Cellulase, Fruit Softening and Abscission in Red RaspberryRubus idaeusL. cv Glen Clova

The ripening of raspberry fruit (Rubus ideausL. cv Glen Clova)is associated with a climacteric rise in ethylene production.As the fruit pigments change from green to red there is a progressivesoftening, loss of skin strength and a breakdown of cell wallsin the mesocarp. An increase in cellulase (endo-1,4-ß-D-glucanase)in both drupelets and receptacles accompanies these changes.The localization of cellulase in the regions of the fruit associatedwith abscission zones suggest the enzyme may be involved infruit separation as well as softening. Rubus idaeusL; raspberry; fruit ripening; ethylene; abscission; cell wall breakdown; cellulase; endo-1,4-ß-D-glucanase     

Carotenoid changes in the peel of ripening persimmon (Diospyros kaki) cv Triumph

The changes of the carotenoid pigments in the peel of ripening persimmon (Diospyros kaki) cv Triumph were followed for an entire season. During ripening, the total carotenoid decreased until colour break, then increased gradually and drastically at the last ripening stages. The chloroplast carotenoid pattern of the unripe fruit changed into a chromoplast pattern in which cryptoxanthin was the predominant pigment, reaching a level between 40 and 50% of the total carotenoids. It accumulated continuously at a rate of approximately 10% at each 2 week interval, its percentage being characteristic for each ripening stage. Other major pigments at levels of approximately 10% of the total carotenoids were zeaxanthin, antheraxanthin and violaxanthin. In the fully ripe fruit, ripened both on and off the tree, lycopene which was not present before was found as the second major pigment. This unusual pattern change is discussed.     

A role for jasmonates in climacteric fruit ripening

Jasmonates are a class of oxylipins that induce a wide variety of higher-plant responses. To determine if jasmonates play a role in the regulation of climacteric fruit ripening, the effects of exogenous jasmonates on ethylene biosynthesis and color, as well as the endogenous concentrations of jasmonates were determined during the onset of ripening of apple (Malus domestica Borkh. cv. Golden Delicious) and tomato (Lycopersicon esculentum Mill. cv. Cobra) fruit. Transient (12 h) treatment of pre-climacteric fruit discs with exogenous jasmonates at low concentration (1 or 10 μM) promoted ethylene biosynthesis and color change in a concentration-dependent fashion. Activities of both 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase and ACC synthase were stimulated by jasmonate treatments in this concentration range. The endogenous concentration of jasmonates increased transiently prior to the climacteric increase in ethylene biosynthesis during the onset of ripening of both apple and tomato fruit. The onset of tomato fruit ripening was also preceded by an increase in the percentage of the cis-isomer of jasmonic acid. Inhibition of ethylene action by diazocyclopentadiene negated the jasmonate-induced stimulation of ethylene biosynthesis, indicating jasmonates act at least in part via ethylene action. These results suggest jasmonates may play a role together with ethylene in regulating the early steps of climacteric fruit ripening. Received: 14 August 1997 / Accepted: 4 October 1997     

The Synthesis of Ethylene in Melon Fruit during the Early Stage of Ripening

The levels of mRNA and polypeptide for a 1-aminocyclopropane-1-carboxylate(ACC) oxidase were studied to identify the tissues in whichthe synthesis of ethylene first occurs during the initial stageof ripening. RNA and immunoblot analysis showed that the levelsof the mRNA and polypeptide for ACC oxidase were very low inunripe fruit. They first became detectable in the placentaltissue at the pre-climacteric stage, and then their levels increasedin the mesocarp tissue during the climacteric increase in theproduction of ethylene. Two mRNAs for ACC synthase (transcribedfrom ME-ACS1 and ME-ACS2) were detected in the placental tissueand seeds at the pre-climacteric stage, but only the level ofME-ACS1 mRNA, which has been characterized as the mRNA for awound-inducible ACC synthase, increased in mesocarp, placentaltissues and seeds during ripening. The level of ME-ACS2 mRNAthat was isolated from etiolated seedlings of melon, did notchange markedly during ripening. These results suggest thatthe central region of melon fruit (placental tissue and seeds)plays a major role in the production of ethylene during theearly stage of ripening. ³These three authors made equal contribution to this study.     

Expression of MdCAS1 and MdCAS2, encoding apple beta-cyanoalanine synthase homologs, is concomitantly induced during ripening and implicates MdCASs in the possible role of the cyanide detoxification in Fuji apple (Malus domestica Borkh.) fruits

Fruit ripening involves complex biochemical and physiological changes. Ethylene is an essential hormone for the ripening of climacteric fruits. In the process of ethylene biosynthesis, cyanide (HCN), an extremely toxic compound, is produced as a co-product. Thus, most cyanide produced during fruit ripening should be detoxified rapidly by fruit cells. In higher plants, the key enzyme involved in the detoxification of HCN is β-cyanoalanine synthase (β-CAS). As little is known about the molecular function of β-CAS genes in climacteric fruits, we identified two homologous genes, MdCAS1 and MdCAS2, encoding Fuji apple β-CAS homologs. The structural features of the predicted polypeptides as well as an in vitro enzyme activity assay with bacterially expressed recombinant proteins indicated that MdCAS1 and MdCAS2 may indeed function as β-CAS isozymes in apple fruits. RNA gel-blot studies revealed that both MdCAS1 and MdCAS2 mRNAs were coordinately induced during the ripening process of apple fruits in an expression pattern comparable with that of ACC oxidase and ethylene production. The MdCAS genes were also activated effectively by exogenous ethylene treatment and mechanical wounding. Thus, it seems like that, in ripening apple fruits, expression of MdCAS1 and MdCAS2 genes is intimately correlated with a climacteric ethylene production and ACC oxidase activity. In addition, β-CAS enzyme activity was also enhanced as the fruit ripened, although this increase was not as dramatic as the mRNA induction pattern. Overall, these results suggest that MdCAS may play a role in cyanide detoxification in ripening apple fruits.     

The respiratory climacteric is present in Charentais (Cucumis melo cv. Reticulatus F1 Alpha) melons ripened on or off the plant

Ripening of climacteric fruit is accompanied by an increasein respiration and autocatalytic ethylene synthesis. In harvestedmelons, there is variation in the magnitude and duration ofthe respiratory climacteric depending on the cultivar. It hasrecently been reported that, while the ripening-associated increasein ethylene production is present, the respiratory climactericis absent in ripening melon fruit attached to the plant, leadingto the suggestion that climacteric respiration is an artifactof harvest. To address the universality of this phenomenon,ripening behaviour in the melon cultivar Charentais (Cucumismelo cv. Reticulatus F1 Alpha), was investigated and the resultsshow that the respiratory climacteric occurs in fruit ripenedboth on and off the plant. Key words: Cucumis melo, ethylene, respiratory climacteric     

Acid phosphatase in plant tissues I. Changes in activity and multiple forms in tea leaves and tomato fruit during maturation and senescence

Multiple forms of acid phosphatase varied in tea leaves (Camelliasinensis L.) depending on variety and the age of the leaves.Polyacrylamide gel electrophoresis indicated the presence ofsix multiple forms in extracts of young leaves and seven inextracts of mature leaves of the variety Hatzumomizi. VarietyBenihomare showed five bands in zymograms of young-leaf extractsand six in zymograms of mature-leaf extracts. In addition tothe appearance of the new band, the relative intensity of stainingin two other bands changed during maturation. One decreasedand one increased in intensity. The components were partiallyseparated by CM-cellulose chromatography and isoelectric focusing.Total activity of acid phosphatase did not change greatly ona fresh-weight basis during maturation and aging. Extracts of immature green tomato (Lycopersicon esculentum Mill.)fruit showed six bands on polyacrylamide gels. Isoelectric focusingand subsequent electrophoresis of the fractions obtained atdifferent pH values, produced evidence for seven acid phosphatasecomponents in tomato extracts. Unlike the situation with certainother climacteric fruit, acid phosphatase in tomato extractsdecreased steadily during ripening to a level of activity only25–40% that of immature green fruit extracts. It is thereforeconcluded that the onset of senescence in climacteric fruitsis not dependent on an increase in acid phosphatase. Substratespecificity, response to inhibitors, Km, and pH optima of partiallypurified fractions were similar to those of tea-leaf extracts. (Received November 29, 1972; )     

Alternative Respiration and Heat Production in Ripening Banana Fruits (Musa paradisiaca van Mysore Kadali)

The involvement of alternative respiration in thermogenesisduring the ripening of banana {Musa paradisiaca var. MysoreKadali) fruits, attached to a bunch, has been examined. Thetemperature of the youngest (unripened) banana fruit increasedfrom 27·0 ± 0·2°C to 30·8±0·1°C and the total respiration (in nmo1 oxygen min¹ g¹ drywt.) increased from 1·39·6 ± 5·5to 167·3 ± 7·0 at the fully ripened stage.Although the capacity for alternative respiration showed littlechange, the actual operation of this pathway increased from38 to 73% (p= 0·38 to 0·73) during ripening. Similarresults were obtained in fruits along the central axis at differentstages of ripening. It is suggested that alternative respirationmay contribute to the temperature rise observed in ripeningbanana fruit. Key words: Alternative respiration, tehrmogenesis, fruit ripening     

Changes in Activity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Three Peroxisomal Enzymes during Tomato Fruit Development and Ripening

Ribulose-1,5-bisphosphate carboxylase/oxygenase, catalase, glycolate oxidase, and hydroxypyruvate reductase activities on a protein and fresh weight basis were measured over seven stages of tomato fruit development and ripening. Ribulose-1,5-bisphosphate carboxylase decreased steadily during fruit development from 23 ± 8 nmoles per minute per milligram protein at the mature green stage to 13.4 ± 2 at the table ripe stage. There was no change in partially purified preparations of the enzyme in the ratio of carboxylase to oxygenase activity, which was about 10. Catalase activity reached a maximum during the climacteric, simultaneously with increased ethylene and CO₂ formation. Glycolate oxidase activity decreased during early stages of development and was barely detectable at the climacteric. Hydroxypyruvate reductase, associated with serine formation by the glycerate pathway, increased in specific activity during early stages of tomato fruit ripening. In the fruit of the rin tomato mutant, which does not ripen normally, none of these changes in enzyme activity occurred.     

Candidate genes and QTLs for fruit ripening and softening in melon

Different factors affect the quality of melon fruit and among them long shelf life is critical from the consumer’s point of view. In melon, cultivars showing both climacteric and non-climacteric ripening types are found. In this study we have investigated climacteric ripening and fruit softening using a collection of near-isogenic lines (NILs) derived from the non-climacteric melon parental lines PI 161375 (SC) and “Piel de Sapo” (PS). Surprisingly, we found that QTL eth3.5 in NIL SC3-5b induced a climacteric-ripening phenotype with increased respiration and ethylene levels. Data suggest that the non-climacteric phenotypes from PI 161375 and “Piel de Sapo” may be the result of mutations in different genes. Several QTLs for fruit flesh firmness were also detected. Candidate genes putatively involved in ethylene regulation, biosynthesis and perception and cell wall degradation were mapped and some colocations with QTLs were observed. These results may provide additional data towards understanding of non-climacteric ripening in melon.     

Changes in Adenosine Pyrophosphates in Cantaloupe Fruit Ripening Normally and after Treatment with Ethylene

During the climacteric rise in respiration of cantaloupe fruit(Cucumis melo L., var. reticulatus Naud.) the concentrationper gramme fresh weight of adenosine triphosphate (ATP) increasedand that of adenosine diphosphate (ADP) did not change; thusa net synthesis of adenosine pyrophosphate occurs during therespiratory climacteric. A net synthesis of protein which wasobserved was positively correlated with the concentration ofATP. Ethylene treatment stimulated a climacteric-like rise inthe respiration and in the rate of ripening in fruit harvestedat 9 to 32 days after anthesis. The ratio ATP/ADP increasedin fruit ripened with ethylene only when harvested 20 days ormore after anthesis.     

Maturation and Ripening of Fruit ofAmelanchier alnifoliaNutt. are Accompanied by Increasing Oxidative Stress

The extent of oxidative stress during ripening of saskatoon(AmelanchieralnifoliaNutt.) fruit was examined. Lipid peroxidation duringfruit development from the mature green to the fully ripe (purple)stage was evidenced by the accumulation of ethane and 2-thiobarbituricacid reactive substances. Fruit polar lipid and free fatty acidconcentrations also declined during ripening. Moreover, thedouble bond index of fatty acids in the polar lipid fractionfell during ripening, reflecting a progressive increase in thesaturation of membrane lipids. This increase in saturation waspartly due to a 65% decline in the concentration of linolenicacid. Activities of superoxide dismutase (SOD) and catalase(CAT) fell about 4-fold and 18-fold, respectively, during development,indicating higher potential for the accumulation of cytotoxicH₂O₂. Peroxidase activity remained relatively low and constantfrom the mature green to the dark red stage of development,then increased towards the end of ripening as fruits turnedpurple. Lipoxygenase (LOX) activity increased 2.5-fold fromthe mature green to the fully ripe stage. Tissue prints showedLOX to be present throughout fruit development and Western analysisrevealed that the increase in activity during ripening was dueto increased synthesis of the enzyme. Collectively, these resultsprovide evidence that ripening of this climacteric fruit isaccompanied by a substantial increase in free-radical-mediatedperoxidation of membrane lipids, probably as a direct consequenceof a progressive decline in the enzymatic systems responsiblefor catabolism of active oxygen species. The role of glutathione-mediatedfree-radical scavenging was also examined as a potential systemfor coping with this increased oxidative stress. Concentrationsof reduced and oxidized glutathione (GSSG) increased 2-foldand GSSG increased as a percentage of total glutathione, reflectingthe increase in oxidative status of fruits during ripening.Tissue prints of glutathione reductase (GRase) and transferase(GTase) showed these enzymes to be distributed throughout thepericarp at all stages of fruit development. GRase and GTaseactivities rose sharply during the later stages of fruit ripening,correlating well with substantial increases in the levels ofboth enzymes. Hence, the glutathione-mediated free-radical scavengingsystem was up-regulated towards the end of ripening, perhapsin response to the increasing oxidative stress resulting fromthe accumulation of lipid hydroperoxides from increased LOXactivity, in conjunction with a decline in SOD/CAT activities.Copyright1998 Annals of Botany Company Amelanchier alnifoliaNutt.; saskatoon fruit; ripening; oxidative stress.     

Role of Brassinosteroids, Ethylene, Abscisic Acid, and Indole-3-Acetic Acid in Mango Fruit Ripening

Rapid ripening of mango fruit limits its distribution to distant markets. To better understand and perhaps manipulate this process, we investigated the role of plant hormones in modulating climacteric ripening of ??Kensington Pride?? mango fruits. Changes in endogenous levels of brassinosteroids (BRs), abscisic acid (ABA), indole-3-acetic acid (IAA), and ethylene and the respiration rate, pulp firmness, and skin color were determined at 2-day intervals during an 8-day ripening period at ambient temperature (21?±?1°C). We also investigated the effects of exogenously applied epibrassinolide (Epi-BL), (+)-cis, trans-abscisic acid (ABA), and an inhibitor of ABA biosynthesis, nordihydroguaiaretic acid (NDGA), on fruit-ripening parameters such as respiration, ethylene production, fruit softening, and color. Climacteric ethylene production and the respiration peak occurred on the fourth day of ripening. Castasterone and brassinolide were present in only trace amounts in fruit pulp throughout the ripening period. However, the exogenous application of Epi-BL (45 and 60?ng?g^?1 FW) advanced the onset of the climacteric peaks of ethylene production and respiration rate by 2 and 1?day, respectively, and accelerated fruit color development and softening during the fruit-ripening period. The endogenous level of ABA rose during the climacteric rise stage on the second day of ripening and peaked on the fourth day of ripening. Exogenous ABA promoted fruit color development and softening during ripening compared with the control and the trend was reversed in NDGA-treated fruit. The endogenous IAA level in the fruit pulp was higher during the preclimacteric minimum stage and declined during the climacteric and postclimacteric stages. We speculate that higher levels of endogenous IAA in fruit pulp during the preclimacteric stage and the accumulation of ABA prior to the climacteric stage might switch on ethylene production that triggers fruit ripening. Whilst exogenous Epi-BL promoted fruit ripening, endogenous measurements suggest that changes in BRs levels are unlikely to modulate mango fruit ripening.     

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.     

Changes in the activities of some cell wall-degrading enzymes during development and ripening of Japanese pear fruit (Pyrus serotina Rehder var. culta Rehder)

1. Based on changes in DNA content per whole fruit and wholefruit weight during development, the development of Japanesepear fruit (cultivars Hosui and 93-3) was divided into celldivision, pre-enlargement, enlargement and ripening stages. 2. A climacteric rise in respiration with ethylene evolutionwas recognized although it was not very marked. 3. The ratio of pectinic acid to total pectin content increasedwith ripening. Total pectin content on a DNA content basis increasedclearly in the pre-enlargement and ripening stages, but roughlyremained constant in the cell division and enlargement stages. 4. Changes in the activities of cell wall-degrading enzymes,i.e. endocellulase, exocellulase, polygalacturonase, pectinmethylesterase and ß-galactosidase, were investigatedduring fruit growth. The activities per fresh weight of allenzymes, except exocellulase, were fairly high in the cell divisionand pre-enlargement stages, decreased in the enlargement stage,and increased remarkably with ripening or overripening, exceptfor pectin methylesterase. Endocellulase was present as an acidtype and neutral types. The former was more active than thelatter in the cell division and pre-enlargement stages, butwith ripening the reverse was found. On the other hand, theactivities, on a DNA content basis, in all the enzymes wereroughly constant, not decreasing during cell division, pre-enlargementand enlargement stages, but increasing extensively with ripening.That is, the lowering of these enzyme activities per g freshweight in the enlargement stage seemed not to be due to inactivationor stimulation of enzyme degeneration. The extensive enhancementsof cell wall-degrading enzyme activities with ripening or overripeningseem to be closely related to the softening or pithiness ofthe fruit. ¹ This paper is Contribution A-63, Fruit Tree Res. Sta. (Received June 11, 1976; )     

Downregulation of RdDM during strawberry fruit ripening

Background

Recently, DNA methylation was proposed to regulate fleshy fruit ripening. Fleshy fruits can be distinguished by their ripening process as climacteric fruits, such as tomatoes, or non-climacteric fruits, such as strawberries. Tomatoes undergo a global decrease in DNA methylation during ripening, due to increased expression of a DNA demethylase gene. The dynamics and biological relevance of DNA methylation during the ripening of non-climacteric fruits are unknown.

Results

Here, we generate single-base resolution maps of the DNA methylome in immature and ripe strawberry. We observe an overall loss of DNA methylation during strawberry fruit ripening. Thus, ripening-induced DNA hypomethylation occurs not only in climacteric fruit, but also in non-climacteric fruit. Application of a DNA methylation inhibitor causes an early ripening phenotype, suggesting that DNA hypomethylation is important for strawberry fruit ripening. The mechanisms underlying DNA hypomethylation during the ripening of tomato and strawberry are distinct. Unlike in tomatoes, DNA demethylase genes are not upregulated during the ripening of strawberries. Instead, genes involved in RNA-directed DNA methylation are downregulated during strawberry ripening. Further, ripening-induced DNA hypomethylation is associated with decreased siRNA levels, consistent with reduced RdDM activity. Therefore, we propose that a downregulation of RdDM contributes to DNA hypomethylation during strawberry ripening.

Conclusions

Our findings provide new insight into the DNA methylation dynamics during the ripening of non-climacteric fruit and suggest a novel function of RdDM in regulating an important process in plant development.

     

A novel tomato F‐box protein,SlEBF3, is involved in tuning ethylene signaling during plant development and climacteric fruit ripening

Ethylene is instrumental to climacteric fruit ripening and EIN3 BINDING F‐BOX (EBF) proteins have been assigned a central role in mediating ethylene responses by regulating EIN3/EIL degradation in Arabidopsis. However, the role and mode of action of tomato EBFs in ethylene‐dependent processes like fruit ripening remains unclear. Two novel EBF genes, SlEBF3 and SlEBF4, were identified in the tomato genome, and SlEBF3 displayed a ripening‐associated expression pattern suggesting its potential involvement in controlling ethylene response during fruit ripening. SlEBF3 downregulated tomato lines failed to show obvious ripening‐related phenotypes likely due to functional redundancy among SlEBF family members. By contrast, SlEBF3 overexpression lines exhibited pleiotropic ethylene‐related alterations, including inhibition of fruit ripening, attenuated triple‐response and delayed petal abscission. Yeast‐two‐hybrid system and bimolecular fluorescence complementation approaches indicated that SlEBF3 interacts with all known tomato SlEIL proteins and, consistently, total SlEIL protein levels were decreased in SlEBF3 overexpression fruits, supporting the idea that the reduced ethylene sensitivity and defects in fruit ripening are due to the SlEBF3‐mediated degradation of EIL proteins. Moreover, SlEBF3 expression is regulated by EIL1 via a feedback loop, which supposes its role in tuning ethylene signaling and responses. Overall, the study reveals the role of a novel EBF tomato gene in climacteric ripening, thus providing a new target for modulating fleshy fruit ripening.     

Pathways of Uptake and Accumulation of Sugars in Tomato Fruit

The route of sucrose unloading from the conducting tissue, theregulation of sucrose hydrolysis and the uptake and subsequentmetabolism of sugars were investigated in the rapidly growingtomato fruit. During the first two weeks of fruit enlargement, the vacuoleaccounted for more than 85% of the protoplast volume and theintercellular space accounted for 20% of the fruit placentaltissue. The plasmodesmatal frequency was highest between phloemparenchyma cells and lowest between phloem sieve cells and phloemparenchyma. The total invertase activity was about 8 µmolglucose g^–1 d. wt min^–1 during the rapid growingperiod and increased six-fold at ripening. The wall-bound invertaseaccounted for less than 11% of the total activity. Invertaseactivity increased with increasing sucrose concentrations (upto 50 mM) in the incubation medium, but decreased at higherconcentrations. Sucrose synthase activity could only be detectedwhen fruit was older than 19 d. The uptake and metabolism of sugars by fruit cells were investigatedby incubation of fruit slices with ¹⁴C-sugars for 3 h. The uptakeof sucrose increased with the sucrose concentration up to 200mM. The rate of glucose uptake and its conversion to the ethanol-insolublefraction were higher than those of sucrose. The uptake of sucrosedid not compete with that of glucose or vice versa, providedthe osmotic potential of the incubation solution was maintainedconstant. The uptake of sucrose was not inhibited by metabolicinhibitors such as PCMBS, CCCP, sodium azide or vanadate. TheATPase activity in the fruit tissue was low. These findings did not identify conclusively the mode of sucroseunloading. However, the uptake of sugars by fruit cells is non-specificand does not appear to require a membrane carrier or plasmalemmaATPase to provide energy for sucrose uptake. Fruit, invertase, Lycopersicon esculentum, phloem unloading, plasmodesmata, sucrose