-methoxypsoralen) biosynthesis in Ammi majus have now been demonstrated in vitro. The results suggest that bergaptol and not hydroxymarmesin in the precursor of bergapten.     

Dose responses for Colletotrichum lindemuthianum elicitor-mediated enzyme induction in French bean cell suspension cultures

The induction of L-phenylalanine ammonialyase (PAL, EC 4.3.1.5) and flavanone synthase in French bean cell suspension cultures in response to heat-released elicitor from cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum is highly dependent upon elicitor concentration. The elicitor dose-response curve for PAL induction shows two maxima at around 17.5 and 50 g elicitor carbohydrate per ml culture, whereas the flavanone synthase response shows one maximum at around 100 g ml^-1. The PAL response is independent of the elicitor concentration present during the lag phase of enzyme induction; if the initial elicitor concentration is increased after 2 h by addition of extra elicitor, or decreased by dilution of the cultures, the dose response curves obtained reflect the concentration of elicitor present at the time of harvest. PAL induction is not prevented by addition of methyl sugar derivatives to the cultures; -methyl-D-glucoside, itself a weak elicitor of PAL activity, elicits a multiphasic PAL response when increasing concentrations are added in the presence of Colletotrichum elicitor. Eight fractions with different monosaccharide compositions, obtained from the crude elicitor by gel-filtration, each elicit different dose-responses for PAL induction; the response to unfractionated elicitor is not the sum of the response to the isolated fractions. There is no correlation between the ability of the fractions to induce PAL in the cultures and their ability to act as elicitors of isoflavonoid phytoalexin accumulation in bean hypocotyls.Abbreviations PAL phenylalanine ammonia-lyase - PMS Phytophthora megasperma var sojae     

Fungal Elicitor-Mediated Responses in Pine Cell Cultures : III. Purification and Characterization of Phenylalanine Ammonia-Lyase

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is involved in the lignification of pine suspension cultures in response to an elicitor prepared from an ectomycorrhizal fungus. To elucidate the molecular basis of this response, PAL was purified to homogeneity from jack pine (Pinus banksiana) suspension cultures using anion-exchange and chromatofocussing fast protein liquid chromatography. Physical characterization of the enzyme revealed that pine PAL was similar to PAL from other plant sources. Pine PAL had a pH optimum of 8.8, an isoelectric point of 5.75, and a native molecular mass of 340 kilodaltons. The enzyme appears to be a tetramer composed of 77 kilodalton subunits. Chromatographic and western blot analyses were used to identify possible isoenzymic changes in pine PAL in response to elicitation and to determine the nature of the increase in PAL activity associated with inducible lignification in these cultures. Only one species of PAL was detected in P. banksiana cell cultures and increased quantities of this protein were correlated with the enhanced enzyme activity observed in elicited cultures. P. banksiana PAL was not feedback-inhibited by a wide range of phenolic compounds at micromolar concentrations, including the reaction product cinnamic acid. Our data suggest that a different set of metabolic and molecular controls must be in place for the regulation of PAL in pine.     

Modification of l-phenylalanine ammonia-lyase in soybean cell suspension cultures by 2-aminooxyacetate and l-2-aminooxy-3-phenylpropionate

Suspension-cultured cells of soybean (Glycine max (L.) Merr. cv. Kanrich) produce large amounts of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), the first enzyme of phenylpropanoid metabolism, during growth. 2-Aminooxyacetic acid (AOA) and l-2-aminooxy-3-phenylpropionic acid (l-AOPP) inhibit the enzyme competitively in vitro and have been used for in vivo studies. The amount of extractable enzyme in the cells and their utilization of NO ₃ ^– and NH ₃ ⁺ are reduced upon the addition of AOA. When AOA was added at various times during growth, the appearance of additional enzyme activity was prevented but enzyme already formed was not inhibited. No evidence was obtained for the presence of an inhibitor in the extracts and AOA inhibition in vitro was readily reversible. It is conculded that AOA acts to inhibit the formation of PAL in suspension-cultured soy bean cells. In vitro inhibition of soybean PAL by l-AOPP could not be reversed; in contrast, the inhibition of maize (Zea mays L.) PAL was readily reversible. Added l-AOPP, which was rapidly taken up by the soybean cells, prevented the large increase in enzyme activity. Although PAL activity was blocked in the cultures, no appreciable increase in phenylalanine content could be detected in cell extracts. The response of soybean cell suspensions to l-AOPP addition thus differs from that of other tissues which in presence of l-AOPP show an increase in PAL activity and an accumulation of phenylalanine.Abbreviations AOA 2-aminooxyacetic acid - l-AOPP l-2-aminoxy-3-phenylpropionic acid - PAL l-phenylalanine ammonialyase (EC4.3.1.5)     

Cellular localization of nonhost resistance reactions of parsley (Petroselinum crispum) to fungal infection

The response of parsley seedlings (Petroselinum crispum) inoculated with zoospores of the soybean-pathogenic fungus, Phytophthora megasperma f. sp. glycinea, ranged from immunity to physiological susceptibility depending on the post-inoculation environmental conditions. Typical nonhost resistance reactions, hypersensitive cell death and the formation of small local lesions, occurred under high relative humidity and 16 h illumination per day. Localized biochemical reactions were monitored using fluorescence microscopy combined with histochemical and immunohistochemical methods. The rapid accumulation of furanocoumarin phytoalexins, wall-bound phenolics and callose was detected around infection sites. Indirect antibody staining of frozen tissue sections demonstrated the local accumulation of phenylalanine ammonia-lyase, a key enzyme of general phenylpropanoid metabolism, and S-adenosyl-L-methionine: bergaptol O-methyltransferase, a specific enzyme of the furanocoumarin pathway. The results indicate that phenylpropanoid derivatives are synthesized de novo at infection sites.Abbreviations BMT S-adenosyl-L-methionine:bergaptol O-methyltransferase - PAL phenylalanine ammonia-lyase - PBS phosphate-buffered saline     

Occurrence of phytoalexins and other putative defense-related substances in uninfected parsley plants

Considerable amounts of the following substances were found in uninfected parsley (Petroselinum crispum) cotyledons: furanocoumarins, the putative phytoalexins of this and some related plant species, two enzymes of the furanocoumarin pathway (S-adenosyl-L-methionine: xanthotoxol and S-adenosyl-L-methionine: bergaptol O-methyltransferases), two hydrolytic enzymes (1,3--glucanase, EC 3.2.1.39, and chitinase, EC 3.2.1.14), and pathogenesis-related proteins. The furanocoumarins and the methyltransferase activities reached their highest levels at the onset of cotyledon senescence as the hydrolytic enzymes increased from low to relatively high activity values. The relative amounts of pathogenesis-related proteins 1 and 2, as well as the corresponding mRNAs, also increased markedly. Two enzymes of general phenylpropanoid metabolism, L-phenylalanine ammonia-lyase and 4-coumarate: CoA ligase, decreased in activity in a biphasic fashion during cotyledon development. At all developmental stages, the levels of these putative defense-related agents in total cotyledon extracts were too high to enable detection of, possibly, additional changes upon infection with zoospores of Phytophthora megasperma f. sp. glycinea, a fungal pathogen to which parsley shows a non-host, hypersensitive resistance response.Abbreviations BMT S-adenosyl-L-methionine: bergaptol O-methyltransferase (EC 2.1.1.-) - 4CL 4-coumarate: CoA ligase (EC 6.1.1.12) - CMT S-adenosyl-L-methionine: caffeate O-methyltransferase (EC 2.1.1.-) - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5) - PR pathogenesis-related - XMT S-adenosyl-L-methionine: xanthotoxin O-methyltransferase (EC 2.1.1.-)     

Variation in the growth and biosynthetic activity of cloned cell cultures of Capsicum frutescens and their response to an exogenously supplied elicitor

Twenty clones established from single cells of a suspension culture of Capsicum frutescens were maintained as callus and in suspension over a sixteen week culture period. These clones exhibited marked differences in growth, chlorophyll and chloroform-soluble phenolic content which became more apparent with increasing time in culture. Clones in suspension exhibited a more rapid change in morphology and biosynthetic activity than those cultured as callus. Elicitation increased PAL activity, reduced the incorporation of L-[U-¹⁴C] phenylalanine into the chloroform-soluble fraction of the culture medium and increased incorporation into the methanol-soluble fraction of the cells in ten suspension clones. Differences to elicitation were observed among clones; in particular the faster growing isolates incorporated more radioactive label into soluble phenolics that remain in the cells than those that are released into the medium. The implications of these results are discussed.Abbreviations SH Schenk & Hildebrandt - PAL phenylalanine ammonia-lyase - RGR relative growth rate - TCC total chlorophyll content - HPLC high performance liquid chromatography     

Expression profile of a PAL gene from Astragalus membranaceus var. Mongholicus and its crucial role in flux into flavonoid biosynthesis

An important traditional Chinese medicine herb, Astragalus membranaceus var. Mongholicus, whose dried root is known as Radix astragali (“Huangqi” in Chinese), has high flavonoid content as an essential active constituent. Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) catalyzes the first and also a rate-limiting step in phenylpropanoid pathway, which supplies precursors for a variety of secondary metabolites including flavonoids. A PAL gene, designated AmPAL1 (GenBank accession no. AY986506), was isolated from A. membranaceus var. Mongholicus with a full-length cDNA of 2562 nucleotides and an open reading frame of 2154 bp. Northern blot analysis revealed that AmPAL1 expressed universally in different organs, and its expression was markedly induced by UV irradiation, mechanical wounding, and white light irradiation on etiolated seedlings, with some distinctive responsive properties. Content of a typical flavonoid, quercetin, in A. membranaceus var. Mongholicus of different ages correlated with PAL enzymatic activity. Transgenic tobacco plants harboring AmPAL1 under the control of the CaMV35S promoter showed significantly increased PAL activity and correlatively increased quercetin content than those in non-transformed plants. These results indicate that PAL is maybe a key point for flux into flavonoid biosynthesis in the genetic control of secondary metabolism in A. membranaceus var. Mongholicus.     

Furanocoumarin biosynthesis in Ruta graveolens cell cultures

The biosynthetic routes to four linear furanocoumarins—psoralen, xanthotoxin, bergapten. isopimpinellin-co-occurring in Ruta graveolens cell cultures have been investigated with six ¹⁴C-labelled compounds. Mevalonic acid was only poorly incorporated, in contrast to umbelliferone. In support of previous suggestions, 7-demethylsuberosin and (±)-marmesin were very good precursors of the linear furanocoumarins. 7-O-Prenylumbelliferone also was fairly well utilized, but this was probably owing to a prior ether cleavage yielding umbelliferone. Psoralen was well incorporated into bergapten and xanthotoxin, but not into the dimethoxylated isopimpinellin. Differences exist between the organized plant and its cell culture in terms of metabolic products and, by implication, precursor utilization. S(+)-Marmesin was obtained in small quantity from an acid-hydrolysable conjugate present in the culture medium. Syntheses of [2-¹⁴C]7-demethylsuberosin, [2-¹⁴C]osthenol, [2-¹⁴C]7-O-prenylumbelliferone, [3-¹⁴C] (±)-marmesin, and [3-¹⁴C]psoralen are described, as well as an improved method for separation of furanocoumarin mixtures by TLC and GLC.     

Elicitor-mediated induction of enzymes of lignin biosynthesis and formation of lignin-like material in a cell suspension culture of spruce (Picea abies)

Incubations of photomixotrophic suspension culture cells of spruce (Picea abies) (L.) (Karst) with an autoclaved cell wall preparation of Rhizosphaera kalkhoffii as elicitor led to a rapid increase of the activity of a number of enzymes involved in lignin biosynthesis. l-phenylalanine ammonia-lyase (EC 4.3.1.5) was induced about 10-fold, feruloyl-Coenzyme A reductase (ED 1.2.1.44) 4-fold, cinnamyl alcohol dehydrogenase (NADP⁺) (EC 1.1.1.195) 2-fold and peroxidase (EC 1.11.1.7) about 1.5-fold. The induction of the enzymes, with the exception of the peroxidase, was transient, showing maximal activity within 3 days after elicitation. Extracellular peroxidase activity, determined in the culture medium, rapidly decreased on initiation of elicitation.Concomitant with the increase of activity of the enzymes of lignin synthesis was a rapid clouding of the culture medium. Phloroglucinol-HCl staining revealed the presence of lignin-like material in the medium and also in the cells. The IR-spectrum of this material was identical with the IR-spectrum of authentic spruce lignin.Abbreviations PAL l-phenylalanine ammonia-lyase - FCR feruloyl-Coenzyme A reductase - CAD cinnamyl alcohol dehydrogenase - POD peroxidase     

L-Phenylalanine ammonia-lyase fromPhaseolus vulgaris: Modulation of the levels of active enzyme bytrans-cinnamic acid

The extractable activity ofl-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in cell suspension cultures of bean (Phaseolus vulgaris) is greatly induced following exposure to an elicitor preparation from the cell walls of the phytopathogenic fungusColletotrichum lindemuthianum. Following exogenous application oftrans-cinnamic acid (the product of the PAL reaction) to elicitor-induced cells, the activity of the enzyme rapidly declines. Loss of enzyme activity is accompanied by inhibition of the rate of synthesis of PAL subunits, as determined by [³⁵S]methionine pulse-labelling followed by specific immunoprecipitation; this is insufficient to account for the rapid loss of PAL enzyme activity. Pulse-chase and immune blotting experiments indicate that cinnamic acid does not affect the rate of degradation of enzyme subunits, but rather mediates inactivation of the enzyme. A non-dialysable factor from cinnamicacid-treated bean cells stimulates removal of PAL activity from enzyme extracts in vitro; this effect is dependent on the presence of cinnamic acid. Such loss of enzyme activity in vitro is accompanied by an apparent loss or reduction of the dehydroalanine residue of the enzyme's active site, as detected by active-site-specific tritiation, although levels of immunoprecipitable enzyme subunits do not decrease. Furthermore, cinnamic-acid-mediated loss of enzyme activity in vivo is accompanied, in pulse-chase experiments, by a greater relative loss of³⁵S-labelled enzyme subunits precipitated by an immobilised active-site affinity ligand than of subunits precipitated with anti-immunoglobulin G. It is therefore suggested that a possible mechanism for cinnamic-acid-mediated removal of PAL activity may involve modification of the dehydroalanine residue of the enzyme's active site.Abbreviations AOPP l--aminoxy--phenylpropionic acid - CA trans-cinnamic acid - PAGE polyacrylamide gel electrophoresis - PAL l-phenylalanine ammonia-lyase - SDS sodium dodecyl sulphate     

Polyacetylenes accumulation in <Emphasis Type="Italic">Ambrosia maritima</Emphasis> hairy root and cell cultures after elicitation with methyl jasmonate

Three lines of hairy root culture of Ambrosia maritima induced by Agrobacterium rhizogenes ATCC15834 were established. Thiarubrine A, thiarubrine A epoxide, thiarubrine A diol and their precursor pentayneene were produced by the hairy roots after elicitation with methyl jasmonate, the common signal molecule in the plant defense and development. Thiarubrine A diol was the main form detected in the medium. Maximum yield was achieved when the 13-day-old hairy root cultures were exposed to 40 M methyl jasmonate for 72 h. Callus and cell suspension cultures were established and maintained on Murashige and Skoog medium supplied with -naphthylacetic acid (NAA) and kinetin. When the cell suspension cultures were elicited with methyl jasmonate, pentayneene was the only polyacetylene produced. The yield of pentayneene in hairy root cultures was much higher (9.6 times) than that of cell suspension cultures.     

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.