Influence of time from harvest to 1-MCP treatment on apple fruit quality and expression of genes for ethylene biosynthesis enzymes and ethylene receptors

Postharvest Biology and Technology 43 (2007) 28–35

Influence of time from harvest to 1-MCP treatment on apple fruit quality and expression of genes for ethylene biosynthesis enzymes and ethylene receptors Miho Tatsuki a,∗ , Atsushi Endo b,1 , Hiroshi Ohkawa c b

a National Institute of Fruit Tree Science, NARO, 2-1 Fujimoto, Tsukuba, Ibaraki 305-8605, Japan Fukushima Fruit Tree Experiment Station, 1 Danno Higashi, Hirano, Iisaka, Fukushima 960-0231, Japan c National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan

Received 10 March 2006; accepted 26 August 2006

Abstract A strong potent inhibitor of ethylene action, 1-methylcyclopropene (1-MCP) maintains apple fruit quality during storage. To understand the influence of time after harvest until 1-MCP treatment, we studied expression patterns of genes for ethylene biosynthesis enzymes and ethylene receptors in two apple cultivars, ‘Orin’ and ‘Fuji’, which differ in ethylene production. Ethylene production and expression of MdACS1, MdERS1, and MdERS2 were suppressed in all 1-MCP-treated ‘Fuji’ fruit, but in ‘Orin’, the later 1-MCP was applied after harvest, the less was the suppression of ethylene production and expression of these genes. In fruit in which 1-MCP had low efficacy (e.g., ‘Orin’ treated at 7 DAH), ethylene production and the level of MdERS1 were briefly reduced by 1-MCP treatment at 2 days after treatment, then began to increase. Since ethylene receptors negatively regulate the ethylene signalling pathway, the increased levels of ethylene production and ethylene receptors after 1-MCP treatment might reduce 1-MCP efficacy. © 2006 Elsevier B.V. All rights reserved. Keywords: Malus domestica Borkh.; ACC synthase; ACC oxidase; Endopolygalacturonase

1. Introduction Ethylene plays essential roles in multiple developmental processes, including seed germination, fruit ripening, abscission, and senescence (Abeles et al., 1992). In climacteric fruit, increased ethylene production is required for normal fruit ripening, as demonstrated in transgenic tomato fruit in which ethylene production is suppressed (Hamilton et al., 1990; Oeller et al., 1991; Picton et al., 1993) or ethylene sensing is inhibited (Wilkinson et al., 1997). The pathway of ethylene biosynthesis proceeds from S-adenosyl-l-methionine (SAM) via 1-aminocyclopropane-1-carboxylic acid (ACC) (Adams and Yang, 1979). The first step is catalysed by ACC synthase ∗

Corresponding author. Tel.: +81 29 838 6506; fax: +81 29 838 6437. E-mail address: [email protected] (M. Tatsuki). 1 Present address: Northern Fukushima Agriculture and Forestry Office, Japan. 0925-5214/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2006.08.010

and the second by ACC oxidase. ACC synthase is generally the rate-limiting enzyme in the biosynthetic pathway. ACC synthase and ACC oxidase are encoded by multigene families, and their expressions are regulated by developmental and environmental factors (Kende, 1993; Zarembinski and Theologis, 1994). Ethylene perception and signal transduction have been extensively studied at the genetic and biochemical levels in Arabidopsis and tomato. Crossing experiments with Arabidopsis receptor knockouts indicate that ethylene receptors act as negative regulators of ethylene response (Hua and Meyerowitz, 1998). Ethylene receptor genes have been identified in many plant species, and their expression patterns have been examined; their expressions are regulated differentially according to tissue, developmental stage, and environmental stimuli (Lashbrook et al., 1998; Sato-Nara et al., 1999; Rasori et al., 2002; Cin et al., 2005). A strong potent inhibitor of ethylene action, 1methylcyclopropene (1-MCP), binds ethylene receptors more

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strongly than ethylene and prevents the physiological action of ethylene. The application of 1-MCP has been shown to maintain the freshness of various fruit, vegetables, and flowers (Blankenship and Dole, 2003). Apple is a climacteric fruit, and harvested fruit quality is maintained by 1-MCP treatment (Fan et al., 1999a, 1999b; Rupasinghe et al., 2000; Watkins et al., 2000; DeEll et al., 2002). The efficacy of 1-MCP is influenced by cultivar, storage conditions, treatment temperature and duration, fruit maturity, and time from harvest (Blankenship and Dole, 2003). The effectiveness of 1-MCP declined slightly with later harvest times (Mir et al., 2001). Ethylene production in apple fruit at the time of 1MCP treatment might influence 1-MCP efficacy (Watkins et al., 2000), although 1-MCP has a higher affinity for ethylene receptors than ethylene has. The molecular mechanism of 1-MCP efficacy has not yet been examined. Ethylene biosynthesis in apple fruit differs considerably among cultivars (Abeles et al., 1992). Sunako et al. (1999) reported that an allele of MdACS1, which is expressed predominantly in climacteric fruit (Dong et al., 1991; Cin et al., 2005), accounts for the low level of ethylene production in apple cultivars such as ‘Fuji’, which is homozygous for MdACS1-2, which possesses a retroposon-like insertion in the promoter region. The ACC synthase allelotype has no effect on the fruit softening rate, although it defines the rate of fruit drop (Sato et al., 2004). Three apple ethylene receptor genes (MdETR1, MdERS1, MdERS2) have been isolated (Cin et al., 2005; Tatsuki and Endo, 2006), and their expression patterns were examined in abscising fruitlets (Cin et al., 2005) and in 1-MCP-treated apple fruit (Tatsuki and Endo, 2006). MdETR1, MdERS1, and MdERS2 are expressed in ripening fruit, and expression of MdERS1 and MdERS2 is suppressed by 1-MCP treatment (Tatsuki and Endo, 2006). Endopolygalacturonase (PG) catalyses the hydrolytic cleavage of ␣-(1-4) galacturonan linkages and is a key enzyme involved in the large changes in pectin structure that accompany the ripening of many fruit (Hadfield and Bennett, 1998). In tomato, accumulation of PG mRNA is induced by ethylene (Maunders et al., 1987; Bird et al., 1988; Sitrit and Bennett, 1998), and continuous ethylene perception is required for PG expression (Lincoln et al., 1987; Davies et al., 1988). MdPG1 cDNA (L27743) has been isolated in apple (Atkinson, 1994); it is up-regulated in ripening apple fruit (Atkinson et al., 1998) and is controlled by ethylene (Wakasa et al., 2006). In this study, to understand the influence of the timing of 1-MCP treatment on its efficacy, we treated apple fruit at 1, 3, or 7 days after harvest, and examined fruit quality and the expression patterns of genes for ethylene biosynthesis enzymes (ACC synthase, MdACS1 and MdACS3; and ACC oxidase MdACO1), ethylene receptors (MdETR1, MdERS1, MdERS2), and endopolygalacturonase (MdPG1) as a ripening indicator. We used two apple cultivars, ‘Fuji’ and ‘Orin’, which differ in ethylene production. The MdACS1 genotype of ‘Orin’ is Md-ACS1-1/1-2, and ‘Orin’ produces more ethylene than ‘Fuji’.

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2. Materials and methods 2.1. Plant materials and treatment ‘Orin’ and ‘Fuji’ apple (Malus domestica Borkh.) fruit were harvested at commercial maturity in Fukushima Prefecture. All apples were stored at 20 ◦ C for 1, 3, or 7 days until 1-MCP treatment. For 1-MCP treatment, fruit were placed in 117-L plastic containers and treated with 1 ␮L L−1 of 1MCP (SmartFresh, AgroFresh Inc., Springhouse, PA, USA) for 12 h at 22 ◦ C. For the control, fruit that were stored for 1 day after harvest (DAH) were left in air for 12 h at 22 ◦ C. After 1-MCP treatment, fruit were stored in air for 2, 4, 8, 15, 31, 46, 61, or 91 days at 20 ◦ C. Five fruit per treatments on each day were sampled and used for ethylene measurement, and then fruit firmness and acidity were determined. Part of each fruit was frozen in liquid nitrogen, and stored at −80 ◦ C until RNA extraction. 2.2. Assessment of fruit quality For measurement of ethylene, fruit were placed in a 1.2-L air-tight glass chamber for 1 h at 20 ◦ C. One millilitre of headspace gas was withdrawn from the chamber for each measurement and injected into a gas chromatograph (model GC-14B, Shimadzu, Kyoto, Japan) equipped with an activated alumina column and flame ionization detectors. Firmness was determined on opposite sides of each fruit by using a penetrometer (Italtest, FT011, 8-mm diameter) and expressed in newtons (N). After measuring firmness, we divided each apple into eight equal-sized segments, then measured titratable acidity (TA) in two segments that were diametrically opposite from each other by using an autotitrator (Foodstat FS-51, Toko Chemical Laboratories, Co., Ltd., Tokyo, Japan). 2.3. RNA extraction and isolation of cDNA fragments Total RNA of fruit was extracted from frozen fruit samples by the hot borate method (Wan and Wilkins, 1994). First-strand cDNA was synthesized by reverse transcriptase (SuperScript II, Invitrogen, Carlsbad, CA, USA) from 2 ␮g of the total RNA from ripening fruit of each cultivar. The cDNA fragments of MdACS1 (L31347), MdACS3 (U73816), MdACO1 (X61390), MdETR1 (AF032448), MdERS1 (AY083169), MdERS2 (AB213028), and MdPG1 (L27743) were amplified by RT-PCR (reverse transcription PCR) with specific primer sets (Table 1) using cDNA templates from ‘Orin’ (MdACS1, MdACO1, and MdPG1) and ‘Fuji’ (MdACS3, MdETR1, MdERS1, and MdERS2) fruit. The PCR conditions for MdACS1, MdACO1, and MdACS3 were 95 ◦ C for 12 min, followed by 35 cycles of 94 ◦ C for 0.5 min, 55 ◦ C for 1 min, and 72 ◦ C for 2 min, with a final extension of 7 min at 72 ◦ C. AmpliTaq Gold (Applied Biosystems, Foster City, CA, USA) was used. The PCR conditions for MdETR1, MdERS1, and MdERS2 were 94 ◦ C for 1 min, followed by 30

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Table 1 Oligonucleotide primers used for amplification of cDNAs by RT-PCR Name

Oligonucleotide sequence

Md-ACS1 Md-ACS3 Md-ACO1 Md-ETR1 Md-ERS2 Md-ERS1 Md-PG1

Sense 5 -CTTACAGCTTGTATCCATACACAAG-3 ; antisense 5 -TACACTAATCACATTGTATAGAATC-3 Sense 5 -GCTTTCTTGAAGGCCAAAGTGCTT-3 ; antisense 5 -CCATCGATTATACAAACTGATTGTG-3 Sense 5 -GCAAATACATAAACACAATC-3 ; antisense 5 -AAACAAAATAAACCCAAACT-3 Sense 5 -GTTTAAGTGGCTCAATC-3 ; antisense 5 -AGTAATGCTCCTGTGTC-3 Sense 5 -GCTCGTTCACATTATCCCT-3 ; antisense 5 -ACCCTCGCTTTCAATCCAAAT-3 Sense 5 -CGCGAATTCAAGGAGATGGGGCTTATG-3 ; antisense 5 -CGCCTCGAGTCCACTGGCATCCAAAGA-3 Sense 5 -CGATTTGTTAGGTGTTTCTA-3 ; antisense 5 -TATTTCACAATGCCCATAAT-3

cycles of 94 ◦ C for 0.5 min, 55 ◦ C for 1 min, and 72 ◦ C for 2 min, with a final extension of 7 min at 72 ◦ C. TaKaRa LA Taq polymerase (TaKaRa, Kyoto, Japan) was used. The PCR conditions for MdPG1 were 28 cycles of 94 ◦ C for 20 s, 55 ◦ C for 30 s, and 72 ◦ C for 1.5 min, with a final extension of 5 min at 72 ◦ C. TaKaRa Ex Taq (TaKaRa, Kyoto, Japan) was used. Amplified fragments were cloned into pCR2.1 (Invitrogen) and sequenced.

TA was significantly greater in ‘Orin’ fruit treated at 1 DAH than in fruit treated later (Fig. 2, Table 2). TA was also significantly greater in all 1-MCP-treated ‘Fuji’ fruit than in untreated fruit during storage, and no marked differences were observed among the three 1-MCP treatments (Fig. 2, Table 2).

2.4. Northern blot analysis

Control ‘Orin’ fruit produced a large amount of ethylene (35.9 nL g−1 FW h−1 ) at 1 DAH, and the amount continued increasing to the maximum level (109.9 nL g−1 FW h−1 ) at 9 DAH, and then gradually decreased (Fig. 3, Table 2). In ‘Orin’ fruit treated with 1-MCP at 1, 3 and 7 DAH, ethylene production briefly declined before increasing again. The period and degree of the decline became less as the treatment day advanced.

Five micrograms of total RNA was separated on a 1.0% agarose gel that contained 0.66 M formaldehyde, and was blotted onto a nylon membrane (Hybond N+, Amersham Biosciences). The cDNA probes of MdACS1 and MdACS3, and MdETR1, MdERS1, and MdERS2 did not hybridize with each other (data not shown). The probes were labelled with PCR DIG Labeling Mix (Roche Diagnostics, Mannheim, Germany). Hybridization was performed in 7% SDS, 50% formamide, 5 × SSC, 0.1% N-lauroylsarcosine, 2% blocking buffer (Roche Diagnostics), and 50 mM sodium phosphate (pH 7.0) at 50 ◦ C. Membranes were washed twice for 30 min with 0.1 × SSC, 0.1% SDS at 65 ◦ C, and then exposed to Xray film (Fuji Film, Tokyo, Japan). Northern blot analyses were performed at least three times per probe.

3.2. Effect of 1-MCP on ethylene production

3. Results 3.1. Effects of 1-MCP on flesh firmness and TA of apple fruit The firmness of untreated ‘Orin’ fruit decreased rapidly from 37 to 20 N within 9 DAH (Fig. 1, Table 2). ‘Orin’ fruit treated with 1-MCP at 1 DAH softened from 37 to 33 N within 2 days after treatment (DAT), and then gradually softened to 30 N at 8 DAT, when a constant level of firmness was retained. ‘Orin’ fruit treated with 1-MCP at 3 DAH lost flesh firmness after treatment, but softening stopped at 8 DAT, and a constant level of firmness was retained for a month. ‘Orin’ fruit treated at 7 DAH had already softened to 25 N before 1-MCP treatment, and softening was not stopped by 1-MCP treatment. The firmness of ‘Fuji’ showed no remarkable differences between 1-MCP-treated and untreated fruit except at 91 DAT, when all treated fruit were significantly firmer than the control fruit.

Fig. 1. Flesh firmness of ‘Orin’ and ‘Fuji’ apples during storage. Fruit were harvested at commercial maturity. Harvested fruit were treated with or without (control) 1 ␮L L−1 1-MCP at 1, 3, or 7 days after harvest (DAH) at 22 ◦ C for 12 h, and then stored at 20 ◦ C for 2, 4, 8, 15, 31, 46, 61, or 91 days. Arrows indicate the time of 1-MCP treatment. Vertical bars represent the S.E. (n = 5).

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Table 2 Flesh firmness, titratable acidity and ethylene production of ‘Orin’ and ‘Fuji’ apples during storage DAT

Flesh firmness (N)

Titratable acidity (%)

Ethylene production (nL g−1 FW h−1 )

Control

Control

Control

1-MCP 1 DAH

3 DAH

7 DAH

1-MCP 1 DAH

3DAH

7DAH

Orin 0 2 4 8 15 31 46

36.7a 29.2ab 29.4ab 20.1b 21.0c 18.6b 14.1b

36.7a 32.6a 31.6a 30.2a 29.9a 27.9a 29.8a

31.0b 28.9ab 26.8b 23.3b 25.1b 24.4a 19.2b

25.0c 24.2b 21.7c 23.9b 19.2c 18.7b 14.8b

0.34a 0.26 0.30ab 0.23 0.27a 0.19b 0.17

0.34a 0.32 0.33a 0.29 0.30a 0.25a 0.23

0.31ab 0.30 0.29ab 0.23 0.26a 0.20ab 0.18

0.30b 0.27 0.25b 0.27 0.19b 0.20ab 0.18

Fuji 0 2 4 8 15 31 61 91

31.7 33.4 33.4 31.8 32.3 33.8 29.5 23.8b

31.7 33.1 32.7 33.3 34.2 34.9 30.6 27.6a

33.0 33.0 33.6 32.9 33.4 34.3 30.5 28.5a

32.7 32.3 34.3 34.9 32.8 32.5 28.1 28.9a

0.41 0.42 0.41 0.34 0.33 0.26 0.18b 0.17

0.41 0.36 0.36 0.36 0.35 0.32 0.28a 0.20

0.40 0.37 0.39 0.36 0.37 0.32 0.25a 0.19

0.34 0.40 0.35 0.37 0.33 0.31 0.24ab 0.21

35.9c 78.5a 95.6a 110a 80.2a 40.4b 19.7b 0.47 0.61a 2.42a 5.49a 8.39a 12.45a 7.34 4.03

1-MCP 1 DAH

3 DAH

7 DAH

35.9c 6.20c 11.5c 29.4c 49.9b 60.8a 36.4a

81.3b 29.0b 42.2b 55.4bc 78.6a 44.9b 28.5b

120.6a 70.9a 66.1a 71.4b 65.1ab 35.5b 20.0b

0.47 0.20b 0.23b 0.14b 0.09b 0.05b 0.14 7.35

2.20 0.24b 0.22b 0.36b 0.13b 0.04b 0.11 9.67

3.34 0.52ab 0.33b 0.24b 0.11b 1.59b 6.44 4.17

Different letters indicated significant differences within the same row at p < 0.05.

Control ‘Fuji’ fruit produced little ethylene at 1 DAH (0.47 nL g−1 FW h−1 ), and the amount gradually increased (Fig. 3, Table 2). In ‘Fuji’ fruit treated with 1-MCP at 1 and 3 DAH, ethylene production was suppressed for 61

DAT. Ethylene production in fruit treated at 7 DAH was 3.3 nL g−1 FW h−1 , but then decreased by 1-MCP treatment. Ethylene production began to increase again earlier in fruit treated at 7 DAH than in fruit treated at 1 or 3 DAH.

Fig. 2. Titratable acidity (TA) of ‘Orin’ and ‘Fuji’ fruit during storage. Fruit were harvested at commercial maturity. Harvested fruit were treated with or without (control) 1 ␮L L−1 1-MCP at 1, 3, or 7 days after harvest (DAH) at 22 ◦ C for 12 h, and then stored at 20 ◦ C for 2, 4, 8, 15, 31, 46, 61, or 91 days. Arrows indicate the time of 1-MCP treatment. Vertical bars represent the S.E. (n = 5).

Fig. 3. Ethylene production of ‘Orin’ and ‘Fuji’ fruit during storage. Fruit were harvested at commercial maturity. Harvested fruit were treated with or without (control) 1 ␮L L−1 1-MCP at 1, 3, or 7 days after harvest (DAH) at 22 ◦ C for 12 h, and then stored at 20 ◦ C for 2, 4, 8, 15, 31, 46, 61, or 91 days. Arrows indicate the time of 1-MCP treatment. Vertical bars represent the S.E. (n = 5).

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Fig. 4. Levels of MdACS1, MdACS3 and MdACO1 expression in ‘Orin’ and ‘Fuji’ fruit. Total RNA was extracted from the same sample as in Figs. 1–3. Total RNA (5 ␮g) was subjected to Northern analysis and hybridized with DIG-labelled probes for the genes for ethylene biosynthesis enzymes. Upper number shows the days after 1-MCP treatment, and lower number shows the days after harvest. Ethidium bromide RNA was used to show equivalence of RNA loading.

Fig. 5. Levels of MdETR1, MdERS1, MdERS2 and MdPG1 expression in ‘Orin’ and ‘Fuji’ fruit. Total RNA was extracted from the same sample as in Figs. 1–3. Total RNA (5 ␮g) was subjected to Northern analysis and hybridized with DIG-labelled probes for the genes for ethylene receptors and PG. Upper number shows the days after 1-MCP treatment, and lower number shows the days after harvest. Ethidium bromide RNA was used to show equivalence of RNA loading.

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3.3. Expression patterns of ethylene biosynthesis enzymes In control ‘Orin’ fruit, MdACS1 was already expressed at 1 DAH, and the level increased during storage (Fig. 4). On the other hand, in control ‘Fuji’ fruit, MdACS1 was not expressed until 9 DAH, after which expression increased. Although 1-MCP decreased the level of MdACS1 in ‘Orin’ in all treatments, suppression was greater when 1-MCP was applied sooner after harvest. 1-MCP treatment suppressed the level of MdACS1 in ‘Fuji’ in all treatments for a prolonged time. MdACS1 was expressed earlier in fruit treated at 7 DAH than in the fruit treated at 1 and 3 DAH. The expression of MdACS3 was detected only in ‘Fuji’ fruit, both treated and untreated, and gradually decreased during storage (Fig. 4). The level of MdACO1 was constant during storage in untreated fruit of both cultivars, and it was not affected by 1-MCP in ‘Orin’, but in 1-MCP-treated ‘Fuji’ fruit it gradually decreased until 31 DAT and then began to increase again. 3.4. Expression patterns of ethylene receptor genes The expression of MdETR1 was constant in untreated fruit of both cultivars during storage (Fig. 5). In ‘Fuji’, 1MCP slightly decreased the level of MdETR1. In ‘Orin’ fruit treated at 1 DAH, the level of MdERS1 decreased at 2 DAT, and remained low for up to 46 DAT. On the other hand, in ‘Orin’ fruit treated with 1-MCP at 3 and 7 DAH, the level of MdERS1 was briefly reduced before increasing again. In ‘Fuji’, MdERS1 mRNA was suppressed by 1-MCP more severely than in ‘Orin’. In ‘Fuji’, the expression pattern of MdERS2 was similar to that of MdERS1. In control ‘Orin’, the level of MdERS2 was low, and was reduced by 1-MCP treatment. 3.5. Expression patterns of MdPG1 In control ‘Orin’ fruit, MdPG1 was expressed abundantly during storage (Fig. 5). In control ‘Fuji’, the level of MdPG1 increased during storage to a maximum at 9 DAH, and then it remained constant. In the 1-MCP-treated ‘Orin’ fruit, the level of MdPG1 was reduced briefly. The expression of MdPG1 was suppressed at 2 DAT in all 1-MCP-treated ‘Fuji’ fruit. The expression of MdPG1 was more severely inhibited in ‘Fuji’ than in ‘Orin’.

4. Discussion 1-MCP treatment of apple fruit maintains firmness and TA, slows loss of chlorophyll and starch, reduces respiration, inhibits ethylene production, and prolongs storage life (Blankenship and Dole, 2003). We examined the influence of 1-MCP treatment time after harvest on fruit quality, ethylene production, and expression patterns of genes for ethylene

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biosynthesis enzymes and ethylene receptors in two apple cultivars. In ‘Orin’ fruit, the later 1-MCP was applied after harvest, the less the effect was. 1-MCP maintained firmness and TA in both cultivars (Figs. 1 and 2), showing the greatest effect in ‘Fuji’ fruit. Ethylene production and MdACS1 transcript levels in harvested fruit were significantly different between the cultivars, being higher in ‘Orin’ than in ‘Fuji’ (Figs. 3 and 4). The rate of ethylene production was faster in ‘Orin’ fruit after harvest, and ethylene production at 1-MCP treatment was high. In contrast, the low ethylene production after 1MCP treatment of ‘Fuji’ fruit correlates with the high fruit quality in all 1-MCP treated fruit. Thus, we assume that ethylene production at the time of 1-MCP treatment influences the effect of 1-MCP on fruit quality. The reduction in flesh firmness of ‘Orin’ fruit treated at 1 and 3 DAH did not stop quickly after 1-MCP treatment (Fig. 1). We assume that some cell-wall-modifying enzymes that are controlled by ethylene were produced before 1-MCP treatment, and continued to act after treatment. The softening stopped at 8 DAT (Fig. 1), when the enzymes might have become inactive, or proteolysis and production of new enzymes might have been suppressed by 1-MCP. Wakasa et al. (2006) suggested that the level of MdPG1 transcripts is correlated with the rate of firmness loss. The level of MdPG1 expression was higher in untreated ‘Orin’ fruit than in ‘Fuji’, and was decreased by 1-MCP in both cultivars (Fig. 5). Since MdPG1 was detected in ‘Orin’ fruit treated with 1-MCP at 1 DAH and in untreated ‘Fuji’ fruit (Fig. 5), in which firmness loss was very slow (Fig. 1), the relationship between level of MdPG1 expression and fruit softening is not clear. TA reduction was not suppressed in ‘Orin’ fruit treated at 3 and 7 DAH, although it was suppressed in all treated ‘Fuji’ fruit (Fig. 2). These results imply that to suppress the reduction in TA, 1MCP should be applied before ethylene production increases or quickly after harvest. 1-MCP suppresses ethylene production (Fan et al., 1999a, 1999b; Rupasinghe et al., 2000; Watkins et al., 2000) and MdACS1 expression (Wakasa et al., 2006) in apple fruit. The expression of MdACS1 and ethylene production in 1MCP-treated ‘Orin’ fruit briefly declined at 2 DAT, then increased again (Figs. 3 and 4), so we assume that suppression of ethylene production by 1-MCP was incomplete. Although MdACS1 expression and ethylene production were suppressed in all 1-MCP-treated ‘Fuji’ fruit, ethylene production began to increase at 14 DAT in ‘Fuji’ fruit treated at 7 DAH, earlier than in the other treatments (Fig. 3). This result indicates that the suppression of ethylene production by 1-MCP is lower in ‘Fuji’ fruit treated at 7 DAH than in fruit treated earlier. Thus, ethylene insensitivity caused by 1-MCP treatment differed by cultivar and timing of application. It is thought that 1-MCP binds permanently to receptors present at the time of treatment, and any return of ethylene sensitivity is due to the appearance of new receptors (Blankenship and Dole, 2003). We speculate that the amounts of ethylene

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production and of ethylene receptors present when 1-MCP is applied and the amounts that are induced after 1-MCP treatment are the keys to 1-MCP efficacy. Endogenous ethylene likely competes with 1-MCP for binding (Sisler et al., 1996). Since ethylene receptors act as negative regulators of the ethylene signalling pathway (Hua and Meyerowitz, 1998; Ciardi et al., 2000; Tieman et al., 2000), a greater abundance of ethylene receptors that are not occupied with ethylene or that are bound to 1-MCP might strongly suppress ethylene responses. In ‘Fuji’ fruit of 1 DAH, ethylene was produced at low levels, and most ethylene receptors might be bound by 1-MCP, and then ethylene production would be suppressed and these 1-MCP bound ethylene receptors may negatively regulate ethylene signalling. On the other hand, in ‘Orin’ fruit treated at 3 or 7 DAH, which produced some ethylene, endogenous ethylene already present may have competed with 1-MCP to bind to ethylene receptors, and the amount of 1-MCP bound ethylene receptors may be reduced. Furthermore, in these ethylene-overproducing tissues, ethylene production and the expression of ethylene receptor genes were not completely suppressed after 1-MCP treatment (Figs. 3 and 5). This indicates that ethylene binds to receptors that were produced after 1-MCP treatment, and the amount of ethylene-bound receptors increases, which could increase ethylene sensitivity. Thus, we speculate that high ethylene production at the time of and after 1-MCP treatment and high levels of expression of ethylene receptor genes might reduce the efficacy of 1-MCP. The rate of turnover of ethylene receptors and how long the 1-MCP-bound receptors can suppress ethylene signalling have not been determined. We speculate the rate of turnover of ethylene receptors might be regulated by ethylene binding. To understand 1-MCP efficacy at the molecular level, analysis of the protein level of ethylene receptors (e.g. stability of ethylene receptors) will be required. Ross et al. (1992) reported that MdACO1 increased with ethylene production by the fruit during ripening, and it was enhanced in both ethylene-treated and wounded fruit. Wakasa et al. (2006) reported that expression of MdACO1 mRNA was suppressed by 1-MCP treatment. However, the expression of MdACO1 did not decrease in 1-MCP-treated ‘Orin’ fruit, and it decreased very slowly in 1-MCP-treated ‘Fuji’ fruit. In these tissues, transcripts of ethylene receptor genes and MdPG1 were reduced rapidly by 1-MCP (Fig. 4). Thus, 1-MCP has some efficacy. One possible reason for this difference in results may be a difference in the stage of the fruit at which 1-MCP was applied. That is, MdACO1 was already expressed at the time of 1-MCP treatment in this study, whereas little MdACO1 transcript was detected at the time of 1-MCP treatment by Wakasa et al. (2006). MdACO1 expression showed great differences among apple cultivars, and the levels were not related to the amount of ethylene (Wakasa et al., 2006). Therefore, we assume that although ethylene is one of the essential factors in MdACO1 expression, other factors also regulate MdACO1. We speculate that ethylene might be required to induce and increase expression

of MdACO1 but does not have a crucial role in maintaining the level of MdACO1 expression. Two systems are assumed to regulate ethylene production in higher plants. System 1 functions during normal vegetative growth, and is responsible for producing the basal levels of ethylene detectable in all tissues, including non-climacteric fruit. System 2 is responsible for the upsurge of ethylene production during the ripening of climacteric fruit (Leli`evre et al., 1997). MdACS1 might be involved in system 2 (Dong et al., 1991; Cin et al., 2005). Sunako et al. (1999) reported that MdACS3 was expressed constitutively in fruit. However, we detected MdACS3 only in ‘Fuji’ fruit, where it decreased during storage, not in ‘Orin’ fruit (Fig. 4). Tomato LeACS1A and LeACS3 are expressed in fruit throughout their development, and their expression is independent of ethylene action, so it has been assumed that they mediate system 1 ethylene production (Nakatsuka et al., 1998). MdACS3 might be responsible for system 1 ethylene production in apple fruit, and in ‘Fuji’, MdACS3 might work in the place of MdACS1 in the early ripening stage before MdACS1 expression increases.

5. Conclusion Treatment with 1-MCP delayed fruit softening and reduction of titratable acidity, and suppressed the increase of ethylene production and the expression of MdACS1, MdERS1, MdERS2, and MdPG1 genes. 1-MCP efficacy was dependent on the ethylene production by fruit at the time of 1-MCP treatment: the later 1-MCP was applied after harvest, the less was the efficacy. Ethylene production and expression of ethylene receptor genes were not suppressed in fruit in which 1-MCP had low efficacy. Thus, ethylene production and expression of ethylene receptor genes at and after the time of 1-MCP treatment may have an important effect on deciding 1-MCP efficacy.

Acknowledgements We thank Y. Kashimura and H. Hayama (National Institute of Fruit Tree Science) for their help in 1-MCP treatment and valuable discussions. This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant-in-Aid for Young Scientists no. 15780031 to M.T.).

References Abeles, F.B., Morgan, P.W., Saltveit Jr., M.E., 1992. Ethylene in Plant Biology. Academic Press, San Diego. Adams, D.O., Yang, S.F., 1979. Ethylene biosynthesis: identification of ACC as an intermediate in the conversion of methionine to ethylene. Proc. Natl. Acad. Sci. U.S.A. 76, 170–174. Atkinson, R.G., 1994. A cDNA clone for endopolygalacturonase from apple. Plant Physiol. 105, 1437–1438.

M. Tatsuki et al. / Postharvest Biology and Technology 43 (2007) 28–35 Atkinson, R.G., Bolitho, K.M., Wright, M.A., Iturriagagoitia-Bueno, T., Reid, S.J., Ross, G.S., 1998. Apple ACC-oxidase and polygalacturonase: ripening-specific gene expression and promoter analysis in transgenic tomato. Plant Mol. Biol. 38, 449–460. Bird, C.R., Smith, C.J.S., Ray, J.A., Moureau, P., Bevan, M.W., Bird, A.S., Hughes, S., Morris, P.C., Grierson, D., Schuch, W., 1988. The tomato polygalacturonase gene and ripening-specific expression in transgenic plants. Plant Mol. Biol. 11, 651–662. Blankenship, S.M., Dole, J.M., 2003. 1-Methylcyclopropene: a review. Postharvest Biol. Technol. 28, 1–25. Cin, V.D., Danesin, M., Boschetti, A., Dorigoni, A., Ramina, A., 2005. Ethylene biosynthesis and perception in apple fruitlet abscission (Malus domestica (L.) Borkh). J. Exp. Bot. 56, 2995–3005. Ciardi, J.A., Tieman, D.M., Lund, S.T., Jones, J.B., Stall, R.E., Klee, H.J., 2000. Response to Xanthomonas campestris pv. vesicatoria in tomato involves regulation of ethylene receptor gene expression. Plant Physiol. 123, 81–92. Davies, K.M., Hobson, G.E., Grierson, D., 1988. Silver ions inhibit the ethylene-stimulated production of ripening-related mRNAs in tomato. Plant Cell Environ. 11, 729–738. DeEll, J.R., Murr, D.P., Murray, D., Porteous, H., Rupasinghe, H.P.V., 2002. Influence of temperature and duration of 1-methylcyclopropene (1-MCP) treatment on apple quality. Postharvest Biol. Technol. 24, 349–353. Dong, J.G., Kim, W.T., Yip, W.K., Thompson, G.A., Li, L., Bennett, A.B., Yang, S.F., 1991. Cloning of a cDNA encoding 1-aminocyclopropane1-carboxylate synthase and expression of its mRNA in ripening apple fruit. Planta 185, 38–45. Fan, X., Blankenship, S.M., Mattheis, J.P., 1999a. 1-Methylcyclopropene inhibits apple ripening. J. Am. Soc. Hortic. Sci. 124, 690–695. Fan, X., Mattheis, J.P., Blankenship, S., 1999b. Development of apple superficial scald, soft scald, core flush, and greasiness is reduced by MCP. J. Agric. Food Chem. 47, 3063–3068. Hadfield, K.A., Bennett, A.B., 1998. Polygalacturonases: many genes in search of a function. Plant Physiol. 117, 337–343. Hamilton, A.G., Lycett, G.W., Grierson, D., 1990. Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants. Nature 346, 284–286. Hua, J., Meyerowitz, E.M., 1998. Ethylene responses are negatively regulated by a receptor gene family in Arabidopsis thaliana. Cell 94, 261–271. Kende, H., 1993. Ethylene biosynthesis. Annu. Rev. Plant Physiol. 44, 283–307. Lashbrook, C.C., Tieman, D.M., Klee, H.J., 1998. Differential regulation of the tomato ETR gene family throughout plant development. Plant J. 15, 243–252. Leli`evre, J.M., Latch´e, A., Jones, B., Bouzayen, M., Pech, J.C., 1997. Ethylene and fruit ripening. Physiol. Plant. 101, 727–739. Lincoln, J.E., Cordes, S., Read, E., Fischer, R.L., 1987. Regulation of gene expression by ethylene during Lycopersicon esculentum (tomato) fruit development. Proc. Natl. Acad. Sci. U.S.A. 84, 2793–2797. Maunders, M.J., Holdsworth, M.J., Slater, A., Knapp, J.E., Bird, C.R., Schuch, W., Grierson, D., 1987. Ethylene stimulates the accumulation of ripening-related mRNAs in tomatoes. Plant Cell Environ. 10, 177–184. Mir, N.A., Curell, E., Khan, N., Whitaker, M., Beaudry, R.M., 2001. Harvest maturity, storage temperature, and 1-MCP application Frequency alter firmness retention and chlorophyll fluorescence of ‘Redchief Delicious’ apples. J. Am. Soc. Hortic. Sci. 126, 618–624. Nakatsuka, A., Murachi, S., Okunishi, H., Shiomi, S., Nakano, R., Kubo, Y., Inaba, A., 1998. Differential expression and internal feedback regulation of 1-aminocyclopropane-1-carboxylate synthase, 1-

35

aminocyclopropane-1-carboxylate oxidase, and ethylene receptor genes in tomato fruit during development and ripening. Plant Physiol. 118, 1295–1305. Oeller, P.W., Lu, M.W., Taylor, L.P., Pike, D.A., Theologis, A., 1991. Reversible inhibition of tomato fruit senescence by antisense RNA. Science 254, 437–439. Picton, S., Barton, S.L., Bouzayen, M., Hamilton, J., Grierson, D., 1993. Altered fruit ripening and leaf senescence in tomatoes expressing an antisense ethylene-forming enzyme transgene. Plant J. 3, 469– 481. Rasori, A., Ruperti, B., Bonghi, C., Tonutti, P., Ramina, A., 2002. Characterization of two putative ethylene receptor genes expressed during peach fruit development and abscission. J. Exp. Bot. 53, 2333–2339. Ross, G.S., Knighton, M.L., Lay-Yee, M., 1992. An ethylene-related cDNA from ripening apples. Plant Mol. Biol. 19, 231–238. Rupasinghe, H.P.V., Murr, D.P., Paliyath, G., Skog, L., 2000. Inhibitory effect of 1-MCP on ripening and superficial scald development in ‘McIntosh’ and ‘Delicious’ apples. J. Hortic. Sci. Biotechnol. 75, 271–276. Sato, T., Kubo, T., Akada, T., Wakasa, Y., Niizeki, M., Harada, T., 2004. Allelotype of a ripening-specific 1-aminocyclopropane-1-carboxylate synthase gene defines the rate of fruit drop in apple. J. Am. Soc. Hortic. Sci. 129, 32–36. Sato-Nara, K., Yuhashi, K.I., Higashi, K., Hosoya, K., Kubota, M., Ezura, H., 1999. Stage- and tissue-specific expression of ethylene receptor homolog genes during fruit development in muskmelon. Plant Physiol. 119, 321–329. Sisler, E.C., Dupille, E., Serek, M., 1996. Effects of 1-methylcyclopropene and methylcyclopropene on ethylene binding and ethylene action on cut carnation. Plant Growth Regul. 18, 79–86. Sitrit, Y., Bennett, A.B., 1998. Regulation of tomato fruit polygalacturonase mRNA accumulation by ethylene: a re-examination. Plant Physiol. 116, 1145–1150. Sunako, T., Sakuraba, W., Senda, M., Akada, S., Ishikawa, R., Niizeki, M., Harada, T., 1999. An allele of the ripening-specific 1aminocyclopropane-1-carboxylic acid synthase gene (ACS1) in apple fruit with a long storage life. Plant Physiol. 119, 1297–1304. Tatsuki, M., Endo, A., 2006. Analyses of expression patterns of ethylene receptor genes in apple (Malus domestica Borkh.) fruits treated with or without 1-methylcyclopropene (1-MCP). J. Jpn. Soc. Hortic. Sci., in press. Tieman, D.M., Taylor, M.G., Ciardi, J.A., Klee, H.J., 2000. The tomato ethylene receptors NR and LeETR4 are negative regulators of ethylene response and exhibit functional compensation within a multigene family. Proc. Natl. Acad. Sci. U.S.A. 97, 5663–5668. Wan, C.Y., Wilkins, T.A., 1994. A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.). Anal. Biochem. 223, 7–12. Wakasa, Y., Kudo, H., Ishikawa, R., Akada, S., Senda, M., Niizeki, M., Harada, T., 2006. Low expression of an endopolygalacturonase gene in apple fruit with long-term storage potential. Postharvest Biol. Technol. 39, 193–198. Watkins, C.B., Nock, J.F., Whitaker, B.D., 2000. Responses of early, mid and late season apple cultivars to postharvest application of 1methylcyclopropene (1-MCP) under air and controlled atmosphere storage conditions. Postharvest Biol. Technol. 19, 17–32. Wilkinson, J.Q., Lanahan, M.B., Clark, D.G., Bleecker, A.B., Chang, C., Meyerowitz, E.M., Klee, H.J., 1997. A dominant mutant receptor from Arabidopsis confers ethylene insensitivity in heterologous plants. Nature Biotech. 15, 444–447. Zarembinski, T.I., Theologis, A., 1994. Ethylene biosynthesis and action: a case of conservation. Plant Mol. Biol. 26, 1579–1597.