JXB Advance Access originally published online on August 27, 2004
Journal of Experimental Botany 2004 55(406):2281-2290; doi:10.1093/jxb/erh250
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RESEARCH PAPER |
European, Chinese and Japanese pear fruits exhibit differential softening characteristics during ripening
1Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan
2Laboratory of Postharvest Agriculture, Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan
3Faculty of Agriculture, Yamagata University, Tsuruoka 977-8555, Japan
To whom correspondence should be addressed. Fax: +81 86 251 8338. E-mail: ykubo{at}cc.okayama-u.ac.jp
Received 19 April 2004; Accepted 15 July 2004
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Abstract |
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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.
Key words: Cell wall modification, enzyme activity, fruit softening, gene expression, pear fruit, polygalacturonase
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferences |
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Softening is a developmentally programmed ripening process in many fruits. The greater part of the process is the consequence of modifications in the structure of the cell walls, providing different textures with different fruits including juiciness, crispness, and stiffness (Seymour et al., 2002
). On the other hand, fruit softening influences infection by post-harvest pathogens, and restricts transportation, storage and shelf-life (Brummell and Harpster, 2001). In plant primary cell walls, cellulose microfibrils are coated with and cross-linked together with hemicellulose and the spaces in these networks are filled with pectins, which also form a network (Brummell and Harpster, 2001). During fruit softening, pectins (Fischer and Bennett, 1991) and hemicelluloses (Wakabayashi, 2000) typically undergo solubilization and depolymerization that are thought to contribute to cell wall loosening and disintegration.
Modifications in cell wall polymers during ripening are complicated and considered to involve the co-ordinated and interdependent action of a range of cell wall-modifying enzymes and proteins such as polygalacturonase (PG), pectin methylesterase, ß-galactosidase, 
-L-arabinofuranosidase, endo-(1,4) ß-D-glucanase (EGase), expansin, and xyloglucan endotransglycosylase (XET) (Brummell and Harpster, 2001). In tomato, the role of individual cell wall-modifying enzymes in fruit softening has been extensively studied using genetic engineering and various mutants. For instance, softening in transgenic tomato fruit with suppressed PG activity was similar to that of control fruit (Smith et al., 1988) and overexpression of the PG gene using rin tomato fruit did not facilitate fruit softening, although there was solubilization of cell wall polyuronides (Giovannoni et al., 1989). By contrast, suppression of ß-galactosidase activity and expansin early in ripening reduced fruit softening (Brummell and Harpster, 2001). These reports in tomato could be utilized in elucidating softening in other fruits. However, the pectin and xyloglucan contents and composition in fruit cell walls are different in various fruit species (Redgwell et al., 1997; Wakabayashi, 2000), and the nature, timing, and extent of the modification of cell wall polysaccharides vary between species (Brummell and Harpster, 2001; Rose et al., 1998). Therefore, the role of individual cell wall-modifying enzymes in fruit softening would be dissimilar in different fruit species.
In European pear, as in other softening fruits, an increase in some cell wall-modifying enzyme activities and the degradation of cell wall polysaccarides have been reported (Ahmed and Labavitch, 1980a, b; Yoshioka et al., 1992). In ‘La France’ pears, it was found that application of 1-MCP, a powerful inhibitor of ethylene perception, even after the commencement of fruit softening, interrupted further softening with reduced gene expression of two PGs and one XET, exclusive of no significant changes in two EGases and another XET expression (Hiwasa et al., 2003a, b).
Among the genus Pyrus, there are several species having fruits with different softening characteristics during ripening. European pear (Pyrus communis L.) ‘La France’ fruit, a climacteric fruit, undergoes dramatic softening with ethylene production during ripening, resulting in an attractive melting texture. Chinese pear (Pyrus bretschneideri Rehd.) ‘Yali’ shows a massive climacteric ethylene production, but does not exhibit fruit softening and its flesh texture remains crisp even at the late ripening stage. Japanese pear (Pyrus pyrifolia Nakai) ‘Nijisseiki’, which is classified as a non-climacteric fruit, does not exhibit the striking change in flesh firmness during ripening. This may be due to the low level of ethylene production in ‘Nijisseiki’ during ripening (Itai et al., 1999). Until now, few comparative studies on ripening and softening using fruit from the same genus have been reported. Therefore, a comparison of differences in softening-related factors among the three pear fruits can provide more profound insight on the softening mechanism. In the present report, the degradation in cell wall polysaccharides, the expression of two PG and one XET genes, and PG activities during ripening in the three pears are described.
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Materials and methods |
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Plant materials and treatments
European pear (P. communis L. cv. ‘La France’), Chinese pear (P. bretschneideri Rehd. cv. ‘Yali’), and Japanese pear (P. pyrifolia Nakai cv. ‘Nijisseiki’) were obtained at commercial maturity from orchards in Yamagata, Okayama, and Tottori, Japan, respectively. ‘La France’ and ‘Yali’ pears were treated at 20 °C with 5000 µl l–1 propylene. In order to synchronize the onset of ripening, both fruits were treated for 7 d until sufficient autocatalytic ethylene production was detected, and then held in ambient conditions at 20 °C. ‘Nijisseiki’ pears were treated with propylene throughout the experiment.
Rates of ethylene production and flesh firmness were measured as described previously (Hiwasa et al., 2003b) at appropriate intervals using three fruits for each measuring point. The flesh tissues were subsequently frozen in liquid nitrogen and stored at –80 °C until used for the extraction of cell wall polysaccharides, total RNA, and enzyme.
Isolation and fractionation of cell wall polysaccharides
Approximately 20 g of frozen flesh tissue was ground to powder in liquid nitrogen using a mortar and pestle and then boiled with 80 ml of 100% ethanol for 30 min. After filtration through a glass fibre filter (Whatman GF/C), the insoluble material was washed sequentially with 80% boiling ethanol, with 100% ethanol and 100% acetone, and then dried. The alcohol-insoluble solids (AIS) were sequentially fractionated into water-, 50 mM CDTA-, 50 mM Na2CO3-, and 4 M KOH-soluble fractions, and residual materials as described by Murayama et al. (2002), but modified by using 50 mM CDTA (pH 6.5) containing 50 mM Na-acetate instead of 50 mM EDTA (pH 6.5).
Total uronic acids (UA) and total sugar (TS) contents of each fraction were determined using the m-hydroxydiphenyl method (Blumenkrantz and Asboe-Hansen, 1973) and the phenol-sulphuric acid method (Dubois et al., 1956), respectively. Xyloglucan (XG) content was determined as described by Nishitani and Masuda (1981).
The remaining neutralized solution of each fraction, except that of the water-soluble fraction, was dialysed against distilled water for 2 d at 4 °C. The dialysed fractions the and water-soluble fraction were lyophilized and stored in a desiccator.
Size-exclusion chromatography of fractionated polysaccharides
Aliquots (0.5–1.2 mg of galacturonic acid equivalents and/or approximately 1 mg of glucose equivalents) of lyophilized products of water- or Na2CO3-soluble fractions were chromatographed on a size-exclusion column (XK 16/70, Amercham Pharmacia Bioteck AB, Uppsala, Sweden) of Sephacryl S-400 (Amercham Pharmacia Bioteck AB). Elution buffers for water- and Na2CO3-soluble fractions were 30 mM Na-acetate (pH 5.0) containing 10 mM EDTA, and 50 mM Na-acetate (pH 5.0) containing 100 mM NaCl and 10 mM EDTA, respectively. Four ml fractions were collected at a flow rate of 18 ml h–1 and assayed for UA and TS as described above. Size separation of the KOH-soluble fraction was done in a similar manner, except that 0.1 N NaOH was used as the elution buffer. TS and XG contents of the fractions collected were assayed as described above.
RNA and genomic DNA gel blot analyses
RNA and genomic DNA gel blot analyses were performed as described previously (Hiwasa et al., 2003a, b). For PC-PG1 (AB084461
[GenBank]
), PC-PG2 (AB084462
[GenBank]
), and PC-XET1 (AB095368
[GenBank]
), DIG-labelled probes containing untranslated regions of the cDNAs were synthesized.
Cell wall protein extraction and PG activity assay
Frozen flesh tissue (10 g) was powdered in liquid nitrogen and thawed in 3 vols ice-cold buffer [50 mM Na-acetate (pH 5.0), 15 mM EDTA, 5 mM ß-mercaptoethanol, and 0.5% (w/v) PVPP (polyvinyl polypyrrolidone)]. The homogenate was centrifuged at 10 000 g and 4 °C for 30 min and the pellets were resuspended in ice-cold buffer. Thereafter, the pellets were suspended in 10 ml of cell-wall-protein extraction buffer [50 mM Na-acetate (pH 5.0), 15 mM EDTA, 5 mM ß-mercaptoethanol, and 1.5 M NaCl] and then stirred for 2 h on ice. The suspensions were centrifuged at 10 000 g for 30 min at 4 °C and the supernatants were dialysed overnight at 4 °C (Mr cut-off 8000 kDa) against dialysis buffer [10 mM Na-acetate (pH 5.0) and 15 mM EDTA].
Following dialysis, the cell wall protein solution was used for the detection of PG activity by measuring the release of reducing sugars from polygalacturonic acid (PGA, Sigma, USA) using 2-cyanoacetamide method (Gross, 1982). The reaction mixture contained 140 µl cell wall enzyme preparation, 4 µl 1 M K-acetate (pH 5.5), 8 µl 2.5 M KCl, 40 µl 1% PGA, and 8 µl distilled water. The reaction mixtures were incubated at 37 °C for 60 min followed by measurement of reducing sugar using D--galacturonic acid as a standard.
For viscometric measurement of PG activity, the cell wall proteins were extracted from propylene-treated day 7 fruit of ‘La France’ and ‘Nijisseiki’ and day 15 fruit of ‘Yali’. The protein fractions were dialysed and concentrated using Apollo® Concentrators (Orbital Biosciences, Topsfield, MA, USA), and the aliquots of concentrates equivalent to 60 units (1 unit=1 nmol reducing sugar h–1) of total PG activity in the reducing sugar assay described above, were used for analysis. The reaction mixtures contained 0.5 ml 1% PGA, 40 µl 2.5 M KCl, and 20 µl 1 M K-acetate (pH 5.5), and 440 µl of the protein extract, adjusted with distilled water, were incubated at 37 °C for up to 8 h, and the viscosity of each reaction mixture was determined at multiple time points in semi-micro viscometers (Cannon-Manning, State College, PA).
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Results |
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Ethylene production and flesh firmness
Figure 1 shows the changes in the rate of ethylene production and flesh firmness during ripening in three pears. In ‘La France’, ethylene production in both control and propylene-treated fruits increased during ripening (Fig. 1A). Concomitant with the increase in ethylene production, flesh firmness decreased dramatically (Fig. 1B), resulting in a melting and juicy texture until day 11 in propylene-treated fruit and day 15 in the control.
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‘Yali’ fruit produced the highest amount of ethylene (>150 nl g–1 h–1) during ripening with or without propylene treatment (Fig. 1C). In parallel with this high ethylene production, the peel colour turned yellow and an aroma developed. However, flesh firmness did not change throughout the experiment, irrespective of propylene treatment (Fig. 1D).
In ‘Nijisseiki’, only the basal level of ethylene and a slight decline in firmness were detected throughout the experiment in control fruit (Fig. 1E). However, continuous propylene treatment induced ethylene production slightly (<0.3 nl g–1 h–1), and reduced flesh firmness to some extent (Fig. 1F). In propylene-treated ‘Nijisseiki’, texture did not turn into melting but rather become mealy and the fruit finally showed senescence symptoms such as partially water-soaked appearance and crack of peel with separation from flesh after day 11.
Since no differences in ripening behaviour between the control and propylene-treated fruit were detected in both ‘La France’ and ‘Yali’, only propylene-treated fruits were used for subsequent experiments.
Solubility of pectic and hemicellulosic polysaccharides
To determine the relationship between softening behaviours and modes of cell wall solubilization in the three pear types, AIS isolated from pre-ripe and ripe fruit of each pear was sequentially extracted into five fractions, and TS, UA, and XG contents were determined in each fraction.
In ‘La France’ fruit, both TS and UA contents in the Na2CO3-soluble fraction decreased dramatically during ripening (Fig. 2A, B), whereas water-soluble polysaccharides increased considerably, suggesting the solubilization of pectic polysaccharides. On the other hand, in ‘Yali’ fruit, TS and UA contents of either the water- or the Na2CO3-soluble fractions did not change during ripening (Fig. 2D, E). In ‘Nijisseiki’, control fruit did not show any distinct changes in the amount of polysaccharides in these pectic fractions during storage (Fig. 2G, H). In propylene-treated ‘Nijisseiki’ fruit, UA and TS contents of Na2CO3-soluble fraction decreased during storage, whereas the UA content of the water-soluble fraction increased. The extent of the changes, however, was not as dramatic as that for ‘La France’ fruit.
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In all pears, the CDTA-soluble fraction contained less pectic polysaccharides than the water- and Na2CO3-soluble fractions and no remarkable changes in UA and TS content were detected during ripening. In the KOH-soluble fraction, TS content in ‘La France’ and ‘Yali’ fruits increased slightly during ripening, while XG content in ‘Yali’ and propylene-treated ‘Nijisseiki’ decreased (Fig. 2C, F, I). However, these changes in TS and XG content did not seem to influence the softening behaviour of fruits from the three pears.
Molecular mass distribution of pectic and hemicellulosic polysaccharides
Changes in the molecular mass distribution of the constituent polysaccharides during ripening were determined in the two main pectic fractions, the water- and Na2CO3-soluble fractions (Figs 3, 4), and the KOH-soluble hemicellulosic fraction (Fig. 5).
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In the water-soluble fraction for ‘La France’ fruit, low molecular weight polysaccharides for both TS and UA profiles increased with ripening (Fig. 3A, B). In ‘Yali’ fruit, the UA profile barely changed with ripening and the TS profile shifted slightly to the low molecular weight region (Fig. 3C, D). In ‘Nijisseiki’, propylene-treated fruit exhibited a downshift of UA in the water-soluble fraction but non-treated fruit did not (Fig. 3E, F). However, no downshift in TS was observed, irrespective of propylene treatment.
In the Na2CO3-soluble fraction of ‘La France’ fruit, in addition to a large decrease in the amount of polymers in the TS and UA components, the elution profiles of the TS component showed a marked decrease in molecular weight during ripening (Fig. 4A, B). In ‘Yali’ fruit, the TS and UA profiles hardly changed (Fig. 4C, D). In ‘Nijisseiki’ fruit, the downshift in TS and UA profiles due to continuous propylene treatment was observed (Fig. 4E, F). Interestingly, downshifts in the molecular mass profiles of the Na2CO3-soluble polysaccharides with softening were more apparent in propylene-treated ‘Nijisseiki’ than ‘La France’ (Fig. 4A, B, E, F).
In the KOH-soluble fraction, only slight downshifts in both TS and XG profiles with ripening were observed in ‘La France’ and ‘Yali’, while no changes were observed in either profile in ‘Nijisseiki’, regardless of propylene treatment (Fig. 5).
DNA and RNA gel blot analyses of PG and XET genes
Using the gene-specific regions of two PG (PC-PG1 and PC-PG2) and one XET (PC-XET1) cDNAs isolated from ‘La France’ fruit as probes, hybridized bands were detected in DNA gel blots of all three pears (data not shown), suggesting that homologues of PC-PGs and PC-XETs exist in ‘Yali’ and ‘Nijisseiki’ genomes.
RNA gel blot analysis using the same probes showed that the accumulation of both PC-PG1 and PC-PG2 mRNAs was detectable at harvest in ‘La France’ fruit (Fig. 6). The mRNA abundance of PC-PG1 increased with ripening, reaching a peak at day 7 and then declined. The mRNA abundance of PC-PG2 also increased during the first 3 d of ripening in ‘La France’ and then remained at a high level throughout the experiment. In ‘Yali’, no accumulation of mRNAs that hybridized with PG probes was detected at any stage. In ‘Nijisseiki’, PC-PG1 mRNA did not accumulate in either the control or propylene-treated fruit. PC-PG2 mRNA was slightly detected at harvest in ‘Nijisseiki’ fruit and increased gradually during storage in non-treated control fruit. This slight detection was significantly enhanced by propylene treatment and a high level was maintained until the end of the experiment. PC-XET1 mRNA accumulation increased slightly in ‘La France’ fruit during ripening and the level was almost constant in ‘Yali’ and ‘Nijisseiki’ fruits throughout the experiment, irrespective of the propylene treatment.
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Polygalacturonase activity determined by reducing sugar assay and viscometric measurement
Figure 7 shows the changes in PG activity during ripening in three types of pear determined by measuring the release of reducing sugars from PGA. In ‘La France’ fruit, PG activity increased dramatically within the first 7 d of ripening and then showed a slight decrease at day 15, consistent with the expression patterns of the PG genes. In ‘Nijisseiki’ fruit, PG activity increased during storage to a lesser extent and was stimulated slightly by propylene treatment. Despite the lack of accumulation of mRNA that hybridized to the two PG genes, the enzyme activity increased significantly during ripening in ‘Yali’ fruit. Thus, patterns of PG activity measured by the reducing sugar assay did not correspond with the expression of PG genes in terms of a comparison among the three pear varieties.
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To understand this discrepancy, the endo-acting PG activities in the propylene-treated pears were compared by viscometric measurement (Fig. 8). For the measurement, proteins of three pears were prepared to have equivalent PG activities as determined by the reducing sugar assay. The results showed large differences among the three pears in the ability to reduce the viscosity of PGA. The viscosity of the protein solution from ‘La France’ fruit decreased drastically and exponentially with incubation time, while that from ‘Yali’ fruit hardly decreased in viscosity. The protein solution from ‘Nijisseiki’ decreased in viscosity, exponentially to a much lower extent than that from ‘La France’.
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Discussion |
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Difference of softening behaviour and cell wall polymers among three types of pear fruit
The three types of pear used in this study showed different softening behaviours during ripening. The European pear ‘La France’ softened rapidly (Fig. 1A, B) and developed the typical melting texture with a burst of ripening ethylene. The Chinese pear ‘Yali’ did not show any change in flesh firmness even after 35 d of storage at room temperature despite a high level of ethylene production (Fig. 1C, D). The Japanese pear ‘Nijisseiki’ showed a decrease in flesh firmness when the fruit were continuously treated with propylene (Fig. 1E, F). However, the decrease was not as dramatic as that observed in ‘La France’. The flesh texture of propylene-treated ‘Nijisseiki’ was not melting but rather mealy. Therefore, factors that distinguish ‘Yali’ from propylene-treated ‘Nijisseiki’ and ‘La France’ would be related to the decrease in firmness, while factors that separate ‘La France’ and propylene-treated ‘Nijisseiki’ would be linked to textural difference between melting and mealiness.
In order to clarify these factors, the modes of cell wall modifications with softening in three pear fruits were compared first. The degradation of pectic polysaccharides corresponded well with softening behaviours in the three pear fruits. Depolymerization and solubilization of pectic polysaccharides were observed in ‘La France’ and propylene-treated ‘Nijisseiki’ but not in ‘Yali’ (Figs 2, 3, 4). However, some differences between ‘La France’ and propylene-treated ‘Nijisseiki’ were observed in the degree and pattern of solubilization and depolymerization. In the water-soluble fraction, the UA content of ‘La France’ increased 3-fold during ripening while the content of propylene-treated ‘Nijisseiki’ increased 1.5-fold (Fig. 2). In a comparison of water- and Na2CO3-soluble fractions, strong solubilization and depolymerization were observed in ‘La France’ while propylene-treated ‘Nijisseiki’ showed stimulated depolymerization rather than solubilization during ripening (Fig. 4). Moreover, in the UA profile of the CDTA-soluble fraction, distinct depolymerization was only observed in ‘La France’ fruit (data not shown). These differences might be related to the degree of decrease in flesh firmness and to qualitative differences in texture between propylene-treated ‘Nijisseiki’ and ‘La France’ fruits. Huber and O'Donoghue (1993) have demonstrated that depolymerization of pectic polysaccharides in avocado fruit is more extensive than that of tomato, and correlates with the differences in flesh texture between these fruits. Similar relationships between modes of pectin degradation and particular textural properties have also been revealed in other species (Redgwell et al., 1997; Yoshioka et al., 1992). These findings may suggest that the mode of pectin disassembly is one of the important factors determining fruit firmness and texture.
Depolymerization of hemicellulosic polysaccharides during fruit softening has also been reported in various fruits, such as tomatoes, melons, strawberries, and avocados (Huber, 1983, 1984; Rose et al., 1998; Sakurai and Nevins, 1997). In this study, depolymerization of hemicellulosic polysaccharides with ripening was observed in ‘La France’ and ‘Yali’ but not in ‘Nijisseiki’ even in the fruit treated with propylene (Fig. 5). This suggests that the modification of the hemicellulosic polysaccharides is not sufficient to decrease fruit firmness in pear fruits. However, since degradation of pectins occurred in both ‘La France’ and propylene-treated ‘Nijisseiki’ fruits, but hemicellulosic polysaccharides depolymerized only in ‘La France’, degradation of both pectins and hemicellulose may be required to develop the melting texture in ‘La France’ fruit.
Difference of PG activity among three types of pear fruit
Endo-PG activity has been identified in a number of ripening fruits and has been shown to correlate with an increase in soluble pectins during ripening (Fischer and Bennett, 1991; Hadfield and Bennett, 1998). In peach, a remarkable increase in endo-PG activity during ripening was detected in melting flesh cultivars, but not in non-melting flesh cultivars, while the level of exo-PG activity was similar in both cultivars suggesting a possible involvement of endo-PG in the development of the melting texture (Pressey and Avants, 1978). In European pear, activities of both exo- and endo-PGs were identified and characterized in ripening fruit (Pressey and Avants, 1976), although the relationship between each type of PG activity and softening was not established.
In this study, PG activity in three types of pear fruit was analysed by two methods, the reducing sugar assay and the viscometric method respectively (Figs 7, 8). The reducing sugar assay does not differentiate between the two types of PG, while the viscometric method shows endo-PG activity exclusively even in the presence of exo-PG (Bartley et al., 1982). Unexpectedly, PG activity determined by the reducing sugar assay increased with ripening to a greater or lesser extent in all the three pears. However, in the viscometric measurement, ‘La France’ exhibited the highest endo-PG activity and ‘Nijisseiki’ to a lesser extent, whereas ‘Yali’ showed almost no detectable endo-PG activity (Fig. 8). Therefore, PG activity detected in ripening ‘Yali’ fruit by reducing sugar assay could be due to exo-PG activity.
Difference of expression of PG and XET genes among three types of pear fruit
The softening process and the modification of pectic polymers in fruit has been shown to correlate with the expression of PG genes (Rose et al., 1998; Hiwasa et al., 2003b). However, transgenic trials in tomato have indicated that PG-dependent pectin degradation is not essential for fruit softening to occur (Smith et al., 1988; Giovannoni et al., 1989). Therefore, the contribution of PG to fruit softening has still not been established.
The involvement of the expression of PG genes in the differences in softening behaviour and PG activities among the three types of pear fruit were investigated. The PC-PG1 gene was expressed only in the ‘La France’ pear while expression of PC-PG2 was detected in both ‘La France’ and ‘Nijisseiki’ (Fig. 6). However, no transcript hybridizing with both PG genes was detected in ‘Yali’ fruit. Taken together with the result of PG activity, these results suggest that PC-PG1 and PC-PG2 could be transcripts for endo-PG and an additional PG gene for exo-PG could exist, at least in ‘Yali’. Genomic Southern blot analysis using PC-PG1 and PC-PG2 as probes indicated the existence of a counterpart for each gene in ‘Yali’ and ‘Nijisseiki’ (data not shown). It has previously been demonstrated that expression of both genes in ‘La France’ fruit during ripening is ethylene-dependent (Hiwasa et al., 2003b). In this study, expression of PC-PG2 in ‘Nijisseiki’ was stimulated by propylene treatment while no transcript of the two PG genes was detected in ‘Yali’, despite the high level of ethylene production. In ‘Yali’ fruit, exogenous ethylene at the preclimacteric stage stimulated ethylene production, respiration, and the development of flavour and colour, but not softening (data not shown), suggesting that the fruit is sensitive to ethylene like other pear fruits. Therefore, there is speculation that there has been a possible mutation of the promoter region or modification of the transcriptional factor of both PG genes in ‘Yali’ and PC-PG1 in ‘Nijisseiki’.
Since the exo-PG activity was detected in ‘Yali’ fruit during ripening in this study, an attempt was made to clone another PG gene by RT-PCR from ripening ‘Yali’ fruit using degenerate primers based on the conserved domain of PG genes registered in the database. However, this attempt was unsuccessful and led to speculation that the gene encoding exo-PG in ‘Yali’ could be very divergent from the known PGs.
Hemicellulose modification is brought about by cell wall-degrading enzymes such as XET and EGase. Previously, two XETs, and two EGases were cloned from ‘La France’ fruit and it was demonstrated that only PC-XET1 expression was detected at the preclimacteric stage, increased during fruit softening, and was affected in part by treatment with 1-MCP, a potent inhibitor of ethylene perception (Hiwasa et al., 2003a, b). This suggests that the expression of PC-XET1 is regulated by both developmental factors and ethylene. The increase of XET activity and its mRNA accumulation have also been shown to correlate with fruit softening in tomato and kiwifruit (Arrowsmith and de Silva, 1995; Schröder et al., 1998). In this study, expression of PC-XET1 in ‘La France’ fruit increased slightly during ripening; however, in ‘Yali’ and ‘Nijisseiki’ its expression was constitutive, suggesting that transcriptional regulation of PC-XET1 would not be a factor to discovering the difference in hemicellulose depolymerization among the three types of pear fruit.
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Conclusion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferences |
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The sequential results presented in this study indicate that the diversity in softening behaviour and textural changes among the three types of pear fruit could be related to solubilization and depolymerization of pectic and hemicellulosic polymers. Furthermore, the high endo-PG activity and expression of PC-PG1 and PC-PG2 in ‘La France’ pears could be responsible for the remarkable degradation of pectic polymers, which leads to drastic fruit softening and the development of a melting texture. The low endo-PG activity and expression of PC-PG2 in ‘Nijisseiki’ could relate to the moderate modification of pectins, leading to a gradual decrease in fruit firmness and a mealy texture. In spite of the huge amount of ethylene production, in ‘Yali’ fruit, the small change in firmness and pectic polymers during ripening could be due to a lack of detectable endo-PG activity and transcripts that hybridized with PC-PG1 and PC-PG2.
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Acknowledgements |
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The authors thank Dr Francis M Mathooko (Jomo Kenyatta University of Agriculture and Technology, Kenya) and Dr Willis O Owino (Graduate School of Natural Science and Technology in Okayama University, Japan) for their helpful advice and critical reading of this manuscript. This work was supported in part by Grants-in-Aid for Scientific Research (grant no. 11660032 to YK and 14360023 to AI), JSPS Research Fellowships for Young Scientists (grant no. 6509 to KH) from the Japanese Society for the Promotion of Science, the Sasakawa Scientific Research Grant from the Japan Society (to KH), and Okayama University COE program ‘Establishment of Plant Health Science’ (to YK).
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Footnotes |
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* Present address: Department of Food Systems, Faculty of Food Culture, Kurashiki-Sakuyo University, Kurashiki 710-0292, Japan.
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References |
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