Changes in hemicellulose-degrading enzymes during development and ripening of Japanese pear fruit

Seasonal changes in some hemicellulose-degrading enzymes inJapanese pear fruit were studied in connection with fruit development,softening and over-ripening. These enzyme activities per fruitfresh weight were very high during the cell division and preenlargementstages, and greatly decreased in the enlargement stage. Thereafter,they again exhibited clear increase with ripening. These enzymeactivities per cell (DNA content basis), however, were roughlyconstant throughout the cell division, pre-enlargement and enlargementstages. These cell wall degrading enzymes were divided intothe following three groups by their alteration patterns withripening, i.e. the first group including polygalacturonase,exocellulase and mannanase which seems to be associated withfruit softening, the second one including arabanase, ß-glucosidaseand endocellulase and the third one including xylanase, ß-xylosidaseand ß-galactosidase, both of which seem to functionin cellular breakdown with over-ripening. ¹ This paper is contribution A-87, Fruit Tree Research Station. (Received August 4, 1978; )     

Changes in cell wall polysaccharides and monosaccharides during development and ripening of Japanese pear fruit

1. Changes in polysaccharide and monosaccharide components in thecell wall were studied during cell division, cell enlargmementand softening in Japanese pear fruit. Wall polysaccharides werefractionated into water soluble carbohydrate, NaClO₂ solublecarbohydrate, EDTA soluble carbohydrate, acid soluble hemicellulose,alkali soluble hemicellulose and cellulose. These polysaccharideswere composed of glucose, uronic acid, xylose, arabinose, galactose,rhamnose, mannose and fucose.
2. The total polysaccharide contentof the cell wall per cell (DNAcontent basis) remained constantduring the cell division period(S1). But during the pre-enlargementperiod (S2) it began toincrease rapidly in spite of the slightnessof cell enlargement.Thereafter, during the enlargement period(S3) the polysaccharidesremained almost constant although thefruits enlarged dramatically,and the polysaccharides increasedsomewhat with ripening. Thequality of the polysaccharides,however, seemed to change activelyat each stage. This suggestedthat the extensive fruit enlargementdid not require an increasein polysaccharide content, and wasrather accompanied by thepartial breakdown or partial interconversionof polysaccharidecomponents already present.
3. The loss of arabinose and galactosein acid soluble hemicellulosewas prominant in fruit softeningoccurring in the ripening stage.The cellulose component decreasedwith overripening. Water solublepectin increased parallel tothe increase in total pectin withripening. On the other hand,xylose and non-cellulosic glucoseresidues did not alter withripening or overripening. Non-cellulosicglucose continued toaccumulate during cell enlargement.

¹ This paper is Contribution A-88, Fruit Tree Research Station. (Received August 4, 1978; )     

Molecular cloning of beta-galactosidase from Japanese pear (Pyrus pyrifolia) and its gene expression with fruit ripening

We have cloned a cDNA fragment encoding a beta-galactosidase from Japanese pear (Pyrus pyrifolia) fruit (JP-GAL). It contained an untranslated sequence of 182 nucleotides at the 5' end, a presumptive coding sequence of 2,193 nucleotides and an untranslated sequence of 268 nucleotides including a polyadenylation signal and a poly (A) tail at the 3' end. It encoded a protein with a calculated molecular weight of 80.9 kDa which consists of 731 amino acids. Both the nucleotide and the deduced amino acid sequences showed a 98% sequence identity with that obtained from the apple beta-galactosidase cDNA. The peptide sequence obtained from the purified Japanese pear beta-galactosidase III matched the deduced amino acid sequence of SVSYDHKAIIINGQKRILISG (amino acid 25-45). Northern blot analysis showed that the probe derived from JP-GAL hybridized to a single 2.6 kb RNA. The mRNA was detected solely in the fruit; none was detected in the buds, leaves, roots or shoots of the Japanese pear. The steady-state level of the beta-galactosidase mRNA was measured during fruit ripening in three cultivars, Housui, Kousui (early ripening) and Niitaka (late ripening). The results showed that regardless of the cultivar, no JP-GAL mRNA was detected in the immature fruit. Increment of the mRNA level with fruit ripening coincided with the increase in the beta-galactosidase III activity. Our results showed that the expression of JP-GAL correlated with fruit softening and JP-GAL may be beta-galactosidase III.     

Lupeol esters from the twig bark of Japanese pear (Pyrus serotina Rehd.) cv. Shinko.

Five new lupeol esters, lup-20(29)-ene-3beta-yl eicosanoate, docosanoate, tetracosanoate, hexacosanoate and octacosanoate, were isolated as a mixture from the twig bark of Japanese pear (Pyrus serotina Rehd.) cv. Shinko, together with lupeol and epifriedelinol. Their structures were determined by spectral analyses including 2D-NMR experiments.     

Changes in the phosphorylation state of sucrose synthase during development of Japanese pear fruit

Changes in the protein level and phosphorylation state of sucrose synthase (SS) were studied throughout the development of Japanese pear fruit. The level of SS protein was high at the young stage, dropped with fruit enlargement and increased again with fruit maturation. Antibody against phospho-Ser reacted with SS from young fruit, but did not react with SS that had been dephosphorylated by alkaline phosphatase (AP). The activities of SS isozymes were separated by ion-exchange chromatography. It was found that the fluctuation in SS activity was caused by two SS isozymes (SSI and SSII); (SSI reacted with antibody against phospho-Ser, while SSII did not. Phosphorylation of SS affected its kinetic parameters, that is, the affinity of phosphorylated SS for UDP was higher than that of dephosphorylated SS, while it was the contrary for UDP-glucose. The reaction of dephosphorylated SS was inclined toward sucrose synthesis more than that of phosphorylated SS. Phosphorylated SS protein was most abundant in young fruit, but decreased with fruit development, while non-phosphorylated SS protein increased in mature fruit. These results suggest that SS isoforms may be affected by post-translational modifications such as phosphorylation, and that the regulation of phosphorylation may potentially control the properties and functions of SS throughout the development of Japanese pear fruit.     

Changes in peel pigmentation during ripening of mango fruit (Mangifera indica var. Tommy Atkins)

The pigments in the peel of Tommy Atkins mango were analysed at six stages during ripening at 22 ^oC. The loss of green colour and the development of yellow colouration was associated with an almost complete loss of chlorophyll and an increase in carotenoids. Anthocyanin content showed a slight decrease during ripening. An ultrastructural study showed plastids in green fruit with a well developed grana network system. On ripening the chloroplasts underwent extensive disorganisation which was associated with the development of large osmiophilic globules.     

Identification of 1-aminocyclopropane-1-carboxylic acid synthase genes controlling the ethylene level of ripening fruit in Japanese pear (Pyrus pyrifolia Nakai)

The shelf life of Japanese pear fruit is determined by its level of ethylene production. Relatively high levels of ethylene reduce storage potential and fruit quality. We have identified RFLP markers tightly linked to the locus that determines the rate of ethylene evolution in ripening fruit of the Japanese pear. The study was carried out using sequences of two types of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase genes (PPACS1 and pPPACS2) and a ACC oxidase gene (PPAOX1) as probes on 35 Japanese pear cultivars expressing different levels of ethylene (0.0∼300 μl/kg fresh weight/h) in ripening fruit. When total DNA was digested with HindIII and probed with pPPACS1, we identified a band of 2.8 kb which was specific to cultivars having very high ethylene levels (≧10 μ1/kg f.w./h) during fruit ripening. The probe pPPACS2 identified a band of 0.8 kb specific to cultivars with moderate ethylene levels (0.5 μl/kg f.w./h–10 μl/kg f.w./h) during fruit ripening. The cultivars that produce high levels of ethylene possess at least one additional copy of pPPACS1 and those producing moderate levels of ethylene have at least one additional copy of pPPACS2. These results suggest that RFLP analysis with different ACC synthase genes could be useful for predicting the maximum ethylene level during fruit ripening in Japanese pear. Received: 1 July 1998 / Accepted: 6 October 1998     

Activities of antioxidant enzymes during strawberry fruit development and ripening

The analyses of some antioxidant enzyme activities were carried out in the course of strawberry fruits development and ripening. The catalase activity was maximum in small-sized green fruits, it decreased in middle-sized green fruits and increased again during the ripening stages. The highest superoxide dismutase and peroxidase activities were observed in white fruits.     

Behaviour of some mitochondrial enzymes in ripening tomato fruit

The activities of four mitochondrial enzymes were studied in four stages of ripening tomato fruit. The highest enzyme activity was recorded for malate dehydrogenase followed by cytochrome c oxidase. Succinate dehydrogenase and NADH oxidase levels were low and could only be determined in the green stage of the fruit. However, peaks of various enzyme activities coincided in identical mitochondrial fractions on the sucrose density gradient. Moreover, the levels of malate dehydrogenase and cytochrome c oxidase were constant during the ripening process while the other two enzymes, succinate dehydrogenase and NADH oxidase, declined. This might indicate that mitochondria retain some of their essential functions through the ripening process.     

Ultrastructural changes in the cell walls of ripening apple and pear fruit

Ultrastructural changes in the cell walls of “Calville de San Sauveur” apples (Malus sylvestris Mill) and “Spadona” pear (Pyrus communis L.) fruit were followed during ripening. In apple, structural alterations in cell walls became apparent at advanced stages of softening and showed predominantly dissolution of the middle lamella. In pears softening was also associated with the dissolution of the middle lamella, and in addition a gradual disintegration of fibrillar material throughout the cell wall. In fully ripe fruit almost all of the fibrillar arrangement in the cell wall was lost. Application of enzyme solutions containing polygalacturonase and cellulase to tissue discs from firm pear fruit led to ultrastructural changes observed in naturally ripening pears. In apple polygalacturonase alone was sufficient to dissolve the middle lamella region of the cell walls, as was also found to occur in naturally ripening fruit. In both apple and pear the cell wall areas containing plasmodesmata maintained their structural integrity throughout the ripening process. At advanced stages of ripening vesicles appeared in the vicinity of plasmodesmata.     

Partitioning of (13)C-photosynthate from spur leaves during fruit growth of three Japanese pear (Pyrus pyrifolia) cultivars differing in maturation date

BACKGROUND AND AIMS: In fruit crops, fruit size at harvest is an important aspect of quality. With Japanese pears (Pyrus pyrifolia), later maturing cultivars usually have larger fruits than earlier maturing cultivars. It is considered that the supply of photosynthate during fruit development is a critical determinant of size. To assess the interaction of assimilate supply and early/late maturity of cultivars and its effect on final fruit size, the pattern of carbon assimilate partitioning from spur leaves (source) to fruit and other organs (sinks) during fruit growth was investigated using three genotypes differing in maturation date. METHODS: Partitioning of photosynthate from spur leaves during fruit growth was investigated by exposure of spurs to (13)CO(2) and measurement of the change in (13)C abundance in dry matter with time. Leaf number and leaf area per spur, fresh fruit weight, cell number and cell size of the mesocarp were measured and used to model the development of the spur leaf and fruit. KEY RESULTS: Compared with the earlier-maturing cultivars 'Shinsui' and 'Kousui', the larger-fruited, later-maturing cultivar 'Shinsetsu' had a greater total leaf area per spur, greater source strength (source weight x source specific activity), with more (13)C assimilated per spur and allocated to fruit, smaller loss of (13)C in respiration and export over the season, and longer duration of cell division and enlargement. Histology shows that cultivar differences in final fruit size were mainly attributable to the number of cells in the mesocarp. CONCLUSIONS: Assimilate availability during the period of cell division was crucial for early fruit growth and closely correlated with final fruit size. Early fruit growth of the earlier-maturing cultivars, but not the later-maturing ones, was severely restrained by assimilate supply rather than by sink limitation.     

Development of microsatellite markers in the Japanese pear (Pyrus pyrifolia Nakai)

Thirteen polymorphic microsatellite loci were developed in the Japanese pear (Pyrus pyrifolia Nakai) by using an enriched genomic library. The obtained microsatellite loci showed a high degree of polymorphism in the Japanese pear with 3–6 alleles per locus. The average values of observed and expected heterozygosities among these 13 loci were 0.69 and 0.71, respectively. Ten microsatellites could successfully amplify loci in the European pear (Pyrus communis L.), which were highly polymorphic as well.     

Self-Incompatibility-Related RNases in Styles of Japanese Pear (Pyrus serotina Rehd.)

Isoelectric focusing revealed that S-genotypes of Japanese pearvarieties with gametophytic self-incompatibility were correlatedwith the isoforms of their stylar RNases. In a self-compatiblemutant, the S-allele-related band was much less intense thanin the original variety. These observations suggest that theRNase isoforms may be the stylar products of S-genes. (Received February 21, 1992; Accepted June 19, 1992)     

Style-specific and developmentally regulated accumulation of a glycosylated thaumatin/PR5-like protein in Japanese pear (Pyrus serotina Rehd.)

The stylar proteins of Japanese pear (Pyrus serotina Rehd.) were analyzed by two-dimensional gel electrophoresis, and a 32-kDa protein with an isoelectric point of 4.8 was found to be a major component in the style. The 32-kDa protein was a soluble glycoprotein which reacted with concanavalin A. The 32-kDa protein specifically accumulated in the style in a developmentally regulated manner, but was not detected in the other floral organs and leaves. An oligonucleotide representing the N-terminal amino acid sequence of the 32-kDa protein was used to amplify a cDNA fragment by polymerase chain reaction (PCR). The generated PCR product was used to screen a style cDNA library. The selected cDNA clone encoded 244 amino acid residues containing the N-terminal sequence of the 32-kDa protein. The N-terminus of the protein was preceded by putative signal peptide of 22 amino acid residues. The 32-kDa protein showed significant homology with the thaumatin/PR5-like proteins, and was named PsTL1 (Pyrus serotina thaumatin-like protein 1). The possible biological role of PsTL1 in the styles is discussed. Received: 27 November 1997 / Accepted: 19 January 1998     

Activity of cell wall-associated enzymes in ripening olive fruit

Enzymatically active cell wall isolaled from olive (Olea europaea) fruit was employed Hi investigate some hydrolytic enzymes bound to the cell wall and the changes in these during ripening. Seven glycosidases. β-glucosidase (EC 3.2.1.21) α-galactosidase (EC 3.2.1.22). β-galactosidase (EC 3.2.1.23). α-arabinosidase (EC 3.2.1.55), α-mannosidase (EC 3.2.1,24). β-xylosidase (EC 3.2.1.37) and β-N-acetylglucosamidase (EC 3.2.1.30). as well as Cx-cellulase (EC 3.2.1.4) and endo-polygalacturonase (EC 3.2.1.15). were identified in the cell wall preparation, at four stages of ripeness (mature green. changing colour, black and black-ripe). Activities of all these cell wall-associated enzymes fionicallv and covalently linked) were determined either by cell wall incubation with artificial substrate or after extraction from the cell wall with buffers of high salt concentration (Cx-cellulase). and were compared to those of forms solubilized from acetone powders with 500 nM citrate buffer (cytoplasmic and/or apoplastic plus ionically hound to cell wall) In general, the activities of low ionic strength buffer-soluble enzymes were found to be much higher than those of the bound enzymes. The bound enzymes are present in the fruit at the green colour stage, whereas the activities of the soluble enzymes only increased from the changing colour stage onwards. The tenacity of binding of enzymes to the wall was investigated by treating the walls with high salt and measuring residual activity. The nature of the ionic and covalent binding and the changes during ripening were also established for wall-hound glycosidase During ripening there was a marked change in the percentages of covalently- and tonically linked activities of β-glucosidase and β-galaclosidase: al the changing colour stages about 75–80% of the bound active in was present in high ionic strength buffer while al the black-ripe stage it was only 15–20. A possible role for these cell wall degradative enzymes in olive softening is discussed.