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Induction of disease resistance providing new insight into sulfur dioxide preservation in Vitis vinifera L.
Scientia Horticulturae 225 (2017) 567–573
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Research Paper
Induction of disease resistance providing new insight into sulfur dioxide preservation in Vitis vinifera L. Meizhao Xue, Huilan Yi
MARK
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School of Life Science, Shanxi University, Taiyuan, 030006, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Table grape SO2 preservation PAL Phenolic compounds PPO PR proteins
The effect of a postharvest treatment with sulfur dioxide (SO2) under commercial conditions on table grapes (Vitis vinifera L. ‘Muscat Hamburg’) quality and the possible action mechanisms were investigated. The results indicated that SO2 treatment resulted in an improved control of infections in grapes during storage. The fresh fruit rate of SO2 treatment group was significantly higher than that of the control group. Furthermore, SO2 markedly enhanced activity of phenylalanine ammonia lyase (PAL), promoted the accumulation of phenolic compounds including total phenol, flavonoid and lignin, and improved the polyphenol oxidase (PPO) activity. Moreover, SO2 significantly elevated transcription levels of pathogenesis-related (PR) proteins genes chitinase3 (CHI3), CHI1b and β-1,3-glucanase (PR2). Consistently, the activities of chitinase and β-1,3-glucanase remarkably increased in SO2 treatment group. Our founding indicates that SO2 activates defense responses associated with secondary metabolism and PR proteins for enhanced disease resistance and thereby thus extending the storage life of table grapes.
1. Introduction
Awad, 2015), salt solutions (Youssef and Roberto, 2014), methyl jasmonate (Jiang et al., 2015), spermine (Harindra Champa et al., 2015) and polyamines (Mirdehghan and Rahimi, 2016). (iii) physical means; Physical methods of controlling gray mold include UV-C irradiation (Romanazzi et al., 2006), ozone (Gabler et al., 2010), CO2 (SanchezBallesta et al., 2006; Teles et al., 2014) and hyperbaric treatment (Romanazzi et al., 2008). Although these methods have shown good effect on the control of Botrytis cinerea, they are limited to laboratory scale experiments. They have not yet begun to be adopted on a commercial scale. Until now, no alternative could be effective as preservative in the table grape industry except SO2 (Meng et al., 2008; Liu et al., 2010; Sun et al., 2013). After SO2 enters the cell, it can rapidly hydrolyze and dissociate into sulfites ions (SO32−) and bisulfite ions (HSO3−) (Zhao and Yi, 2014; Considine and Foyer, 2015). Among these, SO32− not only can be oxygenated to be sulfate finally stored in plant cells via oxidative pathway, but also can be converted into cysteine and other sulfhydrylcontained compounds through sulfur reductive pathway (Giraud et al., 2012). Elemental sulfur, the intermediate of sulfur metabolism, is a kind of phytoalexin, which is effective against a broad spectrum of fungal pathogen (Cooper and Williams, 2004). Moreover, cysteine, glutathione and thioredoxin are involved in antioxidant response and resistance against pathogens (Kruse et al., 2007; Höller et al., 2010; Li et al., 2010). During postharvest storage, sulfur assimilation and
Grapevine (Vitis vinifera L.) is a major and valuable fruit crop worldwide that is highly susceptible to fungi infections. Gray mold caused by Botrytis cinerea is one of the major destructive postharvest diseases of grapes (Qin et al., 2015). Sulfur compounds have been used as preservatives in the viticultural industry since Roman times (Freedman, 1980; Smilanick et al., 1990; Crisosto et al., 2002). But these days it comes with a health warning, causing more and more people to call for lower-sulphite fruits. The table grape industry allows the highest concentration of residual sulfites is to be no more than 10 mg/kg. The study of alternative means to control postharvest decay has progressed over the past several decades. These alternative approaches were grouped as follows: (i) biological control; Bacillus circulans (PauL et al., 1997), Muscodor albus (Mlikota et al., 2006), Cryptococcus laurentii (Meng et al., 2010), Saccharomyces cerevisiae (Nally et al., 2012), Hanseniaspora uvarum (Qin et al., 2015), and Metschnikowia pulcherrima (Parafati et al., 2015) have been explored to approach good effectiveness to control postharvest gray mold of table grapes. (ii) chemical treatments; Some chemical compositions have been applied with preharvest or/and postharvest to maintain quality and extend postharvest life of table grapes, including ethanol (Lurie et al., 2006; Yu et al., 2006), plant extract (Xu et al., 2007), acetic acid (Venditti et al., 2008), chitosan (Meng et al., 2008; Al-Qurashi and
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Corresponding author. E-mail address: [email protected] (H. Yi).
http://dx.doi.org/10.1016/j.scienta.2017.07.055 Received 22 February 2017; Received in revised form 26 July 2017; Accepted 28 July 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.
Scientia Horticulturae 225 (2017) 567–573
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reduction pathways were activated, and some defense-related genes were up-regulated. Meanwhile, redox poise mediating by reactive oxygen species (ROS) altered dramatically in SO2 treatment group (Giraud et al., 2012). UV radiation and ozone also caused oxidative responses (Willekens et al., 1994; Gabler et al., 2010). However, the efficacy of commercial to grape was poor compared with SO2 (Gabler et al., 2010). These results suggested that SO2 preservation is a complex biological process. Knowledge of these pathways will not only help understand the mode of action of SO2, but may also identify other compounds that have similar effects and act as more appropriate food preservatives. Phenylpropanoid pathway is one of the most important secondary metabolic pathways in plants. PAL, the key enzyme of phenylpropanoid pathway, is responsible for the synthesis of a wide variety of secondary metabolites (Sangeetha and Sarada, 2015). Secondary metabolites are not directly involved in growth or reproduction but they are often involved with plant defense. Phenolic compounds are a large class of secondary metabolites produced by plants to defend themselves against pathogens (Freeman and Beattie, 2008). They are produced primarily via the shikimic acid pathway in higher plants, and include a wide variety of defense-related compounds including flavonoids, lignin, etc. (Dixon and Paiva, 1995; Humphreys and Chapple, 2002). Except for secondary metabolites, PR proteins were also considered as key enzymes related to defense reaction against pathogen infections (Chong et al., 2008; Sels et al., 2008). Our previous study proved that numerous enzymes required for the phenylpropanoid pathway and coding genes for PR proteins, such as β-1,3-glucanase (PR2), chitinase (PR3), hevein (HEL), OSM34 and phytoalexin (PDF1.4), were highly activated on SO2 fumigation in Arabidopsis (Li and Yi, 2012; Zhao and Yi, 2014). However, it is not clear whether the efficacy of SO2 preservative is related to secondary metabolism and PR proteins. In present study, the effect of SO2 preservative on secondary metabolism and PR proteins in table grapes were explored.
2.3. Enzyme assays The grape skins for enzyme assays, including phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), β-1,3-glucanase and chitinase, were harvested from the control and SO2 treatment group during storage. PAL activity was assayed following the method of Romero et al. (2008). Skin samples (0.1 g) were homogenized in 1 mL ice cold extracting solution containing 0.05 M borate buffer, 5 mM 2-Mercaptoethanol, 1 mM ethylenediaminetetraacetic acid disodium salt (EDTANa2), 5% (w/v) glycerol (pH 8.3) and 5% (w/v) polyvinyl-polypyrrolidon (PVP). The homogenate was centrifuged at 10000g for 10 min at 4 °C. The supernatant was used for enzyme analysis. PAL activity was determined as the rate of the conversion of L-phenylalanine to trans-cinnamic acid at 290 nm. One unit of PAL activity was defined as the amount of enzyme causing an increase of 0.01 in absorbance per hour. PPO activity was measured according to the method of catechol (Sangeetha and Sarada, 2015). The skin samples (0.15 g) were ground in 1 mL of 0.2 M sodium phosphate buffer (pH 6.5) containing 1% (w/ v) polyvinyl-polypyrrolidon (PVP). The homogenate was centrifuged at 10000g for 20 min at 4 °C, and the supernatant was used as the enzyme source. The reaction mixture consisted of 600 μL of crude extract and 300 μL of 0.2 M sodium phosphate buffer. To start the reaction, 300 μL of 0.05% catechol was added and the activity was expressed as change in absorbance at 420 nm. The increase in absorbance at 420 nm was recorded every 30 s one time and lasted for 5 min. One unit of PPO activity was defined as the amount of enzyme causing a change of 0.01 in absorbance per minute. The β-1,3-glucanase activity was assayed referred to Jiang et al. (2015). Grape skins (0.1 g) were extracted with 1 mL 0.1 M sodium citrate buffer (pH 5.0) and centrifuged at 12000g for 15 min at 4 °C. The supernatant was used for the enzyme assay of β-1,3-glucanase. The reaction mixture containing 10 μL enzymic extract and 100 μL of 0.04% fucoidan was incubated at 50 °C for 10 min, then added 200 μL of 3,5Dinitrosalicylic acid (DNS) at boiling water for 5 min. The absorbance was measured at 540 nm after natural cooling. The activity was expressed as the rate of catalyzing fucoidan to glucose per hour per gram fresh weight of sample (mg/g·h). The chitinase activity assay was analyzed according to the method of Jiang et al. (2015). The activity was expressed as the rate of releasing N-acetyl glucosamine from chitin per hour per gram fresh weight of sample (μg/g·h).
2. Materials and methods 2.1. Plant material and treatment ‘Muscat Hamburg’ grapes (Vitis vinifera L.) were harvested at commercial maturity (16.38% total soluble solids; 0.43% titratable acidity) periods from vineyards and immediately transported to the laboratory. The bunches of grape were selected on the basis of uniform color, size and firmness. Any damaged berries were discarded. The grapes were randomly distributed into different plastic packages (5 kg per package) and precooled for 12 h at 0 °C. After that, the packages with table grapes were divided into two groups. One group served as SO2 treatment group, and another group served as the control group. For SO2 treatment group, SO2 quick release sheets (containing Na2S2O5 1.2 g) and sustained release tablets (containing Na2S2O5 6.8 g) were put into the packages for SO2 dual release. Then, tie up the packages with a string. Control group had the same experimental conditions only without Na2S2O5. All packages were stored at (0 ± 0.5) °C. On days 0, 20, 40 and 60, thirty berries from each treatment were sub-sampled, snap-frozen in liquid nitrogen and stored at −80 °C.
2.4. Estimation of secondary metabolites The extracting solution for total phenol and flavonoid content estimation were the same. Grape skins (0.1 g) were grounded in 1.5 mL methanol including 2% hydrochloric acid. The homogenate was extracted in dark for 24 h at 28 °C and then centrifuged at 12000g for 10 min at 4 °C. The supernatant was used as sample. Content of total phenol was measured according to the method of Folin-Phenol (Chamkha and Cathala, 2003). Sample (20 μL) was added to 80 μL of extracting solution (methanol including 2% hydrochloric acid). 0.1 mL diluted sample, 0.5 mL of Folin-Phenol and 0.4 mL of 7.5% sodium carbonate was kept at 28 °C for 2 h. The absorbance was measured using spectrophotometer at 765 nm. Gallic acid was used as standard. The amount of total phenol was expressed as milligrams of gallic acid per gram of sample (mg/g). Flavonoid content was measured according to the NaNO2AlCl3eNaOH colorimetric method (Wolfe et al., 2003). Rutin was used as standard. The amount of flavonoid was expressed as milligrams of rutin per gram of sample (mg/g). The content of lignin was extracted and measured by the method of Chen et al. (2002). The amount of lignin was calculated as the absorbance at 280 nm per gram of sample (OD280 nm/g).
2.2. Measurement of fresh fruit rate Fresh fruit rate of table grapes during storage was measured by monitoring weight of dropped and rotten fruit at 0, 20, 40 and 60 d, respectively. Fresh fruit rate was calculated using the following formula.
Fresh fruit rate (%) total weight − (dropped weight = total weight
+
rotten weight)
× 100
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2 μg total RNA (in 20 μL total volume) was reverse transcribed using Oligo(dT18) primer and RT enzyme mix according to the manufacturer’s instructions (TransGen, Beijing). The expression patterns of PR genes (CHI3, CHI1b and PR2; coding genes for chitinase and β-1,3-glucanase) were analyzed. Actin1 was used as a reference. PCR amplification was performed using the following gene-specific primers listed in Table 1.
Table 1 Primer sequences used in this work. Gene name
Primers sequence
Tm (°C)
PR2
5′5′5′5′5′5′5′5′-
57
CHI1b CHI3 Actin1
TGCTGTTTACTCGGCACTTG-3′ CTGGGGATTTCCTGTTCTCA-3′ ATGCTGCAGCAAGTTTGGTT-3′ CATCCTCCTGTGATGACATT-3′ AGATGGCATAGACTTCGACA-3′ GTACTTTGACCACAGCATCA-3′ CCCCATGCTATCCTTCG-3′ AGGCAGCTCATAGTTCTTCTC-3′
60 58
2.6. Statistical analysis
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All experiments were repeated at least three times. All results were presented as mean ± SE (standard error). For statistical analysis, oneway analysis of variance (ANOVA) was used to evaluate the statistical significance in different treatment groups. Different letters indicate significant difference (P < 0.05). All data analyses were performed using SPSS version 17.0.
2.5. RNA isolation and RT-PCR analysis Transcriptional levels of selected genes were verified by RT-PCR using the RNA samples isolated from grape skins (0.15 g) obtained from the control and SO2 treatment group at 0, 20, 40, and 60 d during storage. Total RNA was extracted and isolated by using CTAB method, as described by Carra et al. (2007) with modifications. The RNA was finally dissolved in 30 μL RNase free water and kept at −80 °C. Quality and integrity of prepared RNA samples were checked by spectrophotometric analysis for A260:A280 and A260:A230 ratios and electrophoresis in 2.0% agarose gel. For the synthesis of first-strand cDNA,
3. Results 3.1. SO2 maintained fruit quality of table grapes Fresh fruit rate of table grapes during storage is shown in Fig. 1A. Fruits both in control and SO2 treatment groups were intact during 20 d of storage. Thereafter the fresh fruit rate in control group was reduced rapidly with the prolonged storage time. The fresh fruit rate of the
Fig. 1. Effect of SO2 on fruit quality of table grapes. (A) Effect of SO2 on fresh fruit rate; (B) Effect of SO2 on grape appearance quality at 60 days. Red arrows indicate the infection fruit. CK: the control group; SO2: SO2 treatment group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
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Fig. 2. Effects of SO2 on the activities of PAL (A), PPO (E) and contents of total phenol (B), flavonoid (C), lignin (D) in table grapes during storage.
plump in the SO2 treatment group. In addition, the SO2-treated fruit had no occurred bleaching at 60 d. However, the fruit appeared obvious infection symptoms in the control group (Fig. 1B). The fruits quality of SO2 treatment group was significantly higher than that of the control.
control group was significantly lower than that of the SO2 treatment group at 40 d. After 60 days, the fresh fruit rate in the control group declined to 75% while still maintained 95% in SO2 treatment group. The stem of grape was more fresh and green, and the fruit was more 570
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Fig. 3. Effect of SO2 on transcription levels of PR proteins coding genes in table grapes during storage.
The results showed that SO2 could effectively improve the fruit quality and extend postharvest life of table grapes.
3.2. SO2 activated secondary metabolism pathway SO2 treatment markedly enhanced activities of PAL and PPO, promoted the accumulation of total phenol, flavonoid and lignin during storage (Fig. 2A–E). PAL activity in SO2 group was significantly higher than that in control group (Fig. 2A), suggesting that secondary metabolism were activated by SO2 during storage. Within 20 days, there was no obviously difference of PAL activity in control and SO2 group but significant increase could be found after that. PAL activity in the control group decreased quickly while the SO2 treatment group still kept the high level at 40 d. It was worth noting that change of PAL activity was consistent with fresh fruit rate during storage. As the key and limited enzyme in phenylpropanoid metabolic pathway, the increase of PAL activity is conducive to the synthesis of secondary metabolites. The changes in the content of total phenol, flavonoid and lignin are shown in Fig. 2(B-D). During storage, higher and earlier accumulation of phenolic compounds was observed in SO2 treatment group. The content of total phenol showed an increasing trend in both control and SO2 treatment group during storage and SO2 significantly enhanced the elevated levels. Flavonoid and lignin contents obviously increased in the SO2 group, whereas they increased slightly or remained unchanged in the control group. As shown in Fig. 2(E), PPO activity was initially higher and rapidly decreased both in the control and SO2 treatment group during storage. PPO activity in SO2 group was noticeably higher than that in control group during 20 d to 40 d.
Fig. 5. A schematic model of SO2-induced disease resistance in Vitis vinifera L. during storage.
3.3. SO2 activated the expression of PR genes and proteins The Transcript levels of CHI3, CHI1b and PR2 in SO2 treatment group was overall increased (Fig. 3). CHI3 responded faster than other two genes. The expression of CHI3 was up-regulated at 20 d in the SO2 group, and then gradually increased with the extension of SO2 treatment. PR2 gradually increased and peaked at 40 d, and then declined at 60 d. CHI1b expressed almost no change before 40 d and up-regulated until 60 d. In SO2 treatment group, chitinase and β-1,3-glucanase activity increased rapidly. Meanwhile, SO2 maintained significantly higher activities of chitinase and β-1,3-glucanase during the whole storage period compared with the control (Fig. 4), which is consistent with transcript levels of both CHI3 and PR2. In addition, the activities of chitinase and β-1,3-glucanase remarkably increased in control group at 60 d, which is highly likely to be associated with decline of fruit
Fig. 4. Effects of SO2 on the activities of chitinase (A) and β-1,3-glucanase (B) in table grapes during storage.
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markedly induced in SO2 treatment group. The two enzymes can hydrolyze chitins or glucans which are essential components of the cell wall of pathogens, leading to the death of pathogens and further infection. These results further confirmed that the SO2-induced disease resistance against pathogens infection in table grapes during storage is also associated with priming of PR proteins defense responses. In conclusion, our results demonstrated that SO2 can effectively induce disease resistance and maintain fruit quality of table grapes during postharvest storage. Moreover, the SO2 induced disease resistance is associated with priming of defense responses through enhancing (i) secondary metabolism pathway: SO2 significantly enhanced activity of PAL, promoted the accumulation of phenolic compounds, and improved the PPO activity; (ii) PR proteins coding genes expression and enzymes activity (Fig. 5). The results provide a new insight into SO2 preservation and scientific foundation for searching for more effective and safe food preservatives. The authors declare that they have no conflict of interest.
qualities at this time. 4. Discussion Although table grapes are a non-climacteric type of fruit with a low rate of physiological activity following harvest, berries are highly susceptible to fungal infection during storage. To date the only effective preservative is SO2 in the viticultural industries. The SO2 dual release technique, which is the most effective commercial preservation method so far, was used in our experiment. In the early stage of storage (the first stage), the high concentration SO2 could kill the pathogens carried from the field, and in the late stage (second stage), the low concentration of SO2 could maintain the environment of suppress pathogens (Suppl Fig. 1). The previous research found that the sulfite residue content in grape was only 3.9 mg/kg after SO2 dual release treatment for 180 d (Zhao et al., 2011). The view that sulphites act directly on bacterial and fungal pathogens may be simplistic. Mechanisms of sulfur-enhanced defense are largely unknown. In this respect, the current study evaluated the effects of SO2 on fruit quality, PAL activity, contents of phenolic compounds, PPO activity, and PR proteins activity and transcript levels of coding genes of ‘Muscat Hamburg’ table grapes during storage. Our present study showed that SO2 resulted in higher activities of PAL, PPO, and the contents of total phenol, flavonoid and lignin. Induction of PAL activity is a reliable indicator in grape defense against Botrytis cinerea during postharvest storage (Duarte-Sierra et al., 2015; Jiang et al., 2015; Qin et al., 2015). PAL is critically involved in the biosynthesis of phenolic compounds that contribute to plant disease resistance (Nguyen et al., 2003; Freeman and Beattie, 2008). Phenolic compounds in grapes could improve the quality and nutritional value because of antibacterial activities and strong antioxidant (Donnini et al., 2016; Cheng et al., 2017). Eshghi et al. (2014) found that there was a strong correlation between total phenolic content and antioxidant activity in different grapevine cultivars. Flavonoids are a class of bioactive compounds having antioxidant properties. Flavonoids greatly affect the sensory and nutritional quality of grapes (Brillante et al., 2015). Lignin is involved in the formation of structural barriers that can block pathogen invading and spread (Cartea et al., 2011). In the current experiment, SO2 enhanced PAL activity in table grapes during storage, resulting in accumulation of total phenolic, flavonoid and lignin, which collectively contribute to resistance against pathogens. Besides PAL, increased levels of PPO are also important for defense against pathogen infections of table grapes during storage (Al-Qurashi and Awad, 2015; Awad et al., 2015; Jiang et al., 2015; Qin et al., 2015). PPO is involved in lignification of host plant cells (Jiang et al., 2015) and could produce antimicrobial quinones through oxidizing phenolic compounds (Melo et al., 2006). In this study, PPO activities in both control group and SO2 treatment group were significantly decreased under low temperature storage. The variation trend is conducive to alleviate the browning of grape pulp during storage (Guardo et al., 2013; Hu et al., 2014). As an important terminal oxidase, the decrease of PPO activity might be caused by low temperature inhibiting fruit respiration rate of grapes (Chitbanchong et al., 2009). Therefore, PPO has a dual role during storage: resisting pathogen infection and preventing flesh browning. All these results suggested that SO2 effectively primes the secondary metabolism for enhanced disease resistance in table grapes during storage. Induction of PR proteins is also believed an indicator of plant induced defense responses (Vleeshouwers et al., 2000). Usually, chitinase and β-1,3-glucanase were induced synergistically to enhance plants disease resistance (Pozo et al., 2002; Duarte-Sierra et al., 2015; Jiang et al., 2015; Song et al., 2015). Duarte-Sierra et al. (2015) found that SO2 treatment primed defense responses against Botrytis cinerea in Redglobe and Sugraone grapes through inducing higher expression of chitinase and β-1,3-glucanase. Our study showed that SO2 significantly enhanced expression levels of CHI3, CHI1b and PR2 in the table grapes. Meanwhile, the activities of chitinase and β-1,3-glucanase were
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Research Paper
Induction of disease resistance providing new insight into sulfur dioxide preservation in Vitis vinifera L. Meizhao Xue, Huilan Yi
MARK
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School of Life Science, Shanxi University, Taiyuan, 030006, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Table grape SO2 preservation PAL Phenolic compounds PPO PR proteins
The effect of a postharvest treatment with sulfur dioxide (SO2) under commercial conditions on table grapes (Vitis vinifera L. ‘Muscat Hamburg’) quality and the possible action mechanisms were investigated. The results indicated that SO2 treatment resulted in an improved control of infections in grapes during storage. The fresh fruit rate of SO2 treatment group was significantly higher than that of the control group. Furthermore, SO2 markedly enhanced activity of phenylalanine ammonia lyase (PAL), promoted the accumulation of phenolic compounds including total phenol, flavonoid and lignin, and improved the polyphenol oxidase (PPO) activity. Moreover, SO2 significantly elevated transcription levels of pathogenesis-related (PR) proteins genes chitinase3 (CHI3), CHI1b and β-1,3-glucanase (PR2). Consistently, the activities of chitinase and β-1,3-glucanase remarkably increased in SO2 treatment group. Our founding indicates that SO2 activates defense responses associated with secondary metabolism and PR proteins for enhanced disease resistance and thereby thus extending the storage life of table grapes.
1. Introduction
Awad, 2015), salt solutions (Youssef and Roberto, 2014), methyl jasmonate (Jiang et al., 2015), spermine (Harindra Champa et al., 2015) and polyamines (Mirdehghan and Rahimi, 2016). (iii) physical means; Physical methods of controlling gray mold include UV-C irradiation (Romanazzi et al., 2006), ozone (Gabler et al., 2010), CO2 (SanchezBallesta et al., 2006; Teles et al., 2014) and hyperbaric treatment (Romanazzi et al., 2008). Although these methods have shown good effect on the control of Botrytis cinerea, they are limited to laboratory scale experiments. They have not yet begun to be adopted on a commercial scale. Until now, no alternative could be effective as preservative in the table grape industry except SO2 (Meng et al., 2008; Liu et al., 2010; Sun et al., 2013). After SO2 enters the cell, it can rapidly hydrolyze and dissociate into sulfites ions (SO32−) and bisulfite ions (HSO3−) (Zhao and Yi, 2014; Considine and Foyer, 2015). Among these, SO32− not only can be oxygenated to be sulfate finally stored in plant cells via oxidative pathway, but also can be converted into cysteine and other sulfhydrylcontained compounds through sulfur reductive pathway (Giraud et al., 2012). Elemental sulfur, the intermediate of sulfur metabolism, is a kind of phytoalexin, which is effective against a broad spectrum of fungal pathogen (Cooper and Williams, 2004). Moreover, cysteine, glutathione and thioredoxin are involved in antioxidant response and resistance against pathogens (Kruse et al., 2007; Höller et al., 2010; Li et al., 2010). During postharvest storage, sulfur assimilation and
Grapevine (Vitis vinifera L.) is a major and valuable fruit crop worldwide that is highly susceptible to fungi infections. Gray mold caused by Botrytis cinerea is one of the major destructive postharvest diseases of grapes (Qin et al., 2015). Sulfur compounds have been used as preservatives in the viticultural industry since Roman times (Freedman, 1980; Smilanick et al., 1990; Crisosto et al., 2002). But these days it comes with a health warning, causing more and more people to call for lower-sulphite fruits. The table grape industry allows the highest concentration of residual sulfites is to be no more than 10 mg/kg. The study of alternative means to control postharvest decay has progressed over the past several decades. These alternative approaches were grouped as follows: (i) biological control; Bacillus circulans (PauL et al., 1997), Muscodor albus (Mlikota et al., 2006), Cryptococcus laurentii (Meng et al., 2010), Saccharomyces cerevisiae (Nally et al., 2012), Hanseniaspora uvarum (Qin et al., 2015), and Metschnikowia pulcherrima (Parafati et al., 2015) have been explored to approach good effectiveness to control postharvest gray mold of table grapes. (ii) chemical treatments; Some chemical compositions have been applied with preharvest or/and postharvest to maintain quality and extend postharvest life of table grapes, including ethanol (Lurie et al., 2006; Yu et al., 2006), plant extract (Xu et al., 2007), acetic acid (Venditti et al., 2008), chitosan (Meng et al., 2008; Al-Qurashi and
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Corresponding author. E-mail address: [email protected] (H. Yi).
http://dx.doi.org/10.1016/j.scienta.2017.07.055 Received 22 February 2017; Received in revised form 26 July 2017; Accepted 28 July 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.
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reduction pathways were activated, and some defense-related genes were up-regulated. Meanwhile, redox poise mediating by reactive oxygen species (ROS) altered dramatically in SO2 treatment group (Giraud et al., 2012). UV radiation and ozone also caused oxidative responses (Willekens et al., 1994; Gabler et al., 2010). However, the efficacy of commercial to grape was poor compared with SO2 (Gabler et al., 2010). These results suggested that SO2 preservation is a complex biological process. Knowledge of these pathways will not only help understand the mode of action of SO2, but may also identify other compounds that have similar effects and act as more appropriate food preservatives. Phenylpropanoid pathway is one of the most important secondary metabolic pathways in plants. PAL, the key enzyme of phenylpropanoid pathway, is responsible for the synthesis of a wide variety of secondary metabolites (Sangeetha and Sarada, 2015). Secondary metabolites are not directly involved in growth or reproduction but they are often involved with plant defense. Phenolic compounds are a large class of secondary metabolites produced by plants to defend themselves against pathogens (Freeman and Beattie, 2008). They are produced primarily via the shikimic acid pathway in higher plants, and include a wide variety of defense-related compounds including flavonoids, lignin, etc. (Dixon and Paiva, 1995; Humphreys and Chapple, 2002). Except for secondary metabolites, PR proteins were also considered as key enzymes related to defense reaction against pathogen infections (Chong et al., 2008; Sels et al., 2008). Our previous study proved that numerous enzymes required for the phenylpropanoid pathway and coding genes for PR proteins, such as β-1,3-glucanase (PR2), chitinase (PR3), hevein (HEL), OSM34 and phytoalexin (PDF1.4), were highly activated on SO2 fumigation in Arabidopsis (Li and Yi, 2012; Zhao and Yi, 2014). However, it is not clear whether the efficacy of SO2 preservative is related to secondary metabolism and PR proteins. In present study, the effect of SO2 preservative on secondary metabolism and PR proteins in table grapes were explored.
2.3. Enzyme assays The grape skins for enzyme assays, including phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), β-1,3-glucanase and chitinase, were harvested from the control and SO2 treatment group during storage. PAL activity was assayed following the method of Romero et al. (2008). Skin samples (0.1 g) were homogenized in 1 mL ice cold extracting solution containing 0.05 M borate buffer, 5 mM 2-Mercaptoethanol, 1 mM ethylenediaminetetraacetic acid disodium salt (EDTANa2), 5% (w/v) glycerol (pH 8.3) and 5% (w/v) polyvinyl-polypyrrolidon (PVP). The homogenate was centrifuged at 10000g for 10 min at 4 °C. The supernatant was used for enzyme analysis. PAL activity was determined as the rate of the conversion of L-phenylalanine to trans-cinnamic acid at 290 nm. One unit of PAL activity was defined as the amount of enzyme causing an increase of 0.01 in absorbance per hour. PPO activity was measured according to the method of catechol (Sangeetha and Sarada, 2015). The skin samples (0.15 g) were ground in 1 mL of 0.2 M sodium phosphate buffer (pH 6.5) containing 1% (w/ v) polyvinyl-polypyrrolidon (PVP). The homogenate was centrifuged at 10000g for 20 min at 4 °C, and the supernatant was used as the enzyme source. The reaction mixture consisted of 600 μL of crude extract and 300 μL of 0.2 M sodium phosphate buffer. To start the reaction, 300 μL of 0.05% catechol was added and the activity was expressed as change in absorbance at 420 nm. The increase in absorbance at 420 nm was recorded every 30 s one time and lasted for 5 min. One unit of PPO activity was defined as the amount of enzyme causing a change of 0.01 in absorbance per minute. The β-1,3-glucanase activity was assayed referred to Jiang et al. (2015). Grape skins (0.1 g) were extracted with 1 mL 0.1 M sodium citrate buffer (pH 5.0) and centrifuged at 12000g for 15 min at 4 °C. The supernatant was used for the enzyme assay of β-1,3-glucanase. The reaction mixture containing 10 μL enzymic extract and 100 μL of 0.04% fucoidan was incubated at 50 °C for 10 min, then added 200 μL of 3,5Dinitrosalicylic acid (DNS) at boiling water for 5 min. The absorbance was measured at 540 nm after natural cooling. The activity was expressed as the rate of catalyzing fucoidan to glucose per hour per gram fresh weight of sample (mg/g·h). The chitinase activity assay was analyzed according to the method of Jiang et al. (2015). The activity was expressed as the rate of releasing N-acetyl glucosamine from chitin per hour per gram fresh weight of sample (μg/g·h).
2. Materials and methods 2.1. Plant material and treatment ‘Muscat Hamburg’ grapes (Vitis vinifera L.) were harvested at commercial maturity (16.38% total soluble solids; 0.43% titratable acidity) periods from vineyards and immediately transported to the laboratory. The bunches of grape were selected on the basis of uniform color, size and firmness. Any damaged berries were discarded. The grapes were randomly distributed into different plastic packages (5 kg per package) and precooled for 12 h at 0 °C. After that, the packages with table grapes were divided into two groups. One group served as SO2 treatment group, and another group served as the control group. For SO2 treatment group, SO2 quick release sheets (containing Na2S2O5 1.2 g) and sustained release tablets (containing Na2S2O5 6.8 g) were put into the packages for SO2 dual release. Then, tie up the packages with a string. Control group had the same experimental conditions only without Na2S2O5. All packages were stored at (0 ± 0.5) °C. On days 0, 20, 40 and 60, thirty berries from each treatment were sub-sampled, snap-frozen in liquid nitrogen and stored at −80 °C.
2.4. Estimation of secondary metabolites The extracting solution for total phenol and flavonoid content estimation were the same. Grape skins (0.1 g) were grounded in 1.5 mL methanol including 2% hydrochloric acid. The homogenate was extracted in dark for 24 h at 28 °C and then centrifuged at 12000g for 10 min at 4 °C. The supernatant was used as sample. Content of total phenol was measured according to the method of Folin-Phenol (Chamkha and Cathala, 2003). Sample (20 μL) was added to 80 μL of extracting solution (methanol including 2% hydrochloric acid). 0.1 mL diluted sample, 0.5 mL of Folin-Phenol and 0.4 mL of 7.5% sodium carbonate was kept at 28 °C for 2 h. The absorbance was measured using spectrophotometer at 765 nm. Gallic acid was used as standard. The amount of total phenol was expressed as milligrams of gallic acid per gram of sample (mg/g). Flavonoid content was measured according to the NaNO2AlCl3eNaOH colorimetric method (Wolfe et al., 2003). Rutin was used as standard. The amount of flavonoid was expressed as milligrams of rutin per gram of sample (mg/g). The content of lignin was extracted and measured by the method of Chen et al. (2002). The amount of lignin was calculated as the absorbance at 280 nm per gram of sample (OD280 nm/g).
2.2. Measurement of fresh fruit rate Fresh fruit rate of table grapes during storage was measured by monitoring weight of dropped and rotten fruit at 0, 20, 40 and 60 d, respectively. Fresh fruit rate was calculated using the following formula.
Fresh fruit rate (%) total weight − (dropped weight = total weight
+
rotten weight)
× 100
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2 μg total RNA (in 20 μL total volume) was reverse transcribed using Oligo(dT18) primer and RT enzyme mix according to the manufacturer’s instructions (TransGen, Beijing). The expression patterns of PR genes (CHI3, CHI1b and PR2; coding genes for chitinase and β-1,3-glucanase) were analyzed. Actin1 was used as a reference. PCR amplification was performed using the following gene-specific primers listed in Table 1.
Table 1 Primer sequences used in this work. Gene name
Primers sequence
Tm (°C)
PR2
5′5′5′5′5′5′5′5′-
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CHI1b CHI3 Actin1
TGCTGTTTACTCGGCACTTG-3′ CTGGGGATTTCCTGTTCTCA-3′ ATGCTGCAGCAAGTTTGGTT-3′ CATCCTCCTGTGATGACATT-3′ AGATGGCATAGACTTCGACA-3′ GTACTTTGACCACAGCATCA-3′ CCCCATGCTATCCTTCG-3′ AGGCAGCTCATAGTTCTTCTC-3′
60 58
2.6. Statistical analysis
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All experiments were repeated at least three times. All results were presented as mean ± SE (standard error). For statistical analysis, oneway analysis of variance (ANOVA) was used to evaluate the statistical significance in different treatment groups. Different letters indicate significant difference (P < 0.05). All data analyses were performed using SPSS version 17.0.
2.5. RNA isolation and RT-PCR analysis Transcriptional levels of selected genes were verified by RT-PCR using the RNA samples isolated from grape skins (0.15 g) obtained from the control and SO2 treatment group at 0, 20, 40, and 60 d during storage. Total RNA was extracted and isolated by using CTAB method, as described by Carra et al. (2007) with modifications. The RNA was finally dissolved in 30 μL RNase free water and kept at −80 °C. Quality and integrity of prepared RNA samples were checked by spectrophotometric analysis for A260:A280 and A260:A230 ratios and electrophoresis in 2.0% agarose gel. For the synthesis of first-strand cDNA,
3. Results 3.1. SO2 maintained fruit quality of table grapes Fresh fruit rate of table grapes during storage is shown in Fig. 1A. Fruits both in control and SO2 treatment groups were intact during 20 d of storage. Thereafter the fresh fruit rate in control group was reduced rapidly with the prolonged storage time. The fresh fruit rate of the
Fig. 1. Effect of SO2 on fruit quality of table grapes. (A) Effect of SO2 on fresh fruit rate; (B) Effect of SO2 on grape appearance quality at 60 days. Red arrows indicate the infection fruit. CK: the control group; SO2: SO2 treatment group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
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Fig. 2. Effects of SO2 on the activities of PAL (A), PPO (E) and contents of total phenol (B), flavonoid (C), lignin (D) in table grapes during storage.
plump in the SO2 treatment group. In addition, the SO2-treated fruit had no occurred bleaching at 60 d. However, the fruit appeared obvious infection symptoms in the control group (Fig. 1B). The fruits quality of SO2 treatment group was significantly higher than that of the control.
control group was significantly lower than that of the SO2 treatment group at 40 d. After 60 days, the fresh fruit rate in the control group declined to 75% while still maintained 95% in SO2 treatment group. The stem of grape was more fresh and green, and the fruit was more 570
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Fig. 3. Effect of SO2 on transcription levels of PR proteins coding genes in table grapes during storage.
The results showed that SO2 could effectively improve the fruit quality and extend postharvest life of table grapes.
3.2. SO2 activated secondary metabolism pathway SO2 treatment markedly enhanced activities of PAL and PPO, promoted the accumulation of total phenol, flavonoid and lignin during storage (Fig. 2A–E). PAL activity in SO2 group was significantly higher than that in control group (Fig. 2A), suggesting that secondary metabolism were activated by SO2 during storage. Within 20 days, there was no obviously difference of PAL activity in control and SO2 group but significant increase could be found after that. PAL activity in the control group decreased quickly while the SO2 treatment group still kept the high level at 40 d. It was worth noting that change of PAL activity was consistent with fresh fruit rate during storage. As the key and limited enzyme in phenylpropanoid metabolic pathway, the increase of PAL activity is conducive to the synthesis of secondary metabolites. The changes in the content of total phenol, flavonoid and lignin are shown in Fig. 2(B-D). During storage, higher and earlier accumulation of phenolic compounds was observed in SO2 treatment group. The content of total phenol showed an increasing trend in both control and SO2 treatment group during storage and SO2 significantly enhanced the elevated levels. Flavonoid and lignin contents obviously increased in the SO2 group, whereas they increased slightly or remained unchanged in the control group. As shown in Fig. 2(E), PPO activity was initially higher and rapidly decreased both in the control and SO2 treatment group during storage. PPO activity in SO2 group was noticeably higher than that in control group during 20 d to 40 d.
Fig. 5. A schematic model of SO2-induced disease resistance in Vitis vinifera L. during storage.
3.3. SO2 activated the expression of PR genes and proteins The Transcript levels of CHI3, CHI1b and PR2 in SO2 treatment group was overall increased (Fig. 3). CHI3 responded faster than other two genes. The expression of CHI3 was up-regulated at 20 d in the SO2 group, and then gradually increased with the extension of SO2 treatment. PR2 gradually increased and peaked at 40 d, and then declined at 60 d. CHI1b expressed almost no change before 40 d and up-regulated until 60 d. In SO2 treatment group, chitinase and β-1,3-glucanase activity increased rapidly. Meanwhile, SO2 maintained significantly higher activities of chitinase and β-1,3-glucanase during the whole storage period compared with the control (Fig. 4), which is consistent with transcript levels of both CHI3 and PR2. In addition, the activities of chitinase and β-1,3-glucanase remarkably increased in control group at 60 d, which is highly likely to be associated with decline of fruit
Fig. 4. Effects of SO2 on the activities of chitinase (A) and β-1,3-glucanase (B) in table grapes during storage.
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markedly induced in SO2 treatment group. The two enzymes can hydrolyze chitins or glucans which are essential components of the cell wall of pathogens, leading to the death of pathogens and further infection. These results further confirmed that the SO2-induced disease resistance against pathogens infection in table grapes during storage is also associated with priming of PR proteins defense responses. In conclusion, our results demonstrated that SO2 can effectively induce disease resistance and maintain fruit quality of table grapes during postharvest storage. Moreover, the SO2 induced disease resistance is associated with priming of defense responses through enhancing (i) secondary metabolism pathway: SO2 significantly enhanced activity of PAL, promoted the accumulation of phenolic compounds, and improved the PPO activity; (ii) PR proteins coding genes expression and enzymes activity (Fig. 5). The results provide a new insight into SO2 preservation and scientific foundation for searching for more effective and safe food preservatives. The authors declare that they have no conflict of interest.
qualities at this time. 4. Discussion Although table grapes are a non-climacteric type of fruit with a low rate of physiological activity following harvest, berries are highly susceptible to fungal infection during storage. To date the only effective preservative is SO2 in the viticultural industries. The SO2 dual release technique, which is the most effective commercial preservation method so far, was used in our experiment. In the early stage of storage (the first stage), the high concentration SO2 could kill the pathogens carried from the field, and in the late stage (second stage), the low concentration of SO2 could maintain the environment of suppress pathogens (Suppl Fig. 1). The previous research found that the sulfite residue content in grape was only 3.9 mg/kg after SO2 dual release treatment for 180 d (Zhao et al., 2011). The view that sulphites act directly on bacterial and fungal pathogens may be simplistic. Mechanisms of sulfur-enhanced defense are largely unknown. In this respect, the current study evaluated the effects of SO2 on fruit quality, PAL activity, contents of phenolic compounds, PPO activity, and PR proteins activity and transcript levels of coding genes of ‘Muscat Hamburg’ table grapes during storage. Our present study showed that SO2 resulted in higher activities of PAL, PPO, and the contents of total phenol, flavonoid and lignin. Induction of PAL activity is a reliable indicator in grape defense against Botrytis cinerea during postharvest storage (Duarte-Sierra et al., 2015; Jiang et al., 2015; Qin et al., 2015). PAL is critically involved in the biosynthesis of phenolic compounds that contribute to plant disease resistance (Nguyen et al., 2003; Freeman and Beattie, 2008). Phenolic compounds in grapes could improve the quality and nutritional value because of antibacterial activities and strong antioxidant (Donnini et al., 2016; Cheng et al., 2017). Eshghi et al. (2014) found that there was a strong correlation between total phenolic content and antioxidant activity in different grapevine cultivars. Flavonoids are a class of bioactive compounds having antioxidant properties. Flavonoids greatly affect the sensory and nutritional quality of grapes (Brillante et al., 2015). Lignin is involved in the formation of structural barriers that can block pathogen invading and spread (Cartea et al., 2011). In the current experiment, SO2 enhanced PAL activity in table grapes during storage, resulting in accumulation of total phenolic, flavonoid and lignin, which collectively contribute to resistance against pathogens. Besides PAL, increased levels of PPO are also important for defense against pathogen infections of table grapes during storage (Al-Qurashi and Awad, 2015; Awad et al., 2015; Jiang et al., 2015; Qin et al., 2015). PPO is involved in lignification of host plant cells (Jiang et al., 2015) and could produce antimicrobial quinones through oxidizing phenolic compounds (Melo et al., 2006). In this study, PPO activities in both control group and SO2 treatment group were significantly decreased under low temperature storage. The variation trend is conducive to alleviate the browning of grape pulp during storage (Guardo et al., 2013; Hu et al., 2014). As an important terminal oxidase, the decrease of PPO activity might be caused by low temperature inhibiting fruit respiration rate of grapes (Chitbanchong et al., 2009). Therefore, PPO has a dual role during storage: resisting pathogen infection and preventing flesh browning. All these results suggested that SO2 effectively primes the secondary metabolism for enhanced disease resistance in table grapes during storage. Induction of PR proteins is also believed an indicator of plant induced defense responses (Vleeshouwers et al., 2000). Usually, chitinase and β-1,3-glucanase were induced synergistically to enhance plants disease resistance (Pozo et al., 2002; Duarte-Sierra et al., 2015; Jiang et al., 2015; Song et al., 2015). Duarte-Sierra et al. (2015) found that SO2 treatment primed defense responses against Botrytis cinerea in Redglobe and Sugraone grapes through inducing higher expression of chitinase and β-1,3-glucanase. Our study showed that SO2 significantly enhanced expression levels of CHI3, CHI1b and PR2 in the table grapes. Meanwhile, the activities of chitinase and β-1,3-glucanase were
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