Induction of Phenylalanine Ammonia-lyase in <i>Xanthium</i> Leaf Disks. Photosynthetic Requirement and Effect of Daylength

Zucker, Milton

doi:10.1104/pp.44.6.912

Cited by 104 publications

(38 citation statements)

References 27 publications

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“…A number of factors lead to enhanced activity of both enzymes. The available evidence suggests that the increase in activity is due to synthesis of new protein and not to activation of a pre-existing protein (1,24). Recently, evidence has accumulated that the loss of activity is also an active process (14,20,23,24).…”

Section: Methodsmentioning

confidence: 99%

“…The reduction in activity, however, was smaller and less consistent than that of the other enzymes studied. Both nitrate reductase and PAL are reported to be enzymes formed and inactivated within a few hours and subject to control mechanisms which as yet are only poorly understood (3, 1 1,17,23,24). A number of factors lead to enhanced activity of both enzymes.…”

Section: Methodsmentioning

confidence: 99%

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Effects of Water Stress on the Activities of Three Enzymes in Maize Seedlings

Bardzik¹,

Marsh²,

Havis³

1971

Plant Physiol.

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Changes in the activities of three enzymes (nitrate reductase, L-phenylalanine ammonia-lyase, and a dehydronicotinamide adenine dinucleotide-oxidase complex) were measured during development of water stress in young maize (Zea mays) plants.L-Phenylalanine ammonia-lyase and nitrate reductase activities decreased markedly with water deficits of 10 to 20%. 'Abbreviation: PAL, L-phenylalanine ammonia-lyase. MATERIALS AND METHODSThe dominant tall form of the d1 dwarf maize (Zea mays) was grown in vermiculite in the greenhouse. Nutrient requirements were provided with a modified Hoagland's solution 1 (9). Moisture stress was initiated by withholding water for varying periods beginning approximately 10 days after germination. In early experiments, the plants remained in the greenhouse until sampled for assay, but in later experiments they were transferred to a growth chamber when water stress was initiated. The chamber environment was 27 C, 30 to 50% relative humidity, and had a light period of 12 hr/day of 1000 ft-c. The plants were sampled at the middle of the light period. In the rehydration experiments, the vermiculite base was saturated with water 24 hr prior to sampling.Water deficit was estimated by the method of Weatherly (21). Duplicate or triplicate 5-cm sections were taken from the midportion of the third leaf of two or three plants. The original weight was determined immediately after harvest, and the tissue was floated in deionized water for 4 hr. At that time, the saturated weight was taken and the tissue was dried at 95 C for 24 hr for dry weight determination. The water deficit was expressed as the ratio of water taken up divided by the final water content, multiplied by 100.The midportion of the leaf blades from four to six seedlings was used as the source of tissue for nitrate reductase and NADH oxidase. PAL was prepared from the leaf sheaths of the same or comparable seedlings. Nitrate reductase activity was determined by the method of Hageman and Flesher (6), except that the tissue was ground in a chilled mortar with pestle and sand. PAL was prepared and assayed as described by Reid and Marsh (15). NADH oxidase activity was determined in the same extract used for nitrate reductase. The reaction mixture for the oxidase assay contained 200 ymoles of tris buffer, pH 8.2; 20 ,tmoles of (NH4)2SO4; 0.66 umole of NADH; plus enzyme in a final volume of 3 ml. The reaction was run at 30 C and was initiated by the addition of NADH. Changes in absorbancy at 340 nm were followed spectrophotometrically for 5 min.One unit of activity for all three enzymes was defined as the amount of enzyme which catalyzed the formation of 1 ,tmole of product in 1 min under standard conditions. Protein was determined by standard biuret or Lowry methods (12) with bovine serum albumin as a standard. RESULTSThe results of water deficit measurements (Fig. 1)

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Effects of Water Stress on the Activities of Three Enzymes in Maize Seedlings

Bardzik¹,

Marsh²,

Havis³

1971

Plant Physiol.

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“…It is known that detached leaves of some plants, when floated on water in the light, exhibit increased activity of glucose-6-phosphate dehydrogenase (Farkas et al 1964;Lewington, Talbot, and Simon 1967) and also of phenylalanine ammonia-lyase (Creasy 1968;Engelsma 1968;Zucker 1969). These two enzymes are associated with the synthesis of phenolic compounds.…”

Section: Resultsmentioning

confidence: 99%

Changes in the Formononetin Content of Detached Mature Leaves of Trifolium Subterraneum

Rossiter¹

1970

Aust. Jnl. Of Bio. Sci.

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Anthocyanin formation is stimulated in the leaves of some plants when the leaves are detached and floated on water or on sucrose solutions and exposed to light (e.g. Eberhardt 1954;Creasy, Maxie, and Chichester 1965). The synthesis of other flavonoids, e.g. flavans, can be increased under these conditions (Creasy and Swain 1966).We have observed that anthocyanin formation can be greatly stimulated by detaching fully expanded leaves of subterranean clover (T. subterraneum) and holding them for several days in diffuse daylight. We then wondered whether the oestrogenic isoflavones formononetin, genistein, and biochanin A, which occur in subterranean clover (Beck 1964), were also increased. If they were, the point would be of some interest, since with entire plants the net increase in the amounts of these three isoflavones after the leaves reach full expansion is negligible (Rossiter and Beck 1967).Several simple experiments were done with detached leaves, mostly using the Northam A strain of T. subterraneum. Increases in biochanin A were usually small, while for genistein the increases were seldom more than 50% and usually much less than this. Results for these two compounds are not presented here, since treatment differences rarely reached statistical significance. However, for formononetin there were large relative increases, and these are reported below. Materials and MethodsThe clover plants were grown in shallow boxes in an open-sided glasshouse during the winter growing season (average day and night temperatures usually more than 15°e and 10 0 e, respectively). Mature leaves showing no visible senescence were harvested between 1100 and 1500 hr as required, mixed before sorting in a cold room, and weighed. Sufficient leaves were used for each sample to provide a total fresh weight of c. 500 mg. The leaves (laminae only) were floated on 30 ml of solution in covered Petri dishes (9 cm diameter). In all instances the dry matter content of the leaves was within the range 20-25%.Unless specified otherwise, incubations were done in diffuse daylight near a laboratory window where the mean temperature was 21°e, and the range about ±2°e. Light intensity at the leaf surface seldom exceeded 350 f.c., as measured with an EEL light-meter. Some incubations were done in an artificially lit type LB cabinet (Morse and Evans 1962).Formononetin was estimated by the thin-layer chromatographic method of Beck (1964) with modifications based on the microtechnique of Francis and Millington (1965).In the first two experiments the treatments were unreplicated, but three replications were used thereafter. Isoflavone contents are expressed on the basis of original fresh weight, rather than per leaf, since leaf size varied between strains and between experiments.

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“…The wavelength of 290 nm was chosen in order to avoid the absorption of phenylalanine that interferes with X maximum of cinnamate at 268 nm (17). In later work Zucker replaced the acetone powder preparation by direct aqueous extract because of the rapidity of this procedure, as well as the decrease in variation between samples as compared with the acetone powder method (18,19). In crude aqueous extracts, it may be expected that a-keto acids will be present, thus furthering the activity of PT in the reaction mixture determining PAL.…”

Section: Methodsmentioning

confidence: 99%

Possible Errors in Quantitative Determination of Phenylalanine Ammonia-Lyase Activity by Spectrophotometric Methods

Erez¹

1973

Plant Physiol.

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A possible error in spectrophotometric determination of cinnamate, the product of phenylalanine ammonia-lyase activity, using nonpurified protein extracts has been shown.Under optimal conditions for phenylalanine ammonia-lyase activity, with borate buffer and in the presence of ao-keto acids, phenylpyruvate is produced and its enol tautomer-borate complex formed, strongly absorbs at 290 nanometers.idoxal phosphate to the reaction system (4) was not confirmed for phenylalanine (5) or the related tryptophan (14) transaminases. PLP will interfere in,the estimation of trans-cinnamate at 290 nm only under conditions that will drive the keto-enol equilibrium of PLP towards the enol tautomer. This will happen under acidic conditions, under very basic conditions (12), or in the presence of borate (8). The complex enol-borate formed absorbs strongly at 290 nm with X maximum at 298 nm. The possible interference between the two enzymes has therefore been investigated in crude extracts of peach apices in borate buffer, pH 8.8. MATERIALS AND METHODSPhenylalanine ammonia-lyase, first described in plant tissue by Koukol and Conn (9), has received much attention during the last decade mainly because of its strategic position in the biosynthetic pathway of polyphenols and lignin (10) and because of the strong effect light has on its synthesis (3,17). Physiological interest in this enzyme led to the development of rapid methods for determination of PAL2 activity. The method used by Zucker (17) of determining the absorption of the trans-cinnamate formed at 290 nm in the reaction mixture has been followed in most cases (2,11,15). His reaction mixture consisted of borate buffer pH 8.8, L-phenylalanine, and enzyme extract. The wavelength of 290 nm was chosen in order to avoid the absorption of phenylalanine that interferes with X maximum of cinnamate at 268 nm (17). In later work Zucker replaced the acetone powder preparation by direct aqueous extract because of the rapidity of this procedure, as well as the decrease in variation between samples as compared with the acetone powder method (18,19). In crude aqueous extracts, it may be expected that a-keto acids will be present, thus furthering the activity of PT in the reaction mixture determining PAL. Purifying crude extracts with active carbon (11) did not remove a-keto acids from the enzyme preparation. In these cases PLP and an amino acid corresponding to the keto acid present will be produced. PT is widespread in plants (5, 6, 10). Its pH optimum is 8.0. Furthermore, PT has a rather broad substrate specificity for the keto acid-amino group receptor, and it transaminates phenylalanine with a-KG, pyruvate, and oxaloacetate (4, 5). The Two-year-old plants of the peach cultivar Redhaven, grown in 15-liter containers outdoors, were used. Apices consisting of the meristem and the three youngest leaves were taken for enzymic analyses.Enzymic extract has been prepared essentially according to Wightman and Cohen (16) in phosphate buffer, pH 8.5. The grinding medium included 10 mm...

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Induction of Phenylalanine Ammonia-lyase in Xanthium Leaf Disks. Photosynthetic Requirement and Effect of Daylength

Cited by 104 publications

References 27 publications

Effects of Water Stress on the Activities of Three Enzymes in Maize Seedlings

Effects of Water Stress on the Activities of Three Enzymes in Maize Seedlings

Changes in the Formononetin Content of Detached Mature Leaves of Trifolium Subterraneum

Possible Errors in Quantitative Determination of Phenylalanine Ammonia-Lyase Activity by Spectrophotometric Methods

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