Messenger RNA-controlled Increase of Phenylalanine Ammonia-Lyase Activity in Parsley: Light-Independent Induction by Dilution of Cell Suspension Cultures into Water

A specific antiserum, raised against purified phenylalanine ammonialyase from irradiated cell suspension cultures of parsley (Petroselinum hortense Hoffm.), was used to compare the enzyme species induced either by dilution or by irradiation of the cell suspensions, to investigate the effect of dilution on the rate of synthesis of the enzyme protein in vivo, and to analyze the changes in specific activity of polyribosomal mRNA for the enzyme subunits in vitro. The mRNA activity in vitro was measured by translation of the polyribosomal RNA in a rabbit reticulocyte lysate.     

Phenylalanine Ammonia-Lyase from Tomato Cell Cultures Inoculated with Verticillium albo-atrum

Tomato (Lycopersicon esculentum Mill.) cell suspension cultures accumulated wall-bound phenolic materials in response to inoculation with Verticillium albo-atrum Reinke et Berth. in a fashion analogous to that observed in whole plants. Both monomeric and polymeric materials were recovered. Deposition of phenolics into the cell walls of inoculated tomato cell cultures was inhibited by the phenylalanine ammonia-lyase (PAL) inhibitor, 2-amino-2-indanephosphate. Tomato PAL activity was induced over 12-fold by fungal inoculation, with a concomitant increase in the corresponding mRNA. The enzyme was purified >3400-fold, to apparent homogeneity, by anion-exchange chromatography, chromatofocusing, and gel filtration. The holoenzyme had a molecular mass of 280 to 320 kilodaltons, comprising 74-kilodalton subunits, and displayed an isoelectric point of 5.6 to 5.7. Induced PAL displayed apparent Michaelis-Menten kinetics (K_m = 116 micromolar) and was not appreciably inhibited by its product cinnamic acid. Chromatographic analysis did not reveal multiple forms of the enzyme in either inoculated or uninoculated cultures.     

Regulation of Glutathione Synthesis in Suspension Cultures of Parsley and Tobacco*

Experiments in vitro have shown that γ-EC synthesis, the first step in GSH formation, is subject to feedback inhibition by physiological GSH concentrations. In order to evaluate the role of this feedback inhibition on γ-EC synthetase in vivo GSH synthesis was modulated in suspension cultures of P. crispum and N. tabacum by administration of cadmium. The alterations in the thiol contents were measured and in addition the effect of Cd exposure on γ-EC synthetase (E.C. 6.3.2.2) and GSH synthetase (E.C. 6.3.2.3) was studied. Decreasing cellular GSH concentrations by cadmium induced PC synthesis caused 7–10 fold increase in the rate of glutathione synthesis as measured by the accumulation of (γ-EC)_nG. This increase was not linked to an increase in extractable activities of γ-EC- or GSH synthetase in parsley. In tobacco the activities of γ-EC- and GSH synthetase increased by a factor of 1.6 and 1.8, respectively, after 3 d of Cd exposure. In both species the exposure to Cd resulted in an increased cellular γ-EC content that reached a plateau within 24 h, and in a doubling of the cysteine content. In vitro experiments showed that GSH synthetase activity is inhibited by cadmium concentrations that have no effect on γ-EC synthetase activity. This may explain the accumulation of γ-EC in Cd exposed cells. Incubation with 0.25 mM cysteine did not effect the γ-EC- and GSH content in tobacco cells. In parsley the cellular GSH content increased threefold and the y-EC content twofold and stayed constant thereafter at the elevated levels. Taken together the results show that GSH synthesis in vivo is controlled by feedback inhibition as well as by the supply with cysteine. In the latter case the feedback inhibition may act as a kind of safety valve and prevent the accumulation of unphysiological GSH concentrations if the supply of cysteine is too large.     

Phytochrome Control of Phenylalanine Ammonia-Lyase Levels and the Regulation of Cell Division in Artichoke Callus Cultures

Phytochrome controls phenylalanine ammonia-lyase (PAL) levelsin synchronously-dividing tuber tissue of the Jerusalem artichokeduring S but not during G₁. Red light enhances extractable PALlevels during S and the effect is far-red reversible. Howeverit is concluded that the effect of phytochrome on PAL levelsis only secondary since this effect is manifest many hours afterthe light treatments. Consequently, the relationship betweenphytochrome, PAL levels and cell division cannot be a simpleone.     

Regulation of Phytoalexin Synthesis in Jackbean Callus Cultures: Stimulation of Phenylalanine Ammonia-Lyase and o-Methyltransferase

Jackbean, Canavalia ensiformis (L.), callus tissues synthesized the phytoalexin, medicarpin (3-hydroxy-9-methoxypterocarpan), when treated with spore suspensions of Pithomyces chartarum (Berk. and Curt.) M. B. Ellis, a nonpathogen of jackbean. Medicarpin was isolated from treated callus tissue and identified by its ultraviolet and mass spectra. The minimum spore concentration found to elicit medicarpin synthesis after 26 hours was 1 × 10⁵ spores/ml; levels of medicarpin in callus tissue increased linearly up to 1 × 10⁷ spores/ml, indicating that the recognition sites for presumed elicitors were not saturated. Medicarpin was first detected in callus treated with 1 × 10⁷ spores/ml, 6 to 12 hours after application, and the concentration reached a maximum at 48 hours, slowly declining thereafter to 72 hours. In callus treated with 3.15 mm HgCl₂, medicarpin concentrations were also maximum by 48 hours. Phenylalanine ammonia-lyase (EC 4.3.1.5) activity increased 2-fold in spore-treated callus after 36 hours. Isoliquiritigenin, daidzein, and genistein o-methyltransferase (EC 2.1.1.6) activities were increased 3- to 4-fold in treated callus. Caffeic acid and naringenin were more efficient substrates for o-methyltransferase activity than the other flavonoids or apigenin, but there was no increase in these o-methyltransferase activities in spore-treated callus. The phytoalexin response in this callus tissue culture system compares well with natural plant systems and should be an excellent system for investigating regulation of phytoalexin synthesis.     

Phytochrome-mediated Induction of Phenylalanine Ammonia-Lyase in Mustard Seedlings: A Contribution to Eliminate Some Misconceptions

The present report shows that the effect of red and far red light on the synthesis of phenylalanine ammonia-lyase can be ascribed to the action of phytochrome Pfr. This is true not only for short term irradiation but also for the action of continuous far red light. In the latter case, the model worked out by Hartmann implicating some excited species of phytochrome has to be taken into account.     

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

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

Ethylene-enhanced Synthesis of Phenylalanine Ammonia-Lyase in Pea Seedlings

The effect of ethylene on the development of phenylalanine ammonia-lyase activity in segments excised from the epicotyl apex of pea seedling was studied. Although there was some increase in phenylalanine ammonia-lyase activity in segments not treated with ethylene, a marked increase in phenylalanine ammonia-lyase activity occurred in ethylene-treated tissues during the incubation. The induction period was estimated to be about 6 hours. The activity reached a maxmum at 30 hours and then declined. On withdrawal of ethylene, the increase was sustained for a short period and then stopped. After retreatment with ethylene, the increase was resumed. Addition of CO₂ reduced the effect of ethylene. Administration of cycloheximide or actinomycin D at an early period almost completely suppressed the increase in phenylalanine ammonia-lyase activity. However, if these inhibitors were administered at a later period, while phenylalanine ammonia-lyase activity was approaching a maximum, they not only failed to reduce but rather stimulated the activity. These results are consistent with the view that there exist both phenylalanine ammonia-lyase-synthesizing and -inactivating systems, and that the development of both systems may involve de novo synthesis of protein.     

Phenylalanine Ammonia-Lyase Genes Involved in Anthocyanin Synthesis and the Regulation of its Expression in Suspension Cultured Carrot Cells

D and is also induced rapidly and transiently by transfer of cells to fresh medium and lowering the cell density. From the carrot genomic library, four clones of PAL genes, gDcPAL1,2,3 and 4, were obtained. Analyses of nucleotide sequences revealed that only the gDcPAL3 gene is responsible for the induction of anthocyanin synthesis by 2,4-D. Several cis-elements, boxes M, P, A, L, and G, exist in the proximal promoter region of gDcPAL3. Transient expression experiments in carrot protoplasts using deletion mutants of the proximal promoter region of gDcPAL3 gene showed that boxes M and L, both of which contain core sequences of the Myb binding sites, might play an important role in gDcPAL3 promoter activity. Four myb cDNAs, Dcmyb8,10,12 and 14 were obtained from a carrot subtracted cDNA library and their structure and expression patterns were analyzed. In addition to the analysis of the proximal region of gDcPAL3 promoter, the possibility of the regulation of gene expression by genomic DNA structure and chromatin modification in metabolic differentiation is discussed. Received 10 June 2000/ Accepted in revised form 1 July 2000     

Stress Responses in Alfalfa (Medicago sativa L.): II. Purification, Characterization, and Induction of Phenylalanine Ammonia-Lyase Isoforms from Elicitor-Treated Cell Suspension Cultures

l-Phenylalanine ammonia-lyase has been purified from elicitor-treated alfalfa (Medicago sativa L.) cell suspension cultures using two protocols based on different sequences of chromatofocusing and hydrophobic interaction chromatography. Three distinct forms of the intact enzyme were separated on the basis of affinity for Octyl-Sepharose, with isoelectric points in the range pH 5.1 to 5.4. The native enzyme was a tetramer of M_r 311,000; the intact subunit M_r was about 79,000, although polypeptides of M_r 71,000, 67,000 and 56,000, probably arising from degradation of the intact subunit, were observed in all preparations. Two-dimensional gel analysis revealed the presence of several subunit isoforms of differing isoelectric points. The purified isoforms of the native enzyme had different K_m values for l-phenylalanine in the range 40 to 110 micromolar, although mixtures of the forms in crude preparations exhibited apparent negative rate cooperativity. The enzyme activity was induced approximately 16-fold within 6 hours of exposure of alfalfa cells to a fungal elicitor or yeast extract. Analysis by hydrophobic interaction chromatography revealed different proportions of the different active enzyme isoforms, depending upon either time after elicitation or the elicitor used. The elicitor-induced increase in enzyme activity was associated with increased translatable phenylalanine ammonia-lyase mRNA activity in the polysomal fraction.     

Phenylalanine Ammonia-Lyase in Sliced Sweet Potato Roots

A marked rise in the phenylalanine ammonia-lyase activity and the polyphenol synthesis was observed in sliced roots of a sweet potato. The enzyme activity was found to be localized in the root tissue adjacent to the sliced surface. In this region, the synthesis of polyphenols was much higher compared to the inner tissues. When the specific inhibitors for the protein and nucleic acid biosynthesis such as an actinomycin D and blasticidin S were added to the tissues by vacuum infiltration technique, both the development of phenylalanine ammonia-lyase activity and the synthesis of polyphenols were severely prevented. These results suggest the important role of phenylalanine ammonia-lyase in the polyphenol synthesis and de novo synthesis of the enzyme protein molecule in the sliced tissues.     

Ethylene-induced Phenylalanine Ammonia-Lyase Activity in Carrot Roots

Ethylene enhanced the activity of phenylalanine ammonialyase in carrot (Daucus carota L., var. “Nauty”) root tissue. Slight increase in enzyme activity was exhibited by root discs incubated in ethylene-free air. It was probably due to the ethylene formed within the sliced tissue. Addition of ethylene to the air stream increased phenylalanine ammonia-lyase activity and the total protein content of the discs until maximum activity was reached after 36 to 48 hours of incubation. The continuous presence of ethylene was required to maintain high level of activity. Ethylene, at a concentration of 10 microliter per liter induced higher activity than at lower or higher concentrations. CO₂ partially inhibited the ethylene-induced activity. Cycloheximide or actinomycin D effectively inhibited the ethylene-induced activity in discs that had not previously been exposed to ethylene. The results appear to support the hypothesis that the mode of action of ethylene may involve both de novo synthesis of the enzyme protein and protection or regulation of activity of the induced enzyme.     

Reduced Phenylalanine Ammonia-Lyase and Tyrosine Ammonia-Lyase Activities and Lignin Synthesis in Wheat Grown under Low Pressure Sodium Lamps

Wheat (Triticum aestivum L. cv Fremont) grown in hydroponic culture under 24-hour continuous irradiation at 560 to 580 micromoles per square meter per second from either metalhalide (MH), high pressure sodium (HPS), or low pressure sodium (LPS) lamps reached maturity in 70 days. Grain yields were similar under all three lamps, although LPS-grown plants lodged at maturity. Phenylalanine ammonia-lyase (PAL) and a tyrosine ammonia lyase (TAL) with lesser activity were detected in all extracts of leaf, inflorescence, and stem. Ammonia-lyase activities increased with age of the plant, and plants grown under the LPS lamp displayed PAL and TAL activities lower than wheat cultured under MH and HPS radiation. Greenhouse solar-grown wheat had the highest PAL and TAL activities. Lignin content of LPS-grown wheat was also significantly reduced from that of plants grown under MH or HPS lamps or in the greenhouse, showing a correlation with the reduced PAL and TAL activities. Ratios of far red-absorbing phytochrome to total phytochrome were similar for all three lamps, but the data do not yet warrant a conclusion about specific wavelengths missing from the LPS lamps that might have induced PAL and TAL activities in plants under the other lamps.     

A Phytochrome-mediated Sequence of Reactions Regulating Cell Division in Developing Callus Cultures

Radiation at the red end of the visible spectrum is active inreducing the extent of the first synchronous division in explantsremoved from Jerusalem artichoke tubers and cultured in a liquidmedium containing 2,4-D. The inhibitory effect of short redlight exposures may be overcome by subsequent irradiation withfar red light. However, prolonged exposures to red light mayonly partially be reversed by far red light. The addition ofcoconut milk to the medium completely overcomes the effect ofshort light exposures but cannot prevent the effect of prolongedlight treatments. These results are consistent with a hypothesiswhich postulates that a phytochrome-mediated reaction initiatesa chain of events which results in the depletion of an essentialmetabolite, also present in coconut milk, below a critical levelrequired for cell division. There is evidence to suggest thatthe essential metabolite is L-phenylalanine.     

Properties of Phenylalanyl-tRNA Synthetase and Phenylalanine Ammonia-lyase of Pine Suspension Cultures

Phenylalanyl-tRNA synthetase and phenylalanine ammonia-lyase activities were demonstrated in partially purified extracts of pine (Pinus elliottii) suspension cultures. The optimum pH for the phenylalanyl-tRNA synthetase reaction was 7.5 and the optimum ATP and Mg²⁺ concentrations were 1.0 and 15 mM respectively. Pine, calf liver and yeast tRNA were inadequate substitutes for pea tRNA in the synthetase reaction mixtures. The optimum pH for the phenylalanine ammonia-lyase reaction was 9.0. The K_m for phenylalanine was approximately 6.6 × 10^?5M. The activity of both enzymes in the partially purified extracts was unstable on storage.     

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)     

Incorporation of [C]Phenylalanine into Flavan-3-ols and Procyanidins in Cell Suspension Cultures of Douglas Fir

L-[¹⁴C]Phenylalanine, fed to cell suspension cultures of Douglas fir, (Pseudotsuga menziesii Franco) was incorporated simultaneously, but at different rates, into (+)-catechin, (−)-epicatechin, and procyanidins of increasing molecular weight. Asymmetric labeling of dimers and polymers was demonstrated, with more label appearing in the upper than in the lower or terminal unit. In addition, the total pool of free monomers was 10 to 30 times more highly labeled than was this lower, terminal unit of dimers and higher oligomers. Since the dimer, epicatechin-catechin, contained more label than catechin-catechin, it is concluded that the carbocation with the 2,3-cis stereochemistry of (−)-epicatechin was formed more rapidly than was that of the 2,3-trans type of (+)-catechin.     

Callus and Cell Suspension Cultures of Carnation

Callus cultures of carnation, Dianthus caryophyllus L. ev. G. J. Sim, were grown on a synthetic medium of half strength Murashige and Skoog salts, 3 % sucrose, 100 mg/l of myo-inositol, 0.5 mg/l each of thiamin, HCl, pyridoxin, HCl and nicotinic acid and 10 g/l agar. Optimal concentrations of growth regulators were observed to be 3 × 10^?6M indoleacetic acid (JAA) combined with 3 × 10^?6M benzylaminopurin (BAP) or 10^?6M 2,4-dichlorophenoxy acetic acid (2,4-D) alone. IAA + BAP caused a 100 fold increase in fresh weight over 4 weeks at 25°C. Addition of casein hydrolysate increased growth further. Cell suspension cultures worked best in media containing 2,4-D in which they had a doubling time of about 2 days. Filtered suspensions were successfully plated on agar in petri dishes, but division was never observed in single cells. The cultures initiated roots at higher concentrations of IAA or NAA, but all attempts to induce formation of shoots or em-bryoids gave negative results.