Deferral of senescence and abscission by chemical inhibition of ethylene synthesis and action in bean explants

Three compounds known to inhibit ethylene synthesis and/or action were compared for their ability to delay senescence and abscission of bean explants (Phaseolus vulgaris L. cv Contender). Aminoethoxyvinyl-glycine (AVG), AgNO₃, and sodium benzoate were infiltrated into the petiole explants. Their effect on abscission was monitored by measuring the force required to break the abscission zone, and their effect on senescence was followed by measuring chlorophyll and soluble protein in the distal (pulvinus) sections. AVG at concentrations between 1 and 100 micromolar inhibited ethylene synthesis by about 80 to 90% compared to the control during sampling periods of 24 and 48 hours after treatment. This compound also delayed the development of abscission and senescence. Treatment with AgNO₃ at concentrations between 1 and 100 micromolar progressively reduced ethylene production, but to a lesser extent than AVG. The effects of AgNO₃ on senescence and abscission were quite similar to those of AVG. Sodium benzoate at 50 micromolar to 5 millimolar did not inhibit ethylene synthesis during the first 24 hours, but appreciably inhibited ethylene synthesis 48 hours after treatment. It also delayed the development of abscission and senescence. The effects of AVG, Ag⁺, and sodium benzoate suggest that ethylene could play a major role in both the senescence induction phase and the separation phase in bean explants.     

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.     

Abscission: the phytogerontological effects of ethylene

The role of ethylene in the aging of bean (Phaseolus vulgaris L. cv. Red Kidney) petiole abscission zone explants was examined. The data indicate that ethylene does accelerate aging in addition to inducing changes in break strength. Application of ethylene during the aging stage (stage 1) promoted abscission when followed by a second ethylene treatment during the cell separating stage (stage 2). The half-maximal effective concentration of ethylene to induce aging was around 0.3 microliter per liter; 10 microliters per liter was a saturating dose. CO₂ reversal of ethylene action during stage 1 was incomplete and gave ambiguous results. CO₂ (10%) reversed the effect of 10 microliters per liter ethylene but not 1 microliter per liter ethylene. The possibility that ethylene not only accelerated aging but was also a requirement for it was tested, and experimental evidence in favor of this idea was obtained. It was concluded that ethylene plays a dual role in the abscission of bean petiole explants: a phytogerontological effect and a cellulase-inducing effect.     

Patterns of Ethylene and Carbon Dioxide Evolution during Cotton Explant Abscission

The relationship between abscission and the evolution of ethylene and CO₂ was examined in explants and explant segments of cotton seedlings (Gossypium hirsutum L. cv. Acala SJ-1) under both static and flow system conditions, and in the presence and absence of mercuric perchlorate. Explant excision was immediately followed by increased ethylene evolution (wound ethylene); senescence was also accompanied by increased ethylene evolution (senescence ethylene). One or two ethylene peaks were found to interrupt the low background rate of ethylene evolution during the period between excision and senescence. The first intermediate ethylene peak coincided with a rise in CO₂ evolution; however, precedence could not be established. No statistical correlations were discovered between either intermediate ethylene peak and abscission. The best statistical correlation was found between wound ethylene and abscission at 12 hr after excision. No positive correlations were found between senescence ethylene and abscission. Implications of these results for the understanding of the role of ethylene in explant abscission are discussed.     

Characterization of a non-abscission mutant in Lupinus angustifolius. I. Genetic and structural aspects

A spontaneous mutant, Abs, that does not abscise any organs despite an apparently normal pattern of growth and senescence was isolated from among plants of Lupinus angustifolius cv. 'Danja'. Abs was found to be a recessive single gene mutation, and it was proposed that the gene for the original mutant phenotype, referred to as Abs, be designated abs1. An artificially induced mutant allelic to abs1 was also obtained and a non-allelic mutant phenotype, Delabs (delayed abscission), which was designated abs2. Morphological and cytological features of the abscission process under conditions of natural and ethylene-induced senescence were compared in the wild-type parent and Abs mutant. In the parent genotype abscission under natural conditions is similar to many other species, consisting of a stage of cell division forming an abscission zone, activation of the cytoplasm of zone cells, dissolution of the middle lamella, disorganization of fibrillar wall structure, and cell separation. A slightly different pattern of abscission zone development was observed for ethylene-treated explants of the parent, mainly with respect to features of cell division and cell enlargement. In Abs no abscission occurred for any abscission sites under conditions of natural senescence or with ethylene treatment of small shoot explants. However, relatively normal abscission zones were differentiated at all sites in the mutant except that extensive cell wall disorganization did not occur. Ethylene production by leaves or other organs of the mutant was no different from that of Danja. Application of copper salts or hydrogen peroxide, droughting, waterlogging, or application of abscisic acid (ABA) increased ethylene production equally in both genotypes but did not result in abscission in the mutant. Release of root cap border cells, the only other cell separation process examined, was similar in each genotype. The study concludes that the mutation is quite specific to the abscission process and may be due to a lack of or delay in the expression of hydrolytic enzyme(s) associated specifically with abscission zone differentiation and separation.     

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.     

Methyl jasmonate induces the formation of secondary abscission zone in stem of Bryophyllum calycinum Salisb

Methyl jasmonate (JA-Me) at a concentration of 0.5 % induced the formation of secondary abscission zone and senescence in several types of stem explants (only internode segment, internode segment with nodes and without leaves, internode segment with nodes and debladed petioles) of Bryophyllum calycinum when it was applied in various places of the stem or the debladed petiole as lanolin paste. In the presence of small leaves in stem explants methyl jasmonate also induced the formation of secondary abscission zone and senescence but the presence of larger leaves completely inhibited methyl jasmonate-induced processes. Auxin, (indole-3-acetic acid, IAA), at a concentration of 0.1 % extremely prevented the formation of secondary abscission zones and senescence in the stem tissues induced by methyl jasmonate. Similar relationship between auxin and methyl jasmonate to induce the formation of secondary abscission zone and senescence was found in decapitated shoot of the intact plant. Mechanisms of the formation of secondary abscission zone are also discussed in terms of the interaction of methyl jasmonate with auxin.     

Is uronic acid oxidase involved in the hormonal regulation of abscission in explants of citrus leaves and fruits?

The role of uronic acid oxidase in abscission was studied in explants of citrus ( Citrus sinensis L. Osbeck; var. Shamouti) leaves and fruits. In leaf explants, activity of uronic acid oxidase prior to onset of abscission and the rate of abscission were markedly accelerated by ethylene and delayed by 2,4-dichlorophenoxyacetie acid. Similar results were obtained for uronic acid oxidase activity in the exocellular fraction of young fruit explants. In mature fruit explants, treated with ethylene, an immediate increase in activity was evidenty in the non-active shoot/peduncle abscission zone, whereas in the calyx abscission zone the rise in activity occurred after a prolonged exposure to ethylene, when most of the fruits had already abscised. Whenever ethylene enhanced uronic acid oxidase activity, 2,4-dichlorophenoxyacetic acid delayed it. A gradient of decreasing activity or uronic acid oxidase was recorded from both sides of the abscission zone in leaves and fruits toward the separation line, where activity was the lowest as compared with the activity found in adjacent tissues. It is suggested that uronic acid oxidase is involved in senescence and cell wall degradation. However, it is yet questionable whether this enzyme is directly related to the control mechanism of abscission.     

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.     

Transdifferentiation of Mature Cortical Cells to Functional Abscission Cells in Bean

Abscission explants of bean (Phaseolus vulgaris L.) were treated with ethylene to induce cell separation at the primary abscission zone. After several days of further incubation of the remaining petiole in endogenously produced ethylene, the distal two-thirds of the petiole became senescent, and the remaining (proximal) portion stayed green. Cell-to-cell separation (secondary abscission) takes place precisely at the interface between the senescing yellow and the enlarging green cells. The expression of the abscission-associated isoform of β-1,4-glucanhydrolase, the activation of the Golgi apparatus, and enhanced vesicle formation occurred only in the enlarging cortical cells on the green side. These changes were indistinguishable from those that occur in normal abscission cells and confirm the conversion of the cortical cells to abscission-type cells. Secondary abscission cells were also induced by applying auxin to the exposed primary abscission surface after the pulvinus was shed, provided ethylene was added. Then, the orientation of development of green and yellow tissue was reversed; the distal tissue remained green and the proximal tissue yellowed. Nevertheless, separation still occurred at the junction between green and yellow cells and, again, it was one to two cell layers of the green side that enlarged and separated from their senescing neighbors. Evaluation of Feulgen-stained tissue establishes that, although nuclear changes occur, the conversion of the cortical cell to an abscission zone cell is a true transdifferentiation event, occurring in the absence of cell division.     

Citrus leaf abscission. Regulatory role of exogenous auxin and ethylene on peroxidases and endogenous growth substances

Abstract The abscission of citrus leaf explants demonstrates the well-known enhancing effect of ethylene and the delaying one of auxin when treatment is started at excision time. Total peroxidase activity increases differently in tissues of the blade, abscission zone, and petiole. The highest activity at zero time is recovered in abscission zone in which also the response to the abscission regulators is the most visible. Isoperoxidase profiles are modified in opposite directions by ethylene and auxin respectively. Both regulators affect the activity of the same cathodic and anodic isoperoxidases without any qualitative changes. By the same time, auxin-like compounds increase in isolated abscission zones at 24 h from excision and decrease at 48 h. The level of one inhibitor complex undergoes an inverse variation. It is suggested that the increase in auxin during the first stage of abscission is necessary for influencing the growth of cells which is required to cause abscission.     

Abscisic Acid, Auxin, and Ethylene in Explant Abscission

Experiments with explants of Phaseolus vulgaris L., cv. CanadianWonder, show that abscission and the associated rise in oarboxymethyl-cellulaseactivity in the separation zone are initiated by a peak in ethyleneproduction during senescence of pulvinar tissue distal to thezone. Distal applications of abscisic acid (ABA) induce an earlierpeak in ethylene production, increase cellulase activity, andpromote abscission. ABA is more effective in these ways if treatmentis delayed from 0 to 24 h after excision. With increasing concentrations of ABA the maximum rate of ethylene production is achievedsooner. Indol-3yl-acetic acid (IAA) and ABA are antagonisticin this system and have opposing effects. IAA retards the timeof peak ethylene-production and delays abscission. Explantsmay be retained for long periods without abscinding if incubatedin an ethylene-free atmosphere: the addition of ethylene forany one 24-h period (except the first 24 h after excision) willinduce abscission. The initial period of insensitivity to ethyleneis extended by distal applications of IAA. Ethylene-inducedabscission can be inhibited by IAA applied up to 72 h afterexcision provided the ethylene is not applied first. It is proposedthat abscission in the explant is controlled at two levels:(1) an auxin-dependent stage determining the duration of insensitivityto ethylene; (2) the timing of a rise in ethylene productionin senescing tissue distal to the separation zone. An auxin-ethylenebalance-mechanism at the separation zone is discussed.     

Abscission: movement and conjugation of auxin

A 1-hour application of indole-3-acetic acid to bean (Phaseolus vulgaris L. cv. Red Kidney) explants inhibited abscission for an 8-hour aging period. Use of indole-3-acetic acid-¹⁴C showed that the applied indole-3-acetic acid was conjugated within explant tissue and that this conjugation mechanism accounts for loss of effectiveness of indole-3-acetic acid in inhibiting abscission after 8 hours. Reapplication of indole-3-acetic acid to an explant at a later time, before the induced aging requirement was completed reinhibited abscission. 2,4-Dichlorophenoxyacetic acid, which is not destroyed or conjugated by this system, did not lose its ability to inhibit abscission. It was concluded that indole-3-acetic acid destruction is one of the processes involved in the aging stage of abscission in explants.     

Abscission of mango fruitlets as influenced by enhanced ethylene biosynthesis

Experiments were conducted on developing fruitlet explants of two mango (Mangifera indica L.) cultivars to establish the source and dynamics of ethylene production prior to and during fruitlet abscission. Abscission of all fruits in the samples occurred at approximately 86 and 74 hours postharvest in `Keitt' and `Tommy Atkins,' respectively. Increased abscission began 26 hours from harvest and was preceded by enhanced ethylene synthesis. Enhanced ethylene production initiated approximately 48 hours prior to abscission and increased to a maximum near the time of fruitlet abscission. The seed produced the highest amount of ethylene on a per gram fresh weight basis. The pericarp, however, was the main source of ethylene on an absolute basis, since it represented more than 85% of total fruitlet weight. Pedicels containing the abscission zone produced no detectable ethylene prior to or at the moment of abscission. Fumigation of `Tommy Atkins' fruitlets with 1, 15, or 100 microliters per liter ethylene accelerated abscission by 24 to 36 hours in comparison with unfumigated controls. Diffusion of ethylene from distal fruitlet tissues to the abscission zone triggers the events leading to separation of the fruit from the tree.     

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.     

The physiological significance of phenylacetic Acid in abscising cotton cotyledons

The physiological role of phenylacetic acid (PAA) as an endogenous regulator of cotyledon abscission was examined using cotton (Gossypium hirsutum L. cv LG 102) seedlings. Application of 100 micromolar or more PAA to leafless cotyledon abscission-zone explants resulted in the retardation of petiole abscission and a decrease in the rise of ethylene evolution that normally accompanies aging of these explants in vitro. The partial inhibition of ethylene evolution in these explants by PAA was indirect since application of this compound stimulated short-term (<24 hours) ethylene production. PAA treatment partially suppressed the stimulation of petiole abscission elicited by either ethylene or abscisic acid. Both free and an acid-labile, bound form of PAA were identified in extracts prepared from cotyledons. No discernible pattern of changes in free or bound PAA was found during the course of ethylene-induced cotyledon abscission. Unlike indole-3-acetic acid, transport of PAA in isolated petiole segments was limited and exhibited little polarity. On the whole, these results are not consistent with the direct participation of PAA in the endogenous regulation of cotyledon abscission.     

Endogenous ethylene and abscisic Acid relative to phytogerontology

Endogenous production of ethylene and endogenous levels of abscisic acid were measured from Hibiscus rosa-sinensis L. abscission zone explants at six stages of development: tight bud, open flower, closed flower, petal abscission, calyx abscission, and peduncle abscission.     

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.     

Further examination of abscission zone cells as ethylene target cells in higher plants

BACKGROUND AND AIMS: Two aspects of the competence of abscission zone cells as a specific class of hormone target cell are examined. The first is the competence of these target cells to respond to a remote stele-generated signal, and whether ethylene acts in concert with this signal to initiate abscission of the primary leaf in Phaseolus vulgaris. The second is to extend the concept of dual control of abscission cell competence. Can the concept of developmental memory that is retained by abscission cell of Phaseolus vulgaris post-separation in terms of the inductive/repressive control of beta-1,4-glucan endohydrolase (cellulase) activity exerted by ethylene/auxin be extended to the rachis abscission zone cells of Sambucus nigra? METHODS: Abscission assays were performed using the leaf petiole-pulvinus explants of P. vulgaris with the distal pulvinus stele removed. These (-stele) explants do not separate when treated with ethylene and require a stele-generated signal from the distal pulvinus for separation at the leaf petiole-pulvinis abscission zone. Using these explants, the role of ethylene was examined, using the ethylene action blocker, 1-methyl cyclopropene, as well as the significance of the tissue from which the stele signal originates. Further, leaf rachis abscission explants were excised from the compound leaves of S. nigra, and changes in the activity of cellulase in response to added ethylene and auxin post-separation was examined. KEY RESULTS: The use of (-stele) explants has confirmed that ethylene, with the stele-generated signal, is essential for abscission. Neither ethylene alone nor the stelar signal alone is sufficient. Further, in addition to the leaf pulvinus distal to the abscission zone, mid-rib tissue that is excised from senescent or green mid-rib tissue can also generate a competent stelar signal. Experiments with rachis abscission explants of S. nigra have shown that auxin, when added to cells post-separation can retard cellulase activity, with activity re-established with subsequent ethylene treatment. CONCLUSIONS: The triggers that initiate and regulate the separation process are complex with, in bean leaves at least, the generation of a signal (or signals) from remote tissues, in concert with ethylene, a requisite part of the process. Once evoked, abscission cells maintain a developmental memory such that the induction/repression mediated by ethylene/auxin that is observed prior to separation is also retained by the cells post-separation.     

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.