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.     

Respiratory Contribution of the Alternate Path during Various Stages of Ripening in Avocado and Banana Fruits

The respiration of fresh slices of preclimacteric avocado (Persea americana Mill. var. Hass) and banana (Musa cavendishii var. Valery) fruits is stimulated by cyanide and antimycin. The respiration is sensitive to m-chlorobenzhydroxamic acid in the presence of cyanide but much less so in the presence of antimycin. In the absence of cyanide the contribution of the cyanide-resistant pathway to the coupled preclimacteric respiration is zero. In uncoupled slices, by contrast, the alternate path is engaged and utilized fully in avocado, and extensively in banana. Midclimacteric and peak climacteric slices are also cyanide-resistant and, in the presence of cyanide, sensitive to m-chlorobenzhydroxamic acid. In the absence of uncoupler there is no contribution by the alternate path in either tissue. In uncoupled midclimacteric avocado slices the alternate path is fully engaged. Midclimacteric banana slices, however, do not respond to uncouplers, and the alternate path is not engaged. Avocado and banana slices at the climacteric peak neither respond to uncouplers nor utilize the alternate path in the presence or absence of uncoupler.

The maximal capacities of the cytochrome and alternate paths, V_cyt and V_alt, respectively, have been estimated in slices from preclimacteric and climacteric avocado fruit and found to remain unchanged. The total respiratory capacity in preclimacteric and climacteric slices exceeds the respiratory rise which attends fruit ripening. In banana V_alt decreases slightly with ripening.

The aging of thin preclimacteric avocado slices in moist air results in ripening with an accompanying climacteric rise. In this case the alternate path is fully engaged at the climacteric peak, and the respiration represents the total potential respiratory capacity present in preclimacteric tissue. The respiratory climacteric in intact avocado and banana fruits is cytochrome path-mediated, whereas the respiratory climacteric of ripened thin avocado slices comprises the alternate as well as the cytochrome path. The ripening of intact fruits is seemingly independent of the nature of the electron transport path.

Uncouplers are thought to stimulate glycolysis to the point where the glycolytic flux exceeds the oxidative capacity of the cytochrome path, with the result that the alternate path is engaged.

     

Phosphorylation in avocado fruit slices in relation to the respiratory climacteric

The rate of uptake of inorganic phosphate by tissue discs from both preclimacteric and climacteric peak avocados is linear for at least 60 minutes. The loss of ³²P upon excessive washing was much greater from peak than from preclimacteric tissue. Short incubation periods and, most important, rapid washing procedures are essential for meaningful comparisons.

Phosphate esterification proceeded at a much greater rate in climacteric than in preclimacteric tissue. The phosphorylation was sensitive to 2,4-dinitrophenol (DNP). The ADP to ATP ratio decreased materially with the advance of ripening. It was concluded that neither uncoupling nor acceptor control can account for the onset of the respiratory rise. Changes in permeability appear to play an important role in fruit metabolism during the climacteric.

     

钙对香蕉采后果实呼吸的影响

本实验对香蕉成熟过程中水溶性钙的变化及CaCl_2溶液真空浸渗处理对香蕉果实呼吸作用的影响进行研究,结果表明,香蕉成熟期间,水溶性钙含量随成熟度的提高而增加,果实呼吸跃变上升前期,水溶性钙的增加尤为明显,果皮和果肉中水溶性钙含量与采后成熟天数呈正相关。以0.05mol/l和0.1mol/l CaCl_2溶液真空浸渗,可使香蕉果实呼吸跃变高峰延迟,但没有明显降低跃变峰值。     

Respiration during Postharvest Development of Soursop Fruit, Annona muricata L

Fruit of soursop, Annona muricata L., showed increased CO₂ production 2 days after harvest, preceding the respiratory increase that coincided with autocatalytic ethylene evolution and other ripening phenomena. Experiments to alter gas exchange patterns of postharvest fruit parts and tissue cylinders had little success.

The respiratory quotient of tissue discs was near unity throughout development. 2,4-Dinitrophenol uncoupled respiration more effectively than carbonylcyanide m-chlorophenylhydrazone; 0.4 millimolar KCN stimulated, 4 millimolar salicylhydroxamic acid slightly inhibited, and their combination strongly inhibited respiration, as did 10 millimolar NaN₃. Tricarboxylic acid cycle members and ascorbate were more effective substrates than sugars, but acetate and glutarate strongly inhibited.

Disc respiration showed the same early peak as whole fruit respiration; this peak is thus an inherent characteristic of postharvest development and cannot be ascribed to differences between ovaries of the aggregatetype fruit. The capacity of the respiratory apparatus did not change during this preclimacteric peak, but the contents of rate-limiting malate and citrate increased after harvest.

It is concluded that the preclimacteric rise in CO₂ evolution reflects increased mitochondrial respiration because of enhanced supply of carboxylates as a substrate, probably induced by detachment from the tree. The second rise corresponds with the respiration during ripening of other climacteric fruits.

     

Simple brominated phenols in the bluegreen alga Calothrix brevissima West

Summary Changes in the percent apparent free space (%AFS) in pericarp tissue of tomatoes at successive ripening stages were determined, using the climacteric sequence as a standard. A considerable increase in %AFS occurred in the preclimacteric stage. This initial rise was followed by a decline until shortly after the onset of the climacteric rise, apparently due to a temporary stabilization effect, followed by a final increase in permeability.     

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.     

Role of sucrose phosphate synthase in sucrose biosynthesis in ripening bananas and its relationship to the respiratory climacteric

During ripening of bananas (Musa spp. [AAA group, Cavendish subgroup]), there is a massive conversion of starch to sucrose. Also during ripening there is a rise in respiration known as the respiratory climacteric. In this study changes in carbohydrate content, activities of starch and sucrose metabolizing enzymes, and respiration were measured to assess their potential interrelationships. Sucrose phosphate synthase activity increased dramatically during the first 4 days after initiation of ripening by ethylene treatment. Starch concentration decreased and sucrose concentration increased during this time period. Developmental changes in sucrose phosphate synthase activity were measured with limiting substrate (plus Pi) and saturating substrate concentrations. Activities were not parallel under the two assay conditions, providing tentative evidence that kinetically different forms of the enzyme may exist at different stages of ripening. Sucrose accumulation rate was most highly correlated with sucrose phosphate synthase activity assayed with limiting substrate concentrations (plus Pi). The cumulative amount of CO₂ respired during ripening was positively correlated with sugar accumulation (R² = 0.97). From this linear regression it was calculated that a constant 0.605 millimoles of CO₂ was evolved per mole of sucrose formed throughout ripening. Using this quantity, the percentage of the total respiratory ATP produced which was required for the conversion of starch to sucrose was calculated assuming different models for carbon export from the amyloplast. The results suggest that sucrose biosynthesis during ripening constitutes a significant sink for respiratory ATP.     

Cyanide-Resistant Respiration in Relation to Fruit Respiratory Climacteric

Changes in respiratory rate and the effects of respiratory inhibitorson respiration were determined in apple (Malus sylvestris cv. Delicious) and red pepper (Capsicum fructescens) fruits dusting different stages of development and ripening.The results showed that there was an abrupt rise in respiration daring ripening inapple fruit, but the respiration of the red pepper declined continuously throughout theripening period. Thus the apple is climacteric and the red pepper is non-climacteric fruit. The respiration of apple fruit was sensitive to KCN (1 mM) during the period ofdevelopment but changed to CLAM-sensitive and CN-resistant during preclimactericand climacteric phases, indicating that a diversion of respiratory pathways from the cy-tochrome path to the alternative path has occurred. The respiration of the red pepperfruit was CN-sensitive thoughout the whole period of fruit ripening, suggesting thatthe operation of the CN-resistant path was insignificant. Slices from climacteric apple fruits developed induced .respiration after aging, bothKCN and CLAM (1 mM) inhibited the induced respiratic considerably. However, slices from red pepper fruits showed no evidence of induced respiration after aging. Slices from climacteric apple fruits infiltrated with 3 mM CLAM before aging, reducedthe peak of the induced respiration by about 30%, indicating that the development ofinduced respiration was suppressed by the presence of CLAM. The above results indicated that the: climacteric fruits were characterized by diversion of traffic from the cytochrome path to the alternative path during ripening andby the development of induced respiration after slicing and aging. While in nonclimacteric fruits no .diversion of electron transport path was observed during ripening andno induced respiration occurred after aging. Although both the eytochrome and alternative pathways were present in the tissue of red pepper fruits, the alternative pathwas not operating except when the cytochrome path was blocked or was saturated by electron flow.     

Interrelationship of Gene Expression, Polysome Prevalence, and Respiration during Ripening of Ethylene and/or Cyanide-Treated Avocado Fruit

Upon initiation of ripening in avocado fruit (Persea americana Mill. cv Hass) with 10 microliters/liter ethylene, polysome prevalence and associated poly(A)⁺ mRNA increase approximately 3-fold early in the respiratory climacteric and drop off to preclimacteric levels at the peak of the respiratory climacteric. The increase in poly(A)⁺ mRNA on polysomes early in the respiratory climacteric constitutes a generic increase in constitutive mRNAs. New gene expression associated with ripening is minimal but evident after 10 hours of ethylene treatment and continues to increase relative to constitutive gene expression throughout the climacteric. The respiratory climacteric can be temporally separated into two phases. The first phase is associated with a general increase in protein synthesis, whereas the second phase reflects new gene expression and accumulation of corresponding proteins which may be responsible for softening and other ripening characteristics. A major new message on polysomes that arises concomitantly with the respiratory climacteric codes for an in vitro translation product of 53 kilodaltons which is immunoprecipitated by antiserum against avocado fruit cellulase.

Cyanide at 500 microliters/liter fails to affect the change in polysome prevalance or new gene expression associated with the ethylene-evoked climacteric in avocado fruit. Treatment of fruit with 500 microliters/liter cyanide alone initiates a respiratory increase within 4 hours, ethylene biosynthesis within 18 hours, and new gene expression akin to that educed by ethylene within 20 hours of exposure to cyanide.

     

Effect of Infection by Hypomyces solani f. sp. Cucurbitac on Apparent Free Space, Cell Membrane Permeability, and Respiration of Squash Hypocotyls

Initial symptoms and increases in respiration, apparent free space, and rate of leakage of amino acids occurred concomitantly in squash (Cucurbita maxima Dcne) hypocotyls infected by Hypomyces solani f. sp. cucurbitae Snyd. and Hans. Young, rapidly expanding lesions had greater respiratory rates and apparent free space than comparable tissues from healthy plants.

Hypocotyl tissues above (1-45 mm) lesions possessed greater endogenous respiratory rates (2-3 times) and lower respiratory quotients than similar tissues from healthy plants. But no differences were found in membrane permeability to nonelectrolytes and water and in apparent free space between cells above lesions and healthy hypocotyls.

Host cells contiguous to fungal hyphae at lesion margins were completely permeable to solutes and failed to accumulate neutral red or exhibit cyclosis.

     

Upsurge of particulate peroxidase in ripening banana fruit

Peroxidase was isolated from the pulp of ripening banana fruit and assayed with o-dianisidine as hydrogen-donor. Cell macerates contained soluble and particle-bound peroxidase. Soluble peroxidase levels did not appreciably differ in pre-climacteric, climacteric and post-climacteric fruit. Particulate peroxidase levels increased 3-fold with the initiation of the respiration climacteric and gradually declined with the onset of senescence. Bound peroxidase was released from cell wall and membrane fractions with washing in 0–8 M CaCl₂.     

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.     

Senescense: association of synthesis of Acid phosphatase with banana ripening

During ripening of banana (Musa sapientum L., var. Gros Michel or Valery) acid phosphatase activity increases 13-to 26-fold in the precipitate and 2- to 4-fold in the supernatant fraction of tissue homogenates. These increases are closely correlated with the onset and peak of the climacteric. The precipitate enzyme may be extracted with Triton X-100, CaCl₂ or NaCl; about 80% of it is in a 500g precipitate. Studies on effect of tonicity of the grinding medium indicate that the precipitate enzyme is desorbed from membrane or cell wall surfaces, and is not released as a result of lysis of membranes. The development of acid phosphatase during aging of tissue slices is the same as in intact fruit. Short term studies of tissue slices with cycloheximide and actinomycin D indicate that the increase in activity is owed to new enzyme synthesis, which is dependent upon synthesis of RNA. The possible effects of the increase in acid phosphatase on ripening are discussed.     

The climacteric in ripening tomato fruit

Phosphofructokinase is identified as the regulator reaction activated at the onset of the climacteric rise in respiration of the ripening tomato fruit (Lycopersicon esculentum Mill). The concentration of ATP in the fruit increases to a maximum value after the climacteric peak of respiration is past. Orthophosphate is proposed as the most probable activator of phosphofructokinase in the ripening fruit.     

Non‐invasive quantification of ethylene in attached fruit headspace at 1 p.p.b. by gas chromatography–mass spectrometry

Ethylene is a gaseous plant hormone involved in defense, adaptations to environmental stress and fruit ripening. Its relevance to the latter makes its detection highly useful for physiologists interested in the onset of ripening. Produced as a sharp peak during the respiratory burst, ethylene is biologically active at tens of nl L^?1. Reliable quantification at such concentrations generally requires specialized instrumentation. Here we present a rapid, high‐sensitivity method for detecting ethylene in attached fruit using a conventional gas chromatography–mass spectrometry (GC‐MS) system and in situ headspace collection chambers. We apply this method to melon (Cucumis melo L.), a unique species consisting of climacteric and non‐climacteric varieties, with a high variation in the climacteric phenotype among climacteric types. Using a population of recombinant inbred lines (RILs) derived from highly climacteric (‘Védrantais’, cantalupensis type) and non‐climacteric (‘Piel de Sapo’, inodorus type) parental lines, we observed a significant variation for the intensity, onset and duration of the ethylene burst during fruit ripening. Our method does not require concentration, sampling times over 1 h or fruit harvest. We achieved a limit of detection of 0.41 ± 0.04 nl L^?1 and a limit of quantification of 1.37 ± 0.13 nl L^?1 with an analysis time per sample of 2.6 min. Validation of the analytical method indicated that linearity (>98%), precision (coefficient of variation ≤2%) and sensitivity compared favorably with dedicated optical sensors. This study adds to evidence of the characteristic climacteric ethylene burst as a complex trait whose intensity in our RIL population lies along a continuum in addition to two extremes.     

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.