Food Chemistry 176 (2015) 27–39

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Analytical Methods

Differential protein profiles of postharvest Gynura bicolor D.C leaf treated by 1-methylcyclopropene and ethephon Li Jiang a, Ruoyi Kang a, Li Zhang b, Juan Jiang c, Zhifang Yu a,⇑ a

College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China Suzhou Academy of Agricultural Sciences, Suzhou 215155, PR China c College of Life Sciences, Qufu Normal University, Jining 273165, PR China b

a r t i c l e

i n f o

Article history: Received 16 April 2013 Received in revised form 1 November 2014 Accepted 15 November 2014 Available online 21 November 2014 Keywords: G. bicolor Senescence 1-Methylcyclopropene Ethephon Proteomics

a b s t r a c t Proteins were extracted from G. bicolor that had been treated with 1-methylcyclopropene and ethephon and then stored at room temperature for 1, 3 and 7 days. More than 300 protein spots were detected by 2-DE and 38 differentially abundant spots (P < 0.05) were excised and analysed by using MALDI-TOF/TOF. Thirty-three proteins were finally confidently identified. According to the Clusters of Orthologous Groups of proteins, the proteins identified were classified into those responsible for metabolism (75.8%), information storage and processing (9.1%) and cellular processes and signaling (12.1%). Compared with ethephon and control treatments, 1-methylcyclopropene specifically increased the abundances of superoxide dismutase, peroxidase, carbonic anhydrase, nucleoside diphosphate kinases, glyceraldehyde 3-phosphate dehydrogenase, RuBisCO and ribulose bisphosphate carboxylase/oxygenase activase. 1-Methylcyclopropene protected leaf chloroplast and cells by enhancing stress response and defense, and delayed senescence by inhibiting substance and energy metabolisms. Therefore, 1-methylcyclopropene allowed better self-defense and delayed senescence of G. bicolor leaf. Ó 2014 Published by Elsevier Ltd.

1. Introduction G. bicolor (Gynura bicolor D.C), a cultivated leafy vegetable, belongs to Composite Gynura Cass and is mostly produced in southern China (Jiang, Hou, Yuan, Jiang, & Yu, 2010). More than 90% of the leaf content is water, and chlorophyll and anthocyanin give a unique green surface and a purple back. Since the living organism with high metabolic activity after harvest is prone to losing nutrients and appearance during senescence, proper post-harvest treatments are in need for G. bicolor production. However, most G. bicolor-related studies have focused on its nutritional value, antioxidant activity, and distilled anthocyanin and flavonoids, while effective preservation technologies were only reported by our group during the last 6 years (Jiang, 2010; Jiang et al., 2010). Up to date, 1-methylcyclopropene (1-MCP), as a highly potent inhibitor of ethylene, has been widely applied to keep vegetables fresh (Blankenship & Dole, 2003). 1-MCP can dramatically slow senescence (Watkins, 2006), lower ethylene production and respiratory rates (Ella, Zion, Nehemia, & Amnon, 2003), reduce membrane lipid peroxidation (Blankenship & Dole, 2003) and maintain good quality of G. bicolor leaf (Jiang, 2010). However, ⇑ Corresponding author. Tel.: +86 25 84399098; fax: +86 25 84395618. E-mail address: [email protected] (Z. Yu). http://dx.doi.org/10.1016/j.foodchem.2014.11.081 0308-8146/Ó 2014 Published by Elsevier Ltd.

most studies hitherto merely focused on the physiological and biochemical changes (Blankenship & Dole, 2003) and gene expression (Ziliotto, Begheldo, Rasori, Bonghi, & Tonutti, 2008). Proteins, which execute physiological functions in an organism and directly reflect life phenomena, have specific activity patterns and provide indirectly genetic information (Komatsu et al., 2011). Evaluating protein structures and functions may clarify the mechanisms under physiological or pathological conditions. Liu, Li, Lai, Lo, and Chen (2013) found that N6-benzylaminopurine decreased the quantity of broccoli proteins involved in energy, carbohydrate and amino acid metabolisms. Zhang et al. (2012) reported that differentially abundant peach proteins, in response to 1-MCP and ethephon, were involved in energy and metabolism, cell structure, protein fate, stress response and defense, and ripening and senescence. A recent study on papaya fruit suggested that differentially accumulated proteins were related to cell wall degrading, oxidative damage protection and protein folding, and cell growth and survival were induced by 1-MCP during fruit ripening (HuertaOcampo et al., 2012). At present, the proteomics of postharvest leafy vegetables is critical, particularly for evaluating the regulatory effects of 1-MCP on the protein files during leafy vegetable senescence. We herein determined biological parameters and protein profiles of postharvest G. bicolor to analyze the functions of

28

L. Jiang et al. / Food Chemistry 176 (2015) 27–39

differentially abundant proteins to explore the relationship between protein profiles and biological parameters, and to elucidate the G. bicolor response to 1-MCP. Since this is the first proteomics study involving postharvest G. bicolor, the results are feasibly beneficial to analogous studies. 2. Materials and methods 2.1. Plant materials and treatments G. bicolor leaf were obtained on 10th Dec 2010 from the markets in Nanjing, Jiangsu, China. Harvested leaves (CK0) were immediately delivered to the laboratory and sorted to remove those with visual defects (pest-damaged, bruised and defective). The leaves were then divided into a control group (900 g), a 1-MCP treated group (900 g) and an ethephon treated group (900 g), and were immediately subjected to the following treatments: (a) control group (CK): without any treatment; (b) 1-MCP treatment (MT): leaves were closed up in desiccators in the presence of 500 lL L1 1-MCP (Agrofresh, USA) for 6 h. KOH (Sinopharm Chemical Reagent Beijing Co., Ltd., China) solution (1% (w/v)) was placed inside the desiccators to absorb CO2 during treatment. After being ventilated for 30 min, the leaves were packaged; (c) ethephon treatment (ET): leaves were dipped in a 200 lL L1 ethephon (Sinopharm Chemical Reagent Beijing Co., Ltd., China) solution for 10 min before being air-dried and packed. Leaves from each treatment group were divided into nine samples, each containing 100 g (100 g  9) and packed in sealed plastic bags. Thereafter they were placed in a room held at 20–25 °C with 80–90% relative humidity for 1, 3 and 7 days. Stalk-free leaves from each treatment group were immediately frozen in liquid nitrogen and stored at 20 °C prior to further analysis. CK0, CK1, CK3, CK7, MT1, MT3, MT7, ET1, ET3 and ET7 are abbreviations of control, 1-MCP and ethephon treatments on Day 0, 1, 3 and 7 respectively. 2.2. Determination of respiratory rate, and anthocyanin and chlorophyll contents The respiratory rate of G. bicolor, which was determined with the small skep method based on CO2 absorption (Wu et al., 2009), was expressed as milligrams of CO2 per kilogram per hour fresh weight (FW). The chlorophyll and anthocyanin contents in the fresh leaf samples was determined by the method of Jiang (2010). The absorbance of the extracts was determined using a spectrophotometer (WFJ UV-2802 PC). The chlorophyll content was expressed as mg g1 FW, and that of anthocyanin was expressed as lg g1 FW. 2.3. Protein sample preparation To minimize errors, the proteomic analysis at each treatment time point was conducted in triplicate. Total proteins of G. bicolor leaf were extracted according to a slightly modified phenol extraction method (Zhang et al., 2011). The frozen leaf tissue (0.5 g + 10% PVPP) was finely powdered in liquid nitrogen and extracted with 5 ml of extraction solution (1.05 mol L1 sucrose, 60 mmol L1 Tris, 10 mmol L1 EGTA, 1 mmol L1 PMSF, 1 mmol L1 DTT and 1% (v/v) TritonX-100, pH 8.35), and left for 2 h. The homogenate was centrifuged at 5000g for 30 min at 4 °C, the supernatant was collected for subsequent extraction using different solvents. After adding Trissaturated phenol (15 ml, pre-ice-cold, pH 7.8), samples were left for 2 h before the mixture was centrifuged at 5000g at 4 °C for 30 min. The collected upper phenolic phase was precipitated overnight with five folds of chilled acetone at 20 °C. After being

washed twice with ice-cold methanol and twice with chilled acetone, the pellets were then air-dried at room temperature and stored at 20 °C. The dried protein powders were finally solubilised overnight at 4 °C in lysis buffer (1 mg pellets in 100 lL of lysis buffer) containing 7 mol/l urea, 2 mol/l thiourea, 4% (w/v) CHAPS, 1% (w/v) DTT and 0.5% (v/v) pH 4–7 IPG buffer. The samples were then centrifuged at 5000g for 10 min at 4 °C. The supernatant protein contents were determined according to the method of Zhang et al. (2012) by using bovine serum albumin as the standard, and the samples were stored at 20 °C until 2-DE.

2.4. 2-DE and staining Sample aliquots (350 lL) containing 300 lg proteins were applied to pH 4–7 IPG (17 cm) strips, and were isoelectrically focused on a PROTEAN IEF system (Bio-Rad, Hercules, CA, USA) for a total run of 60 kV h at 20 °C. Then the focusing was performed on an IPGphor apparatus under the following conditions: 100 V, 200 V, 500 V and 1000 V for 1 h, respectively; 4000 V and 8000 V for 2 h and 8000 V until final volt-hours (60 kV h). Before SDS–PAGE, the strips were equilibrated for two periods of 15 min with 2% (w/v) DTT and 2.5% (w/v) iodoacetamide in equilibration buffer (1.5 mol/l Tris–HCl, pH 8.8, 6 mol/l urea, 20% glycerol, 4% SDS) respectively, after which the strips were run on 12% SDS–PAGE self-cast gels. Electrophoresis was carried out at 20 °C and 1.0 W/gel for 2 h, and at 15 W/gel until the dye front reached about 1 cm from the bottom of the gel using an Ettan Six vertical set (GE Healthcare). The gels were stained with silver nitrate as described by Blum, Beier, and Gross (1987). At least three replicates were performed for each treatment.

2.5. Image acquisition and data analysis All the stained gels were imaged by a Versdoc 3000 scanner (Bio-Rad) at 300 dpi resolution, and analysed by PDQuest Version 8.0 (Bio-Rad) (Zhang et al., 2012). The images were cropped and optimised, and protein spots were matched automatically and then carefully manually edited and confirmed. For each spot, the mean percentage volume (PV) was computed at every stage, and the spots showing a mean PV that increased/decreased at least twofold (statistically significant as calculated by one-way ANOVA (P < 0.05)) in different stages/treatments were considered as differentially abundant proteins.

2.6. Identification of protein in-gel digestion by MALDI-TOF/TOF The differentially abundant protein spots were digested with trypsin as described by Shevchenko, Wilm, Vorm, and Mann (1996). MALDI-TOF/TOF tandem MS analyses were performed by a 4800 MALDI-TOF/TOF Analyzer (Applied Biosystems, USA) in the m/z range of 700–3500 with an accelerating voltage of 20 kV in reflectron mode and a delayed extraction set to 120 ns. Thereafter, a combined search (MS plus MS/MS) was performed using GPS Explorer™ v3.5 (Applied Biosystems) over NCBI databases using the MASCOT search. Only the best matches with high confidence levels (confidence interval = 100%) were chosen when the software gave more than one eligible result. The differentially abundant proteins were functionally classified according to Functional Catalogue (http://mips.helmholtz-muenchen.de/proj/funcatDB) and Clusters of Orthologous Groups of proteins (COG, http://www. ncbi.nlm.nih.gov/COG/). The subcellular localisation predictions of 33 differentially abundant proteins were based on PSORT (http://wolfpsort.org).

L. Jiang et al. / Food Chemistry 176 (2015) 27–39

2.7. Data analysis Each result was determined as the mean of three replicates, and vertical bars indicate the standard deviations. A multivariate analysis was performed over the whole set of spots and on those showing differences. Principal component analysis (PCA) was applied to the correlation matrix to reduce its dimensionality. Using un-rotated principal component (PC) scores, the relationship between different treatment times was studied by determining the spots with the highest load on the variance. Statistical analysis was performed by SPSS (v.18 package). The differences were considered significant when P < 0.05. 3. Results Minimal respiratory rate was found in the MT3 group (P < 0.05) (Fig. 1A). The respiratory rates of G. bicolor in MT and CK groups did not differ significantly, but they remained significantly lower than

CK 1-MCP ethephon

Respiratory rate (CO2 mmol / Kg.h)

A 100 80 60 40 20 0

0

1

2

3

4

5

6

7

storage time (days)

80

Anthocyanin conten （µ g/g.FW）

B

60

CK 1-MCP ethephon

40

20

0

1

2

3

4

5

6

7

storage time (days)

Chlorophyll conte （mg/g.FM）

C 10

CK 1-MCP ethephon

8 6 4 2 0

0

1

2

3

4

5

6

7

29

those in ET group until the end of storage. The contents of anthocyanin in the three groups changed almost identically, but the contents in CK and MT groups remained significantly higher than those in ET group from Day 2 (P < 0.05) (Fig. 1B). The content of chlorophyll decreased sharply on the first day and then decreased slowly until the seventh day. Notably, MT effectively increased the chlorophyll content in contrast to ET from Day 2 to Day 4 (Fig. 1C). Hence, 1-MCP and ethephon had opposing effects on G. bicolor during senescence. After 2-DE, more than 300 protein spots were reproducibly detected at pH 4–7 in the relative molecular mass range of 14–90 kDa (Fig. 2A). Thirty-eight protein spots were selected with the abundance changes more than 2.0-fold (or less than 0.5-fold) in response to 1-MCP and ethephon during senescence, and 33 proteins were finally successfully identified (Fig. 2B and Table 1). Moreover, the influences of MT and ET on the type and quantity of abundant proteins and metabolic pathways were different during leaf senescence (Fig. 2C). The 33 proteins were involved in different metabolic pathways (Table 1 and Fig. 3A), including metabolism (75.8%; 30.3%: carbohydrate transport and metabolism, 21.1%: inorganic ion transport and metabolism, 15.2%: energy production and conversion, 6.1%: amino acid transport and metabolism, and 3.1%: nucleotide transport and metabolism), information storage and processing (9.1%: transcription) and cellular processes and signaling (12.1%; 3.0%: cell wall/membrane/envelope biogenesis, and 12.1%: posttranslational modification, protein turnover and chaperones). Importantly, different treatments (CK, MT and ET) and times (0, 1, 3 and 7 days) altered the types and abundances of these proteins (Fig. 3B). All the 33 identified proteins were assigned to six categories (Table 1 and Fig. 3C), including chloroplast (17, 51.5%), cytoplasm (5, 15.2%), endoplasmic reticulum lumen (3, 9.1%), vacuole (1, 3%), mitochondrion (4, 12.1%) and extra (secreted) (3, 9.1%). The high proportion of chloroplast and cytoplasm proteins (22, 66.7%) could be ascribed to postharvest storage conditions and protein extraction. As shown in Table 1, proteins were regulated both by ET and MT to various extents at different time periods. MT and ET had opposing effects on the same proteins (G3, 8, 22 and 27 on Day 1; G8, 10 and 23 on Day 3; G8, 30 on Day 7). MT3 significantly up-regulated proteins G6 and G14 (2.70-fold and 1.42-fold, respectively) while ET3 barely had any effect. Protein G11 was depressed by MT, while it was up-regulated 1.64-fold by ET3. Protein G15 was depressed by MT7, whereas G25 and G33 were induced 1.67-fold and 1.64-fold by MT3, respectively. Meanwhile, MT specifically down-regulated proteins G2 (MT1), G32 (MT3) and G29 (MT7), while it up-regulated G12, 13 (MT1), G16 (MT3) and G4, 12 and 28 (MT7), respectively. PCA was carried out for differentially abundant protein spots (P < 0.05) (Fig. 4, Supplementary Tables 1, 2). Thirty-three proteins (Fig. 4A) and CK, MT and ET treatments with increasing treatment time (Fig. 4B) were significantly separated using PCA, respectively. In addition, G24, 31, 16, 15, 25 and 33 were the key proteins in PC1 and G9, 20, 13, 22, 3, 16, 8, 12, 23, 26 and 19 were the important proteins in PC2 (Supplementary Table 2). Treatment scores are shown in Supplementary Table 3. CK0, MT3 and CK1 had higher scores in FAC_1, while CK3, MT7 and MT1 scored higher in FAC_2, and CK0, MT3 and MT1 had highest total scores.

4. Discussion

storage time (days) Fig. 1. Respiratory rate (A), anthocyanin content (B) and chlorophyll content (C) of G. bicolor leaf.

Leaf senescence, which involves the regulation of numerous genes, is governed by several stress resistance- and aging-related issues after postharvesting (Watkins, 2006). G. bicolor leaf is

30

L. Jiang et al. / Food Chemistry 176 (2015) 27–39

C

Fig. 2. Representative spot maps (A), identification (B) and differences of protein abundance (C) of all 33 identified proteins. (A) Representative spot maps of G. bicolor leaf. (B) Identification of 33 protein spots from 1-MCP- and ethephon-treated G. bicolor leaf by 2-DE and MALDI-TOF/TOF analysis. (C) Differences of protein abundance among 1-MCPtreated and ethephon-treated G. bicolor leaf. Different storage time periods are shown on the top. Arrows and numbers indicate the differential spots among different treatments.

Table 1 Identification of 33 proteins in 1-MCP- and ethephon-treated G. bicolor leaf stored for 0, 1, 3 and 7 days compared with the control leaves at every corresponding storage time. a Spot No.

b

Protein name

c Accession No.

d Matched peptide sequences (m/z)

e Matched peptides

f Theoretical Mr(kDa)/pI

Observed Mr(kDa)/pI

g Score/ threshold

Coverage rate (%)

h Subcellular localization

i

Spots %volume variations

(p < 0.05) j

MetabolIsm Energy production and conversion G32 Chlorophyll a-b binding protein 6A, chloroplastic; AltName [Solanum lycopersicum]

gi|115764

YPGGAFDPLGYSK (1371.6865)

1

26.8/5.82

23.4/5.61

50/44

4

chlo

a 0.2

b b

Chlorophyll a/b binding protein [Vitis vinifera]

gi|37780998

QYFLGLEK (997.6130) LGLIPPETALPWFK (1582.0415)

2

18.0/7.03

22.7/5.77

67/40

13

chlo

0d

ATPase subunit I [Spinacia oleracea]

gi|307136215

gi|7636090

VALVVVTGDR (1028.6860) KGNSYFLR (984.6003) SDPVIHTLLPLSPK (1516.9943) ALQESLASELAAR (1358.8370)

4

ASSVAQVVTNFQER (1535.7970) HTLIIYDDLSK (1317.7234) EAYPGDVFYLHSR (1553.7563) TNKPEFQEIISSTK (1621.8563)

4

LKLELAQFAELEAFAQFASDLDK (2597.4021)

1

41.3/5.95

47.3/5.47

40.5/5.9

42.5/6.16

244/42

228/43

12

12

chlo

mito

Alpha subunit of ATPase [Nicotiana tabacum]

gi|11769

G9 a b

c 0d

0.25 0.2 0.15 0.1 0.05 0

4

mito

3d

7d

G20

a

b b b

c 0d

1d

3d

7d

G24

0.08 a a

a

b

a

b

ab b

c

0.02

98/38

b

c

a

0.04

41.3/6.42

a b b

1d

0.06

55.5/5.14

7d

a

0

G29

3d

0d

1d

0.25 0.2 0.15 0.1 0.05 0

3d

7d

G29

a b

a

a

b

b

ab

b b

0d

1d

3d

7d

a

1.2 1

G9+G32

a

L. Jiang et al. / Food Chemistry 176 (2015) 27–39

G24

b

1d

1

0

ATP synthase [Cucumis melo subsp. melo]

b

1.5

0.5

G20

a

b

0.1 0

G9

G32 a

a

0.3

a 0.8 0.6

b

b

a

b b b

0.4 0.2 0 0d

Amino acid transport and metabolism gi|242085204 G1 Hypothetical protein SORBIDRAFT_08g006540 [Sorghum bicolor]

GSGHTVPEYKPK (1299.7808)

1

54.9/5.96

20.0/4.86

47/40

2

vaco

1d

0.2

3d

G1

a

0.15

7d

a

a ab b b

b

0.1

c

c

0.05 0 0d

G13

GLN1;5 (glutamine synthetase 1;5); glutamateammonia ligase [Arabidopsis thaliana]

gi|15221198

QHIAAYGEGNER (1344.7427)

1

38.4/6.20

42.1/5.84

50/38

3

cyto

1d

3d

7d

G13

0.2 0.15 0.1 b

0.05

c

a

b

a

b

0 0d

1d

3d

7d

(continued on next page) 31

32

Table 1 (continued) a Spot No.

b

Protein name

c

Accession No.

d

Matched peptide sequences (m/z)

e Matched peptides

f

Theoretical Mr(kDa)/pI

Observed Mr(kDa)/pI

g Score/ threshold

Coverage rate (%)

h Subcellular localization

i

Spots %volume variations

(p < 0.05) Nucleotide transport and metabolism gi|575953 G22 Nucleoside diphosphate kinase [Solanum lycopersicum]

NVIHGSDAVESAR (1354.8060)

1

15.5/6.84

14.3/6.26

109/41

9

chlo

G22

1.5 1

b

a ab

b a

0.5

a a

b c

0 0d

Carbohydrate transport and metabolism gi|11480 G2 Ribulose-1,5-bisphosphatecarboxylase [Euphorbia characias]

LTYYTPEYETK (1407.7885) DTDILAAFR (1021.6189)

2

6.3/4.81

19.8/5.00

100/43

33

chlo

1d

0.3

a

a

gi|11587

DTDILAAFR (1021.6175) [LTYYTPEYETK (1407.7926)] TFQGPPHGIQVER (1465.8871)

3

47.5/6.32

20.2/5.06

139/43

7

chlo

0d

1d

b

0.05

G3 a a b

gi|255547472

VQYGTLCVR (1095.6489) [ENPGCLFIATNR (1391.7899)]

1

39.9/8.76

34.7/5.01

121/40

5

chlo

0d

1d

GDSLIEGVPETLELLR (1741.0764)

1

33.7/5.09

34.2/5.13

49/41

5

cyto

7d

G4

0.15

0

gi|145349451

3d

0.2 a

b b b

0.05

Predicted protein [Ostreococcus lucimarinus CCE9901]

7d

c

0.1

G5

3d

a

0.1

0d

0.2

1d

a

0.15 b

a b

3d

a

0.1

a

c

b

b

7d

G5 a ab

a

b

b

0.05 0

G7

G10

Chloroplast ribulose-1,5bisphosphate carboxylase/ oxygenase small subunit [Musa balbisiana] Ribulosebisphosphate carboxylase [Pandanus tectorius]

gi|76574179

gi|343013

EYPKAFIR (1023.6191)

LTYYTPEYETK (1407.8019) [DTDILAAFR (1021.6288)] TFQGPPHGIQVER (1465.8931)

1

2

13.9/8.36

52.1/6.34

17.0/5.63

19.4/5.65

43/42

116/42

6

7

chlo

chlo

0.25 0.2 0.15 0.1 0.05 0

0d

1d

3d

7d

G7

a a

a b

b c 0d

1d

3d

7d

G10

0.15 a

a

0.1

b 0.05

b c

a b b

c

0 0d

G16

Triosephosphate isomerase, cytosolic [Coptis japonica]

gi|136057

FFVGGNWK (954.4603)

1

27.2/5.54

25.8/5.97

55/39

3

cyto

1d a

Glyceraldehyde-3phosphate dehydrogenase, cytosolic [Hordeum vulgare]

gi|120668

YDTVHGQWK (1133.6620)

1

33.4/6.20

40.4/6.48

44/37

2

cyto

7d

a c

0d

G27

3d

G16

0.25 0.2 0.15 0.1 0.05 0

0.25 0.2 0.15 0.1 0.05 0

b

1d

b

3d

a

1d

b

7d

G27

a c

0d

b

b b

3d

7d

L. Jiang et al. / Food Chemistry 176 (2015) 27–39

4-Nitrophenylphosphatase, putative [Ricinus communis]

b b c

0

G4

G2 a

b

b

0

Unnamed protein product [Hordeum vulgare] (ribulose-1,5-bisphosphate carboxylase/oxygenase)

7d

a

0.2 0.1

G3

3d

G30

Ribulose bisphosphate carboxylase [Oryza sativa]

gi|20341

IIGFDNVR (933.6359)

1

19.7/8.26

16.4/6.80

49/42

4

chlo

G30

a

0.3

b

0.2 a b b

0.1

a

c

b c

0 0d

G31

Glyceraldehyde-3phosphate dehydrogenase (NADP) (phosphorylating) [Spinacia oleracea]

gi|66026

TFAEEVNAAFR (1254.5839) VIAWYDNEWGYSQR (1786.7904)

2

36.5/6.66

39.0/6.88

255/43

7

chlo

1d

3d

7d

G31

0.15 a

a

0.1

a a

b 0.05

b

0 0d

1d

3d

7d

G2+G3+G7+G10+G30

1 0.8

b 0.6

a

b

a

b

a

a

b

0.4

b

0.2 0 0d

GGHELSLSTGNAGGR (1412.8112)

1

20.7/5.94

17.8/5.72

79/41

7

chlo

1d

3d

7d

G8

0.8

a a

a

0.6 0.4

b c

b

b

c

c

0.2 0 0d

G12

Chloroplast ferredoxinNADP+ oxidoreductase precursor [Capsicum annuum]

gi|6899972

LYSIASSALGDFGDSK (1630.9344) LVYTNDKGEEVK (1394.8308)

2

40.7/8.54

35.5/5.91

179/40

7

chlo

1d

0.6

3d

7d

G12 a

a

0.4

b

b

b

b

0.2 0 0d

G17

Carbonic anhydrase [Flaveria bidentis]

gi|1089983

FMVFACSDSR (1219.6125) EAVNVSLGNLLTYPFVR (1892.1370) VCPSHVLDFQPGEAFVVR (2057.1436)

3

QIPLIGAQSIIGR (1365.9521)

1

36.0/5.85

28.0/6.00

145/42

13

chlo

0.5 0.4 0.3 0.2 0.1 0

Superoxide dismutase [CuZn] [Solidago canadensis var. scabra]

gi|3914998

15.5/5.71

16.1/6.27

49/42

8

cyto

3d

7d

G17 a

a c

0 day

G21

1d

b b b

1 day

3 day

0.4

7 day

G21

a a

0.3 a

0.2

a a

c b

0.1

L. Jiang et al. / Food Chemistry 176 (2015) 27–39

Inorganic ion transport and metabolism gi|20900 G8 Cu/Zn superoxide dismutase II [Pisum sativum]

b

c

0 0d

G23

Carbonic anhydrase, chloroplastic

gi|115472

FMVFACSDSR (1219.6580)

1

34.9/6.61

27.8/6.28

46/40

3

chlo

0.25 0.2 0.15 0.1 0.05 0

1d

Carbonic anhydrase 3 [Flaveria bidentis]

gi|27652186

EAVNVSLANLLTYPFVR (1906.0773) VCPSHVLDFQPGEAFVVR (2057.0754)

2

28.2/7.60

32.1/6.50

147/40

13

chlo

7d

G23 b c b

0d

G26

3d

a

0.4

1d

a c

3d

7d

G26

a

0.3 0.2

b

b

0.1 0 0d

1d

3d

7d

(continued on next page) 33

34

Table 1 (continued) a Spot No.

b

Protein name

c Accession No.

d

Matched peptide sequences (m/z)

e Matched peptides

f

Theoretical Mr(kDa)/pI

Observed Mr(kDa)/pI

g Score/ threshold

Coverage rate (%)

h Subcellular localization

i

Spots %volume variations

(p < 0.05) G28

TPA: class III peroxidase 20 precursor [Oryza sativa Japonica Group]

gi|55700907

DVGLAAALLR (998.6503)

1

39.0/6.65

37.9/6.53

49/43

2

mito

G28

0.4 0.3

a

a

a

ab b

0.2

b

b

b b

0.1 0 0d

Information storage and processing Transcription G6 Heat stress-induced protein [Brassica oleracea var. capitata]

gi|3319646

EPAIIIGGER (1054.6936)

1

23.6/8.37

18.0/5.40

70/41

4

extr

1d

3d

7d

G6

0.15 a

0.1

a

b

0.05

a

b b

0 0d

G14

gi|3319646

EPAIIIGGER (1054.6721) IEEATGAHTYK (1219.7107)

2

23.6/8.37

17.3/5.92

56/41

4

extr

0.5 0.4 0.3 0.2 0.1 0

a

Heat stress-induced protein [Brassica oleracea var. capitata]

gi|3319646

EPAIIIGGER (1054.6320)

1

23.6/8.37

39.7/5.99

67/42

4

extr

3d

7d

a

G14

b

b

a

a

b

c b 0d

G19

1d

1d

3d

7d

G19

0.15 a

0.1 0.05

a a

b

b

b

0 0d

Cellular processes and signaling Cell wall/membrane/envelope biogenesis G18 Conserved hypothetical gi|255580317 protein [Ricinus communis]

KAEQYLADSGIPYTIIR (1938.1432) AGGLQDKEGGVR (1186.6759) ADVAEVCIQALQFEEAK (1921.0448)

3

35.0/8.88

29.1/6.08

172/43

14

mito

0.15

1d

3d

7d

G18

a

a a

b

0.1

c

b

b

a

b

0.05 0 0d

Posttranslational modification, protein turnover, chaperones gi|3914605 FYWAPTR (940.5519) VYDDEVRK G11 Ribulose bisphosphate (1023.6002) IGVCIGIFR carboxylase/oxygenase (1034.6598) activase, chloroplastic [lus x domestica]

3

48.2/8.20

35.0/5.81

82/43

5

chlo

0.06

1d

3d

a

a b

0.04

7d

b c

c

0.02

a G11 ab b

0 0d

G15

BiP isoform C [Glycine max]

gi|475598

SQIDEIVLVGGSTR (1473.8975)

1

49.9/8.93

21.3/6.08

53/41

3

endo

0.15

1d

3d

7d

G15

a

0.1 b

0.05

a

a

b

c

0 0d

G25

Predicted protein [Hordeum vulgare subsp. vulgare] (BiP isoform C)

gi|326511992

TKVDEIVLVGGSTR (1473.9542)

1

68.0/6.00

29.3/6.43

49/34

2

chlo

0.2

1d

3d

a

0.15 b

0.1

b

b

7d

G25

a

a b b

c

0.05 0 0d

1d

3d

7d

L. Jiang et al. / Food Chemistry 176 (2015) 27–39

Heat stress-induced protein [Brassica oleracea var. capitata]

7d 3d

b b

1d

b

b

a

0

0.05

0d

a 0.1

0.15

cyto 2 43/34 25.8/6.12 68.0/6.00 1 TKVDEIVLVGGSTR (1473.8954) gi|326511992 Predicted protein [Hordeum vulgare subsp. vulgare] (BiP isoform C) G33

Numbering corresponds to the 2-DE gel in Fig. 2. Names and species of the proteins obtained via the MASCOT software from the NCBInr/EST database. Accession number from the NCBInr/EST database. d The sequences of all identified peptides with the corresponding m/z ratio in parentheses. e The total number of identified peptides. f Experimental molecular mass (Mr) and isoelectric point (pI) estimated in comparison to a 2D gel with marker proteins, theoretical molecular mass (Mr) and isoelectric point (pI) of the homologous protein calculated with a tool available at NCBInr/EST database g MOWSE score probability (Protein score) for the entire protein and for ions complemented by the percentage of the confidence index (C.I.) h The protein subcellular localization prediction of 33 functional differentially abundant proteins according to PSORT software. i X-axis denotes the storage time (day), and Y-axis denotes the relative levels of protein expression (normalized volume of spots), values are expressed as the mean of three replications ± standard deviation. The values which are statistically significantly changed (more than 2-fold and p < 0.05, according to LSD test) in comparison with the control leaves at every corresponding storage time are marked by different letters. j Functional classification. c

b

a

G33

L. Jiang et al. / Food Chemistry 176 (2015) 27–39

35

affected by MT in various ways; our findings provide threshold information regarding the effects of 1-MCP on the biological processes during G. bicolor leaf senescence at the proteomic level (Table 1), and give evidence postulating the mechanism(s) (Fig. 5) concerning the influences of MT and ET on G. bicolor. 4.1. Transcription regulation, protein folding and assembly Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) not only catalyses glycolysis, but also links metabolic state to gene transcription by migrating from the cytosol to the nucleus (Zheng, Roeder, & Luo, 2003). Nucleoside diphosphate kinases (NDK) are prerequisite for the synthesis of nucleoside triphosphates (NTP) other than ATP. Schneider, Gaal, and Gourse (2002) concluded that NTP sensing by rrn P1 promoters linked translation and ribosome synthesis as a direct regulator. Binding immunoglobulin protein (BiP), a heat stress-induced protein (HSP) 70 molecular chaperone, binds newly synthesised proteins and subsequently translocates, folds and oligomerizes them (Cascardo et al., 2000). In this study, the abundances of GAPDH (G27) and NDK (G22) were up-regulated by MT1 but were down-regulated by ET1, and the results were significantly different from those of CK1. MT3 augmented the content of BiP (G25 and G33) but ET3 did not exert an obvious effect. Therefore, with a strong regulatory role, 1-MCP engaged in gene transcription and translation initially and in protein abundance and modification thereafter. 4.2. Respiratory rate and metabolism Chlorophyll is decomposed during leaf senescence, although nutrients may be recovered (Matile, 2000). Typically, most chlorophylls are distributed in leaf protein complexes (chlorophyll a/b binding protein, CAB), but free chlorophylls are extremely vulnerable to decomposition in the presence of acid, alkali, oxidant and oxygen, etc. (Kusabaa et al., 2007). In this study, respiratory rate and chlorophyll content (Fig. 1A and C) were negatively correlated in the first 3 days. In other words, leafy vegetables may delay senescence through decreasing and degrading chlorophyll. MT effectively maintained the CAB content (G9 + G32) on Day 1 (Table 1) and was associated with an increase (G32) on Day 3 (Fig. 1C). The relationship between chlorophyll and CAB contents indicates that the initial stage of storage predominantly retained chlorophyll, and that 1-MCP delayed the degradation of chlorophyll probably by keeping CAB integral. Similarly, Liu and Shen (2004) reported that CAB was reversibly phosphorylated in the presence of light, NaCl and other stresses. Chloroplasts are targeted protein degradation sites during senescence, containing approximately 80% of the total leaf nitrogen originating from RuBisCO proteins (Mae, Kai, Makino, & Ohira, 1984). RuBisCO is the most abundant protein on earth and accounts for about 90% of the degraded proteins during the early stages of senescence (Miller & Huffaker, 1985). MT inhibited the increases in RuBisCO proteins G2 and G3 on Day 1, G10 on Day 3 and G30 on Day 7, while ET down-regulated their abundance. Abiotic stresses (e.g. drought, heat, hypoxia, heavy metal pollution) exert tremendous impacts on senescence and the degradation of chloroplast proteins (Feller, Anders, & Mae, 2008). In plants, carbonic anhydrase (CA) raises the concentration of CO2 within chloroplast to boost the carboxylation of RuBisCO, or transports CO2 out of tissues to maintain acid-base balance. Ribulose bisphosphate carboxylase/oxygenase activase (RCA) directly or indirectly enhances the activity of carbamylated RuBisCO in addition to promoting carbamylation depending on ATP (He et al., 1997). CA (G23) was up-regulated by MT3 and down-regulated by ET3. Similarly, MT3 and ET3 regulated RCA (G11) in opposite directions, and MT3 inhibited the abundance of RCA in dark. Accordingly, 1-MCP

36

L. Jiang et al. / Food Chemistry 176 (2015) 27–39

protein percentage（%）

C

20

51.5%

16 12 8

15.2%

4

12.1%

9.1%

3.0%

9.1%

0 chloroplast Cytoplasm

Vacuole

location of proteins

Mitoch- Endoplasmic ondrion reticulum lumen

extr

Fig. 3. Functional classification (A), hierarchical clustering (B) and protein subcellular localisation prediction (C) of all 33 identified proteins. (A) Functional groups of differentially abundant proteins identified from control, 1-MCP- and ethephon-treated G. bicolor leaf stored for different days. This classification is based on the COGs Functional Catalogue Database (http://www.ncbi.nlm.nih.gov/COG/) and their homologies and literature. (B) The hierarchical clustering analysis was realised using the cluster and java Treeview. (C) The protein subcellular localisation prediction of 33 identified proteins based on the WOLF PSORT database (http://wolfpsort.org/).

L. Jiang et al. / Food Chemistry 176 (2015) 27–39

37

Fig. 4. Representation of the samples based on main principal components found after PCA. 2-D plot of main principal components (PC1 and PC2) of: (A) proteins (Loading Plot) and (B) spot maps (Score Plot). PCA bi-plot of the two first principal components and together both explained 56.28% of the selected spots variability. Colored dots and numbers represent the gels and spots, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

reduced the RuBisCO degradation by controlling CA and RCA abundances. Our results indicate that 1-MCP could protect the integrity of chlorophyll and chloroplast, and slow the senescence of G. bicolor. In contrast, ethephon accelerated degradation and senescence. 4.3. Stress response and defense HSP, which is induced by environment stress, is also involved in protein folding that protects plant cells (Hu, Hu, & Han, 2009). Triosephosphate isomerase (TPI) is capable of improving plant resistance to abiotic stress (Riccardi, Gazeau, de Vienne, & Zivy, 1998). For HSP proteins, MT induced the abundance of G19 (6.23-fold) on Day 1 and those of G6 (2.70-fold) and G14 (1.42fold) on Day 3, while ET was not sufficiently effective. However,

the abundance of TPI (G16) was down-regulated by both MT1 and ET1and up-regulated by MT3. Hence, 1-MCP and ethephon, as the abiotic stresses, could induce tissue resistance of leaves, but 1-MCP was much more effective. Studies in vivo have revealed that suppressing FNR abundance increased susceptibility to photooxidative damage (Palatnik et al., 2003). In contrast, over-expressing FNR accelerated the electron transport from water to NADP+ and increased the tolerance to oxidative stress (Juric´ et al., 2009). The abundance of FNR (G12) in G. bicolor leaf was induced 2.11-fold by MT1 and down-regulated 0.87-fold by ET1, and was also up-regulated 1.51-fold by MT7. Thus, 1-MCP enhanced the antioxidant capacity of chloroplasts by inducing the abundance of FNR. Glutamine synthases (GS) participate in glutamine metabolism that essentially metabolizes nitrogen (Eisenberg et al., 1987).

38

L. Jiang et al. / Food Chemistry 176 (2015) 27–39

Fig. 5. Postulated mechanism concerning the influences of 1-MCP on G. bicolor. Transduction pathways, metabolism processes and substances are indicated with those upregulated markedly by ‘‘"’’, those down-regulated markedly by ‘‘;’’ and those affected markedly by ‘‘?’’ which indicates this process needs to be further validated.

Anthocyanins are coloured flavonoids resisting to oxidation, the contents of which change in a complex fashion during postharvesting, such as unusual accumulation of reactive oxygen intermediates (ROIs) under stress conditions (infections, low temperature, salinity, light intensity and wound) (Mittler, 2002), and the complement of chlorophyll in nitrogen-deficient condition (Politycka & Golcz, 2004). MT induced the abundance of GS (G13) on the first day, suggesting that 1-MCP may shift the leaf nitrogen metabolism towards the direction of anthocyanin synthesis. Maximum and minimum levels of anthocyanins were brought by MT and ET respectively; 1-MCP boosted the antioxidant capacity by elevating the anthocyanin level in G. bicolor tissues, while ethephon accelerated tissue senescence due to excess production of ROIs. The contents of anthocyanin and chlorophyll were negatively correlated on Day 1 (Fig. 1B and C), which fits well with the results of Cevahir, Yententür, Yazgan, Ünal, and Yilmazer (2004). Brennan and Frenkel (1977) verified that stored vegetables were prone to generating active oxygen species such as H2O2 and O 2 , the accumulation of which then undermined cell membrane by peroxidation. Superoxide dismutase (SOD) converts O to 2 H2O2 and peroxidase (POD)/catalase (CAT) catalyses the reduction of H2O2. Jiang (2010) found MT increased the activities of SOD, POD and CAT, and drastically reduced the level of O 2 directly or indirectly during senescence. In this study, MT considerably induced SOD while ET inhibited it, and POD was up-regulated by MT7, suggesting MT scavenged oxygen free radicals more effectively, shielding G. bicolor cells from damage. 4.4. PCA PCA showed that the key factors of PC1 were related to regulation capacity and PC2 was associated with resistance ability. Therefore, PC1 and PC2 could be considered to be regulation and

resistance factors respectively. In this study, MT and ET played the same regulatory role in the abundances of ATPase (G24), TPI (G16), BIP (G15, 25 and 33) and GAPDH (G31). But MT, compared with ET, significantly induced the abundances of TPI (G16) and BiP (G25 and 33) on Day 3. Accordingly, MT had a stronger impact on regulation than ET, which is consistent with FAC_1 score (Supplementary Table 3). In addition, although MT and ET had the same effects on CAB (G9), ATP synthase (G20), CA (G23 and 26) and HSP (G19) on Day 1, the extent of these was significantly different. In contrast, MT and ET exerted opposing effects on GS (G13), NDK (G22), RuBisCO (G3), SOD (G8) and FNR (G12) on Day 1, and SOD (G8) and CA (G23) on Day 3. Taking FAC_2 score (Supplementary Table 3) into consideration, MT1 increased resistance more than MT3 did, and 1-MCP exhibited an increase in resistance while ethephon accelerated senescence. Notably, CK0, MT3 and MT1 had the top three total scores (Supplementary Table 3), and 1-MCP resisted senescence better than ethephon. 5. Conclusions Regulation of proteins in G. bicolor leaf by 1-MCP at the proteomic level is complex, involving identical metabolic pathways as well as other different pathways. Both 1-MCP and ethephon regulated G. bicolor leaf proteins, but 1-MCP delayed senescence while ethephon promoted it through opposing effects on GAPDH, NDK and BiP abundances. Metabolism was slowed by 1-MCP during senescence due to inhibited respiratory, glycolysis, and carbon, nitrogen metabolism. 1-MCP treatment enhanced defense, induced increases in GS, FNR, SOD and POD abundances and anthocyanin content, facilitated the elimination of oxygen free radicals, and maintained metabolic balance of active oxygen system. 1-MCP treatment regulated HSP abundance and maintained chloroplasts by specifically inducing increases in BiP, CA and CAB abundances

L. Jiang et al. / Food Chemistry 176 (2015) 27–39

and decreased RCA abundance, keeping senescence slow. 1-MCP treatment may improve the quality of G. bicolor leaf and extend shelf life via the above-mentioned metabolic pathways. In conclusion, this study provides new evidence for explaining the regulatory effects of 1-MCP and ethephon on G. bicolor leaf senescence at the proteomic level. Some 1-MCP- or ethephon proteins were missing since the relative protein abundances were too low to be identified or they did not change more than 2.0-fold (or less than 0.5-fold). Thus, more molecular and subcellular proteomics analyses concerning physiological and biological changes are still in need. Acknowledgements The reagent of 1-MCP was supplied by Rohm and Haas’ AgroFresh Division. This research was financially supported by the National Natural Science Foundation of China (No. 31301576), the Fundamental Research Funds for the Central Universities (No. KJQN201428 and KYZ201319), Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Nanjing Agricultural University Innovation and Technology Fund for Youths (No. KJ2011015) and Jiangsu Independent Innovation Fund for Agricultural Science and Technology (CX(12)3081). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2014. 11.081. References Blankenship, S. M., & Dole, J. M. (2003). 1-Methylcyclopropene: A review. Postharvest Biology and Technology, 28(1), 1–25. Blum, H., Beier, D. H., & Gross, H. J. (1987). Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis, 8(2), 93–99. Brennan, T., & Frenkel, C. (1977). Involvement of hydrogen peroxide in regulation of senescence in pear. Plant Physiology, 59(3), 411–416. Cascardo, J. C., Almeida, R. S., Buzeli, R. A., Carolino, S. M., Otoni, W. C., & Fontes, E. P. (2000). The phosphorylation state and expression of soybean BiP isoforms are differentially regulated following abiotic stresses. Journal of Biological Chemistry, 275(19), 14494–14500. Cevahir, G., Yententür, S., Yazgan, M., Ünal, M., & Yilmazer, N. (2004). Peroxidase activity in relation to anthocyanin and chlorophyll content in juvenile and adult leaves of ‘‘MINI-STAR’’ Gazania splendens. Pakistan Journal of Botany, 36(3), 603–609. Eisenberg, D., Almassy, R. J., Janson, C. A., Chapman, M. S., Suh, S. W., Cascio, D., et al. (1987). Some evolutionary relationships of the primary biological catalysts glutamine synthetase and RuBisCO Cold Spring Harb. Cold Spring Harbor Symposia on Quantitative Biology, 52(1), 483–490. Ella, L., Zion, A., Nehemia, A., & Amnon, L. (2003). Effect of the ethylene action inhibitor 1-methylcyclopropene on parsley leaf senescence and ethylene biosynthesis. Postharvest Biology and Technology, 30(1), 67–74. Feller, U., Anders, I., & Mae, T. (2008). Rubiscolytics: Fate of Rubisco after its enzymatic function in a cell is terminated. Journal of Experimental Botany, 59(7), 1615–1624. He, Z. L., Caemmerer, S. v., Hudson, C. S., Price, C. D., Badger, M. R., & Andrews, T. J. (1997). Ribulose-1,5-bisphosphate carboxylase/oxygenase activase deficiency delays senescence of ribulose-1,5-bisphosphate carboxylase/oxygenase but progressively impairs its catalysis during tobacco leaf development. Plant Physiology, 115(4), 1569–1580. Hu, W., Hu, G., & Han, B. (2009). Genome-wide survey and expression profiling of heat shock proteins and heat shock factors revealed overlapped and stress specific response under abiotic stresses in rice. Plant Science, 176(4), 583–590.

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