Climacteric Peak Research Articles

チュウゴクナシ‘鴨梨’果実の樹上および収穫後の成熟特性

Ripening characteristics of the Chinese pear 'Yali', Japanese pear 'Nijisseiki', and European pear 'La France' on and off the tree were compared.Respiration rates of all three kinds of pear fruit on the tree increased slightly during the late stage of maturation. A typical climacteric pattern was observed in stored fruit of 'Yali' and 'La France', but not in those of 'Nijisseiki', independent of their maturity.Ethylene production by intact fruit was detected just before the 'Yali' fruit abscised and during the late mature stage of 'La France', but none was detected in 'Nijisseiki' fruit even at the overripe state. Ethylene production in the stored fruit of 'Yali' and 'La France' showed a marked climacteric pattern which was accompanied by corresponding changes in 1-aminocyclopropane-1-carboxylic acid (ACC) and the ethylene-forming enzyme (EFE) activity. Stored 'Yali' fruit evolved a large volume of ethylene, reaching a maximum of 380 nl/g•hr. The rate of ethylene production at the climacteric peak was progressively lower as the harvest date was delayed. Contrarily, stored 'Nijisseiki' fruit disclosed an unusually high ACC content but a low level of EFE activity while evolving no ethylene.A drastic decline in starch and a slight increase of sugar were commonly observed in the three pear cultivars during their maturation process. However, their composition of sugars differed. In 'Yali' and 'La France', fructose was the predominant sugar throughout the maturation and ripening stages, whereas, in a ripe 'Nijisseiki' fruit, sucrose was the dominant sugar.Malic acid was the predominant acid in the three pear cultivars. No sharp changes in acid content occurred in 'Yali' and 'Nijisseiki' fruits while they were maturing, but malic acid content increased briefly in 'Yali' fruit as it ripened. Malic acid level in 'La France' increased steadily while the fruit matured but the level declined rapidly as the fruit ripened.During maturation on the tree fruit firmness decreased gradually in 'Yali' and 'Nijisseiki' but rapidly in 'La France' during maturation. 'Yali' fruit remained relatively firm during storage but those of 'La France' softened rapidly, whereas those of 'Nijisseiki' softened gradually. We found that 'Yali' fruit retained their firmness even though they produced voluminous amounts of ethylene.

Physiological responses of mangoes cv. Kensington Pride to gamma irradiation treatment as affected by fruit maturity and ripeness

SummaryAn investigation was undertaken to examine responses of mangoes cv. Kensington Pride to irradiation treatment.Gamma irradiation of preclimacteric fruit increased fruit respiration immediately after treatment, delayed the time to attain the climacteric respiratory peak and reduced the magnitude of this peak rate. Fruit softening was unaffected whereas skin colour development was strongly inhibited by irradiation treatment. Inhibition of skin degreening was half‐maximal at a dose of 200 Gy. Exposure to ethylene failed to reverse this inhibition. Less mature fruit were more strongly affected by irradiation treatment. Fruit which were partially ripe and in their climacteric rise at time of treatment were largely unaffected by irradiation. These results are discussed in terms of the utility of irradiation technology for commercial disinfestation.

Physiology and firmness determination of ripening tomato fruit

Tomato (Lycopersicon esculentum Mill.) genotypes varying in intrinsic firmness were examined to determine the quantitative relationships between polygalacturonase (EC 3.2.1.15) activity, firmness and other ripening parameters including rate (days from mature‐green to full red) and intensity (rate of ethylene production at climacteric peak) of ripening. Texture, respiration and ethylene production were monitored in the immature‐green through the red (ripe) stages of development. Polygalacturonase activity was measured by direct assay of salt‐extractable wall protein or by monitoring the release of pectins from isolated, enzymically active wall. In all fruit, polygalacturonase activity was highly correlated with pericarp softening, but only moderately correlated with softening of whole fruit (r = 0.920 and 0.757, respectively). Polygalacturonase activity was positively correlated with cell‐wall autolytic activity in pink (r = 0.969) and red (r = 0.900) fruit. Firmer genotypes exhibited lower rates of respiration and ethylene production during ripening. Polygalacturonase activity in isolates prepared from fruit at the climacteric peak was positively correlated with ethylene production and respiration, and negatively correlated with days to ripening (r = 0.929, 0.805, and ‐0.791, respectively). The data demonstrate the importance of selecting the appropriate method of firmness determination and are consistent with the hypothesis that pectin fragments released by polygalacturonase contribute to the production of autocatalytic (system II) ethylene.

Banana Ripening: Implications of Changes in Glycolytic Intermediate Concentrations, Glycolytic and Gluconeogenic Carbon Flux, and Fructose 2,6-Bisphosphate Concentration

In ripening banana (Musa sp. [AAA group, Cavendish subgroup] cv Valery) fruit, the concentration of glycolytic intermediates increased in response to the rapid conversion of starch to sugars and CO(2). Glucose 6-phosphate (G-6-P), fructose 6-phosphate (Fru 6-P), and pyruvate (Pyr) levels changed in synchrony, increasing to a maximum one day past the peak in ethylene synthesis and declining rapidly thereafter. Fructose 1,6-bisphosphate (Fru 1,6-P(2)) and phosphoenolpyruvate (PEP) levels underwent changes dissimilar to those of G 6-P, Fru 6-P, and Pyr, indicating that carbon was regulated at the PEP/Pyr and Fru 6-P/Fru 1,6-P(2) interconversion sites. During the climacteric respiratory rise, gluconeogenic carbon flux increased 50- to 100-fold while glycolytic carbon flux increased only 4- to 5-fold. After the climacteric peak in CO(2) production, gluconeogenic carbon flux dropped dramatically while glycolytic carbon flux remained elevated. The steady-state fructose 2,6-bisphosphate (Fru 2,6-P(2)) concentration decreased to (1/2) that of preclimacteric fruit during the period coinciding with the rapid increase in gluconeogenesis. Fru 2,6-P(2) concentration increased thereafter as glycolytic carbon flux increased relative to gluconeogenic carbon flux. It appears likely that the initial increase in respiration in ripening banana fruit is due to the rapid influx of carbon into the cytosol as starch is degraded. As starch reserves are depleted and the levels of intermediates decline, the continued enhancement of respiration may, in part, be maintained by an increased steady-state Fru 2,6-P(2) concentration acting to promote glycolytic carbon flux at the step responsible for the interconversion of Fru 6-P and Fru 1,6-P(2).

Endogenous levels of abscisic acid in aging carnation flower parts

Aging carnation flower parts were used to determine whether or not any correlation existed between the concentration of abscisic acid (ABA) and a predisposition of the tissue for ethylene synthesis. Levels of ABA were measured using an enzyme-linked immunosorbent assay (ELISA) following purification steps including prepacked silica gel columns. Increased ABA levels paralleled the increase of ethylene and the onset of irreversible wilting in the carnation petals. Neither the green tissue nor the receptacle showed any sign of wilting with the remainder of the flower parts, but increased ABA was detected in both tissues subsequent to, or coincident with, the ethylene climacteric peak in the senescing petals. An increase of ABA in both the styles and the ovary was detected in the preclimacteric flower, and did not appear to be triggered by the production of ethylene. Increased ABA in the gynoecium also did not result in the onset of ethylene production in the preclimacteric flower.

Expression and transport of cellulase in avocado mesocarp during ripening

Cellulase localization in the mesocarp of ripening avocado fruits (Persea americana Mill. cv. Hass) was studied by a variety of immunological methods. As localized by immunodetection on whole fruit tissue blots, cellulase first appeared near the stylar end of the fruit during the late portion of the rise in climacteric respiration. Cellulase appearance subsequently expanded outward and upward, reaching the peduncle end of the fruit by the day after the climacteric peak. Cellulase expression in cells surrounding vascular bundles was delayed relative to expression in the adjacent mesocarp. Immuno-labeled frozen sections, viewed with the light microscope, showed that cellulase appeared in both parenchyma and oil cells concom-mitantly with wall breakdown. Immunogold detection of cellulase by electron microscopy revealed labeling associated with the endoplasmic reticulum, plasmodesmata, and cell wall during the period between the late portion of the climacteric rise and the day after the climacteric peak. Cellulase appeared in the nucleus during all stages of ripening after the early portion of the climacteric rise. Immunoblot analysis of organelle fractions, isolated from avocado fruit at the climacteric peak of respiration, revealed three molecular weight forms of cellulase; a light and a heavy form found in endoplasmic reticulum-enriched fractions, and an intermediate form found in Golgi and plasma membrane-enriched fractions.

Reversible Inhibition of Ethylene Action and Interruption of Petal Senescence in Carnation Flowers by Norbornadiene

The inhibitory effects of the cyclic olefin 2,5-norbornadiene (NBD) on ethylene action were tested in carnation (Dianthus caryophyllus L. cv White Sim) flowers. Treatment of flowers at anthesis with ethylene in the presence of 500 microliters per liter NBD increased the concentration of ethylene required to elicit a response (petal senescence), indicating that NBD behaves as a competitive inhibitor of ethylene action. Transfer of flowers producing autocatalytic ethylene and exhibiting evidence of senescence (petal in-rolling) to an atmosphere of NBD resulted in a rapid reduction in ethylene production, petal 1-aminocyclopropane-1-carboxylic acid synthase activity, 1-aminocyclopropane-1-carboxylic acid content, and ethylene forming enzyme activity. Removal of NBD resulted in recovery of ethylene biosynthesis. These results support the autocatalytic regulation of ethylene production during the climacteric stage of petal senescence and suggest that continued perception of ethylene is required for maintenance of ethylene biosynthesis. The inhibition of ethylene action by NBD after the flowers had reached the climacteric peak was associated with interruption of petal senescence as evidenced by reversal of senescence symptoms. This result is in contrast to the widely held belief that the rate of petal senescence is fixed and irreversible once petals enter into the ethylene climacteric.

Respiration rate, ethylene production and certain chemical changes during the ripening of jujube fruits

SummaryJujube fruits (Zizyphus spina-christi (L) Wild) of cv. Zaytoni were harvested at the mature green stage and their respiration rate, ethylene production, as well as certain chemical changes during ripening at 20°C were investigated. The rates of respiration and ethylene production increased together as the fruit entered the ripening phase, reached a peak value and then declined. Total soluble solids, carotenoids and ascorbic acid contents increased while titratable acidity and total chlorophylls decreased with fruit maturity. Optimum eating quality was reached at the climacteric peak.

Biochemical changes involved in stress response and ripening behaviour of γ-irradiated mango fruit

The stress response of ripening mango fruits treated in the pre-climacteric phase with radiation doses of 0.75, 1.25 and 1.75 kGy was investigated. L-Phenylalanine ammonialyase, polyphenol oxidase, peroxidase and catalase activities were determined and significant differences were observed. Differences in the patterns of total phenolics, flavanols and proteins were also observed. Malic enzyme activity was used as an indicator of the ripening stage (climacteric rise, climacteric peak and post-climacteric phase) as well as a measure of the effect of γ-irradiation on fruit ripening. It seems that radiation treatment causes a stress condition in the fruit which, depending on the dose, may lead to browning of the tissue or necrotic decay.

セイヨウナシ‘テ•フランス’の追熟生理及び品質における収穫日, 追熟温度及びエチレン処理の影響

The purpose of this study was to investigate the influence of harvest date, ripening temperature, and ethylene treatment on the ripening physiology and quality of ′La France′pears. Fruits were harvested on 3 dates (Oct. 14, 21, and 24, 1985), and then were held at 10°C, 15°C, and 20°C, respectively. Weight, flesh firmness, soluble solids, titratable acidity, total suger, soluble pectin, respiration and ethylene production were measured at harvest and during ripening.1. As harvest date was delayed, the climactric rise developed more rapidly. In the preclimactreric fruits at harvest (Oct. 14, and 21), the fruit reached the climacteric peak much earlier when held at 20°C than at 15°C and 10°C, while the magnitude of the peaks in both respiration rate and ethylene emanation were lower in the fruit held at 20°C than at 15°C and 10°C. This trend was striking in ethylene emanation.2. As harvest date was delayed and ripening temperature was raised, the number of days from harvest to full ripeness became short. No clear differences in eating quality of ripe fruits were found among those fruits, but the quality was reduced more rapidly in early-harvested fruits ripend at 20°C than in late-harvested fruits ripened at both 15°C and 10°C.3. As harvest date was delayed, total suger content became high and starch content became low at harvest. There were no differences in total suger content in fruits ripended at 20°C among different harvest dates.4. As harvest date was delayed, the maximum content of soluble pectin was reduced during ripening.5. Fruit treated with 500ppm ethylene for 24 hours at 20°C showed a higher rate of respiration, and became edible 3 to 4 days earlier than untreated fruit.6. Flesh firmness seems to be a useful indicator for harvest of′La France′pears, as it is related to ripening capacity.

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.

Ca2+ effects on ethylene, carbon dioxide and 1‐aminocyclopropane‐1‐carboxylic acid synthase activity

The response of pericarp disks from ripening tomato (Lycopersicon esculentum Mill. cv. Traveler‘76) to CaCl2, additions was studied to determine the effect of Ca2+ on ethylene and CO2 production. Application of 5 mM CaCl2 resulted in a 2, 20, 33, 39, and 50% increase in ethylene production in disks obtained from preclimacteric minimum, climacteric rise, climacteric peak, one, and two days postclimacteric fruit, respectively. CaCl2 concentrations of 10 and 50 mM gave no additional stimulation of ethylene production; CO2 production at 5 mM CaCl2 was not different from controls, but is increased at 10 and 50mM CaCl2. CaCl2 also increased ethylene production in disks treated with 1‐aminocyclopropane‐1‐carboxylic acid (ACC) or aminoethoxy‐vinylglycine. Chloride salts of K+, Na+, Mg2+, Sr2+ and La3+ did not stimulate ethylene production. SrCl2 stimulated ethylene production to a lesser degree than CaCl2. Disks from potato (Solanum tuberosum L. cv. Katahdin) tubers produced greater quantities of ethylene and ACC when 5 mM CaCl2 was included in the incubation medium (K. B. Evensen, 1983. Physiol. Plant. 60:125–128). Ca2+‐treated disks had more than three times as much ACC synthase activity as control disks after 18 to 24 h incubation, when ethylene and ACC were maximal. The apparent Km for S‐adenosylmethionine was 13 μM at 29°C, pH 8.0 in extracts from both Ca2+‐treated and control disks. Inclusion of 1 to 50 mM CaCl2 in the assay medium did not significantly affect enzyme activity. ACC synthase extracted from control and Ca2+‐treated disks had a pH optimum of 8.5 and an apparent molecular weight of 72 kdalton, estimated by gel filtration. It is likely that the presence of Ca2+ in the buffer allows greater synthesis of ACC synthase as part of the wound‐healing response in potato, while in tomato the predominant effect is on membrane stabilization.

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.

Biochemical changes associated with the ripening of hot pepper fruit

Hot pepper (Capsicum annuum L. cv. Chooraehong) fruit underwent a respiratory climacteric during ripening. However, the rate of ethylene production was low, reaching a maximum of approximately 0.7 μl kg−1 h−1 at the climacteric peak when the surface color was 30 to 40% red. Ripening was accompanied by a loss of galactose and arabinose residues from the cell wall. The content of uronic acid and cellulose in the wall changed only slightly during ripening. The average molecular weight of a cell wall hemicellulosic fraction shifted progressively toward a lower molecular weight during ripening. Total β‐galactosidase (EC 3.2.1.23) activity increased 50‐fold from the immature green to the red ripe stage. No polygalacturonase (EC 3.2.1.15) activity was detected at any stage of ripeness. Thus, the loss of galactose and arabinose residues from the cell wall, as well as the observed modification of hemicelluloses during ripening, seem to be unrelated to active polygalacturonase. Soluble polyuronide content remained relatively constant at approximately 60 μg (g fresh weight)−1 as fruit ripended.

Persimmon Pectinmethylesterase: Extraction and Variation during Ripening

ABSTRACTThe optimum conditions for the extraction of pectinmethylesterase (PME) from persimmon fruit (Diospyros kaki cv. Taubaté) were found to be: 1.6M NaCl + 1% (v/v) Triton X‐100 + 5% (w/v) bovine serum albumin (BSA) for astringent fruit and 0.2M NaCl for nonastringent fruit. During ripening with ethephon, PME showed a close variation with respiration up to the climacteric peak when fruits became soft and nonastringent. Untreated astringent fruits showed little variation in either respiration or PME activity.

Preharvest AVG Treatment of ‘Bartlett’ Pear Fruits: Effects on Ripening, Color Change, and Volatiles

Abstract Preharvest treatment with 400 ppm aminoethoxyvinylglycine (AVG) delayed the ripening of ‘Bartlett’ pears kept at 20°C. The effect was not uniform, with the delay in ripening ranging from a few to 112 days. Prolonged storage at 20° was accompanied by a relatively steady, low level of respiration, gradual degreening, and partial suppression of the eventual climacteric. The combined effect of AVG treatment and prolonged ‘nonripening’ storage at 20° led to a marked attentuation of the production of 4 readily measurable volatiles including methyl-, ethyl-, and hexylacetate as identified by cochromatography and combined gas chromatography–mass spectrometry. Surprisingly, prolonged storage resulted in a much increased production of 2 other volatile fractions when AVG inhibition was reversed by C2H4 treatment. The emanation of all measured volatiles was closely coincident with the climacteric peak. These observations confirm prior reports of disuniform effects of preharvest AVG treatment and reveal that metabolic transitions during prolonged, nonripening storage may have adverse effects on fruit quality.

Induced ethylene evolution and climacteric-like respiration in Rhizopus-infected rin and nor tomato mutants

Rhizopus stolonifer infection markedly stimulated ethylene and CO2 evolution from fruits of the two non-ripening tomato mutants, rin and nor, and caused the climacteric-like patterns of respiration typical of normal fruit. These changes were first detected 24 h after inoculation with maximum rates of ethylene production occurring on the second day after inoculation, while maximum respiratory rates, coinciding with total fruit rotting, occurred only on the fourth day. In uninfected mutant fruits, whether wounded or not, the rates of respiration and of ethylene evolution remained low and constant. Normal tomato fruits inoculated at the green fruit stage showed both an acceleration and an overall increase in the two physiological processes but, when inoculated in the postclimacteric red fruit, new climacteric peaks, with rates higher than those of the uninfected fruit followed fungal infection. The highest rates of ethylene production ocourred in the healthy tissues at the margin of the rot and little was produced by the rotted tissues containing actively growing fungus. Thus, ethylene appears to be a product of the host's metabolism, not of the fungurs.

Differences in Biochemical Composition between ‘Beurre d’Anjou’ and ‘Bose’ Pears during Fruit Development and Storage1

Abstract A comparative study in 1979 and 1980 between ‘Anjou’, a long-keeping winter pear and ‘Bose’, a shorter keeping winter pear (both Pyrus communis L.) revealed that ethanol-insoluble matter, titratable acids, soluble solids, proteins, and free amino acids in fruit of both cultivars during fruit development, maturation, and storage period fluctuated from season to season and were not associated with their difference in postharvest life. Malic acid was the major fraction of organic acids in both cultivars, and it declined at a faster rate in ‘Bosc’ than in ‘Anjou’ during storage at −1.1°C. The amounts of citric, oxaloacetic, and fumaric acids were higher in ‘Bosc’ than in ‘Anjou’ and were maintained at constant levels throughout the storage period. Internal ethylene in both cultivars early in fruit development was about 0.3 ppm and decreased rapidly to below 0.07 ppm during late fruit development and harvest period. For 2 seasons, ‘Bosc’ was capable of ripening after less than 20 days of chilling at −1.1°C when its internal ethylene increased to 0.2 ppm, while ‘Anjou’ required at least 50 days of chilling to develop the ripening capacity coincident with an internal ethylene above 2.0 ppm. Internal ethylene accumulated in ‘Bosc’ about 8 times faster than in ‘Anjou’ during the first 60 days of storage at −1.1° and reached an equilibrium at 40 ppm for ‘Bosc’ and only 5 ppm for ‘Anjou’ during the remaining storage period. After any corresponding period of cold storage, both ethylene and CO2 productions of ‘Bosc’ at ripening temperature of 20° were higher than those of ‘Anjou’, and ‘Bosc’ also required fewer days to reach the climacteric peaks than did ‘Anjou’.

Ultrastructure of the Mesocarp of Mature Avocado Fruit and Changes Associated with Ripening

The mesocarp tissue of ripening avocado fruits was studied by freeze fracture, thin section, and scanning electron microscopy. CO2 and ethylene production by individual fruit were monitored, and samples were analyzed at several stages of the ripening process. The tissue is composed primarily of large, isodiametric, lipid-containing parenchyma cells. At maturity these cells contain the normal complement of plant cell organelles, and all membranes appear intact. When ripening begins, several changes in the ultrastructure occur. The most obvious changes are a loosening and eventual breakdown of the cell wall, and swelling and vesiculation of the rough endoplasmic reticulum. In freeze fracture replicas a significant increase in the number of intramembranous particles in the EF face of the plasmamembrane was observed at the climacteric peak. In postclimacteric, soft fruit the particle density of the EF face of the plasmamembrane decreased to the density observed in the membrane of preclimacteric cells. All of the organelles and membranes appear whole and intact whether examined by thin section, freeze fracture, or scanning electron microscopy. However, the cell walls in postclimacteric fruit have almost completely disappeared. These results indicate that the ripening process per se in avocados does not involve a complete loss of compartmentalization nor a breakdown of organelle and membrane integrity. It may, however, lead to these or similar senescence changes as a result of the loss of the cell walls. The variations in particle density of the plasmamembrane during ripening may reflect one or more of several structural, compositional, or functional membrane phenomena, and this aspect of ripening warrants further study.

Identification of multiple forms of phosphofructokinase in ripening dwarf cavendish banana

The levels of six glycolytic intermediates and the activity of phosphofructokinase (PFK) were determined in Dwarf Cavendish banana at different stages of ripening between harvest and senescence. There was a 2.3-fold increase in the level of fructose- 1,6-diphosphate between the preclimacteric and climacteric peak stage. The PFK preparations from preclimacteric and climacteric peak stages were purified ca 15-fold using Blue-Sepharose affinity chromatography. The clectrophoretic studies with the enzyme preparations ofthese two stages ofripening indicated the presence of two forms of PFK at both stages of ripening.