Ethylene production by suspension-cultured pear fruit cells as related to their senescence

Suspension-cultured pear fruit cells produce low levels of ethylene during growth and division in auxin containing medium. When deprived of auxin, division gradually ceases and ethylene production falls to barely discernible levels. However, notable ethylene production can now be induced by indoleacetic acid, CuCl₂, or 1-aminocyclopropane-1-carboxylic acid. If the auxin-deprived cells are transferred to `aging' medium that lacks auxin but contains 0.4 molar mannitol, inducible ethylene production increases several-fold reaching levels of 40 to 60 nanoliters/10⁶ cells per hour. Maximum inducible ethylene productivity is attained at varying times (1-6 days) after transfer to aging medium and appears to be temporally related to cell survival, i.e. the time of subsequent cell death. It is argued that auxin depletion initiates senescence which, in turn, leads to a transient increase in inducible ethylene production and eventual death. The limitations and potentials of the suspension-cultured pear cells as a system for the study of cellular senescence are discussed.     

Ethylene production by growing and senescing pear fruit cell suspensions in response to gibberellin

A pear (Pyrus communis L. cv Passe Crassane) cell suspension was used as a model system to study the influence of gibberellin on processes related to fruit ripening. Growth of the cell cultures was inhibited and their loss of viability was accelerated when 0.5 millimolar gibberllic acid (GA₃) was added to suspensions at two stages of cell development, namely, growth and quiescence. Cell respiration rate was unaffected up to 2 millimolar GA₃ but ethylene production, both basal and 1-aminocyclopropane-1-carboxylic acid-induced, was inhibited at all stages of cell development. However, the degree of inhibition decreased as the cell cultures aged. The site of ethylene inhibition by GA₃ appeared to be related to the ethylene-forming enzyme. The coincident acceleration of cell senescence and inhibition of ethylene production indicate that the pear cell suspension cannot serve as an analogous model for studying the mode of action of gibberellin in delaying ripening and senescence of fruits in its entirety, although certain specific effects might be relevant.     

Effect of high temperature on calcium uptake by suspension-cultured pear fruit cells

Uptake of Ca²⁺ by suspension-cultured pear (Pyrus communis L. cv Passe Crassane) cells and protoplasts was significantly enhanced by exposure to 38°C compared to 25°C. The increased uptake was specific for Ca²⁺ and was not due to cell wall binding. Tissues pretreated at 38°C showed increased uptake even upon return to 25°C. Treatment with carbonylcyanide m-chlorophenylhydrazone, salicylhydroxamic acid + KCN, or arsenite also increased Ca²⁺ content of cells. Results are discussed with regard to membrane permeability changes, the cellular control of Ca²⁺, and heat treatments used to inhibit softening of fruit during postharvest storage.     

Vacuolar yield and alteration with aging of suspension-cultured cells from pear fruit

Vacuoles were isolated from suspension-cultured pear fruit (Pyrus communis L. Passe Crassane) cells of various ages. Yield of vacuoles averaged 17% of protoplasts throughout all the experimental stages. The isolated vacuoles were progressively larger and more likely to stain with neutral red as cells advanced from division to growth and incipient senescence. A relationship between aging and vacuolar or membrane function was implicit in the size and staining characteristics of isolated vacuoles.     

Ethylene and ethane production in response to salinity stress

Abstract Ethylene and ethane production in mung bean hypocotyl sections were evaluated as possible indicators of stress due to contact with four salts that are common in natural sites. Ethylene production decreased with increasing concentrations of applied NaCl and KCl. When CaCl₂ was applied, the ethylene evolution was greater. However, when MgCl₂ was applied, ethylene evolution remained high then decreased and at higher salt concentrations again showed an increase. NaCl (up to 0.1 kmol m^?1) and KCl (up to 0.5 kmol m^?3) caused a concentration-dependent increase in ethane production. The ethane production with CaCl₂ was the lowest among the salts tested and only a minute increase was noticed with the increase of concentration from 0.01 to 1 kmol m^?3. Ethane production showed a distinct maximum at 0.2 kmol m^?3 MgCl₂. The introduction of 0.01 kmol m^?3 CaCl₂, as well as anaerobic conditions obtained by purging vials with N₂, eliminated that high ethane production. Respiratory activity of the mung bean hypocotyl sections in MgCl₂ concentrations from 0 to 0.5 kmol m^?3 was correlated with ethane but not with ethylene production. The ethane/ethylene ratio showed three patterns for the four salts tested.     

Conservation of the alternative oxidase and enhancement of cyanide-resistant respiration in suspension-cultured pear fruit cells by inhibitors of macromolecular synthesis

The contribution of the alternative pathway to the respiration of suspension-cultured pear ( Pyrus communis cv. Passa Crasanne) cells was enhanced, often severalfold, within 2 to 4 days following the addition of cycloheximide, actinomycin D, or 2-(4-methyl-2,6-dinitroanalino)- N -methyl propionamide (D-MDMP). Concomitant inhibition of cellular protein synthesis by cycloheximide and actinomycin D was transient and incomplete. However, inhibition by D-MDMP was virtually complete (>97%) and persisted over several days. [³⁵S]-labelling and polyacrylamide gel separation indicated that cycloheximide precluded the appearance of discernable new proteins in mitochondria. Probes with monoclonal antibodies revealed a conservation of alternative oxidase protein levels in the mitochondria of inhibitor-treated cells. The data, appraised within the complexities of cell-culture dynamics, lead to the conclusion that the observed increases in capacity for cyanide-resistant respiration are the consequence, likely indirect, of inhibited protein synthesis with resultant retention and activation of constitutive alternative oxidase.     

Conservation of the alternative oxidase and enhancement of cyanide-resistant respiration in suspension-cultured pear fruit cells by inhibitors of macromolecular synthesis

The contribution of the alternative pathway to the respiration of suspension-cultured pear ( Pyrus communis cv. Passa Crasanne) cells was enhanced, often severalfold, within 2 to 4 days following the addition of cycloheximide, actinomycin D, or 2-(4-methyl-2,6-dinitroanalino)- N -methyl propionamide (D-MDMP). Concomitant inhibition of cellular protein synthesis by cycloheximide and actinomycin D was transient and incomplete. However, inhibition by D-MDMP was virtually complete (>97%) and persisted over several days. [³⁵S]-labelling and polyacrylamide gel separation indicated that cycloheximide precluded the appearance of discernable new proteins in mitochondria. Probes with monoclonal antibodies revealed a conservation of alternative oxidase protein levels in the mitochondria of inhibitor-treated cells. The data, appraised within the complexities of cell-culture dynamics, lead to the conclusion that the observed increases in capacity for cyanide-resistant respiration are the consequence, likely indirect, of inhibited protein synthesis with resultant retention and activation of constitutive alternative oxidase.     

Ethylene production by wounded tissue of citrus fruit

Segments cut from young immature fruits and albedo discs excisedfrom both immature and mature fruits of Satsuma mandarin ormature fruits of Natsudaidai produced much ethylene during incubationat 26?C in the dark. Ethylene formation was markedly acceleratedby the application of abscisic acid but markedly delayed by3,5-dibromo-4-hydroxybenzoic acid. Both the stimulation andretardation decreased greatly during the course of incubation.Both compounds seem to be associated with the early stages ofethylene formation by wounded citrus fruit tissues. Albedo discs were fed ¹⁴C methionine labeled at one of threedifferent positions. Of the three radioanalogs (carbon-2, carbon-3and methyl carbon), the label at the 3 position was preferentiallyincorporated into ethylene. This agrees with the former observationthat ethylene is derived from carbon-3 and -4 of methionine.Incorporation of label into ethylene from L-[3-¹⁴C] methioninewas strongly inhibited by L-canaline, L-ethionine, 2,4-dinitrophenoland cycloheximide. Ethylene evolution was also strongly inhibitedby 2,4-dinitrophenol, KCN, NaN₃ and cycloheximide, but lesscompletely by L-canaline and L-ethionine. These results supportthe view that ATP and pyridoxal phosphate are utilized in activationof methionine to form ethylene. (Received October 25, 1977; )     

Ethylene production by plant cell cultures: the effect of auxins, abscisic Acid, and kinetin on ethylene production in suspension cultures of rose and ruta cells

Cell suspension cultures of Ruta graveolens (rue) and Rosa sp. produce ethylene. Both cultures grow at a high rate in hormone-free media. The rose cells are undifferentiated while the Ruta cells differentiate and form shoots after extended culture in hormone-free medium. Addition of 2,4-dichlorophenoxyacetic acid stimulated ethylene production in Ruta cells but not in rose cells. Abscisic acid (ABA) inhibited growth and ethylene production in rose, but only ethylene production in Ruta cells. Addition of kinetin reversed the inhibition by abscisic acid in the rose cells but not in the Ruta cells. The results suggested a distinct physiological difference between the two cultures. The Ruta cells responded to the growth regulators in a manner similar to whole plants.     

Ethylene production in relation to the initiation of respiratory climacteric in fruit

Respiration of cherimoya fruit shows two peaks of CO₂ production.The IEC is less than 0.05 ppm until the midpoint of the firstrespiratory rise. The fruit softens, develops pleasant aromaand flavor, and is considered ripe at the beginning of the secondrespiratory rise. Ripening and CO₂ production in cherimoya andavocado fruit are initiated with no increase of internal ethyleneand at ethylene levels which do not normally induce ripeningin fruit. The ratio of the internal concentration of ethylene to its productionrate varied with the stage of the climacteric and was less thanone except for the very early stage in cherimoyas. ¹Present address: Dept. of Plant Sciences, Kasetsart Univ.,Bangkok, Thailand. (Received February 6, 1975; )     

Ethanol production and toxicity in suspension-cultured carrot cells and embryos

The process of carrot (Daucus carota L.) somatic embryogenesis is highly sensitive to exogenously added ethanol, since 5 mM ethanol inhibits this process by 50%, whereas the growth of proliferating carrot cells is inhibited to the same extent by 20 mM ethanol. This is consistent with the fact that proliferating cultures produce ethanol and release it into the medium at concentrations up to 20 mM, whereas embryogenic culture medium contains less than 1 mM ethanol. Data are presented showing the influence of cell density and 2,4-dichlorophenoxyacetic acid on ethanol production and on the presence of an alcohol-dehydrogenase (EC 1.1.1.1.) inactivator in carrot embryos.Abbreviations ADH alcohol dehydrogenase - 6-BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DTT dithiothreitol - FW fresh weight     

Ethylene and carbon dioxide as mediators in the response of the bean hypocotyl hook to light and auxins

Summary Ethylene inhibits hook opening in the bean hypocotyl and at high concentrations induces closure of the hook. Indoleacetic acid and 2,4-dichlorophenoxyacetic acid, whose inhibitory effect on hook opening resembles that of ethylene, stimulate ethylene production from the hook tissue, and this ethylene production is physiologically active in inhibiting hook opening. It is concluded that the inhibition of opening by auxin is due at least in a major part to auxin-induced ethylene production by the hook tissue.Carbon dioxide promotes hook opening, apparently by antagonizing the action of endogenous ethylene. The concentration of respiratory CO₂ in the internal gas space of the hook tissue is high enough to play a role in the regulation of hook opening.Red light causes a decrease in ethylene production and an increase in CO₂ evolution from the hook tissue. These effects are partially reversible by far-red light. It is concluded that both ethylene and CO₂ serve as natural growth regulators which mediate the hypocotyl hook-opening response to light in bean seedlings.     

Changes in the composition of exocellular proteins of suspension-cultured Lupinus albus cells in response to fungal elicitors or CuCl2

Among many aspects of plant defence responses to pathogenicinfection are changes in the composition of the exocellularmatrix. To study potential defences in white lupin (Lupinusalbus L.), suspension-cultured cells were treated for 24 h withone of three different elicitors: CuCl₂, and two fungal elicitorpreparations from purified cell walls of yeast and ColletotrichumIindemuthianum. Two subsets of exocellular proteins: ionicallyboundwall proteins and proteins secreted into culture medium, wereisolated, and their patterns compared following electrophoreticseparation. Only a few proteins were observed in culture filtrateswith dominating bands at 27, 33, and 42 kDa. About 30 proteinswere observed in cell wall extracts. Changes in protein intensitiesevoked by elicitor treatments depended on the type of elicitorused, the age and composition of lupin cell culture, and concentrationof applied fungal elicitor. Based on these observations, tenproteins were chosen for N-terminal sequencing, and sequences5–30 amino acids long for nine proteins were obtained.Three of the major proteins sequenced were identified as acidicexocellular chitinase, polygalacturonaseinhibiting protein,and germin/oxalate oxidase. Key words: Lupinus aibus, defence response, exocellular proteins, elicitation, N-terminal amino acid sequencing, suspension culture     

Boron is required for the stimulation of the ferricyanide-induced proton release by auxins in suspension-cultured cells of Daucus carota and Lycopersicon esculentum

Boron deficiency reduces the ferricyanide-induced net proton release of suspension-cultured carrot ( Daucus carota L.) and tomato ( Lycopersicon esculentum Mill.) cells by more than 50%. This effect is reversed within 60 to 90 min by the addition of B. Vanadate (400 μ M ) completely suppresses the proton release, indicating an ATPasedriven process. The differences between B treatments do not appear when auxins are omitted from the experimental solution, but can be observed within less than 30 min after the addition of auxin to auxin-deficient cell cultures. This suggests, that an adequate supply of B is required for the auxin action to take place. The results are discussed with respect to the primary functions of B in membranes and transport processes, and its possible influence on auxin-induced metabolic events.     

Oxidative turnover of auxins in relation to the onset of ripening in bartlett pear

Pears (Pyrus communis var. Bartlett) kept in 100% O(2) showed an increase in the rate of softening, chlorophyll degradation, and ethylene evolution. The O(2) application could overcome, in part, the inhibition of ripening by 1 mm indoleacetic acid. Ripening of pears was also accelerated by the application of solutions containing indoleacetic acid-oxidation products, obtained by an overnight incubation of 0.1 and 1 mm indoleacetic acid with traces of H(2)O(2) and horseradish peroxidase. Although both treatments stimulated ethylene evolution, the promotion of ripening could not be attributed to an indirect ethylene effect. Indoleacetic acid oxidation products obtained in vivo by high O(2) tensions or in vitro by enzymatic degradation may function in the promotion of fruit ripening and the synthesis of ethylene.     

Ethylene production by loblolly pine seedlings associated with water stress

Effects of water stress on production of ethylene and its precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), by loblolly pine ( Pinus taeda L.) seedlings from a Texas drought-hardy and a Virginia Coastal Plain source were investigated. Ethylene production rates in needles from the Virgnia source increased slightly with initial stress (-1.3 MPa), declined until water potential reached -1.6 MPa and then increased sharply at -2.5 MPa. The ethylene production rates in needles from the Texas also increased slightly with initial stress, then decreased with decreasing water potential. Ethylene production by root tissue was two to three times higher than needle tissue and decreased with decreasing water potential. ACC concentrations in needles of both seed sources decreased as water potential began decreasing. Below -1.4 MPa, ACC levels started increasing (Texas source) or remained constant until -2.8 MPa (Virginia source) at which time its level increased three-fold. Mean ACC levels in root tissue [122 nmol (g dry weight)⁻¹] were slightly higher than the mean levels in the needle tissue [92 nmol (g dry weight) ⁻¹]; roots apparently were more efficient in converting it to ethylene since ethylene production was two to three times higher than needle tissue. The modulation of ethylene synthesis by ACC synthase and ethyleneforming enzyme appeared to be influenced by stress level, organ and seed source.     

Contamination of pear concentrate by Alicyclobacillus from recirculating flume water during fruit concentrate production

Alicyclobacillus and viable aerobic counts were monitored at nine different production stages of pear concentrate, with the functioning of either a recirculating or a one-pass flume water system. Significantly (P < 0.05) higher levels of Alicyclobacillus were detected in the final pasteurized product (102–104°C for 90 s) when the recirculating flume water system was operational. An average of 1.19 Log₁₀ c.f.u. ml⁻¹ vegetative cells and 1.35 Log₁₀ c.f.u. ml⁻¹ endospores were recovered, whereas 0 c.f.u. ml⁻¹ vegetative cells and endospores were detected when the one-pass flume water system was operational. Alicyclobacillus levels did not differ significantly (P > 0.05) in condensate water during the functioning of the two flume water systems, with 1.81 Log₁₀ c.f.u. ml⁻¹ vegetative cells and 1.01 Log₁₀ c.f.u. ml⁻¹ endospores (recirculating system) and 0.78 Log₁₀ c.f.u. ml⁻¹ vegetative cells and 0.42 Log₁₀ c.f.u. ml⁻¹ endospores (one-pass system) recovered, respectively. As a result, water treatment protocols should be established if untreated recirculating flume or condensate water is to be used in order to prevent Alicyclobacillus contamination and accumulation in the processing environment.