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.     

1-substituted cyclopropenes: Effective Blocking Agents for Ethylene Action in Plants

A series of 1-alkane substituted cyclopropenes has been prepared and tested as ethylene antagonists using banana fruits as an assay system. 1-Methyl-, 1-ethyl-, 1-propyl-, 1-butyl-, 1-pentyl-, 1-hexyl-, 1-heptyl-, 1-octyl-, 1-nonyl-, and 1-decylcyclopropene were all very active compounds. 1-Methylcyclopropene protected bananas from ethylene with a minimum concentration of 0.7 nl.l^–1 after a 24 h exposure. As the carbon chain length was extended the minimum requirement increased some, but starting with 1-butylcyclopropene, the minimum concentration requirement declined and many cyclopropenes were required in lower concentrations than 1-methylcyclopropene. The time of protection at ambient temperature (22–23 °C) was 12 d for 1-methyl-, 1-ethyl-, 1-propyl-, and 1-butylcyclopropene. 1-Pentylcyclopropene protected bananas for 14 d, 1-hexylcyclopropene for 20 d, 1-heptylcyclopropene for 21 d, 1-octylcyclopropene for 25 d, 1-nonylcyclopropene for 35 d, and 1-decylcyclopropene for 36 d.     

Ethylene and carbon dioxide concentrations of soils as influenced by rhizosphere of crops under field and pot conditions

A method for collecting low volumes of soil gas from a small region, and a technique for determining small concentrations of ethylene using an enrichment process are described. Using these methods, it was found that ethylene and carbon dioxide (CO₂) concentrations of soils varied considerably depending on the presence or absence of a rhizosphere. Ethylene was much higher (31–375 nL L^–1; mean: 207) in non-cropped areas (i.e., soils without rhizosphere) than in the rhizosphere region (8–136 nL L^–1; mean: 38) of a field in which maize or soybean were grown. On the other hand, CO₂ concentrations were higher in rhizosphere than in non-rhizosphere soil, especially in pot experiments. The rate of ethylene decomposition was, however, much greater in rhizosphere soil (55 nL g^–1 day^–1) than in non-rhizosphere soil (34 nL g^–1 day^–1). Higher microbial activity was presumed to result in the decrease of ethylene concentration and the increase in CO₂ in rhizosphere regions. The implications of these results in relation to the influence of ethylene in rhizosphere on plant growth, and the role of soil microbes on decomposition of ethylene is discussed.     

Variability of responses to 1-methylcyclopropene by banana: influence of time of year at harvest and fruit position in the bunch

To examine the effect of early‐climacteric (postripening) 1‐methylcyclopropene (1‐MCP) exposure on the shelf‐life and quality of green Cavendish bananas (Musa acuminata cv. Williams) from the middle section of the bunch, bananas were harvested bimonthly and treated with 100 μL L^?1 ethylene for 2 consecutive days prior to exposure to 0, 100, 300, 1000, 3000 or 10 000 nL L^?1 1‐MCP for 24 h prior to storage at 22°C. 1‐MCP treatment at a concentration of 300 nL L^?1 or above increased banana shelf‐life significantly compared with the control, regardless of the month in which fruit were harvested except March where a higher concentration was needed (3000 nL L^?1). Fruit harvested in May were the most responsive with a greater than twofold increase in shelf‐life. To examine the effect of fruit position in the bunch on 1‐MCP efficacy, green fruit from the top or bottom of bunches were treated with 100 μL L^?1 ethylene for 2 consecutive days prior to early‐climacteric 1‐MCP (300 nL L^?1) exposure for 24 h at 22°C. In spring and autumn but not in summer, application of 1‐MCP to early‐climacteric fruit was more effective in fruit from the top than in those treated from the bottom of the bunch, increasing shelf‐life. Firmness of 1‐MCP‐treated fruit was up to 19% greater than that of the control across the year, except in fruit from the bottom of the bunch. Given that 1‐MCP is less effective in extending the shelf‐life of summer‐harvested fruit (particularly those from the bottom of the bunch), we conclude that preharvest conditions and fruit position in the bunch affect their responsiveness to ethylene and their behaviour during the ripening process.     

Role of ethylene in chlorophyll degradation in radish cotyledons

The effect of age of radish seedlings on changes in chlorophyll concentration caused by ethylene was examined. Ethylene was produced at 2–4 nl g^–1 h^–1 following excision of cotyledons from 5-to 20-day-old seedlings. The youngest cotyledons maintained this rate, whereas ethylene synthesis declined by as much as 80% during a 24-h period in older cotyledons. The youngest cotyledons continued to accumulate chlorophyll in the dark, but after 7 days cotyledons lost chlorophyll and the proportion of chlorophyll lost increased with age. Ethylene promoted, and norbornadiene inhibited, this loss of chlorophyll; in combined treatments the effects of ethylene and norbornadiene were competitive. The maximal rate of chlorophyll loss occurred in 1l L^–1 ethylene; extrapolation of the response to concentration indicated that half-maximum loss would occur at 0.005–0.01 l L^–1 ethylene. In cotyledons from 20-day-old seedlings, chlorophyll degradation occurred mainly after 24 h from excision and transfer to the dark. Chlorophyll degradation during 48 h in the dark was affected by norbornadiene or ethylene applied from 0–24 h or from 24–48 h.     

Effect of 1-MCP and low-temperature storage on postharvest conservation of camu–camu

Camu–camu, a native fruit from the Amazon region, is a rich source of bioactive compounds. However, its intense metabolic activity and high-water content limit the fruit’s postharvest storage and marketing. The aim of this study, conducted in two parts, was to evaluate the effects of 1-MCP and storage temperature on the physiology and postharvest preservation of camu–camu fruit. In part 1 of the study, fruit harvested at maturity stage 3 were divided into groups: control, 1-methylcyclopropene (1-MCP; 900 nL L^?1; 12 h) and ethylene (1000 µL L^?1; 24 h) and were stored at 22?±?1 °C and 85?±?5% RH for 9 days. In part 2, fruit harvested at maturity stage 3 were stored at 5, 10, 15, 20, or 25?±?1 °C and 85?±?5% RH for 9 days. During storage, fruit were evaluated daily for decay, mass loss, respiratory activity, and ethylene production, and every 3 days they were evaluated for peel color, pulp firmness, soluble solids content, total titratable acidity, ascorbic acid, total chlorophyll, and total anthocyanins. Fruit treated with 1-MCP exhibited delayed ripening due to lower metabolic activity, as evidenced by delay to softening, reduced mass loss and no decay. Storage at 5 °C prevented ethylene production, mass loss, color changes, and maintained pulp firmness, while did not affect soluble solids content. The results indicated that storage of camu–camu fruit at 5 °C or at 25 °C following application of 900 nL L^?1 1-MCP were effective strategies to delay ripening and maintain fruit quality up to 9 days.     

Induction of secondary somatic embryogenesis in hybrid larch (Larix x leptoeuropaea) as related to ethylene

Induction of secondary somatic embryogenesis was studied with hybridlarch (Larix x leptoeuropaea)cotyledonary somatic embryos obtained after 3, 4, 5 and 6 weeks of culture on amaturation medium supplemented with abscisic acid. Almost all 3-week maturedcotyledonary somatic embryos can develop embryonal masses whereas only 78, 27and 12% of them are able to do so after 4, 5 and 6 weeks of maturation,respectively. During the first week of culture on the induction medium, somaticembryos with high embryogenic potential (i.e. 3-weekmatured) release little ethylene (less than 1.5 nL h^–1g^–1 FW), whereas those which have almost completelylosttheir ability to induce embryonal masses (i.e. 6-weekmatured) produce much more ethylene. Thereafter, ethylene production by bothtypes of embryos is very similar at around 5–6 nLh^–1 g^–1 FW. Enrichment of theatmosphere with ethylene, or addition of 2-chloroethylphosphonic acid(ethephon)or ACC in the induction medium strongly reduced the induction of secondarysomatic embryogenesis. Moreover, inhibitors of ethylene action(AgNO₃and 2,5-norbornadiene) improved the development of embryonal masses fromsomaticembryos, particularly from the 6-week maturated ones. The results obtainedclearly suggest that ethylene is involved in the regulation of somaticembryogenesis in hybrid larch. The possible relationship between somaticembryogenic potential and ethylene biosynthesis by the explants or sensitivityof the latter to ethylene is discussed.     

The effects of ancymidol,abscisic acid,uniconazole and paclobutrazol on somatic embryogenesis of asparagus

Summary The effects of ancymidol, abscisic acid (ABA), uniconazole, and paclobutrazol on asparagus somatic embryogenesis were evaluated. Calli induced from seedlings of genotype G447 were transferred to embryo induction medium (MS plus 3% sucrose, 0.1 mg L^–1 NAA, 0.5 mg L^–1 kinetin and 3% gelrite), with different concentrations of these compounds. After 8 weeks, the recovered bipolar or globular embryos were placed on germination medium (MS plus 6% sucrose, 0.1 mg L^–1 NAA, 0.1 mg L^–1 kinetin, 0.75 mg L^–1 ancymidol, 40 mg L^–1 adenine sulphate dihydrate, 0.17 mg L^–1 sodium phosphate monobasic and 3% gelrite) for conversion to plantlets. Inclusion of ancymidol, ABA, uniconazole and paclobutrazol in the embryo induction medium did not affect the total number of somatic embryos produced relative to the control without these compounds. However, ancymidol, ABA and uniconazole significantly improved embryo development by increasing the production of bipolar embryos 250–750% and decreasing that of globular embryos 8–35% relative to the control. The bipolar embryos produced with any of the four compounds in the embryo induction medium converted to plantlets at rates 700–1100% greater than the control. None of the globular embryos converted to plantlets. Ancymidol (0.75 mg L^–1) and ABA (0.05 mg L^–1) were the most effective treatments; 61 and 46 bipolar embryos g^–1 callus were produced, and 38% and 37% of the bipolar embryos converted to plantlets, respectively. These results indicated that ancymidol, ABA, uniconazole and paclobutrazol significantly enhanced the production of asparagus somatic embryos and their conversion to plantlets, and ancymidol and ABA were more effective than uniconazole and paclobutrazol.Abbreviations Ancymidol a-cyclopropyl-a(4-methoxyphenyi)-5-pyrimidine methanol - NAA 1-naphthaleneacetic acid - Paclobutrazol I-(4-chlorophenyl)-4,4-dimethyl-2(1H-1,2,4-triazol-1-yl)-pentan-3-ol - Uniconazole (E)-(p-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)-1-pentan-3-ol - ABA abscisic acid - GA gibberellic acid     

Growth inhibition of pollen tubes by blocking the aromatic or branched-chain amino acid pathways of biosynthesis

A number of organic molecules that appear to block the ethylene receptor have been discovered recently. For example, on irradiation with visible light, diazocyclopentadiene (DACP), gives rise to some potent but as yet unidentified inhibitor compounds. Some synthetic cyclopropenes have been shown to bind to the ethylene receptor and prevent the physiological action of ethylene for extended periods. Cyclopropene (CP). 1-methylcyclopropene (1-MCP) and 3,3-dimethylcyclopropene (3,3-DMCP) have been shown to prevent ethylene effects in a number of plants. As low a concentration as 0.5 nl l⁻¹ of 1-MCP is sufficient to protect carnation ( Dianthus caryophyllus ) flowers for several days against ethylene, and 0.7 nl l⁻¹ 1-MCP or CP will prevent the ripening of banana ( Musa sapientum ) for 12 days at 24°C. Some plant organs require higher concentrations of these inhibitors. Complete inhibition of ethylene effects in pea seedlings requires treatment with 40 n1 1⁻¹ of 1-MCP. These novel inhibitors appear to be suitable for many commercial applications including extending the vase life of cut flowers and the display life of potted plants. Since 1-MCP apparently is non-toxic at concentrations that are active, it may in future be available for regulating the ripening of fruits and preventing the deleterious effects of ethylene in vegetables.     

Effect of ethylene and 1-methylcyclopropene on chlorophyll catabolism of broccoli florets

Branchlets of broccoli (Brassica oleracea L.) were used to examine ethylene-stimulated chlorophyll catabolism. Branchlets treated with: 1) air (CK); 2) 1 µL·L^–1 1-methylcyclopropene (1-MCP) for 14 hr at 20 °C; 3) 1000 µL·L^–1 ethylene (C₂H₄) for 5 hr at 20 °C; or 4) 1-MCP then C₂H₄, were stored in the dark at 20 °C for up to 3 d. Chlorophyll (Chl) content and branchlet hue angle decreased during the storage period and 1-MCP treatment delayed this change. Chl degradation in broccoli was accelerated by exposure to C₂H₄, especially for Chl a. Prior treatment with 1-MCP prevented degreening stimulated by C₂H₄. Lipoxygenase activity was not altered by any of the treatments, however, 1-MCP with or without ethylene resulted in reduced activity of chlorophyllase (Chlase) and peroxidase (POD). Exposure to C₂H₄ stimulated Chlase activity and extended the duration of high POD activity. Treatment with 1-MCP followed by C₂H₄ resulted in reduced POD activity and delayed the increase in Chlase activity. The results suggest chlorophyll in broccoli can be degraded via the POD – hydrogen peroxide system. Exposure to C₂H₄ enhances activity of Chlase and extends the duration of high POD activity, and these responses may accelerate degreening. Treatment with 1-MCP delays yellowing of broccoli, an effect that may be due to the 1-MCP-induced reduction in POD and Chlase activities.     

Optimization of eicosapentaenoic acid production byPhaeodactylum tricornutumin the flat panel airlift (FPA) reactor

Biomass and eicosapentaenoic acid (EPA) productivities were investigated in a flat panel airlift loop reactor ideally mixed by static mixers. Growth with ammonium, urea and nitrate as nitrogen source were performed at different aeration rates. Cultures grew on ammonium but the decay of pH strongly inhibited biomass increase. On urea biomass productivity reached 2.35 g L^–1d^–1at an aeration rate of 0.66 vvm (24 h light per day, 1000 mol photon m^–2s^–1). Aeration rates between 0.33 vvm and 0.66 vvm and maximal productivities on urea were linearly dependent. Productivity on nitrate never exceeded 1.37 g L^–1d^–1. In the range of maximum productivity photosynthesis efficiency of 10.6% was reached at low irradiance (250 mol photon m^–2s^–1). Photosynthesis efficiency decreased to 4.8% at 1000 mol photon m^–2s^–1. At these high irradiances the flat panel airlift reactor showed a 35% higher volume productivity than the bubble column. At continuous culture conditions the influence of CO₂concentration in the supply air was tested. Highest productivities were reached at 1.25% (v/v) CO₂where the continuous culture yielded 1.04 g L^–1d^–1(16 h light per day, 1000 mol photon m^–2s^–1). The average EPA content amounted to 5.0% of cell dry weight, that resulted in EPA productivities of 52 mg L^–1d^–1(continuous culture, 16 h light per day) or 118 mg L^–1d^–1(batch culture, 24 h light per day).     

Clonal propagation of Enset (Ensete superbum (Roxb.) Cheesman) through shoot tip cultures

Summary The morphogenetic potential of the shoot tip explants ofEnsete superbum (Roxb.) Cheesman, a wild relative of the cultivated bananas, was investigated and an effective clonal propagation method devised. Shoot tip explants grown in modified MS medium containing 1.5 mg l^–1 BAP and 1 mg l^–1 KIN developed corms which on transfer to medium containing 3 mg l^–1 IBA and 1.5 mg l^–1 BAP, regenerated a large number of shoots from the surface of the corm, the origin of which was traced to single hypodermal cells. Shoots were rooted on a half-strength MS medium salts containing 3 mg l^–1 IBA and 0.1 mg l^–1 BAP. The rooted plantlets were hardened and planted in the field where the plants looked normal.     

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.     

Soy-based medium for ethanol production byEscherichia coli KO11

An optimized soy-based medium was developed for ethanol production byEscherichia coli KO11. The medium consists of mineral salts, vitamins, crude enzymatic hydrolysate of soy and fermentable sugar. Ethanol produced after 24 h was used as an endpoint in bioassays to optimize hydrolysate preparation. Although longer fermentation times were required with soy medium than with LB medium, similar final ethanol concentrations were achieved (44–45 g ethanol L^–1 from 100 g glucose L^–1). The cost of materials for soy medium (excluding sugar) was estimated to be $0.003 L^–1 broth, $0.006 L^–1 ethanol.     

Sources and sinks of dissolved organic carbon in a forested swamp catchment

Concentrations of dissolved organic carbon (DOC) were measured in precipitation, throughfall, stemflow, and soil, peat and stream water in a 50 ha catchment with a central 5 ha swamp at Mont St. Hilaire, Quebec. DOC concentrations in precipitation were low (2.0 mg L^–1), but increased in passage through the tree canopies as throughfall (9.1–14.6 mg L^–1) and stemflow (23.1–30.1 mg L^–1). For the period July 1–November 15, 1987, 0.5 g DOC m^–2 was imported as precipitation, and forest canopies contributed a further 1.4–1.7 g m^–2 2 to the soil surface. DOC concentrations were higher (46.0 and 67.6 mg L^–1) in upland soil organic horizons, but decreased with depth because subsoil mineral horizons acted as a major sink of DOC. A laboratory experiment using leaf leachate revealed that subsoil horizons were able to adsorb DOC, with equilibrium DOC concentrations ranging from 3 to 19 mg L^–1. Soil organic carbon appeared to be an important determinant of equilibrium DOC concentrations. The swamp was a major source of DOC, with an overall average DOC concentration of 58.6 mg L^–1 and showed strong spatial and temporal variations related to hydrologic and thermal regimes. During base flow periods, stream DOC concentrations were small (< 3 mg L^–1), dominated by water fed from springs draining upland soils. During high flows, stream DOC concentrations increased through the contribution of DOC-rich water originating in the swamp. Sources, sinks and transport of DOC are thus a function of a complex set of inter-related biotic and abiotic process.     

Fed-batch fermentation for production of Kluyveromyces marxianusFII 510700 cultivated on a lactose-based medium

A strain of Kluyveromyces marxianus was grown in batch culture in lactose-based media at varying initial lactose concentrations (10–60 g L^–1) at 30°C, pH 5.0, dissolved oxygen concentrations greater than 20%. Increasing the concentration of mineral salts three-fold at 40 g L^–1 and 60 g L^–1 initial lactose concentration showed only a small increase in the yield of biomass, from 0.38 g g^–1 to 0.41 g g^–1, indicating that the initial batch cultures were not significantly nutrient- (mineral salts)-limited. A relatively high biomass concentration (105 g L^–1) was obtained in fed-batch culture following extended lactose feeding. An average specific growth rate (0.27 h^–1), biomass yield (0.38 g g^–1) and overall productivity (2.9 g L^–1 h^–1) were obtained for these fed-batch conditions. This fed-batch protocol provides a strategy for achieving relatively high concentrations and productivities of K. marxianus on other lactose-based substrate streams (e.g., whey) from the dairy industry.     

Production of laccase byBotrytis cinerea and fermentation studies with strain F226

After induction, seven strains ofBotrytis cinerea released into the culture broth considerable amounts of laccase in a brief production time. The set-up of a suitable production process was studied with a selected strain in a 10-L fermenter. The optimum fermentation conditions were a 3% inoculum with a high degree of sporulation, a simple medium containing 20 g L^–1 of glucose and 2 g L^–1 of yeast extract at pH 3.5, 2 g L^–1 gallic acid as inducer, added after 2 days of growth, an agitation speed of 300 rpm, an aeration rate of 1.2 vvm and a temperature of 24°C. By optimizing the culture conditions, the enzyme activity reached 28 U ml^–1 in 5 days with a specific activity of 560 U mg^–1 protein. The best procedure to obtain a suitable crude enzyme preparation was concentration of the supernatant medium to 10% of the initial volume by ultrafiltration, followed by a fractional precipitation with ethanol. The optimum pH and temperature for laccase activity were 5.5 and 40°C, respectively, with syringaldazine as the substrate.     

Effect of Nitrate and Aminoethoxyvinylglycine on Cicer Arietinum L. Nodules

The chickpea (Cicer arietinum L.) cv. HC-1 was raised in earthen pots filled with dune sand in screenhouse. At vegetative stage, i.e. 40 – 45 d after sowing, 10, 20 and 40 mM NO₃ ^– was applied through rooting medium. After 24 h of NO₃ ^– treatments an ethylene biosynthesis inhibitor, aminoethoxyvinylglycine (AVG) in concentration 5 M was given. A conspicuous increase in (5 – 9 fold) ethylene evolution in nodules was noticed after NO₃ ^– treatments. This rise was parallel to the increase in 1-aminocyclopropane-1-carboxylic acid (ACC) content and ACC oxidase activity. On the contrary, a sharp decline in ACC content, ACC oxidase activity and ethylene evolution was observed when AVG was given. A decrease of in acetylene reduction assay (ARA) with NO₃ ^– treatments was associated with decline in cytosolic pH (from 6.12 to 5.45), leghemoglobin (Lb) content, accumulation of H₂O₂ and with the loss of membrane integrity. The lipid peroxidation, followed as MDA production and electrolyte leakage increased with NO₃ ^– treatments, however, the level of MDA was brought down in AVG-treated nodules. Results confirm that ethylene might be involved in mechanism by which the functioning of nodules is adversely affected by NO₃ ^–.     

Fluxes of carbohydrate metabolism in ripening bananas

The major fluxes of carbohydrate metabolism were estimated during starch breakdown by ripening bananas (Musa cavendishii Lamb ex Paxton). Hands of bananas, untreated with ethylene, were allowed to ripen in the dark at 21° C. Production of CO₂ and the contents of starch, sucrose, glucose and fructose of intact fruit were determined for a period of 10 d that included the climacteric. The detailed distribution of label was determined after supplying the following to cores of pulp from climacteric fruit: [U-¹⁴C]-, [1-¹⁴C]-, [3,4-¹⁴C]-and [6-¹⁴C]glucose, [U-¹⁴C]glycerol, ¹⁴CO₂. The data obtained were used to estimate the following fluxes, values given as mol hexose · (g FW)^–1 · h^–1 in parenthesis: starch to hexose monophosphates (5.9) and vice versa (0.4); hexose monophosphates to sucrose (7.7); sucrose to hexose (4.7); hexose to hexose monophosphate (3.8); glycolysis (0.5–1.6); triose phosphate to hexose monophosphates (0.14); oxidative pentose-phosphate pathway (0.48); CO₂ fixation in the dark (0.005). These estimates are related to our understanding of carbohydrate metabolism during ripening.We both thank Mr Richard Trethewey for his constructive criticism: S.A.H. thanks the Managers of the Broodbank Fund for a fellowship.