Effects of Glyphosate on the Shikimate Pathway and Regulation of Phenylalanine Ammonia-lyase in Cryptomeria and Perilla Cell Suspension Cultures

Treatment of Cryptomeria and Perilla cell suspension cultureswith glyphosate resulted in a marked suppression of the formationof flavans and caffeic acid derivatives, respectively, whileit caused only a slight decline in the cell growth. In contrastwith 3-deoxy-D-arabino-heptulosonate (DAHP) synthase-Mn isozyme,DAHP synthase-Co isozyme from Cryptomeria and Perilla cellswas much more sensitive to inhibition by glyphosate. The additionof 1 to 2 mM glyphosate caused an accumulation of shikimateand quinate and a reduction of L-phenylalanine in both cellcultures. The inhibition of phenylalanine ammonia-lyase (PAL)activity by glyphosate was reversed by exogenously suppliedL-phenylalanine to near the control level. Cycloheximide andactinomycin D nullified the recovery by exogenous L-phenylalanineon PAL activity. L-Phenylalanine itself promoted PAL activityto some extent. No recovery of PAL activity in L--aminooxy-ß-phenylpropionate(L-AOPP)-treated cell cultures could be observed by the additionof L-phenylalanine. Therefore, L-AOPP seems to inhibit the formationof PAL, though it has been considered a competitive inhibitor. ³Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai 980, Japan. (Received October 28, 1985; Accepted March 13, 1986)     

Effects of Glyphosate on Metabolism of Phenolic Compounds: V. l-alpha-AMINOOXY-beta-PHENYLPROPIONIC ACID AND GLYPHOSATE EFFECTS ON PHENYLALANINE AMMONIA-LYASE IN SOYBEAN SEEDLINGS

The phenylalanine ammonia-lyase (PAL) inhibitor l-alpha-aminooxy-beta-phenylpropionic acid (AOPP) was root-fed to light-exposed soybean seedlings alone or with glyphosate [N-(phosphonomethyl)glycine] to test further the hypothesis that PAL activity is involved in the mode of action of glyphosate. Extractable PAL activity was increased by 0.01 and 0.1 millimolar AOPP. AOPP reduced total soluble hydroxyphenolic compound levels and increased phenylalanine and tyrosine levels, indicating that in vivo PAL activity was inhibited by AOPP. The increase in extractable PAL caused by AOPP may be a result of decreased feedback inhibition of PAL synthesis by cinnamic acid and/or its derivatives. AOPP alone had no effect on growth (fresh weight and elongation) at either concentration, but at 0.1 millimolar it slightly alleviated growth (fresh weight) inhibition caused by 0.5 millimolar glyphosate after 4 days. Reduction of the free pool of phenylalanine by glyphosate was reversed by AOPP. These results indicate that glyphosate exerts some of its effects through reduction of aromatic amino acid pools through increases in PAL activity and that not all growth effects of glyphosate are due to reductions of aromatic amino acids.     

Effects of Glyphosate on Metabolism of Phenolic Compounds VIII. Comparison of the Effects of Aminooxyacetate and Glyphosate

Aminooxyacetate (AOA), an in vitro inhibitor of phenylalanineammonia-lyase (PAL) and of some transaminases, was tested forcomparison with glyphosate's [N-(phosphonomethyl)glycine] effectson plant growth, PAL activity, and accumulation of hydroxyphenoliccompounds of three-day-old, dark-grown soybean [Glycine max(L.) Merr.] seedlings. Root-fed AOA (50 µM) and glyphosate(0.5 mM) caused similar decreases in growth rate and in accumulationof hydroxyphenolics, anthocyanin and chlorophyll. Together,these compounds were neither antagonistic nor synergistic inaffecting these parameters. AOA caused decreases in extractablePAL activity while increasing aromatic amino acid pools—theopposite of glyphosate's effects. In the light, the effect ofglyphosate on PAL and aromatic amino acids predominated overthose of AOA when the chemicals were given together. The effectsof AOA and glyphosate on most free amino acid levels were similar.In those cases in which the effects differed, glyphosate's effectpredominated over that of AOA when the chemicals were giventogether. The similarities and interactions of AOA and glyphosatein their effects on free amino acid profiles indicate that glyphosatesignificantly interferes with non-aromatic as well as aromaticamino acid synthesis. (Received April 6, 1982; Accepted June 28, 1982)     

The formation of catechin and procyanidins in cell suspension cultures ofCryptomeria japonica

Cell suspension cultures, as well as green leaves, ofCryptomeria japonica contained catechin, epicatechin, procyanidins B-1, B-3 and B-4, and polymeric procyanidins. Those compounds in the cell culture were found to increase, midway through the logarithmic phase, but the polymerization of procyanidins seems to proceed in the stationary phase. The dosage of 0.3 mMl-2-aminooxy-3-phenylpropionic acid (l-AOPP), an inhibitor of phenylalanine ammonia-lyase (PAL), to the cells caused inhibition of the flavan formation to a large extent without significant reduction of growth rate, as well as a large increase in the phenylalanine content of the cells. PAL activity in the cell cultures increased, immediately after transfer to a fresh medium, showed its maximum (a first peak) during 15 hr and a second small peak of the activity in the midst of the logarithmic phase. 0.3 mMl-AOPP inhibited remarkably a first peak of PAL activity, but a second peak was nearly unaffected. 2 mMl-AOPP inhibited the PAL activity completely.     

Increases in enzyme levels during the formation of phenolic acids in carrot cell cultures

The levels of glutamic acid dehydrogenase (GDH), phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (CAH) and O-methyltransferase (OMT) were measured during the formation of phenolic acids in carrot cells in suspension culture. Caffeic, ferulic and p-hydroxybenzoic acids were always present as the culture proceded. Total content of these acids increased at the early logarithmic and linear phases. GDH showed high activity at the early logarithmic and stationary phases. PAL activity was much enhanced at the linear and stationary phases. CAH activity was found in actively growing cells, especially at the early and late logarithmic phases OMT behaved similarly to PAL. The increases in GDH and CAH might be responsible for the rapid synthesis of phenolic acid at the early logarithmic phase. The increase in phenolic acid at the linear phase would certainly be due to enhancements of both PAL and OMT. On the other hand, the accumulation of vanillic acid was observed in cells which were transferred and cultured on an agar medium, but not in cells in suspension culture. This accumulation is related to increases in OMT levels and also to changes in the degree of β-oxidation.     

Increased 5-enolpyruvylshikimic acid 3-phosphate synthase activity in a glyphosate-tolerant variant strain of tomato cells

A glyphosate-tolerant variant of cultured tomato cells (Lycopersicon esculentum × L. peruvianum hybrid) was isolated via a single-step selection. Growth of the variant in suspension culture was essentially unaffected by 10 mM glyphosate, 100 times the concentration needed to significantly reduce the growth rate of wild type cells. When treated with glyphosate, variant cells accumulated much less shikimic acid than did the wild type cells. In analyses of 5-enolpyruvyl-shikimic acid 3-phosphate (EPSP) synthase activity in two separate experiments, the variant cells had 8 and 13 times higher specific activity than the wild type cells. The enzyme activities from the two types of cells were equally inhibited by glyphosate. These results suggest that the glyphosate tolerance of the variant results from overaccumulation of a glyphosate-sensitive EPSP synthase. Attempts to regenerate fertile plants from the variant cells were unsuccessful, but abnormal shoots were regenerated and callus from leaves of these shoots retained the tolerance to glyphosate.     

Alcohol Dehydrogenase from Cultured Rice Cells

Rice cells in suspension culture had high alcohol dehydrogenaseactivity during the logarithmic growth phase (3rd to 5th day).Ethanol was accumulated both in the cells and in the medium.The highest amount of ethanol was accumulated on the 4th dayin cells (10 µmoles/g fresh weight) and during the stationarygrowth phase (8th day) (180 mM, ca. 1%) in the medium. The enzymewas isolated from the cell extract and purified 36-fold witha 14% yield by ammonium sulfate fractional precipitation, andchromatography on DEAE-Sephadex, Sephadex G-150 and Blue Dextran-Sepharose.The purified enzyme was homogeneous, as judged by its sedimentationvelocity, and poly acrylamide gel, starch gel and SDS-polyacrylamidegel electrophoreses. Its molecular weight was 76,000 distributedin two, identical 37,000 subunits. The isoelectric point wasat pH 5.5. The enzyme contained 2.1 g atoms of zinc, 12 freeSH groups and 3 to 4 SS bonds per molecule. The pH optimum forethanol oxidatioa was pH 9.5 and for acetaldehyde reductionpH 6.0. The K_m values for ethanol, NAD^$, acetaldehyde and NADHwere 64.5 mM, 47.1 µM, 1.3 mM and 9.5 µM. The aminoacid composition, substrate specificity, and the effects ofchelators, SH reagents and sugar metabolic intermediates alsoare reported. (Received August 25, 1981; Accepted December 7, 1981)     

Production of caffeic,chlorogenic and rosmarinic acids in plants and suspension cultures of Glechoma hederacea

Glechoma hederacea L. (Lamiaceae) is a perennial plant, which is distributed widely in Europe, Asia and America. Important anti-oxidant compounds are caffeic acid esters like rosmarinic acid (RA) and chlorogenic acid (CA). Phenylalanine ammonia-lyase (PAL) and rosmarinic acid synthase (RAS, 4-coumaroyl-CoA:hydroxyphenyllactic acid hydroxycinnamoyltransferase) contribute to the formation of RA. Our aim in this study was to follow the accumulation of RA, CA and caffeic acid in a suspension culture of G. hederacea. Growth, medium and secondary metabolism parameters were determined during a culture period of 14 days. The maximal PAL activity was observed on day 5 and the maximal RAS activity on day 8. The RA content was exceedingly high and reached 25.9% of the dry mass on day 7. Caffeic acid and CA contents remained rather low. Furthermore, the presence of RA, CA and caffeic acid and the expression patterns of RAS and hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (HST), an important enzyme of monolignol formation, in leaves, flowers, stems and roots of naturally grown G. hederacea were assessed. The expression of RAS and HST genes was detectable in all organs except roots. Flowers accumulated 12.5% RA in their dry mass, leaves, stems and roots about 1%. CA was highest in leaves (2.0%), while it was at 1.6% in flowers, 1.3% in stems and almost undetectable in roots. The caffeic acid content remained at or below 0.4% of the dry weight in all organs.     

Changes of shikimate pathway in glyphosate tolerant alfalfa cell lines with reduced embryogenic ability

Glyphosate tolerant cell lines were selected from highly embryogenic cell suspension culture ofMedicago sativa L. Resistant cell lines showed significant reduction of embryogenic ability and during long-term culture in the presence of glyphosate gradual loss of this ability was observed. After glyphosate treatment the increased activity of 5-enolpyruvylshikimate-3-phosphate synthase in tolerant cell lines overcame the block in aromatic amino acid synthesis which was observed in control cell lines. Glyphosate caused marked increase in the content of shikimic acid in both control and tolerant cell lines but the accumulation of shikimic acid was considerably lower in tolerant calli. Significant qualitative and quantitative differences were found in the content of individual phenolic acids. The considerable decrease in the amount of cinnamic acid derivates and broader spectrum of hydroxybenzoic acids suggest in tolerant cell lines the activation of alternative pathway not regulated by phenylalanine ammonia lyase. The possible role of altered pool of phenolic acids on the embryogenic ability is discussed.     

Effects of glyphosate on metabolism of phenolic compounds I. Induction of phenylalanine ammonia-lyase activity in dark-grown maize roots

The herbicide glyphosate, N-(phosphonomethyl)glycine, induced phenylalanine ammonia-lyase (PAL) activity in the roots of dark-grown maize seedlings. This enzymes induction was shown to precede glyphosate-reduced fresh weight gain in the roots by 24–48 h. Feeding aromatic amino acids with glyphosate further inhibited growth while slightly lowering glyphosate-enhanced PAL activity. Soluble protein levels in the enzyme preparations were not affected by glyphosate. These results are discussed in terms of possible glyphosate mode of action through stimulating biosynthesis of growth-inhibiting phenolics.     

2,4-D-Sustained Photomixotrophic Growth of a Chlorophyllous Cell Suspension Culture of Nicotiana tabacum

A chlorophyllous, photomixotrophic cell suspension culture oftobacco (Nicotiana tabacum L.) was established using mediumcontaining 30 g/liter of sucrose and 1.5 µM 2,4-D. The2,4-D-sustained photomixotrophic line was able to show rapidregreening in the light after bleaching in the dark and characterizedwith a much slower and longer growth cycle than a heterotrophicline derived from the same original callus (cell doubling timeof 100 h vs. 40 h and duration of logarithmic phase of 17 daysvs. 7 days). The photomixotrophic line took up sucrose morerapidly than the heterotrophic line and accumulated starch duringthe early logarithmic phase when it showed a maximum photosyntheticcapacity on a chlorophyll basis (6.3µmol O₂/min/mg Chl).Chlorophyll content and photosynthetic capacity on a per cellbasis and on a cell fresh weight basis, on the other hand, decreasedduring this phase and reincreased later to reach maximum levels(310 µg Chl/g fr wt; 1.4 µmol O₂/min/g fr wt) whenthe line exhibited the highest activities of dark respiration(1.0 µmol; O₂/min/g fr wt) and cell division (mitoticindex of 3.0%). These characteristics of the photomixotrophicline were lost if it was grown in the dark to become non-chlorophyllous.Although net O₂ evolution could not be detected in the photomixotrophicline throughout the growth cycle when assayed under suboptimumlight intensity, reaccumulation of starch and a marked increasein cell fresh weight upon addition of minerals, vitamins and2,4-D without sucrose at the late logarithmic phase indicatedthe development of photosynthetic activity under the cultureconditions. ¹The investigations reported were included in the thesis submittedto the Graduate School, Faculty of Agriculture, Kobe University,in partial fulfillment of the requirement for M. Agr. degree. (Received May 30, 1988; Accepted October 5, 1988)     

Effect of l-phenylalanine on PAL activity and production of naphthoquinone pigments in suspension cultures of Arnebia euchroma (Royle) Johnst

The effects of l-phenylalanine (PHE) on cell growth and production of shikonin and its derivatives, acetylshikonin (ACS) and isobutyrylshikonin (IBS), in suspension cultures of Arnebia euchroma were examined. Supplementing media using PHE have been successfully utilized to enhance shikonin production in cell cultures of other species of Boraginaceae. l-Phenylalanine, the key compound in the phenylpropanoid pathway, is converted by phenylalanine ammonia lyase (PAL) to trans-cinnamic acid, which is the precursor of p-hydroxybenzoic acid (PHB). Coupling of PHB and geranyl pyrophosphate (derived from mevalonate pathway) by p-hydroxybenzoate-m-geranyltransferase leads later to biosynthesis of shikonins. The addition of 0.01 or 0.1?mM PHE to the culture medium stimulated cell proliferation, where the highest observed increase in fresh cell biomass (measured as a ratio of final weight to initial weight) was 12-fold, in contrast to an eightfold increase in control cultures. Whereas, growth media supplemented with 1?mM PHE markedly reduced the rate of cell growth (to only twofold). Precursor feeding had detrimental effects on both ACS and IBS production in all PHE-supplemented media. The highest total content (intracellular + extracellular) of the investigated red pigments (9.5?mg per flask) was detected in the control culture without PHE. ACS was the major component of the naphthoquinone fraction determined in cells and post-culture media. Shikonin itself was found only in the post-culture media from cultures supplemented with 0.01 or 0.1?mM PHE. Increases in PAL activity corresponded well with the accumulation of investigated naphthoquinones in control culture. However, peak PAL activity did not directly correlate with maximum production of shikonin derivatives. Cytotoxicity of extracts, prepared from the cells cultivated in the presence of PHE or in control cultures, was tested on three cancer cell lines: HL-60, HeLa, and MCF-7. The extracts prepared from the untreated control cultures proved to be the most potent against the examined cancer cell lines. The mean inhibitory concentration values were 0.3, 13, and 8???g?ml^?1 for the HL-60, HeLa, and MCF-7 cells, respectively.     

Effects of Glyphosate on Metabolism of Phenolic Compounds

Light enhanced the inhibiting effect of root-fed glyphosate (5 × 10^?4M) on dry weight accumulation of soybean [Glycine max. (L.) Merr.] seedling axes. Inhibition of growth by light was greatest in hypocotyls, whereas by glyphosate it was greatest in roots. A synergistic effect of light and glyphosate on stimulation of phenylalanine ammonia-Iyase (PAL, E.C. 4.3.1.5) activity was also demonstrated. In continuous white light PAL activity increased linearly for 4 days in axes of seedlings exposed to glyphosate. Evidence of phytochrome involvement in the light effect was shown. The stimulatory effect of glyphosate on PAL activity was greater in roots than in hypocotyls. Soluble hydroxyphenolic compound levels were reduced by glyphosate but were increased by light on a per axis basis. On a fresh weight basis, hydroxyphenolics were more concentrated in glyphosate-treated than in control tissues in the light. When compared to other amino acids, disproportionate decreases in free pools of phenylalanine and tyrosine occurred in axes of seedlings treated with glyphosate and light. The effect of light on all measured parameters was mainly in the hypocotyl, while that of glyphosate was primarily in the root. In the light, glyphosate caused increases in levels of glutamine and other amino acids that may be the result of amination reactions, protecting from excess ammonia generated by enhanced PAL activity. These results suggest that PAL has a strong influence on its substrate levels in this system and/or that glyphosate inhibits synthesis of aromatic amino acids.     

SYNCHRONIZATION OF BACTERIAL CULTURE BY PREINCUBATION OF CELLS AT HIGH CELL DENSITY

The growth of Escherichia coli strain B in a liquid medium wasfound to cease at a cell density of 5x10⁹ cells per ml. (Thiscritical concentration is designated as the maximum or M-concentration.)Even cells harvested from the logarithmic growth phase couldnot divide at this or higher cell densities. Investigationson the metabolic activities of such cultures, however, showedthat the synthesis of cellular protein and nucleic acid wastaking place under such circumstances, showing that only someprocess (or processes) particularly related to cell divisionwas suppressed at the critical cell concentration in question. This finding led us to devise a new method of synchronizationof E. coli: cells harvested from a logarithmic phase were preincubatedat the critical concentration of 5x10⁹ cells per ml for 45 minutes,and then diluted 100 times with fresh medium. This led to synchronizationof cell division, as shown by a stepwise multiplication in cellnumber. (Received June 20, 1961; )     

The influence of some inhibitors on the formation of caffeic acid in cultures ofPerilla cell suspensions

A cell suspension culture, prepared fromPerilla frutescens var.crispa callus induced by Murashige and Skoog (1962) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 ml/l) and kinetin (0.1 mg/l), contained caffeic acid derivatives as the phenolic components. Fresh and dry weights of the cells increased exponentially for about 11 days after transfer to a fresh medium. The contents of caffeic acid and protein also reached a maximum on the 11th day, but α-amino nitrogen phenylalanine and tyrosine continued to increase in amount until the 20th to 23rd day. Caffeic acid formation in the cells was increased by lowering the concentration of 2,4-D. The administration ofl-2-aminooxy-3-phenylpropionic acid (l-AOPP), 2-aminooxyacetic acid (AOA) andN-(phosphonomethyl)glycine (glyphosate) to the cells inhibited caffeic acid formation to a large extent. An 80% inhibition of caffeic acid formation was caused by 10⁻⁴Ml-AOPP whereas phenylalanine and tyrosine contents of the cells became 7.5 and 2.3 times higher at thisl-AOPP concentration than those in the control. An 85% inhibition of caffeic acid formation was achieved at 10⁻³M glyphosate concentration, while 10⁻³M AOA inhibited caffeic acid formation by 95% and also growth rate by 80%. The influence of inhibitors on caffeic acid formation is discussed in relation to the level of α-amino nitrogen, particularly aromatic amino acids, in the cell suspension cultures.     

Phenylalanine ammonia-lyase,phenolic acids and ethylene in alfalfa (Medicago sativa L.) cell cultures in relation to their embryogenic ability

Phenylalanine ammonia-lyase (PAL) activity, contents of phenolic acids and ethylene production during the lag-phase, and contents of phenolic acids at the late exponential phase, showed significant differences in embryogenic (EC) and non-embryogenic (NEC) suspension cultures of Medicago sativa L. Maximum PAL activity at 6 h after inoculation was followed by an increase in the level of phenolic acids from 9.6 g g^–1 fresh mass to 21 g g^–1 fresh mass in NEC at 12 h. Thereafter the level of phenolic acids decreased to 5.2 g g^–1 fresh mass at 72 h. The decline was caused predominantly by the decrease of ester-bound cinnamic acid derivatives, the decrease ranging from 83 to 20% of total phenolics. Two maxima of ethylene production were observed in NEC: the first one immediately after inoculation and the second at 6 h, coinciding with the peak of PAL activity. In NEC, most of the phenolic acids occurred in esterified form. Ability to form somatic embryos (EC) was associated with the absence of the second peak of ethylene production as well as of the peak of PAL activity at 6 h. The level of phenolic acids during the lag-phase remained low (7.2 g g^–1 FM) and did not change. The proportion of cinnamic acid derivatives was very low (18% of total phenolics), mostly due to the extremely low level of ferulic acid. In EC, phenolic acids bound to methanol insoluble material formed the major fraction. Loss of embryogenic potential of the embryogenic culture (ECL) was associated with qualitative and quantitative changes in the contents of phenolic acids insignificantly increased PAL activity after inoculation was followed by a moderate increase in the contents of phenolic acids from 9.35 g g^–1 fresh mass to 12.42 g g fresh mass. A high rate of ethylene production was observed only immediately after the transfer of the culture to fresh medium. The loss of embryogenicity correlated also with changes in the relative amounts of the investigated fractions of phenolic acids. A distinct increase in the level of methoxy-substituted phenolic acids is a characteristic feature of the ECL culture.Abbreviations NEC non-embryogenic suspension culture - EC embryogenic suspension culture - ECL embryogenic suspension culture after the loss of embryogenic potential - AA anisic acid - CA cinnamic acid - CaA caffeic acid - pCA p-coumaric acid - FA ferulic acid - pHBA p-hydroxybenzoic acid - SA syringic acid - SaA salicylic acid - SiA sinapic acid - VA vanillic acid - PhA phenolic acids - HPLC High-Performance Liquid Chromatography - GC Gas Chromatography - 2,4-D 2,4-dichlorophenoxyacetic acid - kin kinetin - NAA 1-naphthaleneacetic acid - BL-medium medium of Blaydess - FM fresh mass     

Differences in the Composition of the Cell Walls of Two Morphologically Different Lines of Suspension-Cultured Catharanthus roseus Cells

Differences in the composition of cell walls of two morphologicallydifferent lines (A and B) of suspension-cultured Catharanthusroseus cells, which have the same origin, were investigated.The cells of strain A are nearly spherical, while those of strainB are cylindrical. In strain A, the amount of cell wall pergram fresh weight of cells increased during the logarithmicphase. In strain B, the amount of cell wall per cell decreasedduring the logarithmic phase. The level of matrix polysaccharides increased markedly duringthe logarithmic phase in strain A. The amount of cellulose incell wall was relatively larger in strain B than in strain A.The following differences in sugar composition between the twostrains were observed: (a) there was an increase in the relativelevels of 4-linked galactose in the EDTA-soluble fraction andof 3-linked glucose in the 5% KOH-soluble fraction during thelogarithmic phase in strain A; (b) there were significantlyhigher levels of arabinose, probably derived from 2,5- and/or3,5-linked arabinan, in the EDTA-soluble fraction and in theextracellular polysaccharides in strain B; (c) there were decreasesin the relative amounts of some kinds of sugar, probably thosederived from xyloglucan, during the stationary phase in strainB. (Received March 31, 1989; Accepted October 12, 1989)     

Photoautotrophic Culture of Undifferentiated Cells and Shoot-Forming Cultures of Digitalis purpurea L.

Undifferentiated cells and shoot-forming cultures of Digitalispurpurea L. were grown photoautotrophically under 1% CO₂. During3 weeks of culture, the undifferentiated cells multiplied 3-foldand the shoot-forming cultures 2-fold on a fresh weight basis.The chlorophyll content, ribulose 1,5-bisphosphate carboxylaseactivity, Hill reaction activity of the isolated chloroplastsand photosynthetic O₂ evolution of the photoautotrophicallygrown cultures were somewhat higher than the values of the correspondingphotomixotrophic cultures. The digitoxin contents, however,were not improved by photoautotrophic culture. (Received November 9, 1983; Accepted June 11, 1984)     

Regulation of anthocyanin and lignin synthesis in Petunia hybrida cell suspensions

In Petunia hybrida cv. Violet 30 cell suspensions the phenylpropanoid pathway can be induced to produce lignin and anthocyanins. Orthovanadate addition leads to lignin accumulation, subculturing the cells using small inoculum sizes (<2 g fresh weight l^-1) gives rise to both anthocyanin and lignin production. Orthovanadate has a negative effect on cell growth. By replacing the medium, one day after orthovanadate addition, by medium without elicitor, we were able to restore growth without disturbing the lignin accumulation. The activity of phenylalanine ammonia-lyase (PAL) increased immediately after orthovanadate addition; this increase stopped upon medium replacement without affecting the lignin production. Reduction of the NAA concentration from 2 mg l^-1 to 0.1 mg l^-1, subsequent to the elicitation by orthovanadate or dilution stress, gave rise to a further increase in the production of lignin and anthocyanins respectively. Decreasing the NAA concentration without a prior elicitation, didn't have any effect on either PAL activity or product formation.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - BSA bovine serum albumine - FW fresh weight - NAA naphthaleneacetic acid - PAL phenylalanine ammonia-lyase - PPP phenyl propanoid pathway