Ethylene: Modification of peroxidase activity and isozyme complement in cotton (Gossypium hirsutum L.)

Ethylene increased the peroxidase activity in extracts fromcotton plants (Gossypium hirsutum L.) pre-treated with the gas.The response was greatest in young leaves and plant fractionscontaining petioles and abscission zones. Ethylene also increasedthe peroxidase activity of extracts of cotton cotyledonary nodeexplants pre-treated with the gas. These data parallel someearlier reports on IAA-oxidase activity of ethylene-treatedplants. Disc gel electrophoresis of crude extracts of ethylene-treatedcotton seedlings revealed both quantitative and qualitativechanges in peroxidase isozymes from tissues treated with thegas. The possible functional role of peroxidase increased byethylene treatment is discussed in relation to leaf abscission. ¹ Present address: Department of Agronomy, New Mexico StateUniversity, Las Cruces, New Mexico 88001, U.S.A. (Received March 8, 1972; )     

Abscission: role of cellulase

Cellulase (β-1,4-glucan-glucanohydrolase EC 3.2.1.4) activity increased during abscission and was localized in the cell separation layer of Phaseolus vulgaris L. cv. Red Kidney (bean), Gossypium hirsutum L. cv. Acala 4-42 (Cotton) and Coleus blumei Benth. Princeton strain (Coleus) abscission zone explants. Cellulase activity was optimum at pH 7, was reduced by one-half after heating to 55° for 10 min, and was associated with the soluble components of the cell. Explants treated with aging retardants (indoleacetic acid, ⁶N-benzyladenine, and coumarin), CO₂, actinomycin D or cycloheximide had less cellulase activity than untreated controls. Ethylene increased cellulase activity of aged explants after a 3-hr lag period but had no effect on cellulase activity of freshly excised explants. It was concluded that 1 of the roles of ethylene in abscission is to regulate the production of cellulase which in turn is required for cell separation.     

LOCALIZATION OF DEHYDROGENASE AND ACID PHOSPHATASE IN THE ABSCISSION ZONE OF BEAN LEAVES

Leaf abscission in Phaseolus vulgaris L. cv. ‘Contender’ is associated with enzymatic changes during and prior to separation. Deblading resulted in a localized increase in dehydrogenase and acid phosphatase in the abscission zone. Increased enzyme activities were observed 24–48 hr after deblading. In debladed plants separation was complete in 6–8 days. At separation, dehydrogenase activity appeared to decrease and localization was specific to the protective layer, while the petiole side had no activity. In contrast, acid phosphatase activity was observed in some layers of cells on the petiole side after separation. Ethylene treatment promoted abscission and separation occurred in 24–48 hr in both debladed and intact plants. No protective layer was formed during ethylene-induced abscission. Enzymatic changes similar to those observed in debladed control plants were observed with ethylene treatment. Ethylene induced an additional abscission layer between the pulvinus and petiole, where an abscission layer normally does not form. In this ethylene-induced abscission layer, similar enzyme activities were detected.     

Mechanism of Action of Abscission Accelerators

Abscission zone explants of Gossypium hirsutum L., Cassia fistula L., and Coleus blumei Benth. were used to investigate correlations between endogenous rates of ethylene evolution and time of abscission. Additions of 0.1 nl/ml ethylene to the explants markedly accelerated abscission; continuous aeration of the explants, to prevent accumulation of small amounts of endogenously produced ethylene, inhibited abscission compared with that of sealed controls. Substances that stimulated abscission simultaneously accelerated ethylene evolution on all three species and at any position of application. The positional effects of auxin are explained as being due to differences in transport in the explant. Thus, distally applied auxin inhibits abscission, regardless of the accelerated rate of ethylene evolution, by being rapidly transported to the abscission zone. Auxin applied proximally stimulates abscission because it is unable to move as rapidly to the abscission zone and the ethylene effect becomes dominant. Ethylene was found to be most effective on aged tissues, and it is concluded that abscission rates are determined by an increase in sensitivity of the tissue to the ethylene that is already being produced.     

Exo- and Endo-Cellular Cellulase and Polygalacturonase in Abscission Zones of Developing Orange Fruits

The activity of cellulase, cellulase-isoenzymes and polygalacturonase (PG) in the shoot/peduncle and calyx abscission zones (AZ-A and AZ-C, respectively) of young and mature Shamouti orange (Citrus sinensis (L.) Osbeck) fruit explants was tested after extraction of total enzymes from either exo- or endo-cellular fractions from fruits treated with ethylene or 2,4-D. Ethylene enhanced and 2,4-D delayed both abscission and the activity of exo- and endo-cellular cellulase and PG. When tested separately in the exo- and endo-cellular fraction, the effects of both growth regulators on the activity of almost all cellulase isoenzymes were similar, irrespective of their location in the tissue. In mature fruits no abscission occurred in AZ-A, and yet the activity of cellulase and PG was regulated by the hormones as in abscising AZs. This was also true for total activity of exo- and endo-cellular cellulase and PG. Similar effects were observed when the activity of cellulase isoenzymes was tested in AZ-A of non-abscising mature fruits. It is suggested that whenever the increase in activity of the hydrolytic enzymes, and especially cellulase, is not followed by abscission, the substrate is either immune or not available to the enzymes.     

STYLE ABSCISSION IN THE CITRON (CITRUS MEDICA L.) AND OTHER CITRUS SPECIES: MORPHOLOGY,PHYSIOLOGY, AND CHEMICAL CONTROL WITH PICLORAM

Style abscission was studied in detail in citrons (Citrus medica L.) and other citrus varieties. The course of style abscission was followed under orchard conditions and also in an “explant” system consisting of pistils implanted in an agar-sucrose medium and maintained at 25 C in a humid chamber. Morphological and anatomical observations carried out with the explant system revealed a prominent swelling of cell layers proximal to the separation layer prior to abscission. Tests with explants from flowers of different developmental stages showed that before the end of anthesis only the ovaries are capable of performing abscission while style abscission is possible only at a later stage, presumably after fertilization had occurred. Ethylene was able to induce ovary abscission at later stages but could not induce earlier style abscission. Picloram (4, amino-3,5,6-trichloropicolinic acid) increased the percentage of style persistence in citron varieties which naturally tend to retain their styles. Picloram also induced style persistence in Valencia oranges and Eureka lemons, which naturally show 100 % style abscission. Hormonal determinations showed that the style had higher levels of auxin than the ovary but also higher levels of inhibitors, which increased towards the time of style abscission.     

Peroxidase Activity in the Abscission Zone of Bean Leaves during Abscission

Peroxidase activity and localization in the abscission zone of bean leaves were studied histochemically and by gel electrophoresis. Deblading of bean leaves resulted in an increase in peroxidase activity in the abscission zone 2 to 4 days after deblading with highest activity just prior to separation. In debladed plants, the cell division in six to eight layers of cells preceded separation. An ethylene treatment (8 microliters per liter) induced separation of debladed petioles in approximately 24 hours and of intact plants in 36 to 48 hours. Ethylene treatment produced similar results in both debladed and intact plants. In ethylene-treated plants, whether debladed or not, enzyme localization was restricted to only two to three layers of cells with no cell division apparent prior to separation. Infrequent cell divisions were observed after treatment with 2-chloroethylphosphonic acid (1000 micrograms per liter) (Ethephon); however, other changes were similar to those observed with ethylene. Deblading and ethylene treatment resulted in changes in the six peroxidase isozymes observed in the abscission zone. Only four were observed in samples collected 2 centimeters below the abscission zone. Peroxidase bands IV and V increased significantly in debladed and ethylene-treated plants and peroxidase VI decreased only in debladed plants. The changes in peroxidase activity were invariably observed prior to separation in all treatments.     

Enhancement of RNA synthesis, protein synthesis, and abscission by ethylene

Ethylene stimulated RNA and protein synthesis in bean (Phaseolus vulgaris L. var. Red Kidney) abscission zone explants prior to abscission. The effect of ethylene on RNA synthesis and abscission was blocked by actinomycin D. Carbon dioxide, which inhibits the effect of ethylene on abscission, also inhibited the influence of ethylene on protein synthesis. An aging period appears to be essential before bean explants respond to ethylene. Stimulation of protein synthesis by ethylene occurred only in receptive or senescent explants. Treatment of juvenile explants with ethylene, which has no effect on abscission also has no effect on protein synthesis. Evidence in favor of a hormonal role for ethylene during abscission is discussed.     

Optimal and Spatial Analysis of Hormones,Degrading Enzymes and Isozyme Profiles in Tomato Pedicel Explants During Ethylene-Induced Abscission

Activities of degrading enzymes, hormones concentration and zymogram patterns were investigated during control and ethylene-induced abscission of tomato pedicel explants. Exogenous ethylene accelerated abscission of pedicel explants. It was showed that IAA concentration in abscission zone tended to decline at first and then was reduced before separation in control and ethylene-treatment. Moreover, IAA (indole acetic acid) and ABA (abscise acid) concentrations were elevated in each segment when exposing to ethylene, but GA_{1 + 3} (gibberellin1 + gibberellin3) concentration was decreased in abscission zone and the proximal side. Activities of cellulase, polygalacturonase and pectinesterase in the explants were induced in the separating process and strengthened by ethylene. However, comparing with the proximal side, cellulase and polygalacturonase activities in abscission zone and distal side were higher. Electrophoresis of isozymes revealed that at least three peroxidase and three superoxidase isozymes appeared in the explants, respectively. One peroxidase isozyme exhibited differentially among the three positions in control and ethylene-treatment. One esterase isozyme weakened or disappeared in the following hours, but three novel esterase isozymes were detectable from beginning of the process. The data presented support the hypothesis that the distal side, together with abscission zone of explants plays a more important role in separation than does the proximal side. The possible roles of degrading enzymes, hormones and isozymes in three segments during ethylene-induced abscission of tomato pedicel explants are discussed.     

ABSCISIN,AUXIN, AND GIBBERELLIN EFFECTS ON THE DEVELOPMENTAL ASPECTS OF ABSCISSION IN COTTON (GOSSYPIUM HIRSUTUM)

The effects of accelerating and retarding amounts of abscisin (Ab II), auxin (IAA), and gibberellin (GA₃) on abscission in explants of 14-day-old cotton (Gossypium hirsutum L.) seedlings were studied. Applications of Ab II, a potent accelerant (0.025 μg/abscission zone), resulted in a lysigenous breakdown of cells in a weakly defined separation layer in contrast to GA₃, an accelerant (0.01 μg/abscission zone), and IAA, a retardant (0.125 μg/abscission zone), which resulted in a schizogenous type of breakdown of cells in a well-defined separation layer, three or more rows of cells wide. Separation usually commenced adaxially with GA₃, abaxially with IAA and in the controls, and either ad- or abaxially with Ab II. Cell division preceded abscission, the number of cells increasing greatly within 24 hr after GA₃ treatment. Tyloses formed in vessel elements throughout the explant, both distal and proximal to the plane of separation in all treatments and in the controls. The retardant, IAA, appeared to stimulate tyloses formation. Tylosis development was not causal but was secondarily related to abscission.     

Effects of Ethylene and 2,4-D on the Activity of Cellulase Isoenzymes in Abscission Zones of the Developing Orange Fruit

The role of ethylene and 2,4-D in the abscission process, and the induction of cellulase isoenzymes in the abscission zones of Citrus fruit at two physiological stages of fruit development, were studied using a new staining technique for the detection of cellulase isoenzymes in polyacrylamide gels following electrophoretic separation. Four to seven isoenzymes were detected in the shoot-peduncle (zone A) and peduncle-fruit (zone C) abscission zones; at least two of them could be detected at excision time, and of these at least one could not be connected with abscission. In the young fruit, ethylene enhanced and 2,4-D delayed both abscission and the formation of several isoenzymes. In the older fruit, ethylene enhanced and 2,4-D delayed the formation of isoenzymes at a time where no abscission occurred any more in zone A. A slower but significant increase in most of the isoenzyme activity detected was also observed in abscission zone A of untreated older fruit explants after excision. These results fully agree with those reported earlier in relation to total cellulase and polygalacturonase activity (Greenberg et al., Physiol. Plant. 34: 1, 1975) tested at the same stages of fruit development. It is suggested, that the generality of the concept that a rise in hydrolytic enzymes in the abscission zone is necessarily followed by separation of the organ should be re-evaluated.     

Abscission: the role of RNA synthesis

Ethylene stimulated the incorporation of ³²P into RNA in the abscission zone of bean explants (Phaseolus vulgaris L. var. Red Kidney). The enhancement was observed in all fractions separated by methylated albumin kieselguhr column chromatography, although the magnitude of the increase was not the same for each fraction. Differential extraction of the nucleic acids indicated that the ethylene stimulation was confined to the fraction extracted with sodium lauryl sulfate, with the increase mainly in Fraction III (Ribosomal RNA) and Fraction IV (Messenger RNA). Actinomycin D, which blocks ethylene-stimulated abscission, inhibited ³²P incorporation into all column fractions. 5-Fluorouracil, which blocked 50% of the ethylene-enhanced ³²P incorporation, did not inhibit ethylene-enhanced abscission. The results indicate that ethylene may regulate abscission through control of specific RNA's.     

Cell Wall Solubilization in Pedicel Abscission of Begonia Flower Buds

Effects of metabolic inhibitors and growth regulators on the course of abscission and on the activities of cell wall solubilizing enzymes were studied in pedicel explants of Begonia flower buds. Actinomycin D, chloramphenicol and 2,4-dinitrophenol slightly retarded abscission, whereas cycloheximide exerted a strong inhibition if applied until 10.5 h after explant excision. Indoleacetic acid retarded and ethylene promoted abscission and cell wall solubilization. However, the activities of cell wall solubilizing enzymes did not correspond with the course of abscission. No polygalacturonase and pectic acid and pectin transeliminases could be detected in the abscission zone during abscission, whereas a low pectin methylesterase activity did not change. Endo- and exocellulase activities did not increase until about 10 h after the onset of abscission, indicating that they are the result rather than the cause of abscission.     

The Role of Cytokinins and Ethylene in Bean Cotyledon Senescence. The Effect of Free Radicals

During ageing of bean (Phaseolus vulgaris L.) cotyledons in plants with modified life span the time-course of four cytokinins, ethylene, and the end products of free radical attack, lipofuscin-like pigments (LFP), were studied. UV irradiation shortened cotyledon life span, while epicotyl decapitation prolonged it. In controls, LFP increased at the senescence onset but at the end of life span it returned to the initial level. Ethylene increased more than 3-fold at the time of abscission. The content of individual cytokinins (zeatin, zeatin riboside, isopentenyl adenine, isopentenyl adenine riboside) varied differently during ageing but they did not decreased in any case under level observed in young cotyledons at the time of abscission. UV irradiation resulted in 14-fold increase in LFP concentration at the end. Ethylene increased 8-fold 2 h after irradiation. Individual cytokinins increased after UV irradiation to a different extent and time-course, nevertheless cotyledon life span was shortened. Decapitation induced LFP decrease. On day 13, LFP abruptly increased and than decreased and stayed lowered until abscission. Ethylene was maximum on day 24, at the time of abscission, it was above 200 % of control. Decapitation produced transient decrease in some cytokinins namely zeatin and isopentenyl adenine riboside.     

Induced Abscission Sites in Internodal Explants of Impatiens sultani: A New System for Studying Positional Control: with an Appendix: A Mathematical Model for Abscission Sites

Intemodes from Impatiens sultani shoots, explanted into sterileculture, often developed a transverse separation layer afterone to two weeks and the top then abscised from the bottom ofthe explant. Such abscission occurred more rapidly and in agreater proportion of explants when 00001 per cent auxin (IAA)was provided basally and when younger intemodes and shorterexplants were used. The distance of the separation layer fromthe base of the explant varied little with explant length, butincreased with the concentration of auxin applied basally. It seems that in this adventitious abscission the processesof positional definition and differentiation proceed withoutpause, whereas in normal abscission the position is definedearly in development but the final stage of differentiationof the separation layer is delayed until much later when theorgan senesces. To account for the results from the internodal explants andfrom surgical operations on shoots as well as for the characteristicposition of abscission sites of leaves and fruits, we suggestthat the position of abscission is controlled primarily by auxinacting as a morphogen: abscission sites occur at Y-junctionsjust above the base of the arm with the lower activity and auxinstatus, or in single axes above a region of higher auxin status.In both sites, the auxin concentration decreases in the apicaldirection. This hypothesis is supported by a mathematical model (see Appendix)of the interaction of diffusive and polar transport in controllingthe concentration gradient along intemodes with specified auxinconcentrations maintained basally. The model allows predictionsconcerning the site and timing of abscission which accord withobservations on intemodal explants. Impatiens sultani Hook., abscission, auxin, differentiation, diffusion coefficient, IAA, morphogen, polar transport coefficient, positional control, separation layer     

Cherry fruit abscission: evidence for time of initiation and the involvement of ethylene

Initiation of abscission at the pedicel-fruit zone in the sour cherry (Prunus cerasus L. cv. Montmorency) occurs near the transition of Stage II to Stage III of fruit growth. The preinitiation phase is characterized by a high fruit removal force (FRF) and explants prepared from fruits during this period do not undergo abscission as indexed by a reduction in FRF. Ethylene does not cause a significant reduction in FRF either in attached fruit or in explants prepared during this period. By contrast, after initiation (Stage III of fruit growth), there is a marked decrease in FRF with fruit development, explants prepared from fruits during this period undergo abscission, and ethylene markedly promotes the loss in break-strength. Neither the rate of evolution nor the internal concentration of ethylene in the fruit were correlated with fruit abscission. Similar abscission responses, as indexed by FRF and sensitivity to ethylene, were observed in attached fruit and in detached fruit explants.     

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.     

Synthesis of Cellulase during Abscission of Phaseolus vulgaris Leaf Explants

When abscission in leaf explants from Phaseolus vulgaris, cultivar Red Kidney, was allowed to proceed while the explants were in ²H₂O, a 1.25% increase in the buoyant density of cellulase in a cesium chloride gradient was observed. These data indicate that the increase in cellulase activity during abscission is a result of the synthesis of new protein. Two differentially soluble forms of cellulase are present in the abscission zone. The form which is soluble only in a high salt buffer seems more closely related to the abscission process than the form which is soluble in dilute buffer. The correlation between changes in pull force and increase in cellulase activity and the effects of several hormones on cellulase activity are discussed.