Control of synthesis of functional mRNA coding for phenylalanine ammonia-lyase from Rhodosporidium toruloides

The regulation of functional mRNA coding for phenylalanine ammonia-lyase (PAL) from Rhodosporidium toruloides was investigated. Polyadenylic acid [poly(A)]-containing RNA was an efficient template for in vitro translation in rabbit reticulocyte lysate. Non-poly(A)-containing RNA did not stimulate in vitro protein synthesis. Several lines of experimental evidence indicate that mRNA from R. toruloides directs PAL synthesis in reticulocyte lysate: (i) the major radioactive product in immunoprecipitates when lysates, incubated with yeast poly(A)-containing RNA, were reacted with PAL-antiserum had the same molecular weight as native PAL (75,000); (ii) this major radioactive product competes with authentic PAL for binding to PAL-antiserum; and (iii) partial proteolytic peptide maps of the in vitro translation product were very similar to those of native PAL. The levels of functional mRNA coding for PAL, when R. toruloides was grown in different physiological conditions, were determined by quantitation of PAL synthesized in vitro when RNA was added to reticulocyte lysate. Functional PAL mRNA was six times higher in yeast grown on phenylalanine compared with glucose-phenylalanine minimal medium. No functional PAL mRNA was detected in yeast grown on glucose-ammonia minimal medium in the presence or absence of phenylalanine. These observed changes in functional PAL mRNA were similar to levels of PAL catalytic and antigenic activity. The kinetics of functional PAL mRNA synthesis and degradation were studied. Maximum levels of functional PAL mRNA were observed within 60 min of transfer to PAL-inducing growth conditions. Poly(A)-containing RNA and functional PAL mRNA were rapidly degraded when cells were transferred from phenylalanine to glucose-ammonia minimal medium, with half-lives of 25 and 10 min, respectively. Thus, it is suggested that the alterations in the amount of PAL in cells of R. toruloides grown in different physiological conditions primarily result from alteration in the amount of functional mRNA coding for the enzyme.     

Some Biochemical Effects of Glyphosate on Plant Meristems

Glyphosate promoted the activity of phenylalanine ammonia lyase(PAL) in both single node rhizome buds of Agropyron repens androot tips of wheat seedlings, in the latter case correlatinginversely with decline in respiratory activity. Differentialrates of ¹⁴C-amino acid incorporation suggest that glyphosatedepleted the phenylalanine protein precursor pool with resultantinhibition of protein synthesis, as evidenced by a rapid declinein the level of ‘soluble’ protein. Promotion ofPAL is assumed to occur by derepression through decline in end-productlevels. Activities of shikimate: NADP oxidoreductase (SORase),chorismate mutase, and polyphenol oxidase (PPO) were also elevated,but to lesser extents than for PAL.     

Compartmentation of Phenolic Compounds and Phenylalanine Ammonia-Lyase in Leaves of Phyllanthus tenellus Roxb. and their Induction by Copper Sulphate

The compartmentation of phenolic compounds in mature leavesof Phyllanthus tenellus and their induction by copper sulphatewere analysed at histological and subcellular levels. Lightand electron microscopy studies demonstrated that the vacuolesof spongy cells were the main sites of phenolic accumulation.Spraying plants with copper sulphate induced punctated lesionsformed by groups of necrotic cells which accumulated brownishsubstances. Histochemical tests and fluorescence microscopyanalysis of the sprayed leaves indicated that the phenolic compoundsincreased in spongy cells within the lesions. Ultrastructuralanalyses showed that 3 h after elicitation, the organelles ofthe cells within the lesion started to collapse and the contentof phenolic substances increased in the vacuole of spongy cells.Antibody against phenylalanine ammonia-lyase (PAL) from parsleycross-reacted with the crude extract of P. tenellus leaves.Two isoforms, one of 65 kD and the other of 66 kD, were identified.Immunocytochemical studies showed that PAL was synthesized inthe palisade and spongy cells, mainly in the cytoplasm and chloroplasts.The phytotoxicity of Cu²⁺ions induced the accumulation of PALin sub-cellular compartments of palisade cells. PAL accumulationstarted to increase 3 h after elicitation and reached a maximumafter 6 h, decreasing 12 h post-induction. The increase of PALwas more evident in cells within the necrotic punctated regionsthan in surrounding cells. Since the vacuole of palisade cellsdid not accumulate phenolic compounds, the in situ studies suggestedthat the end products of PAL synthesis play a role in palisadecell wall reinforcement or might accumulate in other tissues.The symptoms induced by copper sulphate suggest that this abioticelicitor may be a useful tool in the understanding of the regulationof biosynthetic phenolic pathways inP. tenellus . Copyright2000 Annals of Botany Company Cell death, copper sulphate, heavy metal, immunolabelling, phenolic compounds, phenylalanine ammonia-lyase,Phyllanthus , transmission electron microscopy, ultrastructure     

Immunocytochemical Localization of Phenylalanine Ammonia-Lyase and Cinnamyl Alcohol Dehydrogenase in Differentiating Tracheary Elements Derived from Zinnia Mesophyll Cells

Secondary wall thickening is the most characteristic morphologicalfeature of the differentiation of tracheary elements. Isolatedmesophyll cells of Zinnia elegans L. cv. Canary Bird in differentiationmedium are converted to tracheary elements, which develop lignifiedsecondary wall thickenings. Using this system, we investigatedthe distribution of two enzymes, phenylalanine ammonia-Iyase(PAL) (EC 4.3.1.5 [EC] ) and cinnamyl alcohol dehydrogenase (CAD)(EC 1.1.1.195 [EC] ), by both biochemical and immunological methods.Both PAL and CAD appear to be key enzymes in the biosynthesisof lignin precursors, and they have been shown to be associatedwith the differentiation of tracheary elements. Cultured cellswere collected after various times in culture. The culture mediumwas separated from cells by centrifugation and designated fraction(1), the extracellular fraction. The collected cells were homogenizedand separated into four fractions: (2) cytosol; (3) microsomes;(4) cell walls (loosely bound material); and (5) cell walls(tightly bound material). PAL activity was detected in eachfraction. The extracellular fraction consistently had the greatestPAL activity. Moreover, PAL activity in the cytosolic fractionincreased rapidly prior to lignification, as it did in boththe microsomal and the cell wall (tightly bound) fractions duringlignification. Antisera against PAL and against CAD detectedthe proteins with molecular masses that corresponded to thoseof PAL and CAD in Zinnia. Immuno-electron microscopy revealedthat, in differentiating tracheary elements, PAL was dispersedin the cytoplasmic matrix and was located on Golgi-derived vesiclesand on the secondary wall thickenings. "Cell-free" immuno-lightmicroscopy supported the putative distribution of PAL on lignifyingsecondary walls. The pattern of distribution of CAD was similarto that of PAL. Thus, both PAL and CAD seemed to be localizedin secondary wall thickenings. From the results of both biochemicalassays and immunocytochemical staining, it appeared that atleast two types of PAL and CAD are present in differentiatingcells. One type of each enzyme is distributed in the cytosol,while the other is secreted from the Golgi apparatus and transportedby Golgi-derived vesicles to the secondary wall thickenings. (Received April 19, 1996; Accepted November 18, 1996)     

The Effects of 2,5-Norbornadiene on the Induction of the Activity of 1-Aminocyclopropane-l-carboxylate Synthase and of Phenylalanine Ammonia-lyase in Wounded Mesocarp Tissue of Cucurbita maxima

Activities of both 1-aminocyclopropane-l-carboxylate (ACC) synthaseand phenylalanine ammonia-lyase (PAL) were rapidly induced inexcised mesocarp discs of Cucurbita maxima Duch. The increasein activity of ACC synthase preceded that of PAL. 2,5-Norbornadiene(NBD), an inhibitor of the action of ethylene [Sisler and Yang(1984) Phytochemistry 12: 2765-2768.], enhanced the level ofactivity of ACC synthase in excised mesocarp disc and overcamethe suppression by exogenous ethylene. NBD, by contrast, suppressedthe level of PAL activity induced in the wounded tissue. Theseresults suggest that endogenous ethylene produced in the woundedmesocarp tissue suppresses the induction of ACC synthase butpromotes the induction of PAL. (Received March 9, 1989; Accepted June 14, 1989)     

Herbicide effects on phenolic metabolism in maize (Zea mays L.)and soybean (Glycine max L.) seedling

The activities of phenylalanine ammonia lyase [PAL; EC 4.3.1.5 [EC] ]and chalcone isomerase [Cl; EC 5.5.1.5 [EC] ] as well as the contentsof anthocyanin and total soluble hydroxyphenolic compounds wereinvestigated in maize (Zea mays L.) and soybean (Glycine maxL.) seedlings 120 h after treatment with the field dose of fiveherbicides from different groups (trifluralin, fluometuron,atrazine, alachlor, and rimsulfuron) having varied modes ofaction. The fresh weight of both species was greatly decreasedby trifluralin followed by fluometuron and atrazine. The dryweight was, in general, only slightly decreased by all the herbicideswith the largest response with trifluralin. On the other hand,the activities of PAL and Cl were greatly enhanced in both speciesby alachlor and rimsulfuron, but decreased by trifluralin. Fluometuroninduced decreases in PAL activity of maize only and decreasedCl activity of maize and soybean seedlings. Moreover, hydroxyphenoliccompounds were increased in both species by alachlor and rimsulfuronand decreased by trifluralin and atrazine. Similarly, anthocyanincontent was increased in both seedlings by alachlor and rimsulfuron,but decreased by trifluralin and fluometuron, whereas atrazinedecreased the anthocyanin content in maize only. The presentresults indicate that stress is maintained by the differentherbicides and confirm the controlling action of PAL and Clon the production of anthocyanin and phenolic compounds duringthe induced state of stress. In addition, dry weight reductionappeared to coincide with the changes in the parameters of secondarymetabolism, suggesting a regulatory role of secondary metabolismon seedling growth. Key words: Herbicides, phenylalanine ammonia lyase, chalcone isomerase, anthocyanin, hydroxyphenolics     

Wound-induced PAL activity is suppressed by heat-shock treatments that induce the synthesis of heat-shock proteins

Wounding lettuce leaves induces the de novo synthesis of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), the accumulation of phenolic compounds, and subsequent tissue browning. A brief heat-shock at 45°C reduces the rise in wound-induced PAL, the accumulation of phenolic compounds, and tissue browning. The activity of PAL measured 24 h after wounding and the content of phenolic compounds (absorbance of methanol extract at 320 nm) measured 48 h after wounding was highly correlated (R² > 0.90) in tissue developing the normal wound response and in tissue subjected to 0–180 s of heat-shock after wounding. The synthesis of a unique set of proteins called heat-shock proteins (hsps) is induced by these heat-shock treatments. Western-blot analyses of proteins isolated from wounded and heat-shocked Iceberg and Romaine lettuce mid-rib leaf tissue was done using antibodies against hsp 23. Only those heat-shock treatments that were effective at inducing the synthesis of hsp 23 were effective in reducing the activity of PAL induced by wounding and the subsequent accumulation of phenolic compounds. Hsps induced in non-wounded, whole leaves by exposure to 45°C for 150 s did not significantly interact with PAL previously synthesized in non-heat-shocked wounded leaves to limit its activity. The preferential synthesis of hsps over that of wound-induced PAL, rather than the presence of hsps, may be responsible for the ability of a heat-shock treatment to reduce the wound-induced increase in PAL activity. Our results support this novel concept, and the possibility that heat-shock treatments can have significant physiological effects on the response of the tissue to other stresses, not because of the specific genes they induce or repress, or the products they cause to be synthesized, but by their secondary action of influencing the synthesis of other proteins (e.g. PAL) by the suppression of non-hsps protein synthesis.     

Photoregulation of Phenylalanine Ammonia Lyase is not Correlated with Anthocyanin Induction in Photomorphogenetic Mutants of Tomato(Lycopersicon esculentum)

Photoregulation of phenylalanine ammonia lyase (PAL)(EC 4.3.1.5 [EC] )was analyzed in wild type (WT) and mutants: phytochrome dencient-awrea(au), high pigment exhibiting exaggerated phytochrome response(hp) and the double mutant (au.hp) of tomato (Lycopersicon esculentum(Mill.) cv. Ailsa Craig). Red light, acting via phytochrome,stimulates PAL activity in cotyledons and hypocotyls of tomatoseedlings. The time course of photoinduction of PAL in cotyledonsof the mutants (au and au.hp) and WT seedlings has a peak ofactivity at 4 h, after which the activity falls sharply, exceptin hp seedlings where activity is maintained at a high level.In hypocotyls, photoinduction of PAL also shows an initial rise,reaching a maximum at 3 h, followed by a sharp decline in themutants (au and au.hp) and WT seedlings. However in hp seedlingsphotoinduction of PAL is about 3 fold that in WT. The photoinductionof PAL appears to be dependent on de novo synthesis of proteinand nucleic acids. The use of a PAL specific inhibitor a-aminooxyß-phenylpropionic acid indicated that PAL is an essentialcomponent of the anthocyanin biosyn-thetic pathway in the tomatoseedlings. However, a comparison of anthocyanin biosynthesis[Adamse et al. (1989) Photochem. Photobiol. 50: 107] and PALphotoinduction data revealed that phytochrome mediated inductionof PAL and anthocyanin in the tomato seedlings are not correlated.While au and au.hp mutant seedlings show a similar increasein PAL level as in the WT, there is little formation of anthocyaninin these mutant seedlings. The results indicate that, in contrastto the photoregulation of anthocyanin synthesis which is dependenton the presence of the labile phytochrome (IP) pool in tomatoseedlings, the photoinduction of PAL is mediated via a smallpool of phytochrome in au mutant: stable phytochrome (sP) ora residual /P pool. (Received August 6, 1991; Accepted September 27, 1991)     

Wound-induced ethylene production, phenolic metabolism and susceptibility to russet spotting in iceberg lettuce

Mechanical wounding by cuts or punctures caused a brief increase in ethylene production by iceberg lettuce ( Lactuca sativa L.) leaf tissue. Wounding increased phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity, which was a function of the degree of injury. Wound-induced PAL activity appeared after 4 h and reached maximum activity in about 24 h before slowly declining to normal levels in about a week. A signal for PAL induction was transmitted at about 0.5 cm h⁻¹ from the site of injury to cells up to 2.5 cm away. Treatment with 100 μ2-aminoethoxyvinylglycine prevented wound-induced ethylene production but did not affect induced PAL activity. Injury increased the concentration of several soluble phenolic compounds that were easily oxidized to brown substances by polyphenol oxidase (EC 1.10.3.2) isolated from lettuce tissue. Wounding also increased peroxidase (EC 1.11.1.7) activity and lignin content, with cell wall lignification localized in wounded and adjacent cells. Although wounding alone did not induce russet spotting, it did greatly increase susceptibility to ethylene-induced russet spot development. In the presence of 3 μ1⁻¹ ethylene, the russet spot score increased as the degree of injury increased.     

Temperature Regulation of Anthocyanin Accumulation in Apple Skin

The regulation of anthocyanin accumulation in apple skin (cv.Jonathan) by temperature was studied. In the field the increasein anthocyanin in the skin before harvest coincided with decreasingtemperatures and with increasing ethylene production by thefruit. In detached apples held under continuous white light,the optimum temperatures for anthocyanin accumulation were 12°C in unripe apples and 16–24 °C in ripe apples.These effects were explained by corresponding changes in thelevel of phenylalanine ammonia-lyase (PAL), a key enzyme offlavonoid synthesis. PAL levels were higher at low than at hightemperatures and higher in ripe than in unripe apples. In intermittentlight the effects of temperature were similar but levels ofPAL and anthocyanin were lower, particularly in unripe apples.It is concluded that temperature, in conjunction with ripeningand light, is an important factor regulating anthocyanin accumulationand that its effects are mediated by effects on the level ofPAL activity. Key words: Apple, Anthocyanin, Phenylalanine ammonia-lyase     

Stimulation by Fungal Elicitor of Inositol Phospholipid Turnover in Tobacco Suspension Culture Cells

The effects of fungal elicitor on inositol phospholipid turnoverand induction of phenylalanine ammonia-lyase (PAL) activityin tobacco suspension culture cells were investigated. Tobaccocells labeled by [³H]inositol in vivo were treated with Phytophthoranicotianae elicitor and [³H]metabolites of inositol phospholipidturnover were examined. Stimulation of inositol phospholipidturnover was observed preceding the induction of PAL activity;inositol 1,4-bisphosphate increased 15 times over the control10 min after the elicitor treatment. Increase of inositol 1,4,5-trisphosphatewas only 38% of the control. Phosphatidylinositol and phosphatidylinositol4-phosphate transiently decreased by 21 and 35%, respectively.Phosphatidylinositol 4,5-bisphosphate was not affected significantlyby the elicitor. Inositol 1,4-bisphosphate was preferentiallyelevated by elicitation than 1,4,5-trisphosphate suggestingthat the regulatory mechanism of inositol phospholipid turnoverin tobacco cells is different from that in animal cells. Phosphatidylinositolkinase but not phospholipase C was activated by the elicitorin vitro. Elicitor-dose dependency curves in the induction ofPAL activity and in the stimulation of inositol phospholipidturnover showed a similar feature suggesting that inositol phospholipidturnover is involved in the elicitor-signal transduction intobacco cells. ¹Present address: The Johns Hopkins University School of Hygieneand Public Health, 615 N. Wolfe St., Baltimore, Maryland 21205,U.S.A. ²Present address: Nagoya University BioScience Center and GraduateSchool of Agricultural Sciences, Nagoya University, Chikusa-ku,Nagoya, 464-01 Japan.     

L-Phenylalanine ammonia-lyase from Phaseolus vulgaris. Characterisation and differential induction of multiple forms from elicitor-treated cell suspension cultures

L-Phenylalanine ammonia-lyase (EC 4.3.1.5) has been purified over 200-fold from cell cultures of bean (phaseolus vulgaris L.) exposed to elicitor heat-released from the cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum. Four forms of the enzyme, with identical Mr but differing apparent pI values of 5.4, 5.2, 5.05 and 4.85, were observed following the final chromatofocussing stage of the purification. A preparation (purified 43-fold by ammonium sulphate precipitation, gel-filtration and ion-exchange chromatography) containing all four forms exhibited apparent negative rate cooperativity with respect to substrates. However, the individual forms displayed normal Michaelis-Menten kinetics, with Km values of 0.077 mM, 0.122 mM, 0.256 mM and 0.302 mM in order of decreasing apparent pI value. A preparation purified 200-fold and containing all four forms was used to immunise rabbits for the production of anti-(phenylalanine ammonia-lyase) serum. The antiserum was characterised by: immunotitration experiments; solid phase enzyme-linked immunosorbent assays; comparison of immunoprecipitates of 35S-labelled phenylalanine ammonia-lyase subunits (synthesized both in vivo and in vitro) on both one-dimensional and two-dimensional polyacrylamide gels after immunoprecipitation with the bean antiserum or antisera raised against pea and parsley phenylalanine ammonia-lyase preparations and immune blotting. SDS/polyacrylamide gels and SDS/polyacrylamide gel electrophoresis followed by immune blotting, indicated that the Mr of newly synthesized (in vivo and in vitro) bean phenylalanine ammonia-lyase subunits is 77000; a 70000-Mr form is readily generated as a partial degradation product during purification. Immunoprecipitates of bean phenylalanine ammonia-lyase synthesized both in vivo and in vitro showed the presence of multiple subunit types of identical Mr but differing in pI. Furthermore, treatment of bean cultures with Colletotrichum elicitor resulted in a 10-fold increase in phenylalanine ammonia-lyase extractable activity within 8 h, and chromatofocussing analysis indicated that this was associated with differential increased appearance of the high-pI, low-Km forms as compared to the two higher Km forms. This differential induction was further confirmed by immune blotting of crude extracts subjected to isoelectric focussing.     

Hominoid triosephosphate isomerase: Regulation of expression of the proliferation specific isozyme

Summary Three primary isoforms of the dimeric glycolytic enzyme, triosephosphate isomerase (TPI; EC 5.3.1.1), are detected in proliferating human cells. The electrophoretically separable isoforms result from the three possible combinations of constitutive subunits and subunits expressed only in proliferating cells. Only a single primary isoform is observed in quiescent cells. The two subunits, which differ by covalent modification (s), are products of the single structural locus for this enzyme. Expression of the proliferation specific subunit (TPI-2) is detected within 6–10 hr following mitogen stimulation of quiescent human cells, requires RNA synthesis and is inhibited by agents which inhibit interleukin 2 expression or function. Only the constitutive subunit (TPI-1) is detected in proliferating cells from nonhominoid primate species. A single class of TPI mRNA, which is increased > 10 fold following stimulation of quiescent cells, is detected on northern blot analysis and S1 nuclease digestion analysis of RNA from quiescent and proliferating human cells. It is similar in size to the TPI mRNA from proliferating cells of the African green monkey, a primate species not expressing TPI-2. Comparison of the structure of the TPI gene from rhesus monkey (nonexpressing species) to the gene from expressing species does not suggest a mechanism for generating TPI-2. Thus, the regulation of the expression of the hominoid restricted, proliferation specific subunit of TPI has been further defined, although the mechanism for generating TPI-2 remains elusive.     

Regulation of induction of phenylalanine ammonia-lyase in suspension cultures of Phaseolus vulgaris

Total RNA was extracted from fast growing suspension cells of bean, the mRNA was translated and the products of protein synthesis analysed by gel electrophoresis. Actinomycin D (20 g ml^–1) added to the cultures 12 h before the induction of phenylalanine ammonia-lyase (PAL) activity by naphthylacetic acid (NAA) (1 mg/l) and kinetin (0.2 mg/l) failed to prevent the increased activity of the enzyme usually produced by this ratio of the plant growth hormones. PAL was isolated and purified from suspension cultured bean cells. The purified enzyme ran as a single band on polyacrylamide gel electrophoresis. The protein translated from RNA prepared from induced and non-induced cells was separated by gel electrophoresis and the bands of protein on the gels were compared. There was no evidence for an increase in the amount of PAL synthesised in vitro from the mRNA of induced cells even though these had 5 times the amount of activity of the enzyme compared with that of the non-induced cells. The results indicate that the induction of PAL activity is not immediately preceeded by an increase in the synthesis of PAL-mRNA by the cells. The control of the activity of the enzyme is discussed with respect to this finding.Abbreviations PAL phenylalanine ammonia-lyase - NAA 3naphthylacetic acid - DEAE Diethylamino ethyl - EDTA Ethylenediamine tetraacetate - SDS Sodium dodecyl sulphate     

Response of Glutamine Synthetase and Glutamate Synthase Isoforms to Nitrogen Sources in Rice Cell Cultures

As a model system with no photorespiration and no long distancetransport, rice cell cultures (Oryza saliva L. cv Sasanishiki)were used to investigate the effect of nitrogen sources on thelevels of isoforms of glutamine synthetase (GS) and glutamatesynthase (GOGAT). Isoforms of GS and GOGAT were analyzed byimmunoblotting methods and their activities in early growthphase of the cells. Cytosolic type GS (41 kDa subunit) and NADH-GOGATwere the major isoforms in the rice cells grown in normal R-2medium. However, contents of plastid type GS (44 kDa subunit)and Fd-GOGAT increased in response to NO_–³ supply. NADH-GOGATactivity also increased following the supply of NO_–³.In vitro translated products from poly(A)⁺RNA prepared fromthe cells showed that the precursor of plastid type GS (49 kDa)was detected at 48 h after the inoculation. Supply of NH₊⁴ resultedin an increase in NADH-GOGAT activity but had no effect on thelevels of Fd-GOGAT, of polypeptides of the plastid type GS orof the corresponding mRNAs. (Received May 30, 1990; Accepted August 23, 1990)     

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.