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.     

Calcium distribution in the abscission zone of bean leaves: electron microprobe x-ray analysis

The calcium content and distribution across the abscission zones of (2-chloroethyl) phosphonic acid-treated bean (Phaseolus vulgaris L. var. Contender) leaves were lower and not uniformly distributed as compared to the control. Calcium chloride-treated bean leaves had a higher calcium content, with more calcium localized in the potential abscission layer. Ethephon treatment promoted abscission in both debladed and nondebladed plants; there was a corresponding decrease in calcium in the abscission zone just prior to separation. Deblading of bean leaves under a calcium solution increased the calcium level in the abscission zone and delayed 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.     

A Comparison of Abscission of Rubber (Hevea brasiliensis) Leaves Infected with Microcyclus ulei with Leaf Abscission Induced by Ethylene Treatment, Deblading and Senescence

Studies on the histology and on effects of growth substancesand phenols as well as changes in activities of pectinmethylesterase indicated that the mechanism of abscission of Hevealeaflets infected with Microcyclus ulei differed from the mechanismof abscission of debladed, ethylene treated and senescent leaves.An abscission layer which was formed during abscission of debladed,ethylene-treated and senescent leaves was absent during abscissionof heavily diseased leaves. The ratio of pectinmethyl esteraseactivities in tissues distal to the abscission zone to activitiesin tissues proximal to the zone decreased in debladed and ethylenetreated leaves but such decreases were not detected during abscissionof Hevea leaves infected with M. ulei. Hevea brasiliensis Muell. Arg., rubber, leaf abscission, Microcyclus ulei, ethylene, indol-3-ylacetic acid, kinetin     

Effect of calcium, (2-chloroethyl) phosphonic acid and ethylene on bean leaf abscission

Summary The effects of CaCl₂, (2-chloroethyl) phosphonic acid (Ethephon) and ethylene on leaf abscission of debladed and intact bean plants (Phaseolus vulgaris L.) were studied. Ethephon (1000 g/l) and ethylene (8 l/l) induced abscission in debladed and intact plants in 24–72 h whereas IAA (10^-5M), cycloheximide (10^-5M) and CaCl₂ (0.068M) delayed abscission in debladed plants. CaCl₂ completely inhibited the abscission-enhancing effect of Ethephon in intact bean leaves. When CaCl₂ and Ethephon were applied simultaneously to separate halves of the leaf blade, leaves with Ethephon applied closest to the pulvinus abscised rapidly; when CaCl₂ was applied closest to the pulvinus, abscission was prevented. Calcium pre-treatment prior to ethylene (8 l/l) treatment of debladed plants delayed abscission as compared to those treated with ethylene alone.Michigan Agricultural Experiment Station Journal Article No. 6299.     

Pectin esterase in relation to leaf abscission in coleus and phaseolus

Pectin esterase (PE) activities in abscission zones, other portions of leaves, and adjacent stem tissues were compared in attached leaves and abscissing petioles (previously debladed) of Coleus blumei Benth. and Phaseolus vulgaris L., cv. Canadian Wonder. Earlier findings of Osborne in bean were confirmed and changes in PE activity in coleus were shown to resemble those in bean in some respects. In both plants PE was lower in the distal portion of abscission zones of abscissing petioles than in that portion of attached leaves but this difference was not as large or as consistently clear-cut in coleus as in bean. The general level of PE activity was an order of magnitude lower and changes associated with abscission were smaller in coleus than in bean. Auxin treatment of debladed petioles of coleus prevented abscission and resulted in small increases in PE activity in abscission zones and most of the other regions sampled. The largest increase was observed in the stem tissue adjacent to the attached leaf opposite the debladed, auxin treated one.     

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.     

Effect of Ethylene on Peroxidase Activity

Ethylene increased the peroxidase activity of nine out of ten varieties of sweet potato (Ipomoea batatas (L.) Lam.) root disks tested. The increase which was observed four hours after ethylene treatment was partially overcome by carbon dioxide. The increase was inhibited by actinomycin D and cycloheximide, indicating de novo protein synthesis. Electrophoretic separation on polyacrylamide gels indicated the appearance of two new peroxidase bands. Peroxidase activity in bean petiole explants was localized around the separation layer. Ethylene caused a small increase in peroxidase activity in the petiolar portion of the explant. Phenolic substances had no effect on abscission consistent with their proposed roles as cofactors for auxinoxidase, indicating that auxin-oxidase does not play a role in abscission of Coleus blumei Benth. abscission zone explants.     

ULTRASTRUCTURAL STUDIES OF ABSCISSION IN PHASEOLUS: ETHYLENE EFFECTS ON CELL WALLS

This paper reports light and electron microscope observations of changes in the walls of cortical cells in the laminar abscission region of red kidney bean (Phaseolus vulgaris L.). In intact plants two or three rows of cells comprise the abscission zone. Pectic substances are not present in the walls of these cells when wall breaks occur. The separation cavity involves breaks in both radial and longitudinal cell walls. In ethylene-treated explants pectic substances are present in the cell walls when breaking occurs. The separation cavity involves breaks in longitudinal walls only, and breaking is confined to a single row of cortical cells. Prior to cell wall break the plasma membrane frequently invaginates. In intact plants this may be associated with plasmolysis and with the formation of secondary vacuoles. In ethylene-treated explants it may also be related to plasmolysis. At the time of cell wall break many unidentifiable inclusions of varying sizes and shapes are present in the cell wall region. Chloroplasts and mitochondria are structurally altered but recognizable in the cell at the time of wall break. Plasmodesmata are frequently observed in abscission cells and may be structurally elaborate. The observations of the nature of cell wall changes during abscission in ethylene-treated material fail to confirm physiological studies of other workers suggesting that pectin dissolution is necessary and may be sufficient for formation of a separation layer.     

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.     

C]Ethylene Metabolism during Leaf Abscission in Cotton

Changes in ¹⁴C₂H₄ metabolism in the abscission zone were monitored during cotton (cv. Deltapine 16) leaf abscission. Rates of ¹⁴C₂H₄ oxidation to ¹⁴CO₂ and tissue incorporation in abscission zone segments cut from the second true leaf of nonabscising leaves of intact plants were similar (about 200 disintegrations per minute per 0.1 gram dry weight per 5.5 hours) and relatively constant over a 5-day period. Deblading to induce abscission caused a dramatic rise in ¹⁴C₂H₄ oxidation, but tissue incorporation was not markedly affected. This rise occurred well before abscission, reaching a peak of 1,375 disintegrations per minute per 0.1 gram dry weight per 5.5 hours 2 days after deblading when abscission was 40%. The rate then gradually declined, but on day 5 when abscission reached completion, it was still nearly three times higher than in segments from nonabscising leaves. Application of 0.1 millimolar abscisic acid in lanolin to the debladed petiole ends increased the per cent abscission slightly and initially stimulated ¹⁴C₂H₄ oxidation. In contrast, naphthaleneacetic acid applied in a similar manner delayed and markedly inhibited both abscission and ¹⁴C₂H₄ oxidation.     

Abscission of Azolla branches induced by ethylene and sodium azide

Treatment with ethylene accelerated the abscission of branches of Azolla filiculoides plants. An Azolla plantlet treated with ethylene at 10 microl liter(-1) divided into 4-5 fragments after a lag period of 6-8 h. Ethylene-induced abscission was effectively inhibited by cycloheximide and was associated with an increase in the activities of cellulase and polygalacturonase. At the fracture surface abscised after treatment with ethylene, dissolution of the primary walls of the abscission zone cells was apparent. However, the middle lamella between abscission zone cells was still present. Immunoelectron microscopy using anti-unesterified pectin (JIM5) and anti-methylesterified pectin (JIM7) monoclonal antibodies revealed the presence of both JIM5 and JIM7 epitopes in the wall between abscission zone cells of branches before abscission occurred. In the middle lamella remaining after ethylene-induced abscission, only JIM7 epitopes were observed. The features of ethylene-induced abscission described herein were different from those of the rapid abscission induced by sodium azide, which implies that they are mediated by different mechanisms. The possible mechanisms 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.     

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.     

Effect of Treatments with Growth Retardant Chemicals on Petiole Abscission in Coleus

A bscission of debladed petioles of Coleus was observed following spray applications of growth retardant chemicals and particularly of Phosfon D to the foliage. Sprays were applied to some branches, which were left intact (inducing branches), or to adjacent branches the leaves of which were later debladed (induced branches). In all experiments two applications of growth retardant chemicals were made, after which the induced branches were debladed. Treatments on induced branches accelerated the petiole abscission relative to the controls. Treatments on inducing branches, instead, decreased abscission speed of debladed petioles. The evidence suggests that phosfon D affects abscission by interfering with the indoleacetic acid mechanism.     

A morphometric study on the effects of ethylene treatment in promoting abscission of tobacco flower pedicels

The ultrastructural changes observed in ethylene-induced abscission of tobacco flower pedicels (Nicotiana tabacum L. `Little Turkish') were studied by the techniques of morphometric analysis. The surface area of the membranes, relative volume of the organelles, and the number of organelles were determined for both ethylene-treated and control cells. In pedicels exposed to ethylene for 4.5 to 5 hours, abscission was evident within the separation zone. The most significant change in cell structure was observed in the surface area of the rough endoplasmic reticulum which more than doubled with ethylene treatment of the tissue.     

Peroxidase in Tobacco Abscission Zone Tissue: VII. ULTRAVIOLET ABSORPTION SPECTRA OF ETHYLENE-TREATED TOBACCO FLOWER PEDICEL TISSUE

Peroxidase-containing enzyme supernatant preparations of tobaccoabscission zone flower pedicel tissue sections of ethylene-treatedand untreated Nicotiana tabacum L. plants were assayed for u.v.absorption spectra with and without the addition of IAA. Theu.v. spectral absorption of peroxidase-catalysed IAA oxidationindicated that there is not an appreciable difference in absorbancepeaks in the ethylene-treated plants compared with untreatedcontrols, in the presence of added IAA; however, the ethylene-treatedtissues alone show different absorption peak values in the 1,2, 3, 4, 4.5, and 5 h samples. These results are discussed inrelation to ethylene-induced peroxidase activity in tobaccoflower pedicel abscission zone tissue homogenates.     

FORMATION OF CALLOSE AND LIGNIN DURING LEAF ABSCISSION

Deblading of bean leaves promoted the formation of callose and lignin in the abscission zone. The abscission layer became evident three days after deblading. The greatest increase in callose occurred in about two layers of cells during the development of the abscission layer. Four days after deblading, only a few layers of cells on the distal side of the abscission layer showed an increase in lignin. Lignification continued to expand to 8–10 layers of cells at the time of separation. After separation, the lignified cells remained with the petiole. Sieve elements in the abscission zone were covered with callose plugs and the vessels were occluded with tyloses.     

Abscission: potentiating action of auxin transport inhibitors

Reduction in petiolar auxin transport has been proposed as one of the functional actions of endogenous or exogenous ethylene as it regulates intact leaf abscission. If this hypothesis is correct, auxin-transport inhibitors should hasten the rate or amount of abscission achieved with a given level of ethylene. Evidence presented here indicates that the hypothesis is correct. Three auxin transport inhibitors promoted ethylene-induced intact leaf abscission when applied to specific petioles or the entire cotton plant (Gossypium hirsutum L., cv. Stoneville 213). In addition, the transport inhibitors caused rapid abscission of leaves which usually do not abscise under the conditions employed. No stimulation of abscission occurred during the initial 3 to 5 days after plants were treated with transport inhibitors unless such treatments were coupled with exogenous ethylene or that derived from 2-chloroethylphosphonic acid. However, vegetative cotton plants did abscise some of their youngest true leaves during the 2nd and 3rd weeks of exposure to transport inhibitor alone. Taken as a whole, the results indicate that reducing the auxin supply to the abscission zone materially increases sensitivity to ethylene, a condition which favors a role of endogenous ethylene in abscission regulation. Such a role of ethylene indicates the importance of auxin-ethylene interactions in the over-all hormone balance of plants and specific tissues.     

The Effect of Calcium Nutrition of Ethylene-induced Abscission

The influence of calcium nutrition on ethylene-induced abscission was studied by growing cotton (Gossypium hirsutum L. cv. Stoneville 213) and bean (Phaseolus vulgaris L. cv. Resistant Black Valentine) plants for several weeks in nutrient solutions containing 2, 10 (normal level), 15, or 20 meq/l of calcium, and then treating the plants with ethylene. Increasing the calcium level of cotton from 2 to 20 meq/l resulted in a 9-fold increase in the calcium content of the abscission zone and a maximum reduction of 25% in the amount of leaf abscission induced by ethylene (9 μl/l). Bean plants grown on 10, 15, or 20 meq/l calcium solutions showed corresponding increases in the calcium content of the abscission zone but showed no significant differences in the rate of ethyleneinduced abscission. Only at the lowest calcium level of 2 meq/l, where deficiency symptoms became apparent, was a significant effect observed. These results suggest that under normal cultural practices calcium nutrition has little influence on the rate of ethylene-induced abscission.