Inhibition of sucrose loss during cold storage in Japanese pear (Pyrus pyrifolia Nakai) by 1-MCP

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Postharvest Biology and Technology 48 (2008) 355–363

Inhibition of sucrose loss during cold storage in Japanese pear (Pyrus pyrifolia Nakai) by 1-MCP Akihiro Itai ∗ , Takuro Tanahashi Laboratory of Horticultural Biotechnology, Faculty of Agriculture, Tottori University, Koyama Minami 4-101, Tottori 680-8553, Japan Received 24 July 2007; accepted 22 October 2007

Abstract Maintaining fruit taste during storage is important in many fruit species, including Japanese pear. Since sugars are one of the most important components of fruit taste and quality, we examined changes in sugar metabolism during storage in Japanese pear (Pyrus pyrifolia Nakai cvs. Gold Nijisseiki and Hosui). Storage at room temperature (control) resulted in rapid fruit softening and chlorophyll degradation, whereas these processes were delayed by cold storage. Moreover, additional treatment with 1-MCP (1-methylcyclopropene) extended storage periods for another month compared with cold treatment alone. Thus, cold temperature plus 1-MCP treatment is the most effective method for long storage. Total sugar content was maintained during storage in all treatments. However, cold storage led to an accumulation of hexoses and a decrease of sucrose. After 1 month of cold storage, sucrose content was almost zero, but was maintained in control fruit. However, additional treatment with 1-MCP inhibited sucrose loss and accumulation of hexoses. To investigate whether sucrose loss was related to changes in the expression of sucrose metabolizing enzymes (sucrose synthase, SS; soluble acid invertase, AIV and sucrose phosphate synthase, SPS), we examined the expression of four genes, including SS (PpSUS1), AIV (PpAIV1 and PpAIV2) and SPS (PpSPS1) during storage. Cold treatment increased expression of PpAIV1, especially after 14 days of storage and resulted in a slight decrease in PpSPS1 transcripts. Cold plus 1-MCP inhibited accumulation of PpAIV1 and a decrease in PpSPS1. PpAIV2 and PpSUS1 expression was largely unaffected by cold treatment. The increase in PpAIV1 transcript through cold storage with the concomitant decrease of PpSPS1 transcript may lead to an accumulation of hexoses and a decrease of sucrose. © 2007 Elsevier B.V. All rights reserved. Keywords: Japanese pear; 1-MCP; Cold storage; Sucrose metabolism; Gene expression

1. Introduction Fruit taste depends upon such factors as sugars, organic acids, firmness, amino acids and aromatic compounds. Of these components, sugars are one of the most important affecting fruit taste and quality, since composition and amount of accumulated sugars in fruit directly influences sweetness. Sugars synthesized in source tissues like leaves are transported into sink tissues such as fruit, shoots and other tissues. Sucrose is well known as the main translocated carbohydrate from sources to sinks in many fruit species. However, in the family Rosaceae, including pears, sorbitol is the major carbohydrate form of translocated photosynthate (Webb and Burley, 1962; Bieleski, 1977; Loescher, 1987), with over 80% of sugars exported from leaves translocated in phloem as sorbitol. Sorbitol is converted into fructose and glucose in fruit by NAD+ dependent sorbitol dehydroge-

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nase (NAD-SDH) and sorbitol oxidase (Sox), respectively. Then, sucrose is synthesized or degraded by sucrose phosphate synthase (SPS), sucrose synthase (SS) and acid invertase (AIV) (Yamaki and Moriguchi, 1989; Moriguchi et al., 1992; Tanase and Yamaki, 2000). In general, sugars have been given a relative sweetness of sucrose 100, fructose 150–170, glucose 70–80 and sorbitol 55–70 (Pancoast and Junk, 1980). Therefore, the composition of sucrose, glucose, fructose and sorbitol plays a key role in determining the sweetness of Japanese pear (Nashi) fruit (Kajiura et al., 1979) and differences in sugar composition within cultivars have been reported (Moriguchi et al., 1992). According to these reports, there are considerable differences in sucrose content, and commercially important cultivars such as ‘Nijisseiki’, ‘Kosui’ and ‘Hosui’ accumulate a large amount. Moriguchi et al. (1992) reported that the activity of enzymes catabolizing sucrose differ among Japanese pear cultivars. Maintaining fruit taste during storage is important for many fruit species, including Japanese pear, and fruit storage potential is closely related to the maximum level of ethylene production

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in this kind of pear (Itai et al., 1999). Ethylene production in cultivated Japanese pear fruit varies from 0.1 to 300 nL g−1 h−1 during fruit ripening, suggesting there are both climacteric and non-climacteric cultivars (Downs et al., 1991; Itai et al., 2003a). Climacteric-type fruit exhibit a rapid increase in ethylene production and have a low storage potential, while non-climacteric fruit show no detectable ethylene production, and fruit texture is maintained for over a month in cold storage (Kitamura et al., 1981; Itai et al., 1999). However, the taste rating is altered after long cold storage periods (Beutel, 1990; Ke et al., 1991). The taste of the fruit depends upon the complex interaction of sugars, organic acids, phenolics and more specialized flavor compounds (Tucker, 1993). Furthermore, it is primarily the sugars and organic acids that are attributed to the fruit taste. In the European pear, ethylene-enhanced ripening includes an increase in sucrose, reducing sugars and soluble pectin (Hansen, 1939). However, there are no reports of sugar metabolism, especially sucrose metabolism, in the Japanese pear during storage and thus any correlation between changes in taste and sugar composition during storage is as yet unknown. Here we report that sucrose loss occurred during cold storage via altered gene expression of sucrose metabolizing enzymes and that 1-MCP (1-methylcyclopropene), an inhibitor of ethylene action, prevented sucrose loss. 2. Materials and methods 2.1. Plant materials and treatment Japanese pear (Nashi) (Pyrus pyrifolia Nakai) cvs. Gold Nijisseiki and Hosui were grown at the Tottori University orchard. The skin of ‘Gold Nijisseiki’ fruit is greenish-yellow skin and that of ‘Hosui’ fruit is russet. Over 200 fruit of two cultivars from three trees were used for experiments. Fruit were harvested on September 3 (141 DAF: days after full bloom) and September 7 (147 DAF), 2004, the optimal commercial harvest dates for ‘Gold Nijisseiki’ and ‘Hosui’, respectively. Fruit were divided into three groups for treatments and packed in vented commercial boxes (W 500 mm; D 350 mm; H 275 mm). Packed boxes were then kept at room temperature (25 ◦ C) for up to 30 days or at cold temperature (5 ◦ C) for up to 60 days for the storage experiment. In another experiment, harvested fruit were exposed to 2 ␮L L−1 1-MCP (EthylBloc, Rhome and Hass Co., Springhouse, PA, USA) in a plastic box for 24 h at cold temperature (5 ◦ C). 1-MCP was supplied as a powder and released from vials containing weighed amounts of EthylBloc powder (0.43% active ingredient) by adding 0.1% SDS solution according to the manufacturer’s formula. In addition, 1-MCP treated fruit packed in the vented commercial boxes, were also kept at cold temperature (5 ◦ C) for up to 90 days. 1-MCP treatment was repeated every 2 weeks during cold storage. 2.2. Fruit assessments Fifteen fruit for each treatment (control, cold, cold plus 1MCP) were sampled at 0, 7, 14, 21, 30, 60 and 90 days after

harvest. Fruit color and flesh firmness of the 15 individual fruit were determined on opposite sides of each fruit. Flesh firmness was determined by measuring the maximum force required to penetrate each fruit, with the skin removed to a depth of 1 cm, using a rheometer (model RT-3010D, Sun Scientific Co., Tokyo, Japan). Puncture tests was performed using an 8 mm probe on a drill base with crosshead speed set of 50 mm min−1 . For color measurements, L* a* b* were recorded using a spectrophotometer (Model NF333, Nippon Denshoku Co., Tokyo, Japan) and hue angle (h◦ ) was calculated from arc-tan b* /a* (McGuire, 1992). For ethylene measurement, fruit were placed in 1.5 L sealed jars for 2 h at 20 ◦ C. After 2 h incubation, 2 mL headspace gas samples were withdrawn and analyzed on a gas chromatograph, with a flame ionization detector and a 60/80 mesh activated alumina column. After these measurements, the juice of three fruit was collected as replicates, and a total of five replicates were used for sugar composition analysis. A hand-operated garlic squeezer was used to extract juice from fruit with the skin removed. Juice yield was approximately 70% at harvest and decreased slightly to about 65% over the entire storage period in both cultivars. Extracted juice was centrifuged at 20,000 × g at 4 ◦ C for 15 min. Supernatant was diluted with acetonitrile to 75% acetonitrile solution and filtered with 0.2 ␮m filter unit (Millex-GN, Millipore, Bedford, MA, USA). A 20 ␮L aliquot was injected into a HPLC system (Model L7470, Hitachi, Tokyo, Japan) equipped with a RI detector (Model L7490, Hitachi) and an amino column (Shodex-Ashahi pack NH2P5 4E). Acetonitrile (70%) was used as the solvent, at a flow rate of 1 mL min−1 at 25 ◦ C. Flesh tissue samples from the equatorial region were also frozen in liquid nitrogen and stored at −80 ◦ C until RNA extraction. RNA extraction was conducted in ‘Gold Nijisseiki’ samples. 2.3. RNA extraction and cloning of SPS, SS and AIV Total RNA was extracted by the hot borate method (Wan and Wilkins, 1994). First strand cDNA was synthesized from 1 ␮g of total RNA from ripening (147 DAF) and immature (60 and 90 DAF) ‘Hosui’ fruit using M-MLV reverse transcriptase (ReverTra Ace; Toyobo, Tokyo, Japan). The SPS gene was amplified from cDNA by PCR using the following oligonucleotide primers: sense 5 -GATTCTGATACTGCTGGTCAGGT-3 and antisense 5 -CTGCTTGTGGTGTTTAGGATAAGC-3 , based on the partial SPS sequence (Yamada et al., 2006). The primers for SS were designed from PpSUS1 sequence (Tanase et al., 2002) and were sense 5 -ATTCGGCACGAGACTTGTCGC-3 and antisense 5 GAAACAACCTCTTTGCAAAG-3 . The primers for AIV were designed based on the conserved regions. The sense primer was 5 -TGGCAAMGMACKGCKTWYCAT-3 , while the antisense primer was 5 -GTARAARTCCACRCACTCCCACAT-3 (Yamada et al., 2006). PCR reactions for all clones were performed using 2.5 U of Taq DNA polymerase (Promega, Madison, WI, USA) under the following conditions: hot start 5 min at 94 ◦ C, then 40 cycles at 94 ◦ C (1 min), anneal at 55 ◦ C (1 min) and extension at 72 ◦ C (2 min), with a final extension at 72 ◦ C for 5 min. Amplified fragments were purified

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Fig. 1. Changes in flesh firmness of ‘Gold Nijisseiki’ (A) and ‘Hosui’ (B) pears stored at room temperature (25 ◦ C), cold temperature (5 ◦ C) and cold temperature with 1-MCP treatment (2 ␮L L−1 ). Error bars indicate the standard deviation of each mean value (n = 15). For the same day of storage, values with the same letters are not significantly different (P < 0.05).

and cloned into a pGEM-T easy vector (Promega). Cloned cDNA fragments were used as probes. DNA sequencing was performed using a Big Dye terminator cycle sequencing kit (Applied Biosystems, CA, USA) and an ABI PRISM 310 Genetic Analyzer (Applied Biosystems) and analyzed using DNASIS pro software (Hitachi Software Engineering Co., Tokyo, Japan). Full-length cDNAs were obtained using 3 - and 5 -RACE methods with a GeneRacer Kit (Invitrogen Co., CA, USA) according to the manufacturer’s instructions.

MCP) and shelf-life designated as fixed effects. Data were subjected to analysis of variance (ANOVA) according to treatment. All ANOVAs were performed using treatments as a statistical parameter at a significant level of P < 0.05, with JMP statistical software (version 6.0, SAS Institute, Cary, NC, USA). Means were separated using Tukey’s multiple range test.

2.4. Northern analysis

3.1. Isolation and identification of cDNA clones

For northern blot analysis, 10 ␮g total RNA was denatured and loaded onto a 1.2% (w/v) agarose gel containing 2% formaldehyde in 1× MOPS running buffer (20 mM MOPS, pH 7.0, 8 mM sodium acetate, 1 mM EDTA). RNA was separated at 50 V for 2 h, transferred onto a Hybond N+ nylon membrane (GE Healthcare Bio-science, Buckinghamshire, UK) and fixed by exposing the membrane to UV in a UV cross-linker (Hoefer Pharmacia Biotech., San Francisco, CA, USA). The blots were hybridized overnight in modified Church and Gilbert buffer (0.5 M phosphate buffer, pH 7.2, 7%, w/v, SDS, 10 mM EDTA, 100 ␮g mL−1 denatured and fragmented salmon sperm DNA) at 65 ◦ C with gene-specific probes of SPS, SS and AIV. The probes were labeled with 32 P-dCTP using a random prime labeling kit (GE Healthcare Bio-science). The partial cDNA fragments of PpAIV1, PpAIV2 and PpSPS1 and the full length fragment of PpSUS1 were used as probes. After hybridization, the membranes were washed twice in 2× SSC/0.1% SDS solution at 65 ◦ C for 15 min, then three times in 0.2× SSC/0.1% SDS solution at 65 ◦ C for 15 min and then exposed to an imaging plate (Fuji Film, Tokyo, Japan). Signals were detected with an image analyzer (FLA5000, Fuji Film).

We cloned five fragments from immature and ripe ‘Hosui’ fruit including two different cDNAs for AIV, one for SS and one for SPS genes. The sizes of cDNA fragments for AIV, SPS and SS were 0.6, 1.1 and 2.7 kb, respectively. The amplified fragments were cloned and sequenced. BLAST analysis revealed high identities of these sequences to those of AIV, SPS and SS genes. Thus, the full-length cDNAs for AIV and SPS were subsequently isolated using 3 - and 5 -RACE PCR. The two cDNAs for AIV were designated as PpAIV1 (accession no. AB334115) and PpAIV2 (accession no. AB334116), based on the structural analysis of the deduced amino acid sequences, and the cDNA for SPS was designated as PpSPS1 (accession no. AB334114). The sequences of PpAIV1 and PpAIV2 were almost identical to PsS-AIV1 (AB239589) and PsS-AIV2 (AB239590), respectively (Yamada et al., 2007). The sequence of SS cDNAs isolated by RT-PCR in this study was also identical to that of PpSUS1 (accession no. AB045710). PpSPS1 is 3660 bp long and encodes a protein with 1057 aa and contains a putative F6P binding site, a 14-3-3 regulated phosphoserine and a UDP-glucose binding domain, a light regulated phosphoserine and other domains conserved in other plant species (Langenkamper et al., 2002) (data not shown). PpSPS1 shares relatively high homology with corresponding homologues from citrus (CitSPS 70%) and potato (StSPS 68%).

2.5. Experimental design and statistical analysis The experimental setup was a completely randomized design with treatments (room temperature, cold and cold plus 1-

3. Results

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Fig. 2. Changes in skin color (hue angle) of ‘Gold Nijisseiki’ (A) and ‘Hosui’ (B) pears stored at room temperature (25 ◦ C), cold temperature (5 ◦ C) and cold temperature with 1-MCP treatment (2 ␮L L−1 ). Error bars indicate the standard deviation of each mean value (n = 15). For the same day of storage, values with the same letters are not significantly different (P < 0.05).

3.2. Fruit assessments during storage The flesh firmness of ‘Gold Nijisseiki’ and ‘Hosui’ was 28.9 and 22.9 N at harvest, respectively (Fig. 1A and B). The flesh firmness of ‘Gold Nijisseiki’ and ‘Hosui’ control fruit decreased to 15.8 and 13.6 N at 30 days after harvest. ‘Gold Nijisseiki’ fruit treated with cold and cold plus 1-MCP maintained firmness at over 25 N for 30 days after harvest. Cold storage of ‘Hosui’ fruit resulted in a decrease to 15 N, while firmness was maintained in storage at 23 N in fruit treated with cold plus 1-MCP. Flesh firmness was relatively well maintained through cold storage for 2 months and then decreased rapidly, while the cold plus 1-MCP treatment maintained firmness of ‘Gold Nijisseiki’ fruit for over 90 days. Although firmness was not maintained through cold storage of ‘Hosui’ fruit and it decreased gradually, additional 1-MCP treatment inhibited the gradual loss of flesh firmness. The skin color of ‘Gold Nijisseiki’ control fruit at harvest was greenish, and then turned yellow in 2 weeks, demonstrated by a rapid decline in hue angle value; however, cold and cold plus 1-MCP treatments delayed color changes (Fig. 2A and B). In particular, cold plus 1-MCP treatment delayed the decline in hue angle value more than cold treatment alone. In ‘Hosui’, skin color was russet brown at harvest, and then turned orange brown in 2 weeks but cold plus 1-MCP storage in particular delayed these color changes. Ethylene production of fruit was less than 0.2 nL g−1 h−1 throughout all experiments in both cultivars and there were no differences found between treatments (data not shown). Total sugar content showed few changes in all treatments throughout the experiments in both cultivars (Fig. 3A and B). Of the soluble sugars in the fruit, sucrose was predominant at harvest in ‘Gold Nijisseiki’ and fructose was predominant at harvest in ‘Hosui’ fruit (Fig. 3C–F). In both cultivars, sucrose content increased in control and cold plus 1-MCP treatments until 3 weeks after harvest, while it decreased rapidly in cold storage 2 weeks after harvest (Fig. 3E and F). Also virtually no sucrose was detected in the fruit of both cultivars in cold storage 4 weeks after harvest. The cold plus 1-MCP treatment was

better at preventing sucrose loss than cold alone, and resulted in the presence of sucrose as long as 2 months after harvest. On the other hand, cold storage increased glucose and fructose contents 2 weeks after harvest in both cultivars (Fig. 2C, D, G, H). 1-MCP treatment inhibited the accumulation of glucose and fructose until 4 weeks after harvest. Sorbitol content was largely unaffected by cold or cold plus 1-MCP treatments in either cultivar (Fig. 3I and J). 3.3. Expression of SS, SPS and AIV during storage PpSUS1, PpSPS1, PpAIV1 and PpAIV2 transcripts expressed during storage were detected by northern blots with ‘Gold Nijisseiki’ fruit (Fig. 4). We confirmed that PpAIV1 and PpAIV2 probes did not cross-hybridize (data not shown). The PpSUS1 transcript showed constant high expression at room temperature, and high expression in the cold, although low expression was shown at 7 days. Expression with the cold plus 1-MCP treatment was high at 7 days after treatment, and then remained constant, except at 30 days where low expression was shown. The PpSPS1 transcript level increased gradually during storage at room temperature, while it was barely detectable throughout cold storage. In contrast, the cold plus 1-MCP treatment resulted higher expression at 7 days after harvest, and then decreased until 30 days. Of the two soluble acid invertase genes, PpAIV1 and PpAIV2, the former showed predominant expression in this study. The PpAIV1 transcripts accumulated in response to cold storage and reached maximum abundance at 60 days. In contrast, no expression was detected in control fruit and it accumulated at a much lower rate during later storage in cold plus 1-MCP fruit. PpAIV2 showed faint expression during cold storage. 4. Discussion Fruit are often attractive to the consumer because of their aesthetic qualities of flavor, color and texture. They soften during storage and this is a major quality that often dictates shelflife. Color change is associated with fruit ripening and also

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Fig. 3. Changes in total and specific sugar contents of ‘Gold Nijisseiki’ and ‘Hosui’ pears stored at room temperature (25 ◦ C), cold temperature (5 ◦ C) and cold temperature with 1-MCP treatment (2 ␮L L−1 ). (A) Total sugar content of ‘Gold Nijisseiki’, (B) total sugar content of ‘Hosui’, (C) fructose content of ‘Gold Nijisseiki’, (D) fructose content of ‘Hosui’, (E) sucrose content of ‘Gold Nijisseiki’, (F) sucrose content of ‘Hosui’, (G) glucose content of ‘Gold Nijisseiki’, (H) glucose content of ‘Hosui’, (I) sorbitol content of ‘Gold Nijisseiki’ and (J) sorbitol content of ‘Hosui’. Error bars indicate the standard deviation of each mean value (n = 5). For the same day of storage, values with the same letters are not significantly different (P < 0.05).

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Fig. 3 (Continued ).

represents a major characteristic, along with softening, for the determination of eating quality. The general objective in fruit storage is to slow down the aging processes related to qualities of flavor, color and texture. Cold temperature and 1-MCP, an inhibitor of ethylene action, are widely used for this purpose in many species (Blankenship and Dole, 2003). European pear fruit have been used to study the effects of 1-MCP on ethylene biosynthesis, fruit softening, flavonoid synthesis and superficial scald. Furthermore, 1-MCP is reported to retain fruit firmness and to extend shelf-life (Ekman et al., 2004; Trinchero et al., 2004; MacLean et al., 2007; Spotts et al., 2007). However, there are no reports on the effects of 1-MCP on the ripening characteristics of the Japanese pear. A delay in the softening and chlorophyll degradation was observed through cold storage with 1-MCP in ‘Gold Nijisseiki’ and ‘Hosui’ Japanese pears. 1-MCPtreated ‘Gold Nijisseiki’ fruit maintained more than 20 N after 3 months in storage. In this study, we found the same inhibitory effects on the ripening of Japanese pear as is found in the European pear. The rate of ethylene production ranges from 0.1 to 300 nL g−1 h−1 during ripening of Japanese pear cultivars, and the different ethylene production rates are attributed to different ACC synthase genes (PpACS1 and PpACS2) (Itai et al.,

1999, 2003b). Cultivars used in this study were low ethylene producing non-climacteric types (Downs et al., 1991; Itai et al., 1999) and lack the expression of PpACS1 and PpACS2, which are responsible for high and moderate ethylene production in climacteric cultivars during fruit ripening (Itai et al., 2005). In non-climacteric fruit species, such as orange and strawberry, softening is not affected by 1-MCP (Porat et al., 1999; Tian et al., 2000). In this study, 1-MCP delayed fruit softening in both cultivars, suggesting that non-climacteric cultivars are unable to produce a large amount of ethylene, but that ethylene response in Japanese pear is the same as in climacteric types. Our data indicate that very small amounts of ethylene were sufficient to slowly enhance fruit softening in Japanese pear. The sugar content in fruit depends on the metabolism of unloaded sugars. Pears belong to the Rosaceae family, in which the main translocating sugar is sorbitol (Bieleski, 1977). Sorbitol is converted into glucose, fructose and sucrose in the fruit. The composition of these four sugars plays a key role in determining the sweetness of pear fruit. Differences in sugar composition within Pyrus species have been reported (Kajiura et al., 1979). According to that report, Japanese pears tend to have high sucrose contents, while Chinese pears (Pyrus bretschneideri)

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Fig. 4. Expression analysis of genes encoding for sucrose synthase (PpSUS1), sucrose phosphate synthase (PpSPS1) and soluble acid invertase (PpAIV1 and PpAIV2) in ‘Gold Nijisseiki’ fruit stored at room temperature (25 ◦ C), cold temperature (5 ◦ C) and cold temperature with 1-MCP treatment (2 ␮L L−1 ). Northern blot analysis of total RNA (10 ␮g per lane) extracted from flesh. Ethidium bromide-stained 18SrRNA shown as a control for equal loading.

tend to have low contents, and European pears high fructose and starch contents. Some Japanese pears accumulate high amounts of sucrose in the later stages of fruit development (Moriguchi et al., 1992). The cultivars used in this study had high sucrose contents at harvest. Storage of fruit at cold temperature led to an accumulation of reducing sugars (fructose and glucose) and loss of sucrose. Sucrose content was almost zero after 1 month of treatment in both cultivars. However, on the same test day, control and cold plus 1-MCP fruit had maintained relatively high sucrose contents. A similar phenomenon is observed in potato tubers (Burton, 1969; Zrenner et al., 1996). On the other hand, sucrose content is maintained during cold storage in apples (Ackermann et al., 1992). Furthermore, sucrose loss is not increased by cold storage in loquat (Ding et al., 1998), but is accelerated by storage at room temperature (20 ◦ C) in sugarcane (Mao et al., 2006). These reports indicate that sugar content during storage is regulated by variability in the expression of different enzymes in fruit of various species. Cold storage of potato tubers is known to cause accumulation of reducing sugars and loss of sucrose (Zrenner et al., 1996). This process is called cold sweetening, which leads to dark brown coloring of tuber slices after frying and is a major economic problem (Burton, 1969; Samotus et al., 1974). In Japanese pear, 1-MCP prevented a decline in sucrose content during cold storage. As far as we know, there are no reports of 1-MCP altering sucrose metabolism during cold storage. Zrenner et al. (1996) report that the soluble acid invertase gene (INV-19) is involved in the accumulation of reducing sugars during cold storage in potato tubers. We thus conducted expression analysis of SS, SPS and AIV genes during cold storage in Japanese pear fruit to investigate which enzymes involved in sucrose metabolism are also responsible for hexose accumulation. The pear SS gene, PpSUS1,

showed higher transcript levels regardless of treatment, except at 7 days of cold storage and at 30 days of cold plus 1-MCP storage. However, small changes in the PpSUS1 transcript may not be a determinant for sucrose loss during cold storage. SS is regulated by reversible phosphorylation in Japanese pear (Tanase et al., 2002), maize (Huber et al., 1996), soybean (Zhang and Chollet, 1997) and mungbean (Nakai et al., 1998). Tanase et al. (2002) report that the Ser residue at the N terminal of pear SS is phosphorylated and that dephosphorylated SS is inclined toward sucrose synthesis. We did not check the phosphorylation state of SS in this study and further investigation into the phosphorylation state of SS will be needed to determine the direction of sucrose synthesis or cleavage during storage in Japanese pear. We investigated the expression of two AIV genes (PpAIV1 and PpAIV2) during storage. The transcript level of PsS-AIV1, identical to PpAIV1, was highest at a very early fruit stage (34 DAFB: 34 days after full bloom) and decreased rapidly during fruit development, while the transcript of PsS-AIV2, identical to PpAIV2, increased gradually from a very young stage (34 DAFB) until the middle stage (79 DAFB) (Yamada et al., 2007). Yamada et al. (2007) suggest that PsS-AIV1 and PsS-AIV2 play an important role for cell division and expansion, respectively. Although virtually no expression of PpAIV2 was observed during storage, cold treatment increased PpAIV1 expression, concomitant with sucrose loss. Additional 1-MCP treatment inhibited the accumulation of PpAIV1 transcript resulting in delayed sucrose loss. These data suggest that PpAIV1 plays an important role in sucrose degradation during storage. Cold induction of AIV gene (INV-19) in potato tubers causes accumulation of reducing sugars (Zrenner et al., 1996). PpAIV1 may have the same function as the potato INV-19 gene in response to cold. 1-MCP caused a greater reduction in PpAIV1 expression

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than cold storage. Our data also suggest that PpAIV1 may be under regulation of the ethylene signaling pathway. PpAIV1 is thought to have an important role in supplying substrates for cell division and for responses to cold and ethylene. SPS is also an important enzyme in sucrose biosynthesis. Known SPS genes are classified into six families: 1a, 1b, 2d, 2m, 3 and 4, and PpSPS1 belongs to family 2d (Lutfiyya et al., 2007). We could obtain only one gene (PpSPS1) from ripe fruit in Japanese pear. Other isoforms will be cloned from leaves, young fruit and other tissues. PpSPS1 contains a putative F6P binding site, a 14-3-3 regulated phosphoserine, a UDPglucose binding domain, a light regulated phosphoserine and other domains conserved in other plant species (Langenkamper et al., 2002). Thus, PpSPS1 may have SPS activity. Cold treatment decreased PpSPS1 expression, concomitant with sucrose loss. 1-MCP resulted in the accumulation of PpSPS1 transcript, concomitant with inhibition of sucrose loss. These data suggest that the expression of PpSPS1 and PpAIV1 may be responsible for sucrose degradation during storage in Japanese pear. 5. Conclusion Cold storage caused an accumulation of hexoses and a decrease of sucrose in Japanese pear. Cold treatment only enhanced changes in taste. This may be due to up-regulation of PpAIV1 and down-regulation of PpSPS1. Additional 1-MCP treatment inhibited the accumulation of PpAIV1 transcript and the decline in PpSPS1 transcript, resulting in delayed sucrose loss. This is the first report of the control of AIV and SPS genes by cold and ethylene. Cold plus 1-MCP could prove of considerable benefit to the market by reducing changes in sugar composition as well as those in firmness and appearance in Japanese pear. Acknowledgements This work is supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan (No. 18780021) and from the Japanese Society for the Promotion Science (No. 17380023). References Ackermann, J., Fischer, M., Amado, R., 1992. Changes in sugars, acids, and amino acids during ripening and storage of apples (cv. Glockenapfel). J. Agric. Food Chem. 40, 1131–1134. Beutel, J.A., 1990. Asian pears. In: Janick, J., Simon, J.E. (Eds.), Advances in New Crops. Timber Press, Portland, OR, USA, pp. 304–309. Bieleski, R.L., 1977. Accumulation of sorbitol and glucose by leaf slices of Rosaceae. Aust. J. Plant Physiol. 4, 11–24. Blankenship, S.M., Dole, J.M., 2003. 1-Methylcyclopropene: a review. Postharvest Biol. Technol. 28, 1–25. Burton, W.G., 1969. The sugar balance in some British potato varieties during storage. II. The effects of tuber age, previous storage temperature, and intermittent refrigeration upon low-temperature sweetening. Eur. Potato J. 12, 81–95. Ding, C.-K., Chachin, K., Hamauzu, Y., Ueda, Y., Imahori, Y., 1998. Effects of storage temperatures on physiology and quality of loquat fruit. Postharvest Biol. Technol. 14, 309–315.

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