Journal of Experimental Botany, Vol. 51, No. 347, pp. 1163-1166,
June 2000
© 2000 Oxford University Press
Short Communication Papers |
Isolation of cDNA clones corresponding to genes expressed during fruit ripening in Japanese pear (Pyrus pyrifolia Nakai): involvement of the ethylene signal transduction pathway in their expression
Laboratory of Horticultural Science, Faculty of Agriculture, Tottori University, Tottori 680–8553, Japan
Received 11 November 1999; Accepted 3 March 2000
Abstract
Thirty cDNA clones of genes corresponding to mRNAs up-regulated during fruit ripening of Japanese pear (Pyrus pyrifolia Nakai cv. Kikusui) were obtained by differential screening of a ripe fruit cDNA library. All of these cDNAs were sequenced and gathered into 11 non-redundant groups after database searches. These clones represented genes associated with stress responses, protein catabolism or pathogenesis. The accumulation of transcripts of 3 out of 11 genes was inhibited by 1-methylcyclopropene (MCP), an inhibitor of ethylene action.
Key words: Japanese pear, Pyrus pyrifolia, fruit ripening, differential screening, 1-methylcyclopropene (MCP).
Introduction
Fruit ripening is a complex developmental process that involves changes in gene expression and enzyme activity (Brady, 1987
; Fischer and Bennett, 1991). Understanding the fruit ripening process is of prime importance in improving fruit quality and storage potential. A useful approach to investigate such changes is to isolate the transcripts encoding proteins associated with the ripening process, using differential screening or differential display techniques. Progress in sequencing technology has enabled a large body of sequence data to become available in the databases related to ripening of Japanese pear. A long-term goal of the research in this laboratory has been to identify abundantly expressed transcripts and to utilize upstream sequences from these genes to regulate the production of foreign proteins in transgenic Japanese pear plants. Due to the interest in genes encoding abundant ripening-associated transcripts, differential screening was used with database searches to identify the putative function of the cDNAs. Fruit ripening is considered to have both ethylene-dependent and ethylene-independent components (Leliévre et al., 1997). Using 1-methylcyclopropene (MCP), an inhibitor of ethylene action (Sisler and Serek, 1997), it has been shown that the up-regulation of isolated mRNAs can be classified as either dependent or independent of ethylene action.
Materials and methods
Unripe preclimacteric Japanese pear fruit (Pyrus pyrifolia Nakai. cv. Kikusui) were harvested 132 d after flowering (DAF) from the orchard of Tottori University in Japan. Fruits were kept at 20 °C up to 14 d after harvest (DAH). Total RNA was extracted from the unripe (132 DAF) and ripe (14 DAH) fruit by the hot borate method (Wan and Wilkins, 1994). Poly(A)+ RNA was isolated from total RNA by Oligotex dT30 (Takara Shuzo, Kyoto, Japan). The double strands of the cDNAs were synthesized from the poly(A)+ RNA using a cDNA synthesis kit (Time Saver cDNA synthesis kit, Pharmacia, Uppsala, Sweden). The ripe fruit cDNA library was constructed with an efficiency of 0.9x106 pfu µg-1 of cDNA. For library screening, one filter was hybridized to radiolabelled cDNA from unripe fruit, and the other one was hybridized to radiolabelled cDNA from ripe fruit. A total of 1x104 plaques were screened. Filters were prehybridized at 42 °C for 3 h in 40% formamide, 5x SSPE, 5x Denhardt's, 0.1% SDS, and 0.2% Blocking Reagent (Boehringer Manheim), and hybridized overnight at 42 °C in the same solution containing the radiolabelled probe. Hybridized filters were washed twice for 15 min each at 65 °C in 2x SSC and 0.1% SDS, and three times for 15 min at 65 °C in 0.2x SSC and 0.1% SDS. Then, the filters were exposed to X-ray film for 72 h at -80 °C with an intensifying screen. Ripening-related clones selected from the library screening were in vitro excised from the 
Excell vector into the pExcell phagemid vector, according to the manufacturer's instructions (Pharmacia). Thirty putative ripening-related cDNA clones were sequenced, using M13 universal and reversal primers, on an ALF Express DNA sequencer (Pharmacia). The sequences obtained were edited to remove any vector sequence and poly A sequence, and compared with all known DNA sequences using BLASTN or TBLASTN programes on a network server (www.blast.genome.ad.jp). MCP was synthesized according to the methods of Sisler and Serek (Sisler and Serek, 1997) and fruits (4 DAH) were treated with 1–2 ppm. MCP in 3 l jars for 12 h and then allowed to ripen at 20 °C for 2 d. Northern analysis of transcript levels during fruit ripening and the effect of MCP on the expression were determined by 32P-labelled isolated cDNAs and ACC oxidase cDNA (PPAOX1) (Itai et al., 1999) as probes with FLA2000 Bio Imaging Analyzer (Fuji Film, Tokyo).
Results and discussion
The 30 differentially expressed clones that were selected were classified by screening the ripe fruit library into 11 non-redundant groups of cDNAs after sequence alignment and homology searches (Table 1). There were many redundant isolates. For example, nine identical clones encoded a glycine-rich protein, five identical clones encoded a methallothionein (MT)-like protein, four identical clones encoded a stress-related ripening protein, and four identical clones encoded an endo-chitinase class III were isolated. Identified up-regulated cDNAs were mainly associated with pathogenesis (PPFRU 9 and 19), protein metabolism (PPFRU7, 11 and 32) or stress responses (PPFRU8, 13, 16 and 21). Transcripts encoding two MT-like proteins (PPFRU 8 and 16) were abundant in Japanese pear during fruit ripening (Fig. 1A). PPFRU 8 shared 78% identity with PPFRU 16. Both clones belong to Type-2 MT-like proteins (Reid and Ross, 1997). MTs are small Cys-rich proteins isolated from animals, fungi, and plants, and are known to be induced by hormones, heavy metals, and stress conditions (Coupe et al., 1995; Foley et al., 1997; Zhou and Goldsbrough, 1994). Clones encoding MT-like proteins have been isolated from developing kiwifruit, ripening banana fruit, cold-stored apple fruit, and developing satuma mandarin fruit (Clendennen and May, 1997; Ledger and Gardner, 1994; Moriguchi et al.; 1998; Reid and Ross, 1997), although their precise function in fruit development and ripening is unknown.
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PPFRU13 shared highest sequence homology with the glycine-rich protein from Arabidopsis thaliana (Lang and Palva, 1992
). PPFRU 21 shared the highest sequence homology with a stress-related ripening protein from Solanum chacoense (Silhavy et al., 1995). These two genes are known to be induced by abscisic acid (ABA), low temperature, and water stress (Chang et al., 1996; Close et al., 1989; Lang and Palva, 1992; Robertson et al., 1995). This suggests that Kikusui pear fruit may be under water stress after harvest.
Other clones (PPFRU7, 11 and 32) that may be involved in protein metabolism are those encoding cysteine protease, aspartic proteinase and asparagine synthetase. These proteins may be associated with proteolysis and senescence programs (Buchanan-Wollaston, 1997; Noodén et al., 1997). Cysteine protease homologous genes have been cloned from various plant species during leaf senescence (Drake et al., 1996; Lohman et al., 1994; Smart et al., 1995). The distinction between ripening and senescence has never been finely drawn (Brady, 1987) and both processes may contribute to the final stages Japanese pear fruit development.
Transcripts that encode proteins associated with plant defence responses (PPFRU 9 and 19) were abundant during fruit ripening in Japanese pear. PPFRU 19 showed 98% identity with a polygalacturonase inhibitor (PGIP) of Bartlett pear (Stotz et al., 1993). PGIPs inhibit the activity of fungal polygalacturonases, accumulate elicitor-active oligosaccharide, and therefore exhibit enhanced resistance to fungi. In Bartlett pear, an increase in both the activity of PGIP and an accumulation of PGIP mRNA has been reported during fruit ripening (Stotz et al., 1993) and similar observations have been made on Japanese pear. PPFRU 9 shared high homology with class III acidic chitinases. Recently, it has been reported that the abundant expression of chitinases is associated with fruit ripening in banana, and grapes (Clendennen et al., 1998; Robinson et al., 1997) and these proteins may have a role to protect against pathogenic attack.
Other clones (PPFRU14 and 36) showed high homology with Calmodulin and F1-ATPase, respectively (Morikami et al., 1992; Nicolas et al., 1998; Okamoto et al., 1995). The expression of these clones was up-regulated during ripening, but the expression levels were lower than those of other clones.
Many of the physiological changes during fruit ripening occur in response to elevated ethylene production. The cDNA clones identified in this study were classified according to ethylene-dependence using MCP, which blocks the ethylene receptor. The expression of three (PPFRU16, PPFRU21, and PPFRU36) of the 11 clones and an ACC oxidase cDNA (PPAOX1) was reduced by the treatment with MCP (Fig. 1B
). The expression of ACC oxidase is considered to be subject to positive feedback regulation (Bouquin et al., 1997; Nakatsuka et al., 1997). These data imply that the expression of PPFRU16, PPFRU21 and PPFRU36 is subject to ethylene control during fruit ripening. As mentioned above, both PPFRU 8 and PPFRU 16 showed highest identity with a type-2 MT-like protein, but the expression of PPFRU 8 was only developmentally regulated, while the expression of PPFRU 16 was subject to ethylene control. A gene encoding a MT-like protein was shown to be up-regulated in leaflet abscission zones of Sambucus nigra in the presence of ethylene (Coupe et al., 1995). As described above, PPFRU 21 shared the highest sequence homology with a stress-related ripening protein and PPFRU36 showed highest homology with an F1-ATPase and this is the first report that the expression of such genes may be regulated by ethylene. These three genes may be good candidates for determining the promoter sequence required for ethylene-stimulated fruit ripening.
Acknowledgments
This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan (No. 10556007 and No. 10660026).
Notes
1 To whom correspondence should be addressed. Fax: +81 857 31 6479. E-mail: itai{at}muses.tottor\|[hyphen]\|u.ac.jp 
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