Two-dimensional Electrophoresis Analysis of Proteins in Response to Cold
Stress in Extremely Cold-resistant Winter Wheat Dongnongdongmai 1
Tillering Nodes
Cang Jing1, Yu Jing1, Liu Li-jie1, 2, Yang Yang1, Cui Hong1, Hao Zai-bin1, and Li Zhuo-fu3
1
College of Life Sciences, Northeast Agricultural University, Harbin, 150030, China
College of Communication and Electronic Engineering, Qiqihar University, Qiqihar 161006, Heilongjiang, China
3
College of Agriculture, Northeast Agricultural University, Harbin, 150030, China
2
Abstract: The overwintering survival ratio of the cultivar Dongnongdongmai 1 with strong cold-resistance in paramos of
Heilongjiang Province in China are over 85%. The tillering nodes are the most important organs for overwintering survival of winter
wheat, because there are more substances associated with cold resistance in tillering nodes than those in leaves and roots. Proteins in
the tillering nodes of the cold-resistant cultivar Dongnongdongmai 1 grown under field conditions with or without any
low-temperature stress were analyzed by 2-dimensional electrophoresis and identified by mass spectrometry. In the range of pH 4-7,
the expression of 37 proteins showed obvious difference (±more than two fold) in the proteomic maps of cold-stressed and
non-stressed tillering nodes, including a new protein spot. All proteins exhibiting the difference in expression were identified using
matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, followed by a database search for protein identification
and function prediction. Five groups of proteins were confirmed, namely stress-related proteins (22%), metabolism-associated
proteins (35%), and signaling molecules (24%), cell wall-binding proteins (5%), unclear proteins (14%). This indicated that
tillering node cells supported the energy requirements of plant growth and stress resistance by signal transduction adapting to
metabolism and structure.
Key words: Dongnongdongmai 1, proteome, two-dimensional electrophoresis, peptide mass fingerprinting
CLC number: S512.1
Document code: A
Article ID: 1006-8104(2012)-01-0027-09
Introduction
Low temperature is a serious abiotic stress that limits productivity of agricultural crops. Many countries and
regions in the world that suffer a severe winter have to adopt a 'one crop per annum' cropping system, which
results in under-utilization of cultivated land. Therefore, the breeding of cold-resistant crop cultivars or increasing
the multiple cropping index of cultivable land are the important strategies to increase crop productivity. Plants
have the potential capability to adapt to adverse environment; for example, in low temperature, synthesis of
stress-related proteins can be induced, soluble sugar and peroxide accumulate, membrane fluidity changes, the
water content of tissues decreases, and the activity of protective enzymes is enhanced (Gana et al., 1997; Minami
et al., 2005). Previous studies have explored physiological and biochemical metabolic aspects, molecular
mechanisms and morphological development of cold stress. In view of the previous work, we investigated
physiological indices of cold resistance in the winter wheat cultivar Dongnongdongmai 1 in a cold-winter area
before overwintering and found that soluble protein content increased significantly following the decrease in
temperature (Yu et al., 2008). Although previous studies had investigated plants' cold resistance at the protein
level (Yin, 2005; Yi, 2006; Tai et al., 2008) the proteomic changes differed among different crops and tissues. In
addition, the changes in protein abundance in winter wheat in response to low-temperature stress have not been
examined using two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). In this paper, proteomic
change in tillering nodes of Dongnongdongmai 1 in response to cold stress was examined by means of 2-DE and
MS analysis. Tillering nodes were used as experimental materials, because they had a high ability to store
cold-resistance-related compound (Li, 2006).
1
1
Received 31 March 2011
Supported by Funding (Topic CXZ003) from the New Ideas Team and the Doctoral Research Foundation of Northeast Agricultural University (2008;
2010); The Scientific Research Fund of the Heilongjiang Provincial Education Department (11551067)
Cang Jing (1963-), female. Ph. D, professor, engaged in the research of plant physiology and molecular biology. E-mail: [email protected]
Materials and Methods
Plant materials and field experiment
Winter wheat (Triticum aestivum L.) cultivar Dongnongdongmai 1 was used in the experiment (an extremely
cold-resistant cultivar with 85% overwintering survival ratio in field even under –30℃ in winter and the only
winter wheat variety grown in Heilongjiang, China), and it was obtained from the Wheat Breeding Institue of
Northeast Agricultural University, Harbin, China. The field trial was carried out in autumn, 2007 (September 9th),
at Northeast Agricultual University's Xiangfang Experimental Site in Harbin, China (45°7'N, 126°6'E).
Conventional fertilization and field management practices were used. The base fertilizer was applied with 21 g •
m-2 nitrogen and 10 g • m-2 phosphorus. The cultivar was planted in multi-row (eight rows) plots with three
replications. The rows were 4 m long and spaced 0.2 m apart with 400 seeds in each row. The seeds were sown in
5 cm in depth, and then watered with 8 L running water. The weather temperature was recorded from trefoil stage.
Seedlings were subjected to cold stress with the decrease in temperature under natural conditions, tillering nodes
of each treatment were collected when the temperature was 5℃ and lasted for 5 days (designated the control),
and when the environmental temperature decreased to –30℃ and was lasted 5 days (designated the cold stress
treatment). After collection, samples were immediately frozen in liquid nitrogen, and stored at –80℃ until use.
Protein extraction and two-dimensional elec-trophoresis
Total soluble proteins were extracted from tillering nodes with phenol and precipitated with ammonium
acetate-methanol following the method described by Yu et al (2009). A clean mortar was chilled at –20℃ and
precooled for 10 min with liquid nitrogen. One gram of tillering node tissue was ground in the mortar for 30 min
with liquid nitrogen, and then 5 mL Tris-equilibrium phenol was added and extracted in an ice bath in a fume
cupboard. The solution was transferred to a 50 mL centrifuge tube that was precooled with liquid nitrogen. The
procedure was repeated and the extraction solution combined and thoroughly shaken with a shaking table rejoined
Tris-equilibrium phenol 2 mL, misce bene, shaken and centrifuged at 4℃ at 18 000 r • min-1 for 10 min. The clear
supernatant was transferred to a new centrifuge tube, 5 mL Tris-equilibrium phenol was added and incubated in an
ice bath. The solution was transferred to a quondam tube and centrifuged at 4℃ at 18 000 r • min-1 for 10 min. The
two supernatants were combined, an equal volume of 0.1 mol • L -1 ammonium acetate/methanol
(–20℃) was added, and proteins allowed to precipitate overnight at –40℃. After centrifugation at 4℃ at
20 000 r • min-1 for 15 min, the supernatant was decant-ed off, and the pellet was washed twice in 10 mL 0.1 mol •
L-1 ammonium acetate/methanol and 10 mL 80% acetone, respectively, and centrifuged at 4 ℃ at
18 000 r • min-1 for 10 min. The pellet was removed on vegetable parchment→albumen dry-power and was
resuspended with extraction buffer.
Protein content was measured with the 2D Quant Kit (GE Healthcare). The first dimension was a solid-phase
pH gradient ampholine electrophoresis, using non-linear immobilized pH gradient (IPG) gel strips (pH 4-7, 24 cm;
Amersham Biosciences). For all samples, 1 500 μg protein (450 μL) was loaded on the basic end of the IPG gel
with adjustable volume pipettor. Isoelectric focusing was performed at room temperature using the following
procedure: 30 V, 8 h; 50 V, 4 h; 100 V, 1 h; 300 V, 1 h; 600 V, 1 h; 1 000 V, 1 h; and 8 000 V, 10 h (a total of 26
h). Prior to the second-dimension analysis, the IPG gels were equili-brated for 15 min in reducing buffer (50
mmol • L-1 Tris-HCl, 6 mol • L-1 urea, 30% glycerin, and 1% DTT), and for a further 15 min in alkylating buffer (50
mmol • L-1 Tris-HCl, 6 mol • L-1 urea, 30% glycerin, and 2.5% iodoacetamide). The IPG gels were positioned on the
stacking gel and sealed with 0.5% agarose. The separation in the second dimension was realized on 12.5%
SDS-polyacrylamide gels at a constant current of 17 mA. After electrophoresis, the gels were stained with
CBB-R250 as described by Yu et al (2009).
Protein identification and mass spectro-graphic analysis
Gel images were analyzed using Melanie 3.02 soft-ware (Genebio, Geneva). Specifically, spot detection, spot
measurement, background subtraction, and spot matching were performed. The spots that were detected in
cold-treated samples and the spots that increased in abundance in response to cold stress were subjected to peptide
mass fingerprinting (PMF) by MS. The target protein spots were excised from gels and transferred to a 96-well
PCR plate containing a small amount of MilliQ water. The matrix-assisted laser desorption ionization
time-of-flight mass spectrometry (MALDI-TOF MS) analysis of peptides was performed by the Peking Huada
Protein Research and Development Center Co. Ltd. To obtain more information about proteins, the conserved
domains were estimated from searches of the MSDB and NCBI databases.
Results
Fig. 1 shows the reference 2-DE electrophoretic maps obtained from control (5℃; Fig. 1a) and cold-stressed
(–30℃; Fig. 1b) tillering nodes. Fig. 2 shows examples of protein spots that exhibited a significant increase or
decrease in abundance between the control and cold-stressed nodes. The protein spots showed a broad distribution
in pH ranged from 4.0 to 7.0. Approximately 800 proteins were detected on the CBB-R250 stained gels and about
735 protein spots were matched between three control gels and three cold-treated gels. Protein spots were
compared by Melaine602 software. Obtaining 735 protein spots were distributed on uppermiddle part of gels.
Fig. 1
Two-dimensional proteomic maps of winter wheat Dongnongdongmai 1 tillering nodes exposed to
(designated the control) and (b) –30℃ (cold stressed)
The circles and numbers indicate proteins differentially responsive to cold stress.
(a) 5℃
Fig. 2 Matrix-assisted laser desorption/ionization time-of-flight mass spectrum of tryptic digest (protein spot 30), obtained
in positive-ion delayed extraction reflector mode for the highest resolution and mass accuracy
Variations in spot intensity between the control and treated samples were quantified by Image Master 2D
software image analysis. More than 80 protein spots showed significant difference (p<0.5) from abundance
values >2 and <0.5. Among these protein spots, 37 well-resolved repetitive spots were selected and excised for
tryptic digestion and MALDI-TOF MS PMF charac-terization. The 37 reclaimed protein spots from the gel whose
expression changed in response to cold stress were identified by mass spectra. These protein spots were obtained
integrity peptid finger print (Fig.3) and their result databases were searched in Table 1. Of the 37 identified
proteins, eight proteins (spot 17, 18, 20, 24, 25, 30, 35, and 37) were induced by abiotic stress (e.g. high
temperature, low temperature, and drought); nine proteins (spot 2, 4, 5, 6, 15, 16, 22, 27, and 28) were signaling
molecules; 13 proteins (spot 1, 7, 8, 11, 12, 13, 19, 23, 26, 31, 32, 34, and 36) were related to metabolic processes;
two proteins (spot 3 and 14) were cell-wall-binding proteins; and five proteins (spot 9, 10, 21, 29, and 33) were
unknown proteins. Among the 37 identified proteins, four proteins (spot 11, 12, 31, and 33) were down-regulated by
cold stress, whereas the other proteins were up-regulated.
Fig. 3 Examples of protein spots showing a marked increase or decrease in abundance between winter wheat
Dongnong-dongmai 1 tillering nodes exposed to 5℃ (control) and –30℃ (cold stress)
The numbers refer to the protein spots in Fig. 1.
Table 1
Low-temperature-responsive proteins in winter wheat Dongnongdongmai 1 tillering nodes identified by peptide
mass fingerprinting
Spot
No.1
Protein
Source
Score
Sequence
Coverage(%)
1
2
Phosphoenolpyruvate carboxylase
Serine-threonine protein kinase
Stipagrostis plumos
34
100%
3
Glycine-rich glycoprotein domain
83
27%
+
+
4
Calmodulin
52
79%
+
5
Protein tyrosine kinase
60
19%
+
6
Protein kinase
Elaeis guineensis
Triticum aestivum
Phaseolus vulgaris
Triticum aestivum
Oryza sativa
72
39
49
12%
+
7
Hydratase
Arabidopsis thaliana
59
28%
+
8
ATP1
71
17%
+
9
Function unclear protein
Schoepfia
Ostreococcus tauri
83
17%
+
10
F6N18.1
Arabidopsis thaliana
69
15%
+
11
Starch synthase II
Hordeum vulgare
54
17%
-
12
5,10-Methylene tetrahydrofolate reductase
144
35%
-
13
6-phosphoglucose dehydrogenase
Triticum aestivum
Triticum aestivum
109
32%
+
14
Hydroxyproline-rich protein
Pinus sylvestris
64
46%
+
15
Core binding factor-type protein
44
11%
+
16
Transcription factor
78
11%
+
17
TIR-NBS-LRR-TIR type resistant protein
Brassica oleracea
Oryza sativa
Populus trichocarpa
52
9%
+
18
Resistant protein
Arabidopsis thaliana
69
18%
+
19
Glyceraldehyde-3-phosphate dehydrogenase B subunit
Ostreococcus tauri
56
30%
+
20
14-3-3 type protein
Hordeum vulgare
203
81%
+
21
Predict protein
Physcomitrella patens
60
30%
+
22
Glycosaminoglycan protein retron
Oryza sativa
78
9%
+
23
Lypoxygenase
Capsicum annuum
71
25%
+
24
23.5 kD Heat-shock protein
58
20%
+
25
Cold-induced protein
Triticum aestivum
Oryza sativa
50
27%
+
26
Dihydrofolic acid FH2 domain
Arabidopsis thaliana
60
22%
+
27
Serine-threonine protein kinase
Fagus sylvatica
64
24%
+
28
Phospholipid D
Agropyron mongolicum
143
42%
+
29
Unclear protein
Oryza sativa
76
19%
+
30
Heat-shock protein 70
Cucurbita maxima
199
42%
+
31
Aepfelsaure-dehydrogenase
Triticum aestivum
162
62%
-
32
Nucleotide-binding protein
Arabidopsis thaliana
69
36%
+
33
Imaginary protein
Oryza sativa
151
42%
-
(+/-)
+
34
F0-F1 ATP carboxylase α subunit
Sorghum bicolor
199
49%
+
35
Small-subunit ribosomal protein
Physcomitrella patens
60
48%
+
36
α-glucosidase
Arabidopsis thaliana
54
19%
+
37
DNAJ heat-shock protein
Nicotiana tabacum
53
42%
+
1
For spot number see Fig. 1.
Discussion
Cryoprotective and antifreeze proteins
At low temperature, the ability of protein synthesis is enhanced, and protein synthesis and changes in gene
expression can be detected by changes in the polyribosome population (Li et al., 2004) After 5℃
cold adaptation, polyribosomes of winter rye increas-ed 63%, and protein synthesis increased to ensure seedlings'
survival at low temperature (Li et al., 2004). Our research found that the small-subunit ribosomal protein (spot 35)
was up-regulated, in accordance with the increased content of soluble proteins in Dongnongdongmai 1 following
the decrease in temperature in field conditions (Yin, 2005). In addition, cold-induced proteins could protect plant
cells and organs from injury resulting from low temperature (Li et al., 2004). These proteins were called
cryoprotective proteins (Li et al., 2004). The COR15a gene, which codes for the chloroplast-target polypeptide,
was expressed constitutively to improve the cold resistance of chloroplasts in non-adaptive plants (Uemura et al.,
1996). In our research, a cold-induced protein (spot 25) up-regulated in response to –30°C cold stress was
indicated to be related to the cold resistance of Dongnongdongmai 1 (Hincha et al., 1992). It was purified a 28.5
ku protein, termed a boiling-stability protein (low-temperature protectin) with cryoprotective activity, which was
related to a 7 ku glycoprotein induced by cold stress. In the present study, heat-shock proteins (spot 24, 30, and 37)
were up-regulated following exposure to –30℃ cold stress, but whether these proteins shared a similar function
with cryoprotective proteins needed further investigation.
Griffith purified antifreeze proteins with high cold-resistance activity from the apoplast of cold-adaptive winter
wheat (Griffith et al., 1992). These proteins had similar functions to those of pathogenesis-related proteins, such
as chitinase, endo-β-glucanase, and thaumatin-like proteins. After accumulating emergent evolution, these
pathogenesis-related proteins acquired the function of binding ice crystals. Stress factors such as ethylene,
ultraviolet radiation, ozone, salt, heat, phytotoxic metals, and wounding are reported to induce the synthesis of
pathogenesis-related proteins in barley, potato, cabbage, and spinach (Hincha et al.,
1997; Griffith et al., 1992; Gatschett et al., 1996). It was not a popular stress reaction, but a specific in grains cold
resistance of apoplast antifreeze proteins (Li et al., 2004). Different proteins that execute cold-protective functions
exist in monocotyledons and dicotyledons (Kuiper et al., 2001). In the present study, two disease-resistance
proteins (spot 17 and 18) were matched with disease-resistance proteins of Arabidopsis thaliana, and were
up-regulated in response to –30℃ cold stress, but whether either belongs to the apoplast proteins and increased
the cold resistance of Dongnongdongmai 1 required further study.
Membrane lipid modification-related proteins and signaling molecules
Low temperature often results in reduction of membrane liquidity and initiation of photoinhibition. Previous
studies reported the relationship between membrane liquidity and cold resistance (Jian, 1983). In recent years,
molecular mechanisms of membrane lipid changes have been studied. For example, 3-P glyceroyl transacylase
can enhance cold resistance of broccoli plants and reduce photoinhibition, and lipid transferase also has a
protective function against cold or cold-related stresses (Pyee and Kolattukudy, 1995). The desaturase protein,
induced by low temperature, can increase the degree of unsaturation of membrane lipids to enhance membrane
liquidity and protect the plant from photoinhibition. For example, cold resistance in tobacco was decreased by
transferal of the phospholipase D (PLD) gene, whereas cold resistance was increased by transferal of the antisense
PLD gene (Li et al., 2004). In our study, in response to –30℃ cold stress, two membrane lipid
modification-related proteins (spot 23, lipoxygenase; and spot 28, PLD) were up-regulated. The relationship
between lipoxygenase and cold stress has not been investigated. PLD can exchange signals with Ca2+ messenger
as a transmembrane signal to regulate cold resistance (Hincha et al., 1997), PLD can be activated by a
variety of environmental stimuli, such as low tempera-ture, water stress, pathogen infection and hormone
stimulation (Lee et al., 1997; Zhang et al., 2005).
Protein kinase, a type of phosphotransferase, is activated by different upstream-signaling molecules in
organisms exposed to a variety of stresses. The signals are transmitted to the cell nucleus by phosphorylating
downstream molecules to regulate the expression of specific genes and ultimately to induce a physiological
reaction to the stress. This mechanism is very important in the stress-resistant physiology of plants (Yin, 2005).
There are five kinds of protein kinase; in our study, the expression of four protein kinases (spot 2, 5, 6, and 27)
increased with exposure to –30℃, including serine-threonine protein kinase and tyrosine kinase.
On the basis of expression characteristics, there are two kinds of transcription factors, namely constitutive and
inducible. Most constitutive transcription factors are expressed only in adversity and are regulated by
self-feedback. For example, CBFs as a transcription factor, binds with CRT/DRE to cis-activate of cold-induced
gene expression (Chinnusamy et al., 2003). In the present study, the expression of two proteins (spot 15 and 16)
belonging to the core-binding factor (CBF) family of transcription factors was increased by exposure to –30℃
stress. Whether these two proteins are involved in the expression of seven identified stress protein genes and how
they produce a marked effect needs further investigation.
Metabolism-related proteins
Many proteins or enzymes have important effects on the cold-stress signal transduction pathway to result in
changes of gene expression and metabolism (Cook et al., 2004). Under cold stress, most of the metabolism-related
proteins or enzymes identified have proved that they had an important role for enhancing cold resistance during
primary or secondary metabolism. Furthermore, soluble sugar and osmotic adjustment compound also have
important effects on cold resistance. Under drought, low temperature, and salt stress, physiological functions of
vegetative cell are changed, and especially dynamic balance of plants is destroyed. In adversity, the changes in
carbon metabolism and amino acid metabolism are adaptations to the stress condition (Thomashow et al., 2001).
Glycometabolism-related proteins
Under –30℃ cold stress, the abundance of some proteins involved in glycometabolism changed; PEP carboxylase,
6-P glucose dehydrogenase, 3-P glyceraldehyde dehydrogenase, α-glucosidase, and ATP carboxylase were
up-regulated, while starch synthase was down-regulated.
Normally, PPP metabolic pathway would be en-hanced under adversity. 6-P glucose dehydrogenase is an
important regulatory enzyme in PPP pathway. Lin reported that the activity of 6-P glucose dehydrogenase is
increased under cold acclimation (Lin et al., 2005). It can increase the activity of superoxide dismutase (SOD) and
peroxidase (POD), and significantly decrease the semilethal temperature (LT50) and malo-naldehyde. In our
previous research, in field conditions the activities of SOD and POD increased following a decrease in
temperature (Yin, 2005) which was in accordance with up-regulation of 6-P glucose dehy-drogenase. 3-P
glyceraldehyde dehydrogenase is one of the key enzymes involved in glycolytic energy metabolism. It has an
important function in the maint-enance of vital processes under adversity, such as anaerobic respiration, heat
shock, and injury (Wang et al., 2005). Expression of 3-P glyceraldehyde dehydrogenase was up-regulated in
response to salt stress, and during cold resistance it prevents sugar degradation and increases soluble sugar content.
Furthermore, the chemical reaction 3-P glyceric acid 1, 3 diphosphoglyceric acid is a reversible reaction catalyzed
by 3-P glyceraldehyde dehydrogenase.
Under cold stress, the reaction would operate in the reverse direction.
In the present study, PEP carboxylase was up-regulated, and malate dehydrogenase was down-regulated, in
Dongnongdongmai 1 tillering nodes exposed to –30℃ cold stress. An association with cold stress has not been
reported previously for either of these proteins. PEP carboxylase catalyzes the synthesis of oxaloacetate (OAA)
from PEP and CO2 up-regulation of PEP carboxylase would decrease the 3-P glyceraldehyde content in the
glycolytic pathway to reduce anaerobic respiration. In the tricarboxylic acid cycle, malic acid is dehydrogenized
to OAA by malate -dehydrogenase, which was down-regulated would similarly reduce anaerobic respiration. Thus,
the changes in expression of these two enzymes are likely to contribute to the greater cold resistance of
Dongnongdongmai 1 by reducing respiratory energy consumption and maintaining a high soluble sugar content.
Nucleic acid and amino acid metabolism-related proteins
In our study, expression of 5, 10-methylene tetrahy-drofolate reductase was down-regulated under
–30℃ cold stress. This indicated that a high content of methyltetrahydrofolic acid was involved in DNA
synthesis or repair resulting from cold stress. Tetrahy-drofolic acid is an important coenzyme involved in the
synthesis of purine, pyrimidine, glycine, and serine. It is the product of dihydrofolic acid reduced by dihydrofolate
reductase. In the present study, under –30℃ cold stress, the expression of dihydrofolate reductase was
up-regulated, which could increase the tetrahydrofolic acid content. Under such a condition, enough tryptophan,
glycine, and pyrimidine would be synthesized to protect plants from desiccation and freezing injury through
osmoregulation. Purine and pyrimidine might be utilized for DNA repair.
Structural proteins and unidentified proteins
In our study, under –30℃ cold stress, hydroxyproline-rich protein (spot 14) and glycine-rich protein (spot 3)
were up-regulated. These groups of proteins are structural components of the cell wall that function to maintain
elasticity and mechanical strength of the cell wall. Previous studies have reported that a change in
hydroxyproline-rich protein content is positively correlated with cold acclimation in wheat (Sun et al., 2004), and
glycine-rich proteins are up-regulated in response to abiotic stress (Rao et al., 2002; Gyrgyey et al., 1997) (these
results are in accordance with our findings in the present study).
Determination of five unidentified proteins (spot 9, 10, 21, 29, and 33) and understanding their relationship
with the cold stress response will be a key task in the future research.
Conclusions
The protein contents in tillering nodes of winter wheat Dongnongdongmai 1 grown under cold stress (–30℃) and
non-stressed (5℃) field conditions were analyzed by 2-DE and identified by MS. In the range of pH 4-7, the
expression of 37 proteins showed significant differences in abundance (± more than 2-fold) in proteomic maps for
the cold-stressed and non-stressed tillering nodes. All proteins exhibiting difference in abundance were identified
using MALDI-TOF MS, and searches of the MSDB and NCBI databases were undertaken for protein
identification and function prediction. Five sorts of protein were confirmed, containing: eight stress-related
proteins associated with abiotic stress (22%), nine membrane lipid modification-related proteins and signaling
molecules (24%), 13 metabolism-related proteins (35%), two cell wall-binding proteins (5%), and unclear
pro-teins (14%). These proteins were involved in physio-logical processes to enhance the cold resistance of
Dongnongdongmai 1. Furthermore, besides these five unclear proteins, several proteins were newly identified as
being associated with cold stress. These findings provide a foundation for further studies of cold stress responses
in plants.
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