Ethylene production and peroxidase activity during tomato fruit ripening

Peroxidase activity and enzymic production of ethylene werestudied in tomato fruits (Lycopersicon esculentum MILL.) at3 ripeness stages. As the fruit ripens, one isoperoxidase disappears,and 3 new ones are formed. Activity of peroxidase and of ethylene-formingenzyme both increased 3 to 4 times as the fruit ripened. Histochemicalstaining showed that peroxidase is confined to the outermostand innermost layers of the pericarp, the placental tissue andvascular tissues; stained particles were neither mitochondrianor plastids. (Received October 27, 1969; )     

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; )     

Ethylene receptor expression is regulated during fruit ripening, flower senescence and abscission

Using theArabidopsis ethylene receptorETR1 as a probe, we have isolated a tomato homologue (tETR) from a ripening cDNA library. The predicted amino acid sequence is 70% identical toETR1 and homologous to a variety of bacterial two component response regulators over the histidine kinase domain. Sequencing of four separate cDNAs indicates that tETR lacks the carboxyl terminal response domain and is identical to that encoded by the tomatoNever ripe gene. Ribonuclease protection showed tETR mRNA was undetectable in unripe fruit or pre-senescent flowers, increased in abundance during the early stages of ripening, flower senescence, and in abscission zones, and was greatly reduced in fruit of ripening mutants deficient in ethylene synthesis or response. These results suggest that changes in ethylene sensitivity are mediated by modulation of receptor levels during development.     

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; )     

Biochemical characterisation of MdCXE1, a carboxylesterase from apple that is expressed during fruit ripening

Esters are an important component of apple (Malus × domestica) flavour. Their biosynthesis increases in response to the ripening hormone ethylene, but their metabolism by carboxylesterases (CXEs) is poorly understood. We have identified 16 members of the CXE multigene family from the commercial apple cultivar, ‘Royal Gala’, that contain all the conserved features associated with CXE members of the α/β hydrolase fold superfamily. The expression of two genes, MdCXE1 and MdCXE16 was characterised in an apple fruit development series and in a transgenic line of ‘Royal Gala’ (AO3) that is unable to synthesise ethylene in fruit. In wild-type MdCXE1 is expressed at low levels during early stages of fruit development, rising to a peak of expression in apple fruit at harvest maturity. It is not significantly up-regulated by ethylene in the skin of AO3 fruit. MdCXE16 is expressed constitutively in wild-type throughout fruit development, and is up-regulated by ethylene in skin of AO3 fruit. Semi-purified recombinant MdCXE1 was able to hydrolyse a range of 4-methyl umbelliferyl ester substrates that included those containing acyl moieties that are found in esters produced by apple fruit. Kinetic characterisation of MdCXE1 revealed that the enzyme could be inhibited by organophosphates and that its ability to hydrolyse esters showed increasing affinity (K_m) but decreasing turnover (k_cat) as substrate acyl carbon length increases from C2 to C16. Our results suggest that MdCXE1 may have an impact on apple flavour through its ability to hydrolyse relevant flavour esters in ripe apple fruit.     

Carbon dioxide action on ethylene biosynthesis of preclimacteric and climacteric pear fruit

Ethylene production in pear fruit was studied at 2 degrees C. Several observations showed that the inhibiting effect of CO2 on ethylene production did not operate only via the binding site of the ethylene binding protein. Ethylene production of freshly harvested pears was stimulated by 1-methylcyclopropene (1-MCP), but unaffected or inhibited by CO2 which points to different action sites for both molecules. In climacteric pears, where ethylene production was strongly inhibited by 1-MCP, a range of applied CO2 partial pressures was able to inhibit ethylene production further, to an extent similar to untreated pears. In the case of pears that had been stored for a period of 25 weeks, CO2 only had a clear effect after 1-MCP pretreatment. Respiration measurements showed that the effect of CO2 on ethylene production did not operate via an effect on respiration. Ethylene production models based on measurements of whole pears were used to study CO2 effects. Kinetic parameters derived from the models point to the conversion from ACC to ethylene by ACC oxidase as a possible action site for CO2 inhibition.     

Internal carbon dioxide and ethylene levels in ripening tomato fruit attached to or detached from the plant

The carbon dioxide and ethylene concentrations in tomato fruit ( Lycopersicon esculentum cv. Castelmart) and their stage of ripeness (characteristic external color changes) were periodically measured in fruit attached to and detached from the plant. An external collection apparatus was attached to the surface of individual tomato fruit to permit non-destructive sampling of internal gases. The concentration of carbon dioxide and ethylene in the collection apparatus reached 95% of the concentration in the fruit after 8 h. Gas samples were collected every 24 h. A characteristic climacteric surge in carbon dioxide (2-fold) and ethylene (10-fold) concentration occurred coincident with ripening of detached tomato fruit. Fruit attached to the plant exhibited a climacteric rise in ethylene (20-fold) concentration during ripening, but only a linear increase in carbon dioxide concentration. The carbon dioxide concentration increases in attached fruit during ripening, but the increase is a continuation of the linear increase seen in both attached and detached fruit before ripening and does not exhibit the characteristic pattern normally associated with ripening climacteric fruit. In tomato fruit, it appears that a respiratory climacteric per se, which has been considered intrinsic to the ripening of certain fruit, may not be necessary for the ripening of "climacteric" fruit at all, but instead may be an artifact of using harvested fruit.     

Ethylene and carbon dioxide: regulation of oat mesocotyl growth

Either ethylene or carbon dioxide stimulated the growth of oat mesocotyls in darkness, although the effect was much greater for carbon dioxide. Maximum elongation was obtained in the presence of both gases. Ethylene also induced lateral expansion of the mesocotyl: the volume of the mesocotyl was increased more in air with added ethylene than in air depleted of ethylene. Ethylene also stimulated mesocotyl growth under red light. Gibberellin only slightly increased mesocotyl length under red light and acted cooperatively with ethylene in the promotion of growth. The oat mesocotyl is a unique organ in terrestrial plants because ethylene simultaneously stimulates not only longitudinal growth but also lateral expansion. The ecological significance of oat mesocotyl growth in relation to the response to ethylene and carbon dioxide is discussed.     

Measurement of oxygen consumption and carbon dioxide production rates by a tracer-bolus method

A new method for measuring oxygen consumption and carbon dioxide production is described. The method is based on the injection of a helium bolus into the inspired gas for repeated breaths; the helium can be delivered through the mouth or through a nostril (the injection system being controlled by the integrated flow signal or by the signal of a thermistor, respectively). Compared with the conventional gas-collection technique, the tracer-bolus method allows more rapid and frequent measurements. The method does not necessarily require an airtight respiratory circuit, so that it can be conveniently applied in less cooperative subjects, including children. The results of validation studies based on a comparison with the standard gas-collection technique are presented; these results indicate that the measurements by the tracer-bolus method are accurate in normal subjects. In patients with obstructive lung disease, the determinations are associated with a systematic error, which is expected, the tracer-bolus method being based on a single-compartment, uniformly ventilated and perfused lung model; the error is, however, predictable from the degree of functional impairment and can be corrected using equations based on routine pulmonary function tests.     

Ethylene, ethanol, acetaldehyde and carbon dioxide released by Prunus avium shoot cultures

Wild cherry ( Prunus avium L.) shoots were cultured in closed vessels on a proliferation medium and the volatile substances released during incubation at photosynthetic photon flux density of 30 μmol m^–2 S^–1 were determined. Ethylene and CO₂ started forming at the beginning of the incubation period and a linear relationship between their formation was observed even at high CO₂ concentrations. After 30 days of culture, CO₂ reached a concentration of 30%. Shoots released elhanol and acetaldehyde after several days of incubation.     

Evidence for light-dependent recycling of respired carbon dioxide by the cotton fruit

Conservation of respired CO₂ by an efficient recycling mechanism in fruit could provide a significant source of C for yield productivity. However, the extent to which such a mechanism operates in cotton (Gossypium hirsutum L.) is unknown. Therefore, a combination of CO₂ exchange, stable C isotope, and chlorophyll (Chl) fluorescence techniques were used to examine the recycling of respired CO₂ in cotton fruit. Respiratory CO₂ losses of illuminated fruit were reduced 15 to 20% compared with losses for dark-incubated fruit. This light-dependent reduction in CO₂ efflux occurred almost exclusively via the fruit's outer capsule wall. Compared with the photosynthetic activity of leaves, CO₂ recycling by the outer capsule wall was 35 to 40% as efficient. Calculation of ¹⁴CO₂ fixation on a per Chl basis revealed that the rate of CO₂ recycling for the capsule wall was 62.2 micromoles ¹⁴CO₂ per millimole Chl per second compared with an assimilation rate of 64.6 micromoles ¹⁴CO₂ per millimole Chl per second for leaves. During fruit development, CO₂ recycling contributed more than 10% of that C necessary for fruit dry weight growth. Carbon isotope analyses (δ¹³C) showed significant differences among the organs examined, but the observed isotopic compositions were consistent with a C₃ pathway of photosynthesis. Pulse-modulated Chl fluorescence indicated that leaves and fruit were equally efficient in photochemical and nonphotochemical dissipation of light energy. These studies demonstrated that the cotton fruit possesses a highly efficient, light-dependent CO₂ recovery mechanism that aids in the net retention of plant C and, therein, contributes to yield productivity.     

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.