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Effects of methyl jasmonate on expression of genes involved in ethylene biosynthesis and signaling pathway during postharvest ripening of apple fruit
Scientia Horticulturae 229 (2018) 157–166
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Research Paper
Effects of methyl jasmonate on expression of genes involved in ethylene biosynthesis and signaling pathway during postharvest ripening of apple fruit ⁎,1
Jingyi Lv
MARK
⁎
, Mengyuan Zhang1, Junhu Zhang, Yonghong Ge, Canying Li, Kun Meng, Jianrong Li
College of Food Science and Project Engineering, Bohai University; National & Local Joint Engineering Research Center of Storage, Processing and Safety Control Technology for Fresh Agricultural and Aquatic Products, Bohai University, Jinzhou, Liaoning 121013, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Apple fruit Ethylene biosynthesis Signal transduction Jasmonates Fruit ripening Gene expression
Ethylene plays a key regulatory role in the ripening of climacteric fruit. In addition to ethylene, jasmonates (JAs) have also been demonstrated to play a role in the regulation of the fruit ripening. Apple (Malus × domestica Borkh.) fruit is a common climacteric fruit. Research has been conducted to illustrate the effects of JAs on ethylene production in apple fruit, but little is known about the molecular mechanisms underlying the role of JAs in ethylene biosynthesis and signaling pathway during the ripening of apple fruit. To better understand the effects of JAs on the expression of key genes involved in the ethylene biosynthesis and signaling pathway during postharvest ripening of apple fruit, apples harvested at commercial maturity were treated with methyl jasmonate (MeJA). Our data indicated that MeJA treatment increased ethylene production during fruit ripening. The expression of MdACS1, MdACS6, MdETR1, MdCTR1-3, MdCTR1-4, MdCTR1-5, MdEIN2A, MdEIN2B, MdEIL4 and MdERF1 was positively regulated by MeJA treatment at the early ripening stage, whereas the expression of MdEIL3 was positively regulated by it at the late ripening stage. MeJA treatment enhanced the expression of MdACS3a, MdACS8, MdACO1, MdACO2, MdETR2, MdERS1, MdERS2 and MdEIL1 during the entire storage period, whereas it had no effect on the expression of MdCTR1-1, MdCTR1-2 and MdEIL2 during fruit ripening. The expression of MdERF2 and MdACS1 was negatively by MeJA treatment during the period when the ethylene peak existed. These results indicated that the expression of genes involved in the ethylene biosynthesis and signaling pathway was differentially regulated by JAs during postharvest ripening of apple fruit.
1. Introduction The gaseous phytohormone ethylene plays critical roles in the regulation of many plant physiological processes, including seed germination, seedling growth, organ abscission, fruit ripening, and senescence (Abeles et al., 1992; Tatsuki, 2010). Apple fruit is a climacteric fruit, whose ripening is characterized by an exponential increase in ethylene production and respiration rate (Varanasi et al., 2013; Yang and Hoffman, 1984). Studies have confirmed that ethylene plays a key regulatory role in the ripening of apple fruit, such as flesh softening (Ireland et al., 2014), volatile biosynthesis (Yang et al., 2016) and anthocyanin accumulation (Whale and Singh, 2007). Biosynthesis of ethylene begins with methionine, which is converted to S-adenosyl-Lmethionine (SAM) by SAM synthetase. SAM is then catalyzed by 1aminocyclopropane-1-carboxylate (ACC) synthase (ACS) to produce ACC, which can be further converted to ethylene by ACC oxidase (ACO)
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(Adams and Yang, 1979). The formation of ACC is generally considered to be the rate-limiting step in the biosynthesis of ethylene (Kende, 1993). In apple, five ACS genes and two ACO genes have been found to be related to fruit ripening, including MdACS1, MdACS3a, MdACS6, MdACS7, MdACS8, MdACO1 and MdACO2 (Dal Cin et al., 2007; Li et al., 2013; Wiersma et al., 2007). After synthesis, ethylene is perceived by receptors that act as negative regulators of ethylene responses in plants (Kevany et al., 2007). Binding of ethylene by receptors inhibits the activity of constitutive triple response 1 (CTR1), a Raf-like serine/threonine (Ser/Thr) kinase, and then promotes the cleavage of the carboxyl end of ethylene insensitive 2 (EIN2) (Kieber et al., 1993; Wen et al., 2012). The carboxyl end of EIN2 (CEND) is a trafficking molecule transported into the nucleus where the transcription factors ethylene insensitive 3 (EIN3) and its homolog ein3-like 1 (EIL1) initiate a transcriptional cascade involving ethylene response factor (ERF) and other ethylene response DNA-
Corresponding authors. E-mail addresses: [email protected] (J. Lv), [email protected] (J. Li). These two authors contributed equally to this work.
http://dx.doi.org/10.1016/j.scienta.2017.11.007 Received 11 August 2017; Received in revised form 26 October 2017; Accepted 1 November 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.
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Fig 1. Fruit firmness, ethylene production and respiration rate of apple fruit during storage at 20 °C after treatment with 0.5 mM MeJA. Fruit firmness values were an average of 12 fruit with three measurements on each fruit. The ethylene production and respiration rate at each time point were calculated from the means of three biological replicates, and each replicate included three technical repeats. Vertical bars represent standard errors of means. The LSD (least significant difference) values were calculated at α-value of 0.05. Different letters above or below each data point indicate significant differences between treatment and control groups for each sampling day (P < 0.05).
By contrast, in peaches (Prunus persica), both early and late JAs treatment during fruit development resulted in the down-regulation of ethylene biosynthetic genes (Ruiz et al., 2013; Ziosi et al., 2008). In recent years, studies have shown that the expression of MdACS1 in the skin of apple fruit was regulated by exogenous application of JAs (Kondo et al., 2009). However, the effects of JAs on the expression of ethylene biosynthetic genes during postharvest ripening of apple fruit are still unclear. By now, very few studies have been reported the effects of JAs on ethylene perception during fruit ripening. Only one report in peaches showed that the expression of ethylene receptor genes was differentially affected by exogenous application of JAs during fruit ripening (Ruiz et al., 2013; Soto et al., 2012). It is still unclear what is the effects of JAs on ethylene signaling pathway in ripening apple fruit. Therefore, the molecular mechanisms of JAs in the regulation of ethylene biosynthesis and signaling pathway during the ripening of apple fruit still need to be further elucidated. The aim of the present work was to investigate the effect of JAs on expression of key genes involved in the ethylene biosynthesis and signaling pathway during postharvest ripening of apple fruit.
binding factors (An et al., 2010; Chao et al., 1997; Ijaz, 2016). To date, six ethylene receptor genes (MdETR1, MdETR1b, MdETR2, MdETR5, MdERS1 and MdERS2) have been reported in apple (Dal Cin et al., 2005; Tatsuki and Endo, 2006; Wiersma et al., 2007), and four of them (MdETR1, MdETR2, MdERS1 and MdERS2) are considered to be related to fruit ripening (Li et al., 2010). Five splicing variants of CTR1 (MdCTR1-5) have been investigated, and their expression was reduced by the ethylene antagonist 1-methylcyclopropene (1-MCP) during fruit ripening (Wiersma et al., 2007; Yang et al., 2013). Two EIN2 genes (MdEIN2A and MdEIN2B), four EIL genes (MdEIL1-4) and two ERF genes (MdERF1 and MdERF2) have now been isolated, and they were differentially expressed during the ripening of apple fruit (Wang et al., 2007; Wiersma et al., 2007; Yang et al., 2013). Although ethylene is the major trigger for climacteric fruit ripening, other plant hormones, such as jasmonates (JAs) and abscisic acid (ABA), are shown to be tightly associated with fruit ripening (Kumar et al., 2014). The term jasmonates (JAs) is often used to refer to jasmonic acid, its volatilized methyl ester (methyl jasmonate, MeJA), and amino acid derivatives (Wasternack, 2007). Many studies have confirmed that exogenous JAs application can promote volatile emission (Kondo et al., 2005), stimulate anthocyanin synthesis (Rudell and Mattheis, 2008), accelerate chlorophyll degradation and β-carotene accumulation (Pérez et al., 1993). Fruit ripening is a complex process involving the dynamic interplay between different phytohormones. Previous studies have indicated a role for JAs in the regulation of ethylene biosynthesis during fruit ripening. In apples (Fan et al., 1997, 1998; Kondo et al., 2009) and pears (Pyrus communis) (Kondo et al., 2007), exogenous application of JAs at pre-climacteric stage may enhance ethylene emission, but opposite results were obtained when exogenous JAs were applied at climacteric and post-climacteric stage.
2. Materials and methods 2.1. Plant materials and treatments Apple fruit (Malus × domestica Borkh., ‘Golden Delicious’) without mechanical damage and free of visible defects or decay were harvested at commercial maturity from a commercial orchard in Jinzhou, China. Fruit were randomly sampled from over 80 individual trees and immediately transported to the postharvest laboratory of Bohai University. The selected fruit were randomly divided into two groups. 158
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Fig. 2. Expression of MdACSs and MdACOs in apple fruit during storage at 20 °C after treatment with 0.5 mM MeJA. Mdactin was used as an internal standard for each gene. The values for each time point represent an average of three PCR runs for three biological replicates. Vertical bars represent standard errors of means. The LSD (least significant difference) values were calculated at α-value of 0.05. Different letters above or below each data point indicate significant differences between treatment and control groups for each sampling day (P < 0.05).
FT327; Effegi, Milan, Italy) equipped with a 11 mm diameter probe and expressed in Newtons. Ethylene production and respiration rate were determined by enclosing four apples in sealed 9.35-L glass desiccators for 1 h at room temperature, and then a 1 mL sample of the headspace gas was collected with a syringe, respectively. To analyze the ethylene production, 1 mL sample was injected into a flame ionisation detection gas chromatograph (GC-14A; Shimadzu, Kyoto, Japan) fitted with a flame-ionisation detector (FID). The oven, detector and injector were operated at 70, 70, and 150 °C, respectively. The carrier gas (N2, H2, and air) flow rates were 0.5, 0.5, and 5 mL s−1, respectively. Respiration rate was recorded as carbon dioxide (CO2) production. To measure the respiration rate, 1 mL sample was injected into gas chromatograph (SP9890; Lunan Ruihong, Shandong, China) fitted with a flame-ionisation detector (FID). The oven, detector and injector were operated at 350, 140 and 120 °C, respectively. The carrier gas (N2, H2, and air) flow rates were 1, 0.5, and 6 mL s−1, respectively. Three biological replicates were included for each treatment. The ethylene concentrations and respiration rate were measured three times for each replicate. The ethylene production rate was expressed as nanomoles per kilogram per second. The respiration rate was expressed as micromoles per kilogram per second.
Each group was then divided into three subgroups, and one subgroup of 50 fruit were selected for control or MeJA treatment. MeJA treatment was performed by immersing fruit into 0.5 mM MeJA (Solarbio, Beijing, China) containing 0.077 % (v/v) triton x-100 for 5 min. Untreated control fruit were dipped for 5 min in a deionized water solution containing 0.077 % (v/v) triton x-100 only. All treated and control fruit were stored in open cardboard boxes and placed in the lab at 20 °C. Fruit were randomly selected at seven-day intervals. At each sampling time, a total of 12 fruit were randomly selected from the three treatment subgroups with four fruit for each subgroup for the measurement of ethylene production, respiration rate and fruit firmness, and the other nine fruit with three fruit for each subgroup were cut into 0.6 cm3 cubes (cortex with skin), pooled together, immediately frozen in liquid nitrogen and stored at −80 °C for subsequent molecular analysis. 2.2. Determination of flesh firmness, ethylene production and respiration rate Flesh firmness was determined at each time point from 12 fruit after removing a small disk of skin and then measuring each fruit three times from three positions located at 120° intervals around the equator. Measurements were conducted with a hand-hold pressure tester (Model 159
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Fig. 3. Expression of ethylene receptor genes in apple fruit during storage at 20 °C after treatment with 0.5 mM MeJA. Mdactin was used as an internal standard for each gene. The values for each time point represent an average of three PCR runs for three biological replicates. Vertical bars represent standard errors of means. The LSD (least significant difference) values were calculated at α-value of 0.05. Different letters above or below each data point indicate significant differences between treatment and control groups for each sampling day (P < 0.05).
cDNA, 0.4 μM of each primer, 0.4 μL of ROX reference dye (50×), 6 μL of ddH2O and 10 μL of 2×SYBR Premix Ex Taq II (Takara, Dalian, China) using the real-time PCR detection system (StepOnePlus™; Applied Biosystems). The amplification conditions were 30 s at 95 °C, 40 cycles of 5 s at 95 °C and 34 s at 60 °C. The primers used for all target genes are described in Supplemental Table S1. Mdactin was used as a reference gene according to Guardo et al. (2013). No-template controls for each primer pair were included in each run. The target gene expression was normalized to that of the internal reference gene (Mdactin) according to the 2−ΔΔCT method (Livak and Schmittgen, 2001). Samples from Day 0 (assigned an arbitrary quantity of “1”) were used as the calibrator for calculating the relative expression level of the target gene. The experiment was performed with three biological replicates.
2.3. Total RNA extraction and screening of specific-expressed gene family members Total RNA from each sample was extracted according to the manufacturer’s instructions for the RNAprep pure plant kit (Tiangen, Beijing, China). All RNA extracts were pretreated with DNase I (Promega, Beijing, China) to remove contamination from genomic DNA. Conventional PCR was used to verify gene expression in the fruit. Approximately the same amount of the treated RNAs from each weekly sample was pooled. First-strand cDNA was synthesized from 1 μg of the RNA mixtures using reverse transcriptase M-MLV (RNase H-) (Takara, Dalian, China) and oligo(dT)18 as the primer. Sequences of primers used in this study were shown in Supplemental Table S1. The PCR reactions were performed according to the 2×Taq PCR Master Mix Kit (Tiangen) in a total volume of 25 μL on a PCR system (GeneAmp® 9700 PCR System; Applied Biosystems, CA, USA). The amplification products were separated on 1% agarose by gel electrophoresis and analyzed with the UV transilluminator (UVItec FireReader; UVItec, Cambridge, UK). A single band with the expected size from amplification by agarose gel electrophoresis indicated the expression of specific genes in ripening apple fruit.
2.5. Statistical analysis All measured values presented as the mean ± standard error of the means. Data were tested by analysis of variance (ANOVA) using SAS software (version 8.0; SAS Institute, Cary, NC, USA). The least significant difference (LSD) values were calculated to compare differences between treatment and control groups for each sampling day. P-values below 0.05 were considered statistically significant (P < 0.05).
2.4. Real-time quantitative PCR analysis 3. Results The RNA concentration from each weekly sample was measured using a spectrophotometer (NanoDrop ND-1000; Thermo Fisher Scientific, Wilmington, DE, USA). First-strand cDNA was synthesized from 0.5 μg of the total RNA using the PrimeScript™ RT Master Mix kit (Takara, Dalian, China). The cDNA was diluted 10 times. PCR reactions were carried out in a total volume of 20 μL, including 2 μL of the diluted
3.1. Effect of exogenous MeJA treatment on fruit firmness, ethylene production and respiration rate during postharvest ripening of apple fruit Fruit firmness, ethylene production and respiration rate are ripening indicators of apple fruit. Fruit firmness decreased upon ripening in both 160
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Fig. 4. Expression of MdCTR1s in apple fruit during storage at 20 °C after treatment with 0.5 mM MeJA. Mdactin was used as an internal standard for each gene. The values for each time point represent an average of three PCR runs for three biological replicates. Vertical bars represent standard errors of means. The LSD (least significant difference) values were calculated at α-value of 0.05. Different letters above or below each data point indicate significant differences between treatment and control groups for each sampling day (P < 0.05).
(MdACS1, MdACS3A, MdACS6 and MdACS8), two ACO genes (MdACO1 and MdACO2), four ethylene receptor genes (MdETR1, MdETR2, MdERS1 and MdERS2), five CTR genes (MdCTR1-5), two EIN genes (MdEIN2A and MdEIN2B), four EIL genes (MdEIL1-4) and two ERF genes (MdERF1 and MdERF2) were confirmed to be expressed in the ripening apple fruit tissues. Expression patterns of these identified genes were analyzed by the subsequent qPCR assay.
groups (Fig. 1A). MeJA-treated fruit showed a higher rate of softening than the control fruit during ripening. Significant differences in fruit firmness were observed between both groups during the entire storage period. The ethylene production exhibited a typical climacteric pattern during ripening (Fig. 1B). Ethylene production increased and peaked at 21 d in both groups. Application of MeJA stimulated the ethylene production during ripening. The maximum ethylene production (21 d) in MeJA-treated fruit was 1.2-fold higher than that in the control fruit. The respiration rate increased after 7 d and peaked at 21 d in both groups during ripening (Fig. 1C). MeJA treatment promoted the respiration rate from 14 d to 21 d. The maximum respiration rate in MeJA-treated fruit was 1.4-fold higher than that in control fruit at 21 d.
3.3. Effects of exogenous MeJA treatment on the expression of MdACSs and MdACOs during postharvest ripening of apple fruit Generally, the expression of MdACS1, MdACS6 and MdACS8 showed an increasing trend in both groups upon ripening (Fig. 2). The expression of MdACS1 showed a 217,030-fold increase in the control fruit upon ripening. The expression of MdACS1 was up-regulated by MeJA treatment before 14 d, whereas it was down-regulated by MeJA treatment from 14 d to 28 d compared with controls (Fig. 2A). In both groups, the expression of MdACS3A peaked at 7 d and 21 d (Fig. 2B),
3.2. Preliminary screening of apple fruit expressed genes after harvest Twenty-four genes involved in the ethylene biosynthesis and signal transduction pathway were selected to verify expression during fruit ripening by the conventional PCR assay. As a result, four ACS genes 161
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Fig. 5. Expression of MdEIN2s and MdEILs in apple fruit during storage at 20 °C after treatment with 0.5 mM MeJA. Mdactin was used as an internal standard for each gene. The values for each time point represent an average of three PCR runs for three biological replicates. Vertical bars represent standard errors of means. The LSD (least significant difference) values were calculated at α-value of 0.05. Different letters above or below each data point indicate significant differences between treatment and control groups for each sampling day (P < 0.05).
by MeJA treatment during ripening (Fig. 3). In MeJA-treated fruit, the expression of MdETR1 was significantly higher than the controls at 7 d. No significant difference in its expression between both groups was observed from 14 d to 28 d (Fig. 3A). The expression of MdETR2 was significantly up-regulated by MeJA treatment compared with the controls from 7 d to 21 d (Fig. 3B). The expression of MdERS1 was significantly up-regulated by MeJA treatment throughout the entire storage period (Fig. 3C). The expression of MdERS2 showed a 4.9-fold increase in the control fruit upon ripening. In MeJA-treated fruit, its expression was significantly higher than the controls from 7 d to 21 d (Fig. 3D).
respectively. Application of MeJA significantly up-regulated the expression of MdACS3A compared with controls during storage, suggesting the positive effects of MeJA on its expression. The expression of MdACS6 was significantly up-regulated by MeJA treatment at 7 d and 14 d compared with controls (Fig. 2C). In MeJA-treated fruit, the expression of MdACS8 was significantly higher than the controls throughout the entire storage period, indicating that it was positively regulated by MeJA treatment during ripening (Fig. 2D). The expression of MdACO1 and MdACO2 peaked at 21 d in both groups (Fig. 2E and F). Their expression was significantly higher than the controls throughout the entire storage period. In MeJA-treated fruit, the expression of MdACO1 and MdACO2 was 1.5-fold and 1.2-fold higher than that in the control fruit at 21 d, respectively, indicating that they were positively regulated by MeJA treatment during ripening.
3.5. Effects of exogenous MeJA treatment on the expression of genes involved in ethylene signal transduction pathway during postharvest ripening of apple fruit
3.4. Effects of exogenous MeJA treatment on the expression of ethylene receptor genes during postharvest ripening of apple fruit
Generally, the expression of MdCTR1s did not show large changes from 14 d to 21 d even though an increasing trend was detected in control fruit during storage (Fig. 4). No readily observable difference in
The expression of ethylene receptor genes was differentially induced 162
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ripening stage. MdEIL4 followed similar expression pattern of MdEIN2B (Fig. 5F). Its expression was significantly up-regulated by MeJA treatment at 7 d compared with controls during ripening. 3.6. Effects of exogenous MeJA treatment on the expression of the two ethylene response factor genes during postharvest ripening of apple fruit The expression of the two ethylene response factor genes, MdERF1 and MdERF2, was differentially regulated by MeJA treatment during ripening (Fig. 6). Generally, the expression of MdERF1 showed an increasing trend in both groups upon ripening, although a slight decrease in its expression was observed from 14 d to 21 d (Fig. 6A). Application of MeJA significantly up-regulated its expression compared with controls at 7 d. No significant difference in the expression of MdERF1 between both groups was observed at 14 d and 21 d, respectively. The expression pattern of MdERF2 was in accordance with the change of ethylene production in the control fruit (Fig. 6B). The expression of MdERF2 was significantly down-regulated by MeJA treatment from 14 d to 21 d when the ethylene climacteric peak existed, and then it increased and recovered to the control level at 28 d, indicating that the expression of MdERF2 was negatively regulated by MeJA at the late ripening stage. 4. Discussion The gaseous plant hormone ethylene is well-known for its major role in the regulation of climacteric fruit ripening. There are two systems of ethylene production in higher plants: system-1 occurs during fruit growth and development, and system-2 is exclusively in climacteric fruit ripening (Barry and Giovannoni, 2007). In apple, previous studies have confirmed that applying MeJA at the pre-climacteric stage increased ethylene production (Fan et al., 1997, 1998; Saniewski et al., 1988), but production decreased when it was applied at the climacteric stage and the post-climacteric stage (Miszczak et al., 1995). In this research, MeJA treatment increased the ethylene production compared with controls during the ripening of apple fruit, and no significant increase in the respiration rate was observed from 0 d to 7 d, indicating that it was applied at the pre-climacteric stage. Four ACS genes were identified and shown to be regulated by MeJA treatment during fruit ripening, including MdACS1, MdACS3a, MdACS6 and MdACS8. Recent studies suggested that MdACS1 was expressed specifically in fruit ripening and responsible for the system-2 ethylene biosynthesis (Tan et al., 2013; Wiersma et al., 2007). In this research, the very low starting level of MdACS1 and 217,030-fold increases in its gene expression were observed upon ripening in the control fruit, this results provide evidence that MdACS1 is expressed specifically in fruit ripening. Kondo et al. (2009) found that application of the synthetic JA derivative n-propyl dihydrojasmonate (PDJ) greatly increased the expression of MdACS1 in the skin of apple fruit at 24 h after commercial harvest. In this study, the expression of MdACS1 was induced at 7 d by exogenous application of MeJA, whereas it was down-regulated by MeJA treatment from 14 d to 28 d when the ethylene peak existed, indicating that MeJA treatment might directly stimulate the expression of MdACS1 at the early ripening stage. We speculated that ethylene might regulate its own biosynthesis through a negative feedback loop that represses the expression of MdACS1 to avoid producing excessive ethylene during the period when the ethylene peak existed in the MeJAtreated fruit. Tan et al. (2013) found that MdACS3a operated in the system-1 ethylene production and played important roles in determining the shelf life of apple fruit (Wang et al., 2009). In the current study, MeJA treatment enhanced the expression of MdACS3a, indicating that it was positively regulated by MeJA treatment during ripening. Previous study has revealed that MdACS6 and MdACS8 might work in the system-2 ethylene biosynthesis (Li et al., 2013). In our study, the expression of MdACS6 and MdACS8 was differentially upregulated by MeJA treatment, indicating that they were positively
Fig. 6. Expression of MdERFs in apple fruit during storage at 20 °C after treatment with 0.5 mM MeJA. Mdactin was used as an internal standard for each gene. The values for each time point represent an average of three PCR runs for three biological replicates. Vertical bars represent standard errors of means. The LSD (least significant difference) values were calculated at α-value of 0.05. Different letters above or below each data point indicate significant differences between treatment and control groups for each sampling day (P < 0.05).
the expression of MdCTR1-1 and MdCTR1-2 was observed between both groups, indicating that their expression was not regulated by MeJA treatment during ripening (Fig. 4A and B). The expression of MdCTR1-3 in MeJA-treated fruit was 1.5-fold and 1.2-fold higher than that in control fruit at 7 d and 28 d, respectively (Fig. 4C). MeJA treatment significantly increased the expression of MdCTR1-4 and MdCTR1-5 at 7 d, whereas after 7 d, no significant differences in their expression were observed between both groups (Fig. 4D and E). The expression of MdEIN2A in MeJA-treated fruit was significantly higher than that in control fruit before 21 d (Fig. 5A), suggesting some degree of the positive effect of MeJA on its expression. Similar to the effect on the expression of MdCTR1-4 and MdCTR1-5, MeJA treatment significantly up-regulated the expression of MdEIN2B at 7 d (Fig. 5B). The expression of MdEIL1 showed an increasing trend in both groups upon ripening (Fig. 5C). Its expression was significantly enhanced by MeJA treatment throughout the entire storage period, indicating that the expression of MdEIL1 was positively regulated by MeJA treatment during ripening. The expression of MdEIL2 showed about 4-fold increase upon ripening in both groups (Fig. 5D). MeJA treatment had no apparent effect on its expression, indicating that the expression of MdEIL2 was not regulated by MeJA treatment during ripening. In control fruit, the expression of MdEIL3 increased from 0 d to 14 d, whereas after 14 d, no large changes in its expression were observed (Fig. 5E). Application of MeJA significantly up-regulated its expression from 21 d to 28 d compared with controls, indicating that the expression of MdEIL3 was positively regulated by MeJA treatment at the late 163
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EIN2 is described as being a bi-functional transducer, which mediates an essential step in the ethylene signal propagation between CTR1 and the downstream components (EIN3/EIL) (Alonso et al., 1999). Kim et al. (2013) found that reducing jasmonic acid levels caused ein2 ethylene-insensitive mutants to become ethylene responsive, and therefore they suggested that there might be an EIN2-independent component in ethylene signaling that was inhibited by JA. In this study, the expression of MdEIN2A and MdEIN2B was positively regulated by MeJA treatment to some extent at the early ripening stage. This observation seems contradictory to the previous findings. Because apples and Arabidopsis (Arabidopsis thaliana) belong to different plant species, it is necessary to further investigate the molecular mechanisms of the interaction between EIN2 and JA signaling in apple fruit. EIN3/EILs are positive regulators within the ethylene signal cascade (Tieman et al., 2001). Generally, the expression of MdEIL1-4 increased upon ripening, except for a little decrease in the expression of MdEIL3 at 21 d, indicating that MdEIL1-4 were ripening-related genes. This is consistent with previous findings in other fruit systems, such as banana (Musa acuminata) (Mbeguie-A-Mbeguie et al., 2008) and melon (Cucumis melo) (Huang et al., 2010). It has been confirmed that EIN3/EIL1 physically interacted with JA-Zim domain (JAZ) proteins, a repressor of JA signaling, and integrated ethylene and JA signaling in Arabidopsis (Zhu et al., 2011). However, very little is known about the specific element in integration of both signals in apple fruit. In our study, the expression of MdEIL1, MdEIL3 and MdEIL4 was differentially induced by MeJA treatment. Similar results were also obtained in Arabidopsis (Zhu et al., 2011) and rice (Oryza sativa) (Hiraga et al., 2009). Further studies, such as yeast two-hybrid assay and pull-down assay, are needed to identify which members of the EIL family that interact with JA signaling pathway in apple fruit. The expression of the two MdERF genes was differentially regulated by MeJA treatment during ripening. The expression of MdERF1 was positively regulated by MeJA treatment at the early ripening stage, whereas the expression of MdERF2 was negatively regulated by it from 14 d to 21 d. Actually, except for EIN3/EILs, ERF proteins also play key roles in the integration of both signals in Arabidopsis (Lorenzo et al., 2003). A recent study on banana fruit showed that MaERF10 physically interacted with MaJAZ3, and the interaction led to deeper repression of JA biosynthetic genes by MaERF10 (Qi et al., 2016). Therefore, we speculate that there are two possible reasons for the down-regulation of MdERF2 expression: i) there exist JA-related regulators that interact with MdERF2 and suppress its gene expression; ii) since ERF acts at the last step of the ethylene signaling pathway and controls the transcription of ethylene responsive genes (El-Sharkawy et al., 2009; Klee and Giovannoni, 2011), the down-regulation of MdERF2 may function to repress ethylene responses which are induced by higher than the normal levels of ethylene, and thus maintain the metabolic balance in the MeJA-treated fruit. Our data confirmed that the expression of genes involved in the ethylene biosynthesis and signaling pathway was differentially regulated by JAs during postharvest ripening of apple fruit. Understanding the expression patterns of these genes involved in the ethylene biosynthesis and signaling pathway in response to JAs may help to decode the complex mechanisms of the crosstalk between the ethylene and JA signaling pathways in apple fruit.
regulated by MeJA treatment during ripening. It has been reported that MdACO1 might be one of the major factors of the system-2 ethylene biosynthesis (Schaffer et al., 2007; Wang and Xu 2012; Wiersma et al., 2007). In this study, the expression of MdACO1 was significant higher in MeJA-treated fruit than that in control fruit, indicating that MdACO1 might be involved in the MeJA-induced ethylene accumulation during ripening. Information about the function of MdACO2 is limited. Wiersma et al. (2007) found that expression of MdACO2 was in a cultivar-dependent manner during fruit ripening. The present study showed that expression of MdACO2 was positively regulated by MeJA treatment during ripening. Taken together, our data discussed above indicated that the expression of key genes involved in the ethylene biosynthesis pathway was differentially regulated by JAs during postharvest ripening of apple fruit. Ethylene receptors function as negative regulators of the ethylene signaling pathway, and reduced or induced levels of the receptors appear to confer high ethylene sensitivity or to counteract the ethylene signal, respectively (Hua and Meyerowitz, 1998; Tieman et al., 2000). The expression of MdETR1, MdETR2, MdERS1 and MdERS2 was differentially up-regulated by MeJA treatment compared with controls during ripening, suggesting that these four genes were positively regulated by MeJA. We speculated that the up-regulated expression of these four ethylene receptor genes might be associated with the enhancement of the MeJA-induced ethylene biosynthesis, which might be a mechanism for adaptation in ethylene responses (Hua and Meyerowitz, 1998). In MeJA-treated fruit, the higher level of ethylene production required more ethylene receptors to induce specific responses by relieving the repression activity of the ethylene receptors, and at the same time, it differentially up-regulated the expression of the ethylene receptor genes. The increased expression of the ethylene receptor genes might lead to the production of more proteins that can repress the ethylene response. However, changes in the transcriptional level do not systematically imply changes in the protein level and function. Tatsuki et al. (2009) observed that MdERS1 and MdERS2 transcription increased rapidly after harvest while their protein levels behaved differently. Therefore, it is necessary to further identify the specific members of ethylene receptors involved in the JAs-induced ethylene responses at the protein level during the ripening of apple fruit. CTR1 negatively regulates downstream ethylene signaling events in the absence of ethylene (Stepanova and Alonso, 2009). To date, only one MdCTR1 gene (AY670703) has been identified in apple fruit (Dal Cin et al., 2005), and its full length cDNA was characterized by several splice variants (Wiersma et al., 2007). Yang et al. (2013) characterized the splice variants as follows: MdCTR1-1 and MdCTR-2 (primers located between the first intron and the start of the kinase domain), MdCTR1-3 and MdCTR-4 (those retain 3′ UTR region), and MdCTR1-5 (the one located within kinase domain). Dal Cin et al. (2006) found that the expression of MdCTR1 was up-regulated in fruit during ripening. Similar results were also obtained from the present study. The expression of MdCTR1s increased and showed similar trends in control fruit upon ripening, indicating that these splice variants remained as a consistent portion of the total. However, they responded differentially to the MeJA treatment during ripening. The expression of MdCTR1-3, MdCTR1-4 and MdCTR1-5 was up-regulated by MeJA treatment at the early ripening stage compared with controls, whereas the expression of MdCTR1-1 and MdCTR1-2 was not significantly affected by MeJA treatment during the entire storage period. Previous studies showed that the expression of MdCTR1-3, MdCTR1-4 and MdCTR1-5 was ethylene inducible (Li and Yuan, 2008; Yang et al., 2013). In this study, no significant difference in the expression of MdCTR1-4 and MdCTR1-5 was observed between both groups during the late storage period, although the levels of ethylene production were higher in the MeJA-treated fruit than that in the control fruit during the same storage period. Further analysis is needed to clarify the relationship among MdCTR1s, ethylene and JA signaling in ripening apple fruit.
5. Conclusion In conclusion, MeJA treatment increased the system-2 ethylene production during ripening. The expression of genes involved in the ethylene biosynthesis and signaling pathway was positively regulated by MeJA treatment at varying stages of ripening, except for MdACS1, MdCTR1-1, MdCTR1-2, MdEIL2 and MdERF2. The expression of MdACS1 and MdERF2 was negatively regulated by MeJA treatment when the ethylene climacteric peak existed, whereas the expression of MdCTR1-1, MdCTR1-2 and MdEIL2 was not regulated by it during fruit 164
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Research Paper
Effects of methyl jasmonate on expression of genes involved in ethylene biosynthesis and signaling pathway during postharvest ripening of apple fruit ⁎,1
Jingyi Lv
MARK
⁎
, Mengyuan Zhang1, Junhu Zhang, Yonghong Ge, Canying Li, Kun Meng, Jianrong Li
College of Food Science and Project Engineering, Bohai University; National & Local Joint Engineering Research Center of Storage, Processing and Safety Control Technology for Fresh Agricultural and Aquatic Products, Bohai University, Jinzhou, Liaoning 121013, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Apple fruit Ethylene biosynthesis Signal transduction Jasmonates Fruit ripening Gene expression
Ethylene plays a key regulatory role in the ripening of climacteric fruit. In addition to ethylene, jasmonates (JAs) have also been demonstrated to play a role in the regulation of the fruit ripening. Apple (Malus × domestica Borkh.) fruit is a common climacteric fruit. Research has been conducted to illustrate the effects of JAs on ethylene production in apple fruit, but little is known about the molecular mechanisms underlying the role of JAs in ethylene biosynthesis and signaling pathway during the ripening of apple fruit. To better understand the effects of JAs on the expression of key genes involved in the ethylene biosynthesis and signaling pathway during postharvest ripening of apple fruit, apples harvested at commercial maturity were treated with methyl jasmonate (MeJA). Our data indicated that MeJA treatment increased ethylene production during fruit ripening. The expression of MdACS1, MdACS6, MdETR1, MdCTR1-3, MdCTR1-4, MdCTR1-5, MdEIN2A, MdEIN2B, MdEIL4 and MdERF1 was positively regulated by MeJA treatment at the early ripening stage, whereas the expression of MdEIL3 was positively regulated by it at the late ripening stage. MeJA treatment enhanced the expression of MdACS3a, MdACS8, MdACO1, MdACO2, MdETR2, MdERS1, MdERS2 and MdEIL1 during the entire storage period, whereas it had no effect on the expression of MdCTR1-1, MdCTR1-2 and MdEIL2 during fruit ripening. The expression of MdERF2 and MdACS1 was negatively by MeJA treatment during the period when the ethylene peak existed. These results indicated that the expression of genes involved in the ethylene biosynthesis and signaling pathway was differentially regulated by JAs during postharvest ripening of apple fruit.
1. Introduction The gaseous phytohormone ethylene plays critical roles in the regulation of many plant physiological processes, including seed germination, seedling growth, organ abscission, fruit ripening, and senescence (Abeles et al., 1992; Tatsuki, 2010). Apple fruit is a climacteric fruit, whose ripening is characterized by an exponential increase in ethylene production and respiration rate (Varanasi et al., 2013; Yang and Hoffman, 1984). Studies have confirmed that ethylene plays a key regulatory role in the ripening of apple fruit, such as flesh softening (Ireland et al., 2014), volatile biosynthesis (Yang et al., 2016) and anthocyanin accumulation (Whale and Singh, 2007). Biosynthesis of ethylene begins with methionine, which is converted to S-adenosyl-Lmethionine (SAM) by SAM synthetase. SAM is then catalyzed by 1aminocyclopropane-1-carboxylate (ACC) synthase (ACS) to produce ACC, which can be further converted to ethylene by ACC oxidase (ACO)
⁎
1
(Adams and Yang, 1979). The formation of ACC is generally considered to be the rate-limiting step in the biosynthesis of ethylene (Kende, 1993). In apple, five ACS genes and two ACO genes have been found to be related to fruit ripening, including MdACS1, MdACS3a, MdACS6, MdACS7, MdACS8, MdACO1 and MdACO2 (Dal Cin et al., 2007; Li et al., 2013; Wiersma et al., 2007). After synthesis, ethylene is perceived by receptors that act as negative regulators of ethylene responses in plants (Kevany et al., 2007). Binding of ethylene by receptors inhibits the activity of constitutive triple response 1 (CTR1), a Raf-like serine/threonine (Ser/Thr) kinase, and then promotes the cleavage of the carboxyl end of ethylene insensitive 2 (EIN2) (Kieber et al., 1993; Wen et al., 2012). The carboxyl end of EIN2 (CEND) is a trafficking molecule transported into the nucleus where the transcription factors ethylene insensitive 3 (EIN3) and its homolog ein3-like 1 (EIL1) initiate a transcriptional cascade involving ethylene response factor (ERF) and other ethylene response DNA-
Corresponding authors. E-mail addresses: [email protected] (J. Lv), [email protected] (J. Li). These two authors contributed equally to this work.
http://dx.doi.org/10.1016/j.scienta.2017.11.007 Received 11 August 2017; Received in revised form 26 October 2017; Accepted 1 November 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.
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Fig 1. Fruit firmness, ethylene production and respiration rate of apple fruit during storage at 20 °C after treatment with 0.5 mM MeJA. Fruit firmness values were an average of 12 fruit with three measurements on each fruit. The ethylene production and respiration rate at each time point were calculated from the means of three biological replicates, and each replicate included three technical repeats. Vertical bars represent standard errors of means. The LSD (least significant difference) values were calculated at α-value of 0.05. Different letters above or below each data point indicate significant differences between treatment and control groups for each sampling day (P < 0.05).
By contrast, in peaches (Prunus persica), both early and late JAs treatment during fruit development resulted in the down-regulation of ethylene biosynthetic genes (Ruiz et al., 2013; Ziosi et al., 2008). In recent years, studies have shown that the expression of MdACS1 in the skin of apple fruit was regulated by exogenous application of JAs (Kondo et al., 2009). However, the effects of JAs on the expression of ethylene biosynthetic genes during postharvest ripening of apple fruit are still unclear. By now, very few studies have been reported the effects of JAs on ethylene perception during fruit ripening. Only one report in peaches showed that the expression of ethylene receptor genes was differentially affected by exogenous application of JAs during fruit ripening (Ruiz et al., 2013; Soto et al., 2012). It is still unclear what is the effects of JAs on ethylene signaling pathway in ripening apple fruit. Therefore, the molecular mechanisms of JAs in the regulation of ethylene biosynthesis and signaling pathway during the ripening of apple fruit still need to be further elucidated. The aim of the present work was to investigate the effect of JAs on expression of key genes involved in the ethylene biosynthesis and signaling pathway during postharvest ripening of apple fruit.
binding factors (An et al., 2010; Chao et al., 1997; Ijaz, 2016). To date, six ethylene receptor genes (MdETR1, MdETR1b, MdETR2, MdETR5, MdERS1 and MdERS2) have been reported in apple (Dal Cin et al., 2005; Tatsuki and Endo, 2006; Wiersma et al., 2007), and four of them (MdETR1, MdETR2, MdERS1 and MdERS2) are considered to be related to fruit ripening (Li et al., 2010). Five splicing variants of CTR1 (MdCTR1-5) have been investigated, and their expression was reduced by the ethylene antagonist 1-methylcyclopropene (1-MCP) during fruit ripening (Wiersma et al., 2007; Yang et al., 2013). Two EIN2 genes (MdEIN2A and MdEIN2B), four EIL genes (MdEIL1-4) and two ERF genes (MdERF1 and MdERF2) have now been isolated, and they were differentially expressed during the ripening of apple fruit (Wang et al., 2007; Wiersma et al., 2007; Yang et al., 2013). Although ethylene is the major trigger for climacteric fruit ripening, other plant hormones, such as jasmonates (JAs) and abscisic acid (ABA), are shown to be tightly associated with fruit ripening (Kumar et al., 2014). The term jasmonates (JAs) is often used to refer to jasmonic acid, its volatilized methyl ester (methyl jasmonate, MeJA), and amino acid derivatives (Wasternack, 2007). Many studies have confirmed that exogenous JAs application can promote volatile emission (Kondo et al., 2005), stimulate anthocyanin synthesis (Rudell and Mattheis, 2008), accelerate chlorophyll degradation and β-carotene accumulation (Pérez et al., 1993). Fruit ripening is a complex process involving the dynamic interplay between different phytohormones. Previous studies have indicated a role for JAs in the regulation of ethylene biosynthesis during fruit ripening. In apples (Fan et al., 1997, 1998; Kondo et al., 2009) and pears (Pyrus communis) (Kondo et al., 2007), exogenous application of JAs at pre-climacteric stage may enhance ethylene emission, but opposite results were obtained when exogenous JAs were applied at climacteric and post-climacteric stage.
2. Materials and methods 2.1. Plant materials and treatments Apple fruit (Malus × domestica Borkh., ‘Golden Delicious’) without mechanical damage and free of visible defects or decay were harvested at commercial maturity from a commercial orchard in Jinzhou, China. Fruit were randomly sampled from over 80 individual trees and immediately transported to the postharvest laboratory of Bohai University. The selected fruit were randomly divided into two groups. 158
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Fig. 2. Expression of MdACSs and MdACOs in apple fruit during storage at 20 °C after treatment with 0.5 mM MeJA. Mdactin was used as an internal standard for each gene. The values for each time point represent an average of three PCR runs for three biological replicates. Vertical bars represent standard errors of means. The LSD (least significant difference) values were calculated at α-value of 0.05. Different letters above or below each data point indicate significant differences between treatment and control groups for each sampling day (P < 0.05).
FT327; Effegi, Milan, Italy) equipped with a 11 mm diameter probe and expressed in Newtons. Ethylene production and respiration rate were determined by enclosing four apples in sealed 9.35-L glass desiccators for 1 h at room temperature, and then a 1 mL sample of the headspace gas was collected with a syringe, respectively. To analyze the ethylene production, 1 mL sample was injected into a flame ionisation detection gas chromatograph (GC-14A; Shimadzu, Kyoto, Japan) fitted with a flame-ionisation detector (FID). The oven, detector and injector were operated at 70, 70, and 150 °C, respectively. The carrier gas (N2, H2, and air) flow rates were 0.5, 0.5, and 5 mL s−1, respectively. Respiration rate was recorded as carbon dioxide (CO2) production. To measure the respiration rate, 1 mL sample was injected into gas chromatograph (SP9890; Lunan Ruihong, Shandong, China) fitted with a flame-ionisation detector (FID). The oven, detector and injector were operated at 350, 140 and 120 °C, respectively. The carrier gas (N2, H2, and air) flow rates were 1, 0.5, and 6 mL s−1, respectively. Three biological replicates were included for each treatment. The ethylene concentrations and respiration rate were measured three times for each replicate. The ethylene production rate was expressed as nanomoles per kilogram per second. The respiration rate was expressed as micromoles per kilogram per second.
Each group was then divided into three subgroups, and one subgroup of 50 fruit were selected for control or MeJA treatment. MeJA treatment was performed by immersing fruit into 0.5 mM MeJA (Solarbio, Beijing, China) containing 0.077 % (v/v) triton x-100 for 5 min. Untreated control fruit were dipped for 5 min in a deionized water solution containing 0.077 % (v/v) triton x-100 only. All treated and control fruit were stored in open cardboard boxes and placed in the lab at 20 °C. Fruit were randomly selected at seven-day intervals. At each sampling time, a total of 12 fruit were randomly selected from the three treatment subgroups with four fruit for each subgroup for the measurement of ethylene production, respiration rate and fruit firmness, and the other nine fruit with three fruit for each subgroup were cut into 0.6 cm3 cubes (cortex with skin), pooled together, immediately frozen in liquid nitrogen and stored at −80 °C for subsequent molecular analysis. 2.2. Determination of flesh firmness, ethylene production and respiration rate Flesh firmness was determined at each time point from 12 fruit after removing a small disk of skin and then measuring each fruit three times from three positions located at 120° intervals around the equator. Measurements were conducted with a hand-hold pressure tester (Model 159
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Fig. 3. Expression of ethylene receptor genes in apple fruit during storage at 20 °C after treatment with 0.5 mM MeJA. Mdactin was used as an internal standard for each gene. The values for each time point represent an average of three PCR runs for three biological replicates. Vertical bars represent standard errors of means. The LSD (least significant difference) values were calculated at α-value of 0.05. Different letters above or below each data point indicate significant differences between treatment and control groups for each sampling day (P < 0.05).
cDNA, 0.4 μM of each primer, 0.4 μL of ROX reference dye (50×), 6 μL of ddH2O and 10 μL of 2×SYBR Premix Ex Taq II (Takara, Dalian, China) using the real-time PCR detection system (StepOnePlus™; Applied Biosystems). The amplification conditions were 30 s at 95 °C, 40 cycles of 5 s at 95 °C and 34 s at 60 °C. The primers used for all target genes are described in Supplemental Table S1. Mdactin was used as a reference gene according to Guardo et al. (2013). No-template controls for each primer pair were included in each run. The target gene expression was normalized to that of the internal reference gene (Mdactin) according to the 2−ΔΔCT method (Livak and Schmittgen, 2001). Samples from Day 0 (assigned an arbitrary quantity of “1”) were used as the calibrator for calculating the relative expression level of the target gene. The experiment was performed with three biological replicates.
2.3. Total RNA extraction and screening of specific-expressed gene family members Total RNA from each sample was extracted according to the manufacturer’s instructions for the RNAprep pure plant kit (Tiangen, Beijing, China). All RNA extracts were pretreated with DNase I (Promega, Beijing, China) to remove contamination from genomic DNA. Conventional PCR was used to verify gene expression in the fruit. Approximately the same amount of the treated RNAs from each weekly sample was pooled. First-strand cDNA was synthesized from 1 μg of the RNA mixtures using reverse transcriptase M-MLV (RNase H-) (Takara, Dalian, China) and oligo(dT)18 as the primer. Sequences of primers used in this study were shown in Supplemental Table S1. The PCR reactions were performed according to the 2×Taq PCR Master Mix Kit (Tiangen) in a total volume of 25 μL on a PCR system (GeneAmp® 9700 PCR System; Applied Biosystems, CA, USA). The amplification products were separated on 1% agarose by gel electrophoresis and analyzed with the UV transilluminator (UVItec FireReader; UVItec, Cambridge, UK). A single band with the expected size from amplification by agarose gel electrophoresis indicated the expression of specific genes in ripening apple fruit.
2.5. Statistical analysis All measured values presented as the mean ± standard error of the means. Data were tested by analysis of variance (ANOVA) using SAS software (version 8.0; SAS Institute, Cary, NC, USA). The least significant difference (LSD) values were calculated to compare differences between treatment and control groups for each sampling day. P-values below 0.05 were considered statistically significant (P < 0.05).
2.4. Real-time quantitative PCR analysis 3. Results The RNA concentration from each weekly sample was measured using a spectrophotometer (NanoDrop ND-1000; Thermo Fisher Scientific, Wilmington, DE, USA). First-strand cDNA was synthesized from 0.5 μg of the total RNA using the PrimeScript™ RT Master Mix kit (Takara, Dalian, China). The cDNA was diluted 10 times. PCR reactions were carried out in a total volume of 20 μL, including 2 μL of the diluted
3.1. Effect of exogenous MeJA treatment on fruit firmness, ethylene production and respiration rate during postharvest ripening of apple fruit Fruit firmness, ethylene production and respiration rate are ripening indicators of apple fruit. Fruit firmness decreased upon ripening in both 160
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Fig. 4. Expression of MdCTR1s in apple fruit during storage at 20 °C after treatment with 0.5 mM MeJA. Mdactin was used as an internal standard for each gene. The values for each time point represent an average of three PCR runs for three biological replicates. Vertical bars represent standard errors of means. The LSD (least significant difference) values were calculated at α-value of 0.05. Different letters above or below each data point indicate significant differences between treatment and control groups for each sampling day (P < 0.05).
(MdACS1, MdACS3A, MdACS6 and MdACS8), two ACO genes (MdACO1 and MdACO2), four ethylene receptor genes (MdETR1, MdETR2, MdERS1 and MdERS2), five CTR genes (MdCTR1-5), two EIN genes (MdEIN2A and MdEIN2B), four EIL genes (MdEIL1-4) and two ERF genes (MdERF1 and MdERF2) were confirmed to be expressed in the ripening apple fruit tissues. Expression patterns of these identified genes were analyzed by the subsequent qPCR assay.
groups (Fig. 1A). MeJA-treated fruit showed a higher rate of softening than the control fruit during ripening. Significant differences in fruit firmness were observed between both groups during the entire storage period. The ethylene production exhibited a typical climacteric pattern during ripening (Fig. 1B). Ethylene production increased and peaked at 21 d in both groups. Application of MeJA stimulated the ethylene production during ripening. The maximum ethylene production (21 d) in MeJA-treated fruit was 1.2-fold higher than that in the control fruit. The respiration rate increased after 7 d and peaked at 21 d in both groups during ripening (Fig. 1C). MeJA treatment promoted the respiration rate from 14 d to 21 d. The maximum respiration rate in MeJA-treated fruit was 1.4-fold higher than that in control fruit at 21 d.
3.3. Effects of exogenous MeJA treatment on the expression of MdACSs and MdACOs during postharvest ripening of apple fruit Generally, the expression of MdACS1, MdACS6 and MdACS8 showed an increasing trend in both groups upon ripening (Fig. 2). The expression of MdACS1 showed a 217,030-fold increase in the control fruit upon ripening. The expression of MdACS1 was up-regulated by MeJA treatment before 14 d, whereas it was down-regulated by MeJA treatment from 14 d to 28 d compared with controls (Fig. 2A). In both groups, the expression of MdACS3A peaked at 7 d and 21 d (Fig. 2B),
3.2. Preliminary screening of apple fruit expressed genes after harvest Twenty-four genes involved in the ethylene biosynthesis and signal transduction pathway were selected to verify expression during fruit ripening by the conventional PCR assay. As a result, four ACS genes 161
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Fig. 5. Expression of MdEIN2s and MdEILs in apple fruit during storage at 20 °C after treatment with 0.5 mM MeJA. Mdactin was used as an internal standard for each gene. The values for each time point represent an average of three PCR runs for three biological replicates. Vertical bars represent standard errors of means. The LSD (least significant difference) values were calculated at α-value of 0.05. Different letters above or below each data point indicate significant differences between treatment and control groups for each sampling day (P < 0.05).
by MeJA treatment during ripening (Fig. 3). In MeJA-treated fruit, the expression of MdETR1 was significantly higher than the controls at 7 d. No significant difference in its expression between both groups was observed from 14 d to 28 d (Fig. 3A). The expression of MdETR2 was significantly up-regulated by MeJA treatment compared with the controls from 7 d to 21 d (Fig. 3B). The expression of MdERS1 was significantly up-regulated by MeJA treatment throughout the entire storage period (Fig. 3C). The expression of MdERS2 showed a 4.9-fold increase in the control fruit upon ripening. In MeJA-treated fruit, its expression was significantly higher than the controls from 7 d to 21 d (Fig. 3D).
respectively. Application of MeJA significantly up-regulated the expression of MdACS3A compared with controls during storage, suggesting the positive effects of MeJA on its expression. The expression of MdACS6 was significantly up-regulated by MeJA treatment at 7 d and 14 d compared with controls (Fig. 2C). In MeJA-treated fruit, the expression of MdACS8 was significantly higher than the controls throughout the entire storage period, indicating that it was positively regulated by MeJA treatment during ripening (Fig. 2D). The expression of MdACO1 and MdACO2 peaked at 21 d in both groups (Fig. 2E and F). Their expression was significantly higher than the controls throughout the entire storage period. In MeJA-treated fruit, the expression of MdACO1 and MdACO2 was 1.5-fold and 1.2-fold higher than that in the control fruit at 21 d, respectively, indicating that they were positively regulated by MeJA treatment during ripening.
3.5. Effects of exogenous MeJA treatment on the expression of genes involved in ethylene signal transduction pathway during postharvest ripening of apple fruit
3.4. Effects of exogenous MeJA treatment on the expression of ethylene receptor genes during postharvest ripening of apple fruit
Generally, the expression of MdCTR1s did not show large changes from 14 d to 21 d even though an increasing trend was detected in control fruit during storage (Fig. 4). No readily observable difference in
The expression of ethylene receptor genes was differentially induced 162
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ripening stage. MdEIL4 followed similar expression pattern of MdEIN2B (Fig. 5F). Its expression was significantly up-regulated by MeJA treatment at 7 d compared with controls during ripening. 3.6. Effects of exogenous MeJA treatment on the expression of the two ethylene response factor genes during postharvest ripening of apple fruit The expression of the two ethylene response factor genes, MdERF1 and MdERF2, was differentially regulated by MeJA treatment during ripening (Fig. 6). Generally, the expression of MdERF1 showed an increasing trend in both groups upon ripening, although a slight decrease in its expression was observed from 14 d to 21 d (Fig. 6A). Application of MeJA significantly up-regulated its expression compared with controls at 7 d. No significant difference in the expression of MdERF1 between both groups was observed at 14 d and 21 d, respectively. The expression pattern of MdERF2 was in accordance with the change of ethylene production in the control fruit (Fig. 6B). The expression of MdERF2 was significantly down-regulated by MeJA treatment from 14 d to 21 d when the ethylene climacteric peak existed, and then it increased and recovered to the control level at 28 d, indicating that the expression of MdERF2 was negatively regulated by MeJA at the late ripening stage. 4. Discussion The gaseous plant hormone ethylene is well-known for its major role in the regulation of climacteric fruit ripening. There are two systems of ethylene production in higher plants: system-1 occurs during fruit growth and development, and system-2 is exclusively in climacteric fruit ripening (Barry and Giovannoni, 2007). In apple, previous studies have confirmed that applying MeJA at the pre-climacteric stage increased ethylene production (Fan et al., 1997, 1998; Saniewski et al., 1988), but production decreased when it was applied at the climacteric stage and the post-climacteric stage (Miszczak et al., 1995). In this research, MeJA treatment increased the ethylene production compared with controls during the ripening of apple fruit, and no significant increase in the respiration rate was observed from 0 d to 7 d, indicating that it was applied at the pre-climacteric stage. Four ACS genes were identified and shown to be regulated by MeJA treatment during fruit ripening, including MdACS1, MdACS3a, MdACS6 and MdACS8. Recent studies suggested that MdACS1 was expressed specifically in fruit ripening and responsible for the system-2 ethylene biosynthesis (Tan et al., 2013; Wiersma et al., 2007). In this research, the very low starting level of MdACS1 and 217,030-fold increases in its gene expression were observed upon ripening in the control fruit, this results provide evidence that MdACS1 is expressed specifically in fruit ripening. Kondo et al. (2009) found that application of the synthetic JA derivative n-propyl dihydrojasmonate (PDJ) greatly increased the expression of MdACS1 in the skin of apple fruit at 24 h after commercial harvest. In this study, the expression of MdACS1 was induced at 7 d by exogenous application of MeJA, whereas it was down-regulated by MeJA treatment from 14 d to 28 d when the ethylene peak existed, indicating that MeJA treatment might directly stimulate the expression of MdACS1 at the early ripening stage. We speculated that ethylene might regulate its own biosynthesis through a negative feedback loop that represses the expression of MdACS1 to avoid producing excessive ethylene during the period when the ethylene peak existed in the MeJAtreated fruit. Tan et al. (2013) found that MdACS3a operated in the system-1 ethylene production and played important roles in determining the shelf life of apple fruit (Wang et al., 2009). In the current study, MeJA treatment enhanced the expression of MdACS3a, indicating that it was positively regulated by MeJA treatment during ripening. Previous study has revealed that MdACS6 and MdACS8 might work in the system-2 ethylene biosynthesis (Li et al., 2013). In our study, the expression of MdACS6 and MdACS8 was differentially upregulated by MeJA treatment, indicating that they were positively
Fig. 6. Expression of MdERFs in apple fruit during storage at 20 °C after treatment with 0.5 mM MeJA. Mdactin was used as an internal standard for each gene. The values for each time point represent an average of three PCR runs for three biological replicates. Vertical bars represent standard errors of means. The LSD (least significant difference) values were calculated at α-value of 0.05. Different letters above or below each data point indicate significant differences between treatment and control groups for each sampling day (P < 0.05).
the expression of MdCTR1-1 and MdCTR1-2 was observed between both groups, indicating that their expression was not regulated by MeJA treatment during ripening (Fig. 4A and B). The expression of MdCTR1-3 in MeJA-treated fruit was 1.5-fold and 1.2-fold higher than that in control fruit at 7 d and 28 d, respectively (Fig. 4C). MeJA treatment significantly increased the expression of MdCTR1-4 and MdCTR1-5 at 7 d, whereas after 7 d, no significant differences in their expression were observed between both groups (Fig. 4D and E). The expression of MdEIN2A in MeJA-treated fruit was significantly higher than that in control fruit before 21 d (Fig. 5A), suggesting some degree of the positive effect of MeJA on its expression. Similar to the effect on the expression of MdCTR1-4 and MdCTR1-5, MeJA treatment significantly up-regulated the expression of MdEIN2B at 7 d (Fig. 5B). The expression of MdEIL1 showed an increasing trend in both groups upon ripening (Fig. 5C). Its expression was significantly enhanced by MeJA treatment throughout the entire storage period, indicating that the expression of MdEIL1 was positively regulated by MeJA treatment during ripening. The expression of MdEIL2 showed about 4-fold increase upon ripening in both groups (Fig. 5D). MeJA treatment had no apparent effect on its expression, indicating that the expression of MdEIL2 was not regulated by MeJA treatment during ripening. In control fruit, the expression of MdEIL3 increased from 0 d to 14 d, whereas after 14 d, no large changes in its expression were observed (Fig. 5E). Application of MeJA significantly up-regulated its expression from 21 d to 28 d compared with controls, indicating that the expression of MdEIL3 was positively regulated by MeJA treatment at the late 163
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EIN2 is described as being a bi-functional transducer, which mediates an essential step in the ethylene signal propagation between CTR1 and the downstream components (EIN3/EIL) (Alonso et al., 1999). Kim et al. (2013) found that reducing jasmonic acid levels caused ein2 ethylene-insensitive mutants to become ethylene responsive, and therefore they suggested that there might be an EIN2-independent component in ethylene signaling that was inhibited by JA. In this study, the expression of MdEIN2A and MdEIN2B was positively regulated by MeJA treatment to some extent at the early ripening stage. This observation seems contradictory to the previous findings. Because apples and Arabidopsis (Arabidopsis thaliana) belong to different plant species, it is necessary to further investigate the molecular mechanisms of the interaction between EIN2 and JA signaling in apple fruit. EIN3/EILs are positive regulators within the ethylene signal cascade (Tieman et al., 2001). Generally, the expression of MdEIL1-4 increased upon ripening, except for a little decrease in the expression of MdEIL3 at 21 d, indicating that MdEIL1-4 were ripening-related genes. This is consistent with previous findings in other fruit systems, such as banana (Musa acuminata) (Mbeguie-A-Mbeguie et al., 2008) and melon (Cucumis melo) (Huang et al., 2010). It has been confirmed that EIN3/EIL1 physically interacted with JA-Zim domain (JAZ) proteins, a repressor of JA signaling, and integrated ethylene and JA signaling in Arabidopsis (Zhu et al., 2011). However, very little is known about the specific element in integration of both signals in apple fruit. In our study, the expression of MdEIL1, MdEIL3 and MdEIL4 was differentially induced by MeJA treatment. Similar results were also obtained in Arabidopsis (Zhu et al., 2011) and rice (Oryza sativa) (Hiraga et al., 2009). Further studies, such as yeast two-hybrid assay and pull-down assay, are needed to identify which members of the EIL family that interact with JA signaling pathway in apple fruit. The expression of the two MdERF genes was differentially regulated by MeJA treatment during ripening. The expression of MdERF1 was positively regulated by MeJA treatment at the early ripening stage, whereas the expression of MdERF2 was negatively regulated by it from 14 d to 21 d. Actually, except for EIN3/EILs, ERF proteins also play key roles in the integration of both signals in Arabidopsis (Lorenzo et al., 2003). A recent study on banana fruit showed that MaERF10 physically interacted with MaJAZ3, and the interaction led to deeper repression of JA biosynthetic genes by MaERF10 (Qi et al., 2016). Therefore, we speculate that there are two possible reasons for the down-regulation of MdERF2 expression: i) there exist JA-related regulators that interact with MdERF2 and suppress its gene expression; ii) since ERF acts at the last step of the ethylene signaling pathway and controls the transcription of ethylene responsive genes (El-Sharkawy et al., 2009; Klee and Giovannoni, 2011), the down-regulation of MdERF2 may function to repress ethylene responses which are induced by higher than the normal levels of ethylene, and thus maintain the metabolic balance in the MeJA-treated fruit. Our data confirmed that the expression of genes involved in the ethylene biosynthesis and signaling pathway was differentially regulated by JAs during postharvest ripening of apple fruit. Understanding the expression patterns of these genes involved in the ethylene biosynthesis and signaling pathway in response to JAs may help to decode the complex mechanisms of the crosstalk between the ethylene and JA signaling pathways in apple fruit.
regulated by MeJA treatment during ripening. It has been reported that MdACO1 might be one of the major factors of the system-2 ethylene biosynthesis (Schaffer et al., 2007; Wang and Xu 2012; Wiersma et al., 2007). In this study, the expression of MdACO1 was significant higher in MeJA-treated fruit than that in control fruit, indicating that MdACO1 might be involved in the MeJA-induced ethylene accumulation during ripening. Information about the function of MdACO2 is limited. Wiersma et al. (2007) found that expression of MdACO2 was in a cultivar-dependent manner during fruit ripening. The present study showed that expression of MdACO2 was positively regulated by MeJA treatment during ripening. Taken together, our data discussed above indicated that the expression of key genes involved in the ethylene biosynthesis pathway was differentially regulated by JAs during postharvest ripening of apple fruit. Ethylene receptors function as negative regulators of the ethylene signaling pathway, and reduced or induced levels of the receptors appear to confer high ethylene sensitivity or to counteract the ethylene signal, respectively (Hua and Meyerowitz, 1998; Tieman et al., 2000). The expression of MdETR1, MdETR2, MdERS1 and MdERS2 was differentially up-regulated by MeJA treatment compared with controls during ripening, suggesting that these four genes were positively regulated by MeJA. We speculated that the up-regulated expression of these four ethylene receptor genes might be associated with the enhancement of the MeJA-induced ethylene biosynthesis, which might be a mechanism for adaptation in ethylene responses (Hua and Meyerowitz, 1998). In MeJA-treated fruit, the higher level of ethylene production required more ethylene receptors to induce specific responses by relieving the repression activity of the ethylene receptors, and at the same time, it differentially up-regulated the expression of the ethylene receptor genes. The increased expression of the ethylene receptor genes might lead to the production of more proteins that can repress the ethylene response. However, changes in the transcriptional level do not systematically imply changes in the protein level and function. Tatsuki et al. (2009) observed that MdERS1 and MdERS2 transcription increased rapidly after harvest while their protein levels behaved differently. Therefore, it is necessary to further identify the specific members of ethylene receptors involved in the JAs-induced ethylene responses at the protein level during the ripening of apple fruit. CTR1 negatively regulates downstream ethylene signaling events in the absence of ethylene (Stepanova and Alonso, 2009). To date, only one MdCTR1 gene (AY670703) has been identified in apple fruit (Dal Cin et al., 2005), and its full length cDNA was characterized by several splice variants (Wiersma et al., 2007). Yang et al. (2013) characterized the splice variants as follows: MdCTR1-1 and MdCTR-2 (primers located between the first intron and the start of the kinase domain), MdCTR1-3 and MdCTR-4 (those retain 3′ UTR region), and MdCTR1-5 (the one located within kinase domain). Dal Cin et al. (2006) found that the expression of MdCTR1 was up-regulated in fruit during ripening. Similar results were also obtained from the present study. The expression of MdCTR1s increased and showed similar trends in control fruit upon ripening, indicating that these splice variants remained as a consistent portion of the total. However, they responded differentially to the MeJA treatment during ripening. The expression of MdCTR1-3, MdCTR1-4 and MdCTR1-5 was up-regulated by MeJA treatment at the early ripening stage compared with controls, whereas the expression of MdCTR1-1 and MdCTR1-2 was not significantly affected by MeJA treatment during the entire storage period. Previous studies showed that the expression of MdCTR1-3, MdCTR1-4 and MdCTR1-5 was ethylene inducible (Li and Yuan, 2008; Yang et al., 2013). In this study, no significant difference in the expression of MdCTR1-4 and MdCTR1-5 was observed between both groups during the late storage period, although the levels of ethylene production were higher in the MeJA-treated fruit than that in the control fruit during the same storage period. Further analysis is needed to clarify the relationship among MdCTR1s, ethylene and JA signaling in ripening apple fruit.
5. Conclusion In conclusion, MeJA treatment increased the system-2 ethylene production during ripening. The expression of genes involved in the ethylene biosynthesis and signaling pathway was positively regulated by MeJA treatment at varying stages of ripening, except for MdACS1, MdCTR1-1, MdCTR1-2, MdEIL2 and MdERF2. The expression of MdACS1 and MdERF2 was negatively regulated by MeJA treatment when the ethylene climacteric peak existed, whereas the expression of MdCTR1-1, MdCTR1-2 and MdEIL2 was not regulated by it during fruit 164
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