Relationship between Ethylene Evolution and Senescence in Morning-Glory Flower Tissue

An excised tissue system consisting of corolla rib segments was developed to study the relationship between senescence and ethylene production in morning-glory flowers (Ipomoea tricolor). Such segments, isolated 1 or 2 days (day −1 or day −2) before flower opening (day 0) passed through the same developmental phases as did the corresponding tissues of the intact organ. When excised on day −1 and incubated overnight, the rib segments turned from purple to blue and changed from a slightly curled to a flat configuration. On day 0, these segments rolled up during the afternoon and turned purple again, as did the ribs of an intact corolla; the rolling up coincided with an increased rate of ethylene production. Premature rolling up and associated ethylene evolution were induced by ethylene or propylene treatment. When segments were excised on day −2 and incubated overnight, there were no changes in color or shape; during day −1, no spontaneous rolling up and little ethylene evolution occurred. Application of ethylene or propylene to these immature segments elicited rolling up but did not stimulate endogenous ethylene production.     

Anti-ethylene Effects of cis-2-butene and cyclic olefins

The ability of butenes and cyclic olefins to induce an ethylene response or to counteract ethylene's action in etiolated pea seedlings was investigated. 1-Butene gave an ethylene-like response at 25 OOOμl/l. cis-Butene exhibited no ethylene-like response but appeared to overcome the action of 0.3 μl/l. ethylene at the same concentration. trans-Butene was without effect. 2,5-Norbornadiene competitively inhibited ethylene's action with a K_i of 170 μl/l. (computed as a gas). The K_i for norbornene was 360 μl/l. This value represents about one-half of the activity given by 2,5-norbornadiene. Other K_i values obtained were 488 μl/l. for 1,3-cyclohexadiene, 870 μl/l. for 1,3-cycloheptadiene, 1100 μl/l. for cyclopentene, 4700 μl/l. for 1,4-cyclohexadiene, and 6 ml/l. for cyclohexene. Cyclohexane and benzene were without efrect.     

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-enhanced ethylene oxidation inVicia faba

The alkene oxygenase (AO) of fababean (Vicia faba L.) converts ethylene to ethylene oxide. Treatment of fababeans with 10μl/liter ethylene increases the activity of this enzyme within 2 hours of ethylene treatment. Though other alkenes were taken up by fababean seedlings, ethylene was the most active in inducing AO activity. The ability of ethylene to increase AO was blocked 60% by cycloheximide, an inhibitor of protein synthesis, and 35% by AgNO₃, an inhibitor of ethylene action.     

Responses of banana fruit to treatment with 1-methylcyclopropene

Experiments were conducted to determine levels of 1-methylcyclopropene (1-MCP) exposure needed to prevent ethylene-stimulated banana fruit ripening, characterise responses of ethylene-treated fruit to subsequent treatment with 1-MCP, and to test effects of subsequent ethylene treatment on 1-MCP-treated fruit softening. Fruit softening was measured at 20°C and 90% relative humidity. One hour exposure at 20°C to 1000 nl 1-MCP/l essentially eliminated ethylene-stimulated ripening effects. Exposure for 12 h at 20°C to just 50 nl 1-MCP/l was similarly effective. Fruit ripening initiated by ethylene treatment could also be delayed with subsequent 1-MCP treatment. However, 1-MCP treatment only slowed down ripening of ethylene-treated fruit when applied at 1 day after ethylene and was ineffective when applied 3 or 5 days after ethylene treatment. The ripening response of fruit treated with 1-MCP and subsequently treated with ethylene varied with interval time between 1-MCP and ethylene treatments. As time increased, the response of 1-MCP-treated fruit to ethylene was enhanced. Responses to 0.1, 1, 10 or 100 µl ethylene/l concentrations were similar. Enzyme kinetic analysis applied to 1-MCP effects on ethylene-induced softening of banana fruit suggested that 1-MCP inhibition is by noncompetitive antagonism of ethylene binding.     

Temperature Sensitivity of the Latent Phase in Ethylene-induced Elongation

The temperature sensitivity is reported for the latent period preceding ethylene-induced elongation in the adaxial half of the leaf petiole of Helianthus annuus. When intact plants were exposed to 10 μl of ethylene/l of air over the temperature range 18 to 35 C, the minimum latent time was 62 minutes at 28 C and the maximum was 132 minutes at 18 C. The temperature coefficient, Q₁₀, changed from 2.1 below 28 C, to 0.7 above. In 100 μl of ethylene/l of air, the latent time was reduced by 14% at 18 C, but was significantly increased at 28 and 38 C. These results show that the latent period in the elongation response of the petiole to ethylene cannot be reduced below about 60 minutes by raising either the leaf temperature or the atmospheric ethylene concentration.     

Autoinhibition of Ethylene Formation in Nonripening Stages of the Fruit of Sycomore Fig (Ficus sycomorus L.)

Differences in the mechanism of ethylene emanation of Ficus sycomorus L. during various stages of the fruit development were investigated by enclosing the figs in jars. Two distinct patterns of ethylene emanation were found. Pattern a. in stages not capable of ripening, neither spontaneously nor as a result of physiological treatment (nonripening stages A and C), ethylene concentration in the jar increased linearly for a short time and then remained constant. Pattern b. in stages capable of ripening (ripening stages B, D, and E), the linear increase in ethylene concentration continued for the entire period of measurement. In nonripening stages, ethylene emanation stopped when ethylene concentration in the jar reached a constant value (0.6 μl/l at stage C). Aeration of the figs and the jar renewed ethylene emanation. CO₂ concentration in the jar never exceeded 0.5%. Treatment of stage C figs with 0.6 to 10 μl/l exogenous ethylene caused immediate and complete cessation of ethylene emanation whereas the same treatment did not cause any change in rate of ethylene emanation from figs at the ripening stages B and D. Gashing (wounding) of stage C figs temporarily changed the pattern of ethylene emanation from pattern a to pattern b.     

Changes during low-oxygen storage in the effects of ethylene on induction of ethylene synthesis and stimulation of respiration of 'Gloster 69' apples

The ability of ethylene to stimulate respiration and advance the onset of rapid ethylene production was investigated at different times during storage of 'Gloster 69' apples in 2 kPa O₂ at 1.5–3.5°C. Ethylene stimulated respiration in apples at 15°C immediately after harvest; maximal rates were recorded at 10–1000 μl I⁻¹ but attainment of these rates was delayed after low O₂ storage until day 3 of treatment at 15°C. The onset of rapid ethylene production at 15°C occurred later in non-ethylene-treated apples after storage than after harvest. Ethylene production was induced in some apples during ethylene treatment for 3 or 6 days; in others it was induced about 20 days after treatment, but a proportion of the fruit showed no induction in the 45-day duration of experiments. An ethylene treatment at 10 μl I⁻¹ led to a near maximal increase in the frequency of induction of ethylene production at all times. After storage apples were mainly induced during treatment or not induced, whereas after harvest induction after treatment was more frequent. The presence of 2000 μl l⁻¹ norbornadiene during ethylene treatment inhibited the stimulation of respiration and the induction of ethylene production; this inhibition was only partly reversed by ethylene at 1000 μl l⁻¹ the experiments suggest that receptors for ethylene were present at all stages but that response capacity changed during storage.     

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.     

Effects of carbon dioxide on ethylene production and action in intact sunflower plants

A continuous flow system was used to study the interactions between carbon dioxide and ethylene in intact sunflower (Helianthus annuus L.) plants. An increase in the concentration of carbon dioxide above the ambient level (0.033%) in the atmosphere surrounding the plants increased the rate of ethylene production, and a decrease in carbon dioxide concentration resulted in a decrease in the rate of ethylene production. The change in the rate of ethylene production was evident within the first 15 minutes of the carbon dioxide treatment. Continuous treatment with carbon dioxide was required to maintain increased rate of ethylene production. The rate of carbon dioxide fixation increased in response to high carbon dioxide treatment up to 1.0%. Further increases in carbon dioxide concentration had no additional effect on carbon dioxide fixation. Carbon dioxide concentrations higher than 0.11% induced hyponasty of the leaves whereas treatment with 1 microliter per liter ethylene induced epinasty of the leaves.     

Regulation of Senescence in Carnation (Dianthus caryophyllus) by Ethylene: Mode of Action

Carnation (Dianthus caryophyllus) flowers were exposed to 2 μl/l ethylene and examined at intervals to determine the time course of wilting, decrease in water uptake, and increase in ionic leakage in response to ethylene. A rapid decrease in water uptake was observed about 4 hours after initiating treatment with ethylene. This was followed by wilting (in-rolling of petals) about 2 hours later. Carbon dioxide inhibited the decline in water uptake and wilting and this is typical of most ethylene-induced responses. Ethylene did not affect closure of stomates. Ethylene enhanced ionic leakage, as measured by efflux of ³⁶Cl from the vacuole. This was judged to coincide with the decrease in water uptake. Gassing flowers with propylene initiated autocatalytic ethylene production within 2.4 hours. Since the increase in ethylene production by carnations preceded the increase in ionic leakage and the decline in water uptake by several hours, it is apparent that the change in ionic leakage does not lead to the initial increase in ethylene production as reported (Hanson and Kende 1975 Plant Physiol 55:663-669) in morning glory but may explain the autocatalytic phase of ethylene production.     

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.     

Effect of the defoliant thidiazuron on ethylene evolution from mung bean hypocotyl segments

The effect of the defoliant thidiazuron (N-phenyl-N′1,2,3-thiadiazol-5-ylurea) on ethylene evolution from etiolated mung bean hypocotyl segments was examined. Treatment of hypocotyl segments with concentrations of thidiazuron equal to or greater than 30 nanomolar stimulated ethylene evolution. Increased rates of ethylene evolution from thidiazuron-treated tissues could be detected within 90 minutes of treatment and persisted up to 30 hours after treatment. Radioactive methionine was readily taken up by thidiazuron-treated tissues and was converted to ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC) and an acidic conjugate of ACC. Aminoethoxyvinylglycine, aminooxyacetic acid, cobalt chloride, and α-aminoisobutyric acid reduced ethylene evolution from treated tissues. An increase in the endogenous content of free ACC coincided with the increase in ethylene evolution following thidiazuron treatment. Uptake and conversion of exogenous ACC to ethylene were not affected by thidiazuron treatment. No increases in the extractable activities of ACC synthase were detected following thidiazuron treatment.     

Ethylene Production and Leaflet Abscission of Three Peanut Genotypes Infected with Cercospora arachidicola Hori

Ethylene can induce abscission of leaves and other plant organs. Increased ethylene production by plant tissues can occur after invasion by microorganisms. The fungus Cercospora arachidicola Hori, attacks peanut leaflets and causes defoliation. Our objective was to determine if ethylene was involved in this defoliation. Leaves of three peanut, Arachis sp., genotypes were inoculated with C. arachidicola. Two genotypes, `Tamnut 74' and PI 109839, produced ethylene and were defoliated. The third genotype, PI 276233, a wild species, did not produce ethylene above control levels and was not defoliated. Increase in ethylene production by Tamnut 74 and PI 109839 coincided with appearance of disease symptoms. Tamnut 74 produced the most ethylene, but PI 109839 was equally defoliated. Thus, less overall ethylene production did not necessarily indicate a more resistant genotype in this system unless ethylene production remained at control levels, as it did for PI 276233. Ethylene sufficient to initiate abscission could have been produced by the seventh day after inoculation when it was similar for both Tamnut 74 and PI 109839, but 3 to 4 times control amounts. This occurred before the rapid increase in ethylene production and before disease symptoms were visible. Silver ion, a potent inhibitor of ethylene action, was sprayed at three concentrations on intact Tamnut 74 plants. All rates reduced abscission and 150 mg/liter Ag(I) decreased abscission to below 10%. The data indicate that ethylene produced by peanut leaves in response to C. arachidicola infection initiates abscission and that ethylene action can be blocked by Ag(I) in such a host-pathogen interaction.     

Ethylene-Enhanced 1-Aminocyclopropane-1-carboxylic Acid Synthase Activity in Ripening Apples

Apples (Malus sylvestris Mill, cv Golden Delicious) were treated before harvest with aminoethoxyvinylglycine (AVG). AVG is presumed to reversibly inhibit 1-aminocyclopropane-1-carboxylic acid (ACC) activity, but not the formation of ACC synthase. AVG treatment effectively blocked initiation of autocatalytic ethylene production and ripening of harvested apples. Exogenous ethylene induced extractable ACC synthase activity and ripening in AVG-treated apples. Removal of exogenous ethylene caused a rapid decline in ACC synthase activity and in CO₂ production. The results with ripened, AVG-treated apples indicate (a) a dose-response relationship between ethylene and enhancement of ACC synthase activity with a half-maximal response at approximately 0.8 μl/l ethylene; (b) reversal of ethylene-enhanced ACC synthase activity by CO₂; (c) enhancement of ACC synthase activity by the ethylene-activity analog propylene.

Induction of ACC synthase activity, autocatalytic ethylene production, and ripening of preclimacteric apples not treated with AVG were delayed by 6 and 10% CO₂, but not by 1.25% CO₂. However, each of these CO₂ concentrations reduced the rate of increase of ACC synthase activity.

     

Membrane Lipids in Senescing Flower Tissue of Ipomoea tricolor

Rib segments excised from flower buds of Ipomoea tricolor Cav. pass through the same phases of senescence as the respective tissue on the intact plant. Such segments were used to correlate changes in lipid content with known symptoms of aging, such as rolling up of the ribs and ethylene formation. It was found that the level of phospholipid had already started to decline before visible signs of senescence were evident. As the segments began to roll up and to produce ethylene, the rate of phospholipid loss accelerated sharply. During the same period, the level of fatty acids esterified to phospholipids also fell by 40%. No qualitative changes in any lipid component could be detected during senescence. Labeling experiments using ³³P as marker showed that the rate at which radioactivity was lost from phospholipids during aging was parallel to the rate at which the level of total phospholipids declined. Exogenously applied ethylene accelerated the loss of phospholipid and the senescence of rib segments while benzyladenine retarded both of these processes.     

Increased mitochondrial DNA and RNA polymerase activity in ethylene-treated potato tubers

A purified mitochondrial fraction was isolated from potato (Solanum tuberosum L.) tubers respiring normally at 23°C or at an accelerated rate in response to treatment with ethylene (10 microliters per liter).

A pronounced increase in various mitochondrial enzymic activities was observed in response to exposure of the whole tubers to ethylene. Cytochrome c oxidase activity increased more than 50%, DNA polymerase activity increased about 2-fold, and RNA polymerase activity increased 2.5-fold. Moreover, DNA or RNA polymerase activities of mitochondria isolated from tubers not treated with ethylene were not affected by ethylene treatment in vitro. Respiratory control ratios decreased from 2.84 to 1.50 with increasing periods of ethylene treatment from 0 to 15 hours. None of these changes were observed in untreated tubers. It is concluded that the stimulation of respiration by ethylene in potato tubers is accompanied in vivo by an enhancement of mitochondrial enzymic activity of both membrane-associated enzymes which participate in the mitochondrial oxidative electron transport as well as soluble enzymes which are not directly involved in respiration.

     

Role of cytokinins in carnation flower senescence

Stem and leaf tissues of carnation (Dianthus caryophyllus) plants appear to contain a natural antisenescence factor since removal of most of these tissues from cut carnation flowers hastened their senescence. However, kinetin (5-10 μg/ml) significantly delayed senescence of flowers with stem and leaf tissues removed. In addition, the life span of cut flowers with intact (30-cm) stems was increased with kinetin treatment. Peak ethylene production by presenescent flowers was reduced 55% or more with kinetin treatment and was delayed by 1 day. Kinetin-treated flowers were less responsive to applied ethylene (100 μl/l for 3 hours) than untreated flowers. Possible natural roles of cytokinins in carnation flower senescence are discussed.     

Ethylene-induced gene expression in carnation petals : relationship to autocatalytic ethylene production and senescence

Exposure of carnation (Dianthus caryophyllus L.) flowers to ethylene evokes the developmental program of petal senescence. The temporal relationship of several aspects of this developmental program following treatment with ethylene was investigated. Exposure of mature, presenescent flowers to 7.5 microliters per liter ethylene for at least 6 hours induced petal in-rolling and premature senescence. Autocatalytic ethylene production was induced in petals following treatment with ethylene for 12 or more hours. A number of changes in mRNA populations were noted in response to ethylene, as determined by in vitro translation of petal polyadenylated RNA. At least 6 mRNAs accumulated following ethylene exposure. The molecular weights of their in vitro translation products were 81, 58, 42, 38, 35, and 25 kilodaltons. Significant increases in abundance of most mRNAs were observed 3 hours following ethylene exposure. Ethylene exposure resulted in decreased abundance of another group of mRNAs. Treatment of flowers with competitive inhibitors of ethylene action largely prevented the induction of these ethylene responses in petals. An increase in flower age was accompanied by an increase in the capacity for ethylene to induce petal in-rolling, autocatalytic ethylene production, and changes in mRNA populations suggesting that these responses are regulated by both sensitivity to ethylene and ethylene concentration. These results indicate that changes in petal physiology resulting from exposure to ethylene may be the result of rapid changes in gene expression.     

Wound-induced Accumulation of Trypsin Inhibitor Activities in Plant Leaves: Survey of Several Plant Genera

Asexual embryogenesis in Daucus carota L. `Queen Anne's Lace' callus was suppressed by Ethephon, ethylene, and 2,4-dichlorophenoxyacetic acid (2,4-D). The Ethephon effect could be attributed to volatile and nonvolatile substances. The volatile component was probably entirely ethylene. Ethylene was liberated in the cultures in direct proportion to Ethephon added to the medium. Autoclaving of Ethephon caused a substantial decrease of measurable ethylene. Continuous exposure of callus to 5 μl/l ethylene depressed somatic cell embryogenesis, but not markedly. Depression of embryogenesis by 2,4-D was unrelated to ethylene evolution.