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.     

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.     

The conversion of 2-hydroxyethylhydrazine to ethylene

It was shown that 2-hydroxyethylhydrazine is a source of ethylene in a chemical model system. Treatment of pea seedlings produces physiological responses similar to that of ethylene. The hypothesis is advanced that 2-hydroxyethylhydrazine effects this response in pea seedlings via the release of ethylene, as was suggested earlier for its effect on abscission and flowering.     

Effect of exposure to subfreezing temperatures on ethylene evolution and leaf abscission in citrus

Citrus leaves exposed to subfreezing temperatures evolved ethylene at rates between 0.1 and 38.3 microliters per kilogram fresh weight per hour whereas untreated leaves evolved between 0.01 and 0.50 microliter per kilogram fresh weight per hour. Leaves not injured by freezing temperatures did not abscise, and ethylene evolution was near normal after 2 days. Freeze-injured leaves continued evolving high ethylene levels 4 or 5 days subsequent to freeze injury, and many of the freeze-killed leaves abscised. Supportive evidence suggested freeze-induced ethylene was involved in freeze-induced leaf abscission; whereas freeze-inhibited abscission was not due to a lack of ethylene but injury to other metabolic systems necessary for abscission.     

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.     

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.     

Effect of ethylene on flower abscission: a survey

The effect of ethylene on flower abscission was investigated in monocotyledons and eudicotyledons, in about 300 species from 50 families. In all species studied except Cymbidium, flower abscission was highly sensitive to ethylene. Flower fall was not consistent among the species in any family studied. It also showed no relationship with petal senescence or abscission, nor with petal colour changes or flower closure. Results suggest that flower abscission is generally mediated by endogenous ethylene, but that some exceptional ethylene-insensitive abscission occurs in the Orchidaceae.     

Lack of ethylene involvement in tulip tepal abscission

The tepals of cut flowers of Tulipa hybrida cv. Golden Apeldoorn and Tulipa kaufmanniana cv. Shakespeare abscise 3–4 days after harvest. The weakening of the abscission zones is accompanied by cell wall breakdown and the separation of 3–4 rows of intact cells at the base of the tepal. During senescence, there is no ethylene climacteric and ethylene production rates remain low, between 0.07 and 0.4 nl g⁻¹ fresh weight h⁻¹. Adding 3–5 μl l⁻¹ ethylene slightly accelerated the weakening of the abscission zones but had no effect on the time of first abscission. Neither 0.5 m M silver thiosulphate nor 5 m M aminoethoxyvinylglycine delayed the time to abscission. It is concluded that tulip tepal fall does not involve primary regulation by ethylene, unlike the majority of other abscission systems that have been studied.     

Abscission: ethylene and light control

The role of ethylene in light control of leaf abscission im mung bean, Vigna radiata (L.) Wilczek cv Jumbo, cuttings was examined. While red light inhibits and far-red light promotes loss of break strength in abscission zones as compared with dark controls, changes in the rate of abscission could not be associated with changes in the rate of ethylene production. Reducing ethylene synthesis in tissue with aminoethoxyvinylglycine did not alter the effects of red or far-red light on abscission. Far-red light appeared to increase and red light appeared to decrease tissue sensitivity to ethylene.     

Molecular and genetic characterization of a non-climacteric phenotype in melon reveals two loci conferring altered ethylene response in fruit

Fruit ripening and abscission are associated with an ethylene burst in several melon (Cucumis melo) genotypes. In cantaloupe as in other climacteric fruit, exogenous ethylene can prematurely induce abscission, ethylene production, and ripening. Melon genotypes without fruit abscission or without ethylene burst also exist and are, therefore, non-climacteric. In the nonabscising melon fruit PI 161375, exogenous ethylene failed to stimulate abscission, loss of firmness, ethylene production, and expression of all target genes tested. However, the PI 161375 etiolated seedlings displayed the usual ethylene-induced triple response. Genetic analysis on a population of recombinant cantaloupe Charentais x PI 161375 inbred lines in segregation for fruit abscission and ethylene production indicated that both characters are controlled by two independent loci, abscission layer (Al)-3 and Al-4. The non-climacteric phenotype in fruit tissues is attributable to ethylene insensitivity conferred by the recessive allelic forms from PI 161375. Five candidate genes (two ACO, two ACS, and ERS) that were localized on the melon genetic map did not exhibit colocalization with Al-3 or Al-4.     

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.     

Endogenous and exogenous ethylene induces needle abscission and cellulase activity in post-harvest balsam fir (Abies balsamea L.)

Post-harvest needle loss is a major problem for balsam fir and other Christmas tree species. Recent evidence has implicated ethylene as a signal responsible for post-harvest needle abscission, but enzymological changes remain unknown. The objective of this study was to identify and quantify cellulase activity associated with endogenous and exogenous ethylene-induced abscission. An experiment was designed with three treatments (control, endogenous ethylene, or exogenous ethylene) with five replicates. Key response variables include needle retention duration, xylem pressure potential, ethylene evolution rate, and cellulase activity. Two complimentary methods were used to assess cellulase activity: a cellulose plate digestion and zymography. The results confirm ethylene as a signal for post-harvest abscission and identify ethylene-induced cellulase. Ethylene evolution was typically between 15 and 16 μL g⁻¹ h⁻¹, but there was no difference among the three treatments. However, exogenous ethylene significantly decreased needle retention by 60% and resulted in a sixfold decrease in xylem pressure potential. In addition, cellulase activity increased by 8- and 12-fold in endogenous and exogenous ethylene-induced abscission, respectively, compared to the control. Identification of ethylene-induced cellulase activity has increased our understanding of the post-harvest needle abscission process and confirms ethylene’s role as a signal molecule.     

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.     

Bean abscission cellulase : characterization of a cDNA clone and regulation of gene expression by ethylene and auxin

The physiology and anatomy of abscission has been studied in considerable detail; however, information on the regulation of gene expression in abscission has been limited because of a lack of probes for specific genes. We have identified and sequenced a 595 nucleotide bean (Phaseolus vulgaris cv Red Kidney) abscission cellulase cDNA clone (pBACl). The bean cellulase cDNA has extensive nucleic and amino acid sequence identity with the avocado cellulase cDNA pAV363. The 2.0 kilobase bean mRNA complementary to pBACl codes for a polypeptide of approximately 51 kilodalton (shown by hybrid-selection followed by in vitro translation). Bean cellulase antiserum is shown to immunoprecipitate a 51 kilodalton polypeptide from the in vitro translation products of abscission zone poly(A)⁺ RNA. Ethylene initiates bean leaf abscission and tissue-specific expression of cellulase mRNA. If ethylene treatment of bean explants was discontinued after 31 h and then 2,5-norbornadiene given to inhibit responses resulting from endogenously synthesized ethylene, polysomal cellulase mRNA hybridizing to pBACl decreased. Thus, ethylene is required not only to initiate abscission and cellulase gene expression but also to maintain continued accumulation of cellulase mRNA. Explants treated with auxin 4 hours prior to a 48 hour treatment with ethylene showed no substantial accumulation of RNA hybridizing to pBACl or expression of cellulase activity.     

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.     

Ethylene- and dark-induced flower abscission in potted Plectranthus: Sensitivity,prevention by 1-MCP,and expression of ethylene biosynthetic genes

Prevention of ethylene- and shipping-induced flower abscission is necessary to maintain the quality of both cut flowers and potted plants during handling, transport and retail display. The aims of the present work were to determine the sensitivity of Plectranthus cultivars to applied ethylene, to alleviate ethylene- and shipping-induced flower abscission in intact potted plants using 1-methylcyclopropene (1-MCP), and to investigate the possible causes of dark-induced flower abscission. All cultivars were sensitive to ethylene in a concentration-dependent manner, and complete abscission occurred within 24 h with 1 and 2 μl l^− 1 ethylene. Unopened buds were more sensitive to applied ethylene, and exhibited greater abscission than open flowers. Ethylene synthesis remained below detection limits at all time points under control and continuous dark conditions. Dark treatment significantly increased flower abscission in Plectranthus cultivars, and like ethylene-induced flower abscission, this could be prevented by continuous 1-MCP treatment. Gene expression of ethylene biosynthetic enzymes ACS and ACO was examined as possible causes for the accelerated flower abscission observed in plants kept in continuous darkness. Expression patterns of ACS and ACO varied between different cultivars of Plectranthus. In some cases, increased expression of ACS and ACO led to increased flower abscission. Gene expression was higher in open flowers when compared to unopened flowers suggesting a cause for the observed preferential shedding of open flowers in some cultivars. Although the cause of dark-induced abscission in Plectranthus remains elusive, it can be effectively controlled by treatment with 1-MCP.     

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.     

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.     

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.