Postharvest Biology and Technology 78 (2013) 5566

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Postharvest Biology and Technology

journal homepage: www.elsevier.com/locate/postharvbio

Effect of ethylene and 1-MCP on expression of genes involved in ethylene biosynthesis and perception during ripening of apple fruit
Xiaotang Yang a , Jun Song b, , Leslie Campbell-Palmer b , Sherry Fillmore b , Zhaoqi Zhang a
a b

College of Horticulture, South China Agriculture University, Guangzhou, PR China Agriculture and Agri-Food Canada, AFHRC, Kentville, Nova Scotia, Canada B4N 1J5

a r t i c l e

i n f o

a b s t r a c t
Ethylene plays an important role in regulating fruit ripening and senescence and directly inuences the development of the eating quality of fresh apples, including appearance, color, texture, and avor. Apple fruit (Malus domestica Borkh.) is a well-known climacteric fruit and a good model system to study fruit ripening and senescence. To better understand fruit ripening and the role of ethylene perception and signal transduction, apples harvested at a pre-climacteric stage were allowed to naturally ripen, or ripening was either stimulated by treatment with 36 L L1 ethylene for 24 h or inhibited by 1-MCP treatment (1.0 L L1 for 24 h), respectively. Postharvest physiological indices including respiration and ethylene production were monitored for 22 d for ethylene treatment and 47 d for 1-MCP treatment. Based on an efciency test, 20 genes in relation to ethylene biosynthesis and perception were investigated using real-time qPCR during the post-treatment period. The ETR2, ETR5, ERSs, EIL4, ERFs genes together with ACS1 and ACO1 genes were signicantly up-regulated in fruit during ripening. Ethylene treatment further enhanced the expression of ACO2, ETR1, CTR1s and EIN2A genes, while the ACS3 and ACO3, and EIN2B genes were only slightly affected. 1-MCP treatment signicantly inhibited expression of ACS1, ACO1 and ACO2 ethylene biosynthesis genes, which coincided with ethylene production. 1-MCP treatment also reduced expression of ETR1, ETR2, ETR5, ERSs, CTR1, EIN2A, EIL4 and ERFs genes, while having a limited effect on ACS3, ACO3, and EIN2B. This study demonstrated the complexity and dynamic changes of transcriptional proles of ethylene perception and biosynthesis in response to fruit ripening, ethylene, and 1-MCP treatment. Understanding of the signicant changes of these genes and their function may help to explore the mechanisms controlling apple fruit ripening and its response to exogenous ethylene stimuli and action inhibition at the receptor level during ripening and senescence. Crown Copyright 2012 Published by Elsevier B.V. All rights reserved.

Article history: Received 21 March 2012 Accepted 26 November 2012 Keywords: Apple (Malus domestica Borkh.) Fruit ripening Ethylene 1-MCP Ethylene biosynthesis Signal transduction

1. Introduction Ethylene is one of the most important plant hormones affecting various plant biological processes. Numerous biological roles of ethylene have been discovered and the most studied effect of ethylene is the promotion of fruit ripening (Bleecker and Kende, 2000). Apple fruit (Malus x domestica Borkh.) is one of the most popular fruits and its consumption is highly recommended for a healthy diet. Apple is a climacteric fruit that is responsive to ethylene and undergoes a signicant increase in respiration and ethylene production during ripening. The discovery of the biosynthetic pathway of ethylene and elucidation of the important elements involved in its perception and downstream signal transduction have been achieved to better understand the role of ethylene and the mechanisms of its action (Stepanova and Alonso, 2009; Yang and Hoffman,

Corresponding author. Tel.: +1 902 679 5607; fax: +1 902 679 2311. E-mail address: jun.song@agr.gc.ca (J. Song).

1984). Genes encoding 1-aminocyclopropane-carboxylase (ACC) synthase (ACS) and ACC oxidase (ACO), the two key enzymes catalyzing the last steps of the ethylene biosynthetic pathway, were early targets of fruit ripening studies and manipulation in tomato and other species (Giovannoni, 2004; Theologis, 1994). Suppression of ethylene production by knocking out the expression of ACO and ACS has resulted in a strong inhibition of the ripening process (Gray et al., 1992; Hamilton et al., 1990), which provided proof of the vital role of ethylene on the regulation of the ripening process. The knowledge of ethylene signal perception, gene expression, and protein synthesis has primarily been established in studies with Arabidopsis thaliana (Alonso and Stepanova, 2004). The ethylene signal is perceived by a family of ethylene receptors (ETR1, ERS1, ETR2, ERS2, and EIN4) localize on the 3.L19 in Arabidopsis., that are similar in sequence and structure to the bacterial two-component histidine kinase (Chang et al., 1993; Kendrick and Chang, 2008). Genetic mutants with a reduced number of ethylene receptors show constitutive ethylene responses, indicating

0925-5214/$ see front matter. Crown Copyright 2012 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2012.11.012

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that ethylene receptors act as negative regulators of the ethylene response, and that ethylene binding inactivates them (Hua and Meyerowitz, 1998; Sakai et al., 1998). Constitutive triple response 1 (CTR1) is a key regulator of ethylene responses acting downstream of ethylene receptors (Kieber et al., 1993). It has been shown to be a Raf-like Ser/Thr protein kinase (MAPKK kinase) and, as with the ethylene receptors, is a negative regulator of the ethylene response by repressing the positive regulator EthyleneInsensitive 2 (EIN2) (Alonso et al., 1999). EIN2 then relays the ethylene signal to the transcription factors Ethylene-Insensitive 3 (EIN3) and Ethylene-Insensitive 3 like (EILs), which in turn activates the ethylene response factor 1 (ERF1) transcription factor. The ERF protein functions as trans-factors at the last step of transduction in the nucleus (Solano et al., 1998). These important ethylene perception elements have been studied in a number of fruit species, with tomato as one of the important models. Six receptor gene homologs have been reported in tomato (LeETR16) (Lashbrook et al., 1998; Tieman and Klee, 1999; Tieman et al., 2000), as well as ve in apple fruit (MdETR1, MdETR2, MdETR5, MdERS1, MdERS2) (Dal Cin et al., 2005, 2006; Lee et al., 1998; Li and Yuan, 2008; Wiersma et al., 2007). Some ethylene perception elements in the downstream signal transduction pathway have also been investigated in multiple fruit species (Leclercq et al., 2002; Lin et al., 2008; Tieman et al., 2001); however, there is still limited information on their abundance at the transcript level in apple fruit during fruit ripening (Wang et al., 2007; Wiersma et al., 2007). 1-Methylcyclopropene (1-MCP), a powerful antagonist of ethylene at receptor binding sites, has been recently used in the postharvest phase. The application of 1-MCP extends the freshness of various fruits, vegetables, and owers, prolongs shelf life and delays ripening and senescence. The ripening of harvested apples is prevented or delayed by 1-MCP treatment (Bleecker and Kende, 2000). Besides preventing ethylene-dependent responses by binding with ethylene receptors, 1-MCP can also inhibit apple ethylene production by inhibiting the expression of the gene for ethylene biosynthesis (Kieber et al., 1993; Tatsuki and Endo, 2006). Application of 1-MCP on plum fruit down-regulated the ETR1 and CTR1 mRNA levels in an early cultivar, but only inhibited the CTR1 transcript in a late cultivar (El-Sharkawy et al., 2007). Apple fruit treated with 1-MCP showed the delay of mRNA transcript of some ethylene receptors (ERS1, ETR1 and ERS2) (Tatsuki et al., 2007, 2009a). Recently, using micro array hybridization enabled transcriptome characterization, genes involved in ethylene biosynthesis and signaling, ACO and ETR were found to be ethylene dependent, as both were up-regulated during ripening and repressed by 1-MCP (Costa et al., 2010). In the present work, experiments from two seasons with treatments of ethylene and 1-MCP, respectively, were combined to investigate gene expression of ethylene biosynthesis and perception in apple fruit during ripening and showed the response of gene expression of ethylene biosynthesis and perception to both ethylene and 1-MCP treatments. The results provide further insight into the changes in ethylene biosynthesis, perception, and its signal transduction in apples during ripening and senescence and the inuence of ethylene or 1-MCP on these changes.

ethylene concentrations were determined on 14 apples (Wakasa et al., 2006). One group was placed in glass chambers and ethylene treatment was carried out by ventilating with ethylene gas at 36 L L1 for 24 h at 20 C to initiate ripening; the control group was placed at 20 C for 24 h without any ethylene treatment. After treatments, fruit were allowed to ripen in air at 20 C for 22 d. The study with ethylene treatment was conducted in the year 2004 and repeated in 2005. Experiments involving treatment with 1MCP were conducted over two seasons, using fruit harvested in 2006 and 2007 at the same pre-climacteric stage and divided into two groups. One group was exposed to 1 L L1 of 1-MCP (EthylBloc, 0.14%, Rohm and Haas Company, Philadelphia, PA) in sealed containers for 12 h; the control group was held at 20 C for 12 h without any 1-MCP treatment. At each sampling time, ve fruit were randomly selected from each group, quickly frozen in liquid N2 , grounded to a powder, and stored immediately at 85 C until further use. For the ethylene treatment, tissue from days 0, 7, 13, and 21 of each year was used for real-time PCR analysis. For the 1MCP treatments, ground tissue from days 0, 7, 14 and 22 for control fruit and days 0, 7, 14, 22, 39 and 43 for treated fruit, was used for real-time PCR analysis.

2.2. Respiration and ethylene production Respiration rates, measured as CO2 production, were determined on samples of ve apples that were sealed in ve 4 L glass jars with a consistent ow rate of 0.58 mL s1 of fresh air and equilibrated overnight at 20 C. CO2 production expressed as g kg1 s1 was directly measured by an infrared CO2 analyzer (Li-CO 625, USA). Ethylene production was determined on samples of ve apples that were sealed in ve 4 L glass jars with a constant ow rate of 0.58 mL s1 of fresh air and equilibrated overnight at 20 C. One milliliter samples of headspace gas were withdrawn and injected into a gas chromatograph equipped with a ame ionization detector (Carle Instruments, Inc., Anaheim, CA) and with a 1.9 mm 3.2 mm (o.d.) activated alumina column with a helium carrier ow of 0.83 mL s1 , for the measurement of ethylene production. Quantication was carried out by comparing the gas chromatography response of the samples with certied standards (Praxair Canada Inc.).

2.3. Total RNA extraction and cDNA synthesis Total RNA was extracted from frozen apple peel and esh tissue according to the hot borate method by Wan and Wilkins (1994) with some modications in the extraction buffer, which contained 200 mM sodium tetraborate decahydrate, 30 mM EGTA, 1% deoxycholic acid sodium salt, 10 mM DTT, 2% PVP 40, 1% Nonidet P-40 (octyl phenoxypolyethoxylethanol). The quality and quantity of RNA were determined spectrophotometrically by measuring the OD260/280 and OD260/230 . RNA integrity was assessed by visual inspection after electrophoresis on a formaldehyde agarose gel in the presence of ethidium bromide. All RNA extracts were then treated with DNase I using a DNA-free Kit following the manufacturers recommendations (Applied Biosystems, USA). First-strand cDNA synthesis was performed on 2 g DNase I-treated total RNA, using oligo dT as a primer (5 M) and reverse transcriptase from a RETROscript Kit (Applied Biosystems, USA) as per the manufacturers instructions. Controls with no RETROscript reverse transcriptase (NRT) were used to determine the potential genomic DNA contamination. The concentration of cDNA used for real-time qPCR was measured and each sample was diluted to 0.248 g L1 with TrisEDTA buffer (pH 8.0).

2. Materials and methods 2.1. Plant materials, treatments and storage Apple fruit (Malus domestica Borkh., Golden Delicious) were harvested from a commercial orchard in Berwick, Nova Scotia before the climacteric stage, with internal ethylene concentration 0.10.2 L L1 (ca. 1 week before commercial harvest). Internal

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2.4. Optimization of real-time qPCR analysis The oligonucleotide primers used for real-time quantitative PCR (qPCR) analysis were adopted from the literature (Li and Yuan, 2008; Vandesompele et al., 2002; Wang et al., 2007; Wiersma et al., 2007) or designed within the gene coding region of 3 UTR (Supplementary Table S1). Conditions for all PCRs were optimized in a Eppendorf Mastercyler EP gradient PCR Thermocycler (Eppendorf, USA) and Stratagene MX3005p (Agilent, USA) with regard to forward and reverse primers, various annealing temperatures (5065 C) and cDNA concentration. Amplication products were separated on 1% agarose by gel electrophoresis and analyzed with the PharosFX Molecular Imager (Bio-Rad, Canada). Optimized results were used on further RT-qPCR analysis for all genes. For measurement of primer efciency, amplication of a 6 log of 10fold serial dilutions made from cDNA of fruit sample on Day 0 and ethylene treated fruit on Day 21 was used. For each dilution, a qPCR was run in triplicate. Only genes with primer efciency above 80% were further used. The efciency value for each gene is shown in Supplementary Table S2. Two reference genes, MdActin and MdUBI were analyzed in each real-time qPCR to normalize the expression patterns. Normalization factors were calculated by taking the geometric mean of the two reference genes (Espley et al., 2007). Raw quantication cycles (Cq ) were converted to quantities representing relative expression levels using a modied comparative cycle threshold method (Fan et al., 2011) and with correction for different amplication efciencies (Wan and Wilkins, 1994). In these methods, samples from Day 0 (assigned an arbitrary quantity of 1) were used as a calibrator to calculate the relative quantity of the results.

linkage hierarchical clustering (Euclidean distance) was conducted for both experiments. 3. Results 3.1. Physiological characterizations of fruit ripening and in response to ethylene as well as to 1-MCP treatment In order to relate the gene expression study on ethylene biosynthesis and signal transduction in apple fruit during ripening and senescence to fruit physiology, biological measurements including fruit respiration and ethylene production were conducted. This revealed physiological changes associated with climacteric changes of fruit ripening and response to ethylene and 1-MCP treatment (Fig. 1). Fruit harvested at the pre-climacteric stage was conrmed by low internal ethylene concentration (<0.1 L/L). At 20 C, typical climacteric peaks in respiration were shown around Day 20. Ethylene treatment accelerated the unset of fruit respiration by at least 7 d (Fig. 1A). Ethylene treatment accelerated the onset of ethylene production and the climacteric peak, and signicantly increased the amount of ethylene production (Fig. 1B). In contrast, 1-MCP treatment signicantly delayed the onset of fruit respiration, as well as decreased the amount of ethylene production (Fig. 1C and D). 1-MCP treatment resulted in delayed the respiration and ethylene climacteric peaks by 20 d as compared with control. 3.2. Expression of ethylene biosynthesis genes during fruit ripening and in response to ethylene as well as to 1-MCP treatment Two ACSs and three ACOs were investigated in the present study. Signicant increase of ACS1 and ACO1 gene expression levels were found during fruit ripening, which was 56,000 and 430 times higher than the expression levels on Day 0 (Fig. 2A). Expression of ACO2 was not signicantly changed. While, expression of ACS3 and ACO3 was decreased as compared with Day 0 (Fig. 2). Ethylene treatment induced the gene expression of ACS1 and ACO1 to 66,000 and 510 times higher, respectively, than the expression levels in the controls (Fig. 2A). Little changed in ACO2, ACS3 and ACO3 expression during the early period of post-treatment with ethylene, as compared with the control (Fig. 2A). In this study, the strong treatment effect of 1-MCP on apple ethylene production and perception was also observed. Among the ethylene biosynthesis genes, ACS1 and ACO1 were signicantly inhibited by 1-MCP (Fig. 2B). 1-MCP treatment had no effect on ACO2, ACO3 and ACS3. 3.3. Expression of ethylene perception and signal transduction genes during fruit ripening and in response to ethylene as well as to 1-MCP treatment Ten ethylene receptors and signal transduction genes in apple fruit have been selected and studied in the present experiment (Figs. 36). Ethylene receptor gene ETR1 showed little change during fruit ripening. Expression of ERS2 increased signicantly during fruit ripening. Expression of ETR1, ETR5, and ERS2 was induced signicantly by ethylene treatment (Fig. 3A). However, among the ethylene perception genes, ETR1, ETR2, ETR5, ERS1 and ERS2 were all reduced signicantly by 1-MCP treatment (Fig. 3B). The expression of two CTR genes (CTR13 and 15) was increased signicantly by ethylene treatment. Little change in expression of other CTRs genes was found during fruit ripening and in response to ethylene treatment (Fig. 4A). A signicant inhibitive effect of 1-MCP on CTRs genes was also observed in this study (Fig. 4B). Gene expression of EIN2 members showed little changes during fruit ripening (Fig. 5A). The expression of EIL4 increased slightly.

2.5. Real-time qPCR analysis Real-time qPCR of 25 L reactions were repeated two times on a Stratagene MX3005p (Angilent, USA) using 1 L of dilute cDNA, 1.5 L 10 mM primers and 12.5 L MaximaTM SYBR Green QPCR Master Mix (Fermentas, USA). For all genes, cycling conditions included an initial hot start at 95 C for 10 min, followed by 40 cycles at 95 C for 30 s, 58 C to 60 C for 1 min and 72 C for 1 min. Each real-time PCR was ended by the addition of a dissociation curve analysis of the amplied product, involving denaturation at 95 C for 1 min, cooling to 55 C for 30 s and then gradual heating at 1 C per cycle to a nal temperature of 95 C. Individual PCR products were separated on 1% agarose gels and stained with SYBR green I to examine their size and ensure that a single PCR product was detected for each primer pair.

2.6. Statistical analysis The ethylene experiment (Study 1) was repeated over two years (2004 and 2005) and analyzed in a randomized block model using an ANOVA procedure. To evaluate treatment differences, orthogonal contrast was used for the difference between control vs. ethylene, linear and quadratic response across control and linear and quadratic response across ethylene. The 1-MCP experiment (Study 2) was a combination of 2 replicates for two years (2006 and 2007). These were analyzed in a randomized block model using ANOVA but the treatments were grouped as just control, just 1-MCP, and an overlap of control and 1-MCP. Each analysis included a polynomial contrast to evaluate the change across the stages for 1-MCP, control and the interaction for the rst 3 stages. EPCLUST software (http://www.bioinf.ebc.ee/EP/EP/EPCLUST/) allowed analysis of clustering genes according to their expression prole. A complete

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Ethylene production ( nmol kg-1 s-1)

CO2 production (g kg-1 s-1)

2.5 2.0 1.5 1.0 .5 0.0 0 10 20 30 40 50 60

14 12 10 8 6 4 2 0 0 10 20 30

A Control Ethylene 2 x SEM, n=8

B Control Ethylene 2 x SEM, n=8

40

50

60

Days after treatment

Days after treatment

Ethylene production ( nmol kg-1 s-1)

CO2 production (g kg-1 s-1)

14 12 10 8 6 4 2 0 0 10 20

Control C MCP 2 x SEM Control, n=10 2 x SEM MCP, n=10

2.5 2.0 1.5 1.0 .5 0.0 0 10 20

Control D MCP 2 x SEM Control, n=10 2 x SEM MCP, n=10

30

40

50

60

30

40

50

60

Days after treatment

Days after treatment

Fig. 1. Respiration and ethylene production of apple fruit during ripening and in response to ethylene or 1-MCP treatment at harvest. (A) Respiration after ethylene treatment; (B) ethylene production after ethylene treatment; (C) respiration after 1-MCP treatment; (D) ethylene production after 1-MCP treatment. The values presented were obtained from 2 biological replicates and within each replicate, each data point is the mean value of ve fruit. (B) and (D) were modied from Yang et al. (2012) with permission.

While, ethylene treatment slightly increased the gene expression of EIN2A at 13 d during fruit ripening, it had no effect on gene expression of EIN2B (Fig. 5A). Similar to the effect on EIN2A, ethylene increased the expression of EIL4 (Fig. 5A). 1-MCP treatment seemed to inhibit the gene expression of EIN2A and EIL4, while it had no effect on EIN2B gene expression (Fig. 5B). Gene expression of two ethylene signal transcription factors, ERF1 and ERF2, were induced signicantly upon ripening and increased during the late stage of fruit ripening (Fig. 6A), indicating a signicant relationship between ethylene signal, ERFs and downstream ripening events. Gene expression of ERF1 was inhibited signicantly by 1-MCP treatment (Fig. 6B). 3.4. Hierarchical clustering analysis of differentially expressed genes Based on the gene expression proles, ethylene production and perception genes have been grouped using hierarchical and Kmeans clustering. Three different clusters were dened for both the ethylene and 1-MCP treatment study (Figs. 7 and 8). Cluster I included ACS1 and ACO1, which showed signicantly increased expression levels during fruit ripening (Figs. 7I and 8I). Ethylene treatment remarkably induced cluster I gene expressions and 1MCP treatment depressed cluster I gene expressions during the early period of post-treatment. Cluster II contained six genes ranging from ethylene biosynthesis gene ACO2, ethylene signal receptors, ERSs and ETR2, as well as ethylene signal transcription factors ERFs (Figs. 7II and 8II). These genes showed increased expression levels upon fruit ripening, in addition, ethylene treatment induced gene expressions compared to Day 0 and fruit without treatment. Gene expression of Cluster II was depressed by 1-MCP treatment compared to Day 0 samples at the early stage of post-treatment. Thirteen genes studied in the present study were

grouped in Cluster III (Figs. 7III and 8III). They presented decreased expression levels during ripening and no signicant changes in response to the treatments. 4. Discussion In this study, the results from two separate studies: involving ethylene and 1-MCP treatment, have been combined in order to elucidate the effect of fruit ripening and effect of ethylene on ethylene biosynthetic and signal transduction pathways in apple fruit. Although we noticed the remarkable increase in publications on ripening and treatment effects on ethylene biosynthetic and perception mechanisms (Tatsuki and Endo, 2006; Tatsuki et al., 2009b; Zheng et al., 2007), the information for other ethylene signal transduction elements at both the transcript and proteomic level is still limited. Recently, increased information on apple genomics allowed us to further explore ethylene biosynthesis and signal transduction at the transcript level. Previous studies mainly focused on the changes of ethylene biosynthesis and perceptions during apple fruit development, and the comparison among different apple varieties or fruit species (Dal Cin et al., 2006; Johnston et al., 2009; Wiersma et al., 2007). The study reported here investigated the changes and regulation of ethylene biosynthesis and signal transduction in apple fruit at the mRNA level with focus not only on postharvest ripening but also on ethylene and 1-MCP treatment to gain a better understanding of the role of ethylene and the mechanisms of ethylene perception and signal transduction. ACS and ACO, the two key enzymes catalyzing the last steps of the biosynthetic pathway of ethylene, have been isolated and extensively studied in apple fruit (Costa et al., 2010; Harada et al., 2000; Huang et al., 2010; Pfaf, 2001; Ramakers et al., 2003; Roseneld et al., 1996; Zheng et al., 2007). Both ACSs and ACOs in apple are encoded by multi-gene families. Two apple ACS genes have been

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120000

ACS1

Control Ethylene Control vs Ethylene; p = 0.001 Linear Control; p = 0.006

1.2

ACS3
1.0 .8 .6 .4

90000

60000

30000 .2

Relative Expression Ratio

ACO1
750

Control vs Ethylene; p < 0.001 Quad Control; p = 0.001

0.0 4

ACO2

3 500 2 250 1

0 .8

ACO3

13

21

Days after Treatment

.6

.4

.2

0.0 7 13 21

Days after Treatment

56000 48000 40000 32000 24000 16000 8000

ACS1

Control MCP Linea r Stage MCP; p < 0.001 Control vs MCP; p = 0.019

2.5 2.0 1.5 1.0 .5 0.0

ACS3

Relative Expression Ratio

0 15000 12000

ACO1

Linea r Stage MCP; p < 0.008 Control vs MCP; p = 0.023

ACO2
4 3

9000 6000 3000 0 1.2 1.0 .8 .6 .4 .2 0.0 7 14 22 39 43 2 1 0

ACO3

14

22

39

43

Days after Treatment

Days after Treatment

Fig. 2. Expressions of apple ethylene biosynthesis genes, MdACOs and MdACSs, during 21 d of ripening at 20 C after ethylene treatment and 43 d of ripening at 20 C after 1-MCP treatments. Sampling details are labeled in Fig. 1. Quantitative real-time PCR was used to analyze the mRNA changes as described in Section 2. The y axis represents the relative fold difference of mRNA level and was calculated using a modied 2DDCt formula (Wan and Wilkins, 1994) with Mdactin and MdUBI as references. The values presented were obtained from 2 biological replicates and within each replicate, each data point is the mean value obtained from qPCR reaction performed in triplicate from a pooled sample of ve fruit. Samples from Day 0 (assigned an arbitrary quantity of 1) were used as a calibrator to calculate the relative quantity of the results. Error bars indicate 2 standard error of means.

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2.0

ETR1

Control Ethylene Control vs Ethylene; p = 0.017

ETR2

1.5

4 1.0 2 .5

Relative Expression Ratio

0.0

ETR5
2.5 2.0 1.5 1.0

Control vs Ethylene; p = 0.005 6

ERS1

2 .5 0.0 0

ERS2
6

Control vs Ethylene; p < 0.001 Linea r Control; p = 0.014

13

21

Days after Treatment

0 7 13 21

Days after Treatment

B
2.5 2.0 1.5 1.0 .5

ETR1

Control MCP Control vs MCP; p = 0.038

ETR2
Control vs MCP; p = 0.012

Relative Expression Ratio

0.0 2.5 2.0 1.5

ETR5

Linea r Stage control; p = 0.018 Control vs MCP; p = 0.03

8 6 4

ERS1

Control vs MCP; p = 0.027

1.0 .5 0.0 8 2 0

ERS2

Linea r Stage control; p = 0.049 Control vs MCP; p = 0.046

14

22

39

43

Days after Treatment

0 7 14 22 39 43

Days after Treatment

Fig. 3. Expressions of apple ethylene signal receptors genes, ETR and ERS, during 21 d of ripening at 20 C after ethylene treatment and 43 d of ripening at 20 C after 1-MCP treatments. Sampling details are labeled in Fig. 1. Quantitative real-time PCR details are as described in Fig. 2. Error bars indicate 2 standard error of means.

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A
1.5

CTR1-1

Control Ethylene 1.5

CTR1-2

1.0

1.0

.5

.5

Relative Expression Ratio

0.0

0.0

CTR1-3
1.5

Control vs Ethylene; p = 0.002 Linea r Ethylene; p = 0.04

CTR1-4
1.5

Control vs Ethylene; p = 0.022

1.0

1.0

.5

.5

0.0

0.0

CTR1-5
1.5

Control vs Ethylene; p = 0.039

13

21

Days after Treatment

1.0

.5

0.0 7 13 21

Days after Treatment

B
1.5

CTR1-1

Control MCP Linea r Stage MCP; p = 0.025 Control vs MCP; p = 0.011

CTR1-2
1.5

Control vs MCP; p = 0.022

1.0

1.0

.5

.5

Relative Expression Ratio

0.0

0.0

CTR1-3
1.5

Quad Stage MCP; p = 0.028 Control vs MCP; p = 0.006

CTR1-4
1.5

Quad Stage MCP; p = 0.035 Linea r Stage MCP; p = 0.047 Control vs MCP; p = 0.006

1.0

1.0

.5

.5

0.0

0.0

CTR1-5
1.5

Control vs MCP; p = 0.014

14

22

39

43

Days after Treatment

1.0

.5

0.0 7 14 22 39 43

Days after Treatment

Fig. 4. Expressions of apple ethylene signal receptors CTR1s genes, during 21 d of ripening at 20 C after ethylene treatment or 43 d of ripening at 20 C after 1-MCP treatments. Sampling details are labeled in Fig. 1. Quantitative real-time PCR details are as described in Fig. 2. Error bars indicate 2 standard error of means.

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2 2.0

EIN2A

Control Ethylene

2.0

EIN2A

Ctrl MCP Linear Stage MCP; p =0.024

1.5

1.5

1.0

1.0

.5

.5

Relative Expression Ratio

0.0 1.4 1.2 1.0 .8 .6 .4 .2 0.0 2.0

Relative Expression Ratio

0.0 1.0 .8 .6 .4 .2 0.0 2.5 2.0 1.5 1.0

EIN2B

EIN2B

Linear Stage Control; p =0.018

EIL4

Control vs Ethylene; p = 0.05 Linear Control; p = 0.046

EIL4

Linear Stage MCP; p =0.032

1.5

1.0

.5 .5 0.0 7 13 21 0.0 7 14 22 39 43

Days after Treatment

Days after Treatment

Fig. 5. Expressions of apple ethylene signal transduction component genes, EIN2A, EIN2B and EIL4 during 21 d of ripening at 20 C after ethylene treatment or 43 d of ripening at 20 C after 1-MCP treatments. Sampling details are labeled in Fig. 1. Quantitative real-time PCR details are as described in Fig. 2. Error bars indicate 2 standard error of means.

Relative Expression Ratio

ERF1
6

Control Ethylene
Control vs Ethylene; p = 0.012

6 5 4

ERF2

Control vs Ethylene; p = 0.002

4 3 2 2 1 0 7 13 21 0 7 13 21

Days after Treatment

Days after Treatment

ERF2

Relative Expression Ratio

ERF1

Control MCP Linear Stage MCP; p = 0.003 Linear Stage Control; p = 0.013 Control vs MCP; p = 0.018

0 7 14 22 39 43

0 7 14 22 39 43

Days after Treatment

Days after Treatment

Fig. 6. Expressions of apple ethylene signal transcription factor genes, ERF1 and ERF2, during 21 d of ripening at 20 C after ethylene treatment and 43 d of ripening at 20 C after 1-MCP treatment. Sampling details are labeled in Fig. 1. Quantitative real-time PCR details are as described in Fig. 2. Error bars indicate 2 standard error of means.

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Fig. 7. Hierarchical cluster analysis (EPCLUST software) of transcript levels from ethylene genes differentially expressed during apple fruit ripening with or without ethylene treatment. Red boxes mean higher levels of expression compared to Day 0, and green boxes mean lower expression levels compared to Day 0. The color brightness is directly proportional to the expression ratio. Black boxes are genes not signicantly differentially expressed compared to Day 0. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of the article.)

Fig. 8. Hierarchical cluster analysis (EPCLUST software) of transcript levels from ethylene genes differentially expressed during apple fruit ripening with or without 1-MCP treatment. Red boxes mean high levels of expression compared to Day 0, and green boxes mean lower expression levels compared to Day 0. The color brightness is directly proportional to the expression ratio. Black boxes are genes not signicantly differentially expressed as compared to Day 0. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of the article.)

detected in the present study (Fig. 2A and B, ACS1 and ACS3). The signicant increase in expression of ACS1 during fruit ripening and as the result of the ethylene treatment was pronounced. ACS1 and ACO1 are correlated with the ethylene climacteric burst of apple fruit. Treatment with 1-MCP generally produced effects opposite to ethylene, which provides additional evidence that regulation of these genes coincided with ethylene production (Fig. 1AC). It has been shown that the mutation of the Md-ACS1 gene caused low level of ethylene production in some apple cultivars, leading to extended storage life (Huang et al., 2010; Pfaf, 2001). The expression of ACS3 was reduced during ripening or ethylene treatment as compared to Day 0 (Fig. 2A). The function of ACS3 is still unclear. Similar to our present study, it was reported that the expression of ACS3 did not change upon ripening and 1-MCP treatment (Shibuya et al., 2004; Tatsuki et al., 2007; Wiersma et al., 2007). However, the relationship is not without controversy; others report a negative feedback regulation mechanism where the expression of ACS3 was stimulated by 1-MCP treatment (Costa et al., 2010; Varanasi et al., 2011). The expression of ACS3 can also be inuenced by different fruit varieties and developmental stages (Wiersma et al., 2007; Tatsuki et al., 2007). Three apple ACO genes were expressed differently throughout the fruit ripening process (Fig. 2A and B). According to the expression mode, which was closely correlated to ethylene production, ACO1 in apple fruit may be one of the major factors of ethylene synthesis. This is supported by the recent report of the transgenic line

of anti-ACO1, which shows the suppression of ACO1 results in low levels of ethylene (Schaffer et al., 2007). Information about the function of ACO2 and ACO3 in apple are limited, but interest has focused on ACO3 since it was negatively regulated by ethylene (Fig. 2A), that was also observed in Sunrise apple (Wiersma et al., 2007). Phylogenetic analysis showed that ACO3 is distinct from ACO1 and ACO2, and appeared to be expressed predominantly in young tissue, which is more likely to be a photosynthesis associated gene (Harada et al., 2000). Therefore, it is possible that ACO3, which has been seen in feedback inhibition to ethylene, is proposed to be responsible for the system I ethylene response and less associated with the ethylene burst upon the initiation of fruit ripening. Overall, expression of ve ethylene receptors genes (ETR1, ETR2, ETR5, ERS1 and ERS2) were induced at the transcriptional level upon ripening and by ethylene treatment and decreased by 1-MCP treatment during fruit ripening (Fig. 3A), although some of them have been found to be constant in apple fruit and other fruit tissues (Dal Cin et al., 2006; El-Sharkawy et al., 2003; Kevany et al., 2007; Lashbrook et al., 1998; Martnez et al., 2001; Tatsuki et al., 2009a; Tieman and Klee, 1999; Wiersma et al., 2007). Ethylene receptors are generally considered negative regulators of ethylene response and, as shown in a number receptor mutants, a reduction of receptors appears to render high-ethylene sensitivity (Tieman et al., 2000). This paradox has been seen in many fruit tissues where the transcription of these receptor mRNAs increased upon ripening, and the increased level of gene expression would be expected to increase the protein level and in turn confer low-ethylene sensitivity. Tatsuki et al. (2009a) showed that in apple fruit, MdERS1

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and MdERS2 protein levels were not changed or decreased with ethylene treatment, although the transcript levels were increased. Our results further showed that 1-MCP negatively inuenced the gene expression of receptors and these results coincided well with ethylene production (Figs. 1 and 3). The delayed ethylene production may be directly caused by lack of an ethylene response due to 1-MCP binding on the receptors, which is in agreement with the ndings by Costa et al. (2010). Stability of ethylene receptor proteins is also important. It has also been shown that the binding of ethylene induced the degradation of receptor proteins of tomato LeETR4 and LeETR6 (Kevany et al., 2007). It might be hypothesized that the binding of receptors with 1-MCP may actually have decreased receptor protein degradation that was triggered by the ethylene binding and therefore decreased the sensitivity of fruit to ethylene signal. In the ethylene signaling system, CTR1 plays a central role in regulating a negative ethylene response in the absence of ethylene (Kieber, 1997). Although there has been one CTR1 identied in apple fruit so far, there are several splice variants existing as reported in Sunrise and Golden Delicious apple (Wiersma et al., 2007). We therefore adopted and characterized the three different forms separately as follows: CTR11, -2 (primers located between the rst intron and the start of the kinase domain), CTR13, -4 (those retain 3 UTR region), and CTR15 (the one located within kinase domain). For all scenarios, there were no signicant changes in the expression of CTR1 during all the stages of postharvest ripening. However, we noticed a signicant expression level induction of CTR13, -4 and -5 in ethylene treated fruit and a decreased level of all CTR1s in 1-MCP treated fruit (Fig. 4), indicating that similar to LeCTR1 in tomato fruit (Adams-Phillips et al., 2004; Leclercq et al., 2002), apple CTR1 is ethylene inducible. It has been consistently seen in fruits from the Rosaceae family (apple, pear and plum) that CTR1 expression is up-regulated in fruit during ripening (Dal Cin et al., 2006; El-Sharkawy et al., 2003, 2007). It was interesting to see that a negative regulator of ethylene response was induced by ethylene during fruit ripening. The most logical explanation is that, in most tissues that produce large amounts of ethylene, the induction of the negative expression regulator may act as a damping mechanism to response and temper the suddenly increased ethylene concentration, in order to slow down the ripening and senescence process (Klee, 2002). Since CTR1 is an important component in the complex of ethylene signal perception, and it interacts closely with ETR or ERS, it would be very interesting to see concomitant changes of CTR1 expression at the protein level upon fruit ripening or upon ethylene treatment in apple fruit. EIN2 has been proved acting as a bi-functional signal transducer, which mediates the signal propagation between CTR1 and downstream components (EIN3/EIL). EIN2 mRNA is not altered in response to ethylene, in contrast, its protein is short-lived and subject to regulation by proteasomes in Arabidopsis (Alonso et al., 1999). No signicant change in expression of EIN2A and EIN2B was found during ripening or in response to ethylene treatment in apple fruit, (Fig. 5A and B). A controversy has been observed in many plant species, regarding the regulation of ethylene at the transcription level of EIN2 genes. In cut ower of carnation, expression of DcEIN2 was enhanced by treatment with ethylene, while in petunia, PhEIN2 was regulated by ethylene in a tissue-specic manner (Shibuya et al., 2004), and in rice (Jun et al., 2004) and tomato fruit (Zhu et al., 2006), the expression of EIN2 was not regulated by ethylene. An EIN-like gene was found, when mining in Genbank using the EST entry, that shares high similarity to EIL3 in Arabidopsis (80%), EIN3 in rose (91%), and PpEIL1 in peach (77%), but was only 76%, 73% and 75% identical to MdEIL1, MdEIL2 and MdEIL3 respectively, in Royal Gala apples (Tacken et al., 2010). This gene was then designated as MdEIL4 and its gene expression

was tested during fruit ripening and under different treatments. According to our study, EIL4 in apple fruit tissue was ripening and ethylene inducible (Fig. 5A), which is consistent with recent ndings in other fruit systems, such as tomato (LeEIL4), banana (Ma-EIL2/AB266318), and melon (CmEIL1 and CmEIL2) (Huang et al., 2010; Mbeguie-A-Mbeguie et al., 2008; Yokotani et al., 2003). In addition, 1-MCP decreased the ethylene signaling and reduced the expression level of EIL4 (Fig. 5B). In contrast to the effect on EIN2, ethylene acts to stabilize EIN3/EIL proteins by preventing degradation through the ubiquitinproteasome pathway and further activate downstream signaling (Guo and Ecker, 2003). It has been proposed that EIN3/EIL are not regulated by ethylene in fruit but rather by ripening signals, since the expression levels were unaffected by ethylene in leaf tissue (Huang et al., 2010). EIN3/EILs are believed to be a positive regulator within the ethylene signal cascade (Solano et al., 1998; Tieman et al., 2001). It was also suggested that EIN3/EILs may function differentially among different family genes or different tissues (Mbeguie-A-Mbeguie et al., 2008; Tacken et al., 2010; Yokotani et al., 2003). Since multiple EIL genes have been discovered in apple fruit, the signicant regulation of ripening from other family genes cannot be ruled out. ERF1 and ERF2 are expressed in apple fruit during ripening and induced by ethylene treatment, while the expression of ERF1 was signicantly reduced by 1-MCP treatment (Fig. 6A and B). Our results support the nding that 1-MCP inhibited the ERF1 transcripts as reported (Wang et al., 2007). However, the effect of 1-MCP on ERF2 was not signicant, which may be caused by differences in fruit maturities in these studies. It was proposed that another transcription factor(s) derived from the maturation cascade appears to function as regulator of ERFs gene expression (Wang et al., 2007). Despite the physiological variation between years, our gene expression results collected over two years for each study indicate that changes in expression of ethylene related genes in apple fruit coincide with ethylene regulated fruit ripening. Expression correlated well with the process of ripening and in response to the treatments by ethylene and by 1-MCP. The main objective was to examine the effect of ethylene and or/1-MCP on gene expression obtained from a short period study at 20 C. Our current study provides additional information on regulation of gene expression of ethylene biosynthesis, perception and signal transduction in apple during fruit ripening or under the inuence of ethylene or 1-MCP treatment that may be helpful in understanding ethylene involvement in fruit ripening. These ndings are based on gene expression data and not on protein expression or function. Our long-term goal was a quantitative proteomic study developed and conducted using the same biological samples that will be reported in a subsequent publication. Acknowledgements We thank Dr. Gordon Braun at AAFC for critical review. We thank the MOE and AAFC for the PhD fellowship provided to X.T. Yang. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.postharvbio.2012.11.012. References
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