JXB Advance Access originally published online on February 17, 2007
Journal of Experimental Botany 2007 58(6):1281-1290; doi:10.1093/jxb/erl283
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
Ethylene regulation of fruit softening and cell wall disassembly in Charentais melon
1Graduate School of Natural Science and Technology, Okayama University, Okayama, 700-8530 Japan
2UMR990 INRA/INP-ENSA Toulouse, Avenue de l'Agrobiopole, BP 32607, F-31326 Castanet-Tolosan, France
3Department of Plant Biology, Cornell University, Ithaca, NY 14853, USA
4Department of Plant Sciences, University of California, Davis, CA 95616, USA
To whom correspondence should be addressed. E-mail: abbennett{at}ucdavis.edu
Received 26 September 2006; Revised 23 November 2006 Accepted 27 November 2006
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Abstract |
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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 ß-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.
Key words: Cell wall modification, enzyme, fruit softening, gene expression, 1-MCP, melon fruit, transgenic plant
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Fruit ripening is a genetically programmed event that is characterized by a number of biochemical and physiological processes that alter fruit firmness, fruit colour, flavour, aroma, and texture (Brady, 1987; Brummell and Harpster, 2001). Fruit softening is a major factor that determines fruit quality and post-harvest life. In melon (Cucumis melo L.), a typical climacteric fruit, some cultivars show an extremely rapid decrease in flesh firmness during ripening, limiting their transport, storage, and shelf-life.
The plant primary cell wall consists of three co-extensive polymer networks: cellulose microfibrils coated with and cross-linked with hemicellulose and embedded in a co-extensive pectin network (Carpita and Gibeaut, 1993). Disassembly of the primary cell wall and reduction in cell–cell adhesion following dissolution of the pectinaceous middle lamella are believed to contribute to fruit softening (Crookes and Grierson, 1983; Wakabayashi, 2000). Xyloglucan, the predominant hemicellulose in the cell walls of most dicotyledons, also typically undergoes degradation during fruit ripening (Rose and Bennett, 1999), and the depolymerization of a specific subset of xyloglucan polymers that are tightly bound to cellulose microfibrils has been proposed as one of the earliest ripening-associated wall changes in some fruit (Rose et al., 1998). These modifications in cell wall polymers during ripening involve the co-ordinated and interdependent action of numerous cell wall-modifying enzymes and proteins.
A range of enzymes that contribute to pectin modification and disassembly, including polygalacturonase (PG), pectate lyase, pectin methylesterase, ß-galactanase/ß-galactosidase (ß-Gal), and 
-arabinase/-arabinofuranosidase (Rose et al., 2003) have the capacity to reduce the apparent molecular size of pectic polymers by cleaving the backbone or side chain residues (Ranwala et al., 1992; De Veau, 1993; Hadfield et al., 1998). Similarly, several classes of proteins have been proposed to participate in ripening-related disassembly and reorganization of the cellulose–xyloglucan network (Rose and Bennett, 1999). For example, endo-1,4-ß-glucanase (EGase) mRNA and enzyme activity levels have been correlated with the onset of ripening and softening of many fruits (Rose and Bennett, 1999) as has the expression of expansins, which have been hypothesized to participate in hemicellulose depolymerization by exposing previously inaccessible xyloglucan polymers to the action of wall hydrolases (Rose et al., 1997). However, the in vivo substrates of plant EGases and expansins have still not been determined, and suppression or overexpression of specific members of these gene families in transgenic tomato has not been sufficient to prevent fruit softening (Brummell and Harpster, 2001). Alternatively, xyloglucan endotransglycosylase/hydrolases (XTHs), which specifically use xyloglucan as a substrate, might contribute to xyloglucan depolymerization in ripening fruit by acting in a hydrolytic mode, or by acting as a transglycosylase and using xyloglucan oligosaccharides as acceptor substrates (Rose et al., 2002; Saladié et al., 2006), although this has yet to be directly tested.
Tomato fruit ripening and softening have been intensively studied, but the underlying molecular mechanisms may not be entirely representative of fruit softening in other species. While it is likely that fruits of most species have similar complements of cell wall enzymes, the corresponding patterns of transcript and protein abundance and enzyme activities vary substantially between fruit species (Brummell and Harpster, 2001). Thus, a comprehensive understanding of fruit softening requires investigation of several species. Charentais melon (Cucumis melo var. cantalupensis Naud.) undergoes remarkably rapid softening in the transition from the onset of ripening to over-ripe stages (Rose et al., 1998), thereby providing an excellent model plant to study fruit ripening and softening (Ayub et al., 1996; Rose et al., 1998; Hadfield et al., 2000; Perin et al., 2002). During ripening, Charentais fruit exhibit modification of both pectic and hemicellulosic polymers (Rose et al., 1998), as has been observed in tomato (Huber, 1983), avocado (Huber and O'Donoghue, 1993; Sakurai and Nevins, 1997), European pears (Hiwasa et al., 2004), and other species. Rose et al. (1998) demonstrated that the initial softening process of Charentais fruit coincides with a loss of galactose from polyuronides and xyloglucan disassembly, with the depolymerization of polyuronides occurring predominantly in the late (over-ripe) stages. However, relatively little is known about the expression of genes encoding cell wall-modifying enzymes in melons, other than those encoding PGs (Hadfield et al., 1998).
Ethylene plays a major role in regulating ripening and softening of climacteric fruit, including Charentais melon (Ayub et al., 1996; Hadfield et al., 2000), and accordingly, the expression of some ripening-related cell wall-associated genes and activities, including those of PGs (Sitrit and Bennett, 1998; Hiwasa et al., 2003, 2004), expansins (Rose et al., 1997), and EGases (Lashbrook et al., 1994). Two approaches have been used to determine the precise role of ethylene in fruit ripening and softening: transgenic suppression of ethylene production and the use of volatile inhibitors of ethylene action. Genetically engineered tomatoes and melons with suppressed ethylene biosynthesis exhibit delayed and reduced fruit softening (Murray et al., 1993; Picton et al., 1993; Ayub et al., 1996; Guis et al., 1997; Flores et al., 2001). However, whether melon fruit softening is totally, or partially, dependent on ethylene has not been clearly established, since transgenic melon with reduced expression of the 1-aminocyclopropane-1-carboxylate oxidase (ACO) gene showed a delayed but significant decrease in flesh firmness during ripening (Ayub et al., 1996; Guis et al., 1997; Flores et al., 2001). The application of a highly potent inhibitor of ethylene perception, 1-methylcyclopropene (1-MCP; Sisler and Sereck, 1997) provides an alternative approach to assess the role of ethylene in fruit softening. It was previously demonstrated that treatment of pear fruit with 1-MCP after the onset of ripening restricted softening and prevented the accumulation of PG mRNA and endo-PG activity (Hiwasa et al., 2003).
In this report, the role of ethylene in Charentais melon fruit softening was assessed using transgenic lines with suppressed ACO expression and by treating fruits with 1-MCP after the onset of ripening. The depolymerization of pectic and hemicellulosic cell wall polymers was then analysed and the expression of genes that may contribute to their degradation was monitored in order to evaluate the potential contribution of each gene to softening and the degree to which they exhibit ethylene regulation.
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Materials and methods |
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Plant materials
For experiments with transgenic fruits exhibiting suppressed ethylene synthesis, a stable homozygous line of Charentais melon (C. melo var. cantalupensis Naud. ‘Vedrantais’) harbouring an antisense ACO gene (Balague et al., 1993; Ayub et al., 1996) and its untransformed wild-type control were used. Flowers were tagged on the date of anthesis and only two fruit were kept on each plant. Maximal ethylene production and abscission of wild-type fruit were observed at 43 d after pollination (DAP) and 48 DAP, respectively. Transgenic fruit exhibited no ethylene peak, and basal ethylene levels remained <0.5% of those of the wild type at the climacteric peak, and the development of the abscission zone was also completely inhibited. Wild-type fruit were harvested at 32, 36, 40, 43, and 46 DAP, and transgenic fruit were harvested at the same stages with an additional harvest at 50 DAP. Some transgenic fruit were treated on the vine at 46 DAP with 50 µl l–1 ethylene for 4 d in 32.0 l vessels containing KOH to absorb respiratory CO2. The vessels were ventilated every 12 h to prevent oxygen deprivation and to adjust ethylene concentrations.
For experiments involving 1-MCP treatments of wild-type fruits, Charentais melon plants were cultivated in a greenhouse in the Kochi Prefecture, Japan. Preclimacteric fruit (25–27 DAP) were harvested and treated with 5000 ppm propylene at 25 °C for 48 h to induce uniform ripening. After the propylene treatment, fruit were either stored at 25 °C in ambient conditions (control) or exposed to 10 ppm of 1-MCP (Rohm and Haas, Philadelphia, PA, USA) for 24 h and then stored in the same conditions as the control fruit. Rates of ethylene production and flesh firmness were determined at 0, 2, 4, 7, 10, and 14 d after the start of the experiment. Ethylene production was determined by using a gas chromatograph (model GC-4CMPE, Shimadzu, Kyoto, Japan) and 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. Fruit mesocarp tissues were flash frozen in liquid nitrogen and stored at –80 °C prior to analysis.
Cell wall extraction and size-exclusion chromatography of cell wall fractions
Isolation and extraction of cell wall fractions was performed as previously described (Rose et al., 1998), with slight modifications. Briefly, 50 g of frozen melon mesocarp was boiled in 95% ethanol, homogenized, and filtered through Miracloth (Calbiochem). After an ethanol, chloroform:methanol (1:1, v/v), and acetone wash, the remaining alcohol-insoluble solid (AIS) residue was oven-dried at 30 °C. The AIS residue from each sample was homogenized for 4 h in water containing 0.02% NaN3 and centrifuged at 6000 g for 20 min. The supernatant was lyophilized and the water-insoluble pellet sequentially extracted with 50 mM 1,2-cyclohexanediaminetetra-acetic acid (CDTA), 50 mM Na2CO3, 4% KOH, and 24% KOH, as previously described (Rose et al., 1998). After centrifugation at 8500 g for 20 min, the solubilized fractions were collected, dialysed against distilled water, and lyophilized. Polymers from the lyophilized water-soluble and CDTA-soluble extracts were separated as previously described (Rose et al., 1998) and each column fraction assayed for uronic acid content (Blumenkrantz and Ashoe-Hansen, 1973). Polymers from 24% KOH-soluble extracts were fractionated and the xyloglucan content of the column fractions measured as before (Rose et al., 1998) and, in all fractions, total sugars were also assayed using a modified phenol–sulphuric acid assay (Rose et al., 1998).
Cloning of gene fragments encoding cell wall-modifying enzymes and probe preparation
Total RNA was extracted from mesocarp tissues using a modified hot borate method (Wan and Wilkins, 1994), and poly(A)+ RNA was isolated from the total RNA using Oligotex dT30 (Takara, Otsu, Japan) for cDNA synthesis. First-strand cDNA was synthesized using reverse transcriptase according to the manufacturer's instructions and used as a template to amplify fragments of genes encoding cell wall-modifying enzymes. Polymerase chain reaction (PCR) primers were designed based on the amino acid sequences conserved among the coding sequences of the corresponding genes in the databases [XTHs, 5'-GA(AG)CA(CT)GA(CT)GA(AG)AT(ACT)GA(CT)TT(CT)G-3' and 5'-TCNGT(GA)CA(GA)TA(GA)TT(GA)TA(TGA)ATNG-3'; expansin, as in Rose et al. (1997); EGase, 5'-GGITA(CT)TA(CT)GCIGGIGA-3' and 5'-NCCNA(AG)(AGT)AT(AG)TA(AG)TCNAC(CT) TG-3'; ß-Gal, 5'-GAYTAYYTITGGTAYATGAC-3' and 5'-CCATAYYTG ICCYTTICCCAT-3']. After PCR amplification under adequate conditions, the amplified fragments of the XTH and expansin genes were cloned into pCR-Script (Stratagene Inc. La Jolla, CA, USA) and the fragments of EGase and ß-Gal genes into pGEM-T Easy (Promega, Madison, WI, USA) according to the manufacturer's instructions, and sequenced. Based on sequence homology, a total of seven unique clones (two for XTH, one each for expansin and EGase, three for ß-Gal) were selected and designated CmXTH1 (accession no. DQ914794), CmXTH3 (accession no. DQ914796), CmExp1 (accession no. DQ914793), CmEGase1 (accession no. AB271851), CmGal1 (accession no. AB270923), CmGal2 (accession no. AB270925), and CmGal3 (accession no. AB270925), respectively.
In the experiment using transgenic fruit, radiolabelled probes were prepared using cloned fragments, random hexamer priming using [-32P]dATP (3000 Ci mmol–1), and Klenow DNA polymerase (UBS, Cleveland, OH, USA). For the PG gene family (MPG1, -2, and -3), full-length cDNA clones were used as probes (Hadfield et al., 1998). In the experiment using 1-MCP, digoxigenin-labelled probes were prepared using cloned fragments and the PCR DIG probe synthesis kit (Roche Diagnostics, Mannheim, Germany).
RNA gel blot analysis
An aliquot of total RNA from each sample was subjected to electrophoresis on a 1.2% agarose denaturing gel containing 0.66 M formaldehyde and transferred to Hybond-N+ membrane (Amersham, Piscataway, NJ, USA). In the experiment using transgenic fruit, hybridization was carried out in 10% (v/v) dextran sulphate, 2% (w/v) SDS, 1 M NaCl, and 100 µg ml–1 sonicated salmon sperm DNA at 65 °C for 16 h. The membranes were washed three times in 5x SSC, 0.1% SDS (w/v), 0.05% NaPPi at 42 °C, and three times in 0.5x SSC, 0.1% SDS (w/v), 0.05% NaPPi at 65 °C, and exposed to film with an intensifying screen (Du Pont, Boston, MA, USA) at –80 °C. In the experiment using 1-MCP, membranes were hybridized overnight in high SDS buffer [50% deionized formamide (v/v), 5x SSC, 7% SDS, 2% Blocking Reagent (Roche Diagnostics), 50 mM sodium phosphate (pH 7.0), and 0.1% N-lauroylsarcosine sodium salt (w/v)] at 42 °C. Membranes were washed at a high stringency condition (twice in 2x SSC with 0.1% SDS for 15 min at 42 °C, and then in 0.1x SSC with 0.1% SDS for 30 min at 60 °C). Signals were detected by a chemiluminescent method using CDP-star (Roche Diagnostics).
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Results |
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Fruit softening and cell wall disassembly in wild-type and transgenic melon with suppressed ACO expression
Maximal ethylene production and abscission of wild-type fruit was observed at 43 DAP and 48 DAP, respectively (data not shown). In wild-type fruit, flesh firmness decreased rapidly following the onset of ethylene production and reached the oversoft stage at 50 DAP (Fig. 1). By contrast, transgenic fruit with suppressed ACO expression exhibited no burst of ethylene synthesis and basal ethylene levels remained at <0.5% of the wild-type climacteric peak level. The development of the abscission zone and softening were also completely inhibited. Treatment of the transgenic fruit with ethylene induced a rapid decrease in flesh firmness within 4 d (Fig. 1).
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Size fractionation of water-soluble polymers indicated a clear decrease in the molecular mass of polyuronides between the immature and over-ripe stages in wild-type fruit, with a decrease in the average molecular mass from 260 kDa to
36 kDa (Fig. 2A, B). The profile of polyuronide size distribution from 46 DAP transgenic ACO-suppressed fruit was similar to that from the immature wild type at 36 DAP (Fig. 2A, C), indicating that pectin degradation was substantially suppressed in the transgenic fruit. However, following ethylene treatment of ACO-suppressed transgenic fruit, a complete loss of high and intermediate molecular mass pectins was observed (Fig. 2D). Fractionation of CDTA-soluble polymers also illustrated a clear shift in the polyuronide profile between the immature and over-ripe stages in wild-type fruit, with a decrease in the average molecular mass and with a greatly reduced portion of polyuronides eluting in the void volume (Fig. 3A, B). High molecular mass pectins were predominant at 46 DAP in ACO-suppressed transgenic fruit, and the distribution appeared essentially identical to that from immature wild-type fruit (Fig. 3C). However, the molecular weight profile of pectins from ethylene-treated transgenic ACO-suppressed fruit was similar to that of the 46 DAP ripe wild-type fruit.
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A molecular weight downshift in the 24% KOH-soluble cell wall polymer fraction, resulting from xyloglucan depolymerization, was observed during the ripening of wild-type melons (Fig. 4A, B), as was previously described (Rose et al., 1998). However, the profile of xyloglucan polymers from mature (46 DAP) ACO-suppressed transgenic fruit (Fig. 4C) was similar to that from immature wild-type fruit (Fig. 4B), and xyloglucan depolymerization was observed only after treatment with ethylene (Fig. 4D).
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Expression of cell wall-associated genes in wild-type and transgenic melon with reduced ACO expression
Fruit softening and both pectin and xyloglucan depolymerization were strongly ethylene-dependent, as revealed by evaluation of the ACO-suppressed transgenic melon fruit, and so the abundance of mRNAs encoding a range of proteins potentially involved in melon wall disassembly was evaluated to assess which gene products might be responsible for the observed polymer depolymerization. The expression of three genes encoding PGs (MPG1, MPG2, and MPG3) was evaluated; MPG1 mRNA accumulated to a high level at the peak of ethylene production in wild-type melon fruit at 43 DAP and then declined, while expression was undetectable at any stage in ACO-suppressed transgenic fruit (Fig. 5), but was markedly induced after 1 d and 4 d of exposure to exogenous ethylene (Fig. 5). The accumulation of MPG2 mRNA in wild-type fruit started slightly before the onset of ethylene production (36 DAP) and reached a peak coincident with maximal ethylene levels. In the ACO-suppressed transgenic fruit, MPG2 mRNA accumulation was slightly delayed (40 DAP) but reached a similar level to that of the wild type at later stages of ripening (46–50 DAP) and was only slightly affected by exogenous ethylene (Fig. 5). The expression of MPG3 mRNA in wild-type fruit, although slightly lower, followed a similar pattern to that of MPG1, with maximum abundance at 43 DAP. In ACO-suppressed transgenic fruit, a basal low level of MPG3 transcripts was detected between 32 DAP and 40 DAP, and a substantial increase occurred at later stages (43–50 DAP) that was enhanced by exposure to exogenous ethylene.
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XTHs have been implicated in xyloglucan metabolism and so the expression of several XTH genes was also examined. In wild-type fruit, mRNA corresponding to the CmXTH1 gene exhibited strong but transient accumulation before the onset of ethylene production (36 DAP), then declined during the increase in ethylene production (40 DAP) and was present at barely detectable levels during the later stages of ripening (Fig. 5). In the ACO-suppressed transgenic fruit, a slight and transient accumulation of CmXTH1 mRNA occurred at 36 DAP and then a clear accumulation started at 46 DAP and continued to increase to 50 DAP. Application of exogenous ethylene further induced CmXTH1 mRNA accumulation after 24 h; however, the CmXTH1 transcripts were barely detectable after 4 d of ethylene treatment. The accumulation of CmXTH3 mRNA in wild-type fruit was detected at 43 DAP, with expression levels declining at 46 DAP (Fig. 5). In the transgenic fruit, CmXTH3 mRNA levels were undetectable until 46 DAP and then accumulation further increased at 50 DAP. Application of exogenous ethylene induced a substantial increase in both CmXTH1 and CmXTH3 mRNA, but their levels declined between 1 d and 4 d of ethylene exposure.
Expansins have also been proposed to act co-operatively with cell wall hydrolases to disassemble cell wall polymers (Rose and Bennett, 1999) and, accordingly, in wild-type fruit, CmExp1 mRNA was first detected at the onset of ethylene production, concomitant with the initiation of the softening, and mRNA levels remained high throughout ripening (Fig. 5). In the ACO-suppressed transgenic fruit, a similar pattern of CmExp1 mRNA accumulation was observed, although at somewhat reduced levels relative to wild-type fruit. Treatment of the transgenic fruit with exogenous ethylene further increased CmExp1 mRNA abundance.
Fruit softening and ethylene production in 1-MCP-treated melon fruit
To test further the role of ethylene and specific gene products in melon fruit softening, wild-type melon fruit were treated with 1-MCP to inhibit ethylene perception. The treatment dramatically inhibited fruit senescence, resulting in minimal evidence of senescence or post-harvest infection, and no development of the abscission zone, even 15 d after harvest (Fig. 7). Untreated control fruit showed a typical climacteric pattern of ethylene production, reaching a peak of 18.2 nl g–1 h–1 at 4 d after harvest, and then decreasing to 20% of its peak 10 d after harvest (Fig. 6). Ethylene production was significantly suppressed by 1-MCP treatment after the onset of ripening, declining to trace levels at 2 d after 1-MCP treatment, and gradually restoring production 5 d after the 1-MCP treatment.
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Softening of untreated control fruit began at the onset of ripening, and fruit firmness decreased to half of the initial level within the first 2 d (Fig. 6) and continued to an oversoft level within the next 2 d. By contrast, loss of fruit firmness in 1-MCP-treated fruits was markedly suppressed, with fruits remaining firm until day 10, at which time softening continued, but at a much slower rate than that observed from day 2 to day 4 in control fruit.
Expression pattern of genes encoding cell wall-modifying enzymes in non-treated and 1-MCP-treated melon
As with the ACO-suppressed transgenic fruit, the abundance of mRNAs encoding several proteins potentially involved in melon cell wall disassembly was examined. MPG1 and MPG2 mRNAs were not detected during the preclimacteric stage but started to accumulate with the increase in ethylene levels and the decrease in flesh firmness (Fig. 8). During ripening, mRNA levels reached a peak at day 2 and then decreased gradually, reaching only trace levels at day 10. MPG1 mRNA abundance was strongly reduced following 1-MCP treatment at day 4 and day 7 and then returned to normal levels at day 10. By contrast, MPG2 mRNA accumulation was largely unaffected by 1-MCP treatment. The mRNA encoding ß-Gal (CmGal1) accumulated to high levels in young expanding fruit, peaked at 15 DAP, and decreased to trace levels towards the mature stage (data not shown). CmGal1 mRNA accumulation was detected during ripening after day 4 in control fruit, while expression was completely suppressed by 1-MCP treatment. The mRNA accumulation of CmGal2 showed a similar pattern to that of MPG2, except for the transient accumulation at the very early growing stage (data not shown). CmGal3 and CmEGase1 showed low levels of transcript abundance throughout growing and ripening stages in both control and 1-MCP-treated fruit.
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Discussion |
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Ethylene and softening in Charentais melon fruit
Softening of climacteric fruit is regulated by ethylene, although it is still not clear whether this ethylene dependence is complete or partial. Flores et al. (2001) observed that in ACO-suppressed transgenic melon fruit, softening was accelerated by exogenous ethylene, but continued, albeit at a reduced rate, after the removal of exogenous ethylene, suggesting that softening was partially ethylene-regulated. Here, no decline in flesh firmness of untreated ACO-suppressed transgenic fruit was observed (Fig. 1) and 1-MCP treatment at 2 d after the onset of ripening inhibited the subsequent softening dramatically (Fig. 6). The results of these two different approaches indicate that softening of Charentais fruit is completely dependent on ethylene, even after the initiation of ripening. Similarly, 1-MCP treatment after the onset of ripening resulted in transient but complete cessation of fruit softening in ‘Galia’ melons (Ergun et al., 2005) and pears (Hiwasa et al., 2003).
The loss of ethylene production in ACO-suppressed transgenic fruit resulted in minimal pectin and xyloglucan depolymerization, unlike the substantial degradation that was observed in normal ripening fruit (Figs 2, 3). Moreover, the application of exogenous ethylene stimulated disassembly of both polymer networks (Fig. 4), indicating that these processes are ethylene-regulated, and therefore that the corresponding wall-modifying genes might be similarly regulated.
Regulation of genes encoding cell wall-modifying enzymes in Charentais melon fruit
Ripening of Charentais melons is characterized by a substantial solubilization and depolymerization of pectins in the later stages of ripening (Rose et al., 1998) that is largely ethylene-regulated, as observed in molecular size analysis of water- and CDTA-soluble pectins from wild-type and ACO-suppressed transgenic melon fruit (Figs 2, 3). An increase in PG activity and mRNA levels has been reported in several fruit species concomitant with the degradation of pectin polysaccharides and softening (Huber, 1983; Fischer and Bennett, 1991). Hadfield et al. (1998) showed that the expression of MPG1, -2, and -3 increased during Charentais fruit ripening and correlated temporally with an increase in pectin-degrading activity. A similar ripening-associated expression pattern of MPG was also observed in non-treated wild-type fruit (Figs 5, 8); however, each MPG gene showed a distinct pattern of ethylene regulation. MPG1 expression was barely detectable in the ACO-suppressed transgenic fruit, but was induced to high levels by exogenous ethylene. In addition, 1-MCP application completely suppressed MPG1 expression for several days. These observations indicate that MPG1 expression is totally dependent on ethylene and suggest that MPG1 is one of the primary candidates responsible for ethylene-dependent pectin disassembly. By contrast, MPG2 expression was only slightly affected by either transgenic ACO suppression or 1-MCP treatment, indicating that regulation of MPG2 expression is ethylene-independent. Expression of MPG3 appeared to be regulated by both ethylene-dependent and -independent factors, as MPG3 mRNA was detected in ACO-suppressed transgenic fruit and expression levels were enhanced by exogenous ethylene. Since substantial pectin depolymerization and fruit softening did not occur in the ACO-suppressed transgenic fruit in the absence of ethylene, MPG2 and -3 are not likely to be major factors in inhibition of ethylene-mediated softening and pectin degradation, or their action in the cell wall may require the activities of other key wall-modifying proteins that were inactive in the absence of ethylene. It is also likely that MPG3 encodes an exo-PG, and this activity would not have been detected in the size exclusion chromatography separations (Hadfield et al., 1998).
The loss of galactose has also been proposed to contribute to pectin mobilization during ripening, and genes encoding ß-Gals have been characterized in several fruit species, such as tomato (Smith and Gross, 2000), Japanese pear (Tateishi et al., 2001), and pear (Mwaniki et al., 2005). Smith et al. (2002) demonstrated that down-regulating the expression of a tomato ß-Gal gene (TBG4) in transgenic plants resulted in fruit that were, on average, 40% firmer than controls at the red-ripe stage. In Charentais melon fruit, the galactose content in the pectin and hemicellulose wall extracts decreased during ripening, especially at late softening stages (Rose et al., 1998), and purified ß-Gals have been shown to have the capacity to catalyse an apparent decrease in the molecular size of pectins in vitro (Ranwala et al., 1992). In this study, three ß-Gal genes were cloned and characterized. While the accumulation of CmGal1 and -2 mRNAs was not detectable at the preclimacteric stage, CmGal2 mRNA accumulated abruptly with the onset of ripening and CmGal1 mRNA levels increased following a 2 d lag after the onset of ripening (Fig. 8). Similarly, 1-MCP treatment completely inhibited mRNA accumulation of CmGal1 but had less effect on CmGal2. These observations suggest that ripening-associated expression of CmGal1 is likely to be dependent on ethylene, but that CmGal2 expression could be ethylene-independent. CmGal1 mRNA was not detected in 1-MCP-treated fruit even at later stages (day 10 and day 4), when MPG1 mRNA levels had returned to normal. This suggests a role for CmGal1 in the gradual reinitiation of softening in 1-MCP-treated fruit, and a potential co-operative action between CmGal1 and MPG1. In addition, CmGal1 could be involved less in the early rapid softening process but more in the later softening process.
Rose et al. (1998) suggested that depolymerization of a xyloglucan fraction that is tightly bound to cellulose microfibrils represents one of the early events in ripening-related wall disassembly. The results described here support the correlation between depolymerization of this xyloglucan fraction and fruit softening, and further suggest that this aspect of wall disassembly is ethylene-regulated (Fig. 4). While the enzymatic basis of this process has not been established, cell wall-modifying proteins, including EGases, XTHs, and expansins, have been suggested to play a synergistic role in the restructuring of the cellulose–xyloglucan network (Rose and Bennett, 1999). Two melon XTH genes (CmXTH1 and CmXTH3) and an expansin gene (CmExp1) showed ripening-related patterns of expression (Fig. 5). No major differences were observed in overall mRNA levels of XTH genes and CmExp1 between wild-type and non-softening ACO-suppressed transgenic fruit, except a temporal delay in the transgenic fruit (Fig. 4), although exogenous ethylene enhanced the mRNA levels of these genes. Therefore, expression of CmXTH1 and -3 and CmExp1 is likely to be ripening-associated but only partially ethylene-dependent, suggesting that they do not play a significant role in ethylene-dependent melon fruit softening. While a potential function for these genes in the softening process is not as apparent for MPG1 and CmGal1, it is still possible that they represent important members of the battery enzymes employed in cell wall disassembly, and it should further be noted that the detection of wild-type levels of mRNAs in the transgenic fruit does not preclude post-transcriptional gene regulation as an important part of the overall control of cell wall modification.
This study indicates that while the overall process of cell wall disassembly in ripening Charentais melons is clearly ethylene-regulated, considerable variation exists between the patterns of regulation of divergent families of wall-modifying proteins and between individual members within those families. This work lays the foundation for further investigation into the role of specific members of these gene families, with the goal of discerning their contribution to the wall disassembly during fruit softening and the mechanisms by which their expression is co-regulated as part of the ripening programme.
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Acknowledgements |
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This work was supported in part by a Grant-in-Aid for Scientific Research (grant no. 17380024 to YK) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.
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Footnotes |
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* These authors contributed equally to the work presented here.
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Abbreviations |
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ACO, 1-aminocyclopropane-1-carboxylate oxidase; AIS, alcohol-insoluble solid; CDTA, 1,2-cyclohexanediaminetetraacetic acid; DAP, days after pollination; EGase, endo-1,4-ß-glucanase; ß-Gals, ß-galactanase/ß-galactosidase; 1-MCP, 1-methylcyclopropene; PCR, polymerase chain reaction; PG, polygalacturonase; XTH, endotransglycosylase/hydrolase.
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