Article Cite This: J. Agric. Food Chem. 2018, 66, 6364−6372
pubs.acs.org/JAFC
1‑Methylcyclopropene Treatment on Phenolics and the Antioxidant System in Postharvest Peach Combined with the Liquid Chromatography/Mass Spectrometry Technique Xiaoqin Wu,† Xiujuan An,† Mingliang Yu,‡ Ruijuan Ma,‡ and Zhifang Yu*,† †
College of Food Science and Engineering, Nanjing Agricultural University, Nanjing, Jiangsu 210095, People’s Republic of China Institute of Horticulture, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu 210014, People’s Republic of China
‡
Downloaded via UNIV OF CALIFORNIA SANTA BARBARA on June 29, 2018 at 13:31:27 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
S Supporting Information *
ABSTRACT: In the present study, the potential effect of 1-methylcyclopropene (1-MCP) treatment on phenolics and antioxidant capacity in postharvest peach was assessed. Peach fruit (cv. Xiahui-8) treated with 1-MCP or without treatment was stored in 25 °C for 2, 4, 6, and 8 days. The phenolic composition and change trend were evaluated by liquid chromatography/ mass spectrometry. The reactive oxygen species production and scavenging capacity against DPPH, O2• −, and HO• were determined. Gene expression of enzymes in the flavonoid biosynthetic pathway was assayed by quantitative reverse transcription polymerase chain reaction analysis. 1-MCP application inhibited the ethylene and CO2 production and stimulated the total phenol and total flavonoid contents. Total anthocyanin formation may be influenced directly or indirectly by the level of ethylene. The scavenging capacities of DPPH, HO•, and O2• − after 1-MCP treatment were enhanced. 1-MCP treatment affected the tissue color change, stimulated gene expression of PpaPAL, PpaCHS, PpaF3H, and PpaUFGT, and promoted the biosynthesis of flavonoids and stability of anthocyanin. PpaDFR and PpaUFGT played crucial roles in rapid color change stages. Kaempferol and kaempferol 3-O-galactoside increased distinctively during storage time. KEYWORDS: 1-MCP, peach, LC/MS, polyphenol, gene and individual phenolic compounds in the peach,7 while other studies found that 1-MCP-treated strawberry8 and loquat9 fruit contained elevated phenolics. Conversely, postharvest apple fruit with 1-MCP application did not show a difference in anthocyanin, flavonol, and flavan-3-ol contents.10 It is evident that phenolic change in response to 1-MCP varies among different fruit cultivars, possibly reflecting fruit-specific requirements for synthesis of secondary metabolites in pathogen resistance.11 The development of methodologies for the identification and quantification of phenols has been discussed extensively in the literature. Traditional liquid chromatography (LC) was limited to standard samples in identification of phenolic compounds and has relatively low separation efficiency. Liquid chromatography/mass spectrometry (LC/MS) is the most promising and popular technique nowadays because of its relatively short analysis time and high separation efficiency; moreover, it makes preliminary identification of samples based on mass spectrometry (MS) data and the corresponding specific fragments.12 On the above basis, the objective of the present study was to investigate the potential effect on individual phenolic change, flavonoid biosynthesis, and antioxidant capacities on both a metabolic and genetic level, which can provide an important molecular basis and reference information for future research.
1. INTRODUCTION Peach [Prunus persica (L.) Batsch] fruit is considered to be one of the most popular commodities and is cultivated widely in appropriate climates around the world.1 As a result of its high nutritional and aesthetic value, such as flavor, vitamins, and phenolics, peach fruit has aroused considerable attention and appreciation from both consumers and researchers. Concerning these favorable nutritional compounds, phenolics have attracted more concern in recent years as a result of their noticeable antioxidant properties. With superb abilities of hydrogen and electron donation as well as metal chelation, phenolic compounds in fruit exhibit the superiority in not only medical application2 but also enhancement of antioxidant capacity.3 In addition, phenolic compounds are also involved in fruit visual appearance (pigmentation and browning) and taste (astringency).4 Therefore, unraveling the regulation of phenolic metabolism has biological, dietary, and medical significance. The postharvest life of peach is limited at ambient temperature as a result of the perishable attribute. To explore new strategies to alleviate the decay of peach products and prolong their shelf life, 1-methylcyclopropene (1-MCP) was widely investigated during the past few years as a result of advantages of high efficacy at relatively low concentrations and short treatment periods.5 Application of 1-MCP has been extensively investigated in various fruits and vegetables.6 However, as a result of the complexities of phenolic metabolism, reports on relationships of phenolics of 1-MCP-treated fruit are still limited and inconsistent. Previous work has shown that 1-MCP treatment delayed the accumulation of total phenolics, anthocyanidins, © 2018 American Chemical Society
Received: Revised: Accepted: Published: 6364
April 5, 2018 June 2, 2018 June 6, 2018 June 6, 2018 DOI: 10.1021/acs.jafc.8b01757 J. Agric. Food Chem. 2018, 66, 6364−6372
Article
Journal of Agricultural and Food Chemistry
chalcone isomerase (CHI), chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) were designed on the sequences reported by Liu et al.23 Total RNA samples were extracted using the MiniBEST Plant RNA Extraction Kit (TaKaRa, Japan) and then purifed using RNase-free DNase (TaKaRa, Japan). The first-strand cDNA was synthesized using the PrimeScript RT Master Mix (TaKaRa, Japan). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis was conducted with a CFX96 instrument (Bio-Rad, Hercules, CA, U.S.A.), consisting of SYBR1 Premix Ex Taq (TaKaRa, Japan) and gene-specific primers (Table S1 of the Supporting Information) in a total volume of 20 μL. The qRT-PCR experiment and the relative intensity of gene expression were conducted according to method of Huan et al.15 2.8. LC/MS Analysis of Phenols. Here, we choose the time point at which the climacteric peak occurred (D4) and the last stage of senescence (D8) for further LC/MS analysis. The phenol extract solution described above at D4 and D8 was evaporated to dryness using a rotary evaporator under a reduced pressure and temperature of 30 °C. The residue was redissolved in 6 mL of methanol, filtered through a 0.22 μL membrane (Millipore) filter, and analyzed using LC/MS. The extract was analyzed by the LC/MS system (G2-XS QTof, Waters). A total of 2 μL of solution was injected into the ultra performance liquid chromatography (UPLC) column (2.1 × 100 mm ACQUITY UPLC BEH C18 column containing 1.7 μm particles) with a flow rate of 0.4 mL/min. Buffer A consisted of 0.1% formic acid in water, and buffer B consisted of 0.1% formic acid in acetonitrile. The gradient was 5% buffer B for 2 min, 5−40% buffer B for 12 min, 40−95% buffer B for 2 min, and 95% buffer B for 2 min. Mass spectrometry was performed using an electrospray source in positive or negative ion mode with MSe acquisition mode, with a selected mass range of m/z 50−1200. The lock mass option was enabled using leucine enkephalin (m/z 556.2771) for recalibration. The ionization parameters were the following: capillary voltage of 2.5 kV, cone voltage of 40 V, source temperature of 120 °C, and desolvation gas temperature of 400 °C. Collision energy was 20−40 eV. Data acquisition and processing were performed using MassLynx 4.1. 2.9. Statistical Analysis. All statistical analyses were performed with the SPSS 18.0 software, using a one-way analysis of variance (ANOVA) by Duncan’s test (p ≤ 0.05). Figures were made with Origin Pro 8.6 software.
2. MATERIALS AND METHODS 2.1. Fruit Materials and Treatment. Fruit of ‘Xiahui-8’ cultivar was harvested in the orchard of Jiangsu Academy of Agricultural Sciences in Nanjing, China. A total of 300 fruit, without mechanical damage, selected for uniform size and appearance, were collected and quickly transported to the laboratory on 125 days after florescence. The harvested fruit (D0) was randomly divided into two groups and subjected to the following treatments: (1) In the 1-MCP treatment group (MT), the fruit was treated with 10 μL L−1 1-MCP for 12 h. 1-MCP powder (Sinopharm Chemical Reagent Beijing Co., Ltd., China) was calculated according to the instructions of the manufacturer and dissolved in water, and then 1-MCP gas will be automatically released from that solution. After treatment, peach fruit was then stored at room temperature (25 ± 5 °C) with 80−90% relative humidity for 2, 4, 6, and 8 days (MT2, MT4, MT6, and MT8). (2) The control group (CK) was directly stored under the same conditions as described above without any treatment for 2, 4, 6, and 8 days (CK2, CK4, CK6, and CK8). Samples taken at different storage time points were peeled and immediately used or frozen in liquid nitrogen and stored at −80 °C for further analysis. There were three replicates in each time point, and only the mesocarp was used for analysis. 2.2. Respiratory Rate, Ethylene Production, and Color Determination. The respiratory rate and ethylene production were measured according to our previous report.13,14 Five peaches from each replicate were evaluated for tristimulus color using a Minolta colorimeter (Konica Minolta CR-400 Co., Ltd., Osaka, Japan) on the basis of the CIELAB (L*, a*, and b*) color system. Two values were taken at both of the opposite sides per peach. We used whole fruit for peel color detection and conducted pulp color measurement after peeling. 2.3. Titratable Acidity (TA) and Total Soluble Solids (TSS) Determination. For TA and TSS, fruit tissue was smashed using a mortar and pestle, and a few drops of the juice were placed in a Pocket Brix-Acidity Meter (PAL-BX/ACID 5, Japan). Results are expressed in percentage (%). 2.4. Electrolyte Leakage, Malondialdehyde (MDA), H2O2, and O2• − Content Determination. Electrolyte leakage was determined according to the method of Huan et al.15 MDA and O2• − contents were measured as described in our previous research.16 H2O2 determination was conducted using the Hydrogen Peroxide Assay Kit (Jiancheng, China). The H2O2, MDA, and O2• − contents were all expressed as mmol kg−1 of fresh weight (FW). 2.5. Phenolic Compound Extraction, Total Phenol, Total Flavonoid, and Anthocyanin Content Determination. Approximately 10 g of peach tissue was ground in liquid nitrogen. From these ground-up peach samples, 5 g was accurately weighed and homogenized in 0.1 L of 95% acidic (0.1 M HCl) methanol. The mixture was shaken in the dark for 4 h and then centrifuged at 10000g for 20 min. The supernatant obtained was collected for further analysis. Total phenols were determined by a colorimetric assay based on the procedure described by Singleton et al.17 The total flavonoid content was measured by the aluminum chloride colorimetric assay.18 The total anthocyanin content was measured using the pH differential method described by Albishi et al.19 2.6. DPPH Radical, Superoxide Radical (O2• −), and Hydroxyl Radical (HO • ) Scavenging Capacity. The DPPH radical scavenging activity was estimated by the method of Bondet et al.,20 with some modifications. A total of 0.5 mL of the sample was added to 2.5 mL of 0.2 mM DPPH solution in ethanol. The mixture was incubated in a dark room for 30 min, and absorbance was measured at 517 nm. The O2• − scavenging assay was measured by the method of Wang and Luo.21 The HO• scavenging assay was measured by the method of Ghiselli et al.22 Percent inhibitions of DPPH, O2• −, and HO• were all calculated as (1 − absorbance of the sample/absorbance of the control) × 100. 2.7. RNA Isolation and Expression Analysis. Genes of polyphenol metabolism involving phenylalanine ammonia lyase (PAL), leucoanthocyanidin reductase (LAR), and flavonoid 3′,5′hydroxylase (F3′5′H) were identified from the Genome Database for Rosaceae (GDR; http://www.rosaceae.org/). Primers used for
3. RESULTS 3.1. Respiratory Rate, Ethylene Production, Color, TSS, and TA. The rate of CO2 production in this study increased and reached the climacteric peak at D4, followed a decrease at D6, and then increased sharply at D8 (Figure 1A). 1-MCP treatment effectively maintained a lower CO2 production compared to the control group. Ethylene release increased continually throughout the storage time, and the application of 1-MCP significantly suppressed ethylene production after D4. Changes of L* and b* of both peel and pulp did not have a statistical difference between treatment most of the time. To not lose focus and to simplify the data, we only highlight the a* value in Table 1, which increased continually during storage time in both peel and pulp. 1-MCP treatment statistically induced the pulp color change compared to CK. TSS and TA contents in 1-MCP-treated fruit were a little lower than those in CK most of the time. 3.2. Electrolyte Leakage (EL) and H2O2, MDA, and O2• − Contents. In our results, EL and O2• −, H2O2, and MDA contents showed a similar trend during the ripening stage, increasing constantly until the end. Fruit with 1-MCP treatment effectively maintained a higher ability of ROS scavenge as well as a lower EL and MDA content. 6365
DOI: 10.1021/acs.jafc.8b01757 J. Agric. Food Chem. 2018, 66, 6364−6372
Article
Journal of Agricultural and Food Chemistry
Figure 1. Physiological and biochemical changes of peach in the control group (CK) and 1-MCP treatment group (MT). Values are the mean ± standard error (SE) from three replicates. Different letters indicate significant differences at the 0.05 level (Duncan’s test).
Table 1. Representative Images and Color Change of Peach and Tissuea
Values are the mean ± SE from 10 replicates, and the letters indicate significant differences at the 0.05 level (Duncan’s test).
a
3.3. Total Phenol, Total Flavonoid, and Anthocyanidin Contents. In this experiment, total phenol and total flavonoid contents demonstrated a similar pattern, which decreased slightly at D2 and then increased with the prolonged ripening period (Figure 1J). 1-MCP elevated the total phenol and flavonoid contents in the early stage, except at D8. The total anthocyanin content generally displayed a continuously increasing trend in both control and 1-MCP-treated samples, as shown in Figure 1L. However, 1-MCP-treated fruit holds a lower anthocyanin level compared to CK. 3.4. Scavenging Capacity of the DPPH Radical, O2• −, and HO•. The DPPH radical scavenging capacity in this experiment increased at D4, followed by a slight decrease, and
then reached a maximum at D8. 1-MCP treatment exhibited significantly higher free radical scavenging activity than control group samples (Figure 1H). As showed in Figure 1I, the O2• − scavenging capacity increased sharply at D2 and, thereafter, decreased constantly until the end, and 1-MCP treatment maintained a higher O2• − scavenging ability. The HO• scavenging capacity was continually decreased throughout the storage time. 1-MCP treatment could slow the decline tendency (Figure 1G). 3.5. Phenolic Profile Using LC/MS Analysis. The extracts were subjected to LC/MS, and 20 phenolic compounds were successfully identified on the basis of their retention times, MS data, and corresponding specific fragment, including anthocyanins, flavanones, flavanols, flavones, flavonols, and phenolic 6366
DOI: 10.1021/acs.jafc.8b01757 J. Agric. Food Chem. 2018, 66, 6364−6372
6367
isorhamnetin 3-O-glucoside 7-O-rhamnoside
gallic acid
4-hydroxybenzoic acid 4-O-glucoside
15
16
17
luteolin 7-O-(2-apiosyl-6-malonyl)-glucoside
13
5,3′,4′-trihydroxy-3-methoxy-6:7-methylenedioxyflavone 4′-O-glucuronide
apigenin 7-O-glucoside
12
14
kaempferol 3-O-galactoside
11
(+)-gallocatechin
7
kaempferol
poncirin
6
10
naringenin 4′-O-glucuronide
5
luteolin 7-O-glucuronide
pelargonidin 3-O-rutinoside
4
9
cyanidin 3-O-xylosyl-rutinoside
3
(+)-catechin
peonidin 3-O-(6′′-p-coumaroyl-glucoside)
2
8
pigment A
proposed compound
1
peak
phenolic acids
phenolic acids
flavonols
flavonols
flavones
flavones
flavones
flavones
flavones
flavanols
flavanols
flavanones
flavanones
anthocyanin
anthocyanin
anthocyanin
anthocyanin
catagory
Table 2. Phenolic Compounds Analyzed by LC/MS
3.38
2.50
4.84
1.32
9.48
4.39
3.95
3.94
0.92
3.46
4.23
9.57
3.94
9.70
9.64
4.82
4.09
RT (min)
323.0734
170.0210
624.1701
520.0844
666.1414
432.1066
448.1006
286.0479
462.0811
290.0788
306.0725
594.1942
448.1003
579.1731
727.2090
609.1622
609.1615
neutral mass (Da)
C15H14O7 C15H14O6
0 −0.3 0.1 −0.2 1.4 0
449.1076b 595.2014b 307.0797b 291.0861b 463.0883b 287.0738b
299.0841
c
169.0156c
625.1774
b
521.0917b
667.1486
b
433.1139b
C7H6O5 C13H16O8
−0.2
C28H32O16 0.1
1.2
C23H20O14
C29H30O18
−2.8 0.2
C21H20O10
C21H20O11
C15H10O6
C21H18O12
C27H31O14
0.1
0
C28H34O14
1.2
580.1804b
449.1079
C21H20O11
0.5
728.2163b
b
C31H29O13
−0.2
610.1695b C32H39O19
C31H29O13
0.1
610.1688b
formula
mass error (ppm)
[M + H]b/[M − H]c (m/z)
10
2
40
9
22
17
23
5
17
3
6
19
27
6
7
37
22
fragment number
1 ± 0.08
1 ± 0.06
1 ± 0.06
1 ± 0.16
1 ± 0.18
1 ± 0.17
1 ± 0.10
1 ± 0.07
1 ± 0.06
1 ± 0.08
1 ± 0.04
1 ± 0.03
1 ± 0.10
1 ± 0.01
1 ± 0.06
0.28 ± 0.24 1.17 ± 0.02
0.18 ± 0.01
1.37 ± 0.02 0.51 ± 0.01
1.39 ± 0.07 1.50 ± 0.12
0.85 ± 0.05 1.21 ± 0.05
1.07 ± 0.09
0.84 ± 0.09
0.64 ± 0.09 0.71 ± 0.08
0.14 ± 0.02 0.29 ± 0.03
0.20 ± 0.03 0.04 ± 0.02
0.21 ± 0.03
0.45 ± 0.07
0.23 ± 0.03 0.15 ± 0.02
0.20 ± 0.02 0.14 ± 0.02
2.35 ± 0.09 0.42 ± 0.07
2.25 ± 0.12
1.48 ± 0.05
2.00 ± 0.08 1.47 ± 0.05
1.99 ± 0.11 1.31 ± 0.04
0.77 ± 0.06 1.45 ± 0.08
0.83 ± 0.14
0.49 ± 0.02 1.08 ± 0.03
0.46 ± 0.07 1.27 ± 0.19
0.65 ± 0.08
0.91 ± 0.02 0.74 ± 0.02
0.84 ± 0.05 0.67 ± 0.04
0.97 ± 0.01
0.43 ± 0.03 0.73 ± 0.03
0.87 ± 0.04
2.35 ± 0.09 0.82 ± 0.03
1.47 ± 0.05
1.58 ± 0.06 2.25 ± 0.12
1.24 ± 0.01 1.48 ± 0.05
1.24 ± 0.05
0.90 ± 0.04 1.42 ± 0.04
1.24 ± 0.03 1.32 ± 0.01
1.68 ± 0.10
1.39 ± 0.04 1.68 ± 0.01
1.33 ± 0.02
1.59 ± 0.04
1.42 ± 0.29
1 ± 0.08
1.34 ± 0.03
1.23 ± 0.05 1.18 ± 0.07
1 ± 0.11
CK8 MT8
CK4 MT4
CK0
fold changea
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/acs.jafc.8b01757 J. Agric. Food Chem. 2018, 66, 6364−6372
Article
The relative content of phenolics is expressed according to the peak value of each compound at D0, which is set to 1. Values are the mean ± SE from three replicates. Positive ionization mode in LC/MS analysis. cNegative ionization mode in LC/MS analysis. b
1.08 ± 0.04
acids (Table 2). Representative mass spectrograms of gallic acid at m/z 169.0156 in negative ionization mode and kaempferol at m/z 287.0738 in positive ionization mode were highlighted in Figures S2-1 and S2-2 of the Supporting Information. qRT-PCR Analysis. Gene expressions of the flavonoid biosynthesis pathway were investigated using qRT-PCR, which were exhibited in Figure 3. In our experiment, 1-MCP treatment remarkably stimulated expressions of PpaCHS, PpaF3H, PpaUFGT, PpaDFR, and PpaPAL. The difference between CK and MT was particularly evident from D2 to D6, but that gap was narrowing with the prolonged ripening period. 1-MCPtreated fruit has a less statistical effect on PpaCHI and PpaF3′5′H expression.
4. DISCUSSION As a climacteric fruit, peach is characterized by an upsurge in the respiration correlated with a burst of ethylene production during the ripening stage. Fruit respiration converts stored sugar to energy in the presence of oxygen substrates, which is crucial to maintain normal metabolic processes. However, while playing a key role in metabolism, mitochondrial electron transport can generate reactive oxygen species (ROS) at the same time.24 As members of ROS, H2O2 and O2• − are inevitably accumulated when serious imbalance occurs between production of ROS and antioxidant defense, consequently leading to cell damage and senescence.25 When tissue damage and degradation occur, the lipid peroxidation process would accelerate and generate more MDA product.15 Moreover, EL can effectively reflect the membrane permeability and is used as an indicator of membrane integrity. In this research, the peach respiratory rate showed a typical pattern of climacteric fruit, reaching the climacteric peak at D4. However, the respiratory activity was still intense at D8, which might be induced by tissue damage in the later stage of fruit senescence.26 1-MCP treatment effectively inhibited CO2 and ethylene production as well as ROS accumulation, coinciding with the result described in plum and apple fruits,6 which suggesting that 1-MCP can alleviate oxidative damage and postpone senescence. Phenolic compounds are secondary metabolites widely found in fruit, mostly represented by flavonoids and phenolic acids.27 Anthocyanin is another core member of total phenols with brightly colored compounds and greatly contributes to important organoleptic properties of fruit, such as color, flavor, and taste.28 The growing interest in these substances is mainly due to their antioxidant potential, which is a kind of antioxidant capacity by a variety of mechanisms, including free radical scavenging, single-electron reduction, and metal ion chelation,29 such as DPPH, O2• −, and HO• scavenging abilities. Total phenolic and flavonoid contents in this study kept rising during storage, and 1-MCP apparently activated the synthesis of these compounds, similar to that in strawberry,8 medlar,30 and loquat9 fruits, which are closely related to the higher quenching ability of DPPH, O2• −, and HO• in MT. Furthermore, as the first key enzyme of phenolic metabolism, PpaPAL expression in MT was much higher than that in CK (Figure 3G), which can provoke the phenolic biosynthesis, in accordance with the higher phenolic content in MT (Figure 1J). It is interesting to note that the total phenol and total anthocyanin contents exhibited a similar pattern to ethylene production, Liu et al. also reported a similar phenomenon between total phenol and ethylene production when studying ‘Yuhualu’ peach fruit.7 However, in our study, we found that the rule of the total anthocyanin content in response to 1-MCP closely coincided
a
1.77 ± 0.02
1.11 ± 0.05
0.85 ± 0.01 0.87 ± 0.01
2.12 ± 0.08 1 ± 0.09 7 1 341.0570c caffeoyl glucose 20
phenolic acids
7.44
342.0935
C17H20O9 −0.4 368.1102 4.36 3-feruloylquinic acid 19
phenolic acids
C15H18O9
1.10 ± 0.02
1.11 ± 0.01 0.97 ± 0.01 1 ± 0.17
0.70 ± 0.02
367.1101c
13
0.64 ± 0.01 0.26 ± 0.01 1 ± 0.05 8 C16H18O9 −0.6 353.1012c 3-caffeoylquinic acid 18
phenolic acids
3.55
354.0945
formula mass error (ppm) proposed compound peak
Table 2. continued
catagory
RT (min)
neutral mass (Da)
[M + H]b/[M − H]c (m/z)
fragment number
fold changea
CK8 MT8 CK4 MT4 CK0
Journal of Agricultural and Food Chemistry
6368
DOI: 10.1021/acs.jafc.8b01757 J. Agric. Food Chem. 2018, 66, 6364−6372
Article
Journal of Agricultural and Food Chemistry
Figure 2. Biosynthetic pathway of flavonoids according to a previous report39 and the KEGG PATHWAY database (http://www.genome.jp/kegg/ pathway.html). F3′H, flavonoid 3′-hydroxylase; ANR, anthocyanidin reductase; LDOX, leucoanthocyanin dioxygenase; LAR, leucoanthocyanidin reductase; DFR, dihydroflavonol 4-reductase; UFGT, UDP-glucose:flavonoid 3-O-glucosyltransferase; FLS, flavonol synthase; LDOX, leucoanthocyanidin reductase; CHS, chalcone synthase; CHI, chalcone isomerase; and PAL, phenylalanine ammonia lyase.
with that of ethylene production (panels B and L of Figure 1). Here, we speculate that anthocyanin synthesis is greatly influenced by ethylene, because 1-MCP treatment suppressed ethylene release and the anthocyanin accumulation decreased correspondingly in MT. Our viewpoint is supported by previous research on grape,31 in which exogenous ethylene stimulates the long-term expression of genes related to anthocyanin biosynthesis. In addition, Saure32 proposed that anthocyanin formation may be promoted directly or indirectly by increasing the level of ethylene in apple. The detailed relationship with anthocyanin and ethylene needs further investigation. In our result, 1-MCP treatment effectively stimulated the scavenge capacity of DPPH, O2• −, and HO•, coinciding with the result in another report,7 which were essential for improving defense systems and ensuring cellular homeostasis. Previous investigations demonstrated a correlation between the total phenol content and DPPH radical quenching activity,33,34 and we observed a uniform change tendency between the DPPH scavenge capacity and total phenol content as well (panels H and J of Figure 1). Furthermore, both O2• − and HO• scavenging trends were consistent with total phenolic tendency. After LC/MS analysis, we obtained four anthocyanin compounds (Table 2), which increased in both CK and MT. This is favorable for color change and enhancement of the antioxidant system during storage.35 Gallocatechin (peak 7) and catechin (peak 8) were identified as the two main flavanols, and the syntheses of these two compounds were both catalyzed by leucoanthocyanidin reductase (LAR), using leucodelphinidin and leucoanthocyanidin as substrates, respectively. Leucoanthocyanidin can also be catalyzed into anthocyanidin by leucoanthocyanidin dioxygenase (LDOX) (Figure 2). In our study,
catechin and gallocatechin contents as well as PpaLAR expression all decreased, which is beneficial for the synthesis of anthocyanin in another branch in Figure 2, associated with the elevated level of the anthocyanin content (Figure 1L) in this study. Kaempferol and kaempferol 3-O-galactoside were found to be the main flavone compounds belonging to flavones (Figure 4). The study showed that kaempferol has antioxidant and antitumor properties.36 Both kaempferol and kaempferol 3-O-galactoside were increased distinctively during peach storage, which is helpful for enhancing the antioxidative ability and delaying the ripening process. Five phenolic acids were successfully detected, as shown in Table 2, among which the contents of gallic acid and 3-feruloylquinic acid took up the main content. 4-Hydroxybenzoic acid 4-O-glucoside and 3-caffeoylquinic acid declined during ripening, and 1-MCP treatment exerted the most evident effect on caffeoyl glucose. The detailed mechanism of specific phenolic acid compounds needs further analysis. Chalcone synthase (CHS), chalcone isomerase (CHI), and flavanone 3-hydroxylase (F3H) are committed enzymes involved in flavonoid biosynthesis. 1-MCP treatment stimulated the PpaCHS and PpaF3H expression during ripening compared to D0, which is beneficial to flavonoid synthesis, and, consequently, enhanced fruit antioxidant and pathogen resistance.37 The hydroxylation of flavonoid is a crucially important reaction in the plant, which can dominant the color, stability, and antioxidant capacity of the plant. Flavonoid 3′,5′-hydroxylase (F3′5′H) is a pivotal enzyme that determines the B-ring hydroxylation pattern of flavonoids by introducing hydroxyl groups at the 3′ and 5′ positions,38 including pathways, such as cyanidinand delphinidin-based anthocyanin, and biosynthesis of 6369
DOI: 10.1021/acs.jafc.8b01757 J. Agric. Food Chem. 2018, 66, 6364−6372
Article
Journal of Agricultural and Food Chemistry
Figure 3. Gene expression of the flavonoid biosynthesis pathway in the control group (CK) and 1-MCP treatment group (MT). Transcript levels are expressed relative to the value of each gene at day 0, which is set to 1. Values are the mean ± SE from three replicates, and the letters indicate significant differences at the 0.05 level (Duncan’s test). CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; UFGT, UDP-glucose:flavonoid 3-O-glucosyltransferase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; PAL, phenylalanine ammonia lyase; LAR, leucoanthocyanidin reductase; and F3′5′H, flavonoid 3′,5′-hydroxylase.
Figure 4. Percentage of each phenol (D0) in a total of 20 phenols using LC/MS analysis. A total of 20 phenols are set to 1. The number on the x axis is consistent with the peak number in Table 2. Values are the mean ± SE from three replicates.
synthesis, and elevating DPPH, O2• −, and HO• scavenging capacity. Furthermore, 1-MCP promoted the biosynthesis of flavonoids and stability of anthocyanins by activating PpaDFR and PpaUFGT expression, provoking an apparent color change in peach. The anthocyanin formation was influenced greatly by the level of ethylene, and kaempferol and kaempferol 3-Ogalactoside possess a critical role in delaying fruit senescence.
flavonols, such as quercetin and myricetin39 (Figure 2). In our result, expression of PpaF3′5′H was distinctively increased during ripening, explicating the major role in hydroxylation of flavonoid and the synthesis of anthocyanin. Dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and UDPglucose:flavonoid 3-O-glucosyltransferase (UFGT) were critical enzymes involved in the production or stability of anthocyanin. Research reported the high and consistent PpaDFR and PpaUFGT expression during the rapid color development stages in peach;23 thus, the gene overexpression of these two enzymes in MT was proposed to be in association with the color change of peach during ripening, which is coordinated with an obvious color change of the peach tissue in Table 1. Overall, 1-MCP effectively postponed peach ripening and prolonged the storage time by inhibiting the respiratory rate and ethylene production, stimulating polyphonel and flavonoid
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b01757. Primers used for quantification by qRT-PCR (Table S1), representative mass spectrogram of phenolic compounds from peach tissue extract from m/z 100 to 1000 (negative ionization mode) (A), mass spectrogram of galic acid at 6370
DOI: 10.1021/acs.jafc.8b01757 J. Agric. Food Chem. 2018, 66, 6364−6372
Article
Journal of Agricultural and Food Chemistry
■
(14) Wu, X. Q.; Yu, M. L.; Huan, C.; Ma, R. J.; Yu, Z. F. Regulation of the protein and gene expressions of ethylene biosynthesis enzymes under diferent temperature during peach fruit ripening. Acta Physiol. Plant. 2018, 40, 52. (15) Huan, C.; Jiang, L.; An, X. J.; Yu, M. L.; Xu, Y.; Ma, R. J.; Yu, Z. F. Potential role of reactive oxygen species and antioxidant genes in the regulation of peach fruit development and ripening. Plant Physiol. Biochem. 2016, 104, 294−303. (16) Wu, X. Q.; Jiang, L.; Yu, M. L.; An, X. J.; Ma, R. J.; Yu, Z. F. Proteomic analysis of changes in mitochondrial protein expression during peach fruit ripening and senescence. J. Proteomics 2016, 147, 197−211. (17) Singleton, V. L.; Orthofer, R.; Lamuela-Raventos, R. M. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin−Ciocalteu reagent. Methods Enzymol. 1999, 299, 152−178. (18) Yasir, M.; Sultana, B.; Amicucci, M. Biological activities of phenolic compounds extracted from Amaranthaceae plants and their LC/ESI−MS/MS profiling. J. Funct. Foods 2016, 26, 645−656. (19) Albishi, T.; John, J. A.; Al-Khalifa, A. S.; Shahidi, F. Antioxidative phenolic constituents of skins of onion varieties and their activities. J. Funct. Foods 2013, 5, 1191−1203. (20) Bondet, V.; Brand-Williams, W.; Berset, C. Kinetics and mechanisms of antioxidant activity using the DPPH* free radical method. LWTFood Sci. Technol. 1997, 30, 609−615. (21) Wang, Z. J.; Luo, D. H. Antioxidant activities of different fractions of polysaccharide purified from Gynostemma pentaphyllum Makino. Carbohydr. Polym. 2007, 68, 54−58. (22) Ghiselli, A.; Nardini, M.; Baldi, A.; Scaccini, C. Antioxidant activity of different phenolic fractions separated from an Italian red wine. J. Agric. Food Chem. 1998, 46, 361−367. (23) Liu, T.; Song, S.; Yuan, Y. B.; Wu, D. J.; Chen, M. J.; Sun, Q. N.; Zhang, B.; Xu, C. J.; Chen, K. S. Improved peach peel color development by fruit bagging. Enhanced expression of anthocyanin biosynthetic and regulatory genes using white non-woven polypropylene as replacement for yellow paper. Sci. Hortic. 2015, 184, 142−148. (24) Kang, S. W.; Lee, S.; Lee, E. K. ROS and energy metabolism in cancer cells: Alliance for fast growth. Arch. Pharmacal Res. 2015, 38, 338−345. (25) Wise, R. R.; Naylor, A. W. Chilling-Enhanced PhotooxidationThe Peroxidative Destruction of Lipids during Chilling Injury to Photosynthesis and Ultrastructure. Plant Physiol. 1987, 83, 272−277. (26) Lu, G. H.; Li, C. J.; Lu, Z. H. Wound-Induced Respiration in Thin Slice of Chinese Jujube Fruit. J. Plant Physiol. 1993, 141, 115− 119. (27) Bursal, E.; Koksal, E.; Gulcin, I.; Bilsel, G.; Goren, A. C. Antioxidant activity and polyphenol content of cherry stem (Cerasus avium L.) determined by LC−MS/MS. Food Res. Int. 2013, 51, 66− 74. (28) Ferrer-Gallego, R.; Hernandez-Hierro, J. M.; Rivas-Gonzalo, J. C.; Escribano-Bailon, M. T. Sensory evaluation of bitterness and astringency sub-qualities of wine phenolic compounds: Synergistic effect and modulation by aromas. Food Res. Int. 2014, 62, 1100−1107. (29) Huang, D. J.; Ou, B. X.; Prior, R. L. The chemistry behind antioxidant capacity assays. J. Agric. Food Chem. 2005, 53, 1841− 1856. (30) Selcuk, N.; Erkan, M. The effects of 1-MCP treatment on fruit quality of medlar fruit (Mespilus germanica L. cv. Istanbul) during long term storage in the palliflex storage system. Postharvest Biol. Technol. 2015, 100, 81−90. (31) El-Kereamy, A.; Chervin, C.; Roustan, J. P.; Cheynier, V.; Souquet, J. M.; Moutounet, M.; Raynal, J.; Ford, C.; Latche, A.; Pech, J. C.; Bouzayen, M. Exogenous ethylene stimulates the long-term expression of genes related to anthocyanin biosynthesis in grape berries. Physiol. Plant. 2003, 119, 175−182. (32) Saure, M. C. External Control of Anthocyanin Formation in Apple. Sci. Hortic. 1990, 42, 181−218.
m/z 169.016 (B), and tandem mass spectrometry (MS/MS) at 2.50 min RT (negative ionization mode) (C) (Figure S2-1), and representative mass spectrogram of phenolic compounds from peach tissue extract from m/z 100 to 1000 (positive ionization mode) (D), mass spectrogram of kaempferol at m/z 287.074 (E), and MS/MS at 3.94 min RT (negative ionization mode) (F) (Figure S2-2) (PDF)
AUTHOR INFORMATION
Corresponding Author
*Telephone: +8613951692350. E-mail:
[email protected]. Funding
This research was financially supported by the Innovation Project of Jiangsu Agricultural Science [CX (14) 2015], the Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement (2014015), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Notes
The authors declare no competing financial interest.
■
REFERENCES
(1) Lurie, S.; Crisosto, C. H. Chilling injury in peach and nectarine. Postharvest Biol. Technol. 2005, 37, 195−208. (2) Middleton, E.; Kandaswami, C.; Theoharides, T. C. The effects of plant flavonoids on mammalian cells: Implications for inflammation, heart disease, and cancer. Pharmacol. Rev. 2000, 52, 673−751. (3) Gil, M. I.; Tomas-Barberan, F. A.; Hess-Pierce, B.; Kader, A. A. Antioxidant capacities, phenolic compounds, carotenoids, and vitamin C contents of nectarine, peach, and plum cultivars from California. J. Agric. Food Chem. 2002, 50, 4976−4982. (4) Tomas-Barberan, F. A.; Gil, M. I.; Cremin, P.; Waterhouse, A. L.; Hess-Pierce, B.; Kader, A. A. HPLC−DAD−ESIMS analysis of phenolic compounds in nectarines, peaches, and plums. J. Agric. Food Chem. 2001, 49, 4748−4760. (5) Sisler, E. C.; Serek, M. Inhibitors of ethylene responses in plants at the receptor level: Recent developments. Physiol. Plant. 1997, 100, 577−582. (6) Blankenship, S. M.; Dole, J. M. 1-Methylcyclopropene: A review. Postharvest Biol. Technol. 2003, 28, 1−25. (7) Liu, H.; Cao, J. K.; Jiang, W. B. Changes in phenolics and antioxidant property of peach fruit during ripening and responses to 1-methylcyclopropene. Postharvest Biol. Technol. 2015, 108, 111−118. (8) Villarreal, N. M.; Bustamante, C. A.; Civello, P. M.; Martinez, G. A. Effect of ethylene and 1-MCP treatments on strawberry fruit ripening. J. Sci. Food Agric. 2010, 90, 683−689. (9) Cai, C.; Chen, K. S.; Xu, W. P.; Zhang, W. S.; Li, M.; Ferguson, I. Effect of 1-MCP on postharvest quality of loquat fruit. Postharvest Biol. Technol. 2006, 40, 155−162. (10) MacLean, D. D.; Murr, D. P.; DeEll, J. R.; Horvath, C. R. Postharvest variation in apple (Malus × domestica borkh.) flavonoids following harvest, storage, and 1-MCP treatment. J. Agric. Food Chem. 2006, 54, 870−878. (11) Singh, R.; Rastogi, S.; Dwivedi, U. N. Phenylpropanoid Metabolism in Ripening Fruits. Compr. Rev. Food Sci. Food Saf. 2010, 9, 398−416. (12) Bajoub, A.; Carrasco-Pancorbo, A.; Ajal, E.; Ouazzani, N.; Fernandez-Gutierrez, A. Potential of LC−MS phenolic profiling combined with multivariate analysis as an approach for the determination of the geographical origin of north Moroccan virgin olive oils. Food Chem. 2015, 166, 292−300. (13) Wu, X. Q.; Mason, A. M.; Yu, M. L.; Ma, R. J.; Yu, Z. F. Quantitative proteomic analysis of pre- and post-harvest peach fruit ripening based on iTRAQ technique. Acta Physiol. Plant. 2017, 39, 181. 6371
DOI: 10.1021/acs.jafc.8b01757 J. Agric. Food Chem. 2018, 66, 6364−6372
Article
Journal of Agricultural and Food Chemistry (33) Yasir, M.; Sultana, B.; Nigam, P. S.; Owusu-Apenten, R. Antioxidant and genoprotective activity of selected cucurbitaceae seed extracts and LC−ESIMS/MS identification of phenolic components. Food Chem. 2016, 199, 307−313. (34) Sultana, B.; Anwar, F.; Przybylski, R. Antioxidant activity of phenolic components present in barks of Azadirachta indica, Terminalia arjuna, Acacia nilotica, and Eugenia jambolana Lam. trees. Food Chem. 2007, 104, 1106−1114. (35) Tulio, A. Z.; Reese, R. N.; Wyzgoski, F. J.; Rinaldi, P. L.; Fu, R.; Scheerens, J. C.; Miller, A. R. Cyanidin 3-rutinoside and cyanidin 3xylosylrutinoside as primary phenolic antioxidants in black raspberry. J. Agric. Food Chem. 2008, 56, 1880−1888. (36) Kashafi, E.; Moradzadeh, M.; Mohamadkhani, A.; Erfanian, S. Kaempferol increases apoptosis in human cervical cancer HeLa cells via PI3K/AKT and telomerase pathways. Biomed. Pharmacother. 2017, 89, 573−577. (37) Sivankalyani, V.; Feygenberg, O.; Diskin, S.; Wright, B.; Alkan, N. Increased anthocyanin and flavonoids in mango fruit peel are associated with cold and pathogen resistance. Postharvest Biol. Technol. 2016, 111, 132−139. (38) Seitz, C.; Ameres, S.; Forkmann, G. Identification of the molecular basis for the functional difference between flavonoid 3′hydroxylase and flavonoid 3′,5′-hydroxylase. FEBS Lett. 2007, 581, 3429−3434. (39) Jeong, S. T.; Goto-Yamamoto, N.; Hashizume, K.; Esaka, M. Expression of the flavonoid 3′-hydroxylase and flavonoid 3′,5′hydroxylase genes and flavonoid composition in grape (Vitis vinifera). Plant Sci. 2006, 170, 61−69.
6372
DOI: 10.1021/acs.jafc.8b01757 J. Agric. Food Chem. 2018, 66, 6364−6372
