Journal of Experimental Botany, Vol. 54, No. 383, pp. 771-779,
February 1, 2003
© 2003 Oxford University Press
Ethylene is required for both the initiation and progression of softening in pear (Pyrus communis L.) fruit
Received 31 May 2002; Accepted 26 September 2002
1 Graduate School of Natural Science and Technology, Okayama University, Tsushima, Okayama 700-8530, Japan
2 Laboratory of Postharvest Agriculture, Faculty of Agriculture, Okayama University, Tsushima, Okayama 700-8530, Japan
3 To whom correspondence should be addressed. Fax: +81 86 251 8338. E-mail: ykubo{at}cc.okayama-u.ac.jp
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In order to investigate the physiological role of ethylene in the initiation and subsequent progression of softening, pear fruit were treated with propylene, an analogue of ethylene or 1-methylcyclopropene (1-MCP), a gaseous inhibitor of ethylene action at the preclimacteric or ripening stages. The propylene treatment at the pre-ripe stage stimulated ethylene production and flesh softening while the 1-MCP treatment at the same stage markedly retarded the initiation of the ripening-related events. Moreover, 1-MCP treatment after the initiation of ripening markedly suppressed the subsequent flesh softening and ethylene production. These results clearly indicate that ethylene is not merely a by-product, but plays a crucial role in both the initiation and maintenance of regulating the softening process during ripening. The observations also suggest that ethylene in ripening is regulated entirely in an autocatalytic manner. The mRNA accumulation of pear polygalacturonases (PG) genes, PC-PG1 and PC-PG2, was in parallel with the pattern of fruit softening in both propylene and 1-MCP treatments. However, the expression pattern of pear endo-1,4-ß-D-glucanases (EGase) genes, PC-EG1 and PC-EG2, was not affected in both treatments. The results suggest that ethylene is required for PGs expression even in the late ripening stage, but not for EGases.
Key words: Endo-1,4-ß-D-glucanase, ethylene, 1-methyl cyclopropene, pear fruit ripening, polygalacturonase, softening.
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Introduction |
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Fruit ripening is an aggregate process of various physiological and physical changes, typically including an increase in respiration and ethylene production, de novo synthesis and degradation of pigments, and textural changes that result principally from cell wall modification. It was originally thought that ethylene represents the trigger for these changes in fruit ripening (Abeles et al., 1992), based on the observations that ripening in climacteric fruit is associated with a large increase in ethylene production and can be induced by exogenous ethylene at a preclimacteric stage.
In addition to a role in initiating ripening, it has also been hypothesized that ethylene is important for maintaining the ripening process. This concept has been examined using inhibitors of ethylene action and biosynthesis and transgenic plants (Lelièvre et al., 1997a). It has been shown that application of silver ions to block ethylene perception in ripening tomato fruit arrests subsequent ripening-related events, such as colour change and the synthesis of ripening-related enzymes (Tucker and Brady, 1987). However, there is no direct evidence to show that ethylene is required for the subsequent softening process after its onset. The application of silver ions demands tissue cutting which limits the possible duration of measurement and accurate determination of textural changes. The cutting induces a wound response and might lead to abnormal metabolism. In addition, during application, there is uneven penetration of inhibitor into the target tissues. The application of other inhibitors of ethylene biosynthesis, such as aminoethoxyvinylglycine, amino-oxyacetic acid (inhibitors of ACS) and cobalt ions (an inhibitor of ACO) involves similar problems.
Transgenic fruits expressing ACC oxidase (ACO) and ACC synthase (ACS) antisense genes exhibit a significant delay in ripening and exogenous ethylene reverses the inhibitory effects (Murray et al., 1993; Picton et al., 1993; Guis et al., 1997). These observations confirm the importance of ethylene in initiating fruit ripening. In ACS antisense tomato, mRNA of polygalacturonase (PG) accumulated to levels similar to those in the wild type, so it had been thought that PG expression is independent of ethylene (Oeller et al., 1991). This has recently been challenged by Sitrit and Bennett (1998), who suggested that very low levels of ethylene induce transcription of the PG gene. The residual ethylene in the transgenic lines, equivalent to 0.5% of the wild type, might therefore be enough to support PG expression. The remaining low levels of ethylene production in the transgenic fruits might similarly complicate any conclusions regarding the physiological roles of ethylene during ripening.
A highly potent inhibitor of ethylene action, 1-methylcyclopropene (1-MCP), has recently been identified (Sisler and Serek, 1997) that is thought to bind irreversibly to, and thus inactivate, ethylene receptors. Since 1-MCP is gaseous at ambient temperature and penetrates fruit tissues easily and uniformly, it provides a powerful tool in the identification of ripening-related processes that are ethylene-dependent. To date, several reports have described the use of this inhibitor, although most have focused on practical applications or on the ability of 1-MCP to inhibit ethylene action when certain fruits and ornamentals are treated at a preclimacteric stage (Ku and Wills, 1999; Feng et al., 2000; Watkins et al., 2000).
Pear fruit is a typical climacteric fruit. It is normally harvested at a preclimacteric stage and stored at a low temperature to ensure uniform ripening through the induction of ethylene production. This chilling treatment is essential for normal ripening in some pear cultivars such as ‘Passe-Crassane’ (Lelièvre et al., 1997b), but not always in other cultivars. In ‘La France’ fruit used for this experiment, the treatment is not always essential but can stimulate the ripening process. Pear fruit have relatively large uniform flesh that undergoes a dramatic softening during ripening, therefore it provides an excellent model system to investigate the regulatory mechanism of fruit softening.
It is generally believed that textural changes during the ripening of most fruit largely arise from the degradation of the primary cell wall. During fruit softening, pectin and hemicellulose in walls typically undergo solubilization and depolymerization that are thought to contribute to wall loosening (Fisher and Bennett, 1991; Rose and Bennett, 1999). A wide range of ripening-related cell wall hydrolases can be identified in fruit tissue; the most studied include pectin esterase (EC 3.1.1.11), PG (EC 3.2.1.15), endo-1,4-ß-D-glucanase (EGase) or cellulase (EC 3.2.1.4), and ß-galactosidase (EC 3.2.1.23). Dramatic increases in PG activity, protein and mRNA levels have been observed during ripening in several climacteric fruits (Hadfield and Bennett, 1998). EGase activity and transcript abundance also increase with ripening in tomato (Lashbrook et al., 1994) and several other fruits (Tonutti et al., 1995; Trainotti et al., 1999). As with tomatoes, the modification of pectin and hemicellulose during ripening in pear fruit coincides with increased PG and EGase activities (Ben-Arie et al., 1979; Yoshioka, 1993), although cloning and characterization of the corresponding genes has not yet been reported.
This paper describes the use of 1-MCP to demonstrate that ethylene perception is required not only for the initiation, but also for the subsequent progression, of pear fruit ripening, in terms of fruit softening and the expression of associated PG and EGase genes.
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Materials and methods |
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Plant material and treatment
Pear (Pyrus communis L. cv. La France) fruit were obtained from a commercial orchard at Yamagata, Japan. For the treatment with propylene, an analogue of ethylene, fruit were harvested at 10 d before commercial maturity and ripened in the presence or absence of 5000 µl l–1 propylene at 20 °C.
For the 1-MCP treatment, fruit at commercial maturity (preclimacteric stage) were held at 1 °C for 3 weeks as a chilling treatment and then transferred to 20 °C for ripening. Some fruit were exposed to 20 µl l–1 1-MCP (Rohm and Haas, Philadelphia, PA, USA) for 12 h at harvest (MCP-0), during the chilling treatment (8 d in cold storage, MCP-1) or 4 d after initiation of ripening (MCP-2). In both experiments, rates of ethylene production and flesh firmness were determined during storage and ripening at appropriate intervals. Flesh samples were taken for analysis and stored at –80 °C until RNA extraction.
Determination of ethylene production and flesh firmness
Ethylene production was measured by enclosing samples in an air-tight chamber for 1 h at 20 °C or 1 °C, withdrawing 1 ml of headspace gas from the chamber for each determination, and injecting it into a gas chromatograph (model GC-4CMPE, Shimadzu, Kyoto, Japan) fitted with a flame ionization detector and an activated alumina column. Fruit flesh firmness was measured at four equatorial regions of the peeled flesh using a penetrometer (model SMT-T-50, Toyo Baldwin, Tokyo, Japan) fitted with an 8 mm plunger.
RNA extraction and RT-PCR
Total RNA was extracted by the hot borate method (Wan and Wilkins, 1994) and poly(A)+ RNA was isolated using Oligotex dT30 (Takara, Kyoto, Japan), according to the manufacturer’s protocol. First-strand cDNAs were synthesized from poly(A)+ RNA isolated from pre-ripe and ripening pear fruit using reverse transcriptase according to the manufacturer’s instructions (Toyobo, Tokyo, Japan) and used as a template to amplify the targeted genes by PCR. The degenerate primers were designed based on conserved amino acid sequences; for PG, 5'-AGYCCIAAYACIGAYGGIRTICA-3' and 5'-CARTARTDYTGRTCDATIAYDATIGG-3', for EGase, 5'-TGYTGGGARMGICCIGARGA-3' and 5'-ICCIARDATRTARTCIACYTG-3'. The conditions for PCR amplification were 35 cycles of 94 °C for 1 min, 40 °C for 2 min, and then 72 °C for 3 min.
cDNA library screening and RACE-PCR
A cDNA library was constructed using mixed poly(A)+ RNA prepared from pre-ripe and ripening pear fruit using a 
-ZAP-cDNA synthesis kit (Stratagene, La Jolla, CA, USA) and Gigapack III Gold packaging extract (Stratagene) according to the manufacturer’s protocols. The primary library was screened using the PC-PG1 and PC-PG2 of PCR fragment as probes. The filters were hybridized with DIG-labelled probes overnight at 68 °C in a solution of 5x SSC, 1.0% (w/v) Blocking Reagent (Roche Diagnostics, Mannheim, Germany), 0.1% N-lauroylsarcosine sodium salt (w/v) and 0.02% SDS. Hybridized filters were washed twice with 5x SSC containing 0.1% SDS for 10 min at room temperature and then twice with 2x SSC containing 0.1% SDS for 30 min at 68 °C. After washing, the colour reaction was performed for 20 min with 5-bromo-4-chloro-3-indolyl-phosphate and 4-nitro blue tetrazolium chloride. Positive plaques were carried through second round screening for purification and in vivo excised to release the phagemid DNA. Positive clones were sequenced using a DNA sequencer (model DSQ-1000, Shimadzu, Kyoto, Japan) with either the RV or M13 sequencing primers according to the manufacturer’s instructions (Amersham Pharmacia Biotech, Little Chalfont, UK).
To determine the full-length nucleotide sequences for PC-EG1 and PC-EG2, RACE-PCR was performed using a MarathonTM cDNA amplification kit (Clontech, Palo Alto, CA, USA) according to the manufacturer’s protocol. The 5'-end fragments of PC-EG1 and PC-EG2 were amplified using gene specific primers, 5'-CGCATCCGTGCTACCACCAGCAC-3' and 5'-CGGGAGTATGCGGGGTTGGAGCGGC-3', respectively, and then the PCR products were used for nested PCR with specific primers, 5'-CTATGGACGAAGCAGCTAGAGCG-3' and 5'-CCCGGCAAGATCGGACCCGGGATTG-3', respectively. To amplify the 3'-end, gene specific primers, 5'-GCTGCTGATACACCTGGGTGG-3' and 5'-CCGGTTGGAGCATGACCGAGTTCG-3' were used for PC-EG1 and PC-EG2, respectively. Each primer was designed based on the nucleotide sequences of the cDNA fragments obtained from the RT-PCR. The PCR products were ligated into pGEM®-T Easy vector (Promega, Madison, WI, USA) and sequenced.
RNA blotting and hybridization
Five-microgram samples of total RNA isolated from pericarp tissues were separated by electrophoresis on 1% agarose gels containing 0.66 M formaldehyde and transferred to nylon membranes (BIODYNE®B, PALL, Tokyo, Japan). Membranes were prehybridized for more than 3 h in SDS buffer [50% deionized formamide (v/v), 5x SSC, 7% SDS, 2% Blocking Regent (Roche Diagnostics), 50 mM sodium phosphate (pH 7.0), and 0.1% N-lauroylsarcosine sodium salt (w/v)] and hybridization was performed overnight in the same buffer containing the gene-specific DIG-labelled probe at 55 °C. Probes were prepared with a PCR DIG Probe synthesis kit (Roche Diagnostics) according to the manufacturer’s instructions. All probes were synthesized from untranslated regions of the genes or from more divergent regions of the coding sequence. Following hybridization, membranes were washed twice with 2x SSC containing 0.1% SDS for 10 min at room temperature, and twice with 0.1x SSC containing 0.1% SDS for 30 min at 68 °C. Signals were detected by chemiluminescence using CDP-StarTM (Roche Diagnostics). The specificity of each probe was confirmed by Southern analysis (data not shown).
Sequence analysis
The deduced amino acid sequence of PC-PG1 and PC-PG2 were aligned with 20 full-length deduced amino acid sequences of PG homologues using Clustal V, multiple sequence alignment software (Higgins et al., 1992). Phylogenetic trees were generated from the aligned sequences using the PAUP software package, version 3.1.1 (Swofford, 1993). Deduced amino acid sequences and GenBank accession numbers are: apple (Malus domestica), pGDPG-1 (L27743); peach (Prunus persica), genomic (X77231) and PRF5 (X76735); kiwi (Actinidia deliciosa), genomic (L12019); tomato (Lycopersicon esculentum), pTOM6 (A24194), TAPG1 (U23053), TAPG2 (U70480), and TAPG4 (U70481); avocado (Persea americana), pAVOpg (L06094); melon (Cucumis melo), MPG1 (AF062465), MPG2 (AF062466) and MPG3 (AF062467); oilseed rape (Brassica napus), SAC66 (X95800) and Sta44–4 (L19879); alfalfa (Medicago sativa), P73 (U20431); cotton (Gossypium hirsutum), G9 (U09717); tobacco (Nicotiana tabacum), NPG1 (X71017); Arabidopsis thaliana (X73222); Aspergillus flavus (U05015).
A phylogenetic comparison of the deduced PC-EG1 and PC-EG2 proteins with the deduced sequences of 21 full-length plant and bacteria EGase deduced proteins were aligned and phylogenetic analysis was performed as described above. Deduced amino acid sequences and GenBank accession numbers are: tomato (Lycopersicon esculentum), Cel1 (U13054), Cel2 (U13055), Cel3 (U78526), cel7 (Y11268), Cel8 (AF098292), and TPP18 (U20590); Arabidopsis thaliana, OR16pep (U37702); strawberry (Fragaria x ananassa Duch.), eg1 (AJ006348) and eg3 (AJ006349); pepper (Capsicum annuum), cel1 (X87323), ccel2 (X97190) and ccel3 (X97189); pine (Pinus radiata), PrCel1 (U76725) and PrCel2 (U76756); avocado (Persea americana), pAV363 (M17634); poplar (Populus alba), cel1 (D32166); peach (Prunus persica), pcel1 (X96853); pea (Pisum sativum), EGL1 (L41046); orange (Citrus sinensis), CEL-b1 (AF000136); bean (Phaseolus vulgaris), BAC (M57400); bacteria (Clostridium thermocellum), CelF (X60545).
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Cloning of gene families encoding putative PGs and EGases
Two full-length cDNAs for PG were cloned from mRNAs extracted from ripening pear fruit and designated PC-PG1 (1759 bp, 461 amino acids in open reading frame) and PC-PG2 (1662 bp, 399 amino acids), respectively. Both predicted proteins contained a predicted N-terminal hydrophobic signal sequence, required for entry into the secretory system and secretion into the cell wall. PC-PG1 and PC-PG2 shared 42% identity at the deduced amino acid level. The phylogenetic tree of PG groups into three major clades (Fig. 1); Clade A includes PGs that are expressed in fruit and/or abscission zones, Clade B includes PGs in fruit or dehiscence zones and Clade C includes exo-type PGs in pollen or anthers (Hadfield et al., 1998). PC-PG1 belonged to Clade B and was closely related to an apple PG (pGDPG-1) which expressed in ripening fruit (Atkinson, 1994; Wu et al., 1993). PC-PG2 belonged to Clade A and was most homologous to a ripening-related peach PG (PRF5) (Lester et al., 1994).
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Two cDNAs for EGase were cloned and designated PC-EG1 (2242 bp, 622 amino acids) and PC-EG2 (2442 bp, 621 amino acids), respectively. The deduced protein of PC-EG2 contained an N-terminal hydrophobic signal sequence, however, the deduced protein of PC-EG1 contained a predicted hydrophobic transmembrane domain instead. PC-EG1 and PC-EG2 shared 38% identity at the amino acid level. Figure 2 shows a phylogenetic tree of EGase comprising two major clades. Clade A includes tomato Cel3 (Brummell et al., 1997) and Arabidopsis OR16pep (Sato et al., 2001), which have been proposed to play a role in cellulose synthesis and are localized mainly in the plasma membrane. All three sequences in Clade A include a putative membrane spanning domain while EGases in Clade B possess a signal sequence. PC-EG2 was closely related to strawberry eg3, that is expressed during fruit maturation (Trainotti et al., 1999) and tomato Cel8. The deduced proteins of these three EGases were approximately 100 amino acids longer than the other members in Clade B.
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Effect of propylene treatment on flesh firmness and ethylene production
The control fruit (minus propylene) began to soften after day 17 with the induction of ethylene production (Fig. 3). By contrast, the propylene treatment rapidly induced fruit softening and the flesh of the treated fruit reached a desirable texture for consumption by day 13 (Fig. 3B). Ethylene production in propylene-treated fruit was induced at day 4, accompanied by the initiation of fruit softening, and reached a peak at day 17 (Fig. 3A). An increase in ethylene production by control fruit was observed after 13 d of storage. These results confirm that ethylene induces softening and ethylene production in pear fruit, as observed in other climacteric fruits.
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Effect of propylene on expression of PG and EGase genes
PC-PG1 mRNA accumulation could not be detected in control fruit, but the accumulation in propylene-treated fruit was induced at day 4 and then increased, reaching a peak on day 9 (Fig. 4). PC-PG2 mRNA had accumulated slightly at harvest and gradually increased during storage and the accumulation was strongly enhanced by propylene treatment. PC-EG1 mRNA was constitutively expressed at all stages, irrespective of propylene treatment, while PC-EG2 mRNA was barely detected and accumulated after 9 d in propylene-treated fruits. These results indicate that expression of PG genes can be enhanced by exogenous ethylene in parallel with fruit softening, but not the expression of the EGase genes cloned in this study.
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Changes in flesh firmness and ethylene production during fruit ripening and the effects of 1-MCP
Immediately after the transfer of fruit to 20 °C, a dramatic loss of firmness was observed in control fruit (Fig. 5B). 1-MCP treatment at harvest or during cold storage inhibited fruit softening during subsequent storage at ambient temperature. The treatment at 4 d after the onset of ripening interrupted further softening. Ethylene production in control fruit was initiated during the final phase of cold storage and showed a typical climacteric pattern (Fig. 5A). Conversely, in the fruit treated with 1-MCP at harvest or during cold storage, no ethylene production was detected throughout the experimental period. The application of 1-MCP to fruit in which the ethylene climacteric rise had started, dramatically suppressed ethylene production and restricted it to a basal level. Thus, ethylene perception is required not only for initiation but also for the subsequent progression of softening and the maintenance of ripening ethylene in pear fruit.
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Expression of PG and EGase genes during fruit ripening and the effect of 1-MCP
Basal levels of PC-PG1 and PC-PG2 mRNA were detected at harvest (Fig. 6), while PC-PG1 mRNA was not detectable after 21 d of cold storage. Upon transfer of fruit to 20 °C, a dramatic increase in accumulation of both PC-PG1 and PC-PG2 mRNAs was observed in parallel with the loss of fruit firmness. The 1-MCP treatment at 4 d after the onset of ripening had little effect on the levels of the two PG transcripts for 1 d, but caused a substantial reduction at 7 d. The abundance of PC-EG1 mRNA did not change from the preclimacteric stage at harvest to the over-ripe stage, nor was it affected by the 1-MCP treatment. A slight accumulation of PC-EG2 mRNA was detected at the early ripening stage (4 d and 7 d after the onset of ripening) and a more distinct increase at the full-ripe stage (13 d after the onset of ripening). This transient expression was not affected by 1-MCP treatment at the ripening stage. These observations suggest that the expression of two PG genes correlates with the progression of fruit softening and is regulated by ethylene, while that of the EGase genes is ethylene-independent.
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Ethylene and fruit ripening
The sharp increase in ethylene production at the onset of ripening is characteristic of climacteric fruit and is considered to be a key regulatory event, controlling the initiation of changes in texture, colour, flavour and other biochemical attributes which characterize fruit ripening (Lelièvre et al., 1997a). In this experiment, application of propylene to pear fruit at a preclimacteric stage induced endogenous ethylene production and fruit softening (Fig. 3). Conversely, the application of 1-MCP at a preclimacteric stage significantly retarded their onset (Fig. 5). Oeller et al. (1991) showed that initiation of ripening in the ACS-antisense tomato, in which ethylene synthesis was reduced greater than 100-fold, was inhibited, but its ripening could be restored by exogenous ethylene. Similar observations have been reported in the ACO-antisense tomato (Hamilton et al., 1990; Picton et al., 1993) and in the ACO-antisense melon (Guis et al., 1997). These results agree well with the concept that ethylene triggers the initiation of ripening in climacteric fruit. However, the significance of ethylene production after the onset of fruit ripening has not been fully verified, that is, it has long been unclear whether the ripening-associated ethylene is a cause or a by-product of ripening.
In order to study the physiological roles of ethylene produced during the climacteric stage, pear fruit was treated with 1-MCP after the onset of the climacteric rise. After treatment, the rate of ethylene production declined to trace levels and was not restored to normal levels even at the end of the experiment. Fruit firmness was not affected by 1-MCP for 2 d, but thereafter reached a steady-state level and remained at that level until the end of the experiment (Fig. 5B). These results clearly demonstrate that ethylene is an essential factor for the progression of fruit softening, even after ripening has commenced. However, in ACO antisense melon, fruit softening accelerated by exogenous ethylene was delayed upon removed of the ethylene, though the softening was not entirely suppressed, suggesting that fruit softening depends only partially on ethylene (Flores et al., 2001). This discrepancy between these results and the ACO-antisense melon (Flores et al., 2001) might be due to difference in species or the presence of residual ethylene in the transgenic fruit.
The ethylene-biosynthetic pathway is well known to be subject to both positive and negative feedback regulation (Kende, 1993). It was previously reported (Nakatsuka et al., 1998) that ethylene production in tomato fruit treated with 1-MCP at an early ripening stage decreased, but remained at a considerable level. Conversely, in peach (Mathooko et al., 2001) and banana fruit (Golding et al., 1998), no reduction in ripening-related ethylene was observed in 1-MCP treated fruits when applied at the ripening stage. These observations indicate that the regulatory mechanism of ripening-ethylene varies even among climacteric fruits. These results indicate that ethylene biosynthesis in pear fruit is regulated entirely by positive feedback during ripening. Pear fruit would be unusual in that a single application of 1-MCP led to long-term and strong inhibition of ethylene production (Fig. 5A). The prolonged inhibition of ethylene production and fruit softening in 1-MCP-treated pear fruit might reflect a very slow recovery of sensitivity to ethylene. Otherwise, ethylene production might be so severely inhibited that autocatalysis cannot occur, even if fruit rapidly recovers sensitivity to ethylene.
Ethylene and gene expression of cell wall modifying enzymes
In general, all ripening-associated events are not always ethylene-dependent. This observation suggests that one or more crucial cell wall modifying enzymes for the softening process are regulated by ethylene in pears. Therefore, ethylene-independent factors, even if they are up-regulated with ripening, can be eliminated in the identification of key factors in the softening process of ripening fruit.
At a preclimacteric stage, low levels of PC-PG1 and PC-PG2 expression were detected (Figs 4, 6). The mRNA abundance of both genes dramatically increased with the onset of ethylene biosynthesis in control fruit (Fig. 6) and was induced by propylene treatment at the preclimacteric stage (Fig. 4), whereas 1-MCP treatment after the onset of fruit ripening strongly suppressed gene expression to preclimacteric levels (Fig. 6). These results indicate that basal levels of PC-PG1 mRNA reflect developmental regulation, but strong expression of both genes is ethylene-dependent. A similar observation has been reported in tomato discs treated with silver ions (Tucker and Brady, 1987; Davies et al., 1988).
The increase of PG activity and mRNA accumulation has been observed in several fruit concomitant with the degradation of pectin polysaccharides and softening (Huber, 1983; Fisher and Bennett, 1991; Yoshioka et al., 1992; Hadfield and Bennett, 1998). However, studies using transgenic tomato plants with altered PG gene expression indicated that PG-dependent pectin degradation is not essential for tomato fruit softening (Smith et al., 1988; Giovannoni et al., 1989). In peach fruit, endo-PG activities increase only in the late softening process and does not always relate linearly to firmness, suggesting that endo-PG and the softening process are not closely linked (Orr and Brady, 1993). In kiwifruit, solubilization of pectin occurs during ripening without changes to their primary structure and PG is implicated in the degradation of solublized pectins, but not in the initial solubilization (Redgwell et al., 1992). In the present work, the expression profiles of PG genes paralleled the apparent suppression of softening in 1-MCP-treated pear fruit and its acceleration in propylene-treated fruit (Figs 3B, 4, 5B, 6), suggesting that PG is still one of the candidate enzymes important in fruit softening. This would encourage a re-evaluation of the contribution of PG to fruit softening by a further approach using transgenic pears with an altered PG gene.
Recent studies have demonstrated that hemicellulose undergoes substantial depolymerization in many ripening fruit, including tomato (Huber, 1983), melon (Rose et al., 1998) and pear (Yoshioka, 1993) with the involvement of EGases in hemicellulose degradation (Hatfield and Nevins, 1986; Rose and Bennett, 1999). In tomato fruit, the accumulation of Cel1 and Cel2 mRNAs during ripening was demonstrated to be regulated by ethylene using 2,5-norbornadiene, a competitive inhibitor of ethylene (Lashbrook et al., 1994). Here, it is shown that PC-EG1 mRNA accumulated constitutively, irrespective of fruit ripening stage and was not affected by the 1-MCP treatment. The predicted PC-EG1 protein contains a putative membrane-spanning domain and this class of EGase genes has been suggested to be involved in cellulose synthesis and cell expansion in tomato (Brummell et al., 1997) and Arabidopsis (Sato et al., 2001). In contrast, PC-EG2 mRNA accumulated transiently in the later stages of ripening, but was not affected by the 1-MCP treatment and the expression levels were similar in soft control and firm 1-MCP-treated fruits (Fig. 6). This result suggests that PC-EG2 expression is not sufficient for fruit softening. However, Ben-Arie et al. (1979) showed that total cellulase activity correlates with fruit softening in pear. Therefore, there may be other EGase genes that were not identified in this study or the involvement of other classes of cell wall modifying enzymes such as xyloglucan-endotransglycosylases.
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Acknowledgements |
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The authors would like to thank Dr JKC Rose (Cornell University, Ithaca, New York) for his helpful advice and critical reading of this manuscript. We also thank Dr JC Pech (ENSAT UMR INRA, France) and Mr WZO Owino (Okayama University, Japan) for their helpful advice. This work was supported in part by Grants-in-Aid for Scientific Research (grant no. 09660029 to YK and 11460013 to AI) and JSPS Research Fellowships for Young Scientists (grant no. 6509 to KH) from the Ministry of Education, Science, Sports and Culture of Japan. The sequences of PC-PG1, PC-PG2, PC-EG1, and PC-EG2 of pear (cv. La France) have been submitted to DDBJ under Accession numbers AB084461, AB084462, AB084463, and AB084464, respectively.
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