Density-labelling evidence for the phytochrome-mediated activation of phenylalanine ammonia-lyase in mustard cotyledons

When dark-grown mustard seedlings are irradiated with far-red light the level of phenylalanine ammonia-lyase (EC 4.3.1.5) activity increases. After ²H₂ O treatment phynlalanine amonia-lyase from seedlings irradiated with far-red light is density-labelled to a lesser extent than enzyme from dark-grown tissue. Theoretical arguments are advanced and data presented which show that this result cannot be explained in terms of an increase in de novo synthesis of phenylalanine ammonia-lyase and that the increase most likely involves activation of existing enzyme.     

1,4-Dihydro-l-phenylalanine-its synthesis and behavior in the phenylalanine ammonia-lyase reaction

1,4-Dihydro-l-phenylalanine, a nonaromatic derivative of l-phenylalanine, has been isolated for the first time. It was synthesized as a yet unobserved minor product in the Birch reduction of l-phenylalanine. This is unexpected because it has an electron donor substituent at a reduced sp(3)-carbon atom of the ring system. Kinetic measurements with phenylalanine ammonia-lyase showed that 1,4-dihydro-l-phenylalanine is no substrate but a moderately good competitive inhibitor of the enzymatic reaction. This is in agreement with its predicted behavior and provides further evidence for the plausibility of the recently proposed mechanism of action of phenylalanine ammonia-lyase.     

Phytochrome-mediated induction of phenylalanine ammonia-lyase in the cotyledons of tomato (Lycopersicon esculentum Mill.) plants

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5.) induction in cotyledons from 96-h dark-grown Lycopersicon esculentum Mill. was studied in response to continuous light and hourly light pulses (blue, red, far red). The increases of PAL promoted by blue and red pulses are reversed completely by immediately following 758 nm irradiations. The response to continuous red light could be substituted for by hourly 6-min red light pulses. The effect of continuous red treatments is mainly due to a multiple induction effect of phytochrome. In contrast to red light, hourly light pulses with far red and blue, light can only partially substitute for continuous irradiation. The continuous blue response could be due to a combination of a multiple induction response and of a high irradiance response of phytochrome. The continuous far red response, could represent a high irradiance response of phytochrome. Dichromatic irradiations indicate that phytochrome is the photoreceptor controlling the light response (PAL) in tomato seedlings.Abbreviations Norflurazon NF-4-chloro-5-(methylamino)-2-(,,,-trifluoro-m-tolyl)-3 (2H) pyridazinone - PAL phenylalanine ammonia-lyase - phytochrome photoequilibrium P_fr/P_tot - P_fr far-red absorbing form of phytochrome - P_r red absorbing form of phytochrome - P_tot total phytochrome: P_r+P_fr     

Phenylalanine-dependent de novo synthesis of phenylalanine ammonia-lyase from Rhodotorula glutinis

A synthetic medium was developed in which the presence of phenylalanine ammonialyase (PAL) in the yeast Rhodotorula glutinis was dependent on the addition of l-phenylalanine. The appearance of PAL activity occurred during mid- to late log phase regardless of the time of l-phenylalanine introduction into the medium. Maximum levels of PAL activity were followed by a rapid decline in both total and specific activity. These changes were accompanied by comparable fluctuations in PAL antigen levels as measured by rocket immunoelectrophoresis. Proteins of yeast grown in the presences of l-phenylalanine were radiolabeled in vivo with l-[³H]leucine. The labeled protein was immunoprecipitated with anti-PAL serum and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A labeled protein comigrated with subunits of authentic PAL. These data support the hypothesis that de novo synthesis of PAL in R. glutinis is l-phenylalanine dependent.     

Polysome assembly and RNA synthesis during phytochrome-mediated photomorphogenesis in mustard cotyledons

The effect of phytochrome (high irradiance reaction, elicited by continuous far-red light) on cellular polysome levels was investigated using ribosome-isolation procedures which prevent the methodological artifacts inherent in previous studies on polysomes. By including the large pool of ribosomal subunits in the analysis and using the ratio (polysomes: monomers + subunits) as a quantitative estimate of the translational capacity of the ribosomes in mustard (Sinapis alba L.) cotyledons, we found the following results: 1) After a lag-phase of less than 30 min, phytochrome induces a massive increase in the relative amount of cytosolic (free) polysomes at the expense of ribosomal subunits. 2) Cytosolic and membrane-bound polysomes are increased by phytochrome in constant proportions (constant ratio of 65:35 in light and darkness). 3) Simultaneously with the light-mediated increase of the polysome level there is an increased incorporation of newly synthesized (labeled) non-ribosomal RNA, presumably mRNA, into the polysomes which can be kinetically discriminated from the slower incorporation of newly synthesized (labeled) rRNA. 4) Cordycepin strongly inhibits the synthesis of RNA and completely prevents the light-mediated increase of polysomes. 5) The electrophoretic patterns of the in-vitro translation products obtained with polysomal polyadenylated RNA from dark-grown and light-grown cotyledons showed no significant qualitative differences. We conclude from these results that photomorphogenesis of mustard cotyledons is related to a massive increase of newly synthesized mRNA leading to a correspondingly increased recruitment of ribosomal subunits into polysomes. The phytochrome-induced increase of translatable mRNA involves mainly quantitative changes in the production of mRNA species which are also present in the dark-grown cotyledons.     

Time course of signal transduction in phytochrome-mediated anthocyanin synthesis in mustard cotyledons

Abstract. The effectiveness of phytochrome (Pfr) in mediating anthocyanin synthesis in epidermal cells of mustard ( Sinupis alba L.) cotyledons is strongly increased by a light pretreatment of the seedling prior to competence (Mohr et al. , 1979; Johnson, 1980). Here we test the hypothesis that the observed 'sensitivity amplification' is related to a change in the time course of signal transduction from phytochrome to the responsive cell function. It was found that the time course of escape from photoreversibility—considered to reflect the time course of signal transduction—is not changed by a light pretreatment, although the rate of signal transduction increases with age and depends strongly on the level of Pfr.     

Putative cis-elements in the promoter region of the carrot phenylalanine ammonia-lyase gene induced during anthocyanin synthesis

Deletion mutants of the carrot phenylalanine ammonia-lyase gene promoter were used to survey cis-elements for their effect on expression of promoter activity by transient expression. Two putative cis-elements were required to give full activity, but a third might be the most important in regulation of the promoter by 2,4-dichlorophenoxyacetic acid. Electronic Publication     

Further observations on phenylalanine ammonia-lyase in fungi

Phenylalanine ammonia-lyase (PAL) has been detected in an Ascomycete, Nectria cinnabarina. Growth in light increases levels of PAL in some but not all Basidiomycetes.     

Purification to homogeneity and some properties of L-phenylalanine ammonia-lyase of irradiated mustard (Sinapis alba L.) cotyledons.

1. Lyase (L-Phenylalanine ammonia-lyase, EC 4.3.1.5) from far-red light-irradiated mustard cotyledons was purified to a single protein using ammonium sulphate fractionation, column chromatography on L-phenylalanyl-Sepharose 4B and on Sephadex G-200, isoelectric focusing and polyacryalmide gel electrophoresis. 2. The enzyme constituted 0.01% of total cellular protein, did not catalyse the deamination of L-tyrosine, had a pH optimum of pH 8.6 and an isoelectric point of pH 5.6. 3. The sedimentation coefficient was estimated as 11.3 S, the Stokes' radius 4.25 nm, and the molecular weight 240 000 +/- 9000 (S.E.). 4. Electrophoresis on denaturing polyacrylamide gels gave a single stained protein band corresponding to a subunit molecular weight of 55 000 indicating a tetrameric structure of equal (or near-equal) size subunits. 5. Maximum velocity (V) for the purified lyase at 25 degrees C was 3.83--4.10 nkat. 1(-1) enzyme and Km value 0.151--0.154 mM. Negative cooperativity (Hill coefficient, n = 1.08) was not detected over the substrate concentration range tested. 6. A putative non-diffusible inhibitor isolated from dark-grown gherkin hypocotyls inhibited the homogeneously purified mustard lyase.     

Phenolic synthesis and phenylalanine ammonia-lyase activity in suspension cultures of Acer pseudoplatanus L.

Summary Phenolic metabolism is influenced by the levels of sucrose, nitrogen and 2,4 dichlorophenoxyacetic acid (2,4-D) in the growth medium. Chromatographic evidence suggests that the principle products are polymers of leucocyanin, (-) epicatechin and (+) catechin, constituting condensed tannins. Comparison of ethanolic cell extracts with extracts from plant organs shows that although these compounds are present in parts of the plant they are not the major phenolics.Cells maintained in a modified Heller's medium containing 9.0×10^–7 M 2,4-D produce increased levels of tannins from mid passage (day 12) onwards. The presence of 2,4-D at 9.0×10^–6 M supresses this response and increased initial sucrose levels cause the amount of tannins to be greater. At the period when tannin levels increase the standard medium is exhaused of its nitrogen sources, urea and nitrate. Increased initial nitrogen levels delay the beginning of increased tannin production and the addition of urea or 2,4-D to cultures already containing high levels of tannins causes the tannin content per gram fresh weight and per culture to decline. These results indicate an antagonism between tannin synthesis and nitrogen metabolism. The activity of phenylalanine ammonia-lyase EC 4.1.1.5. (PAL) estimated by a spectrophotometric method in acetone powders derived from Acer cells increases three to four fold at the onset of increased tannin synthesis and then declines sharply. The phase of high PAL activity correlates with the exhausion of the medium nitrogen sources.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid One of the authors (R.J.W.) was supported by a Science Research Council Studentship during the course of this work.     

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.     

Protection of phenylalanine ammonia-lyase from proteolytic attack

Phenylalanine ammonia-lyase contained within permeabilized cells of Rhodosporidium toruloides was protected from proteolytic attack by trypsin, chymotrypsin and duodenal juice. The inactivation by the proteases was biphasic. The enzyme contained within the yeast cells had a similar Km for phenylalanine and Ki for cinnamic acid to the protein in free solution. Phenylalanine ammonia-lyase present in the yeast depleted duodenal juice of free phenylalanine, while the enzyme in free solution did not. The possibility of using permeabilized cells of R. toruloides as a vehicle for protecting orally ingested therapeutic enzymes from proteolytic inactivation is discussed.     

The phenylalanine ammonia-lyase gene family in Arabidopsis thaliana

Phenylpropanoid derivatives are a complex class of secondary metabolites that have many important roles in plants during normal growth and in responses to environmental stress. Phenylalanine ammonialyase (PAL) catalyzes the first step in the biosynthesis of phenylpropanoids, and is usually encoded by a multi-gene family. Genomic clones for three Arabidopsis thaliana PAL genes containing the entire protein-coding region and upstream and downstream sequences have been obtained and completely sequenced. Two A. thaliana PAL genes (PAL1 and PAL2) are structurally similar to PAL genes that have been cloned from other plant species, with a single intron at a conserved position, and a long highly conserved second exon. Previously identified promoter motifs plus several additional sequence motifs were found in the promoter regions of PAL1 and PAL2. Expression of PAL1 and PAL2 is both qualitatively and quantitatively similar in different plant organs and under various inductive conditions. A third A. thaliana PAL gene, PAL3, differs significantly from PAL1 and PAL2 and other sequenced plant PAL genes. PAL3 contains an additional intron, and its deduced amino acid sequence is less homologous to other PAL proteins. The PAL3 promoter region lacks several sequence motifs conserved between A. thaliana PAL1 and PAL2, as well as motifs described in other genes involved in phenylpropanoid metabolism. A. thaliana PAL3 was expressed at very low levels under the conditions examined.     

Chitosan and chitin oligomers increase phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities in soybean leaves

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and tyrosine ammonia-lyase (TAL, 4.3.1.), the key enzymes of the phenylpropanoid pathway, are inducible in response to biotic (such as chitin from fungal cell walls) and abiotic cues. Application of chitin and chitosan to soybean leaf tissues caused increased activity of PAL and TAL enzymes. The elevation of enzyme activity was dependent on the chain length of the oligomers and time after treatment. The hexamer of chitin and pentamer of chitosan produced the maximum activities at 36 h after treatment as compared to controls. Total phenolic content of soybean leaves increased following chitosan and chitin oligomer treatments, showing a positive correlation between enzyme activity and total phenolic content.