Reversible inhibition of ethylene action and interruption of petal senescence in carnation flowers by norbornadiene

The inhibitory effects of the cyclic olefin 2,5-norbornadiene (NBD) on ethylene action were tested in carnation (Dianthus caryophyllus L. cv White Sim) flowers. Treatment of flowers at anthesis with ethylene in the presence of 500 microliters per liter NBD increased the concentration of ethylene required to elicit a response (petal senescence), indicating that NBD behaves as a competitive inhibitor of ethylene action. Transfer of flowers producing autocatalytic ethylene and exhibiting evidence of senescence (petal in-rolling) to an atmosphere of NBD resulted in a rapid reduction in ethylene production, petal 1-aminocyclopropane-1-carboxylic acid synthase activity, 1-aminocyclopropane-1-carboxylic acid content, and ethylene forming enzyme activity. Removal of NBD resulted in recovery of ethylene biosynthesis. These results support the autocatalytic regulation of ethylene production during the climacteric stage of petal senescence and suggest that continued perception of ethylene is required for maintenance of ethylene biosynthesis. The inhibition of ethylene action by NBD after the flowers had reached the climacteric peak was associated with interruption of petal senescence as evidenced by reversal of senescence symptoms. This result is in contrast to the widely held belief that the rate of petal senescence is fixed and irreversible once petals enter into the ethylene climacteric.     

Identification and kinetics of accumulation of proteins induced by ethylene in bean abscission zones

A two-dimensional gel electrophoresis system that combines a cationic polyacrylamide gel electrophoresis at pH near neutrality with sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the spectrum of basic polypeptides that accumulate in bean (Phaseolus vulgaris) abscission zones after treatment with ethylene. Results showed that, as abscission progressed, at least seven basic proteins accumulated in the abscission zone prior to the accumulation of 9.5 cellulase. Six of the seven proteins correspond to pathogenesis-related (PR) proteins. Among them, two isoforms of β-1,3-glucanase and multiple isoforms of chitinase were identified. A 22 kilodalton polypeptide that accumulated to high levels was identified as a thaumatin-like protein by analysis of its N-terminal sequence (up to 20 amino acids) and its serological relationship with heterologous thaumatin antibodies. A 15 kilodalton polypeptide serologically related to PR P1 (p14) from tomato was identified as bean PR P1 (p14)-like protein. The kinetics of accumulation of glucanases, chitinases, thaumatin-like and PR P1 (p14)-like proteins during ethylene treatment were similar and they showed that PR proteins accumulated in abscission zones prior to the increase in 9.5 cellulase. Addition of indoleacetic acid, a potent inhibitor of abscission, reduced the accumulation of these proteins to a similar extent (60%). The synchronized accumulation of this set of PR proteins, early in the abscission process, may play a role in induced resistance to possible fungal attack after a plant part is shed. The seventh protein does not correspond to any previously characterized PR protein. This new 45 kilodalton polypeptide accumulated in abscission zones on exposure to ethylene concomitantly with the increase in 9.5 cellulase. Its N-terminal sequence (up to 15 amino acids) showed some homology with the amino terminal sequence of chitinase. Polyclonal antibodies against chitinase recognized the 45 kilodalton polypeptide, but polyclonal antibodies against the 45 kilodalton protein recognized chitinase weakly. When abscission was inhibited by addition of indoleacetic acid, the accumulation of the 45 kilodalton protein was strongly inhibited (80%). This result suggests that the 45 kilodalton polypeptide may play a more direct role in abscission.     

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.     

Role of ethylene in stimulating stylar abscission in pistil explants of lemons

Abscission in styles of excised Citrus limon (cv. Lisbon) pistils was stimulated by addition of 70 μ M 2-chloroethylphosphonic acid (ethephon) or 0.1 m M 1-aminocyclopropane-1-carboxylic acid (ACC) to the defined medium of cultures. To study the relationship between ethylene and abscission, we used gas chromatography to analyze ethylene in cultures containing a test medium plus or minus abscission-active chemicals. In the presence of ethephon or ACC, ethylene levels in sealed tubes increased rapidly, suggesting that these compounds stimulated abscission because they were converted to ethylene. In the presence of test medium or the inhibitor of abscission 2 μ M picloram, the low ethylene levels found in sealed tubes did not differ strikingly in the two treatments. Ethylene production rates measured prior to abscission with test medium or in the presence of picloram were not markedly different either, although picloram completely inhibited abscission. Stylar abscission was delayed but not prevented by 50 μ M aminoethoxyvinylglycine, an inhibitor of ethylene biosynthesis, and by hypobaric conditions (280 mm Hg) which removed ethylene from cultures. We concluded that ethylene is an important factor regulating stylar abscission in vitro and suggest that the inhibitory effect of picloram involves a process other than detectable ethylene production. Our results do not exclude the possibility that picloram affects enodgenous ethylene biosynthesis and/or metabolism and/or tissue retention.     

Effect of 2,5-norbornadiene on abscission and ethylene production in citrus leaf explants

Citrus ( Citrus sinensis L. Osbeck) leaf explants completely abscise within 48 h when exposed to saturating amounts of ethylene at 25°C. When 2,5-norbornadiene was added, 2000 μl 1⁻¹ reduced abscission of explants also exposed to 2 μl 1⁻¹ of ethylene to the level of the control, and 8000 μl 1⁻¹ reduced abscission in explants exposed to 10 μl 1⁻¹ of ethylene to the level of the control, but abscission was complete when 1 000 μl 1⁻¹ of ethylene was used in the presence of 8 000 μl 1⁻¹ of 2,5-norbornadiene. When explants were exposed to 2 μl 1⁻¹ of ethylene, 2000 μl 1⁻¹ of 2,5-norbornadiene prevented abscission if applied up to 10 h after exposure to ethylene. After 18 h, applied 2,5-norbornadiene had little effect on abscission at 48 h. A Lineweaver-Burk plot gave a 1/2 maximum value of 0.12 μl 1⁻¹ for ethylene on abscission, 2,5-Norbornadiene gave competitive kinetics with respect to ethylene with a K₁ value of approximately 120 μl 1⁻¹ of 2,5-norbornadiene. The presence of 2,5norbornadiene stimulated ethylene production, which progressively increased as the 2,5-norbornadiene concentration was increased from 250 to 8 000 μl 1⁻¹ 2,5-Norbornadiene also suppressed the induction of cellulase and polygalacturonase by ethylene. Together, 2,5-norbornadiene and 2,4-dichlorophenoxyacetic acid were more effective than either alone in reducing abscission. 2,5-Norbornadiene also was effective in preventing the reduction of indole-3-acetic acid transport induced by ethylene.     

Effect of ethylene on plasma membrane density in kidney bean abscission zones

Plasma membranes from abscission zones of kidney bean seedlings were isolated on sucrose density gradients using cellulase and KCl-stimulated ATPase as marker enzymes. Following treatment of the seedling explants with 50 microliters per liter ethylene, an increase in the buoyant density of the plasma membrane fraction from 1.165 to 1.175 grams per cubic centimeter was observed. Such a change is consistent with the loss of lipids from the senescent cells of the abscission zone.     

Auxin-induced delay of abscission: The involvement of calcium ions and protein phosphorylation in bean explants

Treatment with Ca²⁺ channel blockers such as lanthanum chloride and verapamil promoted abscission in pulvinar explants of bean ( Phaseolus vulgaris L. ev. Pirate). In addition, auxin-induced delay of abscission was markedly reduced in the presence of Ca²⁺ channel blockers. In vitro phosphorylation studies were performed using membrane preparations (130000 g pellet) from freshly excised as well as auxintreated and control (minus auxin) pulvinar sections. Auxin-treated sections showed a 66 kDa phosphoprotein as well as Ca²⁺ -dependent phosphorylation that were not observed in control explants.
Coomassie blue stammg of soluble proteins (130000 g supernatent) separated on SDS-PAGE revealed the presence of 62. 55 and 47 kDa polypeptides only in the freshly excised pulvini. However. no distinct changes were observed in soluble protein profile between auxin-treated and control explants. When soluble proteins were phosphorylated in vitro, Ca²⁺ promoted the phosphorylation of 92, 55. 40 and I7 kDa polypeptides only in freshly excised pulvmi. Ca²⁺-dependent phosphorylation of soluble proteins was not observed in either the control or auxin-treated explants. In addition. in vivo phosphorylation studies were performed using freshly excised. auxrn-treated and control explants. Freshly excused segments and auxin-treated ex-plants showed similar phosphoproteins, which were different from those observed mcontrol.     

The roles of abscisic acid and ethylene in the abscission and senescence of cocoa flowers

Cocoa flowers have a limited period of longevity; more than 90% of unpollinated flowers abscised within 32 h after anthesis. Abscisic acid (ABA) levels increased significantly prior to abscission. By 21 h after anthesis, ABA levels had increased almost 10-fold, and by 32 h flowers had 20-fold higher levels of ABA than at anthesis. Fluridone completely inhibited both the increase in ABA, the formation of an abscission zone, and the abscission and senescence of flowers. In contrast, ethylene production increased only slightly 21 h after anthesis and was only 2-fold higher after 32 h. Aminoethoxyvinylglycine (AVG) delayed but did not prevent abscission. In cocoa flowers, ABA is the primary regulator of abscission; ethylene accelerates abscission but only in the presence of ABA. Naphthalene acetic acid (NAA) treatment of flowers at anthesis prevented abscission zone formation and flower abscission, but did not induce fruit set. All parts of the NAA-treated flower except the pedicel senesced after 6 days. NAA+AVG treatment only delayed, whereas fluridone treatment completely prevented flower senescence.     

Regulation of senescence in bean leaf discs by light and chemical growth regulators

The senescence of excised discs of primary leaves of Phaseolus vulgaris, L., var. Red Kidney was followed by measuring the net breakdown of protein and chlorophyll. The chemical growth regulators indoleacetic acid, 2,4-dichlorophenoxy-acetic acid, gibberellic acid, kinetin, and 6-benzylaminopurine were relatively ineffective in retarding senescence in this tissue. White light, on the other hand, was very effective in senescence retardation. The response to light did not have the characteristics of a low energy (phytochrome) response and was blocked by concentrations of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea which inhibited photosynthesis in the leaf discs. The light-induced retardation of senescence was concluded to be dependent on photosynthesis.     

Applied auxin gradients and abscission in explants

The influence of applied auxin on abscission in explants (excised cotyledonary nodes) of cotton was investigated.     

Effects of ethylene and inhibitors of ethylene synthesis and action on nodulation in common bean (Phaseolus vulgaris L.)

The ethylene releasing compound, 2-chloroethylphosphonic acid (ethephon) inhibited nodule development in common bean (Phaseolus vulgaris L.) plants. In contrast, inhibitors of ethylene synthesis or its physiological activity enhanced nodulation. In a co-culture of bean seeds and rhizobia, ethephon inhibited rhizobial growth while inhibitors of ethylene synthesis or action did not influence the growth and proliferation of rhizobia. These data emphasize the role of ethylene as a regulator of nodulation in determinate nodulators and indicate that the ethylene signaling pathway involved in the nodulation process is not limited to the plant host but also involves the bacterial symbiont.     

Retardation of abscission of citrus leaf and fruitlet explants by brassinolide

Brassinolide (BR), a novel plant growth-regulating steroidal lactone, markedly retarded the abscission of leaf explants of Calamondin (Citrus madurensis Lour.), when dissolved in water and fed through the petiole. BR was effective at concentrations as low as 0.021 M, and showed a stronger effect than IAA which also retarded abscission. Trifluoperazine (TFP), an inhibitor of the calmodulin-calcium complex, accelerated abscission, and this acceleration could be counteracted by a simultaneous addition of IAA or BR, the effect of IAA being stronger. BR in lanolin applied to the cut surface of the leaf blade of the explant showed a weaker abscission-retarding effect than that applied in water via the petiole. BR and IAA also markedly retarded the abscission of fruitlet explants of Calamondin.     

Cytokinin stimulation of abscission in lemon pistil explants

The stylar abscission bioassay was used to identify five stimulators of lemon (Citrus limon cv. Lisbon) abscission in pistil explants. The stimulators (N-phenyl-N′-1,2,3-thiadiazol-5-ylurea, N6-benzyladenine, kinetin, zeatin, and N6-isopentenyladenine), which are all cytokinins, accelerated the timing of expiant abscission when they were added as supplements (100 μM) to the test medium. To study possible relationships between cytokinins, ethylene, and abscission, we measured accumulating ethylene concentrations in sealed cultures and endogenous 1 -aminocyclopropane-1-carboxylic aicd (ACC) in explants incubated on test medium plus or minus 100 μM N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (thidiazuron), 100 μM N6-benzyladenine (bzl⁶Ade), or 2 μM picloram, an inhibitor of stylar abscission. Although ethylene accumulated to similar levels in all treatments, the concentrations obtained with picloram and thidiazuron were, respectively, higher and lower than those obtained in control cultures. The accumulation of ethylene in cultures with bzl⁶Ade, on the other hand, was not significantly different from controls. ACC concentrations in explants remained fairly constant in all treatments during the incubations, except in explants on thidiazuron, in which case the ACC concentration declined slightly. We conclude that cytokinins can stimulateCitrus abscissionin vitro and that this stimulation is not accompanied by marked effects on either measurable ethylene or ACC concentrations. Our finding that 100 μM aminoethoxyvinylglycine, an ethylene biosynthesis inhibitor, counteracts the stimulation of abscission by bzl⁶Ade suggests that a minimum level of ethylene production is required for the cytokinin effect. The possibility that cytokinins affect other aspects related to ethylene, such as biosynthetic rates, metabolism, or tissue retention, is not excluded by our results.     

Role of polygalacturonase in bean leaf abscission

The role of polygalacturonase in leaf abscission was studied in explants of Phaseolus vulgaris L. cv. Red Kidney. Bean polygalacturonase was partially characterized and comparisons were made between the bean enzyme and previously reported higher plant polygalacturonases. Polygalacturonase isolated from bean leaf abscission zones has a pH optimum between 4.5 and 5.0 and hydrolyzed polygalacturonides in an exo-fashion. Activity was found to be higher with a deesterified substrate than with an esterified pectin. No correlation between polygalacturonase activity and abscission was observed. Activity remained virtually constant over the course of abscission in explants aged either in air or in ethylene. The enzyme was primarily localized in the abscission zone, however, indicating a possible involvement in the abscission process. A theoretical model which could explain the relationship between polygalacturonase and bean leaf abscission is discussed.     

Role of ethylene action in ethylene production and poststorage leaf senescence and survival of pelargonium cuttings

This study investigated the role of ethylene action in ethylene production and in poststorage performance of pelargonium cuttings. Cuttings of zonal pelargonium (Pelargonium x hortorum L.H. Bailey) of the cultivars Isabell and Mitzou were treated with ethylene and with the ethylene action inhibitors 1-methylcyclopropene (MCP), silver-thiosulfate (STS) and silver nitrate (SN) and were stored in the dark at different temperatures (5, 12, and 20 °C) for 48 h. Ethylene concentrations in the storage boxes were monitored and poststorage leaf senescence, survival and root formation of cuttings were determined. Applications of MCP resulted in a significant increase of ethylene evolution by cuttings of both cultivars which was more pronounced with increasing storage temperature. After 48 h of storage at 20 °C, ethylene concentrations were more than 20-fold higher for the MCP-treated cuttings as compared to those of the untreated controls. Also preharvest applications of STS and SN increased ethylene evolution by cuttings, even though these effects were less pronounced. However, application of these inhibitors caused severe poststorage leaf injury. Application of ethylene during storage had no effect on subsequent leaf damage. Leaf senescence during rooting and decay of cuttings, which raised with increasing storage temperature, could only partially been reduced by MCP. The results strongly support the conclusion, that in zonal pelargonium cuttings, ethylene production is controlled by autoinhibition, and clearly indicate, that temperature dependent processes other than ethylene action are substantially involved in storage-induced leaf senescence and decay.     

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.     

Effects of gibberellins on abscission in cotton seedling explants

Summary Gibberellic acid (GA₃) accelerated abscission when applied, in a wide concentration range, to excised abscission zones of cotton. Abscission was promoted equally by distal or proximal applications of from 10^-3 to 100 g. A slight, but inconsistent, abscission retardation was obtained with distal applications of 10^-6 and 10^-7 g.Seven different gibberellins accelerated abscission equally when applied distally at amounts of 5×10^-4 to 5×10^-1 g per abscission zone. At 5×10^-5 g there were great differences in effectiveness; their activities can be ranked: A₃>A₅A₄>A₇=A₈>A₁=A₉.The ready translocatability of GA₃ was suggested when 1.0 or 0.01 g was applied to one petiole, and the opposite untreated petiole abscised at the same time as the treated one. However, 0.001 g was not effective in moving across the stem and inducing abscission of the untreated petiole.The rate of abscission of petioles treated with 1.0 g GA₃ was not affected by increasing the length of the petiole from 3 to 9 mm. However, abscission of petioles treated with smaller amounts is inversely proportional to petiole length.The rate of abscission of petioles treated with GA₃ decreased with increasing seedling age; there was a simultaneous increase in abscission rate of the controls.Part of this research was based on a portion of a thesis submitted by the senior author to the Graduate Division, University of California, Davis, in partial fulfillment of the requirements for the M.S. degree.