Food Chemistry 218 (2017) 285–293

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

iTRAQ analysis of low-phytate mung bean sprouts treated with sodium

citrate, sodium acetate and sodium tartrate
Xiaolin Jin a,b, Runqiang Yang a, Liping Guo c, Xinkun Wang a, Xiaokun Yan a, Zhenxin Gu a,⇑
a
College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, People’s Republic of China
b
College of Life Science, Shandong Normal University, Jinan 250000, People’s Republic of China
c
College of Food Science and Engineering, Qingdao Agricultural University, Qingdao 266109, People’s Republic of China

a r t i c l e i n f o a b s t r a c t

Article history: The effects of sodium citrate (SC), sodium acetate (SA) and sodium tartrate (ST) spraying on mung bean
Received 30 January 2016 germination were investigated. Exogenous SC, ST and SA treatments significantly reduced the phytic acid
Received in revised form 4 September 2016 content and increased the antioxidant enzyme activities. In this study, an iTRAQ-based proteomic
Accepted 5 September 2016
approach was employed to explore the proteomes of mung bean sprouts, and 81, 101 and 90 differen-
Available online 7 September 2016
tially expressed proteins were identified in 4-day-old SC-, SA- and ST-treated mung bean sprouts, with
38 proteins present in all samples. Functional classification analysis showed that most of the differen-
Keywords:
tially expressed proteins in mung bean sprouts subjected to the three treatments were involved in
Low-phytate
Organic acid salt treatments
carbohydrate and energy metabolism. The inhibitory effect of the SA treatment was probably due to
Mung bean sprouts impairments in protein biosynthesis, whereas enhanced energy metabolism, accelerated reserve hydrol-
iTRAQ analysis ysis and protein processing were very important strategies for growth stimulation in response to ST and
Antioxidant enzymes SC treatments.
Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction stimulus to induce physiological changes and defence or stress-

induced responses (Baenas, García-Viguera, & Moreno, 2014).
Mung beans are consumed in large quantities throughout the Gebaly, Fatma, and Alia (2013) reported that spraying cotton plants
world (Fery, 2002). During germination, the decomposition of with citrate could increase catalase and peroxidase activities in
mung bean reserves, such as proteins and polysaccharides, con- comparison with the control. Lipid oxidation in sliced salmon
tributes to the formation of biofunctional compounds. Meanwhile, (Sallam, 2007) and rainbow trout (Haghparast, Kashiri,
as an anti-nutrient, phytic acid is degraded during mung bean Shabanpour, & Pahlavani, 2010) was delayed by the application
sprouting, thus increasing the nutritive values of the sprouts of sodium acetate and sodium citrate. On the other hand, citrate
(Vayupharp, 2013). In addition, the process of sprouting is very treatment could enhance phytase activity, thus promoting phytic
simple and does not require sunlight nor soil and is not limited acid degradation (Porres, Etcheverry, Miller, & Lei, 2001). However,
by seasonal factors (Jom, Frank, & Engel, 2011). In addition, the influence of exogenous organic acid salt spraying on phytic acid
germination has positive enhancement effects on antioxidant degradation in mung bean sprouts has not been reported. In addi-
compounds in mung bean sprouts (Guo, Li, Tang, & Liu, 2012). tion, although the antimicrobial effects of these organic salts help
However, the consumption of raw mung bean sprouts might prevent bacterial growth, few studies on the overall physiological
constitute potential hazard with respect to microbial contamina- profile and growth of mung bean sprouts in response to organic
tion (Mohle-Boetani et al., 2009). It has been reported that organic acid salt treatments are available.
acid salts, such as sodium acetate and sodium citrate, could inhibit A considerable number of studies have focused on the effects of
the growth of pathogenic microorganisms in the germination of germination time on the nutrients and anti-nutrients in mung bean
zinnia (Szopińska, 2013) and lettuce (Arin & Balci, 2014). In addi- sprouts (Jom et al., 2011; Vayupharp, 2013). Recently, comparative
tion, exogenous organic acid salt spraying might act as a type of proteomic analyses were conducted on mung bean cotyledons at
different germination times (Ghosh & Pal, 2012; Mubarak, 2005).
Considering that proteomics approaches can provide greater
⇑ Corresponding author.
molecular insights into the overall metabolism during germina-
E-mail addresses: 2013208009@njau.edu.cn (X. Jin), Yangrq@njau.edu.cn (R.
Yang), 2012208006@njau.edu.cn (L. Guo), 2012208007@njau.edu.cn (X. Wang), tion, the proteomic profiles of mung bean sprouts treated with
2014108023@njau.edu.cn (X. Yan), Guzx@njau.edu.cn (Z. Gu). different organic acid salts were investigated in this study.

http://dx.doi.org/10.1016/j.foodchem.2016.09.029
0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
286 X. Jin et al. / Food Chemistry 218 (2017) 285–293

In this study, we performed an iTRAQ-based quantitative pro- (2009). The CO2 content during seed germination was measured
teomic analysis on seed germination and the growth of mung bean using an infrared gas analyser LB-BZ (Qingdao Loobo Environmen-
sprouts following sodium acetate (SA), sodium citrate (SC) and tal Protection Technology Co., Ltd., China). The respiratory rate was
sodium tartrate (ST) spraying treatments. The objective was to expressed as mg CO2/g DW h.
reveal the response strategies and the adaptive mechanisms of
mung bean sprouts in response to different organic acid salt 2.4. Measurement of lipid peroxidation and relative electrolyte leakage
treatments.
Membrane lipid peroxidation was measured as the amount of
2. Materials and methods malondialdehyde (MDA) produced in the presence of 5% (w/v) tri-
chloroacetic acid (TCA) (Dhindsa, Plumb-Dhindsa, & Thorpe, 1981).
2.1. Seed germination and exogenous organic acid salt treatments Briefly, the mung bean material (1.0 g) was homogenized in 5 mL
of 5% (w/v) trichloroacetic acid. After centrifugation, 2 mL of the
The mung bean seeds (Phaseolus radiatus L. cv. Qindou 20) were supernatant was mixed with 2 mL of 0.67% (w/v) thiobarbituric
germinated using the protocol reported by Jin et al. (2016) with acid, and then the mixture was placed in a 100 °C water bath for
minor modifications. Initially, the mung bean seeds were surface- 30 min. The absorbance was measured at 440 nm, 532 nm and
sterilized by soaking them in 5% (v/v) sodium hypochlorite for 600 nm and the result was calculated using the following
15 min, and then washed with distilled water until a neutral pH equation: MDA equivalents (lmol/l) = [6.45  (A532
A600)

was achieved. After being steeped in distilled water for 4 h at 0.56  A440]. The result was expressed as lmol g
1 DW.
30 ± 1 °C, the soaked seeds were put into a sprouter (BX-801, The electrolyte leakage was determined using the method
Beixin Hardware Electrical Factory, Zhejiang, China) and germi- described by Li et al. (2014) on a DDS-370 electrical conductivity
nated at 30 °C in the dark. The seeds were watered by an automatic meter (Shanghai Electronics Scientific Instruments, China). Electri-
spraying system that provided a 2 min spray every hour. Based on cal conductivity was first determined in the 25 °C bath solution
previous results (Supplementary Fig. 1), 0.6 mM solutions of SC, SA (C1), and then the samples were heated at 100 °C for 30 min and
and ST (all from Sigma-Aldrich, St. Louis, USA) were freshly pre- conductivity was determined again in the bath solution (C2). Rela-
pared as the spraying solutions and refreshed every 24 h. The con- tive ion leakage was expressed as a percentage of the total conduc-
trol was watered with deionized water. The 4-day-old sprouts tivity after heating at 100 °C (i.e., relative ion leakage % = C1/
were rapidly and randomly sampled for the subsequent experi- C2  100%).
ments using the proteomic approach.
2.5. Determination of the antioxidant enzyme activities
2.2. Determination of the phytic acid content and phytase activity
The germinated sprouts (0.5 g) were homogenized in a mortar
The phytic acid content was determined using the procedure with 5 mL of 50 mM phosphate buffer (pH 7.0) containing 1 mM
reported by Ma et al. (2005) with some modifications. Briefly, dried EDTA and 2% PVP at 4 °C. After centrifugation at 12,000g for
samples were extracted with 30 mL of 1.2% HCl and shaken vigor- 20 min at 4 °C, the supernatant was used for the enzyme activity
ously for 2 h at 30 °C. The supernatant was centrifuged (5000g for assays. The SOD, CAT and POD activities were determined using
10 min) before filtration. A total of 10 mL filtered extract was an SOD Assay Kit (A001, Nanjing Jiancheng Bioengineering Insti-
diluted to 30 mL with distilled water after mixing with 1 mL tute, China), a CAT Assay Kit (A007, Nanjing Jiancheng Bioengineer-
30 g/L NaOH and then passed through an anion resin column ing Institute, China), and a Plant POD Assay Kit (A084-3, Nanjing
(AG1-X4 resin, 100–200 mesh, Alfa Aesar, Germany). The column Jiancheng Bioengineering Institute, China), respectively. GPX was
was washed before use with 20 mL of 0.5 mol/L NaCl solution measured spectrophotometrically as the decrease in the A340 of
and deionized water till no Cl
can be detected. The column was NADPH according to the method reported by Drotar, Phelps, and
washed with 15 mL of distilled water and 20 mL of 0.05 mol/L NaCl Fall (1985). The reaction mixture consisted of 1.49 mL of phos-
solution after sample application. The eluate from the resin was phate buffer (50 mM, pH 7.0), 0.1 mL of 1 mM EDTA, 0.1 mL of
eluted with 0.7 mol/L NaCl to 25 mL. Five millilitres of the collected 1 mM NaN3, 0.1 mL of 1 mM glutathione (GSH), 0.1 mL of 0.2 mM
eluate were mixed with 4.0 mL of Post-column reagent (0.03% NADPH, 0.01 mL of 0.25 mM H2O2, and 0.1 mL of supernatant in
FeCl3 solution + 0.3% sulphosalicylic acid). After centrifugation at a final volume of 2.0 mL. The reaction was initiated by the addition
3000g for 10 min, the absorbance of the supernatant was measured of H2O2.
at 500 nm. The standard curve was prepared using phytic acid as
the standard (Sigma, USA) in concentrations ranging from 0 to 2.6. Determination of the reducing sugar, soluble protein and free
100 mg/100 g. amino acid contents
Phytase was extracted using the method reported by Hegeman
and Grabau (2001) with some modifications. Briefly, mung bean The reducing sugar contents were determined using the
sprouts (1.0 g) were ground with 5 mL of pre-cooled buffer 3,5-dinitrosalicylic acid method (Miller, 1959), with some modifi-
(50 mM sodium acetate, pH 5.5), and then the supernatant was cations. Glucose was used as a standard, which contains 1 g of
used to measure the enzyme activity. Phytase activity was mea- 3,5-dinitrosalicylic acid, 0.2 g of phenol, 0.05 g of sodium sulphite,
sured using the method reported by Heinonen and Lahti (1981). 1 g of NaOH and 20 g of potassium sodium tartrate in a sufficient
One phytase unit corresponded to 1 mmol of inorganic phosphorus amount of water to make 100 mL of reagent. Three millilitres of
formed per minute. The specific activity was expressed as units per the reagent and the sample (1 mL) were mixed, heated in a boiling
gram of dry matter. water bath for 15 min, and the absorbance was read at 550 nm.
Free amino acids were extracted with 5 mL of distilled water in
2.3. Measurement of sprout length and respiration 85 °C water bath for 30 min using the ninhydrin method and leu-
cine as the standard (Moore & Stein, 1954). Approximately 0.2 g
Twenty sprouts from each treatment group were sampled and of mung bean sprouts were homogenized in 5 mL of phosphate
their lengths were measured using a calliper. Respiration rates buffer (pH 7.0), and then centrifuged at 10,000g for 15 min at
were determined using the method reported by Zheng et al. 4 °C. The supernatant was used to determine the soluble protein
 X. Jin et al. / Food Chemistry 218 (2017) 285–293 287

content using the Bradford method (Bradford, 1976) and bovine 2.10. Database search and iTRAQ quantification
serum albumin as the standard.
The samples were analysed by nano-LC-MS/MS. The peak lists
2.7. Protein extraction and iTRAQ labelling were generated with Proteome Discoverer 1.3 (Thermo Fisher Sci-
entific, CA, USA). The proteins were identified using the SEQUEST
Protein extracts were obtained using the P-PERTM Plant Protein search engine by searching against the NCBI Glycine max database
Extraction Kit (89803, Thermo Fisher Scientific, CA, USA) according (Apr 20th, 2015). For protein identification, a mass tolerance of
to the manufacturer’s recommendations. The extracted protein 10 ppm was permitted for the intact peptide masses and 0.02 Da
samples (250 lg each) were reduced with 10 mM DTT, alkylated for the fragmented ions, with an allowance for one missed cleavage
with 55 mM iodoacetamide and digested with sequencing-grade in the trypsin digests. Oxidation at methionine was set as the
trypsin (V5111, Promega, CA, USA) using the Filtered aide sample potential variable modifications, whereas carbamidomethylation
preparation (FASP) method. After trypsin digestion, the peptides at cysteine was set as the fixed modification. The charge states of
were dried by vacuum centrifugation, reconstituted in iTRAQ dis- the peptides were set to +2 and +3. Specifically, an automatic decoy
solution buffer, and labelled using iTRAQ 8-plex kits (AB Sciex database search was performed in SEQUEST by choosing the decoy
Inc., MA, USA), according to the manufacturer’s protocol. Briefly, checkbox in which a random sequence of the database is generated
one unit of iTRAQ reagent was thawed and reconstituted in 70 lL and tested for the raw spectra, as well as the real database. Only
of isopropanol. The control and SC, SA and ST spraying treatment peptides identified at the 99% confidence interval by a SEQUEST
samples were labelled with the 113, 114, 115 and 116 tags, respec- probability analysis were counted as identified and each identified
tively. Three biological replicates were performed. After labelling protein included at least one unique peptide to reduce the proba-
and quenching, the samples were combined and dried by vacuum bility of false identification. Only proteins that were identified in
centrifugation. all three independent experiments were considered. In this study,
a protein was considered differentially abundant when it had a fold
2.8. Fractionation by high pH RP chromatography change of >1.5.

High pH RP chromatography was performed on an UltiMateTM 2.11. Bioinformatic analysis of proteins

3000 HPLC Pump system (Thermo Fisher Scientific, CA, USA). The
iTRAQ-labelled peptide mixtures were reconstituted with RP buffer Functional annotations were generated with the Blast2GO soft-
A (98% H2O and 2% ACN, pH 10.0) and loaded onto a 2.1  100 mm ware (http://www.geneontology.org) using the non-redundant
ACQUITY UPLC BEH C18 column containing 1.7-lm particles protein database and Clusters of Orthologous Groups of Proteins
(Waters, MA, USA). The peptides were eluted with a gradient of System (COG) software (http://www.ncbi.nlm.nih.gov/COG/). We
97% RP buffer A for 12 min and 3–98% RP buffer B (98% ACN and used the KEGG databases (http://www.genome.jp/kegg/pathway.
2% H2O, pH 10.0) for 40 min at a flow rate of 0.2 mL/min. The sys- html) to take advantage of the current knowledge of biochemical
tem was then maintained in 98% RP buffer B for 15 min before pathways and other types of molecular interactions.
equilibrating with RP buffer A for 10 min prior to the next injec-
tion. Elution was monitored by measuring the absorbance at
2.12. Statistical analysis
214 nm, and fractions were collected every 1 min. The eluted
peptides were pooled into 12 fractions and vacuum-dried.
The experiments were performed with three biological repli-
cates. The results were presented as the means ± SD. Statistical
2.9. MS/MS analysis using an LTQ-Orbitrap XL
analyses of the data were performed using ANOVA tests. The
means were separated by the least significant difference (LSD) test
Each fraction was reconstituted in buffer A (2% ACN and 0.1%
at the p < 0.05 level.
FA) and centrifuged at 20,000g for 10 min. Using an auto sampler,
20 lL of supernatant was loaded onto a 2 cm C18 trap column
(inner diameter 200 lm) on a nano HPLC. The peptides were eluted 3. Results and analysis
onto a resolving 10 cm analytical C18 column (inner diameter
75 lm) that was assembled in-house. The samples were loaded 3.1. Changes in the phytic acid content and phytase activity
at 4 lL/min for 8 min and eluted with an 88 min gradient at
400 nL/min from 3 to 28% B (98% ACN, 0.1% FA), 20 min from 28 During germination, there was a decrease in phytic acid content
to 55% B, followed by a 5 min linear gradient to 98% B, maintenance accompanied by an increase in phytase activity (Fig. 1). The phytic
at 98% B for 20 min, and finally a return to 3% B over 1 min. The acid content was decreased by 55.4%, 57.1% and 65.6% after the ST,
peptides were subjected to nano electrospray ionization followed SA and SC spraying treatments, respectively (Fig. 1A). Increased
by tandem mass spectrometry (MS/MS) in an LTQ-Orb iTRAQ XL phytase activity was also observed in response to these three treat-
(Thermo Fisher Scientific, CA, USA) coupled online to the HPLC. ments on the 4th day of germination, although the stimulatory
Intact peptides were detected in the Orbitrap with a resolution of effect was not observed on the 2nd day for the ST and SA treat-
60,000. Peptides were selected for MS/MS using the high-energy ments (Fig. 1B).
collision dissociation (HCD) operating mode with a normalized col-
lision energy setting of 40.0; ion fragments were detected in the 3.2. Growth performance, respiratory rate, MDA content and electrical
orbitrap at a resolution of 7500. A data-dependent procedure that conductivity in germinated mung bean sprouts
alternated between one MS scan followed by 5 MS/MS scans was
applied for the 5 most abundant precursor ions above a threshold Compared with the control, the exogenous ST and SC treat-
ion count of 5000 in the MS survey scan, with duration of 60 s. The ments significantly increased the length of the mung bean sprouts
electrospray voltage applied was 2.4 kV. Automatic gain control on the 4th day of germination (p < 0.05) (Fig. 2A). The respiratory
(AGC) was used to optimize the spectra generated by the orbitrap. rate of mung bean was also increased by the ST and SC treatments
The AGC target for the full MS was 1e6 and 1e5 for MS2. For the MS (Fig. 2B) (p < 0.05). The highest MDA content was observed after
scans, the m/z scan range was 350–1800 Da. For the MS2 scans, the the SA spraying treatment (p < 0.05), followed by the control and
m/z scan range was 100–1800. ST and SC spraying treatments, with no significant difference
288 X. Jin et al. / Food Chemistry 218 (2017) 285–293

1.2 35
2d
A B

Phytic acid content (mg/g DW)

4d
a

Phytase activity (U/g DW)

1.0 a
a 28
b
a
0.8 b
c 21
b b
0.6
c c
a 14
0.4 d
b
c c 7
0.2

0.0 0
CK ST SA SC CK ST SA SC
Germination treatment Germination treatment

Fig. 1. Changes in the phytic acid content (A) and phytase activity (B) of mung bean sprouts in response to ST, SA and SC treatments. Different letters indicate significant
differences among the samples on each day (p < 0.05). ST, SA and SC represent sodium tartrate, sodium acetate and sodium citrate, respectively. CK is sprayed with distilled
water.

Fig. 2. Changes in the patterns of growth performance (A), respiratory rate (B), MDA content (C) and cellular membrane damage (D) of mung bean sprouts in response to ST,
SA and SC treatments. Figure A showed the growth performance on the 4th day of germination. Different letters indicate significant differences among the samples on each
day (p < 0.05).

between the ST and SC spraying treatments (Fig. 2C). In terms of mung bean sprouts during the first two days of germination, with
the electrolyte leakage rate, it is noted that SA spraying produced SA producing the lowest soluble protein content on the 4th day of
the highest relative electrolyte leakage rate, whereas ST and SC germination (Fig. 3A). A corresponding trend was also observed in
spraying showed significantly reduced relative electrolyte leakage the free amino acid content (Fig. 3B); the highest free amino acid
rates compared with the control (p < 0.05), indicating that SA content was observed after SA spraying, followed by ST, SC and
spraying may cause membrane injury to some extent (Fig. 2D). the control, with no significant difference in SC and ST. In contrast,
a consistent increase in the reducing sugar content was observed
3.3. Changes of storage substances in mung bean sprouts during the 4-day germination (Fig. 3C). These three spraying treat-
ments increased the reducing sugar content compared with the
As shown in Fig. 3, the soluble protein content was initially control, with the exception of the 4th day. A significant decrease
increased and then decreased during the 4-day germination, show- in the reducing sugar content was observed after SA spraying,
ing the maximum value on the 3rd day (p < 0.05). These three but no significant difference was observed between ST, SC and
spraying treatments increased the soluble protein contents in the control on the 4th day.
 X. Jin et al. / Food Chemistry 218 (2017) 285–293 289

320 illustrated in a Venn diagram analysis (Fig. 5A). Only proteins that
Free amino acid content (mg/g DW) Soluble protein content (mg/g DW)

a were identified in all three independent experiments were consid-

1d 3d
A
2d 4d
b ered. A protein was considered differentially expressed when the
c c protein had both a fold-change of more than 1.5 and a P-value of
240
a less than 0.05. Based on the two criteria, we identified a total of
a a a 143 differentially expressed proteins. There were 81 differentially
a a expressed proteins in SC treatment, 101 proteins in SA treatment,
160 b b
90 proteins in ST treatment, and 38 proteins in all of the samples
a (Fig. 5A). These 38 common differentially expressed proteins were
b b mainly involved in carbohydrate and energy metabolism, cell wall
80 c and cytoskeleton metabolism, and protein metabolism, and
included glucan endo-1,3-b-glucosidase, phosphoenolpyruvate car-
boxykinase, malate dehydrogenase, a-xylosidase and phenylala-
nine ammonialyase. In particular, some functionally important
0
proteins were phosphorylated, suggesting their key roles in sprout
B
development (Supplementary Tables 1 and 2).
7.5 a a
According to the molecular functions listed on the UniProt and
ab Gene Ontology website, the iTRAQ-quantified proteins in SC treat-
6.0 a ment group were functionally classified into 12 functional classes,
b b b i.e., carbohydrate and energy (30.7%), protein metabolism (13.6%),
4.5 c a biological regulation and signal transduction (11.1%), cell wall and
cytoskeleton metabolism (8.7%), inositol phosphate (3.7%), lipid
b b b metabolism (2.5%), cell transport (3.7%), nucleic acid (6.2%), sec-
3.0 a
ondary metabolism (8.6%), response to stress (4.9%), etc. (Fig. 5B).
b b A similar trend was observed in the SA and ST treatment groups,
1.5 c with the carbohydrate and energy metabolism comprising the
highest percentage of differentially expressed proteins in both
0.0 a treatment groups (Fig. 5C and D).
ab b C Fifty-three proteins were up-regulated and 28 proteins were
Reducing sugar content (mg/g DW)

down-regulated in response to SC treatment (Fig. 5E). Fifty-two

a c proteins were up-regulated and 49 proteins were down-
150 ab
b regulated in response to SA treatment (Fig. 5F). Fifty-eight proteins
b
a a were up-regulated and 32 proteins were down-regulated in
b response to ST treatment (Fig. 5G). The responses of the proteins
100 c
to the different organic acid salts were substantially different. In
a a a mung bean sprouts, only 8 (21.1%) of the down-regulated proteins
b and 30 (79.9%) of the up-regulated proteins were commonly regu-
50 lated by the three different treatments and involved in the same
biological process (Supplementary Table 2). Therefore, the results
showed that the effects of these three treatments on mung bean
0 sprouts have some commonalities but also have additional, specific
characteristics, including exchangeable sodium and organic acid
CK ST SA SC
anion reactions in a complex network involving multiple physio-
Germination treatment
logical and metabolic pathways. The list of peaks used to identify
Fig. 3. Levels of storage materials, including soluble protein (A), free amino acids the proteins is shown in the Supplementary Table 3.
(B) and reducing sugars (C), in mung bean sprouts subjected to the ST, SA and SC
treatments. Different letters indicate significant differences among the samples on
each day (p < 0.05).
4. Discussion
3.4. Changes in antioxidant enzyme activities
Our results showed that mung bean sprout growth was signifi-
As shown in Fig. 4, significant increases in the GPX, POD, CAT and cantly increased by the SC and ST treatments and was accompa-
SOD activities were observed on the 4th day of germination com- nied by a higher respiratory rate, whereas the SA treatment
pared with the 2nd day (p < 0.05). The ST, SA and SC treatments inhibited mung bean growth on the 4th day. The slightly higher
resulted in similar patterns of POD, CAT and SOD activities. All treat- MDA content and electrical conductivity in SA treatment indicated
ments reduced the GPX activity on the 4th day of germination, with that excess free radicals were generated (Li, Zhang, Jiang, & Liu,
SA spraying showing the lowest activity among all of the treatments 2013). In response to the SA-induced stress, significant increases
(Fig. 4A). However, the highest CAT activity was observed in mung in the antioxidant enzymes activities of CAT, POD, SOD were
bean sprouts treated with SA spraying (Fig. 4C), followed by SC, ST detected, which was further confirmed by the increased abundance
and the control. Meanwhile, the highest POD and SOD activities were of POD 52-like (GI: 734397902) and CAT-4 (GI: 17865456) in our
also observed after SA treatment, which were 1.84- and 1.59-fold iTRAQ analysis (Supplementary Table 2). In addition, SA treatment
higher than the control, respectively (Fig. 4B and D) (p < 0.05). significantly increased the expression of isoflavone synthase (GI:
170783768) and isoflavone 20 -hydroxylase (GI: 571487382) com-
3.5. Differentially expressed proteins detected by iTRAQ pared with the control, which might indicate that isoflavone accu-
mulated in response to SA treatment. The accumulation of
The numbers of differentially expressed proteins and their over- isoflavones might play an essential role in scavenging reactive
lap in response to the different organic acid salt treatments were oxygen species, which might occur in response to the high MDA
290 X. Jin et al. / Food Chemistry 218 (2017) 285–293

4500 6000
2d a A B
4d
a

POD activity (U/g DW)

GPX activity (U/g DW)

3600
b 4500
b
2700 c
d c
3000
d
1800
a a
b b 1500
900 c d c
d
0 0
C D 5000
3000
CAT activity (U/g DW)

SOD activity (U/g DW)

a
a 4000
2400 b
b c
c d 3000
1800

a b 2000
1200 d a
c
600 1000
a
b b b
0 0
CK ST SA SC CK ST SA SC

Germination treatment Germination treatment

Fig. 4. Changes in the activities of the antioxidant enzymes GPX (A), POD (B), CAT (C) and SOD (D) in mung bean sprouts in response to SC, SA and ST treatments. Different
letters indicate significant differences among the samples on each day (p < 0.05).

content induced by SA (Fig. 2C). However, the exogenous SC and ST alpha (GI: 94730464) and malate dehydrogenase (GI: 169977)
treatments also produced higher antioxidant enzymes activities, were up-regulated by the SC and SA treatments, whereas increased
which agreed with the existing results showing that SC and ST ferredoxin-NADP reductase expression (GI: 356538289) was
could decrease the formation of free radicals and increase the induced by the SA and ST treatments; all of these up-regulated
antioxidant activities in refrigerated sliced salmon (Sallam, 2007). enzymes were involved in the respiratory electron transport chain.
Meanwhile, phytic acid degradation was promoted by these This result indicated that these three organic acid salts promoted
three organic acid salt treatments due to the increased phytase the activity of the respiratory electron transport chain, thus
activity. Moreover, increased expression of inositol-phosphate increasing sprout respiration. This result was also supported by
phosphatase (GI: 734423661) and purple acid phosphatase (GI: the data that mung bean sprouts treated with these three organic
734348105 and 351720816) were observed in SC-, SA- and ST- acid salts had higher respiratory rates (Fig. 2B).
treated mung bean sprouts (Supplementary Tables 1 and 2). These
findings suggested that these three treatments might be feasible 4.2. Enhanced carbohydrate degradation
ways to stimulate phytic acid degradation and antioxidant enzyme
activities. However, the underlying molecular mechanisms by The common differentially expressed proteins in the sprouts
which the organic acid salts mediate mung bean metabolism needs treated with these three organic acid salts were mainly up-
to be discussed further. regulated proteins that are involved in starch and non-starch
polysaccharides hydrolysis, glycolysis, tricarboxylic acid cycle
4.1. Stimulation of the respiratory electron transport chain and other processes, including the a-1,4-glucan phosphorylase L
(GI: 356551144) and L2 isozymes (GI: 571563869), a-glucan phos-
In our studies, the expression of the identified enzymes associ- phorylase (GI: 356527232), glucan endo-1,3-b-glucosidase (GI:
ated with energy metabolism was increased by these exogenous 356569016), a-xylosidase 1-like (GI: 356495935), and malate
organic acid salt treatments. ATP synthase b-subunit, mitochon- dehydrogenase (GI: 169977) (Supplementary Table 2). This result
drial (GI: 356536246), NADH dehydrogenase (ubiquinone) 1 a- indicated that these organic acid salts might promote carbohydrate
subcomplex subunit 5 (GI: 351726335), and F-type H+- degradation. Phosphoenolpyruvate carboxykinase (PEPCK) (GI:
transporting ATPase c-subunit (GI: 351723485) belong to the 356496541) catalyses a key step in the conversion of fats to sugars
oxidative phosphorylation process, in which ATP is formed as a during the germination of fat-storing seeds, and its activity is clo-
result of the transfer of electrons from NADH or FADH2 to O2. sely paralleled with the gluconeogenic flux (Bryce & Hill, 1993).
The expression of these enzymes was increased by the SC, ST and This enzyme replenished oxaloacetate in the tricarboxylic acid
SA treatments, except for F-type H+-transporting ATPase c- cycle when operating in the reverse direction, and this step is catal-
subunit in the ST treatment group (Supplementary Table 2), indi- ysed by another enzyme - phosphoenolpyruvate carboxylase
cating that ATP synthesis was promoted by these exogenous (PEPC) (GI: 1705587). In our results, the PEPCK (GI: 356496541)
organic acid salt treatments. In addition, cytochrome b 559 subunit levels were reduced by the three organic acid salt treatments
 X. Jin et al. / Food Chemistry 218 (2017) 285–293 291

Sodium citrate/CK Sodium acetate /CK

A B

11
16 26
Carbohy drate and energy metabolism 30.86%
Cell transp ort 3.70%
38 Cell wall and cy toskeleton metabolism 8.64%
Inositol p hosp hate metabolism 3.70%
Lip id metabolism 2.47%
16 26 Nucleic acid metabolism 6.17%
Protein metabolism 13.58%
Biological regulation and signal transduction 11.11%
Resp onse to stress 4.94%
Secondary metabolism 8.64%
10 Other metabolism 4.94%
Unknown metabolism 2.47%

Sodium citrate/ CK
Sodium tartrate/CK
C D

Carbohy drate and energy metabolism 22.77% Carbohy drate and energy metabolism 25.56%
Cell transp ort 3.96% Cell transp ort 6.67%
Cell wall and cy toskeleton metabolism 6.93% Cell wall and cy toskeleton metabolism 7.78%
Inositol p hosp hate metabolism 2.97% Inositol p hosp hate metabolism 3.33%
Lipid metabolism 3.96% Lip id metabolism 5.56%
Nucleic acid metabolism 2.97% Nucleic acid metabolism 4.44%
Protein metabolism 18.81% Protein metabolism 14.44%
Biological regulation and signal transduction 8.91% Biological regulation and signal transduction 7.78%
Resp onse to stress 7.78%
Response to stress 7.92%
Secondary metabolism 6.67%
Secondary metabolism 5.94%
Other metabolism 4.44%
Other metabolism 8.91% Unknown metabolism 5.56%
Unknown metabolism 5.94%

Sodium acetate/ CK Sodium tartrate/CK

Sodium citrate/CK Sodium acetate/CK G Sodium tartrate/CK

E F
Carbohydrate and energy metabolism
Protein metabolism
Response to stress
Inositol phosphate metabolism
Nucleic acid metabolism
Cell wall and cytoskeleton metabolism
Biological regulation and signal transduction
Cell transport
Secondary metabolism up-regulated
Lipid metabolism down-regulated
Other metabolism
Unknown metabolism

0 5 10 15 20 25 0 4 8 12 16 20 24 0 4 8 12 16 20 24

Number of proteins Number of proteins Number of proteins

Fig. 5. Venn diagrams of the differentially expressed proteins (A) and functional classifications of these proteins in mung bean sprouts treated with sodium citrate (B and E),
sodium acetate (C and F) and sodium tartrate (D and G). The numbers of differentially expressed protein spots in response to a given treatment are shown in different
segments.

compared with the control, whereas PEPC (GI: 1705587) showed carbohydrate catabolism was promoted during early germination.
the opposite trend, suggesting that the conversion of lipids to sug- In our study, these three organic acid salt treatments all acceler-
ars was inhibited by the organic acid salt treatments on the 4th day ated the carbohydrate hydrolysis, which was responsible for
of germination. The inhibition might have resulted from a suffi- sustaining post-germinative growth.
cient sugar supply for mung bean growth. On one hand, these It was noted that most of the differentially expressed proteins
organic acid salts might have acted as a carbon source to sustain associated with carbohydrate metabolism were up-regulated in
the sprout growth (Casati, Drincovich, Edwards, & Andreo, 1999). the SC-treated mung bean sprouts, except for acid beta-
On the other hand, the up-regulation of a-glucan phosphorylase fructofuranosidase-like (GI: 356540502), which catalyses the con-
(GI: 356527232) and the a-1,4-glucan phosphorylase L (GI: version of sucrose to fructose and glucose. The difference in the
356551144) and L-2 isozymes (GI: 571563869), could have expression of this protein might be the result of the feedback inhi-
resulted in a significant increase in the reducing sugar content bition due to the high reducing sugars content (Fig. 3C), which was
(Fig. 3C). Yang et al. (2007) also determined that most TCA cycle consistent with the results reported by Zhang and Wang (2002).
and glycolysis-related proteins were up-regulated, such as UDP- Increased expression of probable beta-D-xylosidase 2 (GI:
glucose dehydrogenase, fructokinase, phosphoglucomutase, and 356556038) was also observed in response to ST spraying. Mean-
pyruvate decarboxylase. The accumulation of succinyl-CoA ligase while, pyruvate kinase isozyme A (GI: 356535329) was up-
and cytoplasmic malate dehydrogenase had been observed in rice regulated in response to the SC and SA treatments compared with
embryos during germination (Kim et al., 2009), suggesting that the control, suggesting that glycolysis metabolism was increased.
292 X. Jin et al. / Food Chemistry 218 (2017) 285–293

4.3. Stimulation of cell wall extension and lignin synthesis Post-translational modifications (PTMs), such as glycosylation,
phosphorylation and ubiquitination, are reported to play important
Fibre growth is a key developmental process, which undergoes roles in the regulation of abiotic stress responses. Our results showed
four major discrete developmental stages (differentiation, elonga- that ST spraying increased levels of proteins involved in phosphory-
tion/primary cell wall synthesis, secondary cell wall synthesis, lation (such as receptor-like protein kinase FERONIA-like, GI:
and maturity) and eventually produces a thick cell wall consisting 571496773, probable serine/threonine protein phosphatase 2A reg-
of more than 94% cellulose (Kim & Triplett, 2001). Expansin pro- ulatory subunit, GI: 571569749 and calcium-dependent protein
teins (EXPS) loosen the cell walls and modulate cell and plant kinase isoform, GI: 356521991) in the sprouts, whereas SC spraying
growth. In the present study, the expression of several enzymes also increased the level of receptor-like protein kinase FERONIA-like.
involved in cell wall extension and lignin synthesis (expansin, GI: However, the SC and SA spraying treatments decreased the sprout
330858331 and annexin with 2 isoforms, GI: 356548893 and levels of ubiquitination (ubiquitin-conjugating enzyme E2
356548895) was increased, indicating that the three organic acid 5-like isoform X2, GI: 571525370 and SKP1-like protein 1A, GI:
salt treatments stimulated lignin synthesis and thus affected radi- 356496612, respectively) (Supplementary Table 2). This result
cal extension and sprout elongation to some extent (Daines, shows that the PTMs of some proteins might be impaired in response
Minocha, & Kudakasseril, 1983). to SA treatment. However, the sprout levels of phosphotyrosine pro-
tein phosphatase-like (GI: 571495059) were increased in response
4.4. Increased phenolic biosynthesis to the SA spraying treatment.

Phenylalanine ammonialyase (PAL) is considered the key

4.6. Opposite response of biological regulation and signal transduction
enzyme in phenolic biosynthesis. It catalyses the reductive amina-
tion of L-phenylalanine to form transcinnamic acid, which is the first
Ca2+ may act as a messenger in signal transduction in plants and
step in the biosynthesis of plant phenylpropanoid compounds. In
it depends on a sensor to convey changes in its concentration. The
this study, the expression of several enzymes involved in phenolic
main groups of proteins that bind Ca2+ and confer Ca2+-mediated
synthesis (caffeic acid 3-O-methyltransferase-like, GI: 356518669
responses are EF-hand proteins and C2-domain proteins (Reddy
and phenylalanine ammonialyase class 2-like, GI: 356548301) was
& Reddy, 2004). In this study, the differentially expressed proteins
increased in response to the three organic acid salt treatments. How-
related to Ca2+ sensors, such as calcium-dependent protein kinase
ever, anthocyanidin 3-O-glucosyltransferase-like (GI: 356576401)
11 (GI: 257638616), were down-regulated by these organic acid
was down-regulated in response to SC treatment, indicating that
salt treatments. We also found that calcium-binding EF-hand pro-
glycosylation modification was affected by SC treatment.
teins, calcium-dependent protein kinase SK5 (GI: 356508898) and
calmodulin (GI: 255644599) were down-regulated by the SA treat-
4.5. Opposite response of protein metabolism
ment. Because calcium-dependent protein kinase is primarily
involved in signalling cascades involved in osmotic, developmental
The differentially expressed ribosomal proteins, which are
and nutritional changes (Valmonte, Arthur, Higgins, & MacDiarmid,
known to play an essential role in ribosome assembly and protein
2014), it was reasonable to conclude that Ca2+-related proteins
translation, were mainly down-regulated in response to the SA
may be involved in the responses of mung bean sprouts to organic
spraying treatment, indicating that SA treatment impaired protein
acid salt treatments.
biosynthesis. However, the ribosomal proteins were insignificantly
During the germination process, storage reserves, particularly
affected by the SC and ST spraying treatments (Supplementary
starches, proteins and triglycerides, are broken down in the seed.
Table 2). These results were consistent with our observation that
These reserves are mobilized during germination and seedling
the SA-treated sprouts had the lowest total soluble protein content
growth to supply the energy and metabolic intermediates needed
(Fig. 3A). The inhibition of protein synthesis by the SA treatment
by the seedling prior to the establishment of photosynthetic
can also be confirmed by the decrease in the expression of eukary-
autotrophism (Wilson, Rightmire, Chen, & Tan-Wilson, 1986).
otic translation initiation factor 5A3 (GI: 351721220), which is
b-conglycinin (GI: 356535993), a main type of seed storage
involved in the initiation phase of eukaryotic translation. Further-
protein, accumulates during seed development and is hydrolysed
more, the nascent polypeptide-associated complex (NAC) (GI:
after germination to provide a carbon and nitrogen source for the
356509137) is considered to be involved in protein transport for
developing seedling (Angelovici, Fait, Fernie, & Galili, 2011). How-
the appropriate targeting of ribosome-nascent polypeptide com-
ever, b-conglycinin expression showed different trends in response
plexes (Rospert, Dubaquie, & Gautschi, 2002). Proteins containing
to the organic acid salt treatments. Compared with the control, the
a pentatricopeptide repeat (PPR) (GI: 356512038) may act as
b-conglycinin levels were significantly increased by the SA treat-
sequence-specific adaptors for a variety of other RNA-associated
ment (p < 0.05), but its levels were significantly reduced by the
proteins (Diniz et al., 2010). As shown in Supplementary Table 2,
SC treatment, with no significant difference between the ST spray-
the NAC and PPR-like superfamily proteins were down-regulated
ing treatment and the control. This result indicated that SC spray-
in mung bean sprouts following the SA spraying treatment. These
ing treatment stimulated b-conglycinin degradation during storage
results further showed that SA spraying impaired protein biosyn-
protein mobilization, whereas the degradation of b-conglycinin
thesis in mung bean sprouts.
was inhibited by the SA treatment. The increased degradation of
On the other hand, SC treatment significantly increased the levels
b-conglycinin ensures that a sufficient amount of amino acids will
of proteasome subunit beta type and several other proteases (also
be available for new protein synthesis, and the low levels of
termed proteinase and peptidase-like serine carboxypeptidase-like
b-conglycinin in mung bean sprouts subjected to the SC treatment
51, GI: 356550133, putative serine protease EDA2, GI: 363814290)
were general responses that increased plant growth.
(Supplementary Table 2) that played important roles in protein pro-
cessing, particularly in modifying proteins and remodelling
polypeptides during germination (Lepelley et al., 2012). This result 5. Conclusions
indicates that protein modification and processing might be
enhanced in SC-treated mung bean sprouts. Hence, the proteins pos- The exogenous SC, ST, SA treatments significantly affected
sessing enzymatic activity that were up-regulated by SC spraying mung bean sprout growth, whereas the SC and ST treatments stim-
ensured the specific functions of the proteins. ulated growth and the SA treatment slightly inhibited growth. The
 X. Jin et al. / Food Chemistry 218 (2017) 285–293 293

sprouts subjected to the SC, ST and SA treatments exhibited higher oxidation in rainbow trout (Onchorhynchus mykiss) sticks during refrigerated
storage (4°C). Iranian Journal of Fisheries Sciences, 9, 73–86.
phytic acid degradation compared with the control. The up-
Hegeman, C. E., & Grabau, E. A. (2001). A novel phytase with sequence similarity to
regulation of phytase might due to the increased expression of purple acid phosphatases is expressed in cotyledons of germinating soybean
carbohydrate-degrading enzymes and ATP-synthesising enzymes, seedlings. Plant Physiology, 126, 1598–1608.
which resulted in an increase in phosphate consumption. More- Heinonen, J. K., & Lahti, R. J. (1981). A new and convenient colorimetric
determination of inorganic orthophosphate and its application to the assay of
over, the inhibitory effect of the SA treatment is probably due to inorganic pyrophosphatase. Analytical Biochemistry, 113, 313–317.
impaired protein biosynthesis. Jin, X., Yang, R., Yan, X., Zhou, Y., Wang, X., & Gu, Z. (2016). Malic acid and oxalic acid
spraying enhances phytic acid degradation and total antioxidant capacity of
mung bean sprouts. International Journal of Food Science & Technology, 51,
Conflict of interest 370–380.
Jom, K. N., Frank, T., & Engel, K. H. (2011). A metabolite profiling approach to follow
the sprouting process of mung beans (Vigna radiata). Metabolomics, 7, 112–117.
The authors declare no conflict of interest.
Kim, H. J., & Triplett, B. A. (2001). Cotton fiber growth in planta and in vitro. Models
for plant cell elongation and cell wall biogenesis. Plant Physiology, 127,
Acknowledgement 1361–1366.
Kim, S. T., Wang, Y., Kang, S. Y., Kim, S. G., Rakwal, R., & Kim, Y. C. (2009). Developing
rice embryo proteomics reveals essential role for embryonic proteins in
We are grateful for the financial support from National Natural regulation of seed germination. Journal of Proteome Research, 8, 3598–3605.
Science Foundation of China (31471596) and the Research and Lepelley, M., Amor, M. B., Martineau, N., Cheminade, G., Caillet, V., & McCarthy, J.
Innovation Project for College Graduates of Jiangsu Province (No. (2012). Coffee cysteine proteinases and related inhibitors with high expression
during grain maturation and germination. BMC Plant Biology, 12. http://dx.doi.
KYLX_0526). org/10.1186/1471-2229-12-31.
Li, T., Hu, Y., Du, X., Tang, H., Shen, C., & Wu, J. (2014). Salicylic acid alleviates the
adverse effects of salt stress in Torreya grandis cv. Merrillii seedlings by
Appendix A. Supplementary material activating photosynthesis and enhancing antioxidant systems. PLoS One, 9,
e109492.
Supplementary data associated with this article can be found, in Li, Y., Zhang, S., Jiang, W., & Liu, D. (2013). Cadmium accumulation, activities of
antioxidant enzymes, and malondialdehyde (MDA) content in Pistia stratiotes L..
the online version, at http://dx.doi.org/10.1016/j.foodchem.2016.
Environmental Science and Pollution Research, 20, 1117–1123.
09.029. Ma, G., Jin, Y., Piao, J., Kok, F., Guusje, B., & Jacobsen, E. (2005). Phytate, calcium, iron,
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