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.     

Rapid induction of ethylene biosynthesis in cultured parsley cells by fungal elicitor and its relationship to the induction of phenylalanine ammonia-lyase

The biosynthesis of ethylene was examined in suspension-cultured cells of parsley (Petroselinum hortense) treated with an elicitor from cell walls of Phytophthora megasperma. Untreated cells contained 50 nmol g^-1 of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), and produced ethylene at a rate of about 0.5 nmol g^-1 h^-1. Within 2 h after addition of elicitor to the culture medium, the cells started to produce more ethylene and accumulated more ACC. Exogenously added ACC did not increase the rate of ethylene production in control or elicitor-treated cells, indicating that the enzyme converting ACC to ethylene was limiting in both cases. The first enzyme in ethylene biosynthesis, ACC synthase, was very rapidly and transiently induced by the elicitor treatment. Its activity increased more than tenfold within 60 min. Density labelling with ²H₂O showed that this increase was caused by the denovo synthesis of the enzyme protein. Cordycepin and actinomycin D did not affect the induction of ACC synthase, indicating that the synthesis of new mRNA was not required. The peak of ACC-synthase activity preceded the maximal phenylalanine ammonia-lyase (PAL) activity by several hours. Exogenously supplied ethylene or ACC did not induce PAL. However, aminoethoxyvinylglycine, an inhibitor of ACC synthase, suppressed the rise in ethylene production in elicitor-treated cells and partially inhibited the induction of PAL. Exogenously supplied ACC reversed this inhibition. It is concluded that induction of the ethylene biosynthetic pathway is a very early symptom of elicitor action. Although ethylene alone is not a sufficient signal for PAL induction, the enhanced activity of ACC synthase and the ethylene biosynthetic pathway may be important for the subsequent induction of PAL.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - PAL phenylalanine ammonia-lyase     

On the Mechanism of the Changes in Phenylalanine Ammonia-lyase Activity Induced by Ultraviolet and Blue Light in Gherkin Hypocotyls

Irradiation with ultraviolet light causes in the hypocotyl of dark-grown gherkin seedlings the partial conversion of trans-hydroxycinnamic acids to the cis-isomers. The trans-hydroxycinnamic acids inhibit the development of phenylalanine ammonia-lyase activity, and the transformation of these compounds to the much less inhibitory cis-isomers forms a ready explanation for the increase in phenylalanine ammonia-lyase activity in the hypocotyl of gherkin seedlings irradiated with ultraviolet light. Arguments are advanced that the increase in phenylalanine ammonia-lyase activity caused by irradiation with blue light is also (at least in part) initiated by trans-cis isomerisation of the hydroxycinnamic acids.     

Specific effects on enzyme activities upon ditution of petroselinum hortense cell cultures into water.

Large increases in the specific activities of phenylalanine ammonia-lyase (EC 4.3.1.5) and p-coumarate:CoA ligase (EC 6.2.1.-) occurred within a few hours after dilution of cultured Petroselinum hortense cells into water. No significant changes in the total amount of extractable protein and in the activities of chalcone isomerase (EC 5.5.1.6) and glutamate dehydrogenase (EC 1.4.1.2) were observed under the same conditions. The time course for the change in phenylalanine ammonia-lyase activity included a lag period of 2–3 h, a peak about 13 h after the onset of induction, and a subsequent period of rapid decline. The inducible amount of enzyme activity was greatly dependent upon the degree of dilution of the cells into water. Simultaneous induction by dilution and irradiation of the cells with white, fluorescent light resulted in an increase in the phenylalanine ammonia-lyase level of activity which exceeded that calculated from the sum of the separately induced levels. Consecutive inductions, first by dilution and then 5 h later by irradiation, each required a lag period of 2–3 h. Actinomycin D or cycloheximide were inhibitors of the induction. While the total protein-synthesizing capacity in vitro was not significantly influenced by the dilution of cells, analysis of the labeled products on polyacrylamide gels demonstrated small but significant changes in the radioactivity profiles. The results are consistent with the hypothesis that dilution of the cells into water reduces the concentration of one or more compounds of cellular origin, thereby stimulating the rate of de novo synthesis of a limited number of proteins.     

Induction of phenylalanine ammonia-lyase and increase in phenolics in lettuce leaves in relation to the development of russet spotting caused by ethylene

Russet spotting (RS), consisting of numerous small brown spots on the midrib of head lettuce (Lactuca sativa), is a physiological disorder induced by exposure to ethylene. In leaves suffering RS, the increase in spotting was accompanied by a parallel increase in the amount of phenolic compounds. Of these, chlorogenic acid and isochlorogenic acid were identified. Ethylene induced high phenylalanine ammonia-lyase (PAL) activity and RS formation in the susceptible cultivar Salinas, but not in the resistant cultivar Calmar. In the absence of ethylene neither significant PAL induction nor RS occurred. No correlation was found between the increase in polyphenol oxidase or peroxidase and the development of RS. The increase in PAL activity, however, was closely correlated with the development of RS. The increase in PAL activity preceded the development of RS, and the extent of RS was directly related to the level of PAL. Three temperatures (0.5, 5.5, and 12.5 C) were compared on the basis of their influence on both RS and PAL induction. At the lowest temperature (0.5 C) neither PAL induction nor RS occurred to a significant extent. At the highest temperature (12.5 C) an initial rapid increase in PAL activity and an earlier development of spotting were observed, but subsequently there was a decrease in both PAL activity and the rate of development of RS. At the medium temperature (5.5 C) both PAL activity and RS increased progresively with time. The decline of PAL activity at a higher temperature might be attributed to inactivation of the enzyme. Thus, a temperature favorable for induction of PAL activity by ethylene was also favorable for RS. These observations indicate a close interrelationship between the induction of PAL activity and the development of RS in response to ethylene, and suggest a causal relationship between the two events. PAL serves as a useful biochemical marker for the RS reaction.     

Ethylene production in rice bronzing leaves induced by ferrous iron

Bronzing, a nutritional disorder of rice plants which is widely distributed in tropical lowlands, was induced by dipping the cut end of rice leaves into FeSO₄ solution (pH 3.5). Ethylene production; the activities of peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase; and the effects of Co²⁺, aminoethoxyvinylglycine, Ag⁺, cycloheximide, and 1-aminocyclopropane-1-carboxylate, were investigated in the course of bronzing development. It was found that ethylene production could be stimulated up to about 20 times that of the control by Fe²⁺, and a peak could be reached at about 24 h after incubation. The Fe²⁺-treated leaves also had 10-fold higher peroxidase activity than the control, whereas in vitro enzyme activity was inhibited by Fe²⁺. Cycloheximide retarded in vivo stimulation of peroxidase, indicating that in vivo stimulation resulted from inducing de novo synthesis of the enzyme. No changes in the activities of phenylalanine ammonia-lyase and polyphenol oxidase were observed. The results, obtained from the incubation of leaves with Co²⁺, aminoethoxyvinylglycine, Ag⁺, cycloheximide, or 1-aminocyclopropane-1-carboxylate, showed that ethylene production was the effect of Fe²⁺ stress and that it was not involved in the process of bronzing development, which is probably an acclimation process to enable plants to cope with stress. The accelerated peroxidase activity may be associated with bronzing development.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - EFE ethylene forming enzyme - PAL phenylalanine ammonia-lyase - POD peroxidase - PPO polyphenol oxidase - SE standard error     

Superinduction of phenylalanine ammonia-lyase in gherkin hypocotyls caused by the inhibitor,L-α-aminooxy-β-phenylpropionic acid

The extractable activity of l-phenylalanine ammonia-lyase (EC 4.3.1.5) and the concentration of sugar esters of p-coumaric and ferulic acids in the hypocotyls of etiolated gherkin seedlings increase upon irradiation with white light. Treatment of intact seedlings with the phenylalanine ammonia-lyase inhibitors α-aminooxyacetic acid and l-α-aminooxy-β-phenylpropionic acid during illumination causes enhanced formation of the lyase and reduces the accumulation of hydroxycinnamic acids. Enzyme activity in excised hypocotyl segments floating on buffer increases in the dark as well as in the light, while hydroxycinnamic acids accumulate only in the light. Phenylalanine ammonia-lyase formation in the segments is inhibited by cinnamic acid and, to a lesser extent, p-coumaric acid, while it is slightly enhanced by caffeic acid and is not affected by ferulic acid.Aminooxyphenylpropionate dramatically promotes phenylalanine ammonialyase formation in the segments in darkness and light and prevents the accumulation of hydroxycinnamic acids in the light. Aminooxyphenylpropionate does not, however, affect the time course of apparent lyase formation and decay. Cinnamic acid, the product of the lyase reaction, antagonizes the effect of aminooxyphenylpropionate. It is proposed that the reaction product(s) are involved to some extent in the regulation of the pool of actively lyase in the hypocotyl tissue.     

Shoot Inversion Inhibition of Stem Elongation in Pharbitis nil: A Possible Role for Ethylene-Induced Glycoprotein and Lignin

Inversion of the upper shoot of Pharbitis nil results in the inhibition of elongation in the inverted stem. The objective of the present study was to determine how shoot inversion-induced gravity stress inhibited elongation and to elucidate the possible role of ethylene-induced glycoprotein and lignin in this process. Determinations of hydroxyproline, peroxidase, phenylalanine ammonia-lyase (PAL), phenol, and lignin content/activity were carried out by appropriate spectrophotometric methods. It was found that inversion and Ethrel treatments of upright shoots caused significant increases in hydroxyproline content, peroxidase, and PAL activity in 12 hours and in phenol and lignin contents in 24 hours. All of these increases except for that of cytoplasmic peroxidase activity were partially reversed by AgNO₃, the ethylene action inhibitor. It is concluded that possible cross-linking associated with the accumulation of ethylene-induced hydroxyproline-rich glycoprotein and lignin may be responsible for the later stages of cessation of elongation in the inverted Pharbitis shoot.     

Circadian Rhythmicity in the Activities of Phenylalanine Ammonia-Lyase from Lemna perpusilla and Spirodela polyrhiza

The oscillations in phenylalanine ammonia-lyase activity from Spirodela polyrhiza and phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities from Lemna perpusilla displayed a circadian rhythm under continuous light. Rhythmicity in enzymic activity could not be detected in continuous darkness since under this condition phenylalanine ammonia-lyase activity remains at a fairly constantly low level. Results from our studies of the oscillatory pattern of the respective activities of phenylalanine and tyrosine ammonia-lyase support their “inseparability.”     

The estimation of phenylalanine ammonia-lyase (PAL)-activity in intact cells of higher plant tissue

Summary A procedure is described which permits the estimation of the relative activity of phenylalanine ammonia-lyase (E.C. 4.3.1.5.) in intact plant cells, exemplified by buckwheat hypocotyls. Hypocotyl segments are incubated at pH 5.5 with L-[3-³H]phenylalanine. N³HH₂, which is liberated from phenylalanine by the action of phenylalanine ammonia-lyase, equilibrates with tissue water to yield ³HOH, which is recovered by sublimation. Participation of phenylalanine transaminase in the reactions leading to ³HOH formation is excluded, and it is conclusively shown that ³HOH is formed intracellularly and not by enzymatic activity leaking out of wounded tissue.Abbreviation PAL phenylalanine ammonia-lyase (E.C. 4.3.1.5.)     

Peroxidases in Tobacco Abscission Zone Tissue: II. Time Course Studies of Peroxidase Activity during Ethylene-induced Abscission

Ethylene-induced abscission in flower pedicels of Nicotiana tabacum L. cv. Little Turkish causes a progressive increase in peroxidase activity during the first 4 hours of a 5-hour time course ethylene treatment period, with decrease in peroxidase activity occurring between 4 hours and 5 hours, when the supernatant extracts of abscission zone segments are tested spectrophotometrically for peroxidase activity, using guaiacol and hydrogen peroxide. Nonethylene-treated tissue has a much lower level of peroxidase activity over the same time course period. In ethylene-treated tissue the decline in break-strength correlates with the beginning of increase in peroxidase activity (3 hours). When the abscission zone area of the pedicel is further divided into proximal, abscission zone, and distal portions, respectively, the ethylene-treated tissue has the highest peroxidase activity in the abscission zone portion, with the maximum peak occurring at 4 hours and decreasing between 4 hours and 5 hours. Acrylamide gel electrophoresis of enzyme breis from ethylene-treated aand nonethylene-treated plants reveals that no new peroxidase isozymes are formed in response to ethylene, indicating an increase in the amount of one or in both of the two already existing isozyme banding patterns. The measurement of protein in the proximal, abscission zone, and distal segments, over a 5-hour ethylene treatment period, indicates that it is being translocated in a distal to proximal direction in the abscission zone pedicel. The possible participatory role for peroxidase in ethylene-induced tobacco flower pedicel abscission are discussed.     

Light-induced changes of enzyme activities in parsley cell suspension cultures: Increased rate of synthesis of phenylalanine ammonia-lyase

When dark-grown cell suspension cultures of parsley (Petroselinum hortense) were illuminated for increasing periods of time, increasing amounts of phenylalanine ammonialyase activity were obtained 5 hr after the onset of light.Pulses of [³⁵S]methionine of varying duration from 1 to 150 min were given to cell cultures in the dark period subsequent to a light period of 2.5 hr. The cells were harvested 5 hr after the onset of light. Analysis of the soluble proteins by polyacrylamide gel electrophoresis revealed a distinct peak of radioactivity coinciding with the activity of phenylalanine ammonia-lyase. The results of experiments in which radioactive methionine was administered for 10 min to dark-grown or light-induced cells at different times after the light period were compared. An efficient incorporation of radioactivity into the fractions possessing the enzyme activity was observed 5 hr after induction, while no significant labeling was detected either after 1.5 or 25 hr, or in extracts from nonilluminated cells. The radioactive fractions containing the enzyme activity were further analyzed by sodium dodecyl sulfate-disc gel electrophoresis. Significant amounts of radioactivity at the molecular weight of the subunits of phenylalanine ammonia-lyase (84,000) were found only in the extracts from cells which had been labeled 5 hr after induction. These results suggest that the light-induced increase in phenylalanine ammonia-lyase activity is due to de novo synthesis, but not to an activation of preformed, inactive enzyme.     

Phenylalanine ammonia-lyase: Purification and characterization from soybean cell suspension cultures

Soybean cell suspension cultures (Glycine max L. cv. Kanrich) grown on high-nitrogen medium produce 50 mU/g fresh wt of phenylalanine ammonia-lyase [EC 4.1.3.5] 7–9 days after inoculation. Nitrate was not limiting when the peak of enzyme activity was reached. Phenylalanine ammonia-lyase was purified 53-fold to essentially electrophoretic homogeneity from cell extracts with 10% recovery. The enzyme was stable in crude extracts and through most stages of purification. No activity could be detected with tyrosine as substrate in either crude extracts or purified enzyme. The electrophoretic mobility was somewhat less than that of the enzyme from maize but both eluted from an agarose column at the same position and the molecular weight of the subunit was similar for both enzymes. Thus the soybean enzyme is composed of four subunits and the native enzyme is ~330,000 M_r. The variation in structure and/or size and availability of hydrophobic regions among phenylalanine ammonia-lyases from four sources (potato, maize, Rhodotorula glutinis, and soybean) was shown by the different elution patterns they exhibited on columns of ω-aminoalkyl agarose (agarose-C_n-NH₂, n = 0 to 8). The order of increasing hydrophobicity is soybean, potato, maize, R. glutinis. The soybean enzyme exhibited negative cooperativity before hydroxylapatite chromatography and positive cooperativity afterward. This is the first example of positive cooperativity observed for phenylalanine ammonia-lyase.     

Wound-induced phenylalanine ammonia-lyase in potato tuber tissue. Development of enzyme activity and effects of antibiotics.

Phenylalanine ammonia-lyase [EC 4.3.1.5.] activity increased rapidly after a 3-hr lag period in potato tuber (Solanum tuberosum L. cv. May Queen) disks incubated in a suitable medium in the dark at 25 degrees. The activity reached a maxinum after incubation for about 40 hr. The effects of actinomycin D, 6-methylpurine, cycloheximide, chloramphenicol, and mitomycin C on the induction of phenylalanine ammonia-lyase were investigated during incubation of the disks. Actinomycin D, 6-methylpurine, and cycloheximide all inhibited the formation of phenylalanine ammonia-lyase, though cycloheximide was the most effective at low concentrations. Application of actinomycin D for the initial lag period (3 hr) caused strong inhibition; however, if it was supplied later it did not inhibit but actually increased phenylalanine ammonialyase formation. In contrast, cycloheximide was effective over most of the incubation period. Chloramphenicol and mitomycin C did not inhibit phenylalanine phenylalanine ammonialyase induction, but markedly stimulated it. Light was not an essential factor for phenylalanine ammonia-lyase induction in the wounded tissue.     

Effects of Light and of Fusarium solani on Synthesis and Activity of Phenylalanine Ammonia-Lyase in Peas

Phenylalanine ammonia-lyase was purified from peas, and a specific antiserum against the enzyme was produced in rabbits. The antiserum was used to study the first 8 hours of the phenylalanine ammonia-lyase activity response in two different organs of the pea from different developmental stages and in response to two different stimuli. Etiolated seedlings were pulse-labeled with l-[(35)S]methionine after either no light exposure or after specific periods of irradiation with blue light. Immature pods were pulse labeled with mixed l-[(3)H]amino acids after specific time periods following inoculation of the pod endocarp surfaces with macroconidia of Fusarium solani. Immunoprecipitates isolated from extracts of each group were analyzed with sodium dodecyl sulfate disc gel electrophoresis and were found to contain a radioactive protein with an electrophoretic mobility identical to that of the phenylalanine ammonia-lyase subunit (M(r) 81,000). The radioactivity contained in the subunit band was interpreted as being due to de novo synthesis of the enzyme. The net rate of phenylalanine ammonia-lyase labeling, found to be initially low in both tissue types, rose dramatically, peaking at approximately a six- to ten-fold greater level at 4 hours after the beginning of the stimulus. Thereafter, the rate of labeling declined slowly. Inoculation with F. solani f. sp. pisi, a true pathogen of peas, caused a fifty per cent greater rate of peak labeling than did inoculation with a nonpathogen, F. solani f. sp. phaseoli. The time profile of the changing rate of labeling correlates with the changing activity level of the enzyme which peaks at 12 hours after the onset of the stimulus. The data presented favor a model which explains the changing activity of phenylalanine ammonia-lyase as being due to a changing rate of synthesis or degradation (or both) of the enzyme rather than due to the activation of a preformed zymogen.     

Involvement of phenylalanine ammonia-lyase in the development of pollen in broccoli (Brassica oleracea L.)

Summary To determine whether phenylalanine ammonia-lyase (EC 4.3.1.5) is involved in the maturation of microspores to fertile pollen, anthers of a fertile strain of broccoli (Brassica oleracea L.) were studied in a comparison with anthers of a cytoplasmic male sterile strain. In the normal fertile strain, immature anthers of about 2 mm in length exhibited higher phenylalanine ammonia-lyase activity than mature anthers or those shorter than 2 mm. The 2-mm-long anthers corresponded to the mononucleate stage, just after release of the microspores during pollen development. Immunohistochemical localization of phenylalanine ammonia-lyase in the anthers indicated that the protein was present predominantly in the tapetal cells. The immature anthers of cytoplasmic male sterile broccoli had a lower phenylalanine ammonia-lyase activity than those of the normal fertile strain. The level of phenylalanine ammonia-lyase activity in the immature anthers was positively correlated with the number of fertile pollen grains at the flowering stage in both strains. It seems possible, therefore, that phenylpropanoid metabolism, which involves phenylalanine ammonia-lyase, may play an important role in the maturation of microspores in flowering plants.Abbreviations CHS chalcone synthase - CMS cytoplasmic male sterility - DAPI 4, 6-diamidmo-2-phenylindole dihydrochloride - PAL L-phenylalanine ammonia-lyase     

Plant hormone interaction and phenolic metabolism in the regulation of russet spotting in iceberg lettuce

Russet spotting (RS) is a physiological disorder induced in iceberg lettuce (Lactuca sativa L.) by exposure to parts per million levels of ethylene at 5 ± 2°C. Ethylene induced phenylalanine ammonia-lyase and ionically bound peroxidase activities that correlated with development of RS symptoms. The ethylene-treated tissue had significantly higher lignin content than air control tissue with lignification localized in walls of RS-affected cells. Ethylene also caused the accumulation of the flavonoids (+)catechin and (−)epicatechin and the chlorogenic acid derivatives 3-caffeoyl-quinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid. These soluble phenolic compounds were readily oxidized to brown substances by polyphenol oxidase isolated from RS tissue. Ethylene substantially increased ionically bound indole-3-acetic acid (IAA) oxidase activity, while IAA application greatly reduced ethylene-induced phenylalanine ammonia-lyase, peroxidase, and IAA oxidase activities, soluble phenolic content, and RS development.     

Red and far red effects on phenylalanine ammonia-lyase in raphanus and sinapis seedlings do not correlate with phytochrome spectrophotometry

In seedlings of Raphanus sativus (radish) and Sinapis alba (mustard), irradiation for 6 hours with far red light significantly increases the extractable activity of phenylalanine ammonia-lyase by the end of the light period. A schedule of 10 minutes red light-110 minutes darkness-10 minutes red-110 minutes darkness-10 minutes red-110 minutes darkness has no effect as compared to dark controls. However, the red light program maintains a level of far red-absorbing phytochrome always measurable by in vivo spectrophotometry during the 6-hour experimental period. We conclude that the far red effect on this enzyme and for this specific material cannot be explained solely by formation and maintenance of far red-absorbing phytochrome.