β-Galactosidase, polygalacturonase and pectinesterase in differential softening and cell wall modification during papaya fruit ripening

Papaya ( Carica papaya L. cv. Eksotika) fruit softens differentially in relation to position of the tissue. The inner mesocarp tissue is softer, and its firmness decreases more rapidly during ripening than that of the outer mesocarp tissue. As the fruit ripens, pectin solubility and depolymerisation increase. Hemicellulose, too, appears to be depolymerised but, unlike pectins, this apparent degradation of hemicellulose is associated with an increase rather than a decrease in its level. Pectin and hemicellulose depolymerisation began in the inner mesocarp tissue at about the same time as β-galactosidase (EC 3.2,1.23) activity started to increase and tissue firmness began to decrease more rapidly. In contrast, pectin solubilisation in both outer and inner mesocarp tissues occurred steadily throughout ripening at a comparable rate and paralleled closely the increase of polygalacturonase (PG; EC 3.2.1.67) and pectinesterase (EC 3.1.1.11). In general, irrespective of enzyme distribution, tissue softening during ripening was more closely related to changes in β-galactosidase activity than to PG or pectinesterase activity. Papaya, β-galactosidase appears to be an important wall degrading enzyme and may contribute significantly to differential softening, perhaps by complementing the action of polygalacturonase. Polygalacturonase activity increased with increasing depth of the mesocarp tissue, as did softening of the fruit.     

Metabolism of cell wall polysaccharides from persimmon fruit. Pectin solubilization during fruit ripening occurs in apparent absence of polygalacturonase activity

Pectins from persimmon ( Diospyros kaki L.) fruit pericarp were sequentially extracted with 0. 05 M trans -1,2-diaminocyclohexane-N,N, N', N'-tetraacetic acid (CDTA), 0. 05 M Na₂CO₃ (1°C) and Na₂CO₃ (20°C) and the carbohydrate composition and metabolism during development determined. Young persimmon fruits contained a large proportion of pectins, 46% by dry weight, that decreased to 20% with ripening. This decrease occurred in the CDTA and Na₂CO₃ (1°C) fractions, mainly composed of uronic acids, and represents a net loss of uronic acids, arabinose and galactose. The amount of non-cellulosic neutral sugars was especially high in the Na₂CO₃ (20°C) fraction. The loss of pectins was also accompanied by a depolymerisation of the polysaccharides extracted in the three pectic fractions. However, none of these changes can be attributed to the action of polygalacturonase activity. Proteins were extracted from the pericarp tissue, but endopolygalacturonase (EC 3. 2. 1. 15) activity, determined as a decrease in viscosity of polygalacturonic acid, was not observed in the extract. Determination of exopolygalacturonase (EC 3. 2. 1. 67) activity by measuring the release of reducing groups from polygalacturonic acid was also negative. The results presented indicate that polygalacturonase is not responsible for the metabolism of pectins during persimmon fruit ripening.     

Polygalacturonase: a candidate gene for the soft flesh and deciduous fruit mutation in Capsicum

The soft flesh and deciduous fruit of pepper (Capsicum spp.) originated from the wild C. frutescens BG 2816 accession is a complete dominant trait controlled by the S gene. We constructed an F₂ population from a cross of BG 2816 (SS) and the bell-type C. annuum cultivar Maor (ss) and determined that S cosegregated with the tomato fruit-specific endo-polygalacturonase (PG) gene. The soft flesh and deciduous fruit phenotypes were observed together in all F₂ individuals, indicating a pleiotropic effect of PG on the two traits. We mapped S to pepper chromosome 10 in the region corresponding to that in which PG was previously mapped in tomato. Northern, RT-PCR and western analyses and enzyme activity assays, collectively, indicated that PG is not detected in green, breaker or red fruits of Maor, nor in green fruits of BG 2816. Accumulation of PG mRNA and protein was detected in the fruits of BG 2816, and it increased during ripening from breaker to red stages. The sequence analysis of partial PG cDNA isolated from BG 2816 revealed high homology (87% identity) with the tomato PG. The resemblance of the soft flesh and deciduous fruit phenotypes to PG-associated phenotypes in other fruit crops, the complete linkage between Sand PG, and the greater expression of PG in the fruits of BG 2816 than in those of Maor, all strongly indicate that PG is a candidate gene for S.     

Polygalacturonase gene expression in kiwifruit: relationship to fruit softening and ethylene production

In kiwifruit, much of the softening process occurs prior to the respiratory climacteric and production of ethylene. This fruit therefore represents an excellent model system for dissecting the process of softening in the absence of endogenous ethylene production. We have characterized the expression of three polygalacturonase (PG) cDNA clones (CkPGA, B and C) isolated from fruit of Actinidia chinensis. Expression of CkPGA and B was detected by northern analysis only in fruit producing endogenous ethylene, and by RT-PCR in other tissues including flower buds, petals at anthesis, and senescent petals. CkPGA promoter fragments of 1296, 860 and 467 bp fused to the -glucuronidase (uidA) reporter gene directed fruit-specific gene expression during the climacteric in transgenic tomato. CkPGC gene expression was observed in softening fruit, and reached maximum levels (50-fold higher than for CkPGA and B) as fruit passed through the climacteric. However, expression of this gene was also readily detected during fruit development and in fruit harvested prior to the onset of softening. Using RT-PCR, expression of CkPGC was also detected at low levels in root tips and in senescent petals. These results suggest that PG expression is required not only during periods of cell wall degeneration, but also during periods of cell wall turnover and expansion.     

Function of the polygalacturonase convertor in ripening tomato fruit

In extracts from pericarp tissue of ripening tomato ( Lycopersicon esculentum Mill. cv, Sonato) fruits, two isoenzymes of polygalacturonase (E.C. 3.2.1.15), PG1 and PG2, are usually found. Also in such extracts, or as part of PG1, a convertor (CV) occurs. Incubation of PG2 with this CV gives rise to PG1 or a different isoenzyme, PGx, that is also stable at 65°C but differs in _pH optimum and size from PG1. It appears that CV has two affinity sites that can bind with PG2 or with a polydextran. PG1 is an extraction artifact, consisting of one molecule of CV and two molecules of PG2. PGx is made up of one molecule of CV and one molecule of PG2. It is the CV part of PGx that binds to polydextrans such as Blue Dextran 2000, Sephadex G-100, and cell wall preparations. In this last form PGx is the physiologically active form of the enzyme, solubilizing demethylated pectin.
On Sephacryl S-300, CV appears to have a molecular weight of 81 kDa, but because of its heat stability and partial leakage through a 10 kDa cut-off membrane, it might be a much smaller, rod-like molecule. The polygalacturonase convertor might be a lectin without intrinsic enzyme activity, with a function to immobilize, stabilize and activate enzymic proteins in the cell wall.     

Identification of mRNAs with enhanced expression in ripening strawberry fruit using polymerase chain reaction differential display

Fruit ripening is a complex developmental process that involves specific changes in gene expression and cellular metabolism. In climateric fruits these events are coordinated by the gaseous hormone ethylene, which is synthesized autocatalytically in the early stages of ripening. Nonclimacteric fruits do not synthesize or respond to ethylene in this manner, yet undergo many of the same physiological and biochemical changes associated with the production of a ripe fruit. To gain insight into the molecular determinants associated with nonclimacteric fruit ripening, we examined mRNA populations in ripening strawberry fruit using polymerase chain reaction (PCR) differential display. Five mRNAs with ripening-enhanced expression were identified using this approach. Three of the mRNAs appear to be fruit-specific, with little or no expression detected in vegetative tissues. Sequence analysis of cDNA clones revealed positive identities for three of the five mRNAs based on homology to known proteins. These results indicate that the differential display technique can be a useful tool to study fruit ripening and other developmental processes in plants at the RNA level.     

Calcium in plant senescence and fruit ripening

Abstract. Calcium has long been associated with regulation of the ripening process of fruit and post-harvest storage life. Specifically, maintenance of relatively high calcium concentrations in fruit tissue results in a slower rate of ripening, as seen in lower respiration rates, reduced ethylene production, and slower softening of fruit flesh. There are also some specific fruit disorders such as bitter pit, which can be prevented if sufficient calcium is present. Senescence of other plant tissues such as leaves and flowers has also been shown to be retarded by the application of calcium.
Work leading to the above information is reviewed and discussed in the context of what is currently known of cellular regulation of calcium in plants. The major sites for the action of calcium in senescence and ripening are suggested to be in membrane structure and function, and in cell wall structure. Although high external concentrations of calcium are an advantage in reducing the rate of senescence or ripening, it is emphasized that normal cell function requires the maintenance of low concentrations of free calcium in the cell cytosol. It is suggested that one possible feature of senescence is a breakdown in such cellular regulation.     

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.     

Cloning and characterization of avocado fruit mRNAs and their expression during ripening and low-temperature storage

Differential sereening of a cDNA library made from RNA extracted from avocado (Persea americana Mill cv. Hass) fruit stored at low temperature (7°C) gave 23 cDNA clones grouped into 10 families, 6 of which showed increased expression during cold storage and normal ripening. Partial DNA sequencing was carried out for representative clones. Database searches found homologies with a polygalacturonase (PG), endochitinase, cysteine proteinase inhibitor and several stress-related proteins. No homologies were detected for clones from six families and their biological role remains to be elucidated. A full-length cDNA sequence for avocado PG was obtained and the predicted amino acid sequence compared with those from other PGs. mRNA encoding PG increased markedly during normal ripening, slightly later than mRNAs for cellulase and ethylene-forming enzyme (EFE). Low-temperature storage delayed ripening and retarded the appearance of mRNAs for enzymes known to be involved in cell wall metabolism and ethylene synthesis, such as cellulase, PG and EFE, and also other mRNAs of unknown function. The removal of ethylene from the atmosphere surrounding stored fruit delayed the appearance of the mRNAs encoding cellulase and PG more than the cold storage itself, although it hardly affected the expression of the EFE mRNA or the accumulation of mRNAs homologous to some other unidentified clones.AFRC Research Group in Plant Gene Regulation     

Analysis on virus resistance and fruit quality for T4 generation of transgenic papaya

Molecular biological characterization,fruit characters,and nutrients were analyzed for T4 generation of transgenic papaya.All transgenic papaya plants with the mutated replicase (RP) gene from papaya ringspot virus (PRSV) showed high resistance or immunity against PRSV in the field.The RP transgene can be steadily inherited to,and expressed at RNA level,the progenies.The growth characteristics of transgenic papaya were much better than nontransgenic papaya in the field.The non-transgenic papaya seedlings began to show typical symptoms caused by PRSV after being inoculated with PRSV.They died quickly and never grew to produce fruit.The adult trees developed yellow leaves and produced smaller fruits and were doomed to a slow death after some time,while most oftransgenic papaya plants (about 91.8%) did not show any symptoms caused by PRSV,and produced more,bigger,and high quality fruits.Compared with non-transgenic plants,the fresh fruit length of T4 generation of transgenic papaya increased 2.6%-5%,and the diameter decreased 0.6%-1.5%.The flesh thickness of fresh fruit increased 12%-15%,which made it fitter for eating.Although the fresh fruit quality changed,there was no significant difference between transgenic and non-transgenic papaya.The quality characteristics of dry fruit including the contents of water,lipid,N,protein,reduced sugar,vitamin A,vitamin C,and carotene in the T4 generation of transgenic papaya were all the same as their non-transgenic parents.This means that transgenic plants and non-transgenic plants are substantially equivalent,and the transgene has no effect on dry fruit quality.In this study,we found that vitamin A and vitamin C in red-fleshed papaya were 1.4-1.8 and 1.78-2.07 times more than the yellow-fleshed ones,respectively,while N and protein were only 84.2%-92.1% and 82.1%-98.9% of the yellow-fleshed ones.     

Analysis on virus resistance and fruit quality for T4 generation of transgenic papaya

Molecular biological characterization, fruit characters, and nutrients were analyzed for T4 generation of transgenic papaya. All transgenic papaya plants with the mutated replicase (RP) gene from papaya ringspot virus (PRSV) showed high resistance or immunity against PRSV in the field. The RP transgene can be steadily inherited to, and expressed at RNA level, the progenies. The growth characteristics of transgenic papaya were much better than non-transgenic papaya in the field. The non-transgenic papaya seedlings began to show typical symptoms caused by PRSV after being inoculated with PRSV. They died quickly and never grew to produce fruit. The adult trees developed yellow leaves and produced smaller fruits and were doomed to a slow death after some time, while most of transgenic papaya plants (about 91.8%) did not show any symptoms caused by PRSV, and produced more, bigger, and high quality fruits. Compared with non-transgenic plants, the fresh fruit length of T4 generation of transgenic papaya increased 2.6%–5%, and the diameter decreased 0.6%–1.5%. The flesh thickness of fresh fruit increased 12%–15%, which made it fitter for eating. Although the fresh fruit quality changed, there was no significant difference between transgenic and non-transgenic papaya. The quality characteristics of dry fruit including the contents of water, lipid, N, protein, reduced sugar, vitamin A, vitamin C, and carotene in the T4 generation of transgenic papaya were all the same as their non-transgenic parents. This means that transgenic plants and non-transgenic plants are substantially equivalent, and the transgene has no effect on dry fruit quality. In this study, we found that vitamin A and vitamin C in red-fleshed papaya were 1.4–1.8 and 1.78–2.07 times more than the yellow-fleshed ones, respectively, while N and protein were only 84.2%–92.1% and 82.1%–98.9% of the yellow-fleshed ones. Translated from Acta Ecologica Sinica, 2005, 25(12): 3301–3306 [译自: 生态学报]     

Molecular biology of fruit ripening and its manipulation with antisense genes

Considerable progress in tomato molecular biology has been made over the past five years. At least 19 different mRNAs which increase in amount during tomato fruit ripening have been cloned and genes for enzymes involved in cell wall degradation (polygalacturonase and pectinesterase) and ethylene synthesis (ACC synthase) have been identified by conventional procedures. Transgenic plants have been used to identify regions of DNA flanking fruit-specific, ripening-related and ethylene-regulated genes and trans-acting factors which bind to these promoters have also been identified.Antisense genes expressed in transgenic plants have proved to be highly effective for inhibiting the specific expression of ripening-related genes. These experiments have changed our understanding of how softening occurs in tomato fruit. Antisense techniques have also been used to identify genes encoding enzymes for carotenoid biosynthesis (phytoene synthase) and ethylene biosynthesis (the ethylene-forming enzyme). The altered characteristics of fruit transformed with specific antisense genes, such as retarded ripening and resistance to splitting, may prove to be of value to fruit growers, processors and ultimately the consumer.     

果实热处理保鲜技术研究(综述)

应用热处理技术控制果实采后病虫害和延缓果实衰老是近年来果实保鲜领域的研究热点.本文概述热处理对果实形态、生理生化和品质的影响,以及热处理的应用现状与应用前景.     

Effect of pre-storage treatments on mango fruit ripening

The effectiveness of pre-storage treatments of nitrogen (low oxygen), heat and ethanol and acetaldehyde vapours were examined for their potential for improving mango storage. Mature green mango fruit (Mangifera indica L. cv. Keitt) were treated with low oxygen (< 3% oxygen, 97% nitrogen) for 72 h, acetaldehyde (0.12%) and ethanol (1%) vapours for 24 h or heat (38 ± 2°C) for 48 h prior to storage at 14°C. The nitrogen and ethanol treatments induced substantial levels of acetaldehyde and ethanol in the fruit. Initially the firmness of the nitrogen treated fruit remained higher than the control although later in storage this effect was lost. Differences in ripening were reflected in the total soluble solids and acidity levels, nitrogen maintaining a higher acidity and lower total soluble solids (less mature) whereas the heat treated fruit had lower acidity and higher total soluble solids (more mature). Ethanol and acetaldehyde treatments showed no effect. The use of a pre-storage treatment of nitrogen therefore had a beneficial effect on retarding ripening, although as storage progressed this effect was lost.     

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.     

Hormonal regulation of ripening in the strawberry,a non-climacteric fruit

Anthocyanin accumulation is one measure of ripening in the strawberry (Fragaria ananassa Duch.), a non-climacteric fruit. Neither aminoethoxyvinylglycine, an inhibitor of 1-aminocyclopropane carboxylic acid synthase, nor inhibitors of ethylene action (silver, norbornadiene) affected anthocyanin accumulation in ripening fruit. When the achenes were removed from one half of an unripe fruit there was an accelerated accumulation of anthocyanin and induction of phenylalanine ammonia lyase on the de-achened portion of the ripening fruit. These effects of achene removal could be prevented by the application of the synthetic auxins 1-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid to the de-achened surface. The introduction of 1-naphthalene acetic acid into intact unripe strawberry fruit through the peduncle delayed their subsequent ripening, as measured by the accumulation of anthocyanin, loss of chlorophyll and decrease in firmness. These findings suggest that the decline in the concentration of auxin in the achenes as strawberry fruit mature modulates the rate of fruit ripening.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - NAA 1-naphthaleneacetic acid - PA1 phenylalanine ammonia-lyase - POA phenoxyacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Malic enzyme activity and related Biochemical aspects during ripening of γ-irradiated mango fruit

The increase in malic enzyme (l-malate: NADP⁺ oxidoreductase (oxalacetate-decarboxylating) EC 1.1.1.40) activity, usually observed during the r     

Storage of Tomatoes in Low Oxygen Atmospheres Inhibits Ethylene Action and Polygalacturonase Activity

The effect of low concentrations of O₂ (1%) with or without the application of exogenous ethylene (10 l/l) on the production of endogenous ethylene, the activity of polygalacturonase (PG), and the ripening of tomato fruits during storage for three weeks at 20°C and four weeks at 10°C, followed by one week under ambient conditions (25°C) was studied. The internal ethylene concentration in the fruits stored under low O₂ at 10 or 20°C was low during storage and increased only when fruits were transferred to ambient conditions. The application of exogenous ethylene to fruits stored under low O₂ at 10 or 20°C did not induce autocatalytic ethylene synthesis. By contrast, the internal ethylene concentration of fruits stored in air was high at 20°C and somewhat lower at 10°C. Under low O₂ conditions, PG activity was low and the fruits remained firm and green throughout storage, whereas, during storage in the air, PG activity increased and the fruits softened and developed their characteristic red color.