Starch Synthetase, Phosphorylase, ADPglucose Pyrophosphorylase, and UDPglucose Pyrophosphorylase in Developing Maize Kernels

Three types of whole plant experiments are presented to substantiate the concept that an important function of ethylene in abscission is to reduce the transport of auxin from the leaf to the abscission zone. (a) The inhibitory effect of ethylene on auxin transport, like ethylene-stimulated abscission, persists only as long as the gas is continuously present. Cotton (Gossypium hirsutum L. cv. Stoneville 213) and bean (Phaseolus vulgaris L. cv. Resistant Black Valentine) plants placed in 14 μl/l of ethylene for 24 or 48 hours showed an increase in leaf abscission and a reduced capacity to transport auxin; but when returned to air, auxin transport gradually increased and abscission ceased. (b) Ethylene-induced abscission and auxin transport inhibition show similar sensitivities to temperature. A 24-hour exposure of cotton plants to 14 μl/l of ethylene at 8 C resulted in no abscission and no significant inhibition of auxin transport. Increasing the temperature during ethylene treatment resulted in a progressively greater reduction in auxin transport with abscission occurring at [unk]27 C where auxin transport was inhibited over 70%. (c) Auxin pretreatment reduced both ethylene-induced abscission and auxin transport inhibition. No abscission occurred, and auxin transport was inhibited only 18% in cotton plants which were pretreated with 250 mg/l of naphthalene acetic acid and then placed in 14 μl/l of ethylene for 24 hours. In contrast, over 30% abscission occurred, and auxin transport was inhibited 58% in the corresponding control plants.     

Abscission: the role of ethylene modification of auxin transport

The role of ethylene-mediated reduction of auxin transport in natural and ethylene-induced leaf abscission was studied in the cotton (Gossypium hirsutum L., cv. Stoneville 213) cotyledonary leaf system. The threshold level of ethylene required to cause abscission of intact leaves was between 0.08 and 1 μl/l with abscission generally occurring 12 to 24 hours following ethylene fumigation. The threshold level of ethylene required to reduce the auxin transport capacity in the cotyle-donary petiole paralleled that required for stimulation of abscission. In plants where cotyledons are allowed to senesce naturally there is a decline in auxin transport capacity of petioles and increase in ethylene synthesis of cotyledons. The visible senescence process which precedes abscission requires up to 11 days, and increases in ethylene production rates and internal levels were detected well before abscission. Ethylene production rates for entire cotyledons rose to 2.5 mμ1 g⁻¹ hr⁻¹ and internal levels of 0.7 μl/l were observed. These levels appear to be high enough to cause the observed decline in auxin transport capacity. These findings, along with those of others, indicate that ethylene has several roles in abscission control (e.g., transport modification, enzyme induction, enzyme secretion). The data indicate that ethylene modification of auxin transport participates in both natural abscission and abscission hastened by exogenous ethylene.     

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.     

Involvement of Abscisic Acid in Ethylene-Induced Cotyledon Abscission in Cotton Seedlings

Cotton (Gossypium hirsutum L. cv LG102) seedlings raised from seeds exposed to 100 [mu]M norflurazon (NFZ) during imbibition contained reduced levels of free abscisic acid (ABA) and were visibly achlorophyllous. Exposure of untreated cotton seedlings to ethylene concentrations >1 [mu]L/L for 24 h resulted in cotyledon abscission. In contrast, exposure of NFZ-treated seedlings to concentrations of ethylene [less than or equal to]50 [mu]L/L elicited no cotyledon abscission. Application of ABA, an ABA analog, or jasmonic acid to NFZ-treated seedlings restored ethylene-induced abscission. Isolated cotyledonary node explants prepared from NFZ-treated seedlings exhibited an altered dose-response pattern of ethylene-induced petiole abscission. Endogenous levels of free IAA were unaltered in NFZ-treated seedlings. Ethylene treatment (50 [mu]L/L, 24 h) had no effect on free indoleacetic acid (IAA) levels in either control or NFZ-treated seedlings. Levels of conjugated (ester plus amide) IAA were substantially increased in NFZ-treated seedlings regardless of ethylene treatment. These results indicate that endogenous ABA plays an essential, but physiologically undefined, role in ethylene-induced cotyledon abscission in cotton.     

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.     

Ethylene-induced leaf abscission in cotton seedlings : the physiological bases for age-dependent differences in sensitivity

The speed of ethylene-induced leaf abscission in cotton (Gossypium hirsutum L. cv LG-102) seedlings is dependent on leaf position (i.e. physiological age). Fumigation of intact seedlings for 18 hours with 10 microliters per liter of ethylene resulted in 40% abscission of the still-expanding third true (3°) leaves but had no effect on the fully expanded first true (1°) leaves. After 42 hours of fumigation with 50 microliters per liter of ethylene, total abscission of the 3° leaves occurred while <50% abscission of the 1° leaves was observed. On a leaf basis, endogenous levels of free IAA in 1° leaves were approximately twice those of 3° leaves. Free IAA levels were reduced equally (approximately 55%) in both leaf types after 18 hours of ethylene (10 microliters per liter) treatment. Ethylene treatment of intact seedlings inhibited the basipetal movement of [¹⁴C]IAA in petiole segments isolated from both leaf types in a dose-dependent manner. The auxin transport inhibitor N-1-naphthylphthalamic acid increased the rate and extent of ethylene-induced leaf abscission at both leaf positions but did not alter the relative pattern of abscission. Abscission-zone explants prepared from 3° leaves abscised faster than 1° leaf explants when exposed to ethylene. Ethyleneinduced abscission of 3° explants was not appreciably inhibited by exogenous IAA while 1° explants exhibited a pronounced and protracted inhibition. The synthetic auxins 2,4-D and 1-naphthaleneacetic acid completely inhibited ethylene-induced abscission of both 1° and 3° explants for 40 hours. It is proposed that the differential abscission response of cotton seedling leaves is primarily a result of the limited abscission-inhibiting effects of IAA in the abscission zone of the younger leaves.     

Anatomical and physiological effects of silver thiosulfate on ethylene-induced abscission inColeus

Petioles of expiants ofColeus blumei Benth. exposed to 20 μl/l ethylene abscised within 36 h. Pretreatment of expiants with 4 mM silver thiosulfate (STS) inhibited ethylene-induced abscission. Delaying treatment with STS reduced its effectiveness in retarding ethylene-promoted abscission, suggesting that some events leading to abscission are initiated during the first hours of ethylene treatment. Microscopic study of abscission zones of ethylene-treated expiants showed greatly increased amounts of rough endoplasmic reticulum, disruptions of the plasma membrane, and some cell separation in the region of the middle lamella. Pretreatment with STS prevented ethylene-induced reorganization of the endomembrane system and the subsequent middle lamellar dissolution.     

Ethylene, a regulator of young fruit abscission

In an earlier study we reported that detached cotton flowers produced sufficient ethylene before the period of natural abscission to suggest that ethylene might be a natural regulator of young fruit abscission. The present report explores this probability further. Intact cotton (Gossypium hirsutum L.) fruits produced ethylene at rates as high as 36 μl ethylene/kg fresh wt·hr during the 2 days before they abscised. Direct measurements of ethylene in gas samples withdrawn from fruits indicated that production of 1 μl ethylene/kg fresh wt·hr is equivalent to an internal concentration of approximately 0.1 μl/l. Fumigation of fruiting cotton plants with only 0.5 μl/l caused 100% abscission of young fruits and floral buds within 2 days. This correlated with the estimated endogenous levels of ethylene. Reduced pressure, which reduced the internal levels of ethylene, delayed abscission of young fruits and leaves, a result which supports our conclusion from this study— that ethylene is one of the regulators of young fruit abscission in cotton.     

Abscission: the initial effect of ethylene is in the leaf blade

The leaf blade of cotton (Gossypium hirsutum L. cv. Stoneville 213) was investigated as the initial site of ethylene action in abscission. Ethylene applied at 14 μl/l to intact 3-week-old plants caused abscission of the third true leaf within 3 days. However, keeping only the leaf blade of this leaf in air during ethylene treatment of the rest of the plant completely prevented its abscission for up to 7 days. This inhibition of abscission was apparently the result of continued auxin production in the blade since (a) the application of an auxin transport inhibitor to the petiole of the air-treated leaf blade restored ethylene sensitivity to the leaf in terms of abscission; (b) repeated applications of naphthaleneacetic acid to the leaf blade of the third true leaf, when the entire plant was exposed to ethylene, had the same preventive effect on abscission of this leaf as keeping its leaf blade in air; and (c) the inhibitory effect of ethylene on auxin transport in the petiole, which is reduced by auxin treatment, was also reduced by placing the leaf blade in air.     

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.     

Fruit age and changes in abscisic Acid content, ethylene production, and abscission rate of cotton fruits

The relationships of fruit age, abscisic acid (ABA) concentration, ethylene evolution, and abscission rates were studied in an effort to determine why cotton (Gossypium hirsutum L., cv. Deltapine 16) fruits rarely abscise more than 15 days after anthesis. Because abscission of cotton fruits is increased by conditions that limit photosynthesis, greenhouse-grown plants with fruits of various ages were placed in dim light for 3 days to induce high rates of fruit abscission. Abscission rates, ABA concentrations, and ethylene evolution rates were determined for fruits of various ages. Almost all of the young fruits abscised, but abscission rate declined with age until almost no abscission was observed in fruits that were 15 or more days past anthesis.     

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.     

Ethylene-induced Leaf Abscission Is Promoted by Gibberellic Acid

Gibberellic acid (GA₃) promoted leaf abscission from cotton (Gossypium hirsutum L.) plants exposed to ethylene. With mature plants, only the rate of abscission was increased, but when vegetative plants were exposed to ethylene for 4 days or less, the amount of abscission was increased markedly. Promotion of abscission occurred at near saturating ethylene levels (10 μl/liter), over a wide range of GA₃ concentrations, and with both GA₃ and GA₇.     

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.     

Involvement of ethylene in the action of the cotton defoliant thidiazuron

The effect of the defoliant thidiazuron (N-phenyl-N′-1,2,3-thiadiazol-5-ylurea) on endogenous ethylene evolution and the role of endogenous ethylene in thidiazuron-mediated leaf abscission were examined in cotton (Gossypium hirsutum L. cv Stoneville 519) seedlings. Treatment of 20- to 30-day-old seedlings with thidiazuron at concentrations equal to or greater than 10 micromolar resulted in leaf abscission. At a treatment concentration of 100 micromolar, nearly total abscission of the youngest leaves was observed. Following treatment, abscission of the younger leaves commenced within 48 hours and was complete by 120 hours. A large increase in ethylene evolution from leaf blades and abscission zone explants was readily detectable within 24 hours of treatment and persisted until leaf fall. Ethylene evolution from treated leaf blades was greatest 1 day posttreatment and reached levels in excess of 600 nanoliters per gram fresh weight per hour (26.7 nanomoles per gram fresh weight per hour). The increase in ethylene evolution occurred in the absence of increased ethane evolution, altered leaf water potential, or decreased chlorophyll levels. Treatment of seedlings with inhibitors of ethylene action (silver thiosulfate, hypobaric pressure) or ethylene synthesis (aminoethoxyvinylglycine) resulted in an inhibition of thidiazuron-induced defoliation. Application of exogenous ethylene or 1-aminocyclopropane-1-carboxylic acid largely restored the thidiazuron response. The results indicate that thidiazuron-induced leaf abscission is mediated, at least in part, by an increase in endogenous ethylene evolution. However, alterations of other phytohormone systems thought to be involved in regulating leaf abscission are not excluded by these studies.     

Anatomical and physiological effects of silver thiosulfate on ethylene-induced abscission inColeus

Petioles of expiants ofColeus blumei Benth. exposed to 20 l/l ethylene abscised within 36 h. Pretreatment of expiants with 4 mM silver thiosulfate (STS) inhibited ethylene-induced abscission. Delaying treatment with STS reduced its effectiveness in retarding ethylene-promoted abscission, suggesting that some events leading to abscission are initiated during the first hours of ethylene treatment. Microscopic study of abscission zones of ethylene-treated expiants showed greatly increased amounts of rough endoplasmic reticulum, disruptions of the plasma membrane, and some cell separation in the region of the middle lamella. Pretreatment with STS prevented ethylene-induced reorganization of the endomembrane system and the subsequent middle lamellar dissolution.     

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.     

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.     

Stimulation of ethylene evolution and abscission in cotton by 2-chloroethanephosphonic Acid

Ethrel, a mixture of 2-chloroethanephosphonic acid and its ethyl ester, hastens abscission of leaves, debladed petioles, and flower buds of cotton plants (Gossypium hirsutum, L.). Both young and old leaves abscissed while still green. Application of Ethrel stimulated evolution of ethylene, and this response preceded abscission. Air concentrations of ethylene around enclosed, treated-plants were adequate to produce abscission in plants. Non-treated plants defoliated when enclosed with plants sprayed with Ethrel. The stimulation of abscission of explant petioles by Ethrel was reversed by naphthalene acetic acid. The stimulation of abscission by Ethrel was concluded to be mediated by ethylene.     

Effect of 1-methylcyclopropene on ethylene-induced abscission in citrus

Pre-treatment of citrus leaves and leaf explants ( Citrus sinensis [L.] Osbeck cv. Shamouti), with 1-methylcyclopropene (1-MCP), induced endogenous ethylene production when leaves were further incubated in air. The induction of ethylene production was 1-MCP concentration-dependent. Abscission was concomitantly delayed. In leaves pre-treated with 1-MCP followed by exposure to ethylene, abscission was significantly delayed in comparison with those without 1-MCP pre-treatment. When leaf explants were co-treated for 24 h with ethylene and 1-MCP, abscission was delayed quite efficiently. The Lineweaver-Burke plot yielded a half-maximal value of 0.234 μl l⁻¹ for the effect of ethylene on abscission. 1-MCP⁻¹ competed kinetically with ethylene with a K_i value of approximately 1.4−5.5 nl l⁻¹ 1-MCP. Under these experimental conditions there was some competition between 1-MCP and ethylene. However, ethylene was not able to completely counteract the inhibitory effect of 1-MCP. Pre-treatment with 1-MCP, followed by exogenous ethylene treatment, suppressed the induction of endo- β -glucanase (EG) activity at the laminar abscission zone. The ethylene-dependent accumulation of the hydrolyse gene was demonstrated by blocking the accumulation of CsCel a1 mRNA by 1-MCP. Six hours of exposure of leaves to 1-MCP at various times during a total of 24 h ethylene treatment efficiently reversed ethylene induction of CsCel a1 gene at mRNA level up to 18 h. The results demonstrate that the induction of abscission by ethylene is controlled at mRNA level at the abscission zone.