Abstract

   The potato is an important food crop worldwide. While potatoes are rich
   in nutrition, the production suffers from yield loss caused by frost
   and freezing. This study used a common potato cultivar, ‘Zhengshu 6’,
   as the study system to measure the changes in the contents of soluble
   protein, malondialdehyde (MDA), proline, and chlorophyll after 1, 3, 5,
   and 7 days of low temperature treatment. We performed two-dimensional
   electrophoresis (2-DE) in combination with liquid
   chromatography-electrospray ionization-tandem mass spectrometry
   (LC-ESI-MS/MS) technology and identified 52 differentially expressed
   protein spots among these timepoints. Results showed that levels of
   soluble protein, MDA, and proline increased as the duration of the low
   temperature treatment increased, and the chlorophyll content decreased.
   The 52 identified protein spots were classified by function as involved
   in defense response, energy metabolism, photosynthesis, protein
   degradation, ribosome formation, signal transduction, cell movement,
   nitrogen metabolism, and other physiological processes, thus allowing
   potato plants to achieve metabolic balance at low temperatures.

   Keywords: Potato, Proteomics, Low temperature stress, Differentially
   expressed
     __________________________________________________________________

   potato; proteomics; low temperature stress; differentially expressed.

1. Introduction

   The potato (Solanum tuberosum L) is one of the world's most important
   food crops. Potato tubers are highly nutritious; they are rich in
   starch, vitamins, minerals, and essential amino acids, and are
   therefore suitable for consumption as a staple food source. Potatoes
   are also served as animal feed and used as feedstock for many
   industrial purposes [[35]1]. Despite the agricultural importance of the
   potato most potato cultivars are not tolerant to frost and freezing
   conditions; cold injury occurs when the temperature falls below −0.8
   °C, and severe freezing injury occurs below −2 °C [[36]2]. Low
   temperature-induced frost and freezing are two of the main natural
   hazards that affect potato production. Frost and freezing can cause
   significant damage to potato plants, leading to reduced yield or even
   complete yield loss when severe. Annually, all potato-growing regions
   across the world experience different degrees of cold or freezing
   damage [[37]3,[38]4,[39]5,[40]6,[41]7,[42]8]. Almost all of the major
   potato cultivation areas in China suffer from low temperatures,
   especially in late spring, when potatoes are in the seedling stage.
   Further damage caused by cold waves and early frosts can occur at the
   mature stage.

   Plant response to low temperature stress is mediated by a series of
   proteins. Changes in gene expression have been demonstrated to occur in
   response to low temperature in a wide range of plant species, including
   both freezing-tolerant and freezing-sensitive plants. Cold response
   pathways have been demonstrated to exist in plants that acclimate to
   low temperatures. Therefore, the characteristic variation analysis of
   proteome differentiation among different genotypes in response to low
   temperatures can facilitate analysis of the mechanism underlying cold
   adaptation [[43]9]. As a result, the study of potato proteomics under
   low temperature stress has great significance for revealing the plant's
   cold tolerance mechanism, thereby providing information that may be
   used to improve its stress-resistance traits in order to reduce the
   losses caused by low temperature damage.

   Upon experiencing low temperature stress, plants display a series of
   physiological and biochemical changes, which are caused by molecular
   changes in the plant cells, i.e., changes in the transcriptome,
   proteome and metabolome. Proline is an important osmoregulation
   substance. Plant cells lose water after stress. By increasing the
   content of proline in vivo, the concentration of cell fluid is
   increased, so as to maintain the water holding capacity of cells.
   Studies have shown that the content of proline is positively related to
   the cold resistance of the potato [[44]10]. Malondialdehyde (MDA) is
   one of the main producany plants. For example, a large number of up-
   and downregulated low temperature-rts of membrane lipid peroxidation in
   plants under low temperature stress. When freezing injury occurs in
   plants, the balance of active oxygen metabolism in cells is disrupted.
   The superoxide radicals produced will attack the cell membrane and
   produce a large amount of malondialdehyde through metabolism.
   Therefore, malondialdehyde content can be an important indicator of the
   degree of membrane damage. The mechanism of low temperature tolerance
   has been studied in responsive genes identified in Arabidopsis, wheat,
   and barley using transcriptomics [[45]11,[46]12,[47]13]. However, due
   to the regulation of protein translation and degradation, changes in
   RNA expression under low temperature stress cannot fully indicate the
   changes at the protein level [[48]14,[49]15,[50]16]. Therefore,
   proteomics technology is needed to reveal the expression of proteins
   and to identify those involved in stress tolerance. In recent years,
   proteomics technology has been developing rapidly along with the
   completion of genome sequencing for many crops. In rice
   [[51]17,[52]18], wheat [[53]19], barley [[54]20], and Arabidopsis
   [[55]21], proteomics technology has been successfully applied to study
   the mechanism underlying low temperature tolerance. Some proteomic
   studies on crops from the Solanaceae family (e.g., tomato, tobacco, and
   pepper) are also available [[56]22,[57]23]. In potato plants,
   proteomics has been used to reveal the physiological mechanism
   underlying tuber development and formation, the pathogenesis of potato
   late blight, and the mechanism underlying salt stress [[58]24,[59]25].
   However, there have been no reports concerning the use of proteomics to
   determine the mechanism underlying cold tolerance in the potato. Thus,
   to understand the protein expression profile in potatoes under low
   temperature stress, we utilized two-dimensional electrophoresis (2-DE)
   in combination with liquid chromatography-tandem mass spectrometry
   (LC-MS/MS) to perform proteomics analysis on potatoes under low
   temperature stress conditions. The results may provide insights into
   the mechanism of cold tolerance in the potato.

2. Materials and methods

2.1. Plant materials and growth conditions

   The potato cultivar ‘Zhengshu 6’ [[60]26], bred at the Zhengzhou
   Vegetable Institute, was used in this study. Potato tubers were planted
   in plastic pots (height 22 cm × diameter 15 cm), one plant per pot,
   with six plants total. After seedling emergence, pots were transferred
   to a growth chamber (22 °C 16 h light, 18 °C 8 h dark, 70% relative
   humidity, and light intensity 600 μmol photons m^−2 s^−1). After
   growing for four weeks, plants were subjected to low temperature
   treatment.

2.2. Experimental treatment

   Four-week old potato plants were placed in the growth chamber for low
   temperature treatment (16 h light, 8 h dark, 4 °C daytime, 2 °C night
   time; 65% relative humidity; light intensity 600–1000 μmol photons m^−2
   s^−1). The expanded 3rd and 4th leaves were collected before the low
   temperature treatment (control) and after 1 d, 3 d, 5 d, and 7 d of the
   low temperature treatment. Samples were frozen in liquid nitrogen and
   then stored at −80 °C.

2.3. Measurements of the physiological and biochemical parameters

   The content of soluble protein (SP) was measured using the
   bicinchonininc acid (BCA) method [[61]27]. Proline (PRO) content was
   measured using the ninhydrin method [[62]28]. Malondialdehyde (MDA)
   content was measured using the thiobarbituric acid (TBA) method
   [[63]29]. Chlorophyll content was measured using an acetone and ethanol
   mixture.

2.4. Protein sample preparation

   Total protein extraction from potato leaves was performed using
   trichloroacetic acid/acetone (TCA/A) precipitation [[64]30]. A modified
   Bradford method was used for protein quantification with bovine serum
   albumin as a standard. Quantified protein was then used for further
   analysis [[65]31].

2.5. 2-DE, gel staining, and image analysis

   Isoelectric focusing in the first dimension was carried out on the
   Bio-Rad PROTEAN IEF Cell. Protein samples were diluted with a
   rehydration solution containing 7 M urea, 2 M thiourea, 4% CHAPS (W/V),
   50 mM dithiothreitol (DTT), and 0.5% (V/V) IPG buffer (pH 4–7) to a
   concentration of 800 μg/mL for sample loading. After centrifugation at
   10,000 g for 15 min, 300 μL supernatant was loaded onto the 17 cm gel
   strip. The isoelectric focusing program was as follows: active
   rehydration at 50 V for 13 h (20 °C) followed by a rapid increase of
   the voltage to 100 V applied for 1 h, 500 V for 1 h, and 1000 V for 2
   h, followed by a linear increase of the voltage to 8,000 V for 4 h.
   Subsequently, isoelectric focusing was operated at 8,000 V for 7.5 h,
   reaching 60,000 Vh. Finally, a rapid decrease of voltage to 500 V was
   applied and maintained for 10 h. Following the first-dimension
   isoelectric focusing (IEF) separation, the strips were equilibrated in
   a buffer containing 6 M urea, 0.375 M Tris-HCl (pH 8.8), 2% (W/V)
   sodium dodecyl sulfate (SDS), 20% glycerol (V/V), and 2% (W/V) DTT for
   15 min to unfold the proteins. In the second step, an equilibration
   buffer with 2% (W/V) DTT replaced by 2.5% (w/V) iodoacetamide was used
   for re-equilibration and was applied for 15 min to remove extra DTT.

   The second-dimension sodium dodecyl sulfate–polyacrylamide gel
   electrophoresis (SDS-PAGE) was performed on the EttanTM DALT SIX System
   (GE Healthcare, Washington, USA) with 12% gels (26 cm × 20 cm).
   SDS-PAGE was performed with 1.0 W/gel for 40 min (20 °C) followed by a
   10 W/gel until the loading dye reached 1 cm above the bottom of the gel
   (20 °C).

   A 2-DE gel staining was performed using the silver nitrate method
   [[66]32]. A gel imaging system, the UMAX PowerLook 2100XL scanner (UMAX
   Systems GmbH, Willich, Germany), was used to scan the gel image.
   PDQuest software version 7.1 (Bio-Rad, Hercules, CA, USA) was used to
   analyze the gel image to determine the protein isoelectric point,
   molecular weight, and relative expression, and to match the set of
   protein spots on the gels with three replicates for each treatment.
   Protein quantity was normalized. Compared to the control, protein spots
   with significantly different expression greater than 2-fold were
   considered differentially expressed protein (DEP) spots (Student's
   t-test, P < 0.05).

2.6. In-gel digestion, mass spectrometry analysis, and database searching

   In-gel enzyme digestion of protein spots was carried out as described
   in [[67]33]. Triple TOF 5600 LC-MS/MS high resolution liquid
   chromatography coupled with a mass spectrometry system (AB Sciex) was
   used for the analysis. The Triple TOF 5600 mass spectrometer was used
   for first and second MS scans. IDA was used for data analysis. LC
   separation conditions were as follows: Thermo C18 column (4.6 × 150),
   mobile phase 90% acetonitrile/0.1% formic acid, speed 0.3 mL min^−1.
   The range of the MS scan was 300–2000 m/z, followed by the secondary
   scan (MS/MS). Maximum charge of the precursor ions was 80 eV. The
   obtained MS or MS/MS data were subjected to database searching using
   ProteinPilot™ 5.0 software (AB SCIEX, Framingham, MA, USA) for protein
   identification. The fixed modification was cysteine acetylation, the
   variable modification was methionine oxidation, the maximum number of
   enzyme cleavage sites allowed was two, and mass tolerance of parent
   ions was 0.05 Da. Potato protein sequences downloaded from the potato
   genome database
   ([68]http://solanaceae.plantbiology.msu.edu/pgsc_download.shtml) were
   used for data searching. Protein spectra with confidence intervals of
   0.95 were considered reliable. Peptide sequences were manually matched
   to the database for protein identification.

2.7. Functional classification and subcellular localization of identified
protein spots

   Functional annotation of the identified protein spots was done as
   described in Bevan et al. [[69]34]. Plant-PLoc software
   ([70]http://www.csbio.sjtu.edu.cn/bioinf/plant/) was used for the
   prediction of subcellular localization [[71]35].

3. Results

3.1. Analysis of physiological and biochemical parameters

   During the process of low temperature treatment, the total contents of
   SP, MDA, PRO, and chlorophyll in the leaves at 0 d, 1 d, 3 d, 5 d, and
   7 d were measured; as the duration of treatment increased, SP content
   rapidly increased. However, from 5–7 d, the rate of increase in SP
   content slowed, and the SP content reached its maximum at 7 d
   ([72]Figure 1A). Similar patterns were observed for MDA and PRO
   content; both increased at first and then decreased, reaching the
   maximum values at 5 d and then beginning to decrease at 7 d
   ([73]Fig. 1B, C). Under low temperature stress conditions the potato
   cell membrane was damaged, causing the MDA content to increase. In
   contrast, the total chlorophyll content decreased as the treatment
   duration increased ([74]Figure 1D), indicating that chlorophyll
   synthesis was inhibited during low temperatures.

Figure 1.

   [75]Figure 1
   [76]Open in a new tab

   Changes in soluble protein, malondialdehyde, proline, and chlorophyll
   during low temperature stress.

3.2. Bioinformatic analysis of the 52 identified protein spots

   [77]Figure 2 shows a representative 2-DE gel image before low
   temperature stress and after 1, 3, 5, and 7 d of low temperature
   stress. 52 DEP spots were identified ([78]Figure 3 and [79]Table 1) and
   classified into eight groups ([80]Figure 4).

Figure 2.

   [81]Figure 2
   [82]Open in a new tab

   Representative spot maps of potato cv. Zhengshu 6 at different stages.

Figure 3.

   [83]Figure 3
   [84]Open in a new tab

   Identification of 52 differentially expressed protein spots from potato
   leaves by 2-DE.

Table 1.

   Identification of 52 differently expressed protein spots from potato
   leaves.
   Spot No.[85]^a) Protein Name[86]^b) AccessionNo.[87]^c) Unused[88]^d)
   MP[89]^e) SC(%)[90]^f) Theor. Mr/pI[91]^g) Exp. Mr/pI[92]^h)
   Subcellular localization[93]^i) Fold changes[94]^j)
     __________________________________________________________________

   FC[T1/CK] FC[T3/CK] FC[T5/CK] FC[T7/CK]
   01 Metabolism
   P11 11 thiamine thiazole synthase 1, chloroplastic [Solanum tuberosum]
   gi|565388645 11.38 7 28.6 37.8/5.40 30.6/5.21 Chloroplast 1.22 1.44
   4.61↑ 3.25↑
   P34 34 magnesium protoporphyrin IX methyltransferase, chloroplastic
   [Solanum tuberosum] gi|565348979 10.05 5 11 35.5/6.61 28.3/5.85
   Extracellular 0.68 0.46↓ 0.43↓ 0.61
   P38 38 ketol-acid reductoisomerase, chloroplastic [Solanum tuberosum]
   gi|565366716 48.19 47 35.6 110.7/6.07 54.0/5.83 Nucleus 1.00 0.68 0.53
   0.44↓
   P40 40 glutamine synthetase [Solanum tuberosum] gi|565371595 10.05 5 11
   53.4/5.26 51.3/5.92 Plasma membrane 1.40 3.11↑ 2.04↑ 2.10↑
   P51 51 ferredoxin-dependent glutamate synthase 1,
   chloroplastic/mitochondrial [Solanum tuberosum] gi|565396313 40.15 23
   19.6 77.2/6.18 33.6/5.97 Plasma membrane 0.89 0.89 0.31↓ 0.49↓
   P54 9 putative dihydroxy-acid dehydratase, mitochondrial[Solanum
   tuberosum] gi|565359581 13.23 9 12.9 66.3/6.17 58.0/5.75 Chloroplast
   0.59 0.44↓ 0.28↓ 0.39↓
   P58 22 fructose-bisphosphate aldolase, cytoplasmic isozyme 1 [Solanum
   tuberosum] gi|565358575 64.47 71 45.7 78.2/5.94 35.9/6.45 Chloroplast
   1.20 0.83 0.41↓ 0.72
   P60 28 fructose-bisphosphate aldolase, cytoplasmic isozyme 1 [Solanum
   tuberosum] gi|565358575 16 8 30 38.2/5.94 44.5/6.49 Chloroplast 0.75
   0.59 0.46↓ 0.37↓
   02 Energy
   P4 4 ATP synthase delta chain, chloroplastic [Solanum tuberosum]
   gi|565389084 25.29 28 38.4 27.2/8.61 21.4/4.68 Chloroplast 0.32↓ 1.57
   1.31 1.09
   P8 8 ruBisCO large subunit-binding protein subunit alpha, chloroplastic
   [Solanum tuberosum] gi|565346319 6 3 6.5 61.9/5.37 59.3/4.68 Nucleus
   0.43↓ 0.49↓ 0.41↓ 0.42↓
   P13 13 ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit
   (chloroplast) [Solanum tuberosum] gi|88656812 6.27 5 12.8 52.7/6.59
   56.7/4.98 Cytoplasm 0.48↓ 0.59 0.86 0.47↓
   P19 19 ATP synthase CF1 beta subunit (chloroplast) [Solanum tuberosum]
   gi|329124676 32.51 26 52.4 53.4/5.26 32.3/5.71 Chloroplast 1.55 1.55
   0.79 1.03
   P23 23 glyceraldehyde-3-phosphate dehydrogenase A, chloroplastic
   [Solanum tuberosum] gi|565386971 16.07 10 22.3 42.7/8.46 42.7/5.50
   Cytoplasm 0.43↓ 0.45↓ 0.24↓ 0.48↓
   P24 24 plastidic phosphoglucomutase [Solanum tuberosum] gi|8250622
   27.68 29 18.5 61.2/5.26 60.6/5.46 Chloroplast 0.29↓ 0.42↓ 0.52 0.40↓
   P25 25 glyceraldehyde-3-phosphate dehydrogenase (chloroplast) [Solanum
   tuberosum] gi|327198779 18.61 16 32.2 48.0/7.06 54.4/5.54 Nucleus 1.58
   0.83 3.09↑ 2.09↑
   P26 26 plastidic phosphoglucomutase [Solanum tuberosum] gi|8250622
   39.33 16 26.9 61.1/5.26 60.7/5.65 Nucleus 1.08 0.66 1.29 1.19
   P27 27 2,3-bisphosphoglycerate-independent phosphoglycerate mutase
   [Solanum tuberosum] gi|565402329 29.45 16 19.9 61.2/5.37 60.7/5.65
   Cytoplasm 0.34↓ 0.54 0.64 0.51
   P30 30 ATP-dependent Clp protease ATP-binding subunit clpA homolog
   CD4B, chloroplastic [Solanum tuberosum] gi|565359707 46.65 25 25.4
   102.2/5.99 80.7/5.65 Nucleus 0.18↓ 0.33↓ 0.56 0.47↓
   P32 32 ribulose bisphosphate carboxylase/oxygenase activase 1,
   chloroplastic isoform X2 [Solanum tuberosum] gi|565343801 18.93 18 26.6
   48.3/8.49 23.1/5.94 Endoplasmic reticulum 0.69 0.61 0.33↓ 0.63
   P35 35 ribulose bisphosphate carboxylase/oxygenase activase 1,
   chloroplastic isoform X2 [Solanum tuberosum] gi|565343801 18.02 15 29.8
   48.3/8.49 33.6/5.83 Nucleus 0.58 0.57 0.49↓ 0.67
   P36 36 glyceraldehyde-3-phosphate dehydrogenase B, chloroplastic
   [Solanum tuberosum] gi|565351180 12.34 8 23.1 47.93/7.53 35.5/5.81
   Nucleus 0.85 0.52 3.7↑ 2.31↑
   P41 41 glyceraldehyde-3-phosphate dehydrogenase, cytosolic [Solanum
   tuberosum] gi|565361313 8.05 5 18.7 36.6/6.98 54.0/5.92 Plasma membrane
   0.71 0.60 0.53 0.55
   P46 46 ATP synthase CF1 beta subunit (chloroplast) [Solanum tuberosum]
   gi|329124676 72.63 73 83.1 53.4/5.26 23.7/6.23 Nucleus 1.26 0.90 2.92
   1.67
   P47 47 ATP synthase CF1 alpha subunit (chloroplast) [Solanum tuberosum]
   gi|88656789 35.55 23 44.8 55.4/5.14 27.0/6.14 Nucleus 0.66 0.41↓ 0.42↓
   0.68
   P48 48 oxygen-evolving enhancer protein 1, chloroplastic [Solanum
   tuberosum] gi|565355906 41.19 29 61.4 35.0/5.89 27.3/5.98 Nucleus 0.49↓
   0.49↓ 0.43↓ 0.80
   P49 49 chlorophyll a-b binding protein 3C, chloroplastic isoform X2
   [Solanum tuberosum] gi|565391644 33.99 32 43.1 28.3/5.47 27.0/6.02 0.93
   0.56 0.44↓ 0.80
   P50 50 ATP synthase CF0 subunit I (chloroplast) [Solanum tuberosum]
   gi|88656790 23.51 17 60.3 20.9/8.76 27.8/6.10 Nucleus 0.59 0.38↓ 0.23↓
   0.52
   P53 52 enolase [Solanum tuberosum] gi|565343656 44.86 29 54.1 47.9/5.79
   47.3/6.06 2.36↑ 0.65 4.83↑ 1.71
   P55 10 NADP-dependent malic enzyme [Solanum tuberosum] gi|565357492 12
   6 10 64.1/5.71 61.3/6.06 Chloroplast 0.65 0.91 0.72 0.96
   P61 31 dihydrolipoyl dehydrogenase 1, mitochondrial [Solanum tuberosum]
   gi|565378559 49 44 41.8 52.9/6.90 54.0/6.64 Cytoplasm 5.41↑ 4.76↑ 3.34↑
   1.69
   P62 39 transketolase, chloroplastic [Solanum tuberosum] gi|565357366
   49.81 48 29.9 80.2/6.22 68.9/6.08 Chloroplast 0.61 0.33↓ 0.39↓ 0.48↓
   05 protein synthesis
   P1 1 28 kDa ribonucleoprotein, chloroplastic [Solanum tuberosum]
   gi|565369617 40.72 49 48.3 33.3/4.67 27.1/4.61 Chloroplast 0.75 0.94
   0.65 0.57
   P15 15 P0 ribosomal protein [Solanum tuberosum] gi|82623393 42.13 41
   44.1 33.9/5.11 37.7/5.23 Chloroplast 4.88↑ 1.70 2.92↑ 3.56↑
   P17 17 elongation factor G, chloroplastic [Solanum tuberosum]
   gi|565378095 13.36 8 10.8 86.4/5.40 78.6/5.31 Chloroplast 0.96 0.50
   0.88 0.48↓
   P18 18 29 kDa ribonucleoprotein A, chloroplastic [Solanum tuberosum]
   gi|565395354 8.29 5 13.4 31.8/6.34 27.8/5.33 Vacuole 0.44↓ 0.30↓ 0.30↓
   0.44↓
   P29 29 elongation factor G, chloroplastic [Solanum tuberosum]
   gi|565378095 58.29 43 40.2 86.4/5.40 78.6/5.42 Mitochondrion 0.49↓ 0.52
   0.53 0.46↓
   06 protein destination and storage
   P2 2 26S proteasome non-ATPase regulatory subunit 4 homolog isoform X3
   [Solanum tuberosum] gi|565347475 28.08 22 48.3 42.7/4.46 55.3/4.59
   Nucleus 0.46↓ 0.26↓ 0.28↓ 0.74
   P7 7 2-Cys peroxiredoxin BAS1, chloroplastic [Solanum tuberosum]
   gi|565344108 6 3 14.2 52.7/4.78 57.5/4.68 Chloroplast 0.27↓ 0.48↓ 0.52
   0.37↓
   P16 16 luminal-binding protein [Solanum tuberosum] gi|565353800 44.4 26
   10.8 70.7/4.99 70.0/5.38 Cytoplasm 0.38↓ 0.31↓ 0.48↓ 0.63
   P45 45 ATP-dependent zinc metalloprotease FTSH, chloroplastic [Solanum
   tuberosum] gi|565350308 62.07 42 56.6 76.0/6.19 82.9/5.79 Plasma
   membrane 0.44↓ 0.27↓ 0.77 0.58
   P57 20 proteasome subunit alpha type-6 [Solanum tuberosum] gi|565356827
   12.63 8 45.7 27.3/6.11 26.5/6.39 Nucleus 0.58 0.48↓ 1.08 0.66
   10 signal transduction
   P3 3 calreticulin [Solanum tuberosum] gi|565355763 43.18 35 33.6
   47.6/4.50 51.3/4.69 Mitochondrion 0.27↓ 0.36↓ 0.21↓ 1.10
   11 Disease/defense
   P5 5 2-Cys peroxiredoxin BAS1, chloroplastic [Solanum tuberosum]
   gi|565344108 10 5 18 29.4/6.34 22.7/4.95 Chloroplast 0.05↓ 0.90 0.75
   0.31↓
   P6 6 2-Cys peroxiredoxin BAS1,chloroplastic [Solanum tuberosum]
   gi|565344108 6 3 14.2 29.4/6.34 22.4/4.86 Cytoplasm 0.12↓ 1.45 1.52
   0.96
   P12 12 trigger factor protein TIG [Solanum tuberosum] gi|565358747
   20.41 11 20.3 61.2/5.08 50.0/5.00 Chloroplast 0.32↓ 0.41↓ 0.48↓ 0.34↓
   P14 14 trigger factor protein TIG [Solanum tuberosum] gi|565358747
   91.59 74 68 61.2/5.08 50.0/5.06 Cytoplasm 0.42↓ 0.25↓ 0.49↓ 0.64
   P42 42 protein TIC 62, chloroplastic [Solanum tuberosum] gi|565365922
   20.92 15 15.7 78.6/5.40 68.7/5.71 Vacuole 0.52 1.44 0.33↓ 0.77
   P43 43 thimet oligopeptidase [Solanum tuberosum] gi|565382894 44.87 27
   24.4 9.54/6.37 74.3/5.75 Vacuole 0.38↓ 0.20↓ 0.30↓ 0.44↓
   P44 44 M1 family aminopeptidase isoform X2 [Solanum tuberosum]
   gi|565354114 97.35 68 50.5 110.7/6.07 82.9/5.75 Vacuole 0.87 0.26↓ 0.59
   0.39↓
   20 Secondary metabolism
   P21 P21 hyoscyamine 6-dioxygenase [Solanum tuberosum] gi|565383140 22
   11 22 37.9/5.54 35.9/6.45 Nucleus 1.05 1.14 3.24↑ 2.70↑
   12 unknown
   P33 33 uncharacterized protein At2g37660, chloroplastic [Solanum
   tuberosum] gi|565348015 28.99 18 50.3 32.5/8.91 28.3/5.75 Plasma
   membrane 0.62 0.46↓ 0.35 0.44↓
   P37 37 uncharacterized oxidoreductase At1g06690, chloroplastic [Solanum
   tuberosum] gi|565383154 41.76 33 35.6 40.4/6.78 37.3/5.92 Nucleus 0.83
   0.82 0.24↓ 0.51
   [95]Open in a new tab
   ^a)

   Spot No, Spot number.
   ^b)

   Names and species of proteins obtained via the ProteinPilot™ 5.0
   software from potato protein sequences downloaded from the NCBInr
   database.
   ^c)

   Accession No, Accession number.
   ^d)

   Score probability (protein score) for the entire protein and for ions
   complemented by the percentage of the confidence index (C.I.).
   ^e)

   MP indicate the number of matched peaks for the PMF data, respectively.
   ^f)

   SC, Sequence coverage.
   ^g)

   The subcellular localization prediction of 52 differently abundant
   proteins based on Plant-PLoc.
   ^h)

   Theor. Mr/pI shows theoretical molecular weight and pH isoelectric
   point.
   ^i)

   Exp. Mr/pI shows experimental molecular weight and isoelectric point.
   ^j)

   Fold change was calculated from T1, T3,T5 and T7 over the CK gels,
   which ‘↑‘, ‘↓’ and ‘ns’ stand for up-regulated, down-regulated and no
   significant chang, respectively.

Figure 4.

   [96]Figure 4
   [97]Open in a new tab

   Functional groups of 52 differently expressed protein spots from potato
   leaves under treatments.

3.3. Analysis of the differences among the 52 DEPs

   [98]Figure 4 shows the diagram of the protein expression profiles at
   the four treatment time points. Compared to the control, samples
   treated for 1 d, 3 d, 5 d, and 7 d exhibited protein spots with
   different abundances ([99]Table 1). At 1 d of low temperature
   treatment, we found 3 upregulated and 19 downregulated DEPs. At 3 d of
   treatment, 2 DEPs were upregulated and 22 were downregulated. At 5 d of
   treatment, 9 DEPs were upregulated and 23 were downregulated. At 7 d of
   treatment, 7 DEPs were upregulated and 19 were downregulated. This
   result indicated that as the duration of low temperature treatment
   increased, more proteins began to respond to low temperature stress.
   The number of downregulated proteins rapidly increased, peaking at 5 d,
   followed by a decrease at 7 d. This pattern was the same as for the
   changes in MDA and PRO content, which leveled off after the plants
   adapted to the low temperature and then began to decrease (see
   [100]Figure 5).

Figure 5.

   [101]Figure 5
   [102]Open in a new tab

   Differences in protein abundance at 1 d, 3 d, 5 d, and 7 d of low
   temperature treatment in comparison with the control.

3.4. GO classification and enrichment analysis of DEPs

   We also carried out a gene ontology (GO) functional enrichment analysis
   on all DEPs ([103]Figure 6). The DEPs in potato leaves under low
   temperature stress at the four different points are widely involved in
   29 subcategories among the three GO categories of biological process
   (BP), cellular component (CC), and molecular function (MF).

Figure 6.

   [104]Figure 6
   [105]Open in a new tab

   Gene ontology classification of the differently expressed proteins
   under low temperature stress.

   As shown in [106]Figure 6, DEPs in potato leaves at 1 d, 3 d, 7 d of
   low temperature stress involved 23 GO subcategories, and those at 5 d
   of low temperature stress involved 24 GO subcategories. These GO
   subcategories included biological regulation, response to stimuli, and
   metabolic processes in the BP category; organelles and cell membrane in
   the CC category; and catalytic activity, antioxidant activity, and
   binding in the MF category. A pathway enrichment analysis using the
   KEGG (Kyoto Encyclopedia of Genes and Genomes) database was performed
   to determine the major biochemical metabolic pathways, photosynthetic
   pathways, and the biosynthesis secondary metabolites that DEPs were
   involved in. The results showed that at all time points during low
   temperature stress ([107]Fig. 7A, B, C, D), the pathways with the
   highest number of DEPs were photosynthesis, amino acid biosynthesis,
   alanine metabolism, and starch and sugar metabolism. DEPs significantly
   enriched in the pathway of photosynthetic ATP synthase. Many DEPs were
   also enriched in pathways related to phosphate metabolism, oxidative
   phosphorylation, amino acid biosynthesis, and starch and sugar
   metabolism. Secondary metabolites produced by these metabolic pathways
   can alleviate or eliminate injuries caused by reactive oxygen species
   (ROS) induced by stresses such as low temperature. These results also
   indicated that the response to low temperature stress in the potato was
   a complex physiological and biochemical process controlled by multiple
   genes, pathways, and metabolites. Consistent with the results of the GO
   enrichment analysis, significantly enriched KEGG metabolic pathways of
   the DEPs were mainly biochemical metabolic pathways, photosynthesis,
   and secondary metabolite biosynthesis.

Figure 7.

   [108]Figure 7
   [109]Open in a new tab

   KEGG analysis of differentially expressed proteins under low
   temperature stress.

4. Discussion

4.1. Changes in physiological characteristics in potatoes upon low
temperature stress

   When plants are challenged by low temperature stress a series of
   changes in physiological characteristics occur, including inhibited
   plant growth and photosynthesis, peroxidation of membrane lipids, cell
   metablism disorders, massive accumulation of free radicals and reactive
   oxygen species, inhibited chlorophyll synthesis, increased permeability
   of the plasma membrane, and a rapid increase in MDA. Studying the
   changes in physiological characteristics could help assess plant cold
   tolerance and elucidate the mechanism of cold tolerance. Potato
   cultivars are not tolerant to frost and freezing. found that under low
   temperature stress, the PRO level increased in potato plants; the more
   tolerant to low temperature stress the plants were, the higher the PRO
   level was found to be [[110]10]. Chen et al. found that after cold
   acclimation the level of SP in potato leaves was high, and the increase
   in SP content was positively correlated with cold resistance [[111]36].
   Changes in the levels of SP, MDA, PRO, and chlorophyll after low
   temperature treatment measured in [[112]37]this study exhibited the
   same pattern as in previous studies. Studies have shown that spraying
   ABA could improve crop resistance to cold [[113]37,[114]38],Therefore,
   spraying a certain amount of exogenous hormones can reduced the content
   of MDA, increase the content of chlorophyll, SP and PRO, enhance the
   activity of enzymes in the potato, Thereby improving the cold
   resistance of potatoes.

4.2. Proteins related to energy metabolism

   Proteins for energy metabolism are crucial for maintaining plant cell
   growth and development; they provide ATP and other metabolic
   intermediates necessary for plants. Abiotic stress affects
   physiological reactions in plants, among which the most important are
   physiological reactions related to energy. ATP synthase is a key enzyme
   for oxidative phosphorylation [[115]39,[116]40] as it catalyzes ATP
   synthesis and hydrolysis. In the present study 31 DEPs were identified
   as related to energy metabolism, accounting for 59% of total proteins
   identified. Among those proteins, ATP synthase δ chain (P4), ATP
   synthase β subunit (P17, P46), ATP synthase α subunit (P47), and ATP
   synthase CF0 subunit (P50) are involved in energy metabolism. Protein
   spots P4, P47, and P50 exhibited downregulated expression by different
   degrees at 5 d of low temperature treatment, indicating that the
   photophosphorylation in potato leaves was affected by low temperature
   stress. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) is a key
   enzyme in glycolysis; it is also an enzyme necessary for the survival
   of organisms. A lack of this enzyme can trigger a metabolic disorder in
   the organism. In this study, we found that GADPH A (P23) was
   downregulated under low temperature stress, while GADPH B (P36) was
   upregulated at 5 d and 7 d of low temperature stress, indicating that
   GADPH A was the main GAPDH involved in the potato's stress response to
   low temperature. Downregulated expression of GADPH A decreases normal
   metabolism, thereby enhancing the adaptivity of the potato to low
   temperature. It has been shown that dilute alcoholase is involved in
   the response of plants to abiotic stresses such as extreme temperature,
   salt stress, hypoxia stress, and drought [[117]41,[118]42]. The
   identified enolase (p53) was downregulated under low temperature
   stress, indicating that enolase participated in the corresponding
   process of low temperature stress. Glutamine synthetase (GS) is
   involved in metabolism and amino acid synthesis. In this study, the
   identified GS (P40) was upregulated in response to low temperature
   stress, indicating that when the potato was under low temperature
   stress, the upregulation of GS promoted the synthesis of amino acids
   related to stress to increase the ability to adapt to the environment
   [[119]43,[120]44]. In this study, protein spots P22 and P28
   corresponded to fructose-bisphosphate aldolase, which exhibited
   downregulated expression during low temperature stress, indicating that
   the sugar metabolism pathway was involved in the response to low
   temperature.

4.3. Proteins related to photosynthesis

   Photosynthesis is the process that fixes light energy and synthesizes
   sugar in higher plants. Abiotic stress can damage the thylakoid
   membrane in the chloroplast and can affect the photosynthetic
   metabolism, thereby affecting normal plant growth and development
   [[121]45,[122]46]. Ribulose-1,5-bisphosphate carboxylase/oxygenase
   (RuBisCO) (P13, P32, and P35) is the key enzyme that fixes CO[2]. This
   enzyme greatly influences the photosynthetic rate in plants. Studies
   have shown that the large subunit of RuBisCo, as a photosynetic
   protein, is prone to degradation under abiotic stress. In this study,
   protein spot P8 was identified as the large subunit of Rubisco. Protein
   spot P8 exhibited downregulated expression upon low temperature stress.
   This indicated that RuBisCo activase had degraded, which led to a
   decrease in photosynthetic rate in the leaf and inhibited growth and
   development. Chlorophyll a/b-binding protein (P49) is a key protein in
   the light-harvesting complex. Plant photosystem I (PSI) and the
   light-harvesting complex of photosystem II (PSII) act as light
   receptors that can harvest light energy and transfer light energy to
   the reaction center. Protein spot P49 exhibited downregulated
   expression under low temperature stress. Its expression stablized at 7
   d, which was helpful for energy transfer to defend against cold damage.
   Oxygen-evolving enhancer protein (P48) is one of the peripheral
   proteins of the oxygen evolution complex; the protein functions in
   stablizing PSII. Under low temperature stress P48 expression was
   downregulated, indicating that the photosynthetic rate in potato leaves
   was reduced and that chloroplasts were damaged, leading to inhibited
   chlorophyll synthesis. Studies have shown that UV-C can mitigate the
   effects of low temperatures on chlorophyll content generated [[123]47].
   Through specific measures to avoid potato damage the photosynthetic
   system to reduce the impact of low temperature on chlorophyll content
   produced.

4.4. Proteins related to protein synthesis and protein destination

   In this study there were 10 protein spots involved in protein synthesis
   and destination, accounting for 20% of all identified proteins. The
   ribosomal protein P0 (P15, P18) exhibited upregulated expression under
   low temperature stress, indicating that low temperature stress
   increased the rate of protein synthesis for adaptation to the low
   temperature environment [[124]48]. This may be due to the fact that
   different proteins have different regulatory pathways, which has been
   reported for abiotic stress responses in other plant species. The 26S
   proteosome involved in protein destination is a multi-subunit complex
   that has the activity of a proteolytic enzyme and is ubiquitin
   dependent. It plays important roles in DNA repair, proteolysis, and the
   regulation of the physiological functions of the cell [[125]49]. 26S
   proteosome exhibited downregulated expression under low temperature
   stress, indicating that the metabolic rate in the potato was reduced
   and that proteolysis was affected.

4.5. Proteins related to signal transduction

   Proteins related to signal transduction identified in this study
   include calreticulin (P3). Calreticulin plays important roles in
   maintaining the dynamic Ca2+ balance in plants and is involved in plant
   growth, development, and stress responses. Recently, many studies have
   confirmed the functions of calreticulin in plant stress resistance and
   disease resistance. Studies have shown that calreticulin is highly
   expressed under stressors, and that this increased expression enhances
   plant stress resistance [[126]50,[127]51,[128]52]. The calreticulin P3
   identified in this study exhibited downregulated expression upon low
   temperature stress followed by a normal expression level, indicating
   that upon low temperature stress, the abundance of water-soluble
   calreticulin in the potato was reduced, while the abundance of
   fat-soluble calreticulin increased. It is possible that after the
   potato was exposed to low temperature stress, calreticulin was
   transported to the endoplasmic reticulum, thereby being involved in low
   temperature stress signal transduction.

4.6. Proteins related to disease defense

   Under low temperature stress high levels of ROS are accumulated in
   plants, which causes the oxidation of protein, lipids, and DNA. This
   can damage the structure of the cell membrane and lead to injuries in
   plants [[129]45,[130]53]. To defend the organism from oxidative stress,
   plants produce a series of ROS-scavenging enzymes. One study showed
   that the antioxidant enzymes SOD, CAT, and APX are crucial to plant
   defense from disease [[131]54]. In this study, the expression of
   2-cysteine peroxidase (P5, P6) related to disease defense was
   downregulated, indicating that during low temperature stress, the
   disease defense system in the potato was enhanced to defend against the
   damage from low temperature. Under low temperature stress, enhanced
   oxygen scavenging activity in vivo potato ability to reduce the
   accumulation of reactive oxygen species, can reduce the impact of cold
   damage caused.

5. Conclusion

   This study investigated the changes in proteins in potato plants during
   low temperature stress via measuring the changes in physiological
   parameters and proteomics. Results from this study provide a reference
   for studying the mechanism of cold tolerance in the potato. Combining
   2-DE and LC-ESI-MS/MS technology, we identified a total of 52 protein
   spots involved in eight categories of biological processes including
   energy, metabolism, and protein synthesis. As the duration of low
   temperature stress increased, the number of upregulated and
   downregulated protein spots increased. After adapting to the low
   temperature stress for some time, the number of upregulated protein
   spots increased, while the number of downregulated protein spots
   decreased, which may be related to stress responses during low
   temperature stress in potato plants. The downregulation of RuBisCO,
   ribonucleoprotein, 26S protease ATP, and calreticulin, as well as the
   upregulation of glyceraldehyde-3-phosphate dehydrogenase and glutamate
   decarboxylase, may be associated with the defense reponses against low
   temperature stress in the potato. The changes in protein expression
   indicated that the adjustment of potato plants to low temperature
   stress involved multiple metabolic pathways.

Declarations

Author contribution statement

   Huawei Li: Conceived and designed the experiments; Performed the
   experiments; Wrote the paper.

   Wenbin Luo: Performed the experiments.

   Rongchang Ji, Guochun Xu: Analyzed and interpreted the data.

   Yongqing Xu: Contributed reagents, materials, analysis tools or data.

   Sixin Qiu, Hao Tang: Conceived and designed the experiments.

Funding statement

   This work was supported by the Fujian-Profit Research Institutes Basic
   Research and Special (2018R1026-4), the foreign cooperation project of
   Fujian Academy of Agricultural Sciences (DEC201821211) and the
   innovation team of Fujian Academy of Agricultural Sciences
   (STIT2017-2-3).

Data availability statement

   Data included in article/supplementary material/referenced in article.

Declaration of interests statement

   The authors declare no conflict of interest.

Additional information

   No additional information is available for this paper.

References