Food Chemistry 289 (2019) 635–644

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

iTRAQ-based proteomic analysis reveals the accumulation of bioactive T

compounds in Chinese wild rice (Zizania latifolia) during germination
Cheng Chua,b, Ning Yana, , Yongmei Dua, Xinmin Liua, Meijun Chua, John Shic, Hongbo Zhanga,
⁎

Yanhua Liua, Zhongfeng Zhanga

a
Tobacco Research Institute of Chinese Academy of Agricultural Sciences, Qingdao 266101, China
b
Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081, China
c
Guelph Food Research Center, Agriculture and Agri-Food Canada, Guelph, Ontario N1G 5C9, Canada

ARTICLE INFO ABSTRACT

Keywords: Polyphenols and γ-aminobutyric acid (GABA) accumulate during seed germination, but the mechanisms in-
Chinese wild rice volved are poorly understood. The objective of this study was to elucidate the accumulation of these bioactive
Germination compounds in Chinese wild rice during germination. The greatest differences in the phenolic content were at 36-
iTRAQ h (G36) and 120-h germination (G120) stages. An iTRAQ-based proteomic analysis revealed 7031 proteins, and a
Proteomics
comparison of the G120 and G36 stages revealed 956 upregulated and 188 downregulated proteins. The KEGG
Phenolics
analysis revealed significant protein enrichment in the “metabolic pathways”, “biosynthesis of secondary me-
γ-Aminobutyric acid (GABA)
tabolites” and “phenylpropanoid biosynthesis”. Four phenylalanine ammonia-lyases, one 4-coumarate-CoA li-
gase, one cinnamoyl-CoA reductase, two cinnamyl alcohol dehydrogenases, and four glutamate decarboxylases
exhibited higher expression at the G120 than at the G36 stage and promoted phenolics and GABA accumulation.
This study revealed bioactive compound accumulation in germinating Chinese wild rice, and the finding may
help develop functional foods derived from this cereal.

1. Introduction
Chinese wild rice is the seed obtained from the caryopsis of Zizania
latifolia. It has been used as a grain in China for over 3000 years (Yan
et al., 2018, 2019). Chinese wild rice is rich in bioactive compounds
including dietary fibre, resistant starch, minerals, vitamins, phenolics,
flavonoids, saponins, anthocyanins, chlorophyll and phytosterols
(Jiang, Zhai, Yang, Zhai, & Zhai, 2016; Yan et al., 2018). In our pre-
vious study, we applied an ultra-high performance liquid chromato-
graphy coupled to triple quadrupole mass spectrometry (UHPLC-QqQ-
MS)-based metabolomics and identified 672 metabolites from Chinese
wild rice (Yan et al., 2019). We also quantitatively analysed 12 phenolic
acids and 9 flavonoids in this cereal (Chu et al., 2018). As it is rich in
bioactive compounds, Chinese wild rice exhibits antioxidant activity,
alleviates insulin resistance and lipotoxicity, protects against cardio-
vascular disease, and possesses other bioactivities and health benefits
(Chu et al., 2018; Han, Zhang, Qin, & Zhai, 2013; Han, Zhang, & Zhai,
2012; Yan et al., 2018). The protein content of Chinese wild rice is 2.16
times higher than that of Indica rice and its protein efficiency ratio
(2.75) is greater than that of rice (2.18) and soybean (2.32) (Jiang et al.,
2016; Yan et al., 2018). Furthermore, the amino acid composition of
Chinese wild rice showed higher amounts of essential amino acids
(EAAs) than those reported in white rice, barley and maize (Jiang et al.,
2016; Zhai, Lu, Zhang, Sun, & Lorenz, 2001). Therefore, Chinese wild
rice is both a health-promoting food and a high-quality protein source.
It may also serve as an excellent food for patients with diabetes, obesity,
and hyperlipidaemia.
Germination increases the nutritional value, bioactive compound
levels and bioactivities of edible seeds (Aguilera et al., 2013; Gan et al.,
2017). Germination has been widely used to produce germinated edible
seeds and sprouts for daily dietary consumption. On the one hand,
germination promotes the hydrolysis and degradation of several dif-
ferent nutrients including carbohydrates, proteins and fats. It also in-
duces the accumulation of simple sugars, free amino acids (FAAs) and
organic acids (Hübner & Arendt, 2013). On the other hand, germination
reduces the levels of non-nutritive and indigestible factors such as
protease inhibitors and lectins, and increases the levels of bioactive
phenolic compounds and antioxidant activities in legumes (Aguilera
et al., 2013). Notably, germinated seeds and sprouts accumulate
bioactive compounds such as polyphenols, γ-aminobutyric acid (GABA)
and vitamins. Furthermore, they exhibit antioxidant, anti-in-
flammatory, antibacterial, antidiabetic, anticancer and other

⁎
Corresponding author.
E-mail address: yanning@caas.cn (N. Yan).

https://doi.org/10.1016/j.foodchem.2019.03.092
Received 11 December 2018; Received in revised form 16 March 2019; Accepted 19 March 2019
Available online 19 March 2019
0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
C. Chu, et al.
bioactivities (Gan et al., 2017). For instance, germination of brown rice
may increase its total phenolics content by 63.2% and significantly
increase its antioxidant activity simultaneously (Ti et al., 2014). Ger-
mination for 72 h can significantly increase the GABA content in red
rice (Ding et al., 2018). Therefore, germination may be a method of
engineering green food with high levels of natural bioactive com-
pounds. Germinated edible seeds and sprouts rich in these substances
may be consumed as functional foods to prevent and treat chronic
diseases (Gan et al., 2017).
Proteomics focuses primarily on the protein expression level, post-
translational modification and protein–protein interaction (Ong &
Mann, 2005; Wang et al., 2015, 2018). In contrast, protein chemistry
mainly identifies complete sequences of a small number of proteins and
cannot be used in integrative studies. Quantitative proteomics precisely
identifies and measures all the proteins expressed in a genome or a
complex system and selects differentially expressed proteins (DEPs)
from various samples (Ong & Mann, 2005). Isobaric tags for relative
and absolute quantification (iTRAQ) is a high-throughput screening
technology that has been applied in quantitative proteomics in recent
years (Ross et al., 2004). With iTRAQ, the protein expression levels of
up to eight samples can be compared simultaneously. The output pre-
sents relatively good quantitative results and has satisfactory reprodu-
cibility (Wang et al., 2015, 2018). The iTRAQ technology has been used
to identify changes in the responses of soybean sprouts to UV-B treat-
ment (Jiao & Gu, 2019), alterations in rice hull development (Wang
et al., 2015), biochemical mechanism of cold stress adaptation in razor
clam during controlled freezing-point storage (Wang et al., 2018) and
proteins associated with the quality traits of frozen mud shrimp (Shi
et al., 2018).
There have been several recent reports on the macronutrient,
polyphenol and GABA content of germinated edible seeds and sprouts
(Gan et al., 2017; Hübner & Arendt, 2013). However, little information
is available on the use of proteomics to elucidate the mechanisms by
which these bioactive compounds accumulate in germinated seeds and
sprouts. Proteomics research in Chinese wild rice has been advanced by
the publication of its genome sequence (Guo et al., 2015). In the present
study, the dynamic changes in the content of total phenolics and FAAs
of Chinese wild rice during germination were investigated. The greatest
difference in the total phenolics content in Chinese wild rice was found
between the 36-h germination (G36) and 120-h germination (G120)
stages. An iTRAQ-based proteomic study was conducted in the G36 and
G120 stages to reveal the accumulation of bioactive compounds in
Chinese wild rice during germination. The Gene Ontology (GO) and
Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment ana-
lyses of the DEPs were also performed. The results of the present study
may promote the research and development of functional food derived
from germinating Chinese wild rice and facilitate functional food pro-
duction in general.
2. Materials and methods
2.1. Samples and preparations
The Chinese wild rice used in this study was collected from Baimahu
Village, Qianfeng Town, Jinhu County, Huai'an City, Jiangsu Province,
China (33°11′9″ N; 119°9′37″ E) on 20 September 2017 according to a
previously described sampling method (Yan et al., 2019). After air-
drying, the collected Chinese wild rice was sifted and purified. Full and
plump seeds were selected and stored at 4 °C. They were then immersed
in 0.5% sodium hypochlorite solution (adjusted to pH 5.5 with 6 M HCl)
for 10 min and rinsed with deionised sterile water until the pH of the
rinsate reached 7.0. The disinfected seeds were used as G0 seeds, which
were placed in a conical flask containing deionised water (1:4 seed to
water ratio) and the flask was immersed in a thermostatic water bath at
30 °C for 5 h with shaking every 0.5 h. The seeds were placed in an
illumination incubator (MGC-250; Yihengyiqi, Suzhou, Jiangsu, China)
636
Food Chemistry 289 (2019) 635–644
at 30 °C in dark and germinated for 12 (G12), 24 (G24), 36 (G36), 48
(G48), 60 (G60), 72 (G72), 84 (G84), 96 (G96), 108 (G108) or 120 h
(G120).
Germinating Chinese wild rice seeds (G0–G120) were dried to a
constant weight in a freeze dryer (Alpha 1–2 LD Plus; Martin Christ
Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany), milled
and passed through a 100-mesh sieve. The seed powders were used in
the subsequent experiments. The preliminary results indicated that the
total phenol content was the lowest at the G36 stage and highest at the
G120 stage. Therefore, germinating Chinese wild rice seeds from the
G36 and G120 stages were chosen to evaluate proteomic changes via
iTRAQ. The phenotypes of G36 and G120 seeds are shown in Fig. S1.
The germ of G36 seeds was obvious, but no radicle was found. The germ
of G120 seeds was much longer than that of G36 seeds, and the radicle
was obviously visible. For all assays, sampling was repeated in triplicate
at each time point.
2.2. Extraction and determination of free and bound phenolics
Free and bound phenolics were extracted according to the method
described previously (Ti et al., 2014). This process started by weighing
0.2 g of germinating Chinese wild rice powder to an accuracy of
0.0001 g. The sample was ultrasonically extracted twice with 5 mL of
methanol at 50 ℃ for 40 min per extraction. The samples were cen-
trifuged at 15,300×g and 4 °C for 10 min (Sorvall RC6+; Thermo
Fisher Scientific, Waltham, MA, USA); the supernatants were pooled in
a 50-mL centrifuge tube and passed through a 0.22-μm filter membrane.
The solution was diluted with methanol up to 10 mL in a volumetric
flask to obtain the free phenolics. The residue was added to 5 mL of 4 M
NaOH solution, vortexed and hydrolysed at 250 rpm for 4 h at 30 °C.
The sample was then centrifuged at 15,300×g for 10 min at 4 °C. The
supernatant was collected and the centrifugation was repeated twice,
and then the supernatants were pooled in a 50-mL centrifuge tube. The
pH was adjusted to 1.5–2.0 with 6 M HCl and the phenolics were ex-
tracted with 25 mL of ethyl acetate. The ethyl acetate extract was de-
siccated in a rotary evaporator at 35 °C, dissolved in 5 mL of methanol
and passed through a 0.22-μm filter membrane. The bound phenolics
solution was stored at 4 °C for the subsequent assay.
The phenolics content was determined by Folin–Ciocalteu colori-
metric method (Dewanto, Wu, Adom, & Liu, 2002). The sample solution
(750 μL) was added to 250 μL of Folin–Ciocalteu reagent, thoroughly
mixed and left to react at 25 °C for 5 min. Subsequently, 500 μL of water
and 250 μL of 20% (w/v) sodium carbonate solution were added and
thoroughly mixed with the sample solution. The reaction was run in
dark for 60 min and the sample was centrifuged at 15,300×g for 10 min
at 4 °C. The absorbance of the reaction mixture was measured in a
microplate reader at 740 nm wavelength (Multiskan FC; Thermo Fisher
Scientific, Waltham, MA, USA). Absolute methanol was used as the
control and gallic acid (GA) was the reference standard. The phenolics
content of the sample was recorded as GA milliequivalents per kilogram
of germinating Chinese wild rice (mg GAE/kg).
2.3. Extraction and determination of free amino acids
The FAAs were extracted following a previously reported method
(Jiang et al., 2016). A total of 0.5 g of germinating Chinese wild rice
powder was mixed with 5 mL of 0.1 M HCl. The mixture was ultra-
sonically extracted for 40 min and centrifuged at 15,300×g for 10 min
at 4 °C. The supernatant was collected and passed through a 0.45-μm
filter. Seventeen FAAs and GABA were quantitated with a Biochrom 30
amino acid analyser (Biochrom, Cambridge, UK).
2.4. Protein extraction and iTRAQ labelling
2.4.1. Total protein extraction and peptide preparation
Samples from the G36 and G120 stages were pulverised in a mortar
C. Chu, et al.
with liquid nitrogen and mixed with lysis buffer containing 50 mM Tris-
HCl (pH 8), 8 M urea and 0.2% SDS. The homogenate was ultra-
sonicated on ice for 5 min and centrifuged at 12,000×g and 4 °C for
15 min. The supernatant was transferred to a clean tube and its protein
content was determined by Bradford assay. Subsequently, 2 mM di-
thiothreitol was added to the sample, and the mixture was incubated at
56 °C for 1 h. Iodoacetic acid was added to the samples. The samples
were then incubated for 1 h at 25 °C in dark. Four volumes of cold
acetone were added to the sample extract. This mixture was vortexed
and stored at −20 °C for at least 2 h. The extracts were centrifuged at
12,000×g for 15 min at 4 °C. The pellets were collected, washed twice
with cold acetone and dissolved in a buffer containing 0.1 M triethy-
lammonium bicarbonate (TEAB, pH 8.5) and 8 M urea. The protein
content was determined by Bradford assay. Supernatant from each
sample containing precisely 0.1 mg of protein was digested with
Trypsin Gold (Promega, Madison, WI, USA) at 37 °C for 16 h. The
peptide was dried by vacuum centrifugation after the removal of urea
from it with a C18 desalting cartridge.
2.4.2. iTRAQ labelling of peptides
The desalted peptides were labelled with iTRAQ reagents (iTRAQ®
Reagent-8PLEX Multiplex Kit; Sigma-Aldrich, Shanghai, China). For
every 0.1 mg of peptides, one unit of labelling reagent was used.
Peptides were dissolved in 20 μL of 0.5 M TEAB and the labelling re-
agent was added to 70 μL isopropanol. After incubation for 1 h, the
reaction was stopped with 50 mM Tris-HCl (pH 8). Differently labelled
peptides were mixed in equal proportions and desalted in peptide-de-
salting spin columns (89852; Thermo Fisher Scientific, Waltham, MA,
USA).
2.5. HPLC fractionation
The TMT-labelled peptide mix was fractionated in a Waters BEH
C18 column (4.6 mm × 250 mm, 5 μm) on a Rigol L3000 HPLC
(Changping, Beijing, China) operating at 1 mL/min. The column oven
temperature was set at 50 °C. Mobile phases A (2% acetonitrile adjusted
to pH 10.0 with ammonium hydroxide) and B (98% acetonitrile ad-
justed to pH 10.0 with ammonium hydroxide) were used to develop a
gradient elution. The solvent gradient was as follows: 3% B, 5 min;
3–8% B, 0.1 min; 8–18% B, 11.9 min; 18–32% B, 11 min; 32–45% B,
7 min; 45–80% B, 3 min; 80% B, 5 min; 80–5%, 0.1 min; and 5% B,
6.9 min. The tryptic peptides were monitored under UV at 214 nm
wavelength. Eluent was collected every minute and 10 fractions were
pooled. The samples were vacuum-dried and reconstituted in 0.1% (v/
v) aqueous formic acid (FA) for the subsequent analyses.
2.6. LC–MS/MS analysis
Shotgun proteomics analysis was performed in an EASY-nLC™ 1200
UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) coupled
to an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher
Scientific, Waltham, MA, USA) operating in the data-dependent ac-
quisition (DDA) mode. Two micrograms of total peptides reconstituted
in 0.1% (v/v) FA were injected into an Acclaim PepMap100 C18 Nano-
Trap column (2 cm × 100 μm, 5 μm). Peptides were separated on a
Reprosil-Pur 120 C18-AQ analytical column (15 cm × 150 μm, 1.9 μm)
using a 60-min linear gradient from 5% to 100% eluent B (0.1% FA in
80% acetonitrile (ACN)) in eluent A (0.1% FA in H2O) at a flow rate of
600 nL/min. The solvent gradient was as follows: 5–10% B, 2 min;
10–30% B, 49 min; 30–50% B, 2 min; 50–90% B, 2 min; and 90–100%
B, 5 min.
For DDA, the Q-Exactive HF-X mass spectrometer was operated in
positive polarity mode with a spray voltage of 2.3 kV and a capillary
temperature of 320 °C. Full MS scans from 350 to 1500 m/z were ac-
quired at a resolution of 60,000 (at 200 m/z), an automatic gain control
(AGC) target value of 3 × 106 and a maximum ion injection time of
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Food Chemistry 289 (2019) 635–644
20 ms. From the full MS scan, up to 40 of the most abundant precursor
ions were selected for higher-energy collisional dissociation fragment
analysis at a resolution of 15,000 (at 200 m/z), an AGC target value of
1 × 105, a maximum ion injection time of 45 ms, a normalised collision
energy of 32%, an intensity threshold of 8.3 × 103 and a dynamic ex-
clusion parameter of 60 s.
2.7. Data quality control, protein function annotation and quantitative
analysis
2.7.1. Data quality control
The raw data obtained from MS detection were uploaded directly
into Proteome Discoverer v. 2.2 (Thermo Fisher Scientific, Waltham,
MA, USA) for database retrieval, peptide mapping and protein quanti-
tation. The database used in this study was the protein sequence of Z.
latifolia (http://ibi.zju.edu.cn/Zlatifolia/). The retrieved results were
filtered in Proteome Discoverer v. 2.2. Peptide Spectrum Matches
(PSMs) with 95% confidence intervals. Proteins with at least one unique
peptide fragment were considered reliable. The reliable PSMs and
proteins were verified with other reliable proteins. Peptide fragments
and proteins with false discovery rates (FDR) of > 5% were excluded.
2.7.2. Protein function annotation
The GO, KEGG, Cluster of Orthologous Groups of proteins (COG)
and other databases were used to annotate the identified proteins. The
GO annotation analysed the identified proteins in InterProScan v.
5.22–61.0 (European Bioinformatics Institute, Cambridge, UK), with
the databases Pfam, PRINTS, ProDom, SMART, ProSite and PANTHER.
The KEGG and COG annotations were used in a BLAST comparison (e-
value ≤ 1e-4) of the verified proteins. The results with the highest score
were annotated. InterPro (IPR) also uses InterProScan with the Pfam,
ProDom and SMART domain databases. IPR was conducted on un-
known proteins using their pattern structure or characteristics.
2.7.3. Protein quantitative analysis
Proteome Discoverer v. 2.2 was used to acquire the relative quan-
titative values of the PSMs of all samples. These were based on the peak
area of the plot generated by the original spectrograph. The relative
quantitative values of the unique peptide fragments determined after
calibration were obtained based on the quantitative data for all unique
peptide fragments in each protein.
2.8. Protein differential analysis and Gene Ontology term and Kyoto
Encyclopaedia of Genes and Genomes enrichment analyses
2.8.1. Protein differential analysis
For the differential analysis of proteins, the ratio of the mean values
of all biological repeated quantitative values for the samples was used
as the fold change (FC). To determine the statistical significance of the
difference, a t-test was conducted on the relative quantitative value of
each protein in the two samples being compared. The corresponding P-
value was computed as the significance indicator. When FC ≥ 1.5 and
P ≤ 0.05, it was assumed that the protein expression increased. When
FC ≤ 0.67 and P ≤ 0.05, it was considered that the protein expression
level was downregulated.
2.8.2. Gene Ontology term enrichment analysis
In the GO term enrichment analysis, all DEPs were first mapped to
each term in the GO database (http://www.geneontology.org/). The
number of each protein per term was computed. A hypergeometric test
was then applied to identify the GO terms with significant enrichment
compared with the background value for all proteins. The P-value was
computed using formulas. The GO term within the threshold of
P ≤ 0.05 was defined as significantly enriched. The main biological
function of the DEPs can be determined by GO significance analysis.
C. Chu, et al. Food Chemistry 289 (2019) 635–644

2.8.3. Kyoto Encyclopaedia of Genes and Genomes pathway enrichment
analysis
A hypergeometric test was applied to identify the KEGG pathway
with significant enrichment compared with the background value for
all proteins (Kanehisa, Sato, Kawashima, Furumichi, & Tanabe, 2015).
The KEGG pathway within the threshold of P ≤ 0.05 was defined as
significantly enriched.
2.9. Assay of phenylalanine ammonia-lyase
The phenylalanine ammonia-lyase (PAL) activity was determined
using the PAL test kit (BC0210, Solarbio; Beijing Solarbio Science &
Technology Co., Ltd., Beijing, China). The absorbance change of 0.1 at
290 nm per minute for 1 g of tissue in 1 mL of reaction system is defined
as one unit of PAL activity, which is expressed as U/g fresh weight
(FW).
2.10. Statistical analyses
The data were reported as mean ± SD of three samplings at each
germination stage. The data were analysed by the analysis of variance
in SAS v. 9.4 (SAS Institute Inc., Cary, NC, USA). Duncan’s multiple
comparison test was used at the P < 0.05 level to determine any sig-
nificant differences.
3.2. Changes in the content of free amino acids in Chinese wild rice during
germination
Changes in the content of FAAs in germinating Chinese wild rice are
presented in Table 2. From the G12 to G120 stages, the levels of 18
FAAs gradually increased and reached their highest levels at the G120
stage (Table 2). GABA is a non-protein amino acid present in both
plants and animals. It has been extensively investigated in germinating
brown rice (Ding et al., 2018; Gan et al., 2017). From the G0 to G120
stages, the GABA content gradually increased from 75.82 to
1465.21 μg/g and reached the maximum at the G120 stage. The GABA
content at the G120 stage was 7.29 times higher than that at the G36
stage. In the present study, the EAAs detected in germinating Chinese
wild rice included threonine, valine, methionine, isoleucine, leucine,
phenylalanine and lysine (Table 2). Their levels at the G120 stage were
14.61, 14.69, 25.61, 28.58, 17.56, 28.96 and 14.06 times higher than
those at the G36 stage, respectively. Other FAAs detected in germi-
nating Chinese wild rice included aspartate, serine, glutamate, glycine,
alanine, cysteine, tyrosine, histidine, arginine and proline (Table 2).
Their levels at the G120 stage were 3.13, 9.02, 1.93, 22.82, 9.34, 3.03,
17.68, 8.86, 8.96 and 11.97 times higher than those at the G36 stage,
respectively
3.3. Quality control of the proteome data of germinating Chinese wild rice

3. Results
3.1. Changes in the free, bound and total phenolics content in Chinese wild
rice during germination
The changes in free, bound and total phenolics content in germi-
nating Chinese wild rice are presented in Table 1. They initially de-
clined, and then increased (Table 1). From the G0 to G36 stages, the
free phenolics content gradually decreased from 1012.17 to 792.43 mg
GAE/kg. From the G36 to G120 stages, the free phenolics content
gradually increased from 792.43 to 1483.81 mg GAE/kg with a peak at
the G120 stage. The free phenolics content at the G120 stage was 0.87
times higher than that at the G36 stage. From the G0 to G48 stages, the
bound phenolics content gradually decreased from 318.79 to 201.10 mg
GAE/kg. From the G48 to G120 stages, the bound phenolics content
gradually increased from 201.10 to 617.11 mg GAE/kg with a peak at
the G120 stage. From the G0 to G36 stages, the total phenolics content
gradually decreased from 1330.96 to 1068.25 mg GAE/kg. From the
G36 to G120 stages, the total phenolics content gradually increased
from 1068.25 to 2100.92 mg GAE/kg with a peak at the G120 stage.
The total phenolics content at the G120 stage was 0.97 times higher
than that at the G36 stage. Therefore, the greatest difference in the total
phenolics content was found between the G36 and G120 stages.
In the present study, 531,964 total spectra, 33,130 peptides and
7031 proteins were identified in germinating Chinese wild rice. Mass
spectrometry data must be subjected to quality control checks, in-
cluding peptide length distribution, precursor ion tolerance, unique
peptide number, protein coverage and protein mass. A detailed quality
control report is shown in Fig. S2. Each MS has its measurement range.
Therefore, determination of the length of identified peptides was lim-
ited to some extent. If the peptide was too short or long, it would escape
detection by the MS. In the present study, the range of peptide length
was 6–25 amino acids, which is characteristic of regular complex
samples (Fig. S2A). The molecular weight of the precursor ion (first-
order MS; peptide fragment ion) detected by the MS has a certain bias
compared to the actual molecular weights of peptides. This property is
fixed in a mass spectrometer, and is used to evaluate the performance of
the device and serves as a reference to determine the quality of results.
In the present study, the distribution of the precursor ion tolerance peak
tended to be close to 0; therefore, the mass bias was relatively small
(Fig. S2B). The peptides and proteins were identified by comparison
with a protein database. Proteins containing exactly the same peptides
were placed in the same protein group. Each group had its unique set of
peptides endowing it with specific properties. Protein detection relia-
bility tended to improve with the number of unique peptides in a
protein group. In the present study, the distribution curve of the unique

Table 1
Changes in the free, bound and total phenolics content in Chinese wild rice during germination.

Germination stage Phenolics (mg GAE/kg)a

Free phenolics Bound phenolics Total phenolics

G0 1012.17 ± 44.09de 318.79 ± 27.57 cd 1330.96 ± 70.59de

G12 999.76 ± 39.36def 277.53 ± 4.22de 1277.28 ± 38.44def
G24 949.58 ± 15.40ef 277.39 ± 27.66de 1226.97 ± 40.26ef
G36 792.43 ± 15.04 g 275.82 ± 21.76de 1068.25 ± 35.75 h
G48 912.39 ± 10.93f 201.10 ± 10.66f 1113.48 ± 15.26gh
G60 971.22 ± 20.11ef 230.83 ± 17.48ef 1202.05 ± 32.16 fg
G72 1067.08 ± 39.83d 315.96 ± 1.78 cd 1383.04 ± 38.10d
G84 1160.13 ± 71.09c 359.56 ± 24.36c 1519.69 ± 82.58c
G96 1301.38 ± 26.94b 469.95 ± 45.31b 1771.33 ± 44.95b
G108 1411.07 ± 46.63a 595.45 ± 58.78a 2006.52 ± 77.51a
G120 1483.81 ± 119.29a 617.11 ± 32.00a 2100.92 ± 126.14a

a
GAE, gallic acid equivalents.

638
 Table 2
Changes in the content of free amino acids (FAAs) in Chinese wild rice during germination.
C. Chu, et al.

FAAs (μg/g) Germination stage

G0 G12 G24 G36 G48

Aspartate 55.44 ± 6.44 h 64.19 ± 3.92 h 113.97 ± 2.26 g 164.90 ± 8.08f 194.67 ± 11.97f
Threonine 26.81 ± 2.02 h 39.57 ± 4.27gh 45.00 ± 1.50gh 59.12 ± 6.31gh 78.55 ± 5.31 g
Serine 100.58 ± 3.47 g 98.07 ± 2.45 g 106.77 ± 10.12 g 162.14 ± 7.69 g 232.38 ± 15.21f
Glutamate 604.50 ± 26.98e 341.09 ± 7.99 g 479.33 ± 47.62f 581.90 ± 11.34e 655.72 ± 42.05e
Glycine 14.24 ± 0.54 g 14.95 ± 0.65 g 19.07 ± 1.17 g 22.64 ± 0.45 g 33.63 ± 2.18 g
Alanine 85.58 ± 1.95ij 50.80 ± 1.91j 131.60 ± 5.87hi 205.70 ± 3.64gh 261.04 ± 21.69 g
Valine 88.69 ± 2.20 h 90.44 ± 2.31 h 92.25 ± 4.47 h 142.01 ± 6.32gh 211.04 ± 13.98 g
Cysteine 16.06 ± 0.26 h 28.44 ± 0.70 g 51.61 ± 0.37f 52.18 ± 0.74f 54.06 ± 5.13f
Methionine 10.65 ± 0.32 g 11.84 ± 0.28 g 16.08 ± 0.51 g 22.49 ± 0.53 g 42.53 ± 5.47 g
Isoleucine 9.45 ± 0.58 h 13.34 ± 0.26 h 24.26 ± 0.75 h 46.39 ± 2.18gh 101.11 ± 10.01 g
Leucine 32.49 ± 0.90 h 36.38 ± 1.27 h 50.86 ± 1.84 h 119.65 ± 3.55gh 197.40 ± 16.87 g
Tyrosine 48.80 ± 3.22 h 49.84 ± 3.78 h 50.41 ± 0.86 h 86.59 ± 2.83gh 144.44 ± 18.33 g
Phenylalanine 21.38 ± 0.32 g 32.11 ± 0.70 fg 35.55 ± 1.41 fg 50.76 ± 4.16 fg 127.66 ± 19.94f
γ-Aminobutyric acid 75.82 ± 1.14 h 80.37 ± 2.71 h 88.10 ± 3.23 h 176.67 ± 4.85 g 228.92 ± 16.83 g
Lysine 73.35 ± 4.40 h 81.71 ± 3.16 h 88.21 ± 1.14 h 117.68 ± 17.94gh 151.36 ± 14.03 g
Histidine 113.70 ± 13.68 h 99.47 ± 7.76 h 112.71 ± 10.96 h 151.74 ± 12.49 h 211.09 ± 7.27 g
Arginine 320.21 ± 17.24 g 270.43 ± 2.13 g 287.61 ± 11.21 g 291.07 ± 2.19 g 341.44 ± 4.81 g
Proline 5.04 ± 0.35i 14.59 ± 0.33i 48.85 ± 0.91 h 68.96 ± 4.13 h 132.19 ± 10.16 g

FAAs (μg/g) Germination stage

G60 G72 G84 G96 G108 G120

639
Aspartate 331.64 ± 19.88e 449.89 ± 9.88d 522.58 ± 16.56c 611.96 ± 1.15b 627.87 ± 66.22b 681.15 ± 22.46a
Threonine 176.44 ± 7.36f 301.87 ± 1.18e 426.16 ± 7.60d 601.21 ± 34.05c 658.66 ± 59.76b 922.84 ± 44.80a
Serine 468.37 ± 28.25e 733.50 ± 5.35d 963.28 ± 37.11c 1259.74 ± 18.39b 1319.49 ± 102.81b 1624.97 ± 35.66a
Glutamate 818.22 ± 42.62d 1007.69 ± 41.19c 1088.65 ± 38.92c 1260.75 ± 40.47b 1333.78 ± 104.84b 1703.43 ± 134.50a
Glycine 82.57 ± 9.10f 159.84 ± 2.92e 244.55 ± 15.68d 365.61 ± 32.05c 408.07 ± 25.82b 539.18 ± 68.10a
Alanine 398.91 ± 19.44f 615.74 ± 43.76e 836.87 ± 55.20d 1220.79 ± 79.11c 1347.11 ± 17.90b 2127.57 ± 106.76a
Valine 442.87 ± 35.36f 779.33 ± 10.28e 1101.86 ± 31.06d 1514.85 ± 55.27c 1657.37 ± 143.34b 2227.74 ± 80.03a
Cysteine 60.68 ± 1.65ef 70.64 ± 6.29e 97.55 ± 5.49d 131.20 ± 6.24c 151.14 ± 17.53b 210.54 ± 9.01a
Methionine 95.87 ± 7.92f 184.25 ± 6.67e 270.77 ± 2.79d 389.67 ± 11.12c 437.59 ± 40.12b 598.56 ± 37.40a
Isoleucine 274.88 ± 8.30f 476.32 ± 3.04e 664.19 ± 20.17d 927.53 ± 33.96c 1009.50 ± 80.53b 1372.13 ± 49.83a
Leucine 520.68 ± 26.17f 854.73 ± 12.48e 1139.71 ± 30.84d 1561.79 ± 37.58c 1690.87 ± 110.24b 2101.20 ± 123.57a
Tyrosine 345.82 ± 28.35f 591.72 ± 10.63e 807.64 ± 21.91d 1152.76 ± 47.91c 1229.94 ± 100.18b 1617.93 ± 19.98a
Phenylalanine 294.17 ± 24.00e 601.55 ± 10.70d 743.36 ± 22.84c 1031.56 ± 16.63b 1126.82 ± 75.33b 1470.15 ± 166.12a
γ-Aminobutyric acid 399.16 ± 15.86f 524.54 ± 28.83e 706.43 ± 10.50d 930.50 ± 85.22c 1172.25 ± 10.47b 1465.21 ± 81.00a
Lysine 319.64 ± 23.08f 542.76 ± 8.29e 811.45 ± 10.23d 1138.09 ± 10.39c 1251.98 ± 98.90b 1654.34 ± 47.86a
Histidine 367.59 ± 29.87f 566.54 ± 15.73e 779.71 ± 10.46d 1038.35 ± 31.50c 1132.43 ± 97.94b 1495.53 ± 24.34a
Arginine 591.12 ± 49.86f 1002.12 ± 3.49e 1422.67 ± 42.49d 1989.42 ± 97.52c 2166.56 ± 148.54b 2899.51 ± 134.45a
Proline 211.48 ± 18.00f 330.85 ± 8.33e 436.96 ± 11.41d 586.73 ± 40.91c 645.68 ± 34.18b 894.65 ± 28.92a
Food Chemistry 289 (2019) 635–644
C. Chu, et al.
number of peptides gradually increased. Therefore, the number of both
unique peptides and reliable proteins was relatively large (Fig. S2C).
The reliability of a detected protein improves with the number of
peptides supporting it. In this study, the protein coverage distribution
was relatively wide. Therefore, the overall accuracy of the detection
result was comparatively high (Fig. S2D). Protein mass distribution is
important in evaluating protein size. In the present study, the range of
both the protein mass distribution and detected proteins was relatively
wide (Fig. S2E).
3.4. Protein function annotation of germinating Chinese wild rice
Gene Ontology is an internationally standardised classification
system for gene function description (Ashburner et al., 2000). In the
present study, GO annotated 4850 proteins. The results are shown in
Table S1. The COG database was built using the classifications of the
phylogenetic relationships among the proteins encoded by complete
bacterial, algal and eukaryotic genomes (Tatusov et al., 2003). The
sequence of a certain protein can be annotated to a certain COG. Each
COG cluster was established on direct homologous sequences from
which the sequence function could be inferred. In this study, there were
4044 proteins annotated to the COG database. The results are presented
in Table S2. The KEGG is the main public pathway database and is used
to determine the main metabolic and signal transduction pathways
(Kanehisa et al., 2015). In the present study, 7013 proteins were an-
notated to the KEGG database. The results are shown in Table S3. In-
terProScan software, developed by the European Bioinformatics In-
stitute, is commonly used for domain and function annotation. It is a
non-redundant database that collects protein families, domains and
functional sites (Finn et al., 2016). In the present study, IPR annotated
6418 proteins. The results are presented in Table S4.
The annotation results of GO, COG, KEGG and IPR are presented in
Fig. S3. Among the proteins identified in germinating Chinese wild rice,
68.98% (4850 of 7031), 57.52% (4044 of 7031), 99.74% (7013 of
7031) and 91.28% (6418 of 7031) were annotated by GO, COG, KEGG
and IPR, respectively (Fig. S3). Therefore, KEGG annotated the highest
number of proteins, followed by IPR and GO, with COG annotating the
fewest. Moreover, only 46.17% (3246 of 7031) of the proteins identi-
fied in germinating Chinese wild rice were annotated in all four data-
bases (Fig. S3).
3.5. Protein differential analysis in Chinese wild rice during germination
Among the 7031 proteins identified in germinating Chinese wild
rice, 7023 were detected in both the G36 and G120 stages (Table S5).
Therefore, upregulation and downregulation of the eight proteins that
are not common to both the G36 and G120 stages could not be de-
termined. For each DEP in the G120 versus G36 stage, the logarithm of
FC was calculated to the base 2, and the logarithm of P-value was
calculated to the base 10. These calculated data were used to plot a DEP
volcano of the G120 versus G36 stage (Fig. 1A). The horizontal axis
shows the FC of each protein in the G120 versus G36 stage (log2 value)
and the vertical axis represents P (-log10 value). The black, red and
green colours represent the unchanged, upregulated and down-
regulated proteins, respectively. In the present study, there were 1144
DEPs between the G36 and G120 stages. Relative to that in the G36
stage, there were 956 upregulated and 188 downregulated proteins in
the G120 stage (Fig. 1A, Table S5). We conducted a cluster analysis on
the expression levels of DEP in the G120 versus G36 stage. We used a
cluster heatmap to compare protein upregulation and downregulation
between the G36 and G120 stages. The results are shown in Fig. 1B. The
red and blue segments indicate relatively high and low protein content,
respectively. A hierarchical cluster analysis of the DEPs at the two
stages showed a clear grouping pattern (Fig. 1B). The expression levels
of DEP in the G120 versus G36 stage, the FC, P-value, and the upre-
gulated and downregulated proteins are presented in Table S6.
640
Food Chemistry 289 (2019) 635–644
3.6. Gene Ontology term enrichment analysis of differentially expressed
proteins in Chinese wild rice during germination
The results of the GO term enrichment analysis of the DEPs between
the G36 and G120 stages are shown in Table S7. A bar graph of the GO
enrichment terms is presented in Fig. S4. Under the ‘biological process’
category, ‘response to oxidative stress’ (GO: 0006979), ‘response to
stress’ (GO: 0006950), ‘photosynthesis’ (GO: 0015979), ‘response to
stimulus’ (GO: 0050896), ‘oxidation-reduction process’ (GO: 0055114)
and ‘metabolic process’ (GO: 0008152) were significantly enriched
(adjusted P ≤ 0.05). There were 40, 60, 15, 71, 116 and 437 DEPs in
each GO term category, respectively (Table S7). Under the ‘cellular
component’ category, ‘photosystem II’ (GO: 0009523), ‘thylakoid’ (GO:
0009579), ‘photosystem II oxygen evolving complex’ (GO: 0009654),
‘photosystem’ (GO: 0009521) and ‘extrinsic component of membrane’
(GO: 0019898) were significantly enriched (adjusted P ≤ 0.05). There
were 9, 12, 8, 11 and 7 DEPs in each GO term category, respectively
(Table S7). Under the ‘molecular function’ category, ‘oxidoreductase
activity, acting on peroxide as acceptor’ (GO: 0016684), ‘antioxidant
activity’ (GO: 0016209), ‘peroxidase activity’ (GO: 0004601), ‘haem
binding’ (GO: 0020037), ‘nutrient reservoir activity’ (GO: 0045735),
‘oxidoreductase activity’ (GO: 0016491) and ‘FMN binding’ (GO:
0010181) were significantly enriched (adjusted P ≤ 0.05). There were
44, 44, 41, 47, 18, 102 and 10 DEPs in each GO term category, re-
spectively (Table S7). Therefore, the GO term enrichment analysis in-
dicated that the DEPs between the G36 and G120 stages were mainly
related to metabolic processes (437 DEPs), oxidation-reduction pro-
cesses (116 DEPs), oxidoreductase activity (102 DEPs), response to
stimulus (71 DEPs), response to stress (60 DEPs) and haem binding (47
DEPs).
3.7. Kyoto Encyclopaedia of Genes and Genomes pathway enrichment
analysis of differentially expressed proteins in Chinese wild rice during
germination
The KEGG pathway enrichment analysis (Yan et al., 2017, 2019)
determined the main metabolic and signal transduction pathways of the
participating DEPs between the G36 and G120 stages. The enrichment
result of the KEGG Pathway in the G120 versus G36 stage is presented
in Table S8. A bubble chart of the KEGG Pathway was also plotted. It
shows only the top 20 pathways and is presented in Fig. 2. The P-values
for ‘phenylpropanoid biosynthesis’, ‘biosynthesis of secondary meta-
bolites’, ‘photosynthesis’, ‘metabolic pathways’, ‘carbon fixation in
photosynthetic organisms’ and ‘linoleic acid metabolism’ were < 0.01.
Therefore, these pathways were significantly enriched for the DEPs
between the G36 and G120 stages (Fig. 2, Table S8). The P-values for
‘alanine, aspartate and glutamate metabolism’ and ‘taurine and hypo-
taurine metabolism’ ranged between 0.01 and 0.05. Therefore, these
pathways were relatively enriched for DEPs between the G36 and G120
stages (Fig. 2, Table S8). The KEGG pathway enrichment analysis in-
dicated that the DEPs between the G36 and G120 stages were mainly
related to metabolic pathways (245 DEPs), biosynthesis of secondary
metabolites (165 DEPs) and phenylpropanoid biosynthesis (46 DEPs).
3.8. Analysis of the proteins involved in phenolic biosynthesis in Chinese
wild rice during germination
The proteins involved in phenolic biosynthesis between the G36 and
G120 stages are shown in Table 3. Four PALs, one 4-coumarate-CoA
ligase (4CL), one ferulate-5-hydroxylase (F5H), one cinnamoyl-CoA
reductase (CCR) and two cinnamyl-alcohol dehydrogenases (CADs)
were identified as DEPs between the G36 and G120 stages (Fig. S5).
Relative to those in the G36 stage, four PALs (Zlat_10020199,
Zlat_10028346, Zlat_10028349 and Zlat_10020197), one 4CL
(Zlat_10028742), one CCR (Zlat_10027807) and two CADs
(Zlat_10022488 and Zlat_10011680) were significantly upregulated
C. Chu, et al. Food Chemistry 289 (2019) 635–644

(FC ≥ 1.5, P ≤ 0.05), and one F5H (Zlat_10030721) was significantly

downregulated (FC ≤ 0.67, P ≤ 0.05) in the G120 stage (Table 3).
Furthermore, we detected the PAL activity in Chinese wild rice during
germination to explain the higher expression of these four PALs at the
G120 than at the G36 stage. As expected, the PAL activity at the G120
stage was significantly higher than that at the G36 stage (P < 0.05)
(Fig. S6).

3.9. Analysis of proteins involved in γ-aminobutyric acid biosynthesis in

Chinese wild rice during germination

GABA accumulated in germinating Chinese wild rice (Table 2). The

proteins involved in GABA biosynthesis between the G36 and G120
stages are shown in Table 4. In the present study, four glutamate dec-
arboxylases (GADs) were identified as DEPs between the G36 and G120
stages. Relative to those in the G36 stage, four GADs (Zlat_10003612,
Zlat_10000241, Zlat_10031009 and Zlat_10002989) were significantly
upregulated in the G120 stage (FC ≥ 1.5, P ≤ 0.05) (Table 4).

4. Discussion

4.1. Changes in the content of phenolics and free amino acids in Chinese
wild rice during germination

In the present study, the content of free, bound and total phenolics
in germinating Chinese wild rice initially decreased, and then increased
(Table 1). From the G0 to G36 stages, the content of free phenolics
gradually decreased possibly because water-soluble polyphenol com-
pounds are lost during seed immersion and imbibition (Wang et al.,
2016). For example, the anthocyanins in black rice are unstable water-
soluble phenolics easily degraded by changes in pH, temperature and
other physicochemical factors in the environment (Hiemori, Koh, &
Mitchell, 2009). From stages G48 to G120, the content of free, bound
and total phenolics gradually increased. Similar observations have been
reported in germinating brown rice (Ti et al., 2014) and canary seed
(Chen, Yu, Wang, Gu, & Beta, 2016). On one hand, the increase in free
phenolics during germination is attributed to de novo synthesis and
transformation via phenylpropanoid biosynthesis and other metabolic
pathways (Gan et al., 2017; Tang, Dong, Guo, Li, & Ren, 2014). On the
other hand, as germination time increases, new plant cells form and the
free phenolics are secreted to the cell walls to form new bound phe-
nolics (Gan et al., 2017).
GABA is an important neurotransmitter that lowers blood pressure,
regulates arrhythmia, alleviates anxiety and controls hormone secretion
(Diana, Quílez, & Rafecas, 2014; Gan et al., 2017). In the present study,
from the G0 to G120 stages, the GABA content gradually increased. At
the G120 stage, it was 18.32 times higher than that at the G0 stage
(Table 2). GABA is synthesised primarily from L-glutamic acid via GAD,
a pyridoxal 5′-phosphate-dependent enzyme (Gan et al., 2017; Ramesh,
Tyerman, Gilliham, & Xu, 2017). In the present study, the expression of
four GADs (Zlat_10003612, Zlat_10000241, Zlat_10031009 and Fig. 1. Differentially expressed proteins (DEPs) in Chinese wild rice between
Zlat_10002989) at the G120 stage was significantly higher than that at the 36-h germination (G36) and 120-h germination (G120) stages. (A) Volcano
the G36 stage (Table 4). From the G12 to G120 stages, the glutamate plot of the DEPs between the G36 and G120 stages. (B) Hierarchical clustering
content gradually increased (Table 2). Therefore, the increased ex- analysis of the DEPs between the G36 and G120 stages. Colours of the scatter
pression of the four GADs at the G120 stage and the gradual accumu- points in Fig. 1A indicate the final screening results. Red indicates significantly
lation of glutamate resulted in the accumulation of GABA in Chinese upregulated proteins. Green indicates significantly downregulated proteins.
Black indicates proteins whose levels did not significantly differ between the
wild rice during germination.
two stages. In Fig. 1B, the horizontal axis represents the sample clusters. Si-
FAAs are nitrogenous nutrients directly absorbed by the human
milarity increases with decreasing cluster length. The clusters on the vertical
body. The content and composition ratio of FAAs reflect food quality axis indicate the expression modes of the clusters of proteins between the G36
(Egydio, Catarina, Floh, & dos Santos, 2013). Of the 18 FAAs analysed and G120 stages. (For interpretation of the references to colour in this figure
in the present study, the glutamate content varied the least, whereas the legend, the reader is referred to the web version of this article.)
change in proline content was the highest. The glutamate and proline
content at the G120 stage was 1.82 and 176.51 times higher than that at
threonine (34.42 times), glycine (37.86 times), methionine (56.20
the G0 stage, respectively. In addition, the FC of the content of FAAs
times), leucine (64.67 times), phenylalanine (68.76 times) and iso-
between the G120 and G0 stages as ˃ 20× for lysine (22.55 times),
leucine (145.20 times) (Table 2). During seed germination, proteins are
alanine (24.86 times), valine (25.12 times), tyrosine (33.15 times),

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C. Chu, et al. Food Chemistry 289 (2019) 635–644

Fig. 2. Results of the Kyoto Encyclopaedia

of Genes and Genomes (KEGG) pathway
enrichment analysis of differentially ex-
pressed proteins (DEPs) in Chinese wild rice
between the 36-h germination (G36) and
the 120-h germination (G120) stages. In
Fig. 2, the horizontal axis represents the
ratios of the number of DEPs in the corre-
sponding pathways to the number of pro-
teins detected. The DEP enrichment level in
each pathway increased with ratio magni-
tude. Dot colour represents the P-value
verified by a hypergeometric test. Test re-
liability and statistical significance in-
creased with decreasing P-value. Dot size
represents the number of DEPs in the cor-
responding pathway. The number of DEPs
in the pathway increased with the total
number of DEPs.

enzymatically hydrolysed into low-molecular-weight peptides and
amino acids. Therefore, new small molecules are produced (Wang et al.,
2016) and the content of FAAs increases during germination. Notably,
the arginine, valine, alanine and leucine levels at the G120 stage were
2899.51, 2227.74, 2127.57 and 2101.20 μg/g, respectively.
4.2. Changes in the proteome of Chinese wild rice during germination
To the best of our knowledge, this study is the first to apply iTRAQ-
based proteomics to reveals the accumulation of bioactive compounds
in Chinese wild rice during germination. Two-dimensional electro-
phoresis has been used to analyse the DEPs between Chinese wild rice
and Indica rice (Jiang et al., 2016) and to evaluate the proteome var-
iations associated with the localised formation of smut-gall during Z.
latifolia–Ustilago esculenta interaction (Jose, Bengyella, Handique, &
Talukdar, 2019). However, only a few proteins have been identified
from Z. latifolia in previous studies (Jiang et al., 2016; Jose et al., 2019).
In the present study, 7031 proteins were detected in germinating Chi-
nese wild rice. The GO, COG, KEGG and IPR databases annotated 4850,
4044, 7013 and 6418 proteins, respectively (Table S2–S5). Moreover,
there were 1,144 DEPs between the G120 and G36 stages. Of these, 956
were upregulated and 188 were downregulated (Fig. 1, Table S5). The
hierarchical cluster analysis of the DEPs suggested that those in the G36
and G120 stages had distinctly different grouping patterns (Fig. 1, Table

Table 3
Relative differences in the expression levels of the proteins involved in phenolic biosynthesis between the 36-h germination (G36) and 120-h germination (G120)
stages.

Protein ID Description Abbreviation Fold Change (G120 versus G36) P-value

Zlat_10020199 Phenylalanine ammonia-lyase PAL 1.66 4.62E−03

Zlat_10028346 Phenylalanine ammonia-lyase PAL 1.76 4.51E−03
Zlat_10028349 Phenylalanine/tyrosine ammonia-lyase PAL 1.77 2.62E−03
Zlat_10020197 Phenylalanine/tyrosine ammonia-lyase PAL 1.51 3.61E−04
Zlat_10028742 4-coumarate-CoA ligase 4CL 1.57 1.25E−05
Zlat_10030721 Ferulate-5-hydroxylase F5H 0.55 5.61E−04
Zlat_10027807 Cinnamoyl-CoA reductase CCR 1.99 7.08E−03
Zlat_10022488 Cinnamyl-alcohol dehydrogenase CAD 1.52 5.24E−03
Zlat_10011680 Cinnamyl-alcohol dehydrogenase CAD 1.52 2.09E−02

642
C. Chu, et al.
Table 4
Relative differences in the expression levels of the proteins involved in γ-aminobutyric acid (GABA) biosynthesis between the 36-h germination (G36) and 120-h
germination (G120) stages.
Protein ID Description Abbreviation
Zlat_10003612 Glutamate decarboxylase GAD
Zlat_10000241 Glutamate decarboxylase GAD
Zlat_10031009 Glutamate decarboxylase GAD
Zlat_10002989 Glutamate decarboxylase GAD
S6). Therefore, proteins with similar sequence and expression char-
acteristics are often functionally similar. The KEGG pathway enrich-
ment analysis indicated that the DEPs between the G36 and G120 stages
were associated mainly with metabolic pathways and the biosynthesis
of secondary metabolites and phenylpropanoids (Fig. 2, Table S8).
4.3. Changes in the expression levels of the proteins involved in phenolic
biosynthesis in Chinese wild rice during germination
Phenolic compounds in plants include phenolic acids, flavonoids,
condensed tannins, lignins, and lignans (Soto-Vaca, Gutierrez, Losso,
Xu, & Finley, 2012). Phenolic acids are a class of secondary metabolites
containing phenolic hydroxyl and carboxyl groups. They are widely
distributed in higher plants and are bound via ester and ether linkages
to the structural components of the cell walls in plant food materials
(Shao & Bao, 2015; Vanholme, Demedts, Morreel, Ralph, & Boerjan,
2010). Phenolic acids have antioxidant, antimicrobial, anti-in-
flammatory and anticancer activities. They have gained wide interest in
recent years and are major hotspots in food research (Heleno, Martins,
Queiroz, & Ferreira, 2015). The phenolic acids and their derivatives
identified in Chinese wild rice were gallic acid, protocatechuic acid, p-
hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, syringic
acid, vanillin, protocatechuic acid ethyl ester, p-coumaric acid, o-cou-
maric acid, ferulic acid, sinapic acid, 3,4,5-trimethoxycinnamic acid
and dihydroferulic acid 4-O-glucuronide (Chu et al., 2018). Phenyla-
lanine ammonia-lyase is a key and rate-limiting enzyme involved in
phenolic acid biosynthesis (Fig. S5). It transforms L-phenylalanine into
cinnamic acid (Shao & Bao, 2015; Vanholme et al., 2010). In the pre-
sent study, the expression of four PALs (Zlat_10020199, Zlat_10028346,
Zlat_10028349 and Zlat_10020197) at the G120 stage was significantly
higher than that at the G36 stage (Table 3). Notably, these two PALs
(Zlat_10028349 and Zlat_10020197) also had the function of tyrosine
ammonia-lyase, which can directly convert L-tyrosine to p-coumaric
acid without the non-oxidative deamination of cinnamic acid-4-hy-
droxylase in plants (Rosler, Krekel, Amrhein, & Schmid, 1997). From
the G0 to G120 stages, the phenylalanine and tyrosine content gradu-
ally increased (Table 2). Therefore, the increased expression of the four
PALs at the G120 stage and the gradual accumulation of phenylalanine
accounted for the accumulation of phenolics in Chinese wild rice during
germination (Table 1).
Except for four PALs, the DEPs involved in phenolic biosynthesis
between the G36 and G120 stages are one 4CL, one F5H, one CCR and
two CADs (Table 3). The enzyme 4CL converts 4-coumaric acid, caffeic
acid, ferulic acid, 5-hydroxyferulic acid and sinapic acid into hydro-
xycinnamoyl-CoA esters (Shao & Bao, 2015). The hydroxycinnamoyl
CoA esters are then converted by CCR into hydroxycinnamaldehydes,
which in turn are converted by CAD into hydroxycinnamyl alcohols
(Fig. S5). In the present study, the expression of one 4CL
(Zlat_10028742), one CCR (Zlat_10027807) and two CADs
(Zlat_10022488, and Zlat_10011680) at the G120 stage was sig-
nificantly higher than that at the G36 stage (Table 3). Therefore, the
upregulation of these enzymes promoted the accumulation of phenolics
in Chinese wild rice during germination (Table 1). F5H forms 5-hy-
droxyferulic acid from ferulic acid through C-5 hydroxylation. This step
is critical in the reciprocal transformation between hydroxycinnamic
643
Food Chemistry 289 (2019) 635–644
Fold Change (G120 versus G36) P-value
1.77 2.85E−05
2.25 2.35E−04
2.02 1.01E−03
1.85 3.15E−03
acids (Shao & Bao, 2015; Vanholme et al., 2010). In the present study,
the expression of one F5H (Zlat_10030721) significantly decreased at
the G120 stage relative to that at the G36 stage (Table 3). Therefore,
reciprocal transformation between phenolic acid monomers might
occur during Chinese wild rice germination, and future studies should
endeavour to elucidate these dynamic changes.
5. Conclusions
During Chinese wild rice germination, the content of free, bound
and total phenolics initially declined, and then increased, and the
content of GABA and 17 FAAs increased. To the best of our knowledge,
this study is the first to apply iTRAQ-based proteomics to reveal the
accumulation of these bioactive compounds in Chinese wild rice during
germination. The iTRAQ analysis revealed 7031 proteins from germi-
nating Chinese wild rice and the GO, COG, KEGG and IPR databases
annotated 4850, 4044, 7013 and 6418 of them, respectively. A com-
parison of the G120 and G36 stages revealed 956 upregulated and 188
downregulated proteins. The KEGG pathway analysis of the DEPs be-
tween the G36 and G120 stages revealed significant enrichment of
proteins of the “metabolic pathways”, “biosynthesis of secondary me-
tabolites”, and “phenylpropanoid biosynthesis” categories. The hier-
archical cluster analysis revealed that 1144 DEPs showed a distinct
grouping pattern. Of these, the expression of four PALs, one 4CL, one
CCR, two CADs and four GADs was significantly higher at the G120
stage than at the G36 stage. Consequently, these upregulated enzymes
promoted the accumulation of phenolics and GABA during germination.
Overall, the present study elucidated the biochemical and functional
change patterns and their mechanisms in germinating Chinese wild rice
and may facilitate the development of functional foods derived from it.
Acknowledgements
This work was supported by the Science Foundation for Young
Scholars of the Tobacco Research Institute of the Chinese Academy of
Agricultural Sciences (Grant No. 2018A01), the Fundamental Research
Funds for the Central Non-Profit Scientific Institution (Grant No.
1610232018003), and the Agricultural Science and Technology
Innovation Program (Grant No. ASTIP-TRIC05).
Conflict of interest statement
The authors declare no conflicts of interest.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodchem.2019.03.092.
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