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.     

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.     

Comparison of cyclopropene, 1-methylcyclopropene, and 3,3-dimethylcyclopropene as ethylene antagonists in plants

A comparison has been made of cyclopropene (CP), 1-methylcyclopropene (1-MCP), and 3,3-dimethyl-cyclopropene (3,3-DMCP) in their ability to protect plants against ethylene. In bananas, both CP and 1-MCP are effective around 0.5 nL L^–1, and 3,3-DMCP was effective at 1 L L^–1. Bananas treated with CP and 1-MCP again become sensitive to ethylene at 12 days and those treated with 3,3-DMCP at 7 days. Mature green tomatoes are protected by 5–7 nL L^–1 of 1-MPC for 8 days at 25°C and tomatoes treated with 3,3-DMCP at 5–10 L L^–1 are protected for 5 days. Carnation flowers are protected with CP or 1-MCP after exposure to 0.5 nL L^–1 for 24 hours and by 1 L L^–1 of 3,3-DMCP. The display life of Campanula flowers is increased from 3.3 to 5.4 days by 10 L L^–1 of 3,3-DMCP and to 9 days by 20 nL L^–1 of 1-MCP. Ethylene inhibition of pea seedlings is reduced by treatment with 1-MCP at 10 L L^–1 of ethylene but as ethylene is increased to 3000 L L^–1 growth inhibition increases. 3,3-DMCP treatment causes very little reduction of the ethylene effect even at very low concentrations.     

Stamen abscission and water balance in Metrosideros flowers

Cymules (3-flowered units borne on single pedicels) were cut from inflorescences of Metrosideros collina J.R. & G. Forst. cv. Tahiti and used to test the effects of ethephon and ethylene on stamen abscission in the presence of silver thiosulphate (STS) and 1-methylcyclopropene (1-MCP), and to test the effects of holding solutions on cymule water balance and the progression of floral development. Flower bud and stamen abscission occurred in response to 0.5–5.0 and 0.1 μl l⁻¹ ethylene, respectively. Ethylene effects were partially negated by scrubbing exogenous ethylene, and more completely negated by STS (2.0 m M ). 1-MCP caused greater ethylene production and inhibited stamen abscission for only 1–2 days after treatment. Ethephon (10-10 000 mg l⁻¹) induced stamen wilting rather than abscission, an effect that was not negated by STS. Stamen wilting was negatively correlated with stamen relative water content, and the increase in stamen wilting was generally reduced by treatments that enhanced cymule mass. Stamen wilting was least using a 100 g l⁻¹ sucrose pulse or holding solutions containing 30–40 g l⁻¹ sucrose, with hydroxyquinoline citrate (200 mg l⁻¹) maintained at pH 5. Our results indicate that 1-MCP may be relatively ineffective in blocking the effects of ethylene on the abscission of organs, such as the stamens of M . collina , which are highly sensitive to this hormone.     

Compounds Interacting with the Ethylene Receptor in Plants

Abstract: Some of the compounds binding to the ethylene receptor induce an ethylene response, but others prevent it. The compounds preventing an ethylene response have been developed into a means for protecting plants against ethylene and extending the life of some plant material. 1-Methylcyclopropene (1-MCP), a compound now commercially available under the names EthylBloc and SmartFresh™, is currently being used on flowers, fruit and vegetables with great success. In ethylene sensitive flowers, among other responses, it prevents senescence and abscission of plant organs; in fruit and vegetables it slows down the ripening process. Other similar compounds are now being developed for a range of methods of application.     

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.     

Electron spin resonance evidence for the formation of free radicals in plants exposed to ozone

Electron spin resonance (ESR) spectroscopy was used to demonstrate that free radicals are formed in O₃-fumigated plant leaves prior to the formation of visible leaf injury. ESR signals with a g-value of 2.0037 to 2.0043, were observed in pea ( Pisum sativum L. cv. Feltham first) and bean ( Phaseolus vulgaris L. cv. Pinto) plants that had been fumigated for 4 h with 70–300 nl l⁻¹ of ozone after they had been treated with the spin-trap N- t -butyl-α-phenylnitrone (PBN). The size of the ESR signals increased with the concentration of ozone used but the nature of the trapped radicals could not be identified. However, further experiments using an inhibitor of ethylene biosynthesis, arninoethoxyvinyl glycine (AVG), showed that the reaction between ozone and ethylene is the cause for ozone toxicity.     

Ethylene-associated phase change from juvenile to mature phenotype of daylily (Hemerocallis) in vitro

Hemerocallis plantlets maintained in vitro for extended periods of time in tightly closed culture vessels frequently show a phenotype, albeit on a miniaturized scale, typical of more mature, field-grown plants. The positive relationship of elevated ethylene in the headspace of such vessels to the phase shift from juvenile to mature form is established. Rigorous restriction in air exchange with the external environment by means of silicone grease seals hastens the phase change and improves uniformity of response. Although some plantlets may take longer to accumulate enough ethylene in sealed jars to undergo change, added ethylene and ethylene-releasing agents promote it. Ethylene adsorbants (e.g. mercuric perchlorate) block the shift of juvenile to mature form. Critical ambient ethylene level for the shift is ca 1 μl l⁻¹. Levels up to 1000 μl l⁻¹ do not hasten the response but are not toxic. The phase change is fully reversible when air exchange permits ethylene to drop below 1 μl l⁻¹. At least 1 μl l⁻¹ ethylene is required to sustain the mature phenotype. The ethylene synthesis inhibitor aminoethoxyvinylglycine (AVG) prevents the phase change, while the ethylene biosynthesis intermediate 1-aminocyclopropanecarboxylic acid (ACC) improves it. KOH, as a CO₂ absorbant, does not prevent the phase change. Histology sections demonstrate subtle changes in the form of shoot tips of plantlets undergoing phase change.     

The role of ethylene in photoperiodic control of bulbing in Allium cepa

Bulb formation in Allium cepa L. cv. Dorata di Parma, was promoted by ethylene (Ethrel 120 mg l⁻¹) treatments. Application of the antiethylene complex silverthiosulphate (0.01 m M ) inhibited ethylene-induced bulb formation, but did not affect photoperiod-induced bulb formation. When the inhibitor of ethylene biosynthesis, aminoethoxyvinylglycine, was applied at 0.04 to 4 mg l⁻¹ to onion plants under inductive photoperiodic conditions the bulb promotion was unaffected. Therefore, Ethrel-induced bulb formation does not appear to be related to endogenous control of bulbing.     

Survival and recovery of perennial forage grasses under prolonged Mediterranean drought: II. Water status, solute accumulation, abscisic acid concentration and accumulation of dehydrin transcripts in bases of immature leaves

Effects of ozone on spring wheat ( Triticum aestivum L. cv. Satu) were studied in an open-top chamber experiment during two growing seasons (1992–1993) at Jokioinen in south-west Finland. The wheat was exposed to filtered air (CF), non-filtered air (NF), non-filtered air+35 nl l⁻¹ ozone for 8 h d⁻¹ (NF⁺) and ambient air (AA). Each treatment was replicated five times. Two wk after anthesis, after 4 wk of ozone treatment (NF⁺, 45 nl l⁻¹ 1000–1800 hours, seasonal mean) the net CO₂ uptake of wheat flag leaves was decreased by c . 40% relative to CF and NF treatments, both initial and total activity of Rubisco and the quantity of protein-bound SH groups were decreased significantly. Added ozone also significantly accelerated flag leaf senescence recorded as a decrease in chloroplast size. The effect was significant 2 wk after anthesis, and senescence was complete after 4 wk. In the CF and NF treatments senescence was complete 5 wk after anthesis. The significant effect of ozone on the chloroplasts and net CO₂ uptake 2 wk after anthesis did not affect the grain filling rate. However, since the grain filling period was shorter for ozone fumigated plants, kernels were smaller. The decrease in 1000-grain weight explained most of the yield reduction in the plants under NF⁺ treatment. The results indicate that wheat plants are well buffered against substantial decrease in source activity, and that shortened flag leaf duration is the major factor causing ozone-induced yield loss.     

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.     

Net CO2 exchange of Pinus taeda shoots exposed to variable ozone levels and rain chemistries in field and laboratory settings

Net CO₂ exchange rates (CERs) were measured in seedlings of two loblotly pine ( Pinus taeda L.) families following 6- or 13-week exposures to ozone (charcoalfiltered or ambient air + O₃) and acid rain treatments (pH 3.3, 4.5 and 5.2). Ozone exposures (14 or 170 nl l⁻¹) were made in open-top chambers, and in continously stirred tank reactors (14, 160 or 320 nl l⁻¹) located in the field and laboratory, respectively. The CERs of whole shoots were measured in an open infrared gas analysis system at 6 levels of photosynthetic photon flux density (0, 33, 60, 410, 800 and 1660 μmol m⁻² s⁻¹). Treatment effects were not consistent between field- and laboratory-exposed seedlings. Ozone-treated field seedlings exhibited statistically significant reductions in light-saturated CER of 12.5 and 25% when measured at 6 and 13 weeks, respectively. Laboratory seedlings exhibited mixed responses to O₃, with one family showing reduced CER only after 6 weeks of O₃ exposure and the other only after 13 weeks (O₃ >160 nl l⁻¹ for both). After 13 weeks of exposure, pH 3.3, and 4.5 rain treatments enhanced light-saturated CER by an average of 52% over that observed in seedlings exposed to the pH 5.2 treatment. Enhanced CERs due to acid rain were of the same magnitude (3–5 μmol CO₂g⁻¹ s⁻¹) as ozone-induced CER reductions. No differences in dark respiration were detected between treatments. Although ozone and acid rain treatments altered seedling CER, the differences were not translated into altered final plant dry weights over the 13-week exposure period.     

Effect of 1-methylcyclopropene and methylenecyclopropane on ethylene binding and ethylene action on cut carnations

1-Methylcyclopropene (1-MCP), formerly designated as Sis-X, has been shown to be an effective inhibitor of ethylene responses in carnation flowers in either the light or the dark. The binding appears to be to the receptor and to be permanent. A 6 h treatment at 2.5 nl l^–1 is sufficient to protect against ethylene, and 0.5 nl l^–1 is sufficient if exposure is for 24 h. As carnation flowers age, a little higher concentration appears to be needed. Most of the natural increase in ethylene production during senescence is prevented by treatment with 1-MCP. A closely related compound, methylenecyclopropane shows ethylene activity. A tritium labelled 1-MCP (60 mCi mmol^–1) has been prepared. A higher specific activity is needed for more critical studies.     

In vitro growth and ripening of strawberry fruit in the presence of ACC, STS or propylene

Strawberry ( Fragaria ananassa Duch.) fruit exhibit limited capacity for continued development following harvest. This problem can be circumvented by maintaining harvested strawberry fruit in solutions containing sucrose and a bactericide. In this study, we investigated the respiratory and ethylene production kinetics and ethylene responsiveness in strawberry fruit harvested immature and ripened in vitro in the presence of propylene. The effects of 1-amino-cyclopropane-1-carboxylic acid (ACC) and silver thiosulfate (STS) alone and in combination were also examined. Respiration and ethylene patterns of fruit harvested green and developed in vitro declined with maturation and ripening, as did those of field-grown fruit harvested at different stages of ripeness. Exposure of detached green strawberry fruit to 5000 μl litre^-1 propylene failed to stimulate respiration or ethylene production, but advanced pigmentation changes and fresh-weight gain significantly. Excised fruit provided with 1 mol^-3 ACC exhibited increased ethylene production, enhanced fresh-weight gain, and accelerated anthocyanin accumulation, but showed no change in respiration. The developmental response of harvested strawberry fruit to propylene or ACC was dependent on fruit maturity at harvest, with white fruit exhibiting greater insensitivity compared with green fruit. Silver thiosulfate (0.5 mol^-3) applied alone or in combination with ACC failed to delay ripening in excised strawberry fruit. These experiments demonstrate that ripening in detached strawberry fruit can be modified by ethylene only in green fruit that are provided with a carbohydrate source. Ethylene, when applied exogenously as ACC or propylene to green fruit, can slightly increase fruit growth and the rate of colour development.     

Induction of secondary dormancy in sunflower seeds by high temperature. Possible involvement of ethylene biosynthesis

High temperature (45°C) inhibits seed germinition and seedling sunflower ( Helianthus annuus L. cv. Mirasol). Treatment of imbibed seeds at 45°C for more than 48 h induces a secondary dormancy, which is associated with progressive decrease of germination ability at optimal temperature (25°C) as well as with abnormal seedling growth. Ethylene (55μl l⁻¹) and 2-chloroethylphosphonic acid (ethephon) (2.5 m M ) improve germination of thermodormant seeds at 25°C. but the abnormal growth of the seedlings remains. O₂-enriched atmosphere and dry storage improve germination and normal seedling growth. The induction of thermodormancy in sunflower seeds seems associated with loss of their ability to convert 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. Possible effects of high temperature on membranes and ethylene forming enzyme (EFE) are discussed.     

Effect of long term fumigation with ozone on the turnover of the D-1 reaction center polypetide of photosystem II in spruce (Picea abies)

Long term fumigation of 4-year-old spruce trees with ozone concentrations up to 200 nl l⁻¹ has only minor effects on the photosynthetic activities measured as chlorophyll a fluorescence. Nevertheless, it drastically changes the turnover of the D-1 reaction center polypeptide of photosystem II. During summer, fumigation with ozone for 2 weeks resulted in an almost 4-fold stimulation of the light dependent incorporation of [¹⁴C] leucine into the D-1 protein in the exposed trees. The amount of immunodetectable D-1 protein remained constant when based on chlorophyll. This indicates that exposure to ozone stimulates both the synthesis and the degradation of the D-1 protein. When spruce trees were exposed during winter for 4 weeks to 100 and 200 nl l⁻¹ ozone, respectively, an almost 3-fold increase of the amount of immunodetectable D-1 protein per chlorophyll in the exposed trees was observed. This can be explained by a varying stimulation of D-1 protein synthesis and degradation depending on the different physiological conditions. Since so far the D-1 protein has been found only as a component of photosystem II reaction centers, one has to assume that the relative content of photosystem II reaction centers also increases under certain stress conditions. The increased turnover of the D-1 protein in trees exposed to ozone explains the synergistic effects of stress conditions and high light intensities often observed in the field.     

Biosynthesis of stress ethylene in soybean seedlings: Similarities to endogenous ethylene biosynthesis

The similarity of stress ethylene biosynthesis in whole plants to endogenous ethylene biosynthesis was investigated using two inhibitors of ethylene biosynthesis, aminoethoxyvinylglycine (AVG) and cobalt chloride (Co²⁺); and the intermediates, methionine, S -adenosylmethionine (SAM), and 1-aminocyclopropane-1-carboxylic acid (ACC), of basal ethylene biosynthesis. Stress ethylene production induced by ozone, cadmium, or 2,4-dichlorophenoxyacetic acid was inhibited in hydroponically-grown soybean seedlings in a concentration-dependent manner by both AVG and CO²⁺. The ethylene intermediates evoked responses in intact seedlings similar to that described for endogenous ethylene production in isolated vegetative tissue. The addition of SAM to the hydroponic system relieved AVG inhibition of stress ethylene production. Feeding ACC to the seedlings resulted in increased ethylene production independent of stress application or prior AVG inhibition. Cobalt inhibition of stress ethylene production was relieved by increasing concentrations of ACC. A short lag period of 12–18 min was observed in stress ethylene production following a 30-min ozone exposure. Addition of cycloheximide partially inhibited ozone-induced ethylene production.
These results suggest a common pathway in whole plants for stress ethylene production and endogenous ethylene biosynthesis.     

In vitro regeneration and genetic transformation of the berberine-producing plant,Thalictrum flavum ssp. glaucum

Protocols have been developed for the in vitro regeneration and Agrobacterium -mediated genetic transformation of meadow rue, Thalictrum flavum ssp. glaucum . Ten-day-old seedlings were bisected along the embryonic axis and the cotyledons were co-cultured with various Agrobacterium tumefaciens strains for 3 days. The cotyledons were cultured on a shoot induction medium (B5 salts and vitamins, 30 g l⁻¹ sucrose, 2 mg l⁻¹ kinetin, and 3 g l⁻¹ Gelrite) containing 25 mg l⁻¹ hygromycin B as the selection agent and 250 mg l⁻¹ timentin to facilitate the elimination of Agrobacterium . Only the oncogenic A. tumefaciens strains A281 and C58 produced transgenic T. flavum callus tissues. A281 was the most effective strain producing hygromycin-resistant callus on 85% of the explants. Transgenic callus was subcultured on the shoot induction medium every 2 weeks. After 12 weeks, hygromycin-resistant shoots that formed on explants exposed to strain A281 were transferred to a root induction medium (B5 salts and vitamins, 25 mg l⁻¹ hygromycin B, 250 mg l⁻¹ timentin, and 3 g l⁻¹ Gelrite). Detection of the β -glucuronidase ( GUS ) gene using a polymerase chain reaction assay, the high levels of GUS mRNA and enzyme activity, and the cytohistochemical localization of GUS activity confirmed the genetic transformation of callus cultures and regenerated plants. The transformation process did not alter the normal content of berberine in transgenic roots or cell cultures; thus, the reported protocol is valuable to study the molecular and metabolic regulation of protoberberine alkaloid biosynthesis.     

Do tomatoes on the plant behave as climacteric fruits?

I considered the possibility that changes in fruit photosynthesis obscure the occurrence of the climacteric rise in respiration in tomato fruits attached to the plant. Internal CO₂ and ethylene concentrations in tomatoes ( Lycopersicon esculentum Mill. cv. OH 7814) were analyzed after direct sampling through polyethylene tubes implanted in the external pericarp. Fruits which were shaded with aluminium foil contained up to 60 ml 1⁻¹ CO₂, until the internal ethylene concentration exceeded 1 μl l⁻¹, when CO₂ concentration declined to below 40 ml l⁻¹; the CO₂ concentration in fruits exposed to light only occasionally exceeded 40 ml 1⁻¹. The internal CO₂ concentration of detached fruits first declined and then increased along with ethylene concentration, as expected for the climacteric. Detached green fruits under continuous low photosynthetic photon flux density (100 μmol m⁻² s⁻¹) contained almost no internal CO₂ and produced no CO₂. Changes in photosynthesis and an associated CO₂-generating system in green fruits are thought to obscure the climacteric rise in tomato fruits developing on the plant.