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Postharvest Biology and Technology 78 (2013) 55–66

Contents lists available at SciVerse ScienceDirect

Postharvest Biology and Technology

jou rna l h omepa g e: www.elsev ier .com/ locate /postharvbio

ffect of ethylene and 1-MCP on expression of genes involved in ethyleneiosynthesis and perception during ripening of apple fruit

iaotang Yanga, Jun Songb,∗, Leslie Campbell-Palmerb, Sherry Fillmoreb, Zhaoqi Zhanga

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

r t i c l e i n f o

rticle history:eceived 21 March 2012ccepted 26 November 2012

eywords:pple (Malus domestica Borkh.)ruit ripeningthylene-MCPthylene biosynthesisignal transduction

a b s t r a c t

Ethylene plays an important role in regulating fruit ripening and senescence and directly influencesthe development of the eating quality of fresh apples, including appearance, color, texture, and flavor.Apple fruit (Malus domestica Borkh.) is a well-known climacteric fruit and a good model system to studyfruit ripening and senescence. To better understand fruit ripening and the role of ethylene perceptionand signal transduction, apples harvested at a pre-climacteric stage were allowed to naturally ripen,or ripening was either stimulated by treatment with 36 �L L−1 ethylene for 24 h or inhibited by 1-MCPtreatment (1.0 �L L−1 for 24 h), respectively. Postharvest physiological indices including respiration andethylene production were monitored for 22 d for ethylene treatment and 47 d for 1-MCP treatment. Basedon an efficiency test, 20 genes in relation to ethylene biosynthesis and perception were investigatedusing real-time qPCR during the post-treatment period. The ETR2, ETR5, ERSs, EIL4, ERFs genes togetherwith ACS1 and ACO1 genes were significantly up-regulated in fruit during ripening. Ethylene treatmentfurther enhanced the expression of ACO2, ETR1, CTR1s and EIN2A genes, while the ACS3 and ACO3, andEIN2B genes were only slightly affected. 1-MCP treatment significantly inhibited expression of ACS1, ACO1and 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 alimited effect on ACS3, ACO3, and EIN2B. This study demonstrated the complexity and dynamic changesof transcriptional profiles of ethylene perception and biosynthesis in response to fruit ripening, ethylene,and 1-MCP treatment. Understanding of the significant changes of these genes and their function mayhelp to explore the mechanisms controlling apple fruit ripening and its response to exogenous ethylenestimuli and action inhibition at the receptor level during ripening and senescence.

. Introduction

Ethylene is one of the most important plant hormones affect-ng various plant biological processes. Numerous biological roles ofthylene have been discovered and the most studied effect of eth-lene is the promotion of fruit ripening (Bleecker and Kende, 2000).pple fruit (Malus x domestica Borkh.) is one of the most popular

ruits and its consumption is highly recommended for a healthyiet. Apple is a climacteric fruit that is responsive to ethylene andndergoes a significant increase in respiration and ethylene pro-uction during ripening. The discovery of the biosynthetic pathwayf ethylene and elucidation of the important elements involved

n its perception and downstream signal transduction have beenchieved to better understand the role of ethylene and the mecha-isms 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).

925-5214/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rittp://dx.doi.org/10.1016/j.postharvbio.2012.11.012

Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.

1984). Genes encoding 1-aminocyclopropane-carboxylase (ACC)synthase (ACS) and ACC oxidase (ACO), the two key enzymes cat-alyzing the last steps of the ethylene biosynthetic pathway, wereearly targets of fruit ripening studies and manipulation in tomatoand other species (Giovannoni, 2004; Theologis, 1994). Suppres-sion of ethylene production by knocking out the expression of ACOand ACS has resulted in a strong inhibition of the ripening process(Gray et al., 1992; Hamilton et al., 1990), which provided proof ofthe vital role of ethylene on the regulation of the ripening pro-cess.

The knowledge of ethylene signal perception, gene expression,and protein synthesis has primarily been established in studieswith Arabidopsis thaliana (Alonso and Stepanova, 2004). The eth-ylene signal is perceived by a family of ethylene receptors (ETR1,ERS1, ETR2, ERS2, and EIN4) localize on the 3.L19 in Arabidop-

sis., that are similar in sequence and structure to the bacterialtwo-component histidine kinase (Chang et al., 1993; Kendrick andChang, 2008). Genetic mutants with a reduced number of eth-ylene receptors show constitutive ethylene responses, indicating

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hat ethylene receptors act as negative regulators of the ethyleneesponse, and that ethylene binding inactivates them (Hua andeyerowitz, 1998; Sakai et al., 1998). Constitutive triple response

(CTR1) is a key regulator of ethylene responses acting down-tream of ethylene receptors (Kieber et al., 1993). It has beenhown to be a Raf-like Ser/Thr protein kinase (MAPKK kinase)nd, as with the ethylene receptors, is a negative regulator of thethylene response by repressing the positive regulator Ethylene-nsensitive 2 (EIN2) (Alonso et al., 1999). EIN2 then relays thethylene signal to the transcription factors Ethylene-Insensitive

(EIN3) and Ethylene-Insensitive 3 like (EILs), which in turnctivates the ethylene response factor 1 (ERF1) transcription fac-or. The ERF protein functions as trans-factors at the last step ofransduction in the nucleus (Solano et al., 1998). These impor-ant ethylene perception elements have been studied in a numberf fruit species, with tomato as one of the important models. Sixeceptor gene homologs have been reported in tomato (LeETR1–6)Lashbrook et al., 1998; Tieman and Klee, 1999; Tieman et al.,000), as well as five in apple fruit (MdETR1, MdETR2, MdETR5,dERS1, MdERS2) (Dal Cin et al., 2005, 2006; Lee et al., 1998; Li

nd Yuan, 2008; Wiersma et al., 2007). Some ethylene perceptionlements in the downstream signal transduction pathway havelso been investigated in multiple fruit species (Leclercq et al.,002; Lin et al., 2008; Tieman et al., 2001); however, there is still

imited information on their abundance at the transcript level inpple fruit during fruit ripening (Wang et al., 2007; Wiersma et al.,007).

1-Methylcyclopropene (1-MCP), a powerful antagonist of eth-lene at receptor binding sites, has been recently used in theostharvest phase. The application of 1-MCP extends the fresh-ess of various fruits, vegetables, and flowers, prolongs shelf lifend delays ripening and senescence. The ripening of harvestedpples is prevented or delayed by 1-MCP treatment (Bleecker andende, 2000). Besides preventing ethylene-dependent responsesy binding with ethylene receptors, 1-MCP can also inhibit applethylene production by inhibiting the expression of the gene forthylene biosynthesis (Kieber et al., 1993; Tatsuki and Endo, 2006).pplication of 1-MCP on plum fruit down-regulated the ETR1nd CTR1 mRNA levels in an early cultivar, but only inhibitedhe CTR1 transcript in a late cultivar (El-Sharkawy et al., 2007).pple fruit treated with 1-MCP showed the delay of mRNA tran-cript of some ethylene receptors (ERS1, ETR1 and ERS2) (Tatsukit al., 2007, 2009a). Recently, using micro array hybridizationnabled transcriptome characterization, genes involved in ethyleneiosynthesis and signaling, ACO and ETR were found to be eth-lene dependent, as both were up-regulated during ripening andepressed by 1-MCP (Costa et al., 2010).

In the present work, experiments from two seasons with treat-ents of ethylene and 1-MCP, respectively, were combined to

nvestigate gene expression of ethylene biosynthesis and percep-ion in apple fruit during ripening and showed the response of genexpression of ethylene biosynthesis and perception to both eth-lene and 1-MCP treatments. The results provide further insightnto the changes in ethylene biosynthesis, perception, and its sig-al transduction in apples during ripening and senescence and the

nfluence of ethylene or 1-MCP on these changes.

. Materials and methods

.1. Plant materials, treatments and storage

Apple fruit (Malus domestica Borkh., ‘Golden Delicious’) werearvested from a commercial orchard in Berwick, Nova Scotiaefore the climacteric stage, with internal ethylene concentration.1–0.2 �L L−1 (ca. 1 week before commercial harvest). Internal

d Technology 78 (2013) 55–66

ethylene concentrations were determined on 14 apples (Wakasaet al., 2006). One group was placed in glass chambers and ethyl-ene treatment was carried out by ventilating with ethylene gas at36 �L L−1 for 24 h at 20 ◦C to initiate ripening; the control groupwas placed at 20 ◦C for 24 h without any ethylene treatment. Aftertreatments, fruit were allowed to ripen in air at 20 ◦C for 22 d. Thestudy with ethylene treatment was conducted in the year 2004and repeated in 2005. Experiments involving treatment with 1-MCP were conducted over two seasons, using fruit harvested in2006 and 2007 at the same pre-climacteric stage and divided intotwo groups. One group was exposed to 1 �L L−1 of 1-MCP (Ethyl-Bloc, 0.14%, Rohm and Haas Company, Philadelphia, PA) in sealedcontainers for 12 h; the control group was held at 20 ◦C for 12 hwithout any 1-MCP treatment. At each sampling time, five fruitwere randomly selected from each group, quickly frozen in liquidN2, grounded to a powder, and stored immediately at −85 ◦C untilfurther 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 1-MCP treatments, ground tissue from days 0, 7, 14 and 22 for controlfruit and days 0, 7, 14, 22, 39 and 43 for treated fruit, was used forreal-time PCR analysis.

2.2. Respiration and ethylene production

Respiration rates, measured as CO2 production, were deter-mined on samples of five apples that were sealed in five 4 L glassjars with a consistent flow rate of 0.58 mL s−1 of fresh air and equili-brated overnight at 20 ◦C. CO2 production expressed as �g kg−1 s−1

was directly measured by an infrared CO2 analyzer (Li-CO 625,USA).

Ethylene production was determined on samples of five applesthat were sealed in five 4 L glass jars with a constant flow rateof 0.58 mL s−1 of fresh air and equilibrated overnight at 20 ◦C.One milliliter samples of headspace gas were withdrawn andinjected into a gas chromatograph equipped with a flame ioniza-tion detector (Carle Instruments, Inc., Anaheim, CA) and with a1.9 mm × 3.2 mm (o.d.) activated alumina column with a heliumcarrier flow of 0.83 mL s−1, for the measurement of ethylene pro-duction. Quantification was carried out by comparing the gaschromatography response of the samples with certified standards(Praxair Canada Inc.).

2.3. Total RNA extraction and cDNA synthesis

Total RNA was extracted from frozen apple peel and flesh tissueaccording to the hot borate method by Wan and Wilkins (1994)with some modifications in the extraction buffer, which contained200 mM sodium tetraborate decahydrate, 30 mM EGTA, 1% deoxy-cholic acid sodium salt, 10 mM DTT, 2% PVP 40, 1% Nonidet P-40(octyl phenoxypolyethoxylethanol). The quality and quantity ofRNA were determined spectrophotometrically by measuring theOD260/280 and OD260/230. RNA integrity was assessed by visualinspection after electrophoresis on a formaldehyde agarose gel inthe presence of ethidium bromide. All RNA extracts were thentreated with DNase I using a DNA-free Kit following the manufac-turer’s recommendations (Applied Biosystems, USA). First-strandcDNA synthesis was performed on 2 �g DNase I-treated totalRNA, using oligo dT as a primer (5 �M) and reverse transcrip-tase from a RETROscript Kit (Applied Biosystems, USA) as per themanufacturer’s instructions. Controls with no RETROscript reverse

transcriptase (NRT) were used to determine the potential genomicDNA contamination. The concentration of cDNA used for real-timeqPCR was measured and each sample was diluted to 0.248 g L−1

with Tris–EDTA buffer (pH 8.0).

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.4. Optimization of real-time qPCR analysis

The oligonucleotide primers used for real-time quantitative PCRqPCR) analysis were adopted from the literature (Li and Yuan,008; Vandesompele et al., 2002; Wang et al., 2007; Wiersmat al., 2007) or designed within the gene coding region of 3′UTRSupplementary Table S1). Conditions for all PCRs were optimizedn a Eppendorf Mastercyler EP gradient PCR Thermocycler (Eppen-orf, USA) and Stratagene MX3005p (Agilent, USA) with regardo forward and reverse primers, various annealing temperatures50–65 ◦C) and cDNA concentration. Amplification products wereeparated on 1% agarose by gel electrophoresis and analyzedith the PharosFX Molecular Imager (Bio-Rad, Canada). Optimized

esults were used on further RT-qPCR analysis for all genes. Foreasurement of primer efficiency, amplification of a 6 log of 10-

old serial dilutions made from cDNA of fruit sample on Day 0 andthylene treated fruit on Day 21 was used. For each dilution, a qPCRas run in triplicate. Only genes with primer efficiency above 80%ere further used. The efficiency value for each gene is shown in

upplementary Table S2. Two reference genes, MdActin and MdUBIere analyzed in each real-time qPCR to normalize the expressionatterns. Normalization factors were calculated by taking the geo-etric mean of the two reference genes (Espley et al., 2007). Raw

uantification cycles (Cq) were converted to quantities represent-ng relative expression levels using a modified comparative cyclehreshold method (Fan et al., 2011) and with correction for dif-erent amplification efficiencies (Wan and Wilkins, 1994). In these

ethods, samples from Day 0 (assigned an arbitrary quantity of1”) were used as a calibrator to calculate the relative quantity ofhe results.

.5. Real-time qPCR analysis

Real-time qPCR of 25 �L reactions were repeated two times on Stratagene MX3005p (Angilent, USA) using 1 �L of dilute cDNA,.5 �L 10 mM primers and 12.5 �L MaximaTM SYBR Green QPCRaster Mix (Fermentas, USA). For all genes, cycling conditions

ncluded an initial hot start at 95 ◦C for 10 min, followed by 40 cyclest 95 ◦C for 30 s, 58 ◦C to 60 ◦C for 1 min and 72 ◦C for 1 min. Eacheal-time PCR was ended by the addition of a dissociation curvenalysis of the amplified product, involving denaturation at 95 ◦Cor 1 min, cooling to 55 ◦C for 30 s and then gradual heating at 1 ◦Cer cycle to a final temperature of 95 ◦C. Individual PCR productsere separated on 1% agarose gels and stained with SYBR green

to examine their size and ensure that a single PCR product wasetected for each primer pair.

.6. Statistical analysis

The ethylene experiment (Study 1) was repeated over twoears (2004 and 2005) and analyzed in a randomized block modelsing an ANOVA procedure. To evaluate treatment differences,rthogonal contrast was used for the difference between con-rol vs. ethylene, linear and quadratic response across controlnd linear and quadratic response across ethylene. The 1-MCPxperiment (Study 2) was a combination of 2 replicates forwo years (2006 and 2007). These were analyzed in a ran-omized block model using ANOVA but the treatments wererouped as just control, just 1-MCP, and an overlap of con-rol and 1-MCP. Each analysis included a polynomial contrast

o evaluate the change across the stages for 1-MCP, controlnd the interaction for the first 3 stages. EPCLUST softwarehttp://www.bioinf.ebc.ee/EP/EP/EPCLUST/) allowed analysis oflustering genes according to their expression profile. A complete

d Technology 78 (2013) 55–66 57

linkage hierarchical clustering (Euclidean distance) was conductedfor both experiments.

3. Results

3.1. Physiological characterizations of fruit ripening and inresponse to ethylene as well as to 1-MCP treatment

In order to relate the gene expression study on ethylene biosyn-thesis and signal transduction in apple fruit during ripening andsenescence to fruit physiology, biological measurements includ-ing fruit respiration and ethylene production were conducted. Thisrevealed physiological changes associated with climacteric changesof fruit ripening and response to ethylene and 1-MCP treatment(Fig. 1). Fruit harvested at the pre-climacteric stage was confirmedby low internal ethylene concentration (<0.1 �L/L). At 20 ◦C, typicalclimacteric peaks in respiration were shown around Day 20. Ethyl-ene treatment accelerated the unset of fruit respiration by at least7 d (Fig. 1A). Ethylene treatment accelerated the onset of ethyleneproduction and the climacteric peak, and significantly increased theamount of ethylene production (Fig. 1B). In contrast, 1-MCP treat-ment significantly delayed the onset of fruit respiration, as wellas decreased the amount of ethylene production (Fig. 1C and D).1-MCP treatment resulted in delayed the respiration and ethyleneclimacteric peaks by 20 d as compared with control.

3.2. Expression of ethylene biosynthesis genes during fruitripening and in response to ethylene as well as to 1-MCPtreatment

Two ACSs and three ACOs were investigated in the present study.Significant increase of ACS1 and ACO1 gene expression levels werefound during fruit ripening, which was 56,000 and 430 times higherthan the expression levels on Day 0 (Fig. 2A). Expression of ACO2was not significantly changed. While, expression of ACS3 and ACO3was decreased as compared with Day 0 (Fig. 2). Ethylene treatmentinduced the gene expression of ACS1 and ACO1 to 66,000 and 510times higher, respectively, than the expression levels in the controls(Fig. 2A). Little changed in ACO2, ACS3 and ACO3 expression dur-ing the early period of post-treatment with ethylene, as comparedwith the control (Fig. 2A). In this study, the strong treatment effectof 1-MCP on apple ethylene production and perception was alsoobserved. Among the ethylene biosynthesis genes, ACS1 and ACO1were significantly inhibited by 1-MCP (Fig. 2B). 1-MCP treatmenthad no effect on ACO2, ACO3 and ACS3.

3.3. Expression of ethylene perception and signal transductiongenes during fruit ripening and in response to ethylene as well asto 1-MCP treatment

Ten ethylene receptors and signal transduction genes in applefruit have been selected and studied in the present experiment(Figs. 3–6). Ethylene receptor gene ETR1 showed little change dur-ing fruit ripening. Expression of ERS2 increased significantly duringfruit ripening. Expression of ETR1, ETR5, and ERS2 was induced sig-nificantly by ethylene treatment (Fig. 3A). However, among theethylene perception genes, ETR1, ETR2, ETR5, ERS1 and ERS2 wereall reduced significantly by 1-MCP treatment (Fig. 3B).

The expression of two CTR genes (CTR1–3 and 1–5) wasincreased significantly by ethylene treatment. Little change inexpression of other CTRs genes was found during fruit ripening andin response to ethylene treatment (Fig. 4A). A significant inhibitive

effect of 1-MCP on CTRs genes was also observed in this study(Fig. 4B).

Gene expression of EIN2 members showed little changes duringfruit ripening (Fig. 5A). The expression of EIL4 increased slightly.

58 X. Yang et al. / Postharvest Biology and Technology 78 (2013) 55–66

Days aft er treatment

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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 ethylenet -MCPw nt is tp

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reatment; (B) ethylene production after ethylene treatment; (C) respiration after 1ere obtained from 2 biological replicates and within each replicate, each data poiermission.

hile, ethylene treatment slightly increased the gene expressionf EIN2A at 13 d during fruit ripening, it had no effect on genexpression of EIN2B (Fig. 5A). Similar to the effect on EIN2A, eth-lene increased the expression of EIL4 (Fig. 5A). 1-MCP treatmenteemed to inhibit the gene expression of EIN2A and EIL4, while itad no effect on EIN2B gene expression (Fig. 5B). Gene expressionf two ethylene signal transcription factors, ERF1 and ERF2, werenduced significantly upon ripening and increased during the latetage of fruit ripening (Fig. 6A), indicating a significant relationshipetween ethylene signal, ERFs and downstream ripening events.ene expression of ERF1 was inhibited significantly by 1-MCP treat-ent (Fig. 6B).

.4. Hierarchical clustering analysis of differentially expressedenes

Based on the gene expression profiles, ethylene production anderception genes have been grouped using hierarchical and K-eans clustering. Three different clusters were defined for both

he ethylene and 1-MCP treatment study (Figs. 7 and 8). Cluster included ACS1 and ACO1, which showed significantly increasedxpression levels during fruit ripening (Figs. 7I and 8I). Ethylenereatment remarkably induced cluster I gene expressions and 1-

CP treatment depressed cluster I gene expressions during thearly period of post-treatment. Cluster II contained six genesanging from ethylene biosynthesis gene ACO2, ethylene signaleceptors, ERSs and ETR2, as well as ethylene signal transcrip-ion factors ERFs (Figs. 7II and 8II). These genes showed increasedxpression levels upon fruit ripening, in addition, ethylene treat-

ent induced gene expressions compared to Day 0 and fruitithout treatment. Gene expression of Cluster II was depressed by

-MCP treatment compared to Day 0 samples at the early stage ofost-treatment. Thirteen genes studied in the present study were

treatment; (D) ethylene production after 1-MCP treatment. The values presentedhe mean value of five fruit. (B) and (D) were modified from Yang et al. (2012) with

grouped in Cluster III (Figs. 7III and 8III). They presented decreasedexpression levels during ripening and no significant changes inresponse to the treatments.

4. Discussion

In this study, the results from two separate studies: involv-ing ethylene and 1-MCP treatment, have been combined in orderto elucidate the effect of fruit ripening and effect of ethylene onethylene biosynthetic and signal transduction pathways in applefruit. Although we noticed the remarkable increase in publicationson ripening and treatment effects on ethylene biosynthetic andperception mechanisms (Tatsuki and Endo, 2006; Tatsuki et al.,2009b; Zheng et al., 2007), the information for other ethylene sig-nal transduction elements at both the transcript and proteomiclevel is still limited. Recently, increased information on applegenomics allowed us to further explore ethylene biosynthesis andsignal transduction at the transcript level. Previous studies mainlyfocused on the changes of ethylene biosynthesis and perceptionsduring apple fruit development, and the comparison among differ-ent apple varieties or fruit species (Dal Cin et al., 2006; Johnstonet al., 2009; Wiersma et al., 2007). The study reported here inves-tigated the changes and regulation of ethylene biosynthesis andsignal transduction in apple fruit at the mRNA level with focus notonly on postharvest ripening but also on ethylene and 1-MCP treat-ment to gain a better understanding of the role of ethylene and themechanisms of ethylene perception and signal transduction.

ACS and ACO, the two key enzymes catalyzing the last steps of thebiosynthetic pathway of ethylene, have been isolated and exten-

sively studied in apple fruit (Costa et al., 2010; Harada et al., 2000;Huang et al., 2010; Pfaffl, 2001; Ramakers et al., 2003; Rosenfieldet al., 1996; Zheng et al., 2007). Both ACSs and ACOs in apple areencoded by multi-gene families. Two apple ACS genes have been

X. Yang et al. / Postharvest Biology and Technology 78 (2013) 55–66 59

ACS1

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B

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 after1-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 representsthe relative fold difference of mRNA level and was calculated using a modified 2−DDCt formula (Wan and Wilkins, 1994) with Mdactin and MdUBI as references. The valuespresented were obtained from 2 biological replicates and within each replicate, each data point is the mean value obtained from qPCR reaction performed in triplicate froma pooled sample of five 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 barsindicate 2× standard error of means.

60 X. Yang et al. / Postharvest Biology and Technology 78 (2013) 55–66

ETR1

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B

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-MCPtreatments. 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.

X. Yang et al. / Postharvest Biology and Technology 78 (2013) 55–66 61

CTR1-1

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B

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.

62 X. Yang et al. / Postharvest Biology and Technology 78 (2013) 55–66

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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 ripeningat 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 ofmeans.

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B

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 ◦Cafter 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.

X. Yang et al. / Postharvest Biology and Technology 78 (2013) 55–66 63

Fig. 7. Hierarchical cluster analysis (EPCLUST software) of transcript levels fromethylene genes differentially expressed during apple fruit ripening with or withoutethylene treatment. Red boxes mean higher levels of expression compared to Day0, and green boxes mean lower expression levels compared to Day 0. The colorbrightness is directly proportional to the expression ratio. Black boxes are genes notsignificantly differentially expressed compared to Day 0. (For interpretation of thert

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Fig. 8. Hierarchical cluster analysis (EPCLUST software) of transcript levels fromethylene genes differentially expressed during apple fruit ripening with or without1-MCP treatment. Red boxes mean high levels of expression compared to Day 0, andgreen boxes mean lower expression levels compared to Day 0. The color brightness isdirectly proportional to the expression ratio. Black boxes are genes not significantlydifferentially expressed as compared to Day 0. (For interpretation of the refer-

eferences to color in this figure legend, the reader is referred to the web version ofhe article.)

etected in the present study (Fig. 2A and B, ACS1 and ACS3). Theignificant increase in expression of ACS1 during fruit ripening ands the result of the ethylene treatment was pronounced. ACS1 andCO1 are correlated with the ethylene climacteric burst of apple

ruit. Treatment with 1-MCP generally produced effects oppositeo ethylene, which provides additional evidence that regulation ofhese genes coincided with ethylene production (Fig. 1A–C). It haseen shown that the mutation of the Md-ACS1 gene caused low levelf ethylene production in some apple cultivars, leading to extendedtorage life (Huang et al., 2010; Pfaffl, 2001). The expression of ACS3as reduced during ripening or ethylene treatment as compared toay 0 (Fig. 2A). The function of ACS3 is still unclear. Similar to ourresent study, it was reported that the expression of ACS3 did nothange upon ripening and 1-MCP treatment (Shibuya et al., 2004;atsuki et al., 2007; Wiersma et al., 2007). However, the relation-hip is not without controversy; others report a negative feedbackegulation mechanism where the expression of ACS3 was stimu-ated by 1-MCP treatment (Costa et al., 2010; Varanasi et al., 2011).he expression of ACS3 can also be influenced by different fruit vari-ties and developmental stages (Wiersma et al., 2007; Tatsuki et al.,007).

Three apple ACO genes were expressed differently throughout

he fruit ripening process (Fig. 2A and B). According to the expres-ion mode, which was closely correlated to ethylene production,CO1 in apple fruit may be one of the major factors of ethylene syn-hesis. This is supported by the recent report of the transgenic line

ences to color in this figure legend, the reader is referred to the web version of thearticle.)

of anti-ACO1, which shows the suppression of ACO1 results in lowlevels of ethylene (Schaffer et al., 2007). Information about the func-tion of ACO2 and ACO3 in apple are limited, but interest has focusedon 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 andACO2, and appeared to be expressed predominantly in young tissue,which is more likely to be a photosynthesis associated gene (Haradaet al., 2000). Therefore, it is possible that ACO3, which has beenseen in feedback inhibition to ethylene, is proposed to be responsi-ble for the system I ethylene response and less associated with theethylene burst upon the initiation of fruit ripening.

Overall, expression of five ethylene receptors genes (ETR1, ETR2,ETR5, ERS1 and ERS2) were induced at the transcriptional levelupon ripening and by ethylene treatment and decreased by 1-MCPtreatment during fruit ripening (Fig. 3A), although some of themhave been found to be constant in apple fruit and other fruit tis-sues (Dal Cin et al., 2006; El-Sharkawy et al., 2003; Kevany et al.,2007; Lashbrook et al., 1998; Martínez et al., 2001; Tatsuki et al.,2009a; Tieman and Klee, 1999; Wiersma et al., 2007). Ethylenereceptors are generally considered negative regulators of ethyleneresponse and, as shown in a number receptor mutants, a reductionof receptors appears to render high-ethylene sensitivity (Tiemanet al., 2000). This paradox has been seen in many fruit tissues wherethe transcription of these receptor mRNAs increased upon ripen-

ing, and the increased level of gene expression would be expectedto increase the protein level and in turn confer low-ethylene sen-sitivity. Tatsuki et al. (2009a) showed that in apple fruit, MdERS1

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4 X. Yang et al. / Postharvest Biolo

nd MdERS2 protein levels were not changed or decreased withthylene treatment, although the transcript levels were increased.ur results further showed that 1-MCP negatively influenced theene expression of receptors and these results coincided well withthylene production (Figs. 1 and 3). The delayed ethylene produc-ion may be directly caused by lack of an ethylene response dueo 1-MCP binding on the receptors, which is in agreement withhe findings by Costa et al. (2010). Stability of ethylene receptorroteins is also important. It has also been shown that the bind-

ng of ethylene induced the degradation of receptor proteins ofomato LeETR4 and LeETR6 (Kevany et al., 2007). It might be hypoth-sized that the binding of receptors with 1-MCP may actually haveecreased receptor protein degradation that was triggered by thethylene binding and therefore decreased the sensitivity of fruit tothylene signal.

In the ethylene signaling system, CTR1 plays a central role inegulating a negative ethylene response in the absence of ethyl-ne (Kieber, 1997). Although there has been one CTR1 identifiedn apple fruit so far, there are several splice variants existing aseported in ‘Sunrise’ and ‘Golden Delicious’ apple (Wiersma et al.,007). We therefore adopted and characterized the three differentorms separately as follows: CTR1–1, -2 (primers located betweenhe first intron and the start of the kinase domain), CTR1–3, -4 (thoseetain 3′UTR region), and CTR1–5 (the one located within kinaseomain). For all scenarios, there were no significant changes in thexpression of CTR1 during all the stages of postharvest ripening.owever, we noticed a significant expression level induction ofTR1–3, -4 and -5 in ethylene treated fruit and a decreased levelf all CTR1s in 1-MCP treated fruit (Fig. 4), indicating that simi-ar to LeCTR1 in tomato fruit (Adams-Phillips et al., 2004; Leclercqt al., 2002), apple CTR1 is ethylene inducible. It has been con-istently seen in fruits from the Rosaceae family (apple, pear andlum) that CTR1 expression is up-regulated in fruit during ripen-

ng (Dal Cin et al., 2006; El-Sharkawy et al., 2003, 2007). It wasnteresting to see that a negative regulator of ethylene response

as induced by ethylene during fruit ripening. The most logicalxplanation is that, in most tissues that produce large amounts ofthylene, the induction of the negative expression regulator mayct as a damping mechanism to response and temper the sud-enly increased ethylene concentration, in order to slow downhe ripening and senescence process (Klee, 2002). Since CTR1 isn important component in the complex of ethylene signal per-eption, and it interacts closely with ETR or ERS, it would be verynteresting to see concomitant changes of CTR1 expression at therotein level upon fruit ripening or upon ethylene treatment inpple fruit.

EIN2 has been proved acting as a bi-functional signal trans-ucer, which mediates the signal propagation between CTR1 andownstream components (EIN3/EIL). EIN2 mRNA is not altered inesponse to ethylene, in contrast, its protein is short-lived andubject to regulation by proteasomes in Arabidopsis (Alonso et al.,999). No significant change in expression of EIN2A and EIN2B wasound during ripening or in response to ethylene treatment in appleruit, (Fig. 5A and B). A controversy has been observed in manylant species, regarding the regulation of ethylene at the transcrip-ion level of EIN2 genes. In cut flower of carnation, expression ofcEIN2 was enhanced by treatment with ethylene, while in petu-ia, PhEIN2 was regulated by ethylene in a tissue-specific mannerShibuya et al., 2004), and in rice (Jun et al., 2004) and tomatoruit (Zhu et al., 2006), the expression of EIN2 was not regulatedy ethylene. An EIN-like gene was found, when mining in Gen-ank using the EST entry, that shares high similarity to EIL3 in

rabidopsis (80%), EIN3 in rose (91%), and PpEIL1 in peach (77%),ut was only 76%, 73% and 75% identical to MdEIL1, MdEIL2 anddEIL3 respectively, in Royal Gala apples (Tacken et al., 2010).

his gene was then designated as MdEIL4 and its gene expression

d Technology 78 (2013) 55–66

was tested during fruit ripening and under different treatments.According to our study, EIL4 in apple fruit tissue was ripeningand ethylene inducible (Fig. 5A), which is consistent with recentfindings 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). Inaddition, 1-MCP decreased the ethylene signaling and reduced theexpression level of EIL4 (Fig. 5B). In contrast to the effect on EIN2,ethylene acts to stabilize EIN3/EIL proteins by preventing degra-dation through the ubiquitin–proteasome pathway and furtheractivate downstream signaling (Guo and Ecker, 2003). It has beenproposed that EIN3/EIL are not regulated by ethylene in fruit butrather by ripening signals, since the expression levels were unaf-fected by ethylene in leaf tissue (Huang et al., 2010). EIN3/EILsare believed to be a positive regulator within the ethylene sig-nal cascade (Solano et al., 1998; Tieman et al., 2001). It was alsosuggested that EIN3/EILs may function differentially among differ-ent family genes or different tissues (Mbeguie-A-Mbeguie et al.,2008; Tacken et al., 2010; Yokotani et al., 2003). Since multi-ple EIL genes have been discovered in apple fruit, the significantregulation of ripening from other family genes cannot be ruledout.

ERF1 and ERF2 are expressed in apple fruit during ripening andinduced by ethylene treatment, while the expression of ERF1 wassignificantly reduced by 1-MCP treatment (Fig. 6A and B). Ourresults support the finding that 1-MCP inhibited the ERF1 trans-cripts as reported (Wang et al., 2007). However, the effect of 1-MCPon ERF2 was not significant, which may be caused by differences infruit maturities in these studies. It was proposed that another tran-scription factor(s) derived from the maturation cascade appearsto function as regulator of ERFs gene expression (Wang et al.,2007).

Despite the physiological variation between years, our geneexpression results collected over two years for each study indi-cate that changes in expression of ethylene related genes in applefruit coincide with ethylene regulated fruit ripening. Expressioncorrelated well with the process of ripening and in response to thetreatments by ethylene and by 1-MCP. The main objective was toexamine the effect of ethylene and or/1-MCP on gene expressionobtained from a short period study at 20 ◦C. Our current study pro-vides additional information on regulation of gene expression ofethylene biosynthesis, perception and signal transduction in appleduring fruit ripening or under the influence of ethylene or 1-MCPtreatment that may be helpful in understanding ethylene involve-ment in fruit ripening. These findings are based on gene expressiondata and not on protein expression or function. Our long-term goalwas a quantitative proteomic study developed and conducted usingthe same biological samples that will be reported in a subsequentpublication.

Acknowledgements

We thank Dr. Gordon Braun at AAFC for critical review. We thankthe MOE and AAFC for the PhD fellowship provided to X.T. Yang.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.postharvbio.2012.11.012.

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