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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
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 inﬂuences the 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 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 L−1 ethylene for 24 h or inhibited by 1-MCP treatment (1.0 L L−1 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 efﬁciency 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 signiﬁcantly 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 signiﬁcantly 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 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 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 signiﬁcant 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: firstname.lastname@example.org (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
. Tieman et al. with internal ethylene concentration 0. vegetables. MdETR2. there is still limited information on their abundance at the transcript level in apple fruit during fruit ripening (Wang et al.. 2006.2 L L−1 (ca.83 mL s−1 . Wiersma et al. For the 1MCP treatments. After treatments. perception. Lin et al. was used for real-time PCR analysis. 2001). One group was exposed to 1 L L−1 of 1-MCP (EthylBloc. Wiersma et al. has been recently used in the postharvest phase. ETR1 and ERS2) (Tatsuki et al. 10 mM DTT.1. with tomato as one of the important models. were determined on samples of ﬁve apples that were sealed in ﬁve 4 L glass jars with a consistent ﬂow rate of 0. 1999). as with the ethylene receptors. 39 and 43 for treated fruit. using micro array hybridization enabled transcriptome characterization. 7. and its signal transduction in apples during ripening and senescence and the inﬂuence of ethylene or 1-MCP on these changes.. MdERS2) (Dal Cin et al. CA) and with a 1. a powerful antagonist of ethylene at receptor binding sites. Yang et al. The quality and quantity of RNA were determined spectrophotometrically by measuring the OD260/280 and OD260/230 . 1993).. the control group was placed at 20 ◦ C for 24 h without any ethylene treatment. One milliliter samples of headspace gas were withdrawn and injected into a gas chromatograph equipped with a ﬂame ionization detector (Carle Instruments. Some ethylene perception elements in the downstream signal transduction pathway have also been investigated in multiple fruit species (Leclercq et al. 1993. Recently..3. 1% deoxycholic acid sodium salt. using fruit harvested in 2006 and 2007 at the same pre-climacteric stage and divided into two groups. Inc. respectively.. 1998). 13. 1998. Tieman et al. fruit were allowed to ripen in air at 20 ◦ C for 22 d. ACO and ETR were found to be ethylene dependent. Experiments involving treatment with 1MCP were conducted over two seasons. 1 week before commercial harvest). using oligo dT as a primer (5 M) and reverse transcriptase from a RETROscript Kit (Applied Biosystems.2. First-strand cDNA synthesis was performed on 2 g DNase I-treated total RNA.1–0. For the ethylene treatment.. 30 mM EGTA. 7. Sakai et al. PA) in sealed containers for 12 h. 2007). / Postharvest Biology and Technology 78 (2013) 55–66 that ethylene receptors act as negative regulators of the ethylene response. ﬁve fruit were randomly selected from each group. 2010). 2007). 2007. 2007). ‘Golden Delicious’) were harvested from a commercial orchard in Berwick. and 21 of each year was used for real-time PCR analysis. The study with ethylene treatment was conducted in the year 2004 and repeated in 2005. All RNA extracts were then treated with DNase I using a DNA-free Kit following the manufacturer’s recommendations (Applied Biosystems. experiments from two seasons with treatments of ethylene and 1-MCP. 1998. Tatsuki and Endo. 2% PVP 40. 1998. quickly frozen in liquid N2 . 2005. Tieman and Klee.. MdERS1. Quantiﬁcation was carried out by comparing the gas chromatography response of the samples with certiﬁed standards (Praxair Canada Inc. Total RNA extraction and cDNA synthesis Total RNA was extracted from frozen apple peel and ﬂesh tissue according to the hot borate method by Wan and Wilkins (1994) with some modiﬁcations in the extraction buffer. ethylene concentrations were determined on 14 apples (Wakasa et al. EIN2 then relays the ethylene signal to the transcription factors Ethylene-Insensitive 3 (EIN3) and Ethylene-Insensitive 3 like (EILs). 14. 2. 7. Respiration and ethylene production Respiration rates. 1-Methylcyclopropene (1-MCP). Apple fruit treated with 1-MCP showed the delay of mRNA transcript of some ethylene receptors (ERS1.. USA) as per the manufacturer’s instructions. 2000).58 mL s−1 of fresh air and equilibrated overnight at 20 ◦ C.. Ethylene production was determined on samples of ﬁve apples that were sealed in ﬁve 4 L glass jars with a constant ﬂow rate of 0. 2007.58 mL s−1 of fresh air and equilibrated overnight at 20 ◦ C. for the measurement of ethylene production. The concentration of cDNA used for real-time qPCR was measured and each sample was diluted to 0.. and stored immediately at −85 ◦ C until further use. 2008. 1998). At each sampling time. 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. 2.2 mm (o.). USA).d.9 mm × 3. 2000). One group was placed in glass chambers and ethylene treatment was carried out by ventilating with ethylene gas at 36 L L−1 for 24 h at 20 ◦ C to initiate ripening. Materials and methods 2. 1999. MdETR5. 1% Nonidet P-40 (octyl phenoxypolyethoxylethanol). 2008. 22. In the present work.0)... as both were up-regulated during ripening and repressed by 1-MCP (Costa et al. Philadelphia.14%. and ﬂowers. which contained 200 mM sodium tetraborate decahydrate.. Constitutive triple response 1 (CTR1) is a key regulator of ethylene responses acting downstream of ethylene receptors (Kieber et al. ground tissue from days 0... Nova Scotia before the climacteric stage. 14 and 22 for control fruit and days 0. 2.) activated alumina column with a helium carrier ﬂow of 0. The application of 1-MCP extends the freshness of various fruits.. Six receptor gene homologs have been reported in tomato (LeETR1–6) (Lashbrook et al. but only inhibited the CTR1 transcript in a late cultivar (El-Sharkawy et al. is a negative regulator of the ethylene response by repressing the positive regulator EthyleneInsensitive 2 (EIN2) (Alonso et al. Plant materials. Rohm and Haas Company. prolongs shelf life and delays ripening and senescence. Li and Yuan. 2006). Controls with no RETROscript reverse transcriptase (NRT) were used to determine the potential genomic DNA contamination. It has been shown to be a Raf-like Ser/Thr protein kinase (MAPKK kinase) and. Application of 1-MCP on plum fruit down-regulated the ETR1 and CTR1 mRNA levels in an early cultivar. Lee et al.56 X.. 2002. The ripening of harvested apples is prevented or delayed by 1-MCP treatment (Bleecker and Kende.. These important ethylene perception elements have been studied in a number of fruit species.248 g L−1 with Tris–EDTA buffer (pH 8. genes involved in ethylene biosynthesis and signaling.. which in turn activates the ethylene response factor 1 (ERF1) transcription factor. 2006). RNA integrity was assessed by visual inspection after electrophoresis on a formaldehyde agarose gel in the presence of ethidium bromide. the control group was held at 20 ◦ C for 12 h without any 1-MCP treatment. The results provide further insight into the changes in ethylene biosynthesis. grounded to a powder.. USA). The ERF protein functions as trans-factors at the last step of transduction in the nucleus (Solano et al. Internal . as well as ﬁve in apple fruit (MdETR1. Anaheim. and that ethylene binding inactivates them (Hua and Meyerowitz. 2009a). however. 0. 1-MCP can also inhibit apple ethylene production by inhibiting the expression of the gene for ethylene biosynthesis (Kieber et al. tissue from days 0. measured as CO2 production. treatments and storage Apple fruit (Malus domestica Borkh. CO2 production expressed as g kg−1 s−1 was directly measured by an infrared CO2 analyzer (Li-CO 625. Besides preventing ethylene-dependent responses by binding with ethylene receptors.
Among the ethylene biosynthesis genes. The 1-MCP experiment (Study 2) was a combination of 2 replicates for two years (2006 and 2007). Each analysis included a polynomial contrast to evaluate the change across the stages for 1-MCP.. respectively. 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. and an overlap of control and 1-MCP. cycling conditions included an initial hot start at 95 ◦ C for 10 min. orthogonal contrast was used for the difference between control vs. as well as decreased the amount of ethylene production (Fig. 3B). 2.bioinf. ACO3 and ACS3. ETR5. Raw quantiﬁcation cycles (Cq ) were converted to quantities representing relative expression levels using a modiﬁed comparative cycle threshold method (Fan et al. The expression of two CTR genes (CTR1–3 and 1–5) was increased signiﬁcantly by ethylene treatment.5. 2). ampliﬁcation 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.5 L MaximaTM SYBR Green QPCR Master Mix (Fermentas. 3.3. These were analyzed in a randomized block model using ANOVA but the treatments were grouped as just control. 3. 2007) or designed within the gene coding region of 3 UTR (Supplementary Table S1). control and the interaction for the ﬁrst 3 stages. 4B). ETR5. Wang et al.000 and 510 times higher. Ampliﬁcation products were separated on 1% agarose by gel electrophoresis and analyzed with the PharosFX Molecular Imager (Bio-Rad. 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. Little changed in ACO2. 2008. USA). ERS1 and ERS2 were all reduced signiﬁcantly by 1-MCP treatment (Fig. Gene expression of EIN2 members showed little changes during fruit ripening (Fig. MdActin and MdUBI were analyzed in each real-time qPCR to normalize the expression patterns. Signiﬁcant increase of ACS1 and ACO1 gene expression levels were found during fruit ripening. than the expression levels in the controls (Fig. At 20 ◦ C. 3A). Expression of ETR1. among the ethylene perception genes. various annealing temperatures (50–65 ◦ C) and cDNA concentration. 3–6). 5A). For each dilution. Each real-time PCR was ended by the addition of a dissociation curve analysis of the ampliﬁed product. Ethylene treatment accelerated the unset of fruit respiration by at least 7 d (Fig.4. ACS3 and ACO3 expression during the early period of post-treatment with ethylene. However. ETR2. 1-MCP treatment resulted in delayed the respiration and ethylene climacteric peaks by 20 d as compared with control.ee/EP/EP/EPCLUST/) allowed analysis of clustering genes according to their expression proﬁle. Canada). For all genes. For measurement of primer efﬁciency. 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. ACS1 and ACO1 were signiﬁcantly inhibited by 1-MCP (Fig.5 L 10 mM primers and 12. 2007. A complete . USA) with regard to forward and reverse primers. 1994). Expression of ACO2 was not signiﬁcantly changed. Optimized results were used on further RT-qPCR analysis for all genes. 2011) and with correction for different ampliﬁcation efﬁciencies (Wan and Wilkins.. Expression of ERS2 increased signiﬁcantly during fruit ripening. 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. A signiﬁcant inhibitive effect of 1-MCP on CTRs genes was also observed in this study (Fig. the strong treatment effect of 1-MCP on apple ethylene production and perception was also observed. 1C and D). linear and quadratic response across control and linear and quadratic response across ethylene. The efﬁciency value for each gene is shown in Supplementary Table S2. Wiersma et al. Little change in expression of other CTRs genes was found during fruit ripening and in response to ethylene treatment (Fig. biological measurements including fruit respiration and ethylene production were conducted. as compared with the control (Fig. 2007). which was 56.2. Only genes with primer efﬁciency above 80% were further used. USA) and Stratagene MX3005p (Agilent. The expression of EIL4 increased slightly.. just 1-MCP. In these methods. 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. Real-time qPCR analysis Real-time qPCR of 25 L reactions were repeated two times on a Stratagene MX3005p (Angilent. EPCLUST software (http://www. To evaluate treatment differences. 2. 4A). 1. linkage hierarchical clustering (Euclidean distance) was conducted for both experiments. and ERS2 was induced signiﬁcantly by ethylene treatment (Fig.1.6.. Fruit harvested at the pre-climacteric stage was conﬁrmed by low internal ethylene concentration (<0. 1-MCP treatment had no effect on ACO2. Normalization factors were calculated by taking the geometric mean of the two reference genes (Espley et al. 2002. 3. involving denaturation at 95 ◦ C for 1 min.. Ethylene treatment accelerated the onset of ethylene production and the climacteric peak. samples from Day 0 (assigned an arbitrary quantity of “1”) were used as a calibrator to calculate the relative quantity of the results. While. 2B). ethylene. 1).ebc. expression of ACS3 and ACO3 was decreased as compared with Day 0 (Fig. USA) using 1 L of dilute cDNA. 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. / Postharvest Biology and Technology 78 (2013) 55–66 57 2. typical climacteric peaks in respiration were shown around Day 20. and signiﬁcantly increased the amount of ethylene production (Fig. Conditions for all PCRs were optimized in a Eppendorf Mastercyler EP gradient PCR Thermocycler (Eppendorf.X. cooling to 55 ◦ C for 30 s and then gradual heating at 1 ◦ C per cycle to a ﬁnal temperature of 95 ◦ C. Vandesompele et al. 2A). In contrast. followed by 40 cycles at 95 ◦ C for 30 s. ETR1. 2A). 2A). Two reference genes. This revealed physiological changes associated with climacteric changes of fruit ripening and response to ethylene and 1-MCP treatment (Fig. Ethylene treatment induced the gene expression of ACS1 and ACO1 to 66. 1-MCP treatment signiﬁcantly delayed the onset of fruit respiration. 1A). In this study. 1B).000 and 430 times higher than the expression levels on Day 0 (Fig.1 L/L). Yang et al. Ethylene receptor gene ETR1 showed little change during fruit ripening. 58 ◦ C to 60 ◦ C for 1 min and 72 ◦ C for 1 min. Results 3. a qPCR was run in triplicate.
Tatsuki et al.5 1. the two key enzymes catalyzing the last steps of the biosynthetic pathway of ethylene. Cluster I included ACS1 and ACO1. increased information on apple genomics allowed us to further explore ethylene biosynthesis and signal transduction at the transcript level. 2009. 7II and 8II). Gene expression of two ethylene signal transcription factors. n=8 40 50 60 Days after treatment Days after treatment Ethylene production ( nmol kg-1 s-1) CO2 production (µg kg-1 s-1) 14 12 10 8 6 4 2 0 0 10 20 Control C MCP 2 x SEM Control.. 2003. 5A). 1-MCP treatment seemed to inhibit the gene expression of EIN2A and EIL4. The values presented were obtained from 2 biological replicates and within each replicate. n=10 2. Wiersma et al. Discussion In this study.. n=10 30 40 50 60 30 40 50 60 Days after treatment Days after treatment Fig. (B) and (D) were modiﬁed from Yang et al. 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. Ethylene treatment remarkably induced cluster I gene expressions and 1MCP treatment depressed cluster I gene expressions during the early period of post-treatment.58 X.0 1. Ramakers et al..5 2. Huang et al. ethylene increased the expression of EIL4 (Fig. They presented decreased expression levels during ripening and no signiﬁcant changes in response to the treatments. (A) Respiration after ethylene treatment. 1. Pfafﬂ.. 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. 1996.. (C) respiration after 1-MCP treatment.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. Similar to the effect on EIN2A. ERFs and downstream ripening events. as well as ethylene signal transcription factors ERFs (Figs. 4. (D) ethylene production after 1-MCP treatment. 2007). 3. 6B). ethylene production and perception genes have been grouped using hierarchical and Kmeans clustering. have been isolated and extensively studied in apple fruit (Costa et al. Cluster II contained six genes ranging from ethylene biosynthesis gene ACO2.4. Rosenﬁeld et al. the results from two separate studies: involving ethylene and 1-MCP treatment. 2010. 5B).5 0. n=8 B Control Ethylene 2 x SEM. and the comparison among different apple varieties or fruit species (Dal Cin et al. in addition..5 2. 2006.0 1. 2009b. ERSs and ETR2. Gene expression of Cluster II was depressed by 1-MCP treatment compared to Day 0 samples at the early stage of post-treatment. / Postharvest Biology and Technology 78 (2013) 55–66 Ethylene production ( nmol kg-1 s-1) CO2 production (µg kg-1 s-1) 2.. 2006. Recently. ACS and ACO. Respiration and ethylene production of apple fruit during ripening and in response to ethylene or 1-MCP treatment at harvest.. n=10 2 x SEM MCP. 7 and 8). (2012) with permission. Zheng et al. ethylene treatment induced gene expressions compared to Day 0 and fruit without treatment.. ERF1 and ERF2. Yang et al. (B) ethylene production after ethylene treatment. the information for other ethylene signal transduction elements at both the transcript and proteomic level is still limited. 2000. 7III and 8III). 6A). While. n=10 2 x SEM MCP. 2007).0 0 10 20 Control D MCP 2 x SEM Control. Zheng et al... Thirteen genes studied in the present study were grouped in Cluster III (Figs. were induced signiﬁcantly upon ripening and increased during the late stage of fruit ripening (Fig. Two apple ACS genes have been .5 1.0 . 5A). ethylene treatment slightly increased the gene expression of EIN2A at 13 d during fruit ripening. 7I and 8I). it had no effect on gene expression of EIN2B (Fig. Gene expression of ERF1 was inhibited signiﬁcantly by 1-MCP treatment (Fig. Both ACSs and ACOs in apple are encoded by multi-gene families. Harada et al. Hierarchical clustering analysis of differentially expressed genes Based on the gene expression proﬁles.5 0.0 . 2007). each data point is the mean value of ﬁve fruit. 2010. which showed signiﬁcantly increased expression levels during fruit ripening (Figs. Previous studies mainly focused on the changes of ethylene biosynthesis and perceptions during apple fruit development. 2001. These genes showed increased expression levels upon fruit ripening. indicating a signiﬁcant relationship between ethylene signal. Johnston et al. while it had no effect on EIN2B gene expression (Fig. Three different clusters were deﬁned for both the ethylene and 1-MCP treatment study (Figs. ethylene signal receptors. Although we noticed the remarkable increase in publications on ripening and treatment effects on ethylene biosynthetic and perception mechanisms (Tatsuki and Endo.
006 1. Expressions of apple ethylene biosynthesis genes. The values presented were obtained from 2 biological replicates and within each replicate.2 0.6 . p < 0.8 .4 .2 0.2 ACS3 1.6 .4 90000 60000 30000 . p < 0. Quantitative real-time PCR was used to analyze the mRNA changes as described in Section 2.8 0 ACO3 7 13 21 Days after Treatment .5 1. 1994) with Mdactin and MdUBI as references.0 .8 . Yang et al.4 .023 ACO2 4 3 9000 6000 3000 0 1. during 21 d of ripening at 20 ◦ C after ethylene treatment and 43 d of ripening at 20 ◦ C after 1-MCP treatments.0 ACS3 Relative Expression Ratio 0 15000 12000 ACO1 Linea r Stage MCP. Sampling details are labeled in Fig.001 Control vs MCP.0 7 13 21 Days after Treatment B 56000 48000 40000 32000 24000 16000 8000 ACS1 Control MCP Linea r Stage MCP.001 Linear Control. 1.019 2. p = 0. each data point is the mean value obtained from qPCR reaction performed in triplicate from a pooled sample of ﬁve fruit.008 Control vs MCP.2 1. p = 0.5 2. / Postharvest Biology and Technology 78 (2013) 55–66 59 A 120000 ACS1 Control Ethylene Control vs Ethylene. MdACOs and MdACSs. 2. p = 0.5 0.0 7 14 22 39 43 2 1 0 ACO3 7 14 22 39 43 Days after Treatment Days after Treatment Fig. p = 0.001 0. p < 0. .0 1. p = 0. Error bars indicate 2× standard error of means.2 Relative Expression Ratio 0 ACO1 750 Control vs Ethylene.6 . Samples from Day 0 (assigned an arbitrary quantity of “1”) were used as a calibrator to calculate the relative quantity of the results.001 Quad Control.0 .X.0 4 ACO2 3 500 2 250 1 0 .0 . The y axis represents the relative fold difference of mRNA level and was calculated using a modiﬁed 2−DDCt formula (Wan and Wilkins.
0 Control vs Ethylene.0 1. p = 0. p = 0.005 6 ERS1 4 2 .014 7 13 21 Days after Treatment 4 2 0 7 13 21 Days after Treatment B 2. p = 0. Yang et al.0 0 ERS2 6 Control vs Ethylene.5 Relative Expression Ratio 0.012 4 2 Relative Expression Ratio 0.0 ETR1 Control Ethylene Control vs Ethylene. p = 0. p = 0. p = 0.0 2.017 6 ETR2 1.03 8 6 4 ERS1 Control vs MCP.0 . Error bars indicate 2× standard error of means. ETR and ERS. p = 0. 2. 1. during 21 d of ripening at 20 ◦ C after ethylene treatment and 43 d of ripening at 20 ◦ C after 1-MCP treatments.5 1.5 2.0 .0 1.046 7 14 22 39 43 Days after Treatment 6 4 2 0 7 14 22 39 43 Days after Treatment Fig. Expressions of apple ethylene signal receptors genes.5 2.5 1.049 Control vs MCP.5 0. p = 0.5 4 1.038 6 ETR2 Control vs MCP.5 0 ETR5 Linea r Stage control. 3.5 ETR1 Control MCP Control vs MCP.5 0. p = 0.018 Control vs MCP. / Postharvest Biology and Technology 78 (2013) 55–66 A 2.0 2 .0 0 ETR5 2.5 2.0 8 2 0 ERS2 Linea r Stage control.027 1. Quantitative real-time PCR details are as described in Fig. .0 1.60 X. p = 0. p < 0. Sampling details are labeled in Fig.001 Linea r Control.
during 21 d of ripening at 20 ◦ C after ethylene treatment or 43 d of ripening at 20 ◦ C after 1-MCP treatments.006 CTR1-4 1.0 .0 CTR1-3 1. p = 0.047 Control vs MCP.0 1.5 Quad Stage MCP.0 0. p = 0.0 0.0 CTR1-3 1.0 7 13 21 Days after Treatment B 1.0 .0 1.5 Control vs MCP.5 . 1.5 0. p = 0.5 .5 Quad Stage MCP.5 Control vs Ethylene.0 . p = 0.5 Relative Expression Ratio 0.X.0 . p = 0. p = 0.022 1. 4.5 0.035 Linea r Stage MCP.5 .0 CTR1-5 1. Quantitative real-time PCR details are as described in Fig. p = 0.039 7 13 21 Days after Treatment 1.022 1.5 Control vs Ethylene. 2.011 CTR1-2 1.04 CTR1-4 1.5 . Error bars indicate 2× standard error of means. Yang et al.5 0.0 . . p = 0.0 CTR1-5 1.5 Control vs Ethylene.014 7 14 22 39 43 Days after Treatment 1.0 0.006 1.5 Relative Expression Ratio 0. p = 0.0 0. Expressions of apple ethylene signal receptors CTR1s genes.0 1.0 1.5 CTR1-2 1.5 Control vs MCP. p = 0.0 7 14 22 39 43 Days after Treatment Fig.5 0.0 .002 Linea r Ethylene. p = 0. p = 0.5 CTR1-1 Control MCP Linea r Stage MCP.5 CTR1-1 Control Ethylene 1. p = 0.025 Control vs MCP.028 Control vs MCP. Sampling details are labeled in Fig. / Postharvest Biology and Technology 78 (2013) 55–66 61 A 1.
p = 0. p =0.5 1. 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. Quantitative real-time PCR details are as described in Fig.62 X. Error bars indicate 2× standard error of means.012 6 5 4 ERF2 Control vs Ethylene.0 Relative Expression Ratio 0.018 EIL4 Control vs Ethylene.4 .046 EIL4 Linear Stage MCP.0 .5 .5 1.0 EIN2A Control Ethylene B 2. p = 0.0 1. p = 0. during 21 d of ripening at 20 ◦ C after ethylene treatment and 43 d of ripening at 20 ◦ C after 1-MCP treatment.024 1.0 2.05 Linear Control. / Postharvest Biology and Technology 78 (2013) 55–66 A 2 2.0 7 14 22 39 43 Days after Treatment Days after Treatment Fig. 1. Relative Expression Ratio A ERF1 6 Control Ethylene Control vs Ethylene.0 7 13 21 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 B 8 ERF1 Control MCP Linear Stage MCP. p = 0.5 Relative Expression Ratio 0. 5. 6.8 .8 .018 8 6 6 4 4 2 2 0 7 14 22 39 43 0 7 14 22 39 43 Days after Treatment Days after Treatment Fig. EIN2A.6 . Expressions of apple ethylene signal transcription factor genes.0 1. ERF1 and ERF2. Quantitative real-time PCR details are as described in Fig. 1.4 .6 .2 1. .0 1.5 1. Sampling details are labeled in Fig. p = 0. p = 0. 2.013 Control vs MCP.003 Linear Stage Control.5 2. p =0. Yang et al.0 EIN2A Ctrl MCP Linear Stage MCP.0 1.0 .5 .4 1.032 1.5 0.5 1. Sampling details are labeled in Fig. 2.0 2. p =0. Expressions of apple ethylene signal transduction component genes. p = 0.2 0.2 0.0 .0 EIN2B EIN2B Linear Stage Control.0 . Error bars indicate 2× standard error of means.
This paradox has been seen in many fruit tissues where the transcription of these receptor mRNAs increased upon ripening. ETR5. Tieman and Klee. ACS1 and ACO1 are correlated with the ethylene climacteric burst of apple fruit. 2007). 2000).. ACS1 and ACS3). the reader is referred to the web version of the article. and appeared to be expressed predominantly in young tissue. 2010. but interest has focused on ACO3 since it was negatively regulated by ethylene (Fig. 2007). and the increased level of gene expression would be expected to increase the protein level and in turn confer low-ethylene sensitivity. Three apple ACO genes were expressed differently throughout the fruit ripening process (Fig. 3A).) Fig.. 2011).) detected in the present study (Fig. Black boxes are genes not signiﬁcantly differentially expressed compared to Day 0. 1A–C). Kevany et al. Therefore. it is possible that ACO3.. Varanasi et al.. Ethylene receptors are generally considered negative regulators of ethylene response and. 2007. Tatsuki et al. 2010. Wiersma et al. Tatsuki et al. 1998. 2007). Martínez et al. which shows the suppression of ACO1 results in low levels of ethylene (Schaffer et al.. Similar to our present study. 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. According to the expression mode. 2001). This is supported by the recent report of the transgenic line of anti-ACO1. others report a negative feedback regulation mechanism where the expression of ACS3 was stimulated by 1-MCP treatment (Costa et al. El-Sharkawy et al. Red boxes mean high levels of expression compared to Day 0. / Postharvest Biology and Technology 78 (2013) 55–66 63 Fig. Hierarchical cluster analysis (EPCLUST software) of transcript levels from ethylene genes differentially expressed during apple fruit ripening with or without ethylene treatment. and green boxes mean lower expression levels compared to Day 0.. the relationship is not without controversy.. The signiﬁcant increase in expression of ACS1 during fruit ripening and as the result of the ethylene treatment was pronounced. MdERS1 . Hierarchical cluster analysis (EPCLUST software) of transcript levels from ethylene genes differentially expressed during apple fruit ripening with or without 1-MCP treatment.. Overall. leading to extended storage life (Huang et al. Tatsuki et al. The expression of ACS3 can also be inﬂuenced by different fruit varieties and developmental stages (Wiersma et al. which provides additional evidence that regulation of these genes coincided with ethylene production (Fig. a reduction of receptors appears to render high-ethylene sensitivity (Tieman et al. The function of ACS3 is still unclear. ETR2. Treatment with 1-MCP generally produced effects opposite to ethylene. the reader is referred to the web version of the article. 2007). (For interpretation of the references to color in this ﬁgure legend. which was closely correlated to ethylene production. 2007. 2006.. Tatsuki et al. as shown in a number receptor mutants. it was reported that the expression of ACS3 did not change upon ripening and 1-MCP treatment (Shibuya et al. Information about the function of ACO2 and ACO3 in apple are limited. although some of them have been found to be constant in apple fruit and other fruit tissues (Dal Cin et al. 2007. Yang et al.. Black boxes are genes not signiﬁcantly differentially expressed as compared to Day 0. Pfafﬂ. 1999. (For interpretation of the references to color in this ﬁgure legend. ACO1 in apple fruit may be one of the major factors of ethylene synthesis. that was also observed in ‘Sunrise’ apple (Wiersma et al.. 7. expression of ﬁve ethylene receptors genes (ETR1. Wiersma et al. (2009a) showed that in apple fruit... which is more likely to be a photosynthesis associated gene (Harada et al. 2A). The color brightness is directly proportional to the expression ratio... Phylogenetic analysis showed that ACO3 is distinct from ACO1 and ACO2.. Lashbrook et al.. However. Red boxes mean higher levels of expression compared to Day 0. 2000). and green boxes mean lower expression levels compared to Day 0. The color brightness is directly proportional to the expression ratio. 2001. is proposed to be responsible for the system I ethylene response and less associated with the ethylene burst upon the initiation of fruit ripening. It has been shown that the mutation of the Md-ACS1 gene caused low level of ethylene production in some apple cultivars. 2004. 2A).X. 2007)... 2A and B. 8. 2A and B). 2009a. The expression of ACS3 was reduced during ripening or ethylene treatment as compared to Day 0 (Fig. 2003. which has been seen in feedback inhibition to ethylene.
. which is consistent with recent ﬁndings in other fruit systems. Gordon Braun at AAFC for critical review. 2003). 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. 5A and B).. However. Yang et al. 4). (Fig. This gene was then designated as MdEIL4 and its gene expression was tested during fruit ripening and under different treatments. 2008. since the expression levels were unaffected by ethylene in leaf tissue (Huang et al. EIL4 in apple fruit tissue was ripening and ethylene inducible (Fig. 2002). Barry. at http://dx. we noticed a signiﬁcant expression level induction of CTR1–3. 1-MCP decreased the ethylene signaling and reduced the expression level of EIL4 (Fig. P. -2 (primers located between the ﬁrst intron and the start of the kinase domain). EIN2 mRNA is not altered in response to ethylene. pear and plum) that CTR1 expression is up-regulated in fruit during ripening (Dal Cin et al.. apple CTR1 is ethylene inducible.. Acknowledgements We thank Dr.. but was only 76%. 2003). 2007).. and in rice (Jun et al.. EIN3 in rose (91%). 2008. It was also suggested that EIN3/EILs may function differentially among different family genes or different tissues (Mbeguie-A-Mbeguie et al. 2007). 2004. Supplementary data Supplementary data associated with this article can be found. Expression correlated well with the process of ripening and in response to the treatments by ethylene and by 1-MCP. M. The most logical explanation is that... -4 (those retain 3 UTR region). It has also been shown that the binding of ethylene induced the degradation of receptor proteins of tomato LeETR4 and LeETR6 (Kevany et al. while the expression of ERF1 was signiﬁcantly reduced by 1-MCP treatment (Fig. We therefore adopted and characterized the three different forms separately as follows: CTR1–1. 2010. 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.. 2007). there were no signiﬁcant changes in the expression of CTR1 during all the stages of postharvest ripening. 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. that shares high similarity to EIL3 in Arabidopsis (80%). It has been proposed that EIN3/EIL are not regulated by ethylene in fruit but rather by ripening signals. the expression of EIN2 was not regulated by ethylene. EIN3/EILs are believed to be a positive regulator within the ethylene signal cascade (Solano et al. the effect of 1-MCP on ERF2 was not signiﬁcant. in the online version. when mining in Genbank using the EST entry. Tacken et al. in most tissues that produce large amounts of ethylene. We thank the MOE and AAFC for the PhD fellowship provided to X. 1999). However. El-Sharkawy et al. -4 and -5 in ethylene treated fruit and a decreased level of all CTR1s in 1-MCP treated fruit (Fig. J. 2010. 1 and 3). (2010). the signiﬁcant 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. 2001). 2003). In contrast to the effect on EIN2. and it interacts closely with ETR or ERS. In the ethylene signaling system. Appendix A. and PpEIL1 in peach (77%). 73% and 75% identical to MdEIL1.doi. 2004) and tomato fruit (Zhu et al. Stability of ethylene receptor proteins is also important. regarding the regulation of ethylene at the transcription level of EIN2 genes. Yokotani et al. Leclercq.012. Since CTR1 is an important component in the complex of ethylene signal perception. there are several splice variants existing as reported in ‘Sunrise’ and ‘Golden Delicious’ apple (Wiersma et al.. PhEIN2 was regulated by ethylene in a tissue-speciﬁc manner (Shibuya et al.. In cut ﬂower of carnation.64 X. and CTR1–5 (the one located within kinase domain). 2004. banana (Ma-EIL2/AB266318). 5A). 6A and B). Our current study provides additional information on regulation of gene expression of ethylene biosynthesis. No signiﬁcant change in expression of EIN2A and EIN2B was found during ripening or in response to ethylene treatment in apple fruit. indicating that similar to LeCTR1 in tomato fruit (Adams-Phillips et al. in contrast. in Royal Gala apples (Tacken et al. 2010). 2010)... References Adams-Phillips.. Although there has been one CTR1 identiﬁed in apple fruit so far.. An EIN-like gene was found. which mediates the signal propagation between CTR1 and downstream components (EIN3/EIL). 2006).. the induction of the negative expression regulator may act as a damping mechanism to response and temper the suddenly increased ethylene concentration. L. Yokotani et al. Giovannoni.. The delayed ethylene production may be directly caused by lack of an ethylene response due to 1-MCP binding on the receptors. 2004). 1997). Tieman et al. Bouzayen. J.. Kannan. 5B). 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. CTR1–3. It has been consistently seen in fruits from the Rosaceae family (apple. although the transcript levels were increased..T. CTR1 plays a central role in regulating a negative ethylene response in the absence of ethylene (Kieber.. which is in agreement with the ﬁndings by Costa et al..2012. and melon (CmEIL1 and CmEIL2) (Huang et al.1016/ j. 2002). A controversy has been observed in many plant species. Yang... ethylene acts to stabilize EIN3/EIL proteins by preventing degradation through the ubiquitin–proteasome pathway and further activate downstream signaling (Guo and Ecker. C. Leclercq et al. expression of DcEIN2 was enhanced by treatment with ethylene. In addition. Our results further showed that 1-MCP negatively inﬂuenced the gene expression of receptors and these results coincided well with ethylene production (Figs.org/10. It was interesting to see that a negative regulator of ethylene response was induced by ethylene during fruit ripening. MdEIL2 and MdEIL3 respectively. 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.postharvbio.. Evidence that CTR1-mediated ethylene signal transduction in tomato is . Mbeguie-A-Mbeguie et al. perception and signal transduction in apple during fruit ripening or under the inﬂuence of ethylene or 1-MCP treatment that may be helpful in understanding ethylene involvement in fruit ripening. / Postharvest Biology and Technology 78 (2013) 55–66 and MdERS2 protein levels were not changed or decreased with ethylene treatment. while in petunia.. 2003. EIN2 has been proved acting as a bi-functional signal transducer. 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. Since multiple EIL genes have been discovered in apple fruit. According to our study.11. such as tomato (LeEIL4). 1998. its protein is short-lived and subject to regulation by proteasomes in Arabidopsis (Alonso et al. 2007). in order to slow down the ripening and senescence process (Klee.. For all scenarios. Despite the physiological variation between years. 2007). These ﬁndings are based on gene expression data and not on protein expression or function. Our results support the ﬁnding that 1-MCP inhibited the ERF1 transcripts as reported (Wang et al. 2006..
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