Carrot Cell Suspension Research Articles

Regulation of glutamate dehydrogenase activity by manipulation of nucleotide supply in Daucus carota suspension cultures

The enzyme glutamate dehydrogenase (GDH, EC 1.4.1.2) is ubiquitous in plant species. It is now generally accepted that the primary role of this enzyme is not assimilation of ammonium and it has been suggested that GDH may be important in provision of carbon skeletons under conditions of carbon limitation. In carrot (Daucus carota L. Chantenay) cell suspension cultures carbon starvation results in de‐repression of GDH activity. The regulation of this de‐repression has not been investigated. This paper examines the possibility that the availability of adenosine nucleotides is instrumental in the regulation of GDH activity. In repressed cultures the adenosine nucleotides cAMP (0.2 mM), AMP (0.2 mM) and ADP (0.4 mM) caused an increase in GDH activity of 61, 33 and 7%, respectively. ATP (0.2 mM) had the opposite effect in maintaining repression of GDH. Under de‐repressed conditions only cAMP (0.2 mM) enhanced GDH activity (14%). Inhibition of oxidative phosphorylation using a range of inhibitors resulted in de‐repression of GDH and stimulation of respiration. The results from this work indicate that exogenously applied adenosine nucleotides and electron transport inhibitors alter the GDH repression/de‐repression status. Addition of these compounds alters or disrupts ATP levels, mimicking carbon depletion. This causes an increase in GDH activity, supporting the idea that GDH may provide carbon skeletons for carbon metabolism and suggesting that ATP status is important in regulation of the enzyme activity.

Action spectrum for induction of promoter activity of phenylalanine ammonia-lyase gene by UV in carrot suspension cells.

The full-length promoter (-2335) of the carrot (Daucus carota) phenylalanine ammonia-lyase gene (gDcPAL1) fused to the luciferase reporter gene was transiently transformed to carrot protoplasts by electroporation, and the promoter activity induced by monochromatic UV light of various wavelengths was examined. The action spectrum constructed from the fluence-response curves showed a single peak at around 280 nm, suggesting that the activation of the gDcPAL1 promoter is categorizable as one of the UVB light responses. The same assay system was applied to variously truncated gDcPAL1 promoters and to CaMV35S promoter fusion with various parts 5'-upstream of the gDcPAL1 promoter. The region from -396 to -190 (relative to the transcription start site) fused to the CaMV35S core (-90) promoter showed a 280 nm-dominant response. However, gDcPAL1 promoters truncated above -570 and -396, although they contain the region between -396 and -190, did not show such a typical UVB response, i.e. they responded to 260 nm light as much as to 280 nm light. The promoter truncated to below -190 also responded to 260 nm light as much as to 280 nm light. Therefore we assumed that the gDcPAL1 promoter is composed of three functionally different parts: the upstream above -570 (modulator), the region from -396 to -190 (UVB responsive) and the down-stream below -190 (UVB and C responsive). The overall UVB response of the gDcPAL1 full-length promoter is explained as the result of interaction of these three components.

Attachment of Agrobacterium tumefaciens to carrot cells and Arabidopsis wound sites is correlated with the presence of a cell-associated, acidic polysaccharide.

An early step in crown gall tumor formation involves the attachment of Agrobacterium tumefaciens to host plant cells. A. tumefaciens C58::A205 (C58 attR) is a Tn3HoHo1 insertion mutant that was found to be avirulent on Bryophyllum daigremontiana and unable to attach to carrot suspension cells. The mutation mapped to an open reading frame encoding a putative protein of 247 amino acids which has significant homology to transacetylases from many bacteria. Biochemical analysis of polysaccharide extracts from wild-type strain C58 and the C58::A205 mutant showed that the latter was deficient in the production of a cell-associated polysaccharide. Anion-exchange chromatography followed by 1H nuclear magnetic resonance and gas chromatography-mass spectrometry analyses showed that the polysaccharide produced by strain C58 was an acetylated, acidic polysaccharide and that the polysaccharide preparation contained three sugars: glucose, glucosamine, and an unidentified deoxy-sugar. Application of the polysaccharide preparation from strain C58 to carrot suspension cells prior to inoculation with the bacteria effectively inhibited attachment of the bacteria to the carrot cells, whereas an identical preparation from strain C58::A205 had no inhibitory effect and did not contain the acidic polysaccharide. Similarly, preincubation of Arabidopsis thaliana root segments with the polysaccharide prevented attachment of strain C58 to that plant. This indicates that the acidic polysaccharide may play a role in the attachment of A. tumefaciens to host soma plant cells.

Effect of physiological activities on aluminum uptake in carrot (Daucus carota L.) cells in suspension culture

Responses to aluminum (AI) of carrot (Daucus carota L.) cells in suspension culture (hereafter referred as “carrot suspension cells”) were investigated under different physiological activities. The respiration rate of the cells at the logarithmic growth phase decreased when the external Al concentration increased. A similar tendency was observed for the ATP content of the cells when the external Al concentration of the medium increased. Decrease of about 50% of the respiration rate and of about 35% of the ATP content of the cells was. observed at 300 µm AlCl3 at 24 h, respectively. The Al content (total and citrate-insoluble AI) in cells increased when the external Al concentration increased. Both the respiration rate and the ATP content in cells were reduced when the culture temperature decreased or ATP synthesis inhibitors, namely CCCP (carbonylcyanide-m-chlorophenylhydrazone) and DNP (2,4-dinitrophenol) were added. The addition of SHAM (salicylhydroxamic acid) did not affect the ATP content in cells. The Al content of the cells treated with 50 µm Al together with SHAM was similar to that of the cells treated with only AI. However, the Al content of the cells increased when the cells were treated with 50 µm Al together with CCCP, DNP, or a low temperature (15°C). The content of citrate Al content in cells increased rapidly when ATP synthesis inhibitors were present or the culture temperature was low. There was a negative linear correlation between the Al content and the ATP content in cells. These results suggest that the cells containing a large amount of ATP and showing normal functions of the plasma membranes may display an apparent tolerance to Al by suppressing Al absorption into their cytoplasm at a cell suspension level.

Polyamines and Pectins (I. Ion Exchange and Selectivity).

The ion-binding and -exchange properties of putrescine, spermidine, and spermine on purified walls of carrot (Daucus carota L.) cell suspensions were investigated by producing ion-exchange isotherms and comparing them with the behavior of Na+, Mg2+, and Ca2+. The cation exchange capacity of the carrot cell walls was 0.8 equivalent kg-1 dry matter, and the ionic selectivity sequence of the walls for polyamines followed the sequence spermine4+ > spermidine3+ [almost equal to] Ca2+ > putrescine2+. The polyamines were subjected to only electroselectivity and probably did not induce any favorable supramolecular conformation of pectin like the one induced by Ca2+. Triangular ion exchanges were also performed with three diamines: ethanediamine, butanediamine, and octanediamine. The shorter the diamine, the higher the total adsorption and selectivity of the exchange. The lower selectivity of the cell wall for putrescine was partly attributed to its inability to access and displace Ca2+ from higher affinity sites within dimerized pectic sequences. The polyamine adsorption and exchange on pectic sequences could result in pectic signal modulation in pathogenesis and in differentiation.

Image Analysis Based Quantification of Cells in Suspension Cultures for Producing Somatic Embryos.

A machine vision system which used macroscopic images of carrot cell suspensions was developed. This system was used to non-invasively evaluate cell growth. Packed cell volume (PCV) of the suspension was estimated by two features extracted by image analysis using the B component of the RBG color image. One feature was the number of pixels which were extracted by thresholding the B component at an appropriate value. It represented projected area of cells and cell aggregates, and could be used for PCV estimation. Another feature was the sum total of B values of all pixels in the suspension image. It was also used for PCV estimation having the advantage that it did not require a procedure for extracting pixels corresponding to cells. Using this system, an increase in specific growth rate with a decrease of embryogenic potential was monitored in a carrot embryogenic cell suspension culture. This system showed the potential to be used to select cell lines with desirable growth characteristics for somatic embryo production.

Isolation of microtubule-associated proteins from carrot cytoskeletons: a 120 kDa map decorates all four microtubule arrays and the nucleus.

A method for biochemically isolating microtubule-associated proteins (MAPs) from the detergent-extracted cytoskeletons of carrot suspension cells has been devised. The advantage of cytoskeletons is that filamentous proteins are enriched and separated from vacuolar contents. Depolymerization of cytoskeletal microtubules with calcium at 4 degrees C releases MAPs which are then isolated by association with taxol stabilized neurotubules. Stripped from microtubules (MTs) by salt, then dialysed, the resulting fraction contains a limited number of high molecular weight proteins. Turbidimetric assays demonstrate that this MAP fraction stimulates polymerization of tubulin at concentrations at which it does not self-assemble. By adding it to rhodamine-conjugated tubulin, the fraction can be seen to form radiating arrays of long filaments, unlike MTs induced by taxol. In the electron microscope, these arrays are seen to be composed of mainly single microtubules. Blot-affinity purified antibodies confirm that two of the proteins decorate cellular microtubules and fulfil the criteria for MAPs. Antibodies to an antigenically related triplet of proteins about 60-68 kDa (MAP 65) stain interphase, preprophase band, spindle and phragmoplast microtubules. Antibodies to the 120 kDa MAP also stain all of the MT arrays but labelling of the cortical MTs is more punctate and, unlike anti-MAP 65, the nuclear periphery is also stained. Both the anti-65 kDa and the anti-120 kDa antibodies stain cortical MTs in detergent-extracted, substrate-attached plasma membrane disks ('footprints'). Since the 120 kDa protein is detected at two surfaces (nucleus and plasma membrane) known to support MT growth in plants, it is hypothesized that it may function there in the attachment or nucleation of MTs.

Ionic transport in the plasma membrane of carrot protoplasts from embryogenic cell-suspension cultures

Ionic transport properties of protoplasts obtained from embryogenic carrot suspension cells were studied by the patch-clamp technique. In the whole-cell configuration, carrot protoplasts presented macroscopic time-dependent outward currents, showing kinetics of activation which did not depend appreciably on the amplitude of the stimulus. Time- and voltage-dependent whole-cell inward rectifying currents as well as instantaneous non-selective currents were also observed. Both time-dependent inward and outward currents are carried by potassium ions. In a cell-attached configuration, two types of single-channel signals, displaying conductances of 10 and 17 pS, were observed; the instantaneous 10 pS channel was also present in outside-out excised patches.

A kinesin-like protein, KatAp, in the cells of arabidopsis and other plants.

The kinesin-like proteins (KLPs) are a large family of plus- or minus-end-directed microtubule motors important in intracellular transport, mitosis, meiosis, and development. However, relatively little is known about plant KLPs. We prepared an antibody against two peptides in the microtubule binding domain of an Arabidopsis KLP (KatAp) encoded by the KatA gene, one of a family of genes encoding KLPs whose motor domain is located near the C terminus of the polypeptide. Such KLPs typically move materials toward the minus end of microtubules. An immunoreactive band (Mr of 140,000) corresponding to KatAp was demonstrated with this antibody on immunoblots of Arabidopsis seedling extracts. During immunofluorescence localizations, the antibody produced weak, variable staining in the cytoplasm and nucleus of interphase Arabidopsis suspension cells but much stronger staining of the mitotic apparatus during division. Staining was concentrated near the midzone during metaphase and was retained there during anaphase. The phragmoplast was also stained. Similar localization patterns were seen in tobacco BY-2 cells. The antibody produced a single band (Mr of 130,000) in murine brain fractions prepared according to procedures that enrich for KLPs (binding to microtubules in the presence of AMP-PNP but not ATP). A similar fraction from carrot suspension cells yielded a cross-reacting polypeptide of similar apparent molecular mass. When dividing BY-2 cells were lysed in the presence of taxol and ATP, antibody staining moved rapidly toward the poles, supporting the presence of a minus-end motor. Movement did not occur without ATP, with AMP-PNP, or with ATP plus antibody. Our results indicate that the protein encoded by KatA, KatAp, is expressed in Arabidopsis and is specifically localized to the midzone of the mitotic apparatus and phragmoplast. A similar protein is also present in other species.

Purification and characterization of glycosyltransferases involved in anthocyanin biosynthesis in cell-suspension cultures ofDaucus carota L.

The major anthocyanins accumulated by an Afghan cultivar of Daucus carota L. are cyanidin 3-(xylosylglucosylgalactosides) acylated with sinapic or ferulic acid. The formation of the branched triglycoside present as a common structural element requires an ordered sequence of glycosylation events. Two of these enzymic glycosylation reactions have been detected in protein preparations from carrot cell-suspension cultures. The first step is a galactosyl transfer catalyzed by UDP-galactose: cyanidin galactosyltransferase (CGT) resulting in cyanidin 3-galactoside. The putative second step is the formation of cyanidin 3-(xylosylgalactoside) catalyzed by UDP-xylose: cyanidin 3-galactoside xylosyltransferase (CGXT). Both enzyme activities were characterized from crude protein preparations. The CGT was purified 526-fold from the cytosolic fraction of UV-irradiated cell cultures by ion-exchange chromatography on diethylaminoethyl (DEAE)-Sephacel, affinity chromatography on Blue Sepharose CL-6B, gel permeation chromatography on Sephadex G-75 and elution from the gel matrix after non-dissociating PAGE. Its molecular mass was estimated by SDS-PAGE and by calibrated gel permeation chromatography on Sephadex G-75. In both cases a molecular mass of 52 kDa was determined, indicating that the native protein is a monomer of 52 kDa. The galactosyl transfer and the xylosyl transfer are presumed to be catalyzed by separate enzymes.

Callose Synthesis in Spirostanol Treated Carrot Cells is Not Triggered by Cytosolic Calcium, Cytosolic pH or Membrane Potential Changes

Carrot (Daucus carota L.) cell suspensions were treated with a spirostanol saponin from Yucca. This saponin is an elicitor of callose synthesis. Irrespectively of the mode of action of spirostanol on the callose synthase activity itself, the spirostanol-induced callose synthesis in carrot is not preceded by changes in membrane potential, cytosolic free calcium or cytosolic pH. The inability of modulators of cytosolic free calcium content (verapamil, nifedipine and Br-A23187), EGTA and a proton pump inhibitor (vandate) to inhibit or induce callose formation is consistent with a calcium- and pH-independent mechanism for callose deposition.

Evidence for deamination by glutamate dehydrogenase in higher plants: Commentary

Glutamate dehydrogenase (GDH; EC 1.4.1.2) catalyses the interconversion of 2-oxoglutarate and glutamate. For many years it was assumed that GDH established a net flux in the direction of glutamate synthesis in higher plants and that this ubiquitous enzyme was responsible for the assimilation of ammonium (Davies 1968). However, this view changed with the discovery of glutamate synthase (GOGAT; EC 1.4.1.13), and it is now generally accepted that it is the glutamate synthase cycle, involving the successive operation of glutamine synthetase (GS; EC 6.3.1.2) and GOGAT, that is the major pathway for the assimilation of ammonium by higher plants (Lea and Miflin 1974; Stewart et al. 1980; Lea et al. 1990; Oaks 1994). Although it has been argued that there may be circumstances in which GDH could still provide a route for ammonium assimilation (Cammaerts and Jacobs 1985; Srivastava and Singh 1987; Loyola-Vargas et al. 1988; Magalhaes et al. 1990), recent attention has focused on the oxidative deamination of glutamate by GDH and the hypothesis that GDH performs an anaplerotic function in plants (Robinson et al. 1991, 1992). We used a combination of in vivo and in vitro methods to investigate the function of GDH in carrot (Daucus carota L.) cell suspension cultures, and the results were entirely consistent with a role for the enzyme in the deamination of glutamate. Firstly, enzyme assays of cell extracts showed that GDH activity was lowest in cells in the exponential phase of growth and reached a maximum in the stationary phase (Robinson et al. 1991). This suggests that GDH is unlikely to be important in ammonium assimilation, since the exponential phase coincides with a period of rapid nitrogen assimila-

Chapter 16 Methods for Isolation and Analysis of the Cytoskeleton

Publisher Summary This chapter discusses methods for the isolation and analysis of the cytoskeleton (CSK). The plant CSK consists of microfilaments (MFs) and microtubules (MTs) interchanging dynamically with their corresponding monomeric proteins, actin and tubulin, and perhaps of intermediate filaments. Monomeric actin and tubulin have been isolated from many plant tissues and described. The chapter focuses on the isolation of the CSK itself and deals with solubilized monomers. The first methods for isolating relatively intact CSK in amounts sufficient for biochemical analysis employed protoplasts from carrot suspension cells (Hussey et al ). These pioneering methods suffer from two potential drawbacks: (1) they are suitable only for tissues that yield protoplasts easily and (2) the conditions needed to release protoplasts can themselves cause substantial changes in the CSK and thus lead to artifacts (Tan and Boss). Relatively little is known about actin-binding proteins (ABPs) and tubulin-binding proteins (TBPs) and even less about MF-associated proteins (MFAPs) and MT-associated proteins (MTAPs) because the isolation of actin as MFs and tubulin as MTs is a prerequisite for identifying MF-binding proteins (MFBPs), MTAPs, and any other cellular components that might associate in vivo with the CSK.

The Regulation of Gene Expression in Transformed Maize Aleurone and Endosperm Protoplasts (Analysis of Promoter Activity, Intron Enhancement, and mRNA Untranslated Regions on Expression)

Gene expression in the aleurone and endosperm is highly regulated during both seed development and germination. Studies of alpha-amylase expression in the aleurone of barley (Hordeum vulgare) have generated the current paradigm for hormonal control of gene expression in germinating cereal grain. Gene expression studies in both the aleurone and endosperm tissues of maize (Zea mays) seed have been hampered because of a lack of an efficient transformation system. We report here the rapid isolation of protoplasts from maize aleurone and endosperm tissue, their transformation using polyethylene glycol or electroporation, and the regulation of gene expression in these cells. Adh1 promoter activity was reduced relative to the 35S promoter in aleurone and endosperm protoplasts compared to Black Mexican Sweet suspension cells in which it was nearly as strong as the 35S promoter. Intron-mediated stimulation of expression was substantially higher in transformed aleurone or endosperm protoplasts than in cell-suspension culture protoplasts, and the data suggest that the effect of an intron may be affected by cell type. To examine cytoplasmic regulation, the 5' and 3' untranslated regions from a barley alpha-amylase were fused to the firefly luciferase-coding region, and their effect on translation and mRNA stability was examined following the delivery of in vitro synthesized mRNA to aleurone and endosperm protoplasts. The alpha-amylase untranslated regions regulated translational efficiency in a tissue-specific manner, increasing translation in aleurone or endosperm protoplasts but not in maize or carrot cell-suspension protoplasts, in animal cells, or in in vitro translation lysates.

Ammonium Assimilation and the Role of [gamma]-Aminobutyric Acid in pH Homeostasis in Carrot Cell Suspensions.

In vivo 15N NMR spectroscopy was used to monitor the assimilation of ammonium by cell-suspension cultures of carrot (Daucus carota L. cv Chantenay). The cell suspensions were supplied with oxygen in the form of either pure oxygen ("oxygenated cells") or air ("aerated cells"). In contrast to oxygenated cells, in which ammonium assimilation had no effect on cytoplasmic pH, ammonium assimilation by aerated cells caused a decrease in cytoplasmic pH of almost 0.2 pH unit. This led to a change in nitrogen metabolism resulting in the accumulation of [gamma]-aminobutyric acid. The metabolic effect of the reduced oxygen supply under aerated conditions could be mimicked by artificially decreasing the cytoplasmic pH of oxygenated cells and was abolished by increasing the cytoplasmic pH of aerated cells. The activity of glutamate decarboxylase increased as the cytoplasmic pH declined and decreased as the pH recovered. These findings are consistent with a role for the decarboxylation of glutamate, a proton-consuming reaction, in the short-term regulation of cytoplasmic pH, and they demonstrate that cytoplasmic pH influences the pathways of intermediary nitrogen metabolism.

Gibberellic acid decreases anthocyanin accumulation in wild carrot cell suspension cultures but does not alter 3'-nucleotidase activity

Gibberellic acid decreases anthocyanin accumulation in wild carrot cell suspension cultures but does not alter 3'-nucleotidase activity

Gibberellic acid decreases anthocyanin accumulation in wild carrot cell suspension cultures but does not alter 3′‐nucleotidase activity

Gibberellic acid (GA3) applied at different times during the growth of wild carrot (Daucus carota ssp. Carota) cell suspension cultures inhibited anthocyanin accumulation. Application of 3 × 10–6M GA3 to cultures on day 0 or day 4 gave, respectively, 10 or 35% of anthocyanin accumulation relative to levels occurring when GA3 was applied at the end of the growth period. Endogenous GAs were separated by high pressure liquid chromatography, and identified and quantified by gas chromatography‐selected ion monitoring. Gibberellins GA1, GA3 and traces of GA8. GA19 and GA20 were identified in carrot cell suspension cultures of both high and low anthocyanin‐accumulating clones. The concentrations of GA1. GA3 and GA8 in the two clones were similar and were not significantly different after the application of uniconazole which promoted anthocyanin accumulation. This suggests that these endogenous GAs are not the sole factors controlling the accumulation of anthocyanin in these different clones. Exogenous GA3 and uniconazole had no effect on 3′‐nucleotidase and 5′‐nucleotidase activity in the carrot cell suspension cultures. Thus 3′‐nucleotidase does not appear to play a role in the inhibition of anthocyanin accumulation by exogenous GA3.

Effects of gibberellic acid and uniconazole on the activities of some enzymes of anthocyanin biosynthesis in carrot cell cultures

Gibberellic acid (GA3) inhibition of anthocyanin accumulation by carrot cell-suspension cultures was reversed by supplying dihydroquercitin or naringenin to the culture and not by supplying 4-coumaric acid or malonic acid. This suggested that gibberellic acid was inhibiting chalcone synthase, chalcone isomerase, or acetyl CoA carboxylase. Acetyl-CoA-carboxylase specific activity was the same in GA3-treated and untreated cultures and was not detected in cultures treated with uniconazole, an inhibitor of gibberellic acid biosynthesis. Chalcone-isomerase specific activity was lower in GA3-treated cultures than in untreated cultures and was lower in uniconazole-treated cultures than in the GA3-treated cultures. The total chalcone synthase activity in extracts from GA3- and from uniconazole-treated cells was not significantly different from that in extracts of untreated tissue. When these extracts were chromatographed on a Mono Q column, three peaks of chalcone synthase activity were found in extracts of nontreated cells, whereas only two of these peaks were detected in extracts of GA3-treated cells. The extracts from GA3-treated cells did not contain the peak of chalcone synthase activity that, in untreated cells, preceded the main peak. The correlation between the absence of this peak and the inhibition of anthocyanin accumulation suggests that this form of chalcone synthase is responsible for anthocyanin synthesis and that GA3 prevents this form from appearing in the cells.

Voltage-dependent calcium-permeable channels in the plasma membrane of a higher plant cell.

Numerous biological assays and pharmacological studies on various higher plant tissues have led to the suggestion that voltage-dependent plasma membrane Ca2+ channels play prominent roles in initiating signal transduction processes during plant growth and development. However, to date no direct evidence has been obtained for the existence of such depolarization-activated Ca2+ channels in the plasma membrane of higher plant cells. Carrot suspension cells (Daucus carota L.) provide a well-suited system to determine whether voltage-dependent Ca2+ channels are present in the plasma membrane of higher plants and to characterize the properties of putative Ca2+ channels. It is known that both depolarization, caused by raising extracellular K+, and exposure to fungal toxins or oligogalacturonides induce Ca2+ influx into carrot cells. By direct application of patch-clamp techniques to isolated carrot protoplasts, we show here that depolarization of the plasma membrane positive to -135 mV activates Ca(2+)-permeable channels. These voltage-dependent ion channels were more permeable to Ca2+ than K+, while displaying large permeabilities to Ba2+ and Mg2+ ions. Ca(2+)-permeable channels showed slow and reversible inactivation. The single-channel conductance was 13 pS in 40 mM CaCl2. These data provide direct evidence for the existence of voltage-dependent Ca2+ channels in the plasma membrane of a higher plant cell and point to physiological mechanisms for plant Ca2+ channel regulation. The depolarization-activated Ca(2+)-permeable channels identified here could constitute a regulated pathway for Ca2+ influx in response to physiologically occurring stimulus-induced depolarizations in higher plant cells.

Elicitor induction of enzyme activities and 6-methoxymellein production in carrot cell suspension culture

Fusarium moniliforme endo-polygalacturonase and an elicitor preparation derived from the cell walls of Phythophthora megasperma induced in carrot cell suspension cultures both the accumulation of the carrot phytoalexin, 6-methoxymellein (6-MM), and the activity of 6-hydroxymellein O-methyltransferase (6-HM O-MT), an enzyme which specifically methylates the C-6-hydroxyl group in 6-hydroxymellein (6-HM) to give 6-MM. These fungal elicitors also stimulated a marked increase of phenylalanine ammonia-lyase (PAL) activity without affecting chalcone synthase (CHS) activity. A constitutive caffeic acid O-methyltransferase activity was identified in the same cultured carrot cells.