The Ribosomes of Pear Fruit: Their Synthesis during the Climacteric and the Age-related Compensatory Response to Ionizing Radiation 1

Maceration at liquid nitrogen temperatures, use of poly-vinylpyrrolidone, and careful pH control are essential to the isolation of ribosomes and polysomes from deciduous fruit tissue. Characteristics of the ribosomes and constituent RNA are described.The distribution of ribosomes among monomer and polymer forms remains relatively constant until fruit reach the climacteric peak, after which there is a notable decline in the polymeric forms. In contrast to the relative stability in size distribution there is a marked change in ribosomal turnover during the climacteric rise. A transitory increase in rate of ribosomal synthesis is followed by a rapid decline coincident with the final portion of the climacteric rise. No incorporation of radioactive base into ribosomes could be detected once fruit had reached the climacteric peak.Coincidence of radioactivity with the ribosomal RNA on methylated albumin-Kieselguhr chromatograms and complete inhibition of ribosomal RNA synthesis by actinomycin D confirm that radioactive nucleotide was incorporated into newly synthesized ribosomes. Data are presented to distinguish between a cellular response to injury, as may result from the preparation of tissue slices, and the effects of physiological age.Superimposed intracellular radiation injury stimulates the synthesis of new ribosomes and underscores a major transition in the dynamics of the ribosomal system coincident with the climacteric rise.     

Similarities between the Actions of Ethylene and Cyanide in Initiating the Climacteric and Ripening of Avocados

A continuous exposure of intact avocados (Persea americana) to 400 μl/l of cyanide results in a rapid increase in the rate of respiration, followed by a rise in ethylene production, and eventual ripening. The pattern of changes in the glycolytic intermediates glucose 6-phosphate, fructose diphosphate, 3-phosphoglyceric acid, and phosphoenolpyruvate during the rapid rise in respiration in both ethylene and cyanide-treated fruits is similar to that found in fruits made anaerobic where a 2.3- to 3-fold increase in the rate of glycolysis is observed. It is suggested that both during the climacteric and in response to cyanide, glycolysis is enhanced. It is proposed that cyanide implements the diversion of electrons to the cyanide-resistant electron path through structural alterations which are independent of the simultaneous inhibition of cytochrome oxidase.     

Postharvest Variation in Cellulase, Polygalacturonase, and Pectinmethylesterase in Avocado (Persea americana Mill, cv. Fuerte) Fruits in Relation to Respiration and Ethylene Production

Cellulase, polygalacturonase (PG), pectinmethylesterase (PME), respiration, and ethylene production were determined in single “Fuerte” avocado fruits from the day of harvest through the start of fruit breakdown. PME declined from its maximum value at the time of picking to a low level early in the climacteric. PG activity was not detectable in the preclimacteric stage, increased during the climacteric, and continued to increase during the postclimacteric phase to a level three times greater than when the fruit reached the edible soft stage. Cellulase activity was low in the preclimacteric fruit, started to increase just as respiration increased, and reached a level two times greater than at the edible soft stage. Cellulase activity started to increase 3 days before PG activity could be detected. Increased production of ethylene followed the increase in respiration and cellulase activity by about 1.5 days. These results indicate that a close relation exists between the rapid increase in the cell wall-depolymerizing enzymes and the rise in respiration and ethylene production and refocused attention on the role of the cell wall and the associated plasma membrane in the early events of fruit ripening.     

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.     

Regulation of Ethylene Biosynthesis in Avocado Fruit during Ripening

Preclimacteric avocado (Persea americana Mill.) fruits produced very little ethylene and had only a trace amount of l-aminocyclopropane-1-carboxylic acid (ACC) and a very low activity of ACC synthase. In contrast, a significant amount of l-(malonylamino)cyclopropane-1-carboxylic acid (MACC) was detected during the preclimacteric stage. In harvested fruits, both ACC synthase activity and the level of ACC increased markedly during the climacteric rise reaching a peak shortly before the climacteric peak. The level of MACC also increased at the climacteric stage. Cycloheximide and cordycepin inhibited the synthesis of ACC synthase in discs excised from preclimacteric fruits. A low but measurable ethylene forming enzyme (EFE) activity was detected during the preclimacteric stage. During ripening, EFE activity increased only at the beginning of the climacteric rise. ACC synthase and EFE activities and the ACC level declined rapidly after the climacteric peak. Application of ACC to attached or detached fruits resulted in increased ethylene production and ripening of the fruits. Exogenous ethylene stimulated EFE activity in intact fruits prior to the increase in ethylene production. The data suggest that conversion of S-adenosylmethionine to ACC is the major factor limiting ethylene production during the preclimacteric stage. ACC synthase is first synthesized during ripening and this leads to the production of ethylene which in turn induces an additional increase in ACC synthase activity. Only when ethylene reaches a certain level does it induce increased EFE activity.     

Studies in the Nitrogen Metabolism of Apple Fruits: THE CLIMACTERIC RISE IN RESPIRATION IN RELATION TO CHANGES IN THE EQUILIBRIUM BETWEEN PROTEIN SYNTHESIS AND BREAKDOWN

1. A close parallelism in the drift of the rate of respirationand the protein-N/ non-protein-N ratio is shown to occur bothin apple fruits attached to the tree and when detached fromthe tree at various stages of development and stored for severalmonths at 12 C.
2. In detached fruits the fall in respirationwhich occurs immediately(during the first 48 hours) after pickingis only accompaniedby a concomitant fall in net protein invery young fruits inwhich active cell division is taking place.Subsequently, infruit of all ages when a climacteric rise inrespiration occursit is accompanied by a net increase in protein.
3. It is argued that the climacteric rise in respiration is aresultof increase in the level of protein which will be expectedtoreduce the ATP/ADP ratio.
4. Over the climacteric period,although rate of respiration andnet protein content both rise,R rises more rapidly than proteinand, subsequently, falls ata faster rate than P. It is suggestedthat this may be due tothe ‘new’ protein containinga higher proportionof enzyme(s) directly involved in respirationand leading, forexample, to a reduction in the ATP/ADP ratio.

     

Growth and respiration patterns of snap bean fruits

The relationship of respiration and growth of seed, pericarp tissue and whole fruit of snap beans (Phaseolus vulgaris L.) was studied. The whole fruit exhibited an apparent climacteric type of respiration pattern. This pattern resulted from an increase in CO₂ production by the enlarging seed followed by a rapid decrease in CO₂ evolution by the pericarp tissue, and the pattern was not associated with any concomitant increase in ethylene production. Therefore, the apparent climacteric respiration pattern of a developing bean fruit is not comparable to the phenomenon that occurs in other ripening fruits.     

Influence of Plant Hormones on Ethylene Production in Apple, Tomato, and Avocado Slices during Maturation and Senescence

Ethylene production by tissue slices from preclimacteric, climacteric, and postclimacteric apples was significantly reduced by isopentenyl adenosine (IPA), and by mixtures of IPA and indoleacetic acid, and of IPA, indoleacetic acid, and gibberellic acid after 4 hours of incubation. Ethylene production by apple (Pyrus malus L.) slices in abscisic acid was increased in preclimacteric tissues, decreased in climacteric peak tissues, and little affected in postclimacteric tissues. Indoleacetic acid suppressed ethylene production in tissues from preclimacteric apples but stimulated ethylene production in late climacteric rise, climacteric, and postclimacteric tissue slices. Gibberellic acid had less influence in suppressing ethylene production in preclimacteric peak tissue, and little influenced the production in late climacteric rise, climacteric peak, and postclimacteric tissues. IPA also suppressed ethylene production in pre- and postclimacteric tissue of tomatoes (Lycopersicon esculentum) and avocados (Persea gratissima). If ethylene production in tissue slices of ripening fruits is an index of aging, then IPA would appear to retard aging in ripening fruit, just as other cytokinins appear to retard aging in senescent leaf tissue.     

Banana Ripening: Implications of Changes in Internal Ethylene and CO(2) Concentrations, Pulp Fructose 2,6-Bisphosphate Concentration, and Activity of Some Glycolytic Enzymes

In ripening banana (Musa acuminata L. [AAA group, Cavandish subgroup] cv. Valery) fruit, the steady state concentration of the glycolytic regulator fructose 2,6-bisphosphate (Fru 2,6-P₂) underwent a transient increase 2 to 3 hours before the respiratory rise, but coincident with the increase in ethylene synthesis. Fru 2,6-P₂ concentration subsequently decreased, but increased again approximately one day after initiation of the respiratory climacteric. This second rise in Fru 2,6-P₂ continued as ripening proceeded, reaching approximately five times preclimacteric concentration. Pyrophosphate-dependent phosphofructokinase glycolytic activity exhibited a transitory rise during the early stages of the respiratory climacteric, then declined slightly with further ripening. Cytosolic fructose 1,6-bisphosphatase activity did not change appreciably during ripening. The activity of ATP-dependent phosphofructokinase increased approximately 1.6-fold concurrent with the respiratory rise. A balance in the simultaneous glycolytic and gluconeogenic carbon flow in ripening banana fruit appears to be maintained through changes in substrate levels, relative activities of glycolytic enzymes and steady state levels of Fru 2,6-P₂.     

The energy-conserving and energy-dissipating processes in mitochondria isolated from wild type and nonripening tomato fruits during development on the plant

Bioenergetics of tomato (Lycopersicon esculentum) development on the plant was followed from the early growing stage to senescence in wild type (climacteric) and nonripening mutant (nor, nonclimacteric) fruits. Fruit development was expressed in terms of evolution of chlorophyll a content allowing the assessment of a continuous time-course in both cultivars. Measured parameters: the cytochrome pathway-dependent respiration, i.e., the ATP synthesis-sustained respiration (energy-conserving), the uncoupling protein (UCP) activity-sustained respiration (energy-dissipating), the alternative oxidase(AOX)-mediated respiration (energy-dissipating), as well as the protein expression of UCP and AOX, and free fatty acid content exhibited different evolution patterns in the wild type and nor mutant that can be attributed to their climacteric/nonclimacteric properties, respectively. In the wild type, the climacteric respiratory burst observed in vitro depended totally on an increse in the cytochrome pathway activity sustained by ATP synthesis, while the second respiratory rise during the ripening stage was linked to a strong increase in AOX activity accompanied by an overexpression of AOX protein. In wild type mitochondria, the 10-M linoleic acid-stimulated UCP-activity-dependent respiration remained constant during the whole fruit development except in senescence where general respiratory decay was observed.     

Permeability Characteristics and Amino Acid Incorporation during Senescence (Ripening) of Banana Tissue

Changes in free space of banana tissue during ripening were measured using radioisotopes. Free space increased significantly about 44 hours before the onset of, and rose exponentially during the respiratory climacteric. The increase in free space indicates a progressive increase in the proportion of cells which becomes completely permeable to solutes in the ambient solution by simple diffusion. At the respiratory peak the tissue was essentially 100% free space to mannitol, sucrose, fructose and chloride.

The capacity for active uptake of solutes declined about one day before the onset of the respiratory rise and fell to a very low level by the respiratory peak.

There was no change in the level of protein or amino acids during ripening. Assays of tissue sections before and after washing indicated an increased rate of leakage of amino acids during ripening.

Studies of incorporation of 3 concentrations of ¹⁴C-labeled leucine and phenylalanine indicated marked changes in the size and specific activity of the amino acid pool at the site of protein synthesis just prior to and during the climacteric rise, due to a diffusive mixing of the labeled substrate with the previously sequestered endogenous, unlabeled pool of substrate. The use of a high concentration of exogenous substrate (above saturation for uptake) resulted in an apparent constant specific activity of the metabolic pool through the ripening period. Data from these studies indicated a decline in amino acid incorporation during the climacteric.

It was concluded that the initiation of permeability changes marks the onset of senescence in banana. The causative relations between alterations in permeability, the respiratory rise and other chemical changes attending fruit ripening are discussed.

     

Glycolysis at the climacteric of bananas.

This work was carried out to investigate the relative roles of phosphofructokinase and pyrophosphate-fructose-6-phosphate 1-phosphotransferase during the increased glycolysis at the climacteric in ripening bananas (Musa cavendishii Lamb ex Paxton). Fruit were ripened in the dark in a continuous stream of air in the absence of ethylene. CO2 production, the contents of glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, phosphoenolpyruvate and PPi; and the maximum catalytic activities of pyrophosphate-fructose-6-phosphate 1-phosphotransferase, 6-phosphofructokinase, pyruvate kinase and phosphoenolpyruvate carboxylase were measured over a 12-day period that included the climacteric. Cytosolic fructose-1,6- bisphosphatase could not be detected in extracts of climacteric fruit. The peak of CO2 production was preceded by a threefold rise in phosphofructokinase, and accompanied by falls in fructose 6-phosphate and glucose 6-phosphate, and a rise in fructose 1,6-bisphosphate. No change in pyrophosphate-fructose-6-phosphate 1-phosphotransferase or pyrophosphate was found. It is argued that phosphofructokinase is primarily responsible for the increased entry of fructose 6-phosphate into glycolysis at the climacteric.     

Metabolism of 1-aminocyclopropane-1-carboxylic acid in ripening apple fruits

In preclimacteric apple fruits ( Malus × domestica Borkh. cv. Golden Delicious) ethylene production is controlled by the rates of 1-aminocyclopropane-1-carboxylic acid (ACC) synthesis, and by its metabolism to ethylene by the ethylene-forming enzyme and to 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) by malonyl CoA-ACC transferase. The onset of the climacteric in ethylene production is associated with an increase in the activity of the ethylene-forming enzyme in the pulp and with a rise in the activity of ACC synthase. Malonyl transferase activity is very high in the skin of immature fruit, decreases sharply before the onset of the climacteric, and remains nearly constant thereafter. More than 40% of the ACC synthesized in the skin and around 5% in the flesh, are diverted to MACC at early climacteric. At the climacteric peak there are substantial gradients in ethylene production between different portions of the tissue, the inner cortical tissues producing up to twice as much as the external tissues. This increased production is associated with, and apparently due to, increased content of ACC synthase. Less than 1% of the synthesized ACC is diverted to MACC in the flesh of climacteric apples. In contrast, the skin contains high activity of malonyl transferase, and correspondingly high levels [1000 nmol (g dry weight)⁻¹] of MACC.     

Effect of phenolic compounds in Lycopersicon esculeutum on the synthesis of ethylene

Caffeic, coumaric, sinapic and ferulic acids and naringenin were found in green tomato fruit, Chlorogenic acid accounted for 75% of the total phenolics in mature green fruit but only 35% in ripe fruit. There was very little change in the phenolic composition of the flesh of the fruit during ripening, whereas in the skin, naringenin increased markedly at the onset of the climacteric and three unidentified compounds increased during the climacteric rise. The increase in the concentration of naringenin was accompanied by an increase in the production of ethylene in the skin. Investigation of three systems producing ethylene from 4-methylmercapto-2-oxobutyric acid in the presence of peroxidase, showed that only p`-coumaric acid or naringenin were capable of acting as phenolic substrates, the other phenolic compounds being inhibitory.     

Respiratory metabolism during cold storage of apple fruit. I. Sucrose metabolism and glycolysis

This study provides the first report on the occurrence of the respiratory climacteric during cold storage of apple fruit ( Malus domestica Borkh. cv. Reinette du Canada). The respiratory pattern at 4°C was very similar to that observed during postharvest ripening at room temperature, except that shelf life was considerably extended and the onset of the climacteric delayed. Increasing the calcium content of the apple fruit significantly reduced loss of firmness during cold storage, but showed no effect on respiration or on the other parameters determined. A gradual accumulation of soluble sugars occurred during the first 60 days after harvest and was effectively completed before the climacteric peak was reached. This increase in sugars correlated with an increase in the activity of sucrose-phosphate synthase (EC 2.4.1.14), and a marked change in the kinetic properties of the enzyme was observed after sucrose accumulation ceased. Changes in the hexose-phosphate pool and in glycolytic and gluconeogenic activities indicated an initial increase in the gluconeogenic flow at early stages of the climacteric, followed by activation of glycolysis, with the carbon flow being most likely regulated at the reversible phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate (mostly via pyrophosphate:fructose-6-phosphate phosphotransferase, EC 2.7.1.90) and at the pyruvate kinase (EC 2.7.1.40) steps. The results presented indicate that the respiratory climacteric does not occur to accommodate extra ATP requirements during sucrose synthesis nor can it be a consequence of an increased supply of respiratory substrate.     

The Relation of Ethylene Production and Respiration with Maturation and Senescence of Melon during Postharvest-Storage

There are three types of ethylene production and respiration during the postharvest storage of melon fruits: 1. with climacteric rise in both respiration and ethylene production; 2 with climacteric rise in ethylene production but respiration declined gradually; 3 with no appreciable change in both respiration and ethylene production. The last one has good storage quality.     

Effects of aminoethoxyvinylglycine and countereffects of ethylene on ripening of bartlett pear fruits

Pear fruits (Pyrus communis L. var. Bartlett) were treated with solutions containing aminoethoxyvinylglycine (AVG) using a modified vacuum infiltration method that introduced 4.3 milliliters solution per 100 grams tissue. At concentrations of 1 millimolar, AVG strongly inhibited ethylene production and delayed for 5 days the respiratory climacteric and accompanying ripening changes in skin color and flesh firmness. AVG was less effective in inhibiting the ripening of more mature fruits. Fruit infiltrated with 5 millimolar AVG had not begun to ripen 12 days after initiation of ripening in the controls. When treated with ethylene the inhibited fruit exhibited a climacteric rise in respiration, softened, and became yellow. Treatment of the AVG infiltrated fruits with ethyelne for 24 hours resulted in no recovery in endogenous ethylene production, but in a stimulation of protein synthesis measured as a 200% increase in leucine incorporation by excised tissue and a 74% increase in the percentage of ribosomes present as polysomes.     

Control of apple ripening by succinic Acid 2,2-dimethyl hydrazide, 2-chloroethyltrimethylammonium chloride, and ethylene

Ripening of `Tydeman's Early' apples (Malus sylvestris L.) assessed by the occurrence of the respiratory climacteric was delayed by succinic acid 2,2-dimethyl hydrazide (B-9) but not by 2-chloroethyltrimethylammonium chloride (CCC), each applied 14 days after bloom. B-9 also inhibited ethylene production by fruits exhibiting the climacteric rise. Inhibition of the climacteric by B-9 was overcome by exogenous ethylene, but the latter treatment failed to completely reverse the B-9 inhibition of endogenous ethylene production.     

Aging of cylinders excised from pulp tissues of the ;golden delicious' apple

Aging cylinders excised from `Golden Delicious' apple (Pyrus malus L.) pulp, like the intact fruit, exhibit some characteristic phenomena such as rise in respiration (climacteric), ethylene synthesis, enzymic changes, and increase in ribosomes and mRNA. Aging of cylinders of pulp tissues may offer a useful physiological tool for the study of maturation and senescence.