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South African Journal of 75 (2009) 137–144 www.elsevier.com/locate/sajb

Effect of low oxygen, temperature and 1-methylcyclopropene on the expression of genes regulating ethylene biosynthesis and perception during in ⁎ M.H. Asif a, N. Pathak b,c, T. Solomos b, P.K. Trivedi a,

a Plant Gene Expression Lab, National Botanical Research Institute, Lucknow-226001, UP, India b Department of NRSL, Plant Sciences, University of Maryland, College Park, MD-20742, USA c Department of Biotechnology, Integral University, Kursi Road, Lucknow, UP, India Received 25 August 2008; received in revised form 9 September 2008; accepted 10 September 2008

Abstract

Ethylene initiates and controls ripening in climacteric which is developmentally regulated. During this process, ethylene production generates a strong signaling process inducing/suppressing various target genes that are associated with several attributes of fruit ripening. In apple, low temperature, low oxygen and 1-methylcyclopropene (1-MCP) treatments have been used to increase shelf life. In the present study, effort has been made to understand the molecular basis of the increase in shelf life under the influence of temperature, low oxygen and 1-MCP in Granny Smith apple. The apple fruit were exposed to these treatments either individually or in combination and levels of ethylene as well as transcript accumulation of the genes responsible for ethylene biosynthesis and ethylene receptors were measured. A tight regulation of the ethylene production was observed through differential expression of MdACS1 and MdERS1 genes. The ethylene levels were highly dependent on temperature, oxygen concentration and 1-MCP and effects of each were not only additive but associated with the expression of MdACS1 and MdERS1. © 2008 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords: 1-methylcyclopropene; Ethylene biosynthesis; Ethylene receptor; Fruit ripening; Hypoxia; Malus domestica; Temperature

1. Introduction development including fruit ripening, the concept of system-1 and system-2 ethylene was introduced (McMurchie et al., Ethylene plays an important role in many plant processes 1972). System-1 ethylene is produced at low level and including seed germination, organ senescence, stress responses contributes to the basal levels produced by the plant during and fruit ripening (Nath et al., 2006). Depending on the massive various stages of growth and development and is auto- increase in respiration or ethylene production during ripening, inhibitory (Oetiker and Yang, 1995). System-2 ethylene fruit are classified as climacteric and non climacteric (Biale and production is at a high level, and operates during ripening of Young, 1981). At the onset of ripening, climacteric fruit exhibit climacteric and senescence of some flowers and is auto- a peak in respiration followed by a burst in ethylene production. stimulatory (Oetiker and Yang, 1995). The transition from In non-climacteric fruit, no such burst in ethylene production system-1 to system-2 ethylene production is an important step and respiration is observed. Though a basal level of ethylene during fruit ripening and is developmentally regulated. It is now production is present even in non-climacteric fruit, ripening in established that genes responsible for ethylene biosynthesis and these fruits is ethylene independent. Based on the nature and signal transduction are stimulated during a climacteric burst of amount of ethylene produced during plant growth and ethylene production and may be tightly associated during the transition from system-1 to system-2 (Nakatsuka et al., 1998). ⁎ Corresponding author. The ethylene biosynthesis pathway is well characterised in E-mail address: [email protected] (P.K. Trivedi). higher plants with ACC synthase and ACC oxidase playing key

0254-6299/$ - see front matter © 2008 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2008.09.002 138 M.H. Asif et al. / South African Journal of Botany 75 (2009) 137–144 roles in this pathway (Yang and Hoffman, 1984). Both enzymes treatment the desiccators were connected to air cylinder with the are encoded by multi-gene families and differential expression above flow rate. Depending on the experiments done the above of their members has been reported during fruit ripening desiccators sets were kept at 18 °C, 7 °C or 1 °C as mentioned in (Zarembinski and Theologis, 1994; Barry et al., 2000). In the figure legends. Immature green GS apple fruits kept in air at , eight ACS genes have been identified among which 18 °C were also subjected to ethylene (10 ppm) for 24 h and 48 h to LeACS2 and LeACS4 are highly expressed during tomato fruit study influence of exogenous ethylene. Fruits were removed from ripening (Barry et al., 2000). Of the three ACC oxidases in the desiccators at different time points, peeled and the cortex tissue tomato, LeACO1 expresses during fruit ripening while other was cut in small pieces and frozen in liquid N2. The frozen tissue two play role in other aspects of plant development (Alexander was ground to a fine powder and stored at −70 °C until used. and Grierson, 2002). Ethylene produced by plants is perceived by a family of receptors, ethylene receptors, which have 2.2. Measurement of ethylene production similarity to histidine kinases and are negative regulators of ethylene signal transduction (Guo and Ecker, 2004). These Two ml gas sample was withdrawn from continuous airflow receptors also belong to multi-gene family and their differential from 20 L desiccators containing fifteen to twenty and expression during ripening has also been observed in various injected into the gas chromatograph (HP, 6890 series) to measure climacteric fruits (Nath et al., 2006). After ethylene receptors, ethylene every alternate day. The average of three readings from signal passes to downstream components through MAP kinase each desiccators was taken as measure of ethylene production for cascade and induces expression of ethylene responsive factors each set. Data are expressed as means±SD (standard deviation) of (ERFs). These ERFs modulates expression of ethylene at least 3–4replicates. responsive genes through binding to ethylene responsive elements in promoters of the genes (Johnson and Ecker, 1998). 2.3. Total RNA isolation, cloning and sequencing of cDNA Apple is a climacteric fruit and use of controlled atmosphere such as low oxygen and low temperature storage as well as Total RNA from apple fruit was isolated as described by Asif treatment with 1-methylcyclopropene (1-MCP), an ethylene et al. (2006). RTPCR was performed using RTPCR kit (Invitrogen perception inhibitor, are effective for delaying fruit ripening (Fan Inc., USA) as per manufacturer's recommendations. Briefly, 2 μg et al., 1999; Rupasinghe et al., 2000; Watkins et al., 2000; DeEll total RNA was reverse transcribed by MuMLV Reverse et al., 2002). Since controlled atmosphere storage and treatment Transcriptase at 42 °C using oligo(dT) primer. One tenth of the with 1-MCP significantly delays ripening of apple fruit by reaction was used for PCR product using degenerate or gene prolonging the time for ethylene burst, we suspected that these specific primers. The PCR products were cloned using TA Topo treatments may be regulating ethylene biosynthesis and perception cloning kit (Invitrogen Inc., USA). To isolate full-length cDNA of at transcriptional level. To confirm this, we stored Granny Smith the genes 5′-and3′-RACE was performed using gene specific (GS) apples, an export variety which is susceptible to superficial primers and respective kits (Invitrogen Inc., USA). The primers scald formation (Rudell et al., 2005), in either low oxygen or treated used for isolation of partial cDNA and 5′-and3′-RACE are listed with 1-MCP before storing at different temperatures for various in Supplementary Table 1. Nucleotide sequence of each cDNA lengths of time. Ethylene production and transcript accumulation of was established using M13 universal and reverse primers. different genes related to ethylene biosynthesis and perception were Sequencing was carried out on an automated DNA sequencing monitored in various treatments and at different time in order to system (ABI 377A, Applied Biosystems Inc., USA) using the dye elucidate regulatory role of these genes during fruit ripening. terminator cycle sequencing kit. Sequence comparisons against databases were performed using BLAST and BLASTX algo- 2. Materials and methods rithms (Altschul et al., 1990) at the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). 2.1. Plant material and treatments 2.4. Northern blot analysis Mature apple (Malus domestica, Var. Granny Smith) fruit, which were not producing detectable ethylene, were harvested Total RNA was resolved on 1.2% formaldehyde denaturing from the orchard of the University of Maryland in Western agarose gel and transferred to Hybond XL membrane Maryland, MD, USA. Fruits were transported to the University of (Amersham Biosciences, Buckinghamshire, NA, USA) accord- Maryland, College Park and kept at 1 °C in air with 80% humidity. ing to the manufacturer's protocol. The blot was prehybridized For the air and low oxygen treatments, fifteen to twenty fruits were overnight at 42 °C in a mixture containing 50% formaldehyde, placed in 20 L desiccators and connected to air cylinder (Air) or 1% SDS, 5X SSC and 5X Denhardts solution. Denatured 32P low oxygen cylinder (1.5% O2) at a flow rate of 30–40 ml/min. dCTP radiolabelled probes were added to the same but fresh For 1-MCP treatment, fifteen to twenty fruits were placed in 20 L hybridization solution. The probes were prepared to a specific desiccators and exposed to 10 μL/L 1-methylcyclopropene (Ethyl activity of approximately 108 cpm µg− 1 using Invitrogen Bloc from Biotechnologies for Horticulture Inc, Walterboro, SC, RadPrime labeling kit, and at least 1×106 cpm were added per USA) for 18–20 h. 1-MCP was generated in the desiccators by ml hybridization solution. The hybridization was carried out at placing required inert powder in the Eppendroff tube and adding 42 °C for 14–16 h. The washing of the blot was carried out in water through rubber septum using a syringe. After 1-MCP SSC plus 0.1% SDS. The final wash of the blots was done at M.H. Asif et al. / South African Journal of Botany 75 (2009) 137–144 139

tectable ethylene at day 5 at 18 °C (0.63±0.06 nl C2H4/g/h), day 12 at 7 °C (0.515±0.08 nl C2H4/g/h) and day 17 at 1 °C (0.67± 0.05 nl C2H4/g/h). Fruits connected to 1.5% O2 supply started producing ethylene after 37 days (0.178±0.01 nl C2H4/g/h) at 18 °C, 60 days (0.19±0.02 nl C2H4/g/h) at 7 °C and 70 days (0.2±0.01 nl C2H4/g/h) at 1 °C respectively. 1-MCP treated fruits started producing detectable ethylene after 73, and 104 days at 18 °C and 7 °C respectively. 1-MCP treated fruits stored at 1 °C did not produce detectable ethylene even 196 days post harvest.

3.2. Expression pattern of ethylene biosynthesis and perception related genes

Expression pattern of ethylene biosynthesis and signal transduction genes was analyzed in fruits 35 days post harvest Fig. 1. Ethylene production by GS apples stored under different treatments. Ethylene was measured every day from continuous airflow from desiccators containing apples and kept at 1 °C under various conditions (Fig. 2). The using gas chromatography. The detectable ethylene produced for the first day after expression of MdETR1, MdACO, MdACS3 and MdADH was storage was taken as day for ethylene production in different treatments. Average of observed in air, 1-MCP and 1.5% low oxygen treatments. three readings was taken as measure of ethylene for each set. Data are expressed as However, the expression of MdACS1 and MdERS1 could be ⁎⁎ ⁎⁎⁎ means±SD of at least 3–4 replicates. and significantly different from air at observed only fruits which were kept in air and had started P≤0.01 and 0.001, respectively, according to Student's unpaired t-test. producing ethylene. No detectable transcript for MdACS1 and MdRES1 was observed in fruits treated with 1-MCP or those 65 °C for 15 min in 0.1X SSC and 0.1% SDS and then exposed connected to or low oxygen. No detectable transcript for to Kodak XOMAT X-Ray film at −70 °C for 1 week. MdACS2 is observed in our study. MdADH was taken in the experiment to verify that apples were sensing low oxygen as 2.5. Cloning of ERS1 promoter alcohol dehydrogenase is treated as marker for hypoxia. The

Total DNA from young apple leaf was isolated according to Dellaporta et al. (1983) and a genome walker library was constructed using Universal genome walker kit (Clonetech). For the isolation of ERS1 proximal promoter gene specific primers from the 5′ region of ERS1 close to translational start site was designed and used for PCR amplification using the genome walker library as template. The amplified fragment was cloned in plasmid pCR2.1-TOPO (Invitrogen, USA) and sequenced. The search for conserved regulatory regions with other known sequences was carried using http://www.dna.affrc.go.jp/htdocs/ PLACE/ a database of motifs found in cis-acting regulatory DNA elements of vascular plants (Higo et al., 1999).

2.6. Statistical analysis

Each experiment was carried out under completely rando- mized design with three replications repeated at least thrice. The data were analyzed by student's unpaired t-test, and the treatment mean values were compared at P≤0.05–0.001.

3. Results

3.1. Effect of temperature, 1-MCP and low oxygen on storage of GS apple fruits Fig. 2. Accumulation of transcripts for different genes after 35 days post harvest under different treatments stored at 1 °C. C, control fruits connected to Ethylene production was monitored in different sets of continuous air flow; L, fruits under continuous 1.5% O2 air flow; M, fruits pretreated with 1-MCP and connected to continuous air flow. Northern blot experiments every alternate day. At the time of harvest GS analysis was carried out using 30 µg total RNA samples electrophoresed and apples did not produce any detectable ethylene (Fig. 1). The probed with respective radiolabelled probes. Ethidium bromide staining of gel is untreated fruit connected to air supply started producing de- shown below the blot as loading control. 140 M.H. Asif et al. / South African Journal of Botany 75 (2009) 137–144 increased expression of MdADH suggests that apples stored at detectable amount was observed only after 70 days post harvest low oxygen were sensing hypoxia. which continuously increased to 2.25 nl C2H4/g/h after 196 days post harvest. 1-MCP pretreatment to fruits connected 3.3. Expression of MdACS1 and MdERS1 under long term to air completely blocked ethylene production. However, when storage 1-MCP pretreated fruits were transferred to 18 °C, ethylene production increased rapidly and reached 18.3 nl C2H4/g/h Ethylene production by apple fruits stored at 1 °C and within 32 days (data not shown). connected either air, 1-MCP pretreated and connected to air or Expression of differentially expressing genes, MdACS1 and connected to low O2 is shown in Fig. 3. Stages I, II, III and IV MdERS1, was studied through northern blot analysis at different have been designated as day post harvest ethylene production time points during storage (stages I to IV) of the apple fruits by fruits connected to air. Stage I represent 8 days post harvest (Fig. 3). The expression of MdACS1 and MdERS1 could be first when fruit kept in air did not produce any ethylene. Fruit observed only in air treated fruit at stage II. By stage III fruit connected to air started producing ethylene after 15 days post treated with low O2 also showed the accumulation of MdACS1 harvest which increased continuously and reached to 1.55 nl and MdERS1 transcript. By the time the fruits reached stage IV, C2H4/g/h in 35 days post harvest (stage II) and 5.98 nl C2H4/g/h the air treated fruits were removed due to over ripening and in 100 days post harvest (stage III). Ethylene production in fruit severe scald formation. At stage IV, MdERS1 transcript was kept in air started decreasing after 100 days post harvest and observed, however no expression of MdACS1 was observed in reached to 2.25 nl C2H4/g/h after 196 days post harvest (stage 1-MCP treated fruits. Transfer of 1-MCP pretreated fruits IV). Low O2 storage of fruit delayed ethylene production and a connected to air flow from 1 °C to 18 °C initiated ethylene

Fig. 3. Ethylene production (A) and accumulation of MdACS1 and MdERS1 transcripts (B) at different stages of ripening during the storage of apple fruits under different conditions. Different stages of fruit ripening have been defined and described in the text. In (A) ■, ▲ and × represent fruits connected to continuous air flow, ⁎⁎⁎ fruits under continuous 1.5% O2 air flow and fruits pretreated with 1-MCP and connected to continuous air flow respectively. significantly different at different time points from air at P≤0.001 according to Student's unpaired t-test. In (B) I, II, III and IVare different stages from (A) used to isolate RNA from different samples.

C, control fruits connected to continuous air flow; L, fruits under continuous 1.5% O2 air flow; M, fruits pretreated with 1-MCP and connected to continuous air flow. Northern blot analysis was carried out using 30 µg total RNA samples electrophoresed and probed with respective radiolabelled probes. Ethidium bromide staining of gel is shown below the blot as loading control. M.H. Asif et al. / South African Journal of Botany 75 (2009) 137–144 141

production which reached 18.3 C2H4/g/h with in 32 days. These fruits accumulated MdACS1 and MdERS1 transcripts (data not shown).

3.4. Regulation of MdACS1 and MdERS1 by ethylene

Air treated GS fruit stored at 1 °C that started producing ethylene (1.38 nl C2H4/g/h)weretransferredto18°C.The ethylene production increased to 38.4 nl C2H4/g/h within 24h(Fig. 4A). However, when some of these fruits were treated with 1-MCP, the ethylene production decreased to 3.8 nl C2H4/g/h within 24 h of the treatment. Air and 1-MCP treated fruits were harvested at this stage (S1) and 5 days later (S2). Transcripts of both MdACS1 and MdERS1 were abundant in fruits kept in air. The transcript of MdACS1 was

Fig. 5. Ethylene production and accumulation of MdERS1 and MdACS1 transcripts by immature green GS apple fruits treated with ethylene for 24 and 48 h. 1, preclimacteric control fruits; 2, preclimacteric fruit treated with ethylene for 24 h; 3, preclimacteric fruits treated with ethylene for 48 h; 4, 11 days post treatment with ethylene for 24 h; 5, 11 days post treatment with ethylene for 48 h. Northern blot analysis was carried out using 30 µg total RNA samples electrophoresed and probed with respective radiolabelled probes. Ethidium bromide staining of gel is shown below the blot as loading control. ⁎⁎ and ⁎⁎⁎ significantly different from 2 and 3 at P≤0.01 and 0.001, respectively, according to Student's unpaired t-test.

absent in the fruits treated with 1-MCP at S1 which appeared 5 days later, at S2, when the fruit were producing 12.1 nl C2H4/g/h ethylene. Transcripts of MdERS1 were present at both stages of 1-MCP treated fruit, though lower than the controls. Immature green GS apple fruits kept in air at 18 °C were exposed to ethylene for 24 h and 48 h and the status of transcript accumulation of MdACS1 and MdERS1 were monitored (Fig. 5). Neither air nor ethylene treated fruits produced ethylene till 11 days post treatment. After 11 days, fruits treated with ethylene for 24 and 48 h showed an increase in ethylene production i.e., 0.029 nl C2H4/g/h and 0.341 nl C2H4/g/h respectively. The control fruit and the fruit treated for 24 h also did not produce ethylene till 20 days, by which time they were developmentally competent enough to switch to ethylene production. Expression of MdACS1 was observed only in fruits treated with ethylene for 48 h whereas MdERS1 expression was observed in fruits treated with ethylene for both 24 and 48 h.

3.5. Promoter analysis of ACS1 and ERS1

Fig. 4. Ethylene production (A) and accumulation of MdACS1 and MdERS1 transcripts (B) in GS apples fruits. Fruits were stored at 1 °C and then trans- Expression analyses of MdACS1 and MdERS1 gene suggest ferred to 18 °C after 8 days post harvest. One day after transfer to 18 °C, some that both genes are regulated by the levels of ethylene produced fruits were treated with 1 ppm 1-MCP and connected to air supply (M). Time which might be due to a common regulatory mechanism for the points S1 and S2 refer to the time when fruits were harvested for northern expression of both the genes. To determine if promoters of both analysis. Northern blot analysis was carried out using 30 µg total RNA genes posses some common cis-acting elements, we cloned samples electrophoresed and probed with respective radiolabelled probes. Ethidium bromide staining of gel is shown below the blot as loading control. 797 bp proximal promoter of MdERS1 gene (EMBL accession ⁎⁎⁎ significantly different from air at P≤0.001 according to Student's unpaired no. AY359467) and the sequence of the MdACS1 promoter was t-test. downloaded from the genebank (EMBL accession no 142 M.H. Asif et al. / South African Journal of Botany 75 (2009) 137–144

Table 1 cis-acting elements common in proximal region of MdACS1 and MdERS1 cis-acting Consensus Position with respect to ATG in Function element sequence ACS1 promoter ERS1 promoter ERELEE4 AWTTCAAA −1480 to −1487 −309 to −317 Ethylene responsive element of tomato E4 and carnation GST1 genes; motif mediates ethylene-induced gene expression CURECORECR GTAC −1328 to −1324 −675 to −679 Core of a copper response element; involved in oxygen-response of the genes −860 to −864 −478 to −482 −338 to −342 WBOXNTERF3 TGACT −228 to −233 −700 to −705 “W box” found in the promoter region of a transcriptional repressor ERF3 gene in tobacco −1305 to 1310

AY062129). Both the promoters were analysed in silico and the apples (Wang et al., 2007). However, the ethylene production in common cis-acting elements are shown in Table 1. Interestingly, different cultivars varies considerably and since there were no both the promoters contain ethylene responsive element (ERE) reports of expression of these genes from GS apples, we studied similar to tomato E4 (Montgomery et al., 1993) and carnation the expression of these genes in various treatments. There are 3 GST1 (Itzhaki et al., 1994) genes. Apart from ERE, both the ACC synthase genes (MdACS1, MdACS2 and MdACS3) which genes contain core element of a CuRE (copper-response have been reported from apple with differential gene expression element) found in Cyc6 and Cpx1 genes in Chlamydomonas during ripening (Dong et al., 1991; Rosenfield et al., 1996). We for the oxygen-response (Quinn et al., 2000). studied the expression of these genes and transcripts of MdACS1 and MdACS3 could be detected in our study. Similar 4. Discussion to our study, constitutive expression of MdACS3 was also observed in Golden Delicious and Fuji apples (Sunako et al., Granny Smith apples are prone to scald formation during post 1999). In our study, expression of MdACS1 was shown to be harvest storage leading to huge losses. Growers have been markedly affected by the application of 1-MCP and low O2 treating apples with the antioxidant diphenylamine (DPA) to treatments (Fig. 2). The expression of MdACS1 has also been combat this problem (Rudell et al., 2006). It has also been shown shown to be reduced by the application of 1-MCP, however, the that low oxygen stress followed by storage under 1.0 KPa O2 intensity of reduction varies from cultivar to cultivar (Cin et al., could be a valid alternative to DPA against superficial scald 2005; Tatsuki et al., 2007). In the present study, the expression formation in GS apples (Zanella, 2003). 1-MCP treatments also of MdACO1 did not change after 1-MCP or low oxygen controlled complete scald development and substantially treatments. These are in accordance with the observation of improved the quality and appearance of the fruits as compared Tatsuki et al. (2007) in Fuji and Orin apples, where 1-MCP to normal air and low O2 treatments (Tatsuki et al., 2007). All treatment did not affect the expression of MdACO1. these strategies for post harvest storage of fruits are mainly In our study, expression of MdERS1 was down-regulated by concerned with either inhibition of ethylene production, 1-MCP and low oxygen treatments whereas MdETR1 expres- perception, or ethylene scrubbing or dilution. We show here sion was not affected by these treatments. In other apple that a combination of low temperature with 1-MCP treatments cultivars, 1-MCP treatment also decreased MdERS1 expression and low oxygen treatment significantly decreases ethylene (Cin et al., 2006; Tatsuki et al., 2007). This suggests a role for production and increases the post harvest life of these fruits. this gene in ethylene production during ripening. When GS 1-MCP treatments have been carried out at different apples that were producing ethylene were treated with 1-MCP, temperatures (1–20 °C), concentrations (from 20 nl l− 1 to ethylene production was significantly reduced (Fig. 4). This was 40 µl− 1), durations (4–24 h) and ripening stages at harvest of accompanied by a decrease in the abundance of MdERS1 and different fruits (Abdi et al., 1998; Dong et al., 2001a,b, 2002; MdACS1 transcripts, further supporting the notion that their Fan et al., 2002; Gong et al., 2002; Blankenship and Dole, 2003; expression is regulated by ethylene fluctuations. During Martinez-Romero et al., 2003; Valero et al., 2003; Salvador et ripening, transcripts of MdERS1 accumulated first, followed al., 2004). However, its treatment in combination with low by MdACS1 transcripts (Figs. 3 and 5). oxygen treatment has not been studied as yet. In our study, when It could be inferred form these experiments that when the 1-MCP is applied in combination with low temperature, the production of ethylene shifts from system-1 to system-2 to effect of 1-MCP increases the shelf life of apple fruits in initiate ripening, the expression of MdERS1 increases. This comparison to 1-MCP treatment at higher temperature. It affects the downstream processes and triggers induction of appears from our results that the turnover rate of new ethylene ethylene responsive genes, including ethylene biosynthesis receptors is suppressed at low temperature thereby increasing genes like MdACS1 which in turn affect the production of the efficiency of 1-MCP. ethylene during ripening. There have been many reports on the expression of ethylene The promoter analyses of MdACS1 and MdERS1 suggest signal transduction and ethylene biosynthesis genes during post that both the promoters contain ethylene responsive element harvest storage and 1-MCP treatment in different cultivars of (ERE) similar to tomato E4 (Montgomery et al., 1993) and M.H. Asif et al. / South African Journal of Botany 75 (2009) 137–144 143 carnation GST1 (Itzhaki et al., 1994) genes. Apart from ERE, Blankenship, S.M., Dole, J.M., 2003. 1-Methylcyclopropene: a review. – both the genes contain core element of a CuRE (copper- Postharvest Biology and Technology 28, 1 25. Cin, V.D., Rizzini, F.M., Botton, A., Tonutti, P., 2006. The ethylene biosynthetic response element) found in Cyc6 and Cpx1 genes in Chlamy- and signal transduction pathways are differently affected by 1-MCP in apple domonas for the oxygen-response (Quinn et al., 2000). The W and peach fruit. 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