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SlMYC2 are required for methyl jasmonate-induced tomato fruit resistance to Botrytis cinerea
Food Chemistry 310 (2020) 125901
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SlMYC2 are required for methyl jasmonate-induced tomato fruit resistance to Botrytis cinerea
T
Dedong Mina,1, Fujun Lia,1, Xixi Cuia, Jingxiang Zhoua, Jiaozhuo Lia, Wen Aia, Pan Shua, ⁎ ⁎ Xinhua Zhanga,b, , Xiaoan Lia, Demei Mengc, Yanyin Guoa, Jian Lib, a
School of Agricultural Engineering and Food Science, Shandong University of Technology, Zibo 255049, Shandong, PR China Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology & Business University (BTBU), Beijing 100048, PR China c Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: MYC2 transcription factor Methyl jasmonate Tomato fruit Disease resistance
The mechanism of SlMYC2, involved in methyl jasmonate (MJ)-induced tomato fruit resistance to pathogens, was investigated. The data indicated that MJ treatment enhanced the accumulation of total phenolics and flavonoids, as well as individual phenolic acids and flavonoids, which might be caused by the increased phenylalanine ammonia-lyase and polyphenol oxidase activities, induced pathogenesis-related gene (PR) expression, β-1,3-glucanase and chitinase activities, as well as α-tomatine, by inducing GLYCOALKALOID METABOLISM gene expression. These effects, induced by MJ, partly contributed to tomato fruit resistance to Botrytis cinerea. Nevertheless, the induction effects of MJ were almost counteracted by silence of SlMYC2, and the disease incidence and lesion diameter in MJ + SlMYC2-silenced fruit were higher than those in MJ-treated fruit. These observations are the first evidence that SlMYC2 plays vital roles in MJ-induced fruit resistance to Botrytis cinerea, possibly by regulating defence enzyme activities, SlPRs expression, α-tomatine, special phenolic acids and flavonoid compounds.
1. Introduction Botrytis cinerea (B. cinerea), a fungal pathogen that limits fruit longdistance transport and storage and results in considerable economic losses, has been attracting increasing attention (Van Kan et al., 2017). B. cinerea, as a vital plant-pathogenic fungus, has attracted great attention because of its economic and scientific importance (Dean et al., 2012). Utilization of synthetic fungicides is the main strategy at present for managing fungal diseases of postharvest fruit, but it has negative effects on both the environment and human health (Jankowska, Kaczynski, Hrynko, & Lozowicka, 2016). Thus, it is imperative to explore safe and effective alternatives for disease management in order to reduce postharvest losses of fresh vegetables and fruit. Phytohormones have essential regulatory roles in the defence against pathogen invasion or insects attack in plant (Du et al., 2017). Jasmonic acid (JA) and methyl jasmonate (MJ), as endogenous signal molecules, are pivotal for many crop processes from plant growth and development to plant defence against pathogens (Reyes-Diaz et al., 2016). In addition, current reports have pointed out that MJ was more
valid than JA in inducing defence responses (Jiang & Yan, 2018), and it has been widely used for disease control in fruits and vegetables, such as Chinese bayberry (Wang et al., 2014), peach fruit (Jin, Zheng, Tang, Rui, & Wang, 2009) and tomato (Zhu & Tian, 2012). These studies indicated that appropriate doses of MJ could enhance disease resistance and decrease postharvest losses in various horticultural products. Nevertheless, the molecular mechanism of MJ-mediated fruit resistance remains largely unclear. Decades of reports have found that MYC2 is an important transcription factor involved in the regulation of many MJ-dependent physiological processes in plants (Du et al., 2017; Kazan & Manners, 2013). In banana, MaMYC2s participated in MJ-mediated banana fruit cold tolerance by interacting with MaICE1 and regulating the expression levels of cold-relevant genes (Zhao et al., 2013). In rice, OsMYC2 promoted the accumulation of defence-related compounds (Ogawa et al., 2017). All these studies indicate that MYC2 is a vital regulator of MJ signalling transduction. In addition, phenolics and α-tomatine played key roles in plant defence mechanisms against fungi, bacteria and viruses (Zhang et al., 2017; Koh, Kaffka, & Mitchell, 2013). The
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Corresponding authors. E-mail addresses: [email protected] (X. Zhang), [email protected] (J. Li). 1 These authors contributed equally. https://doi.org/10.1016/j.foodchem.2019.125901 Received 3 July 2019; Received in revised form 12 November 2019; Accepted 12 November 2019 Available online 30 November 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
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ethylene diaminetetraacetic acid (EDTA) and 5 mM β-mercaptoethanol (β-ME) for PAL, 0.1 M phosphate buffer (pH 6.8) for PPO, 0.1 M sodium acetate containing 5 mM β-ME, 1 mM EDTA and 8% (w/v) PVP for GLU and CHI. The activities of PAL, PPO, GLU, and CHI were assayed according to our previously reported methods (Zhang et al., 2017) and expressed as U mg−1 of protein which were analyzed as described by Bradford (1976).
researches showed that MYC2 also played an important role in regulating secondary metabolism, such as taxol phenolic acid (Zhang et al., 2018; Zhou et al., 2016); and, SlMYC2 might regulate the α-tomatine biosynthesis by cooperating with GLYCOALKALOID METABOLISM (GAME) genes in tomato (Cardenas et al., 2016). However, there are few reports on the functions of SlMYC2 in MJ-mediated fruit resistance to B. cinerea. We do not know whether SlMYC2 plays a role in MJmediated fruit disease resistance, or how does it work, by regulating phenolics and α-tomatine metabolism or altering other regulatory pathways? To assess the role of SlMYC2 in MJ-mediated fruit disease resistance, the interaction between tomato fruit and B. cinerea acted as a model system, and the fruits with SlMYC2 gene silence established by virusinduced gene silencing (VIGS), served as an excellent material to assay the roles of SlMYC2 in the disease resistance of fruit. The defence enzyme activities, such as polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL), β-1,3-glucanase (GLU) and chitinase (CHI), and the contents of total or individual phenolics and flavonoids, as well as αtomatine, were assayed. Moreover, the transcriptional changes of pathogenesis-related (PR) genes, four GAME genes (SlGAME 1, 17, 18 and 2) and their regulator SlGAME9 were also detected.
2.5. Assay of total phenolic and flavonoid contents Extraction and measurements of total phenolics were performed according to Zheng et al. (2011). 1.5 g of tomato fruit tissue were ground with 95% (v/v) ethyl alcohol and centrifuged at 10000 rpm for 10 min after standing in the dark for 24 h. The supernatant (1 ml) was mixed with 50% Folin-Ciocalteu reagent (0.5 ml), 95% (v/v) ethyl alcohol (1 ml) and distilled water (5 ml). Five minutes later, 5% (w/v) Na2CO3 (1 ml) was added to the above mixture and stored at room temperature in darkness for 60 min. Then, the mixture was photometrically assayed at 685 nm. The content of flavonoids was detected as previously reported by Li et al. (2018) and the result was described as catechin equivalents, based on fresh weight (g kg−1). 2.6. Individual phenolic acids and flavonoids by high-performance liquid chromatography (HPLC).
2. Materials and methods 2.1. Fruit materials and treatments
The individual phenolic and flavonoid compounds were assayed by HPLC, as described by the method of Surjadinata and Cisneros-Zevallos (2012) with some modification. 10 g of tomato fruit tissue were grouped with 20 ml of methanol, followed by storage in darkness at 4 °C overnight. The supernatant obtained after centrifugation (10 000 rpm, 30 min.) was concentrated to 5 ml by vacuum evaporation at 35 °C, followed by C-18 Sep Pak column chromatography. After washing with water (3 ml), the sample was eluted with methanol (5 ml) and filtered through a 0.22 µm PTFE filter (0.22 µm). Finally, the extract (20 µl) was injected into an HPLC system (Ailent 1260 Infinity II, USA) equipped with autosampler, quaternary pump and photodiode array detector. A C18 reverse column (250 × 4.6 mm, 5 µm, Diamonsil) was used to separate the samples and was maintained at 40 °C. The two mobile phases contained (pH 2.3) hydrochloric acid (solvent A) and acetonitrile (solvent B) with the flow rate as 0.6 ml min−1. The gradient programme was: 0–10 min; 15% B, 10–20 min; 20% B, 20–40 min; 40% B, 40–45 min; 80% B, 45–50 min: 15% B. The diode array detector monitored wavelengths of 280 nm. Commercial standards of quercetin, naringin, phloridzin, rutin, kaempferol, hesperidin, p-coumaric acid, gallic acid, chlorogenic acid, catechin, ferulic acid, salicylic acid, caffeic acid and epicatechin were used for peak identification when possible. Data were expressed in µg g−1 of fresh weight.
In this work, the SlMYC2-silenced tomato fruits (Solanum lycopersicum L. cv. Badun) were established according to our previous study (Min et al., 2018a). In order to silence SlMYC2, we employed tobacco rattle virus (TRV) to construct the recombinant vector pTRV2-SlMYC2 and the carpopodium of tomato fruit was infected with the Agrobacterium suspensions containing pTRV1 and pTRV2 (as control) or pTRV1 and pTRV2-SlMYC2 (as SlMYC2-silenced fruit) in a 1:1 ratio after ten days of pollination. We also measured the SlMYC2 expression level and found that the expression level of SlMYC2 in the SlMYC2silenced fruit was significantly inhibited with values less than half that of the control, indicating that SlMYC2 was silenced successfully. The two groups of fruits were harvested at the mature green stage and divided into two subgroups, respectively. Subsequently, each subgroup was treated with MJ at different concentrations of 0 and 0.05 mmol l−1, based on the results of our previous work (Min et al., 2018a). There were three replicates in each treatment (40 fruit / replicate). 2.2. Pathogen incubation B. cinerea was isolated and purified from rotten fruit, and the spore suspension (2 × 105 spores per millilitre) was obtained from the strains after seven days of incubation at 24 °C on the potato dextrose agar medium.
2.7. Evaluation of α-tomatine content
After treatment with MJ, 10 fruits were chosen randomly from each replicate of each group and wounded at two points by a sterile nail (4 mm deep × 2 mm wide) on the fruit equator; then each wound was inoculated with 10 μl of spore suspension. Disease development was evaluated at 3 d intervals during fruit storage at 25 °C with 80–90% relative humidity. Disease incidence (%) = fruits showing disease symptoms / total fruits × 100.
α-Tomatine analysis was carried out as described by Keukens, Hop, and Jongen (1994). Two grammes of fruit were extracted with 5 ml of methanol. After centrifuging, the supernatant was concentrated to 3 ml by vacuum evaporation at 35 °C. The extract was passed through a 0.22 µm PTFE filter. HPLC analysis was carried out. The following conditions were performed for analyses: mobile phase, acetonitrile/ 20 mM KH2PO4 (25/75, v/v); injection volume, 20 µl; flow rate, 0.6 ml min−1. Separation was also carried out in a C18 reverse column and maintained at 20 °C. Chromatograms were analyzed at 208 nm. The results were expressed as µg g−1 of fresh weight.
2.4. Extraction and measurement of enzyme activities
2.8. Quantitative real-time polymerase chain reaction (qRT-PCR) assay.
For different crude enzyme extracts, fruit tissue (1.5 g) was ground with 5 ml of different extraction buffers: 0.2 M sodium borate buffer, pH 8.8) containing 10% (w/v) polyvinylpyrrolidone (PVP), 1 mM
Extraction of total RNA and synthesis of first-strand complementary DNA (cDNA) were carried out according to our previous study (Min et al., 2018a). Subsequently, the expression levels of genes, such as
2.3. Disease incidence and lesion diameter
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Treatment with MJ greatly inhibited the increase of disease incidence as shown in Fig. 1b and with a value of about 34% lower than the control fruit on day 12 of the storage. The disease incidence in SlMYC2-sienced fruit was higher than that in other treatments, whereas no significant differences were observed on disease incidence between control and SlMYC2-sienced fruits treated by MJ throughout the storage periods except on day 12. The similar treatment effects were also shown by the changes of lesion diameter (Fig. 1c) and the typical symptom of B. cinerea invasion (Fig. 1d). MJ treatment effectively inhibited the expansion of lesion diameter and the development of disease symptoms, while the silence of SlMYC2 reduced the inhibitory effects of MJ on fruit disease development.
SlMYC2, SlPR1, SlPR2a, SlPR2b, SlPR3a, SlPR3b, SlGAME1, SlGAME2, SlGAME9, SlGAME17 and SlGAME18, were assayed on a LineGene 9600 detection system (Bioer, HangZhou, China) with SYBR Green I Master Mix (Toyobo, Osaka, Japan) (Min et al., 2018a; Zhang et al., 2017). In addition, the SlUbi3 was used as host gene. All data were calculated according to the 2−ΔΔCt method. Specific primers of each gene used in qRT-PCR are shown in Supplementary Table S1. 2.9. Statistical analysis The experiment was designed randomly with each treatment replicated 3 times. The data were analyzed statistically, using one-way analysis of variance (ANOVA) and Ducan’s multiple range tests with SPSS 19.0 (SPSS Inc., Chicago, IL, USA). Differences at P < 0.05 were considered as significant. Pearson’s correlation analysis was applied to determine the correlations among the lesion diameter, phenolic compounds and α-tomatine, using R software.
3.2. Activities of PAL and PPO and total/special phenolic acids and flavonoid compound contents As shown in Fig. 2a–b, MJ treatment induced the activities of PAL and PPO, while the silence of SlMYC2 inhibited the induction which was always retained at relatively low values in comparison to other treatments. Even so, treatment with MJ could also induce those defence enzymes in SlMYC2-silenced fruit and there were no obvious differences between control and MJ + SlMYC2-silenced fruit during most of the storage periods. The UV spectrophotometric determinations were performed at 280 nm for special phenolic and flavonoid compounds, respectively. The different analytical performance parameters, such as standard curve, R2, limit of detection (LOD) and limit of quantification (LOQ), were determined (Table S2). All compounds showed good linearity. The calculated LOQ and LOD concentrations confirmed that the method was
3. Results 3.1. SlMYC2 expression, disease incidence and lesion diameter As shown in Fig. 1a, the SlMYC2 relative expression was measured by qRT-PCR. MJ treatment rapidly induced the SlMYC2 expression within 3 days, and it was higher than that in other treatments. The SlMYC2 expression in SlMYC2-silenced fruit was 53% lower than that in control fruit on day 0 and was also markedly inhibited throughout storage periods. There was no significant difference with (MJ + SlMYC2-silenced) fruit, except on day 6.
Fig. 1. Effects of MJ and SlMYC2 silence on SlMYC2 expression (a), disease incidence (b), lesion diameter (c) and symptoms (d) of tomato fruit during storage at 25 °C. The transcript levels were measured by qRT-PCR, normalized to the host SlUbi3 gene, and set relative to the control fruit from day 0. Vertical bars represent the standard errors of the means. Means in a column followed by a different letter differ significantly at P < 0.05 by Duncan's multiple range tests. 3
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Fig. 2. Effects of MJ and SlMYC2 silence on activities of PAL (a), PPO (b) and total and special phenolic and flavonoid contents (c) of tomato fruit during storage at 25 °C. Vertical bars represent the standard errors of the means. Means in a column followed by a different letter differ significantly at P < 0.05 by Duncan's multiple range tests. Normalized values are shown on a colour scale, which is proportional to the content of each identified metabolite. Each square represents relative contents of phenolic compounds and α-tomatine with a colour scale (colour scale key at the side of the Figure). Data are mean values of three independent biology replicates.
content, regardless of whether or not fruit were silenced. On day 9, the α-tomatine content of MJ-treated fruit was increased 109% compared with that in MJ + SlMYC2-silenced fruit that showed no significant difference from control fruit. In addition, we also analyzed the relative expression levels of GAME genes (SlGAME1, SlGAME2, SlGAME9, SlGAME17 and SlGAME18) which played important roles in the biosynthesis of α-tomatine. As shown in Fig. 3, MJ treatment could significantly induce the expression levels of SlGAMEs during most of the storage time. However, the silence of SlMYC2 decreased SlGAME gene expressions in comparison to control fruit and inhibited the induction effect of MJ on those genes. In MJ + SlMYC2-silenced fruit, the expressions of SlGAMEs were lower than those in MJ-treated fruit and there were no significant differences from control fruit.
sufficiently sensitive for the quantitative evaluation of the special phenolic and flavonoid compounds. Data of total/special phenolic and flavonoid compounds are shown in Tables S3 and S4. Among them, MJ treatment induced the accumulation of total and special phenolic acids and flavonoid compounds, such as gallic acid, p-coumaric acid, chlorogenic acid, ferulic acid, catechin, salicylic acid, quercetin, naringin and phloridzin, regardless of whether or not fruit were silenced by VIGS. And, most of the time, no significant differences were found between MJ + SlMYC2-silenced fruit and control, and these compounds in SlMYC2-silenced fruit were lower than those in other treatments. But, in some periods, there were no differences between MJ and MJ + SlMYC2-silenced fruit, e.g. on day 6 of chlorogenic acid and on day 9 of ferulic acid. The contents of quercetin and naringin from all treatments were not significantly different at the beginning and end of storage, respectively. In addition, the levels of caffeic acid, epicatechin, rutin, kaempferol and hesperidin did not significantly change after treatment with MJ compared to other treatments at most storage periods. Following hierarchical clustering analysis, the four treatments (each with four samples from different storage periods) could be grouped into two clear clusters (Fig. 2). Cluster I contained the samples from control and MJ + SlMYC2-silenced fruit on day 6 and all MJ-treated fruit, had relatively higher phenolic acid and flavonoid compound contents compared to other samples. Cluster II contained the remaining samples, in which the phenolic and flavonoid compound contents were lower, especially in fruit with SlMYC2 silence.
3.4. Correlation analysis between lesion diameter and the content of total/ special phenolic compounds or α-tomatine The correlation coefficients among lesion diameter, total/special phenolic acids and flavonoid compounds are shown in Fig. 4. In control, MJ and MJ + SlMYC2-silenced tomato fruit, strong relationships between lesion diameter and total phenolics or flavonoids were observed. No matter what treatment, lesion diameter was correlated negatively with gallic acid, chlorogenic acids, and kaempferol. As expected for MJ + SlMYC2-silenced fruit, strong relationships between lesion diameter and ferulic acid were observed. In addition, close relationships were observed among lesion diameter-naringin (r = −0.53), lesion diameter-p-coumaric acid (r = −0.47), and lesion diameter-caffeic acid (r = −0.35) in control tomato fruit. In MJ-treated fruit, lesion diameter was correlated weakly with rutin (r = −0.31). In SlMYC2-silenced fruit, the lesion diameter was also correlated negatively with p-coumaric acid, catechin and caffeic acid (r = −0.96, r = −0.90, and
3.3. α-tomatine content and SlGAME gene expression As shown in Fig. 3, the α-tomatine content exhibited a pattern of decrease in all treatment fruit, especially from day 3 to day 9. MJ treatment also could significantly inhibit the decrease of α-tomatine 4
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Fig. 3. Effects of MJ and SlMYC2 silence on expression levels of SlGAME genes and α-tomatine content in tomato fruit during storage at 25 °C. The transcript levels were measured by qRT-PCR, normalized to the host SlUbi3 gene, and set relative to the control fruit from day 0. Vertical bars represent the standard errors of the means.
r = −0.45, respectively). In addition, there was a strong correlation between lesion diameter and α-tomatine content (r < −0.90) in all treatment fruits.
and GLU activity in MJ + SlMYC2-silenced fruit were still less than those in MJ-treated fruit. MJ treatment did not cause significant change on SlPR3a expression. However, SlPR3b expression in MJ-treated fruit sharply increased and peaked (6.34) on day 3. CHI activity also increased by MJ treatment and peaked on day 9, with a value increased by 26% compared to control fruit. In agreement with the above observation, the silence of SlMYC2 also significantly reduced SlPR3b expression and CHI activity and inhibited the inductive effects of MJ on them. However, SlPR3b expression and CHI activity in MJ + SlMYC2-silenced fruit were largely enhanced in comparison to SlMYC2-silenced fruit during most of the storage periods.
3.5. Expression of SlPR1, SlPR2a, SlPR2b, SlPR3a and SlPR3b and activities of GLU and CHI The relative expression level of SlPR1 in MJ-treated tomato fruit increased rapidly within 6 days, and it was higher than that in other treatments throughout storage periods (Fig. 5). The data analysis showed that SlPR1 expression was increased by 439% compared with the control fruit on day 6. By contrast, the silence of SlMYC2 decreased the transcription of PR-1 compared with other treatments during the storage. In addition, the MJ treatment also significantly induced the expression levels of SlPR1 in SlMYC2-silenced tomato fruit, except on day 6. No significant difference of SlPR1 expression was found between control and (MJ + SlMYC2-silenced) fruit. Transcription of SlPR2a and SlPR2b in MJ-treated fruit increased rapidly to maximal level within day 3, and possessed the highest level in comparison to other treatment throughout the storage periods except on day 12 (Fig. 6a−b). Consequently, the GLU activity was also enhanced by MJ treatment (Fig. 6c). Conversely, the expression levels of SlPR2a and SlPR2b and GLU activity in SlMYC2-silenced fruit always remained at relatively low values in comparison to other treatments during most of the storage periods. The inhibition effects of SlMYC2 silence were largely prevented in MJ + SlMYC2-silenced fruit during most of the storage, whereas the expression levels of SlPR2a and SlPR2b
4. Discussion Fruits and vegetables are susceptible to infection by pathogens which result in enormous losses. Recently, many researchers have suggested that MJ treatment increased the resistance to pathogens and reduced the postharvest losses. For instance, MJ treatment could enhance resistance against pathogens and inhibit the development of decay in sweet cherry fruit (Wang et al., 2015). In our present work, we also discovered that MJ treatment decreased the incidence and development of tomato fruit disease resulting from B. cinerea. However, the detailed molecular mechanisms still need further study. MYC2, an important transcription factor, is involved in the regulation of many MJdependent physiological processes in plants. Previous studies showed that MYC2 had important roles in MJ5
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Fig. 4. Pearson’s correlations analysis. Pearson coefficient among lesion diameter, total and special phenolic acids, flavonoid and α-tomatine content in each treatment fruit were used to create a heat map. Each square represents the correlation among lesion diameter, phenolic compounds and α-tomatine content with a colour scale (colour scale key at the side of the figure).
the silence of SlMYC2 aggravated fruit decay caused by B. cinerea, and MJ treatment significantly inhibited the development of disease in either silenced or non-silenced tomato fruit. In addition, we found that the incidence and symptoms of fruit disease of MJ + SlMYC2-silenced fruit were more severe than in MJ-treated fruit. These findings suggested that the silence of SlMYC2 abated fruit disease resistance induced by MJ. MJ, as an important phytohormone, has pivotal roles in stimulating the biosynthesis of secondary metabolites (Wang et al., 2009). Many studies have revealed that MJ significantly promotes the accumulation of phenolics and flavonoids in plants (Wang et al., 2009). PAL is the main enzyme associated with the biosynthesis of phenolics, which is viewed as an indicator involved in plant defence against pathogens. It could be oxidized by PPO to form toxic quinones, which further influence invading pathogens in plant-pathogen interactions (Zhang et al., 2017; Zhang, Tian, Zhu, Xu, & Qin, 2012). Meanwhile, PAL is also an essential enzyme in biosynthesis of flavonoids which occurs widely in plants and plays important roles in plant resistance (Winkel-Shirley, 2001). Therefore, the activities of PAL and PPO were measured in the present study to investigate the possible biosynthesis mechanism of phenolics and flavonoids. Our results demonstrate that MJ treatment could induce activities of PAL and PPO, and further increase total phenolic and flavonoid contents which strongly relate to disease resistance. In addition, the silence of SlMYC2 partly counteracted the induction effect of MJ on phenolic and flavonoid compound contents by inhibiting the activities of PAL and PPO. More concretely, there was a large increase in the levels of some phenolic acids and flavonoid compounds in MJ-treated fruit, such as gallic acid, chlorogenic acid, p-coumaric acid, ferulic acid, catechin, salicylic acid, naringin, quercetin and phloridzin. Correlation analysis suggested that the lesion diameter correlated negatively with gallic acid, chlorogenic acid, ferulic acid, rutin and kaempferol in MJ-treated fruit. Thus, the MJ treatment might enhance the tomato fruit resistance
Fig. 5. Effects of MJ and SlMYC2 silence on SlPR1 expression of tomato fruit during storage at 25 °C. The transcript levels were measured by qRT-PCR, normalized to the host SlUbi3 gene, and set relative to the control fruit from day 0. Vertical bars represent the standard errors of the means.
dependent physiological processes in plants. For example, OsMYC2 positively regulated the disease resistance in rice (Uji et al., 2016). Shen et al. (2010) suggested that AaMYC2 could markedly induce artemisinin biosynthesis. Our previous study found that SlMYC2 participated in the chilling resistance induced by MJ in tomato fruit (Min et al., 2018a). Although these results had reported that MYC2 was a key regulator in JA response, the function of MYC2 in MJ-induced disease resistance of postharvest fruit is unclear. Thus, in our study, the fruits with SlMYC2 silence were established by VIGS, as described in our previous methods (Min et al., 2018a). The data clearly indicated that 6
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Fig. 6. Effects of MJ and SlMYC2 silence on expression levels of SlPR2a (a), SlPR2b (b), SlPR3a (d) and SlPR3b (e) and activities of GLU (c) and CHI (f) in tomato fruit during storage at 25 °C. The transcript levels were measured by qRT-PCR, normalized to the host SlUbi3 gene, and set relative to the control fruit from day 0. Vertical bars represent the standard errors of the means.
compounds might indirectly induce the tomato fruit resistance to B. cinerea by regulating other metabolic pathways. In turn, the contents of other phenolic acids, e.g. caffeic acid and epicatechin, or other flavonoid compounds, such as kaempferol and hesperidin, did not significant change, and, most interesting was that MJ had no effect on rutin which was important for pathogen suppression and could induce disease resistance (Wei et al., 2017). The result was similar to the discovery of Krol et al. (2015), who found that after a 1 h treatment of 15-day-old tomato seedlings with MJ had no effect on rutin content. Compared with MJ- treated fruit, the contents of some phenolics and flavonoids, including gallic acid, chlorogenic acid, p-coumaric acid, ferulic acid, catechin, salicylic acid, quercetin, naringin and phloridzin, were significantly inhibited in MJ + SlMYC2-silenced fruit. Moreover, the lesion diameter was negatively associated with gallic acid, chlorogenic acid, phloridzin and kaempferol, from which we inferred that SlMYC2 participated in MJ-mediated fruit resistance to B. cinerea by directly regulating the biosynthesis of gallic acid, chlorogenic acid, and phloridzin. In addition, some phenolic and flavonoid compounds, e.g. p-
to B. cinerea by directly regulating the biosynthesis of gallic acid, chlorogenic acid and ferulic acid. Among these, gallic acid is the major phenol in tomato fruit (Liu, Cai, Lu, Han & Ying, 2012). Likewise, Wang et al. (2009) also reported that MJ treatment significantly enhanced the gallic acid content in Chinese bayberries and reduced fruit decay. Konan et al. (2014) indicated that MJ enhanced the disease resistance of cotton by promoting ferulic acid biosynthesis. Unlike our results, Krol, Igielski, Pollmann, and Kepczynska (2015) suggested that MJ treatment enhances tomato disease resistance via salicylic acid, which has critical roles in inducing plant systemic acquired resistance (SAR) (Min et al., 2018b), and promotes the accumulation of quercetin which has antimicrobial effects on pathogens. Krol et al. (2015) also found that MJ had no effect on chlorogenic acid which is an important antifungal agent (Wei, Zhou, Peng, Pan, & Tu, 2017). Although, the correlation coefficients among lesion diameter, p-coumaric acid, catechin, salicylic acid, quercetin, naringin and phloridzin were not observed in our work, MJ treatment significantly promoted the accumulation of these phenolic acids and flavonoid compounds. Therefore, these 7
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defence-related enzyme activities and SlPRs expression, including SlPR1, SlPR2a, SlPR2b and SlPR3b, which further decreased fruit disease development. Conversely, the induction effects of MJ on fruit were almost counteracted by SlMYC2 silence. Compared with MJ-treated fruit, the contents of α-tomatine and total / special individual phenolic and flavonoid compounds, e.g. gallic acid, p-coumaric acid, chlorogenic acid, salicylic acid, catechin, phloridzin, naringin and quercetin, were decreased in MJ + SlMYC2-silenced fruit during most of the storage time. Meanwhile, the incidence and symptoms of fruit disease of MJ + SlMYC2-silenced fruit were more severe than those in MJ-treated fruit. Therefore, our findings have demonstrated that SlMYC2 played a positive role in tomato fruit resistance to B. cinerea.
coumaric acid, catechin, quercetin, salicylic acid, ferulic acid and naringin, might indirectly involve in SlMYC2-induced fruit resistance, which needs to be further studied in the future. Meanwhile, α-tomatine, a resistance substance, plays essential roles in plant defence mechanisms against fungi, bacteria and viruses, and the level of α-tomatine quickly decreases as tomato fruit ripens (Koh et al., 2013). Recently, many reports suggest that MJ induced the αtomatine biosynthetic pathway (Abdelkareem et al., 2017) and further enhanced the accumulation of α-tomatine (Montero-Vargas et al., 2018). Similar to those results, we found that MJ inhibited the decrease of α-tomatine content during storage. Nevertheless, the α-tomatine content in MJ + SlMYC2-silenced fruit was lower than that in MJtreated fruit. And, the correlation analysis showed that lesion diameter was strongly correlated with α-tomatine content. In addition, research shows that SlGAME9 was co-expressed with SlGAME1, SlGAME17 and SlGAME18 and might regulate the α-tomatine biosynthesis by cooperating with SlMYC2 (Cardenas et al., 2016). Those GAME genes (SlGAME1, SlGAME2, SlGAME17 and SlGAME18) are involved in the process of glycosylation (Cardenas et al., 2015), which plays an important role in α-tomatine biosynthesis; thus we further analyzed the relative expression levels of GAME genes, including SlGAME1, SlGAME2, SlGAME17 and SlGAME18. Our findings indicate that MJ could induce expressions of GAME genes (SlGAME1, SlGAME2, SlGAME9, SlGAME17 and SlGAME18). However, the silence of SlMYC2 not only inhibited SlGAME9 expression, but also decreased the expression levels of SlGAME1, SlGAME2, SlGAME17 and SlGAME18. The result was similar to previous researches that JA could upregulate the expression of SlGAME9 which plays an important role in the JA signalling pathway (Abdelkareem et al., 2017) and impacts the expression of SlGAME1, SlGAME17 and SlGAME18 (Cardenas et al., 2016). In addition, high expression of SlGAME2 might contribute to the α-tomatine formation and is closely related to plant’s resistance (Shinde et al., 2017). Thus, SlMYC2 might be involved in MJ-induced fruit resistance to B. cinerea by regulating α-tomatine biosynthesis. PR proteins, including 17 different families, are considered as plant defence proteins that are related to prevention of pathogen infection (Kesari, Trivedi, & Nath, 2010). Among them, SlPR1 is usually regarded as an indicator for SAR (Durrant & Dong, 2006). In addition, PR2 and PR3, encoding GLU and CHI, respectively, play important roles in controlling the disease development in plant-disease interactions (Kesari et al., 2010; Yang, Cao, Cai, & Zheng, 2011). Similar to the result of Ding, Wang, Gross, and Smith (2002), we discovered that MJ significantly induced the expression of SlPR1, SlPR2a (extracellular GLU), SlPR2b (intracellular GLU) and SlPR3b (intracellular CHI), but hardly increased the expression of SlPR3a (extracellular CHI). Moreover, MJ treatment induced higher activities of GLU and CHI. However, the silence of SlMYC2 also inhibited the induction effects of MJ on SlPR1, SlPR2a, SlPR2b and SlPR3b expression and activities of GLU and CHI. To summarize, these results might partly explain the mechanism of SlMYC2 participating in MJ-induced fruit resistance to B. cinerea, possibly by regulating expressions of related SlPRs and activities of CHI and GLU.
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The study was supported by the National Natural Science Foundation of China (No. 31772024), and the fund of the Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology & Business University (BTBU). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.125901. References Abdelkareem, A., Thagun, C., Nakayasu, M., Mizutani, M., Hashimoto, T., & Shoji, T. (2017). Jasmonate-induced biosynthesis of steroidal glycoalkaloids depends on COI1 proteins in tomato. Biochemical and Biophysical Research Communications, 489, 206–210. https://doi.org/10.1016/j.bbrc.2017.05.132. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254. https://doi.org/10.1016/0003-2697(76)90527-3. Cardenas, P. D., Sonawane, P. D., Heining, U., Bocobza, S. E., Burdman, S., & Aharoni, A. (2015). The bitter side of nightshades: Genomics drives discovery in Solanaceae steroidal alkaloid metabolism. Phytochemistry, 113, 24–32. https://doi.org/10.1016/ j.phytochem.2014.12.010. Cardenas, P. D., Sonawane, P. D., Polier, J., Bossche, R. V., Dewangan, V., Weithorn, E., ... Aharoni, A. (2016). GAME9 regulates the biosynthesis of steroidal alkaloids and upstream isoprenoids in the plant mevalonate pathway. Nature Communications, 7, 10654–10670. https://doi.org/10.1038/ncomms10654. Dean, R., Van Kan, J. A. L., Pretorius, Z. A., Hammond-Kosack, K. E., Pietro, A. D., Spanu, P. D., ... Foster, G. D. (2012). The Top 10 fungal pathogens in molecular plant pathology. Molecular Plant Pathology, 13, 414–430. https://doi.org/10.1111/j.13643703.2011.00783.x. Ding, C. K., Wang, C. Y., Gross, K. C., & Smith, D. L. (2002). Jasmonate and salicylate induce the expression of pathogenesis-related-protein genes and increase resistance to chilling injury in tomato fruit. Planta, 2002(214), 895–901. https://doi.org/10. 2307/23386920. Du, M. M., Zhao, J. H., Tzeng, D. T. W., Liu, Y. Y., Deng, L., Yang, T. X., ... Li, C. Y. (2017). MYC2 orchestrates a hierarchical transcriptional cascade that regulates jasmonatemediated plant immunity in tomato. Plant Cell, 29, 1883–1906. https://doi.org/10. 1105/tpc.16.0095. Durrant, W. E., & Dong, X. (2006). Systemic acquired resistance. Annual Review Phytopathology, 1, 179–184. Jankowska, M., Kaczynski, P., Hrynko, I., & Lozowicka, B. (2016). Dissipation of six fungicides in greenhouse-grown tomatoes with processing and health risk. Environmrntal Science and Pollution Research, 23, 11885–11900. https://doi.org/10. 1007/s11356-016-6260-x. Jiang, D., & Yan, S. C. (2018). MeJA is more effective than JA in inducing defense responses. Larix olgensis Arthropod-Plant Interactions, 12, 49–56. https://doi.org/10. 1007/s11829-017-9551-3. Jin, P., Zheng, Y. H., Tang, S. S., Rui, H. J., & Wang, C. Y. (2009). Enhancing disease resistance in peach fruit with methyl jasmonate. Journal of the Science of Food and Agriculture, 89, 802–808. https://doi.org/10.1002/jsfa.3516. Kazan, K., & Manners, J. M. (2013). MYC2: The master in action. Molcular Plant, 6, 686–703. https://doi.org/10.1093/mp/sss128. Kesari, R., Trivedi, P. K., & Nath, P. (2010). Gene expression of pathogenesis-related
5. Conclusion MJ treatment promoted the accumulation of α-tomatine by inducing the expressions of GAME genes (SlGAME1, SlGAME2, SlGAME9, SlGAME17 and SlGAME18), and enhanced total phenolics and flavonoids by inducing the activities of PAL and PPO. HPLC results indicated that MJ treatment increased the contents of such individual phenolic and flavonoid compounds, as gallic acid, chlorogenic acid, p-coumaric acid, ferulic acid, catechin acid, salicylic acid, quercetin, naringin and phloridzin, in comparison with control fruit. The correlation coefficients indicated that lesion diameter was correlated with gallic acid (-0.81), chlorogenic acid (-0.46), kaempferol (-0.64), ferulic acid (-0.80) and rutin (-0.31) in MJ-treated fruit. Besides, it also induced 8
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SlMYC2 are required for methyl jasmonate-induced tomato fruit resistance to Botrytis cinerea
T
Dedong Mina,1, Fujun Lia,1, Xixi Cuia, Jingxiang Zhoua, Jiaozhuo Lia, Wen Aia, Pan Shua, ⁎ ⁎ Xinhua Zhanga,b, , Xiaoan Lia, Demei Mengc, Yanyin Guoa, Jian Lib, a
School of Agricultural Engineering and Food Science, Shandong University of Technology, Zibo 255049, Shandong, PR China Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology & Business University (BTBU), Beijing 100048, PR China c Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: MYC2 transcription factor Methyl jasmonate Tomato fruit Disease resistance
The mechanism of SlMYC2, involved in methyl jasmonate (MJ)-induced tomato fruit resistance to pathogens, was investigated. The data indicated that MJ treatment enhanced the accumulation of total phenolics and flavonoids, as well as individual phenolic acids and flavonoids, which might be caused by the increased phenylalanine ammonia-lyase and polyphenol oxidase activities, induced pathogenesis-related gene (PR) expression, β-1,3-glucanase and chitinase activities, as well as α-tomatine, by inducing GLYCOALKALOID METABOLISM gene expression. These effects, induced by MJ, partly contributed to tomato fruit resistance to Botrytis cinerea. Nevertheless, the induction effects of MJ were almost counteracted by silence of SlMYC2, and the disease incidence and lesion diameter in MJ + SlMYC2-silenced fruit were higher than those in MJ-treated fruit. These observations are the first evidence that SlMYC2 plays vital roles in MJ-induced fruit resistance to Botrytis cinerea, possibly by regulating defence enzyme activities, SlPRs expression, α-tomatine, special phenolic acids and flavonoid compounds.
1. Introduction Botrytis cinerea (B. cinerea), a fungal pathogen that limits fruit longdistance transport and storage and results in considerable economic losses, has been attracting increasing attention (Van Kan et al., 2017). B. cinerea, as a vital plant-pathogenic fungus, has attracted great attention because of its economic and scientific importance (Dean et al., 2012). Utilization of synthetic fungicides is the main strategy at present for managing fungal diseases of postharvest fruit, but it has negative effects on both the environment and human health (Jankowska, Kaczynski, Hrynko, & Lozowicka, 2016). Thus, it is imperative to explore safe and effective alternatives for disease management in order to reduce postharvest losses of fresh vegetables and fruit. Phytohormones have essential regulatory roles in the defence against pathogen invasion or insects attack in plant (Du et al., 2017). Jasmonic acid (JA) and methyl jasmonate (MJ), as endogenous signal molecules, are pivotal for many crop processes from plant growth and development to plant defence against pathogens (Reyes-Diaz et al., 2016). In addition, current reports have pointed out that MJ was more
valid than JA in inducing defence responses (Jiang & Yan, 2018), and it has been widely used for disease control in fruits and vegetables, such as Chinese bayberry (Wang et al., 2014), peach fruit (Jin, Zheng, Tang, Rui, & Wang, 2009) and tomato (Zhu & Tian, 2012). These studies indicated that appropriate doses of MJ could enhance disease resistance and decrease postharvest losses in various horticultural products. Nevertheless, the molecular mechanism of MJ-mediated fruit resistance remains largely unclear. Decades of reports have found that MYC2 is an important transcription factor involved in the regulation of many MJ-dependent physiological processes in plants (Du et al., 2017; Kazan & Manners, 2013). In banana, MaMYC2s participated in MJ-mediated banana fruit cold tolerance by interacting with MaICE1 and regulating the expression levels of cold-relevant genes (Zhao et al., 2013). In rice, OsMYC2 promoted the accumulation of defence-related compounds (Ogawa et al., 2017). All these studies indicate that MYC2 is a vital regulator of MJ signalling transduction. In addition, phenolics and α-tomatine played key roles in plant defence mechanisms against fungi, bacteria and viruses (Zhang et al., 2017; Koh, Kaffka, & Mitchell, 2013). The
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Corresponding authors. E-mail addresses: [email protected] (X. Zhang), [email protected] (J. Li). 1 These authors contributed equally. https://doi.org/10.1016/j.foodchem.2019.125901 Received 3 July 2019; Received in revised form 12 November 2019; Accepted 12 November 2019 Available online 30 November 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
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ethylene diaminetetraacetic acid (EDTA) and 5 mM β-mercaptoethanol (β-ME) for PAL, 0.1 M phosphate buffer (pH 6.8) for PPO, 0.1 M sodium acetate containing 5 mM β-ME, 1 mM EDTA and 8% (w/v) PVP for GLU and CHI. The activities of PAL, PPO, GLU, and CHI were assayed according to our previously reported methods (Zhang et al., 2017) and expressed as U mg−1 of protein which were analyzed as described by Bradford (1976).
researches showed that MYC2 also played an important role in regulating secondary metabolism, such as taxol phenolic acid (Zhang et al., 2018; Zhou et al., 2016); and, SlMYC2 might regulate the α-tomatine biosynthesis by cooperating with GLYCOALKALOID METABOLISM (GAME) genes in tomato (Cardenas et al., 2016). However, there are few reports on the functions of SlMYC2 in MJ-mediated fruit resistance to B. cinerea. We do not know whether SlMYC2 plays a role in MJmediated fruit disease resistance, or how does it work, by regulating phenolics and α-tomatine metabolism or altering other regulatory pathways? To assess the role of SlMYC2 in MJ-mediated fruit disease resistance, the interaction between tomato fruit and B. cinerea acted as a model system, and the fruits with SlMYC2 gene silence established by virusinduced gene silencing (VIGS), served as an excellent material to assay the roles of SlMYC2 in the disease resistance of fruit. The defence enzyme activities, such as polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL), β-1,3-glucanase (GLU) and chitinase (CHI), and the contents of total or individual phenolics and flavonoids, as well as αtomatine, were assayed. Moreover, the transcriptional changes of pathogenesis-related (PR) genes, four GAME genes (SlGAME 1, 17, 18 and 2) and their regulator SlGAME9 were also detected.
2.5. Assay of total phenolic and flavonoid contents Extraction and measurements of total phenolics were performed according to Zheng et al. (2011). 1.5 g of tomato fruit tissue were ground with 95% (v/v) ethyl alcohol and centrifuged at 10000 rpm for 10 min after standing in the dark for 24 h. The supernatant (1 ml) was mixed with 50% Folin-Ciocalteu reagent (0.5 ml), 95% (v/v) ethyl alcohol (1 ml) and distilled water (5 ml). Five minutes later, 5% (w/v) Na2CO3 (1 ml) was added to the above mixture and stored at room temperature in darkness for 60 min. Then, the mixture was photometrically assayed at 685 nm. The content of flavonoids was detected as previously reported by Li et al. (2018) and the result was described as catechin equivalents, based on fresh weight (g kg−1). 2.6. Individual phenolic acids and flavonoids by high-performance liquid chromatography (HPLC).
2. Materials and methods 2.1. Fruit materials and treatments
The individual phenolic and flavonoid compounds were assayed by HPLC, as described by the method of Surjadinata and Cisneros-Zevallos (2012) with some modification. 10 g of tomato fruit tissue were grouped with 20 ml of methanol, followed by storage in darkness at 4 °C overnight. The supernatant obtained after centrifugation (10 000 rpm, 30 min.) was concentrated to 5 ml by vacuum evaporation at 35 °C, followed by C-18 Sep Pak column chromatography. After washing with water (3 ml), the sample was eluted with methanol (5 ml) and filtered through a 0.22 µm PTFE filter (0.22 µm). Finally, the extract (20 µl) was injected into an HPLC system (Ailent 1260 Infinity II, USA) equipped with autosampler, quaternary pump and photodiode array detector. A C18 reverse column (250 × 4.6 mm, 5 µm, Diamonsil) was used to separate the samples and was maintained at 40 °C. The two mobile phases contained (pH 2.3) hydrochloric acid (solvent A) and acetonitrile (solvent B) with the flow rate as 0.6 ml min−1. The gradient programme was: 0–10 min; 15% B, 10–20 min; 20% B, 20–40 min; 40% B, 40–45 min; 80% B, 45–50 min: 15% B. The diode array detector monitored wavelengths of 280 nm. Commercial standards of quercetin, naringin, phloridzin, rutin, kaempferol, hesperidin, p-coumaric acid, gallic acid, chlorogenic acid, catechin, ferulic acid, salicylic acid, caffeic acid and epicatechin were used for peak identification when possible. Data were expressed in µg g−1 of fresh weight.
In this work, the SlMYC2-silenced tomato fruits (Solanum lycopersicum L. cv. Badun) were established according to our previous study (Min et al., 2018a). In order to silence SlMYC2, we employed tobacco rattle virus (TRV) to construct the recombinant vector pTRV2-SlMYC2 and the carpopodium of tomato fruit was infected with the Agrobacterium suspensions containing pTRV1 and pTRV2 (as control) or pTRV1 and pTRV2-SlMYC2 (as SlMYC2-silenced fruit) in a 1:1 ratio after ten days of pollination. We also measured the SlMYC2 expression level and found that the expression level of SlMYC2 in the SlMYC2silenced fruit was significantly inhibited with values less than half that of the control, indicating that SlMYC2 was silenced successfully. The two groups of fruits were harvested at the mature green stage and divided into two subgroups, respectively. Subsequently, each subgroup was treated with MJ at different concentrations of 0 and 0.05 mmol l−1, based on the results of our previous work (Min et al., 2018a). There were three replicates in each treatment (40 fruit / replicate). 2.2. Pathogen incubation B. cinerea was isolated and purified from rotten fruit, and the spore suspension (2 × 105 spores per millilitre) was obtained from the strains after seven days of incubation at 24 °C on the potato dextrose agar medium.
2.7. Evaluation of α-tomatine content
After treatment with MJ, 10 fruits were chosen randomly from each replicate of each group and wounded at two points by a sterile nail (4 mm deep × 2 mm wide) on the fruit equator; then each wound was inoculated with 10 μl of spore suspension. Disease development was evaluated at 3 d intervals during fruit storage at 25 °C with 80–90% relative humidity. Disease incidence (%) = fruits showing disease symptoms / total fruits × 100.
α-Tomatine analysis was carried out as described by Keukens, Hop, and Jongen (1994). Two grammes of fruit were extracted with 5 ml of methanol. After centrifuging, the supernatant was concentrated to 3 ml by vacuum evaporation at 35 °C. The extract was passed through a 0.22 µm PTFE filter. HPLC analysis was carried out. The following conditions were performed for analyses: mobile phase, acetonitrile/ 20 mM KH2PO4 (25/75, v/v); injection volume, 20 µl; flow rate, 0.6 ml min−1. Separation was also carried out in a C18 reverse column and maintained at 20 °C. Chromatograms were analyzed at 208 nm. The results were expressed as µg g−1 of fresh weight.
2.4. Extraction and measurement of enzyme activities
2.8. Quantitative real-time polymerase chain reaction (qRT-PCR) assay.
For different crude enzyme extracts, fruit tissue (1.5 g) was ground with 5 ml of different extraction buffers: 0.2 M sodium borate buffer, pH 8.8) containing 10% (w/v) polyvinylpyrrolidone (PVP), 1 mM
Extraction of total RNA and synthesis of first-strand complementary DNA (cDNA) were carried out according to our previous study (Min et al., 2018a). Subsequently, the expression levels of genes, such as
2.3. Disease incidence and lesion diameter
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Treatment with MJ greatly inhibited the increase of disease incidence as shown in Fig. 1b and with a value of about 34% lower than the control fruit on day 12 of the storage. The disease incidence in SlMYC2-sienced fruit was higher than that in other treatments, whereas no significant differences were observed on disease incidence between control and SlMYC2-sienced fruits treated by MJ throughout the storage periods except on day 12. The similar treatment effects were also shown by the changes of lesion diameter (Fig. 1c) and the typical symptom of B. cinerea invasion (Fig. 1d). MJ treatment effectively inhibited the expansion of lesion diameter and the development of disease symptoms, while the silence of SlMYC2 reduced the inhibitory effects of MJ on fruit disease development.
SlMYC2, SlPR1, SlPR2a, SlPR2b, SlPR3a, SlPR3b, SlGAME1, SlGAME2, SlGAME9, SlGAME17 and SlGAME18, were assayed on a LineGene 9600 detection system (Bioer, HangZhou, China) with SYBR Green I Master Mix (Toyobo, Osaka, Japan) (Min et al., 2018a; Zhang et al., 2017). In addition, the SlUbi3 was used as host gene. All data were calculated according to the 2−ΔΔCt method. Specific primers of each gene used in qRT-PCR are shown in Supplementary Table S1. 2.9. Statistical analysis The experiment was designed randomly with each treatment replicated 3 times. The data were analyzed statistically, using one-way analysis of variance (ANOVA) and Ducan’s multiple range tests with SPSS 19.0 (SPSS Inc., Chicago, IL, USA). Differences at P < 0.05 were considered as significant. Pearson’s correlation analysis was applied to determine the correlations among the lesion diameter, phenolic compounds and α-tomatine, using R software.
3.2. Activities of PAL and PPO and total/special phenolic acids and flavonoid compound contents As shown in Fig. 2a–b, MJ treatment induced the activities of PAL and PPO, while the silence of SlMYC2 inhibited the induction which was always retained at relatively low values in comparison to other treatments. Even so, treatment with MJ could also induce those defence enzymes in SlMYC2-silenced fruit and there were no obvious differences between control and MJ + SlMYC2-silenced fruit during most of the storage periods. The UV spectrophotometric determinations were performed at 280 nm for special phenolic and flavonoid compounds, respectively. The different analytical performance parameters, such as standard curve, R2, limit of detection (LOD) and limit of quantification (LOQ), were determined (Table S2). All compounds showed good linearity. The calculated LOQ and LOD concentrations confirmed that the method was
3. Results 3.1. SlMYC2 expression, disease incidence and lesion diameter As shown in Fig. 1a, the SlMYC2 relative expression was measured by qRT-PCR. MJ treatment rapidly induced the SlMYC2 expression within 3 days, and it was higher than that in other treatments. The SlMYC2 expression in SlMYC2-silenced fruit was 53% lower than that in control fruit on day 0 and was also markedly inhibited throughout storage periods. There was no significant difference with (MJ + SlMYC2-silenced) fruit, except on day 6.
Fig. 1. Effects of MJ and SlMYC2 silence on SlMYC2 expression (a), disease incidence (b), lesion diameter (c) and symptoms (d) of tomato fruit during storage at 25 °C. The transcript levels were measured by qRT-PCR, normalized to the host SlUbi3 gene, and set relative to the control fruit from day 0. Vertical bars represent the standard errors of the means. Means in a column followed by a different letter differ significantly at P < 0.05 by Duncan's multiple range tests. 3
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Fig. 2. Effects of MJ and SlMYC2 silence on activities of PAL (a), PPO (b) and total and special phenolic and flavonoid contents (c) of tomato fruit during storage at 25 °C. Vertical bars represent the standard errors of the means. Means in a column followed by a different letter differ significantly at P < 0.05 by Duncan's multiple range tests. Normalized values are shown on a colour scale, which is proportional to the content of each identified metabolite. Each square represents relative contents of phenolic compounds and α-tomatine with a colour scale (colour scale key at the side of the Figure). Data are mean values of three independent biology replicates.
content, regardless of whether or not fruit were silenced. On day 9, the α-tomatine content of MJ-treated fruit was increased 109% compared with that in MJ + SlMYC2-silenced fruit that showed no significant difference from control fruit. In addition, we also analyzed the relative expression levels of GAME genes (SlGAME1, SlGAME2, SlGAME9, SlGAME17 and SlGAME18) which played important roles in the biosynthesis of α-tomatine. As shown in Fig. 3, MJ treatment could significantly induce the expression levels of SlGAMEs during most of the storage time. However, the silence of SlMYC2 decreased SlGAME gene expressions in comparison to control fruit and inhibited the induction effect of MJ on those genes. In MJ + SlMYC2-silenced fruit, the expressions of SlGAMEs were lower than those in MJ-treated fruit and there were no significant differences from control fruit.
sufficiently sensitive for the quantitative evaluation of the special phenolic and flavonoid compounds. Data of total/special phenolic and flavonoid compounds are shown in Tables S3 and S4. Among them, MJ treatment induced the accumulation of total and special phenolic acids and flavonoid compounds, such as gallic acid, p-coumaric acid, chlorogenic acid, ferulic acid, catechin, salicylic acid, quercetin, naringin and phloridzin, regardless of whether or not fruit were silenced by VIGS. And, most of the time, no significant differences were found between MJ + SlMYC2-silenced fruit and control, and these compounds in SlMYC2-silenced fruit were lower than those in other treatments. But, in some periods, there were no differences between MJ and MJ + SlMYC2-silenced fruit, e.g. on day 6 of chlorogenic acid and on day 9 of ferulic acid. The contents of quercetin and naringin from all treatments were not significantly different at the beginning and end of storage, respectively. In addition, the levels of caffeic acid, epicatechin, rutin, kaempferol and hesperidin did not significantly change after treatment with MJ compared to other treatments at most storage periods. Following hierarchical clustering analysis, the four treatments (each with four samples from different storage periods) could be grouped into two clear clusters (Fig. 2). Cluster I contained the samples from control and MJ + SlMYC2-silenced fruit on day 6 and all MJ-treated fruit, had relatively higher phenolic acid and flavonoid compound contents compared to other samples. Cluster II contained the remaining samples, in which the phenolic and flavonoid compound contents were lower, especially in fruit with SlMYC2 silence.
3.4. Correlation analysis between lesion diameter and the content of total/ special phenolic compounds or α-tomatine The correlation coefficients among lesion diameter, total/special phenolic acids and flavonoid compounds are shown in Fig. 4. In control, MJ and MJ + SlMYC2-silenced tomato fruit, strong relationships between lesion diameter and total phenolics or flavonoids were observed. No matter what treatment, lesion diameter was correlated negatively with gallic acid, chlorogenic acids, and kaempferol. As expected for MJ + SlMYC2-silenced fruit, strong relationships between lesion diameter and ferulic acid were observed. In addition, close relationships were observed among lesion diameter-naringin (r = −0.53), lesion diameter-p-coumaric acid (r = −0.47), and lesion diameter-caffeic acid (r = −0.35) in control tomato fruit. In MJ-treated fruit, lesion diameter was correlated weakly with rutin (r = −0.31). In SlMYC2-silenced fruit, the lesion diameter was also correlated negatively with p-coumaric acid, catechin and caffeic acid (r = −0.96, r = −0.90, and
3.3. α-tomatine content and SlGAME gene expression As shown in Fig. 3, the α-tomatine content exhibited a pattern of decrease in all treatment fruit, especially from day 3 to day 9. MJ treatment also could significantly inhibit the decrease of α-tomatine 4
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Fig. 3. Effects of MJ and SlMYC2 silence on expression levels of SlGAME genes and α-tomatine content in tomato fruit during storage at 25 °C. The transcript levels were measured by qRT-PCR, normalized to the host SlUbi3 gene, and set relative to the control fruit from day 0. Vertical bars represent the standard errors of the means.
r = −0.45, respectively). In addition, there was a strong correlation between lesion diameter and α-tomatine content (r < −0.90) in all treatment fruits.
and GLU activity in MJ + SlMYC2-silenced fruit were still less than those in MJ-treated fruit. MJ treatment did not cause significant change on SlPR3a expression. However, SlPR3b expression in MJ-treated fruit sharply increased and peaked (6.34) on day 3. CHI activity also increased by MJ treatment and peaked on day 9, with a value increased by 26% compared to control fruit. In agreement with the above observation, the silence of SlMYC2 also significantly reduced SlPR3b expression and CHI activity and inhibited the inductive effects of MJ on them. However, SlPR3b expression and CHI activity in MJ + SlMYC2-silenced fruit were largely enhanced in comparison to SlMYC2-silenced fruit during most of the storage periods.
3.5. Expression of SlPR1, SlPR2a, SlPR2b, SlPR3a and SlPR3b and activities of GLU and CHI The relative expression level of SlPR1 in MJ-treated tomato fruit increased rapidly within 6 days, and it was higher than that in other treatments throughout storage periods (Fig. 5). The data analysis showed that SlPR1 expression was increased by 439% compared with the control fruit on day 6. By contrast, the silence of SlMYC2 decreased the transcription of PR-1 compared with other treatments during the storage. In addition, the MJ treatment also significantly induced the expression levels of SlPR1 in SlMYC2-silenced tomato fruit, except on day 6. No significant difference of SlPR1 expression was found between control and (MJ + SlMYC2-silenced) fruit. Transcription of SlPR2a and SlPR2b in MJ-treated fruit increased rapidly to maximal level within day 3, and possessed the highest level in comparison to other treatment throughout the storage periods except on day 12 (Fig. 6a−b). Consequently, the GLU activity was also enhanced by MJ treatment (Fig. 6c). Conversely, the expression levels of SlPR2a and SlPR2b and GLU activity in SlMYC2-silenced fruit always remained at relatively low values in comparison to other treatments during most of the storage periods. The inhibition effects of SlMYC2 silence were largely prevented in MJ + SlMYC2-silenced fruit during most of the storage, whereas the expression levels of SlPR2a and SlPR2b
4. Discussion Fruits and vegetables are susceptible to infection by pathogens which result in enormous losses. Recently, many researchers have suggested that MJ treatment increased the resistance to pathogens and reduced the postharvest losses. For instance, MJ treatment could enhance resistance against pathogens and inhibit the development of decay in sweet cherry fruit (Wang et al., 2015). In our present work, we also discovered that MJ treatment decreased the incidence and development of tomato fruit disease resulting from B. cinerea. However, the detailed molecular mechanisms still need further study. MYC2, an important transcription factor, is involved in the regulation of many MJdependent physiological processes in plants. Previous studies showed that MYC2 had important roles in MJ5
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Fig. 4. Pearson’s correlations analysis. Pearson coefficient among lesion diameter, total and special phenolic acids, flavonoid and α-tomatine content in each treatment fruit were used to create a heat map. Each square represents the correlation among lesion diameter, phenolic compounds and α-tomatine content with a colour scale (colour scale key at the side of the figure).
the silence of SlMYC2 aggravated fruit decay caused by B. cinerea, and MJ treatment significantly inhibited the development of disease in either silenced or non-silenced tomato fruit. In addition, we found that the incidence and symptoms of fruit disease of MJ + SlMYC2-silenced fruit were more severe than in MJ-treated fruit. These findings suggested that the silence of SlMYC2 abated fruit disease resistance induced by MJ. MJ, as an important phytohormone, has pivotal roles in stimulating the biosynthesis of secondary metabolites (Wang et al., 2009). Many studies have revealed that MJ significantly promotes the accumulation of phenolics and flavonoids in plants (Wang et al., 2009). PAL is the main enzyme associated with the biosynthesis of phenolics, which is viewed as an indicator involved in plant defence against pathogens. It could be oxidized by PPO to form toxic quinones, which further influence invading pathogens in plant-pathogen interactions (Zhang et al., 2017; Zhang, Tian, Zhu, Xu, & Qin, 2012). Meanwhile, PAL is also an essential enzyme in biosynthesis of flavonoids which occurs widely in plants and plays important roles in plant resistance (Winkel-Shirley, 2001). Therefore, the activities of PAL and PPO were measured in the present study to investigate the possible biosynthesis mechanism of phenolics and flavonoids. Our results demonstrate that MJ treatment could induce activities of PAL and PPO, and further increase total phenolic and flavonoid contents which strongly relate to disease resistance. In addition, the silence of SlMYC2 partly counteracted the induction effect of MJ on phenolic and flavonoid compound contents by inhibiting the activities of PAL and PPO. More concretely, there was a large increase in the levels of some phenolic acids and flavonoid compounds in MJ-treated fruit, such as gallic acid, chlorogenic acid, p-coumaric acid, ferulic acid, catechin, salicylic acid, naringin, quercetin and phloridzin. Correlation analysis suggested that the lesion diameter correlated negatively with gallic acid, chlorogenic acid, ferulic acid, rutin and kaempferol in MJ-treated fruit. Thus, the MJ treatment might enhance the tomato fruit resistance
Fig. 5. Effects of MJ and SlMYC2 silence on SlPR1 expression of tomato fruit during storage at 25 °C. The transcript levels were measured by qRT-PCR, normalized to the host SlUbi3 gene, and set relative to the control fruit from day 0. Vertical bars represent the standard errors of the means.
dependent physiological processes in plants. For example, OsMYC2 positively regulated the disease resistance in rice (Uji et al., 2016). Shen et al. (2010) suggested that AaMYC2 could markedly induce artemisinin biosynthesis. Our previous study found that SlMYC2 participated in the chilling resistance induced by MJ in tomato fruit (Min et al., 2018a). Although these results had reported that MYC2 was a key regulator in JA response, the function of MYC2 in MJ-induced disease resistance of postharvest fruit is unclear. Thus, in our study, the fruits with SlMYC2 silence were established by VIGS, as described in our previous methods (Min et al., 2018a). The data clearly indicated that 6
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Fig. 6. Effects of MJ and SlMYC2 silence on expression levels of SlPR2a (a), SlPR2b (b), SlPR3a (d) and SlPR3b (e) and activities of GLU (c) and CHI (f) in tomato fruit during storage at 25 °C. The transcript levels were measured by qRT-PCR, normalized to the host SlUbi3 gene, and set relative to the control fruit from day 0. Vertical bars represent the standard errors of the means.
compounds might indirectly induce the tomato fruit resistance to B. cinerea by regulating other metabolic pathways. In turn, the contents of other phenolic acids, e.g. caffeic acid and epicatechin, or other flavonoid compounds, such as kaempferol and hesperidin, did not significant change, and, most interesting was that MJ had no effect on rutin which was important for pathogen suppression and could induce disease resistance (Wei et al., 2017). The result was similar to the discovery of Krol et al. (2015), who found that after a 1 h treatment of 15-day-old tomato seedlings with MJ had no effect on rutin content. Compared with MJ- treated fruit, the contents of some phenolics and flavonoids, including gallic acid, chlorogenic acid, p-coumaric acid, ferulic acid, catechin, salicylic acid, quercetin, naringin and phloridzin, were significantly inhibited in MJ + SlMYC2-silenced fruit. Moreover, the lesion diameter was negatively associated with gallic acid, chlorogenic acid, phloridzin and kaempferol, from which we inferred that SlMYC2 participated in MJ-mediated fruit resistance to B. cinerea by directly regulating the biosynthesis of gallic acid, chlorogenic acid, and phloridzin. In addition, some phenolic and flavonoid compounds, e.g. p-
to B. cinerea by directly regulating the biosynthesis of gallic acid, chlorogenic acid and ferulic acid. Among these, gallic acid is the major phenol in tomato fruit (Liu, Cai, Lu, Han & Ying, 2012). Likewise, Wang et al. (2009) also reported that MJ treatment significantly enhanced the gallic acid content in Chinese bayberries and reduced fruit decay. Konan et al. (2014) indicated that MJ enhanced the disease resistance of cotton by promoting ferulic acid biosynthesis. Unlike our results, Krol, Igielski, Pollmann, and Kepczynska (2015) suggested that MJ treatment enhances tomato disease resistance via salicylic acid, which has critical roles in inducing plant systemic acquired resistance (SAR) (Min et al., 2018b), and promotes the accumulation of quercetin which has antimicrobial effects on pathogens. Krol et al. (2015) also found that MJ had no effect on chlorogenic acid which is an important antifungal agent (Wei, Zhou, Peng, Pan, & Tu, 2017). Although, the correlation coefficients among lesion diameter, p-coumaric acid, catechin, salicylic acid, quercetin, naringin and phloridzin were not observed in our work, MJ treatment significantly promoted the accumulation of these phenolic acids and flavonoid compounds. Therefore, these 7
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defence-related enzyme activities and SlPRs expression, including SlPR1, SlPR2a, SlPR2b and SlPR3b, which further decreased fruit disease development. Conversely, the induction effects of MJ on fruit were almost counteracted by SlMYC2 silence. Compared with MJ-treated fruit, the contents of α-tomatine and total / special individual phenolic and flavonoid compounds, e.g. gallic acid, p-coumaric acid, chlorogenic acid, salicylic acid, catechin, phloridzin, naringin and quercetin, were decreased in MJ + SlMYC2-silenced fruit during most of the storage time. Meanwhile, the incidence and symptoms of fruit disease of MJ + SlMYC2-silenced fruit were more severe than those in MJ-treated fruit. Therefore, our findings have demonstrated that SlMYC2 played a positive role in tomato fruit resistance to B. cinerea.
coumaric acid, catechin, quercetin, salicylic acid, ferulic acid and naringin, might indirectly involve in SlMYC2-induced fruit resistance, which needs to be further studied in the future. Meanwhile, α-tomatine, a resistance substance, plays essential roles in plant defence mechanisms against fungi, bacteria and viruses, and the level of α-tomatine quickly decreases as tomato fruit ripens (Koh et al., 2013). Recently, many reports suggest that MJ induced the αtomatine biosynthetic pathway (Abdelkareem et al., 2017) and further enhanced the accumulation of α-tomatine (Montero-Vargas et al., 2018). Similar to those results, we found that MJ inhibited the decrease of α-tomatine content during storage. Nevertheless, the α-tomatine content in MJ + SlMYC2-silenced fruit was lower than that in MJtreated fruit. And, the correlation analysis showed that lesion diameter was strongly correlated with α-tomatine content. In addition, research shows that SlGAME9 was co-expressed with SlGAME1, SlGAME17 and SlGAME18 and might regulate the α-tomatine biosynthesis by cooperating with SlMYC2 (Cardenas et al., 2016). Those GAME genes (SlGAME1, SlGAME2, SlGAME17 and SlGAME18) are involved in the process of glycosylation (Cardenas et al., 2015), which plays an important role in α-tomatine biosynthesis; thus we further analyzed the relative expression levels of GAME genes, including SlGAME1, SlGAME2, SlGAME17 and SlGAME18. Our findings indicate that MJ could induce expressions of GAME genes (SlGAME1, SlGAME2, SlGAME9, SlGAME17 and SlGAME18). However, the silence of SlMYC2 not only inhibited SlGAME9 expression, but also decreased the expression levels of SlGAME1, SlGAME2, SlGAME17 and SlGAME18. The result was similar to previous researches that JA could upregulate the expression of SlGAME9 which plays an important role in the JA signalling pathway (Abdelkareem et al., 2017) and impacts the expression of SlGAME1, SlGAME17 and SlGAME18 (Cardenas et al., 2016). In addition, high expression of SlGAME2 might contribute to the α-tomatine formation and is closely related to plant’s resistance (Shinde et al., 2017). Thus, SlMYC2 might be involved in MJ-induced fruit resistance to B. cinerea by regulating α-tomatine biosynthesis. PR proteins, including 17 different families, are considered as plant defence proteins that are related to prevention of pathogen infection (Kesari, Trivedi, & Nath, 2010). Among them, SlPR1 is usually regarded as an indicator for SAR (Durrant & Dong, 2006). In addition, PR2 and PR3, encoding GLU and CHI, respectively, play important roles in controlling the disease development in plant-disease interactions (Kesari et al., 2010; Yang, Cao, Cai, & Zheng, 2011). Similar to the result of Ding, Wang, Gross, and Smith (2002), we discovered that MJ significantly induced the expression of SlPR1, SlPR2a (extracellular GLU), SlPR2b (intracellular GLU) and SlPR3b (intracellular CHI), but hardly increased the expression of SlPR3a (extracellular CHI). Moreover, MJ treatment induced higher activities of GLU and CHI. However, the silence of SlMYC2 also inhibited the induction effects of MJ on SlPR1, SlPR2a, SlPR2b and SlPR3b expression and activities of GLU and CHI. To summarize, these results might partly explain the mechanism of SlMYC2 participating in MJ-induced fruit resistance to B. cinerea, possibly by regulating expressions of related SlPRs and activities of CHI and GLU.
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The study was supported by the National Natural Science Foundation of China (No. 31772024), and the fund of the Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology & Business University (BTBU). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.125901. References Abdelkareem, A., Thagun, C., Nakayasu, M., Mizutani, M., Hashimoto, T., & Shoji, T. (2017). Jasmonate-induced biosynthesis of steroidal glycoalkaloids depends on COI1 proteins in tomato. Biochemical and Biophysical Research Communications, 489, 206–210. https://doi.org/10.1016/j.bbrc.2017.05.132. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254. https://doi.org/10.1016/0003-2697(76)90527-3. Cardenas, P. D., Sonawane, P. D., Heining, U., Bocobza, S. E., Burdman, S., & Aharoni, A. (2015). The bitter side of nightshades: Genomics drives discovery in Solanaceae steroidal alkaloid metabolism. Phytochemistry, 113, 24–32. https://doi.org/10.1016/ j.phytochem.2014.12.010. Cardenas, P. D., Sonawane, P. D., Polier, J., Bossche, R. V., Dewangan, V., Weithorn, E., ... Aharoni, A. (2016). GAME9 regulates the biosynthesis of steroidal alkaloids and upstream isoprenoids in the plant mevalonate pathway. Nature Communications, 7, 10654–10670. https://doi.org/10.1038/ncomms10654. Dean, R., Van Kan, J. A. L., Pretorius, Z. A., Hammond-Kosack, K. E., Pietro, A. D., Spanu, P. D., ... Foster, G. D. (2012). The Top 10 fungal pathogens in molecular plant pathology. Molecular Plant Pathology, 13, 414–430. https://doi.org/10.1111/j.13643703.2011.00783.x. Ding, C. K., Wang, C. Y., Gross, K. C., & Smith, D. L. (2002). Jasmonate and salicylate induce the expression of pathogenesis-related-protein genes and increase resistance to chilling injury in tomato fruit. Planta, 2002(214), 895–901. https://doi.org/10. 2307/23386920. Du, M. M., Zhao, J. H., Tzeng, D. T. W., Liu, Y. Y., Deng, L., Yang, T. X., ... Li, C. Y. (2017). MYC2 orchestrates a hierarchical transcriptional cascade that regulates jasmonatemediated plant immunity in tomato. Plant Cell, 29, 1883–1906. https://doi.org/10. 1105/tpc.16.0095. Durrant, W. E., & Dong, X. (2006). Systemic acquired resistance. Annual Review Phytopathology, 1, 179–184. Jankowska, M., Kaczynski, P., Hrynko, I., & Lozowicka, B. (2016). Dissipation of six fungicides in greenhouse-grown tomatoes with processing and health risk. Environmrntal Science and Pollution Research, 23, 11885–11900. https://doi.org/10. 1007/s11356-016-6260-x. Jiang, D., & Yan, S. C. (2018). MeJA is more effective than JA in inducing defense responses. Larix olgensis Arthropod-Plant Interactions, 12, 49–56. https://doi.org/10. 1007/s11829-017-9551-3. Jin, P., Zheng, Y. H., Tang, S. S., Rui, H. J., & Wang, C. Y. (2009). Enhancing disease resistance in peach fruit with methyl jasmonate. Journal of the Science of Food and Agriculture, 89, 802–808. https://doi.org/10.1002/jsfa.3516. Kazan, K., & Manners, J. M. (2013). MYC2: The master in action. Molcular Plant, 6, 686–703. https://doi.org/10.1093/mp/sss128. Kesari, R., Trivedi, P. K., & Nath, P. (2010). Gene expression of pathogenesis-related
5. Conclusion MJ treatment promoted the accumulation of α-tomatine by inducing the expressions of GAME genes (SlGAME1, SlGAME2, SlGAME9, SlGAME17 and SlGAME18), and enhanced total phenolics and flavonoids by inducing the activities of PAL and PPO. HPLC results indicated that MJ treatment increased the contents of such individual phenolic and flavonoid compounds, as gallic acid, chlorogenic acid, p-coumaric acid, ferulic acid, catechin acid, salicylic acid, quercetin, naringin and phloridzin, in comparison with control fruit. The correlation coefficients indicated that lesion diameter was correlated with gallic acid (-0.81), chlorogenic acid (-0.46), kaempferol (-0.64), ferulic acid (-0.80) and rutin (-0.31) in MJ-treated fruit. Besides, it also induced 8
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