Metabolomics-guided investigations of unintended effects of the expression of the hydroxycinnamoyl quinate hydroxycinnamoyltransferase (hqt1) gene from Cynara cardunculus var. scolymus in Nicotiana tabacum cell cultures.

Chlorogenic acids (CGAs) are phenolic compounds biosynthesized in the phenylpropanoid pathway, with hydroxycinnamoyl quinate hydroxycinnamoyltransferase (HQT) as the key enzyme. Variation of CGAs has been noted in different plants, with globe artichoke (Cynara cardunculus var. scolymus L.) producing high amounts and a diverse spectrum of CGAs in its leaves. In the current study, the effect of overexpression of the hqt1 transgene from globe artichoke in tobacco was evaluated at the metabolome level. Here, metabolomic approaches based on ultra-high performance liquid chromatography coupled to mass spectrometry, together with chemometric models such as principal component analysis and orthogonal partial least square discriminant analysis, were employed to evaluate altered metabolic changes due to hqt1 overexpression. CGA profiles (caffeoylquinic acids: 3-CQA, 4-CQA and 5-CQA; p-coumaroylquinic acids: 4-pCoQA and 5-pCoQA; and 4,5-di-caffeoylquinic acid) of transgenic tobacco cell cultures were detected at lower concentrations than in the wild type. Interestingly, the cells were found to rather accumulate, as an unintended effect, abscisic acid - and benzoic acid derivatives. The results suggest that insertion of hqt1 in tobacco, and overexpression in undifferentiated cells, led to rechannelling of the phenylpropanoid pathway to accumulate benzoic acids. These findings proved to be contrary to the results shown elsewhere in leaf tissues, thus indicating differential metabolic control and regulation in the undifferentiated cell culture system.

Untargeted metabolomics analysis reveals dynamic changes in azelaic acid- and salicylic acid derivatives in LPS-treated Nicotiana tabacum cells.

To counteract biotic stress factors, plants employ multilayered defense mechanisms responsive to pathogen-derived elicitor molecules, and regulated by different phytohormones and signaling molecules. Here, lipopolysaccharide (LPS), a microbe-associated molecular pattern (MAMP) molecule, was used to induce defense responses in Nicotiana tabacum cell suspensions. Intracellular metabolites were extracted with methanol and analyzed using a liquid chromatography-mass spectrometry (UHPLC-qTOF-MS/MS) platform. The generated data were processed and examined with multivariate and univariate statistical tools. The results show time-dependent dynamic changes and accumulation of glycosylated signaling molecules, specifically those of azelaic acid, salicylic acid and methyl-salicylate as contributors to the altered metabolomic state in LPS-treated cells.

Chlorogenic Acids Biosynthesis in Centella asiatica Cells Is not Stimulated by Salicylic Acid Manipulation.

Exogenous application of synthetic and natural elicitors of plant defence has been shown to result in mass production of secondary metabolites with nutraceuticals properties in cultured cells. In particular, salicylic acid (SA) treatment has been reported to induce the production of phenylpropanoids, including cinnamic acid derivatives bound to quinic acid (chlorogenic acids). Centella asiatica is an important medicinal plant with several therapeutic properties owing to its wide spectrum of secondary metabolites. We investigated the effect of SA on C. asiatica cells by monitoring perturbation of chlorogenic acids in particular. Different concentrations of SA were used to treat C. asiatica cells, and extracts from both treated and untreated cells were analysed using an optimised UHPLC-QTOF-MS/MS method. Semi-targeted multivariate data analyses with the aid of principal component analysis (PCA) and orthogonal projection to latent structures-discriminant analysis (OPLS-DA) revealed a concentration-dependent metabolic response. Surprisingly, a range of chlorogenic acid derivatives were found to be downregulated as a consequence of SA treatment. Moreover, irbic acid (3,5-O-dicaffeoyl-4-O-malonilquinic acid) was found to be a dominant CGA in C. asiatica cells, although the SA treatment also had a negative effect on its concentration. Overall SA treatment was found to be an ineffective elicitor of CGA production in cultured C. asiatica cells.

Perturbation of pharmacologically relevant polyphenolic compounds in Moringa oleifera against photo-oxidative damages imposed by gamma radiation.

Oxidative stress is a physiological state associated with almost all biotic and abiotic stresses in plants. This phenomenon occurs due to imbalances which result from the overproduction of reactive oxygen species (ROS). Plants, however, have developed sophisticated mechanisms to mitigate the effect of ROS. In this regard, plant polyphenolic metabolites such as flavonoids are known to possess high antioxidant activities. In the current study, changes in the levels of phenolic compounds from Moringa oleifera after gamma radiation treatment were investigated with reverse phase liquid chromatography and mass spectrometric techniques in combination with multivariate data models such as principal component analysis and orthogonal projection to latent structures discriminant analysis. Our results revealed several polyphenolic compounds such as hydroxycinnamoyl derivatives and flavonoid molecules to be down-regulated post-radiation treatment. Interestingly, other flavonoid molecules were found to be up-regulated post-radiation treatment, thereby suggesting a possible compensatory phenomenon. The existence and involvement of structurally similar metabolites (such as regio-isomers of chlorogenic acids) in M. oleifera towards mitigating photo-oxidative damages are in support of the proposed evolutionary existence of a large pool of polyphenolics which contribute to the state of readiness, aptly described as a "better safe than sorry" phenomenon. Our study thus reaffirms the involvement of phenolic compounds as a first line of constitutive/preformed protection against oxidative stress. Furthermore, the obtained data supports M. oleifera as a source of versatile and pharmacologically relevant metabolites that may be exploited for ameliorating the oxidative damages imposed by several metabolic disorders in humans.

Secondary metabolite perturbations in Phaseolus vulgaris leaves due to gamma radiation.

Oxidative stress is a condition in which the balance between the production and elimination of reactive oxygen species (ROS) is disturbed. However, plants have developed a very sophisticated mechanism to mitigate the effect of ROS by constantly adjusting the concentration thereof to acceptable levels. Electromagnetic radiation is one of the factors which results in oxidative stress. In the current study, ionizing gamma radiation generated from a Cobalt-60 source was used to induce oxidative stress in Phaseolus vulgaris seedlings. Plants were irradiated with several radiation doses, with 2 kGy found to be the optimal, non-lethal dose. Metabolite distribution patterns from irradiated and non-irradiated plants were analyzed using UHPLC-qTOF-MS and multivariate data models such as principal component analysis (PCA) and orthogonal projection to latent structures discriminate analysis (OPLS-DA). Metabolites such as hydroxycinnamic phenolic acids, flavonoids, terpenes, and a novel chalcone were found to be perturbed in P. vulgaris seedlings treated with the aforementioned conditions. The results suggest that there is a compensatory link between constitutive protectants and inducible responses to injury as well as defense against oxidative stress induced by ionizing radiation. The current study is also the first to illustrate the power of a metabolomics approach to decipher the effect of gamma radiation on crop plants.

Metabolomic fingerprinting of primed tobacco cells provide the first evidence for the biological origin of cis-chlorogenic acid.

Previous studies suggest that only trans-isomers of chlorogenic acid (CGA) are naturally produced. Cis-isomers have been noted in some plant tissues exposed to different mechanical processes as well as untreated tobacco leaves exposed to sunlight. Very little, however, is known about the biological significance and origin of cis-isomers. Here we show for the first time the accumulation of cis-5-caffeoylquinic acid in cultured tobacco cells treated with different inducers of plant defence (lipopolysaccharides, flagellin peptide-22, chitosan, acibenzolar-S-methyl and isonitrosoacetophenone), without exposure to UV light and with a 2-fold (on average) increase in the concentration of the pool in comparison to non-stimulated cells. Our UHPLC-Q-TOF-MS and multivariate statistical results suggest the presence of a possible biological pathway responsible for the production of cis-CGAs in tobacco plants.

Biotransformation of isonitrosoacetophenone (2-keto-2-phenyl-acetaldoxime) in tobacco cell suspensions.

Nicotiana tabacum cell suspensions, 2 g wet wt/ml, rapidly took up 1 mM isonitrosoacetophenone (INAP), a plant-derived stress metabolite with anti-oxidative and anti-fungal properties, producing 4'-hexopyranosyloxy-3'-methoxyisonitrosoacetophenone in 54 % yield over 18 h. Unconverted INAP was at 33 µM. UPLC-MS/MS analyses with MassFragment software were used for metabolite identification. INAP had been hydroxylated at its meta- and para-positions as well as undergoing subsequent methoxylation and glycosylation. INAP is thus recognized by the enzymatic machinery of the phenylpropanoid pathway and is converted to a molecule with a substitution pattern similar to ferulic acid.

Lipopolysaccharide-responsive phosphoproteins in Nicotiana tabacum cells.

Mounting evidence is merging to affirm the effectiveness of bacterial lipopolysaccharides (LPS) as biological control agents, inducers of innate immunity, and to stimulate/potentiate the development of defense responses in plants through protein phosphorylation-mediated signal perception/transduction responses. In vivo labeling of protein phosphorylation events during signal transduction indicated the rapid phosphorylation of several proteins. Substantial differences and de novo LPS-induced phosphorylation were also observed with two-dimensional analysis. In this study, qualitative and quantitative changes in phosphoproteins of Nicotiana tabacum suspension cells during elicitation by LPS from the Gram-negative bacteria, Burkholderia cepacia, were analyzed using two-dimensional electrophoresis in combination with a phosphoprotein-specific gel stain. Trypsin digested phosphoproteins were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and nano-electrospray-ionization liquid chromatography tandem mass spectrometry (nano-ESI-LC/MS/MS). A total of 27 phosphoproteins were identified from 23 excised gel spots. The identified phosphoproteins indicate that LPS(B.cep)-induced signal perception/transduction involves G-protein coupled receptor signaling, Ca(2+)/calmodulin-dependent signaling pathways, H(+)-ATPase regulation of intracellular pH, thioredoxin-mediated signaling and phosphorylation of 14-3-3 regulatory proteins. Other targets of LPS(B.cep)-responsive phosphorylation included NTP pool maintenance, heat shock proteins, protein biosynthesis and chaperones as well as cytoskeletal tubulin. The results add novel insights into the biochemical process of LPS perception and resulting signal transduction.

Identification and quantification of gossypol in cotton by using packed micro-tips columns in combination with HPLC.

Self-packed micro-tip columns containing a C18-bonded silica stationary phase, based on the same principles as solid-phase extraction methods, were used to obtain gossypol and related sesquiterpenoid aldehyde-enriched fractions. The enriched metabolite fractions were then analyzed by optimized high-performance liquid chromatography (HPLC) with a C18 column (4.6 mm x 25 cm) eluted with the binary mobile phase acetonitrile-0.1% aqueous TFA solution (80:20). This method has proven to be highly reproducible. The precision and accuracy, as %RSD and %RME values, were determined to be less than 15% for the method. The minimum detection limit of gossypol was determined to be 10 ng (absolute gossypol). Absolute recovery was greater than 94% with a standard deviation of +/-3.68%. This is a simple, fast, and cost-effective method for isolation, identification, and quantification of gossypol and related secondary metabolites. Comparative analysis of gossypol content was performed on different parts of the cotton plant (seeds, stems and leaves) of two different cultivars of Gossypium hirsutum L. (Acala(1517-70) and OR19). The results indicate that the OR19 cv naturally contains higher gossypol levels than the Acala cv. It was also found that treatment of leaves with a Verticillium dahliae-derived elicitor induced production of deoxyhemigossypol rather than gossypol.

Assessment of a simple, non-toxic Alamar blue cell survival assay to monitor tomato cell viability.

The Alamar Blue (AB) assay, which incorporates a medox indicator that changes colour or fluorescence in response to metabolic activity, is commonly used to assess quantitatively the viability and/or proliferation of mammalian cells and micro-organisms. In this study the AB assay was adapted for the determination of the viability of plant cells. Cell suspension cultures of tomato, Lycopersicon esculentum, L., with differing viabilities, served as the experimental model for a comparison of the AB assay with the conventional 2,3,5-triphenyltetrazolium chloride (TTC) viability assay. The AB assay showed a sigmoidal relationship between cell viability and AB reduction (as quantified by spectrofluorometry or spectrophotometry), which was similar to that obtained using the TTC assay. Both assays detected a significant reduction in cell viability after 48 h exposure to virulent Ralstonia solanacearum (biovar III), while the TTC assay, in addition, revealed cell proliferation in control cells from 24 to 72 h. The TTC assay detected cell proliferation over a wider range of cell densities, while the AB assay was more rapid and versatile whilst being non-toxic and thus allowing subsequent cell analysis.

Inhibition of polygalacturonase from Verticillium dahliae by a polygalacturonase inhibiting protein from cotton.

An extracellular endo-polygalacturonase (PGase) [E.C. 3.2.1.15] was isolated from 18-day-old culture filtrates of Verticillium dahliae and partially purified using gel permeation chromatography. The band responsible for PGase activity was electrophoretically characterized as having a molecular mass of approximately 29 500 and an isoelectric point of 5.4. Kinetic studies indicate a Km of 3.3 mg ml(-1) and Vmax of 0.85 micromol reducing units min(-1) ml(-1) with polygalacturonic acid as substrate. Polygalacturonase inhibitor protein (PGIP) in cotton seedlings was induced by 5 mM salicylic acid and immunochemical analysis indicated high levels in the hypocotyl tissues. PGIP was purified from roots and stems using affinity chromatography with endo-PGase from Aspergillus niger as an immobilised ligand. The purified PGIP contained monomeric and dimeric molecules with molecular masses of 34 and 66 kDa respectively. Purified cotton PGIP inhibited endo-polygalacturonase from A. niger in a non-competitive or mixed manner with an inhibition constant. K(I) of 15 nM. The isolated V. dahliae PGase was, however, inhibited in a positive cooperative manner, indicative of allosteric interactions between the enzyme and the inhibitor protein. In addition to reducing the reaction rate, decreased substrate affinity may contribute to the accumulation of elicitor-active oligouronides.

Early responses in methyl jasmonate-preconditioned cells toward pathogen-derived elicitors.

Early, signal transduction-related responses in cultured tobacco cells due to methyl jasmonate (MeJa), a cell-wall-derived elicitor from Phytophthora nicotianae and chitosan, were investigated. MeJa was an effective inducer of lipid peroxidation and lipoxygenase (LOX) activity with maximum levels reached within 2 h and 4-8 h, respectively. Chitosan and the elicitor induced a transient increase (1-4 h) in lipid peroxidation. Conditioning with MeJA, followed by secondary elicitation, led to a significant increase in malondialdehyde concentration after 1 h. Chitosan and the elicitor induced transient activation of LOX with maximal values between 8 and 12 h, with preconditioning resulting in a rapid increase in LOX activity at 4 h post elicitation. MeJA did not effect phosphoprotein accumulation but conditioning led to the potentiation and differential induction of phosphoproteins due to chitosan and elicitor. The results indicate that cells are sensitized by the exposure to MeJa to respond more intensely and rapidly toward secondary elicitation by fungal pathogen derived elicitors.

Panama Disease: Cell Wall Reinforcement in Banana Roots in Response to Elicitors from Fusarium oxysporum f. sp. cubense Race Four.

ABSTRACT The biochemical basis of tolerance in banana to Fusarium wilt, caused by the pathogen Fusarium oxysporum f. sp. cubense race four, was investigated. Tissue culture banana plants from tolerant cv. Goldfinger and susceptible cv. Williams were maintained in a hydroponic system and inoculated with conidial suspensions to evaluate the degree of tolerance to susceptibility between the two clones and to investigate the effectiveness of this technique as a potential tool for early screening for resistance in breeding programs. Similarly, defense responses were induced by treatment of the plants with an elicitor preparation from the mycelial cell walls of the pathogen. Differences in the induction of lignin and callose deposition, phenolics, and the enzymes involved in cell wall strengthening; phenylalanine ammonia lyase, cinnamyl alcohol dehydrogenase, peroxidase, and polyphenol oxidase were determined. Root tissue of the tolerant cv. Goldfinger responded to the fungal elicitor through the strong deposition of lignin, preceded by the induction or activation of the enzyme activities involved in the synthesis and polymerization thereof, whereas only slight increases were observed for the susceptible cv. Williams. No increase in callose content was observed for either clone. These results indicate an important role for cell wall strengthening due to the deposition of lignin as an inducible defense mechanism of banana roots against F. oxysporum f. sp. cubense race four.

Synthesis and evaluation of 4-(3-methyl-2-butenoxy) isonitrosoacetophenone, a radiation-induced stress metabolite in Citrus.

The time- and dose-dependent occurrence of 4-(3-methyl-2-butenoxy)isonitrosoacetophenone, a gamma-irradiation-induced stress metabolite was investigated. The chemical synthesis of the compound is reported. The compound exhibits antifungal activity, as well as antioxidant activity, as indicated by its ability to scavenge reactive oxygen radicals in a chemiluminescence assay.

Protein kinase activities in ripening mango, Mangifera indica L., fruit tissue. III. Purification and characterisation of a calcium-regulated protein kinase.

A calcium-dependent protein kinase (PK-III), not requiring calmodulin for activity, was purified from extracts of ripening mango fruit tissue. Purification was achieved by ammonium sulfate fractionation and sequential anion exchange-, hydrophobic interaction-, dye ligand affinity- and gel filtration chromatography; which allowed a recovery of 1-5% of the total available kinase activity. The final specific activity in the presence of 1 mM Ca2+ was consistently 9 nmol min-1mg-1. The purified enzyme was a monomer with a Mr of 49000, but was resolved by denaturing electrophoresis into two related protein bands of 49 and 45 kDa. Enzyme activity was activated >30-fold by micromolar amounts of free calcium and was dependent upon millimolar Mg2+ or Mn2+ concentrations. Calmodulin (1 microM) had no effect on PK-III activity but the calmodulin antagonists, calmidazolium and chlorpromazine, inhibited PK-III in a dose-dependent manner over a range of 0 to 100 microM. The results suggest a regulatory domain that is similar to calmodulin. PK-III phosphorylated histone III-S and to a lesser extent casein, but did not phosphorylate histone II-S, phosvitin or protamine sulfate. The enzyme phosphorylated substrate proteins on either serine or threonine but not tyrosine. Some endogenous substrates and the ability to autophosphorylate were revealed by autoradiographic studies. PK-III displayed a broad pH optimum (pH 6.6-9.5), and the optimum reaction temperature with histone III-S as substrate was 35 degreesC. The kinetic reaction mechanism of PK-III was studied by using casein as substrate. The KmATP and Kmcasein of PK-III were determined as 10 microM and 1.0 mg ml-1, respectively.

Protein kinase activities in ripening mango, Mangifera indica L., fruit tissue. I: Purification and characterization of a calcium-stimulated casein kinase-I.

A Ca(2+)-stimulated protein kinase (PK-I), active with dephosphorylated casein as exogenous substrate, was purified from ripening mango fruit. The purification procedure involved 30-70% ammonium sulphate fractionation and sequential anion exchange-, affinity-, hydrophobic interaction- and gel filtration chromatography. PK-I was purified ca. 40-fold with an overall yield of < 1%. The final specific activity in the presence of 0.1 mM Ca2+ was 55 nmol min-1 mg-1. Analysis of the most highly purified preparations revealed a monomeric enzyme with an M(r) of 30.9 kDa and pI of 5.1. PK-I efficiently phosphorylated casein and phosvitin, but did not phosphorylate histone II-S, histone III-S, protamine sulphate or bovine serum albumin. PK-I activity was stimulated by micromolar concentrations of Ca2+ and was dependent on millimolar Mg2+ concentrations, which could not be substituted with Mn2+. PK-I activity was stimulated by, but was not dependent on Ca2+. Calmodulin and calmodulin inhibitors did not affect PK-I activity, but heparin and cAMP acted as inhibitors. The pH and temperature optima of the enzyme under standard reaction conditions were 6.5 and 35 degrees C, respectively. The kinetic reaction mechanism of PK-I was studied by using casein as substrate. Initial velocity and product inhibition studies with ADP as product inhibitor best fit an ordered bi-bi kinetic mechanism with the Mg(2+)-ATP complex binding first to the enzyme followed by binding of the protein substrate. The K(m)ATP and K(m)casein of PK-I were 9 microM and 0.26 mg ml-1, respectively. The KiADP of PK-I was 9 microM.

Induction of defence responses in cultured tobacco cells by elicitors from Phytophthora nicotianae.

Black Shank disease of tobacco, Nicotianae tabacum, caused by Phytophthora nicotianae, is one of the most destructive of soil borne diseases. The present investigation was initiated to study the relative importance of various inducible mechanisms operative in this plant: pathogen interaction. Tobacco cells in culture were treated with heat-released soluble cell-wall elicitors from mycelial walls of the pathogen. The timing and intensity of the induced defence responses of the cells with regard to the synthesis of phytoalexins, lignin and defence hydrolases were evaluated. An elicitor concentration of 40 micrograms ml-1 resulted in the optimal induction of sesquiterpenoid phytoalexin synthesis which was detectable 3-4 hr after elicitation and reached a maximum at 20-28 hr post-elicitation. Increased lignin deposition was detectable 4 hr after elicitation with maximal synthesis between 12 and 96 hr. Induction of extracellular and cellular beta-1,3-glucanases was observed within 4 hr, exhibiting a biphasic response with an initial peak at 8-12 h. Increased cellular and extracellular chitinase activities were detected from 8 and 16 hr onwards, respectively. These results were supported by enzyme staining of electrophoretic separated isoforms. The results obtained indicate that the elicitation of tobacco cells by P. nicotianae derived signal molecules exhibits the properties of a multicomponent dynamic system with different protective mechanisms having complementary roles in the overall expression of the defence response.

Specific binding of a Verticillium dahliae phytotoxin to protoplasts of cotton, Gossypium hirsutum.

The occurrence of specific, high-affinity binding sites for a protein-lipopolysaccharide (PLP) phytotoxin purified from culture filtrates of a virulent Vertidllium dahliae isolate has been demonstrated in cotton protoplasts. Binding of the (125)I-radiolabelled PLP-complex to protoplasts from cotyledon tissue was saturable and with an affinity (Kd = 17.3 nM) comparable with the concentration required for biological activity. A single class of binding site, accessible at the surface of the intact protoplasts, was found and the maximal number of binding sites were estimated as 2.41 × 10(-16) moles per protoplast. The binding affinity to protoplasts proved near identical to that found with purified plasma membrane fractions from roots. When cultivars exhibiting resistance or susceptibility towards the pathogen were compared, no significant differences were found in the affinity of binding, but five times as many binding sites per protoplast and sixteen times as many binding sites per mg membrane protein were found in the resistant cultivar.

Cis elements and potential trans-acting factors for the developmental regulation of the Phaseolus vulgaris CHS15 promoter.

A nuclear factor (SBF-1) has previously been identified in Phaseolus vulgaris L. (bean) suspension cell nuclear extracts that binds in vitro to three DNase I-footprinted elements (SBF-1 boxes I, II, and III, 5' to 3') in the 5' region of the bean CHS15 (chalcone synthase) gene promoter. To define the functional role of the three SBF-1 boxes in development, we examined transgenic tobacco plants carrying a series of nested CHS15 promoter-beta-glucuronidase (GUS) fusions for GUS activity by histochemical staining. We show that the CHS15 promoter deleted to position -173 and lacking all three SBF-1 boxes directs the same qualitative pattern of expression in initiating lateral roots and in developing seeds as the full length promoter (-326). Thus, activation of expression in these organs is mediated by sequence elements located downstream of the three SBF-1 boxes. However, specific deletions within the -326 to -173 region modulate expression. Thus, deletion of box II abolishes GUS activity in initiating lateral roots. Further deletion of box III fails to restore expression but subsequent deletion of an additional 43 bp to position -173 re-establishes expression. We show that sequence-specific DNA-binding activities consistent with these results are present in nuclear extracts of bean roots and seeds. These studies reveal cis elements within the CHS15 promoter, and potential trans factors, that permit organ- and tissue-specific developmental patterns of regulation to be combined with a flexible response to environmental cues.

Phenylalanine ammonia-lyase from cotton (Gossypium hirsutum) hypocotyls: properties of the enzyme induced by a Verticillium dahliae phytotoxin.

Phenylalanine ammonia-lyase (EC 4.3.1.5), induced by a Verticillium dahliae phytotoxin, has been purified to electrophoretic homogeneity from cotton hypocotyls by differential ammonium sulfate fractionation and hydrophobic interaction chromatography, with a yield of 52%. The enzyme is a tetramer with a molecular weight of 332,000 to 337,000. The isoelectric point is 4.6, and no isoforms were observed. The subunits of the enzyme are unstable and breaks down to fragments with M(r)'s of 69,000 and 49,500. The enzyme exhibited only activity with L-phenylalanine as substrate. Deamination was optimal at pH 8.9 and the activation energy was calculated as 100.6 kJ mol-1. Non-Michaelian kinetics were observed with a KmL = 10.0 microM and KmH = 75.0 microM describing the binding of the substrate to the enzyme. Negative cooperative interactions occurred between the substrate binding sites with a Hill coefficient of 0.87. The inhibitors AOPP (S)-2-amino-oxy-3-phenylpropanoic acid), APEP (R)-1-amino-2-phenylethylphosphonic acid) and 2-AIP (2-aminoindan-2-phosphonic acid) strongly inactivated the enzyme, as did various analogues of L-phenylalanine and t-cinnamate. The induced enzyme is also sensitive to inhibition by phenylpropanoid intermediates and precursors involved in lignification such as 4-hydroxycinnamate and 3,4-dihydroxycinnamate.