Papaya fruit softening, endoxylanase gene expression, protein and activity

Papaya ( Carica papaya L.) cell wall matrix polysaccharides are modified as the fruit starts to soften during ripening and an endoxylanase is expressed that may play a role in the softening process. Endoxylanase gene expression, protein amount and activity were determined in papaya cultivars that differ in softening pattern and in one cultivar where softening was modified by the ethylene receptor inhibitor 1-methylcyclopropene (1-MCP). Antibodies to the endoxylanase catalytic domain were used to determine protein accumulation. The three papaya varieties used in the study, 'Line 8', 'Sunset', and 'Line 4-16', differed in softening pattern, respiration rate, ethylene production and showed similar parallel relationships during ripening and softening in endoxylanase expression, protein level and activity. When fruit of the three papaya varieties showed the respiratory climacteric and started to soften, the level of endoxylanase gene expression increased and this increase was related to the amount of endoxylanase protein at 32 kDa and its activity. Fruit when treated at less than 10% skin yellow stage with 1-MCP showed a significant delay in the respiratory climacteric and softening, and reduced ethylene production, and when ripe was firmer and had a 'rubbery' texture. The 1-MCP-treated fruit that had the 'rubbery' texture showed suppressed endoxylanase gene expression, protein and enzymatic activity. Little or no delay occurred between endoxylanase gene expression and the appearance of activity during posttranslational processing from 65 to 32 kDa. The close relationship between endoxylanase gene expression, protein accumulation and activity in different varieties and the failure of the 1-MCP-treated fruit to fully soften, supported de novo synthesis of endoxylanase, rapid posttranslation processing and a role in papaya fruit softening.     

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.     

Proteomic analysis of differentially accumulated proteins during ripening and in response to 1-MCP in papaya fruit

Papaya (Carica papaya L.) is a climacteric fruit susceptible to postharvest losses due to the ethylene-induced ripening. The inhibitor of ethylene action, 1-methylcyclopropene (1-MCP), has been used worldwide as a safe postharvest non-toxic agent, but the physiological and biochemical modifications induced by 1-MCP are not well understood. Using the 2-DE analysis, we report the changes in the protein profiles after 6 and 18 days of postharvest and the effect of the effect of 1-MCP treatment on fruits. Twenty seven protein spots showing differences in abundance during ripening were successfully identified by nano-LC-ESI/MS/MS. Some spots corresponded to the cell wall degrading enzymes related to fruit ripening; others were involved in oxidative damage protection, protein folding, and cell growth and survival that were induced by 1-MCP. This is the first proteomic report analyzing the effect of 1-MCP in papaya ripening. The present data will help to shed light on papaya fruit ripening process.     

Papaya beta-galactosidase/galactanase isoforms in differential cell wall hydrolysis and fruit softening during ripening.

The potential significance of the previously reported papaya (Carica papaya L.) beta-galactosidase/galactanase (beta-d-galactoside galactohydrolase; EC 3.2.1.23) isoforms, beta-gal I, II and III, as softening enzymes during ripening was evaluated for hydrolysis of pectins while still structurally attached to unripe fruit cell wall, and hemicelluloses that were already solubilized in 4 M alkali. The enzymes were capable of differentially hydrolyzing the cell wall as evidenced by increased pectin solubility, pectin depolymerization, and degradation of the alkali-soluble hemicelluloses (ASH). This enzyme catalyzed in vitro changes to the cell walls reflecting in part the changes that occur in situ during ripening. beta-Galactosidase II was most effective in hydrolyzing pectin, followed by beta-gal III and I. The reverse appeared to be true with respect to the hemicelluloses. Hemicellulose, which was already released from any architectural constraints, seemed to be hydrolyzed more extensively than the pectins. The ability of the beta-galactanases to markedly hydrolyze pectin and hemicellulose suggests that galactans provide a structural cross-linkage between the cell wall components. Collectively, the results support the case for a functional relevance of the papaya enzymes in softening related changes during ripening.     

Compositional changes in cell wall polymers during mango fruit ripening

Softening of mango fruit has been investigated by analysis of ripening related changes in the composition of the fruit cell walls. There is an apparent overall loss of galactosyl and deoxyhexosyl residues during ripening, the latter indicating degradation of the pectin component of the wall. The loss of galactose appears to be restricted to the chelator soluble fraction of the wall pectin, whilst loss of deoxyhexose seems to be more evenly distributed amongst the pectin. The chelator soluble pectin fraction is progressively depolymerised and becomes more polydisperse during ripening. These changes are similar to those occurring in other fruit and are related to the action of wall hydrolases during ripening.     

Down-regulation of a ripening-related beta-galactosidase gene (TBG1) in transgenic tomato fruits.

Exo-galactanase/beta-galactosidase (EC 3.2.1.23) activity is thought to be responsible for the loss of galactosyl residues from the cell walls of ripening tomatoes. Transgenic tomato plants (Lycopersicon esculentum Mill cv. Ailsa Craig) with reduced exo-galactanase/beta-galactosidase mRNA were generated to test this hypothesis and to investigate the role of the enzyme in fruit softening. A previously identified tomato beta-galactosidase cDNA clone, TBG1, was used in the experiments. Heterologous expression of the clone in yeast demonstrated that TBG1 could release galactosyl residues from tomato cell wall galactans. Transgenic plants showed a reduction in TBG1 mRNA to 10% of normal levels in the ripening fruits. However, despite the reduction in message, total beta-galactosidase and exo-galactanase activities were unaffected. Furthermore, there was no apparent effect on levels of cell wall galactosyl residues when compared with the control. It was concluded that during the ripening of tomato fruits a family of beta-galactosidases capable of degrading cell wall galactans are active and down-regulation of TBG1 message to 10% was insufficient to alter the degree of galactan degradation.     

Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants

Excessive softening is the main factor limiting fruit shelf life and storage. Transgenic plants modified in the expression of cell wall modifying proteins have been used to investigate the role of particular activities in fruit softening during ripening, and in the manufacture of processed fruit products. Transgenic experiments show that polygalacturonase (PG) activity is largely responsible for pectin depolymerization and solubilization, but that PG-mediated pectin depolymerization requires pectin to be de-methyl-esterified by pectin methylesterase (PME), and that the PG -subunit protein plays a role in limiting pectin solubilization. Suppression of PG activity only slightly reduces fruit softening (but extends fruit shelf life), suppression of PME activity does not affect firmness during normal ripening, and suppression of -subunit protein accumulation increases softening. All these pectin-modifying proteins affect the integrity of the middle lamella, which controls cell-to-cell adhesion and thus influences fruit texture. Diminished accumulation of either PG or PME activity considerably increases the viscosity of tomato juice or paste, which is correlated with reduced polyuronide depolymerization during processing. In contrast, suppression of -galactosidase activity early in ripening significantly reduces fruit softening, suggesting that the removal of pectic galactan side-chains is an important factor in the cell wall changes leading to ripening-related firmness loss. Suppression or overexpression of endo-(1\to4)-d-glucanase activity has no detectable effect on fruit softening or the depolymerization of matrix glycans, and neither the substrate nor the function for this enzyme has been determined. The role of xyloglucan endotransglycosylase activity in softening is also obscure, and the activity responsible for xyloglucan depolymerization during ripening, a major contributor to softening, has not yet been identified. However, ripening-related expansin protein abundance is directly correlated with fruit softening and has additional indirect effects on pectin depolymerization, showing that this protein is intimately involved in the softening process. Transgenic work has shown that the cell wall changes leading to fruit softening and textural changes are complex, and involve the coordinated and interdependent activities of a range of cell wall-modifying proteins. It is suggested that the cell wall changes caused early in ripening by the activities of some enzymes, notably -galactosidase and ripening-related expansin, may restrict or control the activities of other ripening-related enzymes necessary for the fruit softening process.     

Ethylene regulation of fruit softening and cell wall disassembly in Charentais melon

Cell wall disassembly in ripening fruit is highly complex, involving the dismantling of multiple polysaccharide networks by diverse families of wall-modifying proteins. While it has been reported in several species that multiple members of each such family are expressed in the same fruit tissue, it is not clear whether this reflects functional redundancy, with protein isozymes from a single enzyme class performing similar roles and contributing equally to wall degradation, or whether they have discrete functions, with some isoforms playing a predominant role. Experiments reported here sought to distinguish between cell wall-related processes in ripening melon that were softening-associated and softening-independent. Cell wall polysaccharide depolymerization and the expression of wall metabolism-related genes were examined in transgenic melon (Cucumis melo var. cantalupensis Naud.) fruit with suppressed expression of the 1-aminocyclopropane-1-carboxylate oxidase (ACO) gene and fruits treated with ethylene and 1-methylcyclopropene (1-MCP). Softening was completely inhibited in the transgenic fruit but was restored by treatment with exogenous ethylene. Moreover, post-harvest application of 1-MCP after the onset of ripening completely halted subsequent softening, suggesting that melon fruit softening is ethylene-dependent. Size exclusion chromatography of cell wall polysaccharides, from the transgenic fruits, with or without exogenous ethylene, indicated that the depolymerization of both pectins and xyloglucans was also ethylene dependent. However, northern analyses of a diverse range of cell wall-related genes, including those for polygalacturonases, xyloglucan endotransglucosylase/hydrolases, expansin, and beta-galactosidases, identified specific genes within single families that could be categorized as ethylene-dependent, ethylene-independent, or partially ethylene-dependent. These results support the hypothesis that while individual cell wall-modifying proteins from each family contribute to cell wall disassembly that accompanies fruit softening, other closely related family members are regulated in an ethylene-independent manner and apparently do not directly participate in fruit softening.     

European, Chinese and Japanese pear fruits exhibit differential softening characteristics during ripening

Softening characteristics were investigated in three types of pear fruit, namely, European pear 'La France', Chinese pear 'Yali', and Japanese pear 'Nijisseiki'. 'La France' fruit softened dramatically and developed a melting texture during ripening, while 'Yali' fruit with and without propylene treatment showed no change in flesh firmness and texture during ripening. Non-treated 'Nijisseiki' did not show a detectable decrease in flesh firmness, whereas continuous propylene treatment caused a gradual decrease in firmness resulting in a mealy texture. In 'La France', the analysis of cell wall polysaccharides revealed distinct solubilization and depolymerization of pectin and hemicellulose during fruit softening. In 'Nijisseiki', propylene treatment led to the solubilization and depolymerization of pectic polysaccharides to a limited extent, but not of hemicellulose. In 'Yali', hemicellulose polysaccharides were depolymerized during ripening, but there was hardly any change in pectic polysaccharides except in the water-soluble fraction. PC-PG1 and PC-PG2, two polygalacturonase (PG) genes, were expressed in 'La France' fruit during ripening, while only PC-PG2 was expressed in 'Nijisseiki' and neither PC-PG1 or PC-PG2 was expressed in 'Yali'. The expression pattern of PC-XET1 was constitutive during ripening in all three pear types. PG activity measured by the reducing sugar assay increased in all three pears during ripening. However, viscometric measurements showed that the levels of endo-PG activity were high in 'La France', low in 'Nijisseiki', and undetectable in 'Yali' fruits. These results suggest that, in pears, cell wall degradation is correlated with a decrease in firmness during ripening and the modification of both pectin and hemicellulose are essential for the development of a melting texture. Furthermore, the data suggest that different softening behaviours during ripening among the three pear fruits may be caused by different endo-PG activity and different expression of PG genes.     

Cell wall modification in 1-methylcyclopropene-treated post-climacteric fresh-cut and intact papaya fruit

Papaya is a climacteric fruit in which ripening is greatly regulated by ethylene often associated with stress responses such as wounding. The changes in cell wall compositions in papaya fruit at an advanced stage of ripening under stress conditions including chilling temperature of 5°C and wounding employed as fresh-cut and how these changes were affected by an ethylene action inhibitor of 1-methylcyclopropopene (1-MCP) were examined in the study. The recovery of ethanol-insoluble solids, total soluble sugars, water-soluble polyuronides, neutral hemicelluloses, and neutral sugars of rhamnose, arabinose, mannose and glucose were not affected by 1-MCP or fresh-cut processing. The fresh-cut processing, however, caused a higher loss of total polyuronides and the neutral sugar galactose while increasing the recovery of chelator-soluble polyuronides. Few significant differences due to 1-MCP application were recorded in the recoveries of alkali-soluble polyuronides, hemicellulosic polyuronides extracted with 4% KOH, and the neutral sugar xylose. Modifications of cell wall polyuronides and hemicelluloses in ripe fresh-cut papaya fruit exhibited mostly similar patterns to those in intact ripe papaya fruit under the chilling temperature of 5°C while minimally affected by 1-MCP.     

1-甲基环丙烯(1-MCP)对油桃果实软化的影响

1-甲基环丙烯(1-MCP)可延缓油桃果实硬度的下降,阻止引起果实软化的细胞物质(淀粉、纤维素、果胶)的降解,抑制与果实软化相关的酶(淀粉酶、纤维素酶、多聚半乳糖醛酸酶)活性。     

Temporal Sequence of Cell Wall Disassembly in Rapidly Ripening Melon Fruit

The Charentais variety of melon (Cucumis melo cv Reticulatus F1 Alpha) was observed to undergo very rapid ripening, with the transition from the preripe to overripe stage occurring within 24 to 48 h. During this time, the flesh first softened and then exhibited substantial disintegration, suggesting that Charentais may represent a useful model system to examine the temporal sequence of changes in cell wall composition that typically take place in softening fruit. The total amount of pectin in the cell wall showed little reduction during ripening but its solubility changed substantially. Initial changes in pectin solubility coincided with a loss of galactose from tightly bound pectins, but preceded the expression of polygalacturonase (PG) mRNAs, suggesting early, PG-independent modification of pectin structure. Depolymerization of polyuronides occurred predominantly in the later ripening stages, and after the appearance of PG mRNAs, suggesting the existence of PG-dependent pectin degradation in later stages. Depolymerization of hemicelluloses was observed throughout ripening, and degradation of a tightly bound xyloglucan fraction was detected at the early onset of softening. Thus, metabolism of xyloglucan that may be closely associated with cellulose microfibrils may contribute to the initial stages of fruit softening. A model is presented of the temporal sequence of cell wall changes during cell wall disassembly in ripening Charentais melon.     

Responses of banana fruit to treatment with 1-methylcyclopropene

Experiments were conducted to determine levels of 1-methylcyclopropene (1-MCP) exposure needed to prevent ethylene-stimulated banana fruit ripening, characterise responses of ethylene-treated fruit to subsequent treatment with 1-MCP, and to test effects of subsequent ethylene treatment on 1-MCP-treated fruit softening. Fruit softening was measured at 20°C and 90% relative humidity. One hour exposure at 20°C to 1000 nl 1-MCP/l essentially eliminated ethylene-stimulated ripening effects. Exposure for 12 h at 20°C to just 50 nl 1-MCP/l was similarly effective. Fruit ripening initiated by ethylene treatment could also be delayed with subsequent 1-MCP treatment. However, 1-MCP treatment only slowed down ripening of ethylene-treated fruit when applied at 1 day after ethylene and was ineffective when applied 3 or 5 days after ethylene treatment. The ripening response of fruit treated with 1-MCP and subsequently treated with ethylene varied with interval time between 1-MCP and ethylene treatments. As time increased, the response of 1-MCP-treated fruit to ethylene was enhanced. Responses to 0.1, 1, 10 or 100 µl ethylene/l concentrations were similar. Enzyme kinetic analysis applied to 1-MCP effects on ethylene-induced softening of banana fruit suggested that 1-MCP inhibition is by noncompetitive antagonism of ethylene binding.     

Delay ripening of ‘Qingnai’ plum (Prunus salicina Lindl.) with 1-methylcyclopropene

‘Qingnai’ plum fruit were treated with 0, 250, 500 or 1000 nL L⁻¹ of 1-methylcyclopropene (1-MCP) for 6 h and stored at 20 °C. The fruit firmness, peel color, chlorophyll content, titratable acidity (TA), respiration rate and ethylene production, chlorophyllase, pectin methylesterase (PME) and polygalacturonase (PG) activities were monitored during postharvest ripening of ‘Qingnai’ plums. ‘Qingnai’ plums without 1-MCP treatment soften very rapidly at room temperature after harvest, showing a continuing decrease in hue angle, chlorophyll content, TA and increase in chlorophyllase, PME and PG activities during postharvest storage. In contrast, the 1-MCP-treated fruits showed reduced ethylene production and respiration rate and delayed softening, which was associated with the reduction in the activity of PME and PG. The 1-MCP treatment also significantly inhibited the chlorophyllase activity and peel color development in ‘Qingnai’ plums during postharvest ripening at 20 °C. These results suggest that 1-MCP treatment may be useful for maintaining the fruit quality and extending the postharvest shelf-life of ‘Qingnai’ plums.     

Labeling and chemistry of grapefruit pectic substances

The labeling of a number of polysaccharides found in grapefruit (Citrus paradisi) was achieved by feeding labeled myo-inositol to ripening grapefruit through their cut fruit stem, and allowing 4 days for the metabolism of label. The pectic polysaccharides were isolated by successive extraction of the labeled grapefruit with 80% ethanol, chloroform-methanol (1:1) and finally with 0.2 M Na₂ EDTA to solubilize pectic polysaccharides. The incorporation of label from myo-inositol into galacturonosyl, arabinosyl, xylosyl and galactosyl residues of pectic polysaccharides via myo-inositol oxidation pathway was demonstrated. Ion exchange chromatography of these labeled pectic polysaccharides using DE-52 cellulose resulted in the elution of eight totally or partially resolved polysaccharides with increasing salt concentration. The results suggest that, like other plant tissues, the myo-inositol oxidation pathway is also operative in ripening grapefruit and this metabolic pathway could be successfully utilized to achieve labeling of a number of pectic polysaccharides.     

Analysis of Papaya Cell Wall-Related Genes during Fruit Ripening Indicates a Central Role of Polygalacturonases during Pulp Softening

Papaya (Carica papaya L.) is a climacteric fleshy fruit that undergoes dramatic changes during ripening, most noticeably a severe pulp softening. However, little is known regarding the genetics of the cell wall metabolism in papayas. The present work describes the identification and characterization of genes related to pulp softening. We used gene expression profiling to analyze the correlations and co-expression networks of cell wall-related genes, and the results suggest that papaya pulp softening is accomplished by the interactions of multiple glycoside hydrolases. The polygalacturonase cpPG1 appeared to play a central role in the network and was further studied. The transient expression of cpPG1 in papaya results in pulp softening and leaf necrosis in the absence of ethylene action and confirms its role in papaya fruit ripening.