Exposure to alcohol vapours reduces chilling-induced injury of excised cucumber cotyledons, but not of seedlings or excised hypocotyl segments

Cucumber (Cucumis sativus L. cv. Poinsett 76) seedlings withfully expanded cotyledons, and excised cotyledons, first trueleaves, hypocotyl segments and fruit mesocarp discs were exposedto vapours from a series of aqueous alcohol solutions of 0 to320 mM methanol, ethanol, n-propanol, n-butanol, and n-pentanolduring chilling at 2.5C for 5 d. Certain concentrations ofeach alcohol reduced subsequent chilling-induced ion leakagefrom the cotyledons and leaves. Exposure of cotyledons to certainmethanol or ethanol solutions also reduced chilling-inducedethylene production, but not carbon dioxide production. In contrast,exposing cucumber seedlings with fully expanded cotyledons tothe same series of alcohol concentrations that resulted in reducedchilling-induced ion leakage and ethylene production of excisedcotyledons actually increased chilling injury of the seedlings.The hypocotyl region directly below the cotyledons was the siteof chilling-induced injury and contained the most chilling-sensitivehypocotyl tissue. Exposing hypocotyl segments excised from thissensitive region to alcohol solutions did not significantlyreduce chilling-inducedion leakage. Exposing excised cucumbercotyledons or hypocotyl segments to equivalent osmotic nonvolatilesolutions of mannitol and glycerol at 2.5C or to alcohol solutionsat 12.5C had no significant effect on the rate of ion leakage.For the series of alcohols used, the relationship between thelog of the alcohol concentration that minimized chilling-inducedion leakage from cucumber cotyledon discs held at 2.5C for5 d and the log of the partition coefficient of the alcoholinto olive oil or the log of the molecular weight of the alcoholswas highly significant. The same concentrations of alcoholsthat reduced chilling-inducedion leakage also reduced stomatalaperture as measured as decreased porosity of excised cotyledons.The correlation between reduced chilling injury and stomatalconductance of cotyledons exposed to a series of ethanol solutionswas highly significant. It appears that alcohols may reducechilling injury of cucumber cotyledons by inducing stomata closure.Sufficient endogenously synthesized ethanol accumulated in discsheld in N2 at 10C for 1 d to confer tolerance to chilling at2.5C for 5 d. Key words: Anaerobic, Cucumis sativus, ethanol, ion leakage, stomatal conductance     

Prior temperature exposure affects subsequent chilling sensitivity

The chilling sensitivity of small discs or segments of tissue excised from chillingsensitive species was significantly altered by prior temperature exposure subsequent to holding the tissue at chilling temperatures as measured by a number of physiological processes sensitive to chilling. This temperature conditioning was reversible by an additional temperature exposure before chilling, and mature-green and red-ripe tomato tissue exhibit similar chilling sensitivities. Exposing pericarp discs excised from tomato fruit (Lycopersicon esculentum Mill. cv. Castelmart), a chilling-sensitive species, to temperatures from 0 to 37°C for 6 h before chilling the discs at 2.5°C for 4 days significantly altered the rate of ion leakage from the discs, but had no effect on the rate of ion leakage before chilling and only a minimal effect on discs held at a non-chilling temperature of 12°C. Exposing chillingsensitive tissue to temperatures below that required to induce heat-shock proteins but above 20°C significantly increased chilling sensitivity as compared to tissue exposed to temperatures between 10 and 20°C. Rates of ion leakage after 4 days of chilling at 2.5°C were higher from fruit and vegetative tissue of chilling-sensitive species (Cucumis sativus L. cv. Poinsett 76, and Cucurbita pepo L. cv. Young Beauty) that were previously exposed for 6 h to 32°C than from similar tissue exposed to 12°C. Exposure to 32 and 12°C had no effect on the rate of ion leakage from fruit tissue of chilling tolerant species (Malus domestica Borkh. cv. Golden Delicious, Pyrus communis L. cv. Bartlett). Ethylene and CO₂ production were higher and lycopene synthesis was lower in chilled tomato pericarp discs that were previously exposed for 6 h to 32°C than the values from tissue exposed to 12°C for 6 h before chilling. Increased chilling sensitivity induced by a 6 h exposure to 32°C could be reversed by subsequent exposure to 12°C for 6 h.     

The Effect of Exogenous ABA on the Resistance to the Chilling Stress in Cucumber Seedlings

In the cucumber seedlings pretreated with ABA under light and dark or additional photosynthetic inhibitor (DCMU) before chilling, the effect and the regulative role of ABA on the chilling resistance of cucumber seedlings were investigated. After the excised cotyledonary discs were floated on 10-6 mol/l ABA solution for 24 h and the seedlings were sprayed by 10-4 mol/1 ABA in light, it has been found that ABA has an effect of the protection against low temperature injury in cucumber seedlings. The results showed that the leakage of electrolytes in cucumber cotyledory discs was decreased. The content of glutathione and the accumulation of MDA content in cotyledon of cucumber seedlings were decreased, and decline of the photosynthesis of the leaves or the quenching of chlorophyll fluorescence were slowed, thus the rate of survival in cucumber seedlings was raised. The effect of these regulation was able to be limited by dark or DCMU.     

外源ABA对黄瓜幼苗抗低温胁迫的作用

在光照下对黄瓜(Cucuntis sativus L.)离体子叶圆片和幼苗分别使用10~(-6)mol/l 与10~(-4)mol/l ABA 处理,有防御低温伤害的作用,表现为降低子叶圆片的电解质渗漏,阻抑幼苗子叶的谷胱甘肽含量的下降和膜脂过氧化产物丙二醛(MDA)的累积,减缓叶片的光合作用的下降和叶绿素荧光的猝灭,提高幼苗的存活率。但这种作用可被黑暗和光合抑制剂(DCMU)所限制。     

PP333对黄瓜幼苗素质及抗冷能力的影响

研究了ＰＰ３３３对黄瓜幼苗素质和抗冷能力的影响,结果显示ＰＰ３３３处理降低了幼苗高度和子叶面积,但却促进了根系生长、根冠比值和叶绿素含量显增加,明显表现壮苗效果。ＰＰ３３３能有效降低黄瓜幼苗在低温下的相对电导率,维持较高的ＳＯＤ活性和Ｖｃ、ＧＳＨ含量,因而提高了幼苗的抗冷能力。     

黄瓜的冷害及耐冷性

随着蔬菜反季节栽培面积的不断扩大,如何提高黄瓜(Cucumis sativus L.)耐冷性已成为选育新品种的研究重点。系统地综述近几年黄瓜耐冷性的鉴定、获得途径、冷害机理以及遗传和分子遗传学等方面的研究,以促进对黄瓜冷害机制的研究, 加速耐冷品种的培育。耐冷性鉴定时要从耐冷指数、低温发芽能力、MDA (丙二醛)含量和电解质渗漏率等几个方面综合鉴定。耐冷性的获得途径主要有冷驯化、激素处理、热激处理和培育耐低温品种,最重要的途径是耐冷品种选育。黄瓜冷害机理包括细胞膜的流动性降低及透性增加,光合作用被抑制,根系吸收减弱,可溶性糖含量减少,淀粉粒积累增加,微管的稳定性受到破坏等。黄瓜低温发芽能力由非加性基因决定,而幼苗时期主要由加性基因控制。黄瓜 耐冷的分子遗传学研究进展缓慢,目前已克隆出在低温锻炼中特异表达的功能未知的基因CCR18。今后还应研究黄瓜低温胁迫时的信号转导系统,以进一步揭示黄瓜的冷害机理;利用野生资源的抗逆性状,拓宽栽培黄瓜的遗传基础,选育适于保护地栽培的耐低温品种。     

黄瓜的冷害及耐冷性

随着蔬菜反季节栽培面积的不断扩大,如何提高黄瓜(Cucumis sativus L.)耐冷性已成为选育新品种的研究重点.系统地综述近几年黄瓜耐冷性的鉴定、获得途径、冷害机理以及遗传和分子遗传学等方面的研究,以促进对黄瓜冷害机制的研究,加速耐冷品种的培育.耐冷性鉴定时要从耐冷指数、低温发芽能力、MDA(丙二醛)含量和电解质渗漏率等几个方面综合鉴定.耐冷性的获得途径主要有冷驯化、激素处理、热激处理和培育耐低温品种,最重要的途径是耐冷品种选育.黄瓜冷害机理包括细胞膜的流动性降低及透性增加,光合作用被抑制,根系吸收减弱,可溶性糖含量减少,淀粉粒积累增加,微管的稳定性受到破坏等.黄瓜低温发芽能力由非加性基因决定,而幼苗时期主要由加性基因控制.黄瓜耐冷的分子遗传学研究进展缓慢,目前已克隆出在低温锻炼中特异表达的功能未知的基因CCRl8.今后还应研究黄瓜低温胁迫时的信号转导系统,以进一步揭示黄瓜的冷害机理;利用野生资源的抗逆性状,拓宽栽培黄瓜的遗传基础,选育适于保护地栽培的耐低温品种.     

Lipid-protein interactions in thylakoid membranes of chilling-resistant and -sensitive plants studied by spin label electron spin resonance spectroscopy

Lipid-protein interactions in thylakoid membranes from lettuce, pea, tomato, and cucumber have been studied using spin-labeled analogues of the thylakoid membrane lipid components, monogalactosyl diglyceride and phosphatidylglycerol. The electron spin resonance spectra of the spin-labeled lipids all consist of two components, one corresponding to the fluid lipid environment in the membranes and the other to the motionally restricted lipids interacting with the integral membrane proteins. Comparison of the spectra from the same spin label in thylakoid membranes from different plants shows that the overall lipid fluidity in the membranes decreases with chilling sensitivity. Spectral subtraction has been used to quantitate the fraction of the membrane lipids in contact with integral membrane proteins. Thylakoid membranes of cucumber, a typical chilling-sensitive plant, have been found to have a higher proportion of motionally restricted lipids and a different lipid selectivity for lipid-protein interaction, as compared with those of pea, a typical chilling-resistant plant. This correlation with chilling sensitivity holds generally for the different plants studied. It seems likely that the chilling sensitivity in thylakoid membranes is not determined by lipid fluidity alone, but also by the lipid-protein interactions which could affect protein function in a more direct manner.     

Cellular expansion at low temperature as a cause of membrane lesions

Rates of solute leakage from excised discs of cucumber (Cucumis sativus L. cv Straight Eight) cotyledons were altered by temperature during plasmolysis in the manner of a simple diffusion phenomenon; the log of the leakage rate increased in proportion to the temperature. During deplasmolysis, however, leakage rates responded to temperature with a very different pattern: chilling conditions (below about 20°C) caused large increases in leakage rates, indicating disruption of membrane integrity in the tissues. The time course of restoration of normal leakage rates after deplasmolysis/chilling damage indicated a rapid repair of the lesions. A similar sensitivity to low temperatures was found during rehydration after leaf desiccation, with low temperatures again causing high leakage rates. It is suggested that low temperatures interfere with membrane expansion, possibly by lowering elasticity and hindering the incorporation of lipid material into the expanding membrane. The expansion of tissues at low temperatures may cause lesions in cellular membranes, contributing to chilling injury.     

Chilling injury in cotton (Gossypium hirsutum L.) Prevention by abscisic acid

Cotton (Gossypium hirsutum L.) intact seedlings and isolatedcotyledonary discs were exposed to chilling (4?C) under humidconditions which prevented dehydration. The damage resultingfrom chilling was estimated by means of electrolyte leakageand survival in whole seedlings and by the electrolyte leakageand necrotic areas in isolated cotyledonary discs. Also, theeffect of chilling on membrane phospholipids and cellular reducedglutathione was determined. Within the first two and three daysof chilling, there was a marked reduction in the reduced glutathioneand membrane phospholipid levels without electrolyte loss andnecrosis. This reduction was completely prevented by pretreatmentwith abscisic acid. Prolonging the chilling period resultedin decreased survival in whole seedlings and in progressiveincrease in electrolyte leakage and necrosis in isolated cotyledonarydiscs. Pretreatment with abscisic acid prior to chilling almostcompletely prevented this chilling injury when exposure to 4?Cwas less than 5 days. Even with longer chilling periods, theabscisic acid pretreatment greatly reduced the damage. ³Incumbent of the Seagram Chair in Plant Science. (Received July 21, 1979; )     

Protective Role of Thidiazuron Treatment on Cucumber Seedlings Exposed to Heavy Metals and Chilling

The action of thidiazuron, a synthetic growth regulator, was studied on 7-day-old cucumber seedlings (Cucumis sativus L., cv. Monastyrskii) exposed to chilling and sublethal concentrations of lead and copper ions. The extent of injury was assessed from the electrolyte leakage from cotyledonary leaves into distilled water. Separate application of each stress factor induced an increase in membrane permeability; however, their combined application caused a weaker response. A preliminary treatment of seedlings with thidiazuron fully or partly prevented the stress-induced stimulation of electrolyte leakage from cotyledon segments. It is concluded that thidiazuron elevates plant resistance to adverse environments.     

Contribution of Oxidative Stress to the Development of Cold-Induced Damage to Leaves of Chilling-Sensitive Plants: 3. Injury of Cell Membranes by Chilling Temperatures

Changes in permeability of cell membranes (judged from electrolyte leakage) were examined on leaves of 7- to 11-day-old seedlings of maize (Zea mays L.), cucumber (Cucumis sativus L.), millet (Panicum miliaceum L.), and on etiolated shoots of potato (Solanum tuberosum L.) immediately after cooling plants for 1–24 h at 2°C and one day after a 24-h chilling treatment. A gradually increasing leakage of ions from the cells was observed upon prolongation of chilling exposure, with the maximum attained by the end of 24-h chilling treatment. The leakage of electrolyte was slightly reduced in the post-treatment period but it was still higher than the electrolyte leakage from the control samples (untreated plants). The cold treatment of chilling-sensitive plants (but not of potato) revealed a positive correlation between the rates of lipid peroxidation, indicative of chilling injury, and the electrolyte efflux (r = 0.61–0.96). The evaluation of plant susceptibility to injury showed that millet and potato plants recovered from the chilling damage in 24 h after the treatment, whereas maize and cucumber plants did not show such a recovery.     

The Effect of Low Temperature on Ethylene Production by Leaf Tissue of Phaseolus vulgaris L.

When leaf discs cut from primary leaves of Phaseolus vulgarisL. cv. Masterpiece plants grown at 25°C were incubated attemperatures below 25 °C, basal and wound ethylene productioncontinued at reduced rates. In both cases detectable levelsof ethylene were produced at 25 °C. When the rates of ethyleneproduction were plotted according to the Arrhenius equationa marked discontinuity was found at 11.4 °C which is consistentwith a membrane phase-transition at the critical chilling temperatureof the plant. Activation energies for the rate-limiting enzymereaction in ethylene production above and below the criticaltemperature have been calculated and the data interpreted asindicating the involvement of membrane-bound enzyme systemsin the biosynthesis of basal and wound ethylene. ethylene, temperature, Arrhenius plot, activation energy, Phaseolus vulgaris L., bean     

General and local anaesthetics perturb the fusion of phospholipid vesicles

The effects of general and local anaesthetics on Ca2+-induced fusion of negatively charged lipid vesicles have been investigated. Vesicles composed of phosphatidylcholine and phosphatidic acid (2:1 molar ratio) were induced to fuse using 5 mM free Ca2+. Fusion, assessed by an increase in size using gel filtration techniques and confirmed by electron microscopy, displayed a dependence on Ca2+ and Mg2+ concentration and on temperature. The inhalational anaesthetics halothane, methoxyflurane and diethyl ether enhanced fusion as did the uncharged local anaesthetic benzocaine. In contrast, the charged local anaesthetics lignocaine and bupivacaine inhibited the fusion process. It is suggested that the enhancement observed with the inhalational anaesthetics and benzocaine was mediated by an effect on lipid fluidity and the inhibition observed with the charged tertiary amine anaesthetics was due to an antagonism towards Ca2+.     

Impact of postharvest exogenous γ-aminobutyric acid treatment on cucumber fruit in response to chilling tolerance

Low-temperature storage is generally used to extend postharvest lifetime and to inhibit decay of cucumber fruit, but it also enhances the intensity of chilling injury. The capability of γ-aminobutyric acid to enhance antioxidant enzyme activities and reduce chilling injury was studied in cucumber (Cucumis sativus L.) fruit stored at 1 °C for 5 weeks. The purpose of this study was to define if the GABA-induced modification in antioxidant system and phospholipase activity is linked to the reduced chilling injury in cold-stored cucumber fruit. Alleviation of chilling injury by GABA treatment was related to increased content of proline, endogenous GABA and enhanced activities of CAT and SOD, together with reduced activities of PLC, PLD and LOX. We suggest that PLC, LOX and PLD are associated with chilling injury initiation by involvement in a signaling pathway and membrane deterioration. Therefore the results obtained in this study suggest GABA’s potential for postharvest applications for reducing chilling injury symptom in cucumber fruit.     

The Decreased Cold-Resistance of Chilling-Sensitive Plants Is Related to Suppressed CO2 Assimilation in Leaves and Sugar Accumulation in Roots

Tomato (Lycopersicon esculentum L., cv. Sibirskii skorospelyi) and cucumber (Cucumis sativus L., cv. Konkurent) plants were grown in a soil culture in a greenhouse at an average daily temperature of 20°C and ambient illumination until the development of five and eight true leaves, respectively. During the subsequent three days, some plants were kept in a climatic chamber at 6°C in the light, whereas other plants remained in a greenhouse (control). The cold-resistance of cucumber leaves and roots, as assayed from the electrolyte leakage, was reduced after cold exposure stronger than cold-resistance of tomato organs. The ratio photosynthesis/dark respiration was lower in cucumber than in tomato leaves at all measurement temperatures. The concentrations of sugars (sucrose + glucose + fructose) increased in chilled tomato roots but decreased in cucumber roots. Cold exposure changed the activities of various invertase forms (soluble and insoluble acidic and alkaline invertases). The total invertase activity and the ratio of mono- to disaccharides increased. The lower cucumber cold-resistance is related to the higher sensitivity of its photosynthetic apparatus to chilling and, as a consequence, insufficient root supply with sugars.     

Effect of temperature and ripening stages on membrane integrity of fresh-cut tomatoes

The shelf-life of fresh-cut tomatoes mainly depends on loss of tissue integrity and firmness that occurs also in intact fruits after long-term cold storage due to chilling injury. Round-fruit tomatoes (Solanum lycopersicum L.) cv. Jama were stored in 1.1-L plastic (polyethylene) fresh-cut produce containers as 10.0-mm-thick tomato slices and as intact tomatoes at 4 ± 0.5 °C. The aim of this work was to study the loss of membrane integrity and biochemical processes involved in membrane disruption. Electrolyte leakage and lipid peroxidation were studied at different stages of maturity: mature green, pink (PK), fully ripe and two different storage temperatures: 4 and 15 °C. The tomato slices of PK stage stored at 4 °C did not show changes for both parameters, while significant increase in membrane leakage and lipid peroxidation was observed at 15 °C, especially after 24 h of storage. The enzymes showed a simultaneous increase in their activities with a rise in electrolyte leakage and lipid peroxidation after 7 days of storage. Finally, phospholipase C (PLC) and phospholipase D (PLD) were investigated for intact fruit and tomato slices stored at 4 °C. The PLC had higher activity compared with PLD. In conclusion, the loss of membrane integrity in fresh-cut tomatoes is mainly affected by ripening stages, storage temperature and duration. The wounds enhance the PLC and PLD activities and they play a role late during storage.     

Changes in Membrane Fluidity and Protein Composition during Release of Cucumber Seeds from Dormancy by a Higher Temperature Shift

Dormancy of seeds of cucumber (Cucumis sativus L.) was inducedby imbibing in -1.8 MPa polyethylene glycol 6000 (PEG) solutionand pulsing with far red light for 15 min prior to washing anddrying. When re-imbibed with water at 20 °C, dormancy wasbroken by raising the temperature to 30 °C for 6 h. Thistreatment was also effective when -0.9 MPa PEG was present duringre-imbibition and high temperature. Seeds with broken dormancywere found to germinate in water over a smaller temperaturerange than seeds in which dormancy had not been induced. Whenthe duration of the temperature shift to 30 °C was varied,germination percentage increased from 7 to 60% after 6 h, butlonger exposures up to 12 h had no further promoting effect.The time course of germination after transfer to water following6 h at 30 °C in PEG showed piercing of the perisperm-endospermenvelope after 9–12 h and radicle protusion after 12–15h. If PEG was retained after high temperature treatment no visiblegermination was observed. Thus, to study membrane fluidity andthe protein content associated with germination, seeds weresampled 9 h after high temperature treatment. To study the germinablebut not germinating state, seed held in PEG for 9 h rather thanin water was used. Dormant seed was sampled before the hightemperature treatment. Membrane fluidity was assessed usingfluorescence polarization of membrane fractions treated withDPH (1,6-diphenyl-1,3,5-hexatriene) or its derivatives. Membraneproteins were compared using one-dimensional SDS-PAGE electrophoresis.Intracellular membrane fluidity was not increased in the transitionfrom the dormant to germinable state, but did increase in thetransition to germination. There were no detected changes inintracellular membrane proteins during either transition. Inplasma membrane fractions, fluidity increased during both transitions,while a marked increase in 21, 18 and 17 kD proteins was observedin the transition from germinable to germinating state. Thusmodification of plasma membrane fluidity rather than changesin protein profile is associated with the high temperature releaseof cucumber seeds from dormancy. Copyright 2000 Annals of BotanyCompany