General Effects of Ethylene on Enzyme Systems in the Cotton Leaf

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.Ethylene can stimulate peroxidase activity in petiole, stem, and leaf blade tissue of cotton plants (6, 12); in the pulvinus and separation layer of bean explants (3,14); in etiolated peas (1); and in sweet potato root discs (3). It has been suggested that the distribution of peroxidase activity and IAA oxidase content can be correlated with the processes of natural abscission (10,11,13,17 (Fig. 1).Groups of 10 segments (each segment 6 mm in length) from the flower pedicels were used for the spectrophotometric assay to determine peroxidase activity. A separate group of 10 segments was collected, and the 6-mm portion of the flower pedicel was further divided into distal (2 mm), abscission zone (2 mm), and proximal (2 mm) segments. Peroxidase activity in enzyme breis prepared from tissue segments was determined where the tissue had been previously exposed to ethylene for periods up to 5 hr.After excision from the flower pedicel the tissue segments (6-and 2-mm sections) were weighed and immediately put into prechilled mortars at 4 C. The tissue samples were ground with a mortar and pestle at 4 C for 5 min in 4 ml of 0.01 M phosphate buffer, pH 6.0. The tissue homogenates were centrifuged for 10 min at 4 C at 2.000g to remove cellular debris.The supernatant fraction was used for the peroxidase assays (15).Spectrophotometric determinations were measured at a wavelength of 460 nm, using a Beckman model DU s...

mentioning

confidence: 99%

Peroxidases in Tobacco Abscission Zone Tissue

Henry

Valdovinos²,

Jensen³

1974

“…Numerous reports on the biochemical effect of ethylene on protein and nucleic acid metabolism have appeared, although the mechanisms involved remain obscure (1,18,34,39). It has been reported that ethylene enhances the formation of enzymes such as peroxidase (16,20,26,37), cellulase (19), polyphenol oxidase (37), and PAL' (20,31) and also the formation of phenolic compounds in plants (5,20). PAL is known to be a key enzyme in the biosynthetic pathway of phenylpropanoids in plants (7,41).…”

mentioning

confidence: 99%

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

Hyodo

Yang

1971

The effect of ethylene on the development of phenylalanine ammonia-lyase activity in segments excined from the epicotyl apex of pea sling was studied. Although there was some increas in phenylanine ammonia-base 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 CO, 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-ynthesizing and -inactivating systems, and that the development of both systems may involve de novo synthesis of protein.Ethylene is known to be a plant hormone initiating fruit ripening and regulating many aspects of plant growth (3, 28). Numerous reports on the biochemical effect of ethylene on protein and nucleic acid metabolism have appeared, although the mechanisms involved remain obscure (1,18,34,39). It has been reported that ethylene enhances the formation of enzymes such as peroxidase (16,20,26,37), cellulase (19), polyphenol oxidase (37), and PAL' (20,31) and also the formation of phenolic compounds in plants (5,20). PAL is known to be a key enzyme in the biosynthetic pathway of phenylpropanoids in plants (7,41). In addition to ethylene, several factors are known to affect the development of PAL. They are light (2, 8-10, 24, 35, 44, 46), wounding (23, 44), disease (12,14,23,29), gamma-ray irradiation (25,30), germination (38), development and differentiation (17,22,32,42), and the application of certain macromolecules (15 (27,33), are known to act as regulators of plant growth, we have therefore examined the effects of applied ethylene on the development of PAL activity in intact etiolated pea seedlings as well as in excised epicotyl segments. The present paper describes the characteristics of PAL development in pea seedlings as affected by the application of ethylene. MATERIAILS AND METHODSEtiolated pea seedlings (Pisum sativum L., cv. Alaska) were grown at 23 C in vermiculite in plastic pots. For the experiments with intact etiolated pea seedlings, two pots (nine plants per pot) of 5-day-old seedlings were placed in 18-liter glass jars fitted with rubber gaskets and metal plates. Water was added to the bottom of the jars to provide moisture. Each jar contained 50 ml of 20% KOH in a beaker to absorb CO. produced during the incubation. In the control jar, an additional beaker co...

“…One approach aimed at elucidating mechanisms whereby ethylene exerts its effects on plant tissues has been to investigate its effect on RNA and protein synthesis (1), and on the level and activity of various enzymes (5,7,9,10,17,19). In these studies evidence was presented for the induction by ethylene of de novo synthesis of enzymes and increased enzymatic activity.…”

mentioning

confidence: 99%

Ethylene-induced Phenylalanine Ammonia-Lyase Activity in Carrot Roots

Chalutz

1973

Ethylene enhanced the activity of phenylalanine ammonialyase in carrot (Daucus carota L., var. "Nautv") 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. (4,8,10,15,16 'Abbreviation: PAL: phenylalanine ammonia-lyase.MATERIALS AND METHODS Fresh carrot (Daucus carota L., var. "Nanty") roots of uniform size were washed in detergent and water and surface sterilized with 70% ethanol. Uniform discs (1 cm thick and 1.5 cm in diameter) were cut from the roots and placed on moist filter paper in open Petri plates. About 50 discs were incubated at 20 C in the dark, inside 20-liter glass jars through which air, ethylene-air, and other gaseous mixtures saturated with water were continuously passed at a rate of 100 ml/min.For nucleic acid and protein synthesis inhibitor studies, discs were dipped for 1 min into the inhibitor solution, then placed on filter paper in Petri plates which contained 2 ml of the inhibitor solution.Acetone powders were prepared from the root discs by cutting the tissue into small pieces and placing 50-g tissue in 500 ml of acetone previously chilled to -18 C. The acetone was changed twice within a period of 1 hr. The tissue was then homogenized for 3 min, filtered through a Whatman No. 1 filter paper in a Buchner funnel, and rinsed with two 500-ml portions of chilled acetone. Mats were dried for 1 hr at room temperature, then kept overnight under vacuum, and stored at -18 C.PAL activity in acetone powders was assayed by the method of Rahe et al. (14) with slight modifications. PAL was extracted by suspending 0.5 g of powder in 10 ml of cold 0.1 M borate buffer, pH 8.8. The suspension was kept in an ice bath for 1 hr with occasional stirring and centrifuged at 40,000g for 20 min at 1 C. The supernatant served as the enzyme preparation. For the assay, 1.5 ml of the supernatant was added to a test tube containing 1.0 ml of 0.05 M L-phenylalanine and 2.5 ml of 0.1 borate buffer, pH 8.8. The assay mixture was incubated in a water bath at 40 C for 1 hr, the reaction was stopped by the addition of 0.1 ml of 5 N HCl, and the acidified mixture was extracted with 7 ml of ether. Two and one-half ml of the ether phase were evaporated to dryness, and the residue was dissolved in 2.5 ml of 0.05 N NaOH. The concentration of cinnamic acid was determined by absorbance at 269 nm, with 0.05 N NaOH as reference. Two controls were run in each assay; a no-substrate assay in which 1.0 ml of deionized water was substituted in the reaction mixture for the L-phenylalanine solution and a zero time stop reaction assay mixture acidified at zero time of incubation. Both controls were assayed as described above. Values obtained from the zero time incubation were applied in the determination of PAL ac...