Proteomic variation in Vitis amurensis and V. vinifera buds during cold acclimation

Scientia Horticulturae 263 (2020) 109143

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Proteomic variation in Vitis amurensis and V. vinifera buds during cold acclimation

T

Valerie Farai Masochaa,b,c,1, Qingyun Lib,d,1, Zhenfei Zhua,c, Fengmei Chaia, Xiaoming Suna, Zemin Wangc,e, Long Yangd, Qingfeng Wanga,b, Haiping Xina,b,* a

CAS Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Wuhan, 430074 China b Center of Economic Botany, Core Botanical Gardens, Chinese Academy of Sciences, Wuhan, 430074, PR China c University of Chinese Academy of Sciences, Beijing, 100049, PR China d Agricultural Big-Data Research Center and College of Plant Protection, Shandong Agricultural University, Taian, 271018, PR China e Beijing Key Laboratory of Grape Sciences and Enology/Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, PR China

ARTICLE INFO

ABSTRACT

Keywords: Grapevine Vitis amurensis iTRAQ Cold acclimation Phenylpropanoid biosynthesis pathway

Amur grape (Vitis amurensis) is a wild grape species with excellent freezing tolerance compared with the widely cultivated common grapevine (V. vinifera). Here, we investigated the effect of cold acclimation (CA) on the proteomes of V. amurensis (cv. Zuoshan-1) and V. vinifera (cv. Jingzaojing) buds to explore the cold-tolerance mechanisms used by these species. The buds were collected in late fall (October) and early winter (December) and subjected to an iTRAQ-based proteomic analysis. A total of 472 and 713 differentially abundant proteins (DAPs) were identified between the two time points in V. amurensis and V. vinifera, respectively. The two species shared 235 DAPs, which were mainly involved in the protein chaperone and metabolic pathways, particularly carbohydrate metabolism. These DAPs represent the general responses to CA in Vitis species. V. amurensis contained less unique DAPs (237) than V. vinifera (478). A functional category analysis indicated that the phenylpropanoid biosynthesis pathway was enriched in V. amurensis. Among the DAPs identified in this pathway, seven upregulated and three downregulated DAPs were present in V. amurensis, while three upregulated and 20 downregulated DAPs were present in V. vinifera. Contrasting patterns were observed between the two species for phenylalanine ammonia-lyase, cinnamoyl-CoA reductase 1, and shikimate O-hydroxycinnamoyl transferase, which accumulated in V. amurensis but decreased in V. vinifera. The qRT-PCR results indicated that the transcriptional changes of 12 genes encoding selected DAPs were all consistent with the changes observed at the protein level. Our work provides new insights into the mechanisms by which cold hardiness is achieved in V. amurensis buds.

1. Introduction Low temperature is a major abiotic stress that affects the spatial distribution, yield, and quality of agricultural and horticultural crops. Although temperate cereal (winter wheat, barley, oats, and so on) and fruit (apple, pear, strawberries, and so on) crops require vernalization to produce a yield, extremely low temperatures can result in severe damage and losses. Low temperatures cause wilting or even death by

directly inhibiting metabolic reactions, and indirectly by inducing osmotic stress that results in cellular dehydration (Levitt, 1980; Steponkus, 1984). Many plants have therefore developed an adaptation strategy known as cold acclimation (CA) that enhances their freezing tolerance (Steponkus, 1984; Thomashow, 1999). After exposure to low but nonfreezing temperatures, a series of modifications occurs in these plants at the biological and biochemical levels, including changes in the plasma

Abbreviations: 2-DE, Two-dimensional gel electrophoresis; BCA, Bicinchoninic acid; CA, Cold acclimation; COR, Cold regulated; FASP, Filter-aided sample preparation; GO, Gene Ontology; iTRAQ, isobaric tags for relative and absolute quantification; KEGG, Kyoto Encyclopedia of Genes and Genomes; ROS, Reactive oxygen species ⁎ Corresponding author at: CAS Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Wuhan, 430074 China. E-mail address: [email protected] (H. Xin). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.scienta.2019.109143 Received 25 October 2019; Received in revised form 9 December 2019; Accepted 13 December 2019 0304-4238/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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membrane composition, the accumulation of sugars and soluble proteins, and the increased activity of antioxidant enzymes (Hincha and Zuther, 2014; Levitt, 1980; Steponkus, 1984). Transcriptional and translational changes are vital for CA processes, with transcription factors (TFs) such as C-repeat binding factors acting as master switches to regulate the expression of a variety of cold-induced genes (Chinnusamy and Zhu, 2007; Thomashow, 2010). Shifts in the isozymic composition of various enzymes and changes in the quality and quantity of the cellular proteins also contributes to the increased cold tolerance of plants after CA (Guy, 1990; Kosová et al., 2011). Grapevines (Vitis sp.) are one of the most important temperate crops. The most commonly cultivated varieties are derived from Vitis vinifera. This species can tolerate temperatures as low as –15 °C if given time to acclimate to dropping temperatures in the fall (Fennell, 2004); however, extremely low temperatures in winter can cause serious damage to its buds and root tissues. In contrast, the wild Amur grape (Vitis amurensis) is extremely cold tolerant, withstanding temperatures as low as −30 °C (Fennell, 2004). It has therefore become a valuable germplasm resource for breeding new grapevine cultivars with excellent cold hardiness. Several studies have examined the mechanisms underlying cold hardiness in grapevine species (Xiao et al., 2006, 2008), particularly in V. amurensis. Significant differences in gene expression patterns have been identified between V. amurensis and V. vinifera under 4 °C (Xin et al., 2013). Low-temperature-induced genes encoding TFs, such as members of the ERF, NAC, and WRKY families, were identified and analyzed in V. amurensis (Xin et al., 2013; Xu et al., 2014). A functional analysis of VaERF057 revealed that it is involved in an ethylene-dependent signal transduction response under cold stress (Sun et al., 2016), while VaAQUILO, a MYB-like TF, is involved in the low temperature response in V. amurensis by regulating the biosynthesis of raffinose (Sun et al., 2018a,b). Furthermore, VaWRKY33 was reported to be transcriptionally regulated by VaERF092 under low temperatures (Sun et al., 2019). Additionally, metabolites such as ascorbate, putrescine, and galactinol were reported to be specifically and highly abundant in the leaves of V. amurensis during CA (Chai et al., 2019). These findings increased our understanding of the low temperature response in V. amurensis. Due to the accessibility of the materials, most of the research into low temperature responses in grapevine has been performed using leaves; therefore, little is known about the responses of grapevine buds during the CA process. The photoperiod influences bud dormancy and CA in V. labruscana and V. riparia (Fennell and Hoover, 1991), while late embryogenesis abundant (LEA)-like proteins accumulated in overwintering buds of Vitis labruscana during CA (Salzman et al., 1996). Transcriptomic variation throughout bud development and during the fulfillment of the chilling requirement was also detected (DíazRiquelme et al., 2012; Mathiason et al., 2009). Rubio and Perez (2019) studied the involvement of abscisic acid and its signaling pathway during CA, which causes the deacclimation of grapevine buds. Despite these rare insights, little is known about the responses of the grapevine buds during the CA process in the continental climate. Proteins are the direct effectors of the stress responses in plants. Stress-related proteins are activated through post-transcriptional and translational modifications, such as phosphorylation, glycosylation, and ubiquitination. These modifications sometimes result in a poor correlation between the level of accumulated proteins and their corresponding mRNAs (Haider and Pal, 2013; Kosová et al., 2018); therefore, proteomics analyses are a powerful tool for investigating how plants adjust their proteins in response to the altered environment. Traditionally, two-dimensional gel electrophoresis (2-DE) technology has been used to study protein abundance under cold stress (Amme et al., 2006); however, recent advances have led to the use of isobaric tags for relative and absolute quantification (iTRAQ) as one of the most robust techniques for the accurate identification and quantification of proteome profiles (Vanderschuren et al., 2013; Zieske,

2006). iTRAQ has been used across various plant species for studies into the proteomic effects of abiotic stresses such as cold (Xu et al., 2018a,b; Zhang et al., 2017), salt (Chen et al., 2016; Ji et al., 2016; Li et al., 2017), heat (George et al., 2015; Liu et al., 2014; Xu et al., 2018a,b), and drought (Alvarez et al., 2014). George and Haynes (2014) reviewed the use of proteomic analyses in the study of abiotic stress in grapevines. While cold-stress-related proteomics studies are rare in grapes, Deng et al. (2017) performed comparative proteomic analyses of V. amurensis and V. vinifera (cv. Muscat of Hamburg) plantlets under low temperatures, revealing that most proteins decreased in abundance. Most of the research into the proteomes of grapevine buds focus on their dormancy or dormancy release (Fennell et al., 2015; George et al., 2018; Khalil-Ur-Rehman et al., 2017; Parada et al., 2016); thus, the proteomic modification of grapevine buds during the CA process is yet to be elucidated. Here, we report an iTRAQ-based comparative proteomic analysis of V. amurensis and V. vinifera buds during CA in field conditions. The study was designed to identify differentially abundant proteins (DAPs) between two stages of the CA process in V. amurensis and V. vinifera buds exposed to gradually decreasing temperatures in late fall. The results reveal proteomic modifications during CA in both species, and provide clues for further elucidating the molecular mechanisms regulating cold tolerance in V. amurensis. 2. Materials and methods 2.1. Plant materials V. amurensis cv. Zuoshan-1 and V. vinifera cv. Jingzaojing were grown in the vineyard at the Institute of Botany, Chinese Academy of Sciences, Beijing, China. Buds were collected from one-year-old branches at two time points: late fall (October 20th, 2016) and early winter (December 2nd, 2016). The daily maximum and minimum temperatures for October, November, and December are shown in Supplementary Figure S1. For the proteomic analysis, three biological repeats were conducted, with each replication containing ten buds randomly picked from different branches. For the RNA isolation and quantitative reverse transcription PCR (pRT-PCR), three buds were combined into a biological replicate, and three biological replications were performed. The samples were quickly frozen in liquid nitrogen and stored at −80 °C prior to use. 2.2. Protein extraction, in-solution digestion, and peptide extraction The collected buds were ground into powder in liquid nitrogen and transferred into a 5-mL centrifuge tube. Four volumes of lysis buffer (8 M urea, 1 % Triton-100, 10 mM dithiothreitol, and 1 % Protease Inhibitor Cocktail) were added to each sample, followed by three rounds of sonication on ice using a high-intensity ultrasonic processor (Scientz, Ningbo City, China).The debris was removed using a centrifugation at 20,000 g for 10 min at 4 °C. The protein was precipitated with cold 20 % TCA for 2 h at −20 °C. After another centrifugation at 12,000 g for 10 min at 4 °C, the supernatant was discarded. The remaining precipitate was washed three times with cold acetone, after which the protein was resuspended in 8 M urea and its concentration was quantified using the BCA protein assay reagent. The protein solution for each sample was reduced with 10 mM dithiothreitol for 30 min at 56 °C, and then alkylated with 50 mM iodoacetamide in darkness for 15 min at room temperature. Precooled acetone was added, and the samples were incubated at −20 °C for 2 h. After centrifugation at 20,000 g for 10 min, the precipitate was diluted with 0.25 M TEAB and the concentrations of the peptides was measured. For each sample, 150 μg peptides were digested using trypsin (Promega), which was added at a 1:50 trypsin: protein mass ratio for the first overnight digestion, and a 1:100 trypsin: protein mass ratio for 2

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a second 4-h digestion. The digestion was then stopped by adding 50 % acetic acid, and was followed by a centrifugation at 12,000 g for 45 min at 37 °C. The precipitates were rinsed with 0.1 % TFA and dissolved in 20 μL TEAB (0.5 M).

2.6. Functional annotation of the proteins The functional annotation of the proteins was conducted using a BLASTp search of the non-redundant proteins database in NCBI. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations were derived from the grape database and KEGG database (https://www.kegg.jp/; Kanehisa et al., 2017), respectively. The GO and metabolic pathway enrichment analyses of the DAPs were performed using the online software agriGO v2 (http://systemsbiology. cau.edu.cn/agriGOv2/; Tian et al., 2017) and the KEGG Automatic Annotation Server (KAAS) (https://www.genome.jp/tools/kaas/; Moriya et al., 2007), respectively. The statistical significance of any identified enrichment was determined using Fisher’s exact test (corrected p value < 0.05).

2.3. iTRAQ labeling and high-performance liquid chromatography (HPLC) fractionation A 100-μg aliquot of each trypsin-digested peptide sample was labeled using an iTRAQ Reagent-8plex Multiplex Kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. Three biological repeats of four labelled peptide mixtures from two grapevines at the two time points were pooled and dried using vacuum centrifugation. The peptides were subjected to cation exchange fractionation using a high-pH reversed-phase HPLC with an Agilent 300 Extend C18 (5 μm particle size, 4.6 mm internal diameter, 250 mm length). A total of 60 fractions were separated and combined into 18 fractions after desalting, and the combined fractions were concentrated using vacuum centrifugation and stored at −80 °C until required for the liquid chromatography-mass spectrometry (LC–MS)/MS analysis.

2.7. qRT-PCR analysis A qRT-PCR analysis was performed to determine the expression levels of genes encoding selected DAPs. Total RNA was extracted from three independent replications of each sample using a Plant Total RNA Isolation Kit (Tianz Inc., Beijing, China). The RNAs were subjected to a DNase treatment before being used to synthesize first-strand cDNAs using HI Script® II Reverse Transcriptase (Vazyme Biotech Co., Nanjing, China). Two technical replicates were performed for each biological replicate, and the PCR was performed using SYBR Green PCR master mix (Vazyme Biotech Co., Nanjing, China), following the manufacturer’s instructions. VvActin was used as the internal control. The gene-specific primer pairs were designed using Primer-BLAST (http:// www.ncbi.nlm.nih.gov/tools/primer-blast/), and are shown in Supplementary Table S1.

2.4. LC–MS/MS analysis The MS/MS analysis was performed on an EASY-nLC 1000 UPLC system connected to a Q-Exactive mass spectrometer (Thermo Fisher Scientific). The tryptic peptides were dissolved in solvent A (0.1 % formic acid) and directly loaded onto a reversed-phase analytical column (75 μm ×15 cm, 3 μm, 200 Å; Thermo Fisher Scientific). Using a constant flow rate of 400 nL/min and a column temperature of 35 °C, the separation was performed using a linear gradient of solvent B (0.1 % formic acid in 98 % acetonitrile) as follows: an increase of 6%–23% over 26 min, increasing from 23 % to 35 % over 8 min, climbing to 80 % in 3 min, then holding for the last 3 min. The peptide fractions were then subjected to a nanospray ionization source followed by tandem mass spectrometry (MS/MS) using a QExactive Plus (Thermo Fisher Scientific) for 60 min. The mass spectrometer was operated in positive ionization mode, with a m/z scan range of 350–1800 for the full scan. The intact peptides were detected in the Orbitrap at a resolution of 70,000 at 100 m/z, with the automatic gain control (ACG) set at 3.0 × 10–6. Peptides were then selected for the MS/ MS2 scan using NCE setting as 28, and the fragments were detected in the Orbitrap at a resolution of 17,500 at 200 m/z with a 15.0-s dynamic exclusion. Ion fragmentation was performed using a higher energy collision dissociation with normalized collision energies of 50 % and an ion injection time of 30 s. Three biological replicates were performed for this experiment.

2.8. Statistical analysis A series of statistical tests were performed to identify the DAPs in the two grapevines during CA using the t-test function in R (v3.5.1). For the qRT-PCR experiments, the relative expression levels of the selected genes were calculated using a double delta Ct (2–ΔΔCt) analysis, and the corresponding figures were drawn using GraphPad Prism v8.0 software (GraphPad Software Inc., La Jolla, CA, USA) (Berkman et al., 2019). The Venn diagram was drawn using the online software jvenn (http:// jvenn.toulouse.inra.fr/app/index.html) (Bardou et al., 2014). 3. Results 3.1. Overview of the proteomics data The buds were collected in late fall when the grapevines begin to defoliate. The maximum and minimum temperatures on October 20th, 2016, were about 15 °C and 11 °C, respectively. The second time point for sampling was December 2nd, 2016, when the maximum and minimum temperatures dropped to 9 °C and −3 °C, respectively (Supplementary Figure S1). We compared these time points to explore the different responses of the V. amurensis cv. Zuoshan-1 and V. vinifera cv. Jingzaojing flower buds during CA. A total of 7859 distinct proteins were identified in the buds of these two grapevines. After eliminating low-scoring data, 6639 proteins were analyzed (Supplementary Table S2). Their GO annotations showed that the identified proteins cover a wide range of biological processes, including cellular processes (74.1 %), metabolic processes (73.3 %), single-organism processes (65.4 %), responses to stimuli (41.5 %), biological regulation (29.8 %), cellular component organization or biogenesis (27.3 %), localization (23.0 %), and developmental processes (21.3 %) (Supplementary Figure S2). Proteins that underwent a 1.2-fold or greater change (P < 0.05) in abundance between the two time points in all three independent iTRAQ experiments in a particular species were identified as DAPs. A total of

2.5. MS/MS data analysis and quality control The raw MS/MS spectra were analyzed using the Max Quant search engine (v1.5.2.8) with the V. vinifera protein database (55,564 entries; http://genomes.cribi.unipd.it/DATA/V2/V2.1/; Vitulo et al., 2014), concatenated with a reverse decoy database. The search parameters were set as follows: trypsin was specified as the cleavage enzyme, allowing up to two missing cleavages. Cysteine carbamidomethylation was specified as the fixed modification, and the oxidation of methionine was specified as a variable modification. The mass tolerance for the precursor ions was set as 20 ppm in the first search and 5 ppm in the main search. The mass tolerance for the fragment ions was set at 0.02 Da. Using a false discovery rate (FDR) < 0.01, the data were filtered, and the minimum score for peptides was set at > 40. Proteins identified as being differently abundant between the two time points in all three experiments (with a 1.2-fold cut-off and a p value < 0.05) were considered to be DAPs. 3

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Table 1 The number of DAPs (more than 1.2-fold or less than 0.834-fold change, P < 0.05) in V. amurensis and V. vinifera during cold acclimation.

Upregulated Downregulated Unique Total

V. amurensis

V. vinifera

Overlapped

196 276 237 472

308 405 478 713

100 135 – 235

Table 2 The detailed information of top 10 upregulated DAPs in V. amurensis and V. vinifera. Va

1 2

Vv

4

3 4 5 6 7 8 9 10

Fig. 1. Venn diagram shows the DAPs in V. amurensis and V. vinifera during cold acclimation. The up arrow indicates upregulated proteins and the down arrow indicates downregulated proteins. 235 proteins are the total overlapping DAPs in both species.

Protein accession

VIT_208s0007g07780.1 VIT_212s0059g00440.1 VIT_208s0040g02330.2 VIT_204s0023g02900.1 VIT_216s0100g00960.1 VIT_215s0048g02600.1

5

VIT_216s0050g00390.1 VIT_202s0012g00750.3 VIT_202s0025g02790.2

1 2 3

VIT_202s0025g04260.1 VIT_207s0005g01680.1 VIT_208s0007g05580.1 VIT_200s0301g00090.5

6 7

VIT_219s0090g00660.2 VIT_202s0154g00090.1

8

VIT_212s0059g02590.1

9 10

VIT_200s0309g00090.1 VIT_216s0050g02210.1

Description

polygalacturonase keratin-associated protein 6-2 elongation factor 1-alpha cytochrome P450 84A1 stilbene synthase 4-like granule-bound starch synthase 1 oxalate–CoA ligase haloacid dehalogenase-like granule-bound starch synthase 1 thaumatin-like protein stachyose synthase putative methyltransferase NAD(P)H-ubiquinone oxidoreductase A1 GDSL esterase acid betafructofuranosidase cysteine-rich repeat secretory protein 38-like expansin-like B1 acidic endochitinase

Fold changes Va

Vv

2.59 2.26

3.59 –

2.1 1.97 1.96 1.85

0.67 1.99 – 1.48

1.82 1.81 1.79

3.00 0.76 1.77

1.74 – 1.58 1.39

– 6.20 4.03 3.83

– 1.52

2.66 2.63

–

2.56

– –

2.52 2.45

among the identified DAPs in two species, with an up to 6.20-fold increase only in V. vinifera. Methyltransferase (VIT_208s0007g05580.1), NAD(P)Hubiquinone oxidoreductase A1 (VIT_200s0301g00090.5), and acid betafructofuranosidase (VIT_202s0154g00090.1) accumulated in both grapevines during CA. Another four proteins, including GDSL esterase (VIT_219s0090g00660.2) and expansin-like B1 (VIT_200s0309g00090.1), increased in V. vinifera during CA.

928 DAPs was identified in two species, including 382 upregulated DAPs (more abundant in the early winter (December) measurement than in the late fall (October) sample), 524 downregulated DAPs, and 22 DAPs with contrasting patterns in the two grapevine species. The numbers of DAPs and the overlaps between the two grapevines are summarized in Table 1. V. amurensis had fewer DAPs (472) than V. vinifera (713). We observed that 235 DAPs had similar patterns in the two grapevines, including 100 upregulated and 135 downregulated proteins. V. vinifera had more unique DAPs (208 upregulated and 270 downregulated) than V. amurensis (96 upregulated and 141 downregulated), as shown in Fig. 1. Detailed information about the DAPs is provided in Supplementary Table S3.

3.3. Functional categories of the overlapping CA-related DAPs in V. amurensis and V. vinifera The DAPs that could be identified in both V. amurensis and V. vinifera may represent the fundamental responses that occur in all grapevines during the CA process. The functions of the DAPs shared by the two grapevines were explored using a KEGG pathway enrichment analysis, and the results are shown in Fig. 2. A total of 19 pathways were enriched, including 14 upregulated and 13 downregulated pathways. Detailed information about each pathway is provided in Supplementary Table S4. The eight pathways related to the metabolism, carbohydrate metabolism, transport and catabolism, cellular processes, biosynthesis of other secondary metabolites, glycosyltransferases, metabolism of other amino acids, and pentose and glucuronate interconversions were enriched in both the upregulated and downregulated DAPs. The upregulated DAPs were also enriched in the protein processing (chaperones and folding catalysts, protein processing in the endoplasmic reticulum) and energy metabolism (oxidative phosphorylation and starch and sucrose metabolism) pathways. Protein chaperones help to stabilize proteins under stress conditions, while the oxidative phosphorylation and starch and sucrose metabolisms may provide the energy required for the basic metabolic processes and stress responses (Thalmann and Santelia, 2017). The downregulated DAPs indicated a reduced activity in several cytological processes, including those related to the exosome, phagosome, cytoskeleton proteins, and amino sugar and nucleotide sugar metabolism.

3.2. Analysis of the 10 most upregulated DAPs in V. amurensis and V. vinifera during CA The top 10 upregulated DAPs in both grapevines are shown in Table 2. Polygalacturonase (VIT_208s0007g07780.1), which is involved in polyuronide degradation, increased by 2.59-fold in V. amurensis during CA, and by 3.59-fold in V. vinifera. Another three DAPs had similar patterns, including cytochrome P450 84A1 (VIT_204s0023g02900.1), granule-bound starch synthase 1 (VIT_215s0048g02600.1, VIT_202s0025g02790.2), and oxalate-CoA ligase (VIT_216s0050g00390.1). Three DAPs only increased in V. amurensis, including keratin-associated protein 6-2 (VIT_212s0059g00440.1), stilbene synthase 4-like (VIT_216s0100g00960.1), and thaumatin-like protein (VIT_202s0025g04260.1). Two of the upregulated DAPs in V. amurensis, including elongation factor 1-alpha (VIT_208s0040g02330.2) and haloacid dehalogenase-like (VIT_202s0012g00750.3), decreased in V. vinifera during CA. Stachyose synthase (VIT_207s0005g01680.1), which is involved in the biosynthesis of stachyose from raffinose, showed the highest fold change 4

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Fig. 2. Enriched KEGG pathways of overlapped DAPs between V. amurensis and V. vinifera during cold acclimation. The x axis indicates the percentage of DAPs numbers of specific pathway to all overlapped DAPs. The red, blue, and grey bars of y axis display upregulated, downregulated, and no enriched pathways, respectively (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

3.4. Pathways specifically enriched in V. amurensis and V. vinifera during CA

the exception of the pyruvate metabolism and glycolysis/gluconeogenesis pathways, which are related to energy supply (Fig. 3B). A total of 13 pathways were enriched in the upregulated V. vinifera DAPs. Several biological processes, such as ribosome biogenesis, carbon fixation, and mitochondrial biogenesis, were enhanced, as well as the glutathione metabolism pathway, which participates in the stress response. The downregulated V. vinifera DAPs were mainly enriched in the secondary metabolism pathways, such as phenylalanine metabolism, flavonoid biosynthesis, and phenylpropanoid biosynthesis (Fig. 3D). The phenylpropanoid biosynthesis pathway showed a robust decrease in V. vinifera, but was dramatically increased in V. amurensis, as mentioned above.

Among the 928 DAPs identified in this experiment, 671 only had an altered pattern in one species of grapevine during CA, while 22 DAPs displayed contrasting patterns in the two grapevines (Table 1 and Supplementary Table S3). More than twice as many unique DAPs were identified in V. vinifera (478) than in V. amurensis (237). A KEGG analysis was performed for these unique DAPs to further elucidate the pathways enriched in the different grapevines. As shown in Fig. 3, more enriched pathways were identified in V. vinifera than in V. amurensis, probably because of its higher number of unique DAPs. Detailed information about each enriched pathway is presented in Supplementary Table S5. Five pathways were enriched in the upregulated V. amurensis DAPs (Fig. 3A). Four of these pathways (endocytosis, protein processing in the endoplasmic reticulum, oxidative phosphorylation, and chaperones and folding catalysts) were also enriched in V. vinifera (Fig. 3C), while the phenylpropanoid biosynthesis pathway was enriched in the upregulated V. amurensis DAPs (Fig. 3A) but also in the downregulated V. vinifera DAPs (Fig. 3D). Most of the identified pathways enriched in the downregulated V. amurensis DAPs can also be found in V. vinifera, with

3.5. The phenylpropanoid biosynthesis pathway is differently regulated in the two grapevines during CA To further elucidate the different responses in the two grapevines during CA, the identified DAPs involved in phenylpropanoid biosynthesis were analyzed. A total of 39 phenylpropanoid biosynthesisassociated proteins were identified in our experiments (Supplementary Table S6), 26 of which were significantly altered during the CA process in at least one species of grapevine (Table 3). Seven of the upregulated

Fig. 3. Enriched KEGG pathways of uniquely DAPs in V. amurensis and V. vinifera during cold acclimation. (A) Upregulated DAPs in V. amurensis (Va DAPs up), (B) Downregulated DAPs in V. amurensis (Va DAPs down), (C) Upregulated DAPs in V. vinifera (Vv DAPs up) and (D) Downregulated in V. vinifera (Vv DAPs down). 5

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Table 3 DAPs involved in phenylpropanoid biosynthesis in V. amurensis (Va) and V. vinifera (Vv). Protein accession

VIT_204s0023g02900.1 VIT_200s0371g00100.1 VIT_211s0052g01090.1 VIT_208s0040g01710.1 VIT_215s0048g02480.1 VIT_209s0018g01190.3 VIT_209s0070g00240.1 VIT_204s0023g02570.2 VIT_206s0009g00810.5 VIT_214s0006g01790.1 VIT_218s0001g06850.4 VIT_205s0020g00600.1 VIT_202s0025g02920.1 VIT_206s0004g02620.1 VIT_201s0010g03720.1 VIT_211s0016g05280.1 VIT_206s0004g08150.1 VIT_213s0067g02360.1 VIT_206s0004g07770.1 VIT_210s0116g01780.1 VIT_207s0031g00350.1 VIT_213s0019g04460.1 VIT_206s0004g06110.3 VIT_216s0098g00850.1 VIT_210s0003g05420.1 VIT_214s0036g01050.1 1 2

KEGG

K09755 K00083 K01904 K10775 K13066 K13065 K09753 K00430 K01188 K05350 K00430 K11188 K13066 K10775 K01904 K00430 K00487 K00430 K00430 K00430 K00588 K10775 K05349 K13066 K22395 K01188

Fold Change1,

Description

cytochrome P450 84A1 [Vitis vinifera] probable mannitol dehydrogenase [Vitis vinifera] 4-coumarate–CoA ligase 2 [Vitis vinifera] phenylalanine ammonia-lyase [Vitis vinifera] caffeic acid 3-O-methyltransferase [Vitis vinifera] shikimate O-hydroxycinnamoyltransferase [Vitis vinifera] cinnamoyl-CoA reductase 1 [Vitis vinifera] peroxidase 72 [Vitis vinifera] beta-glucosidase BoGH3B-like [Citrus sinensis] beta-glucosidase 44 [Vitis vinifera] cationic peroxidase 1 [Vitis vinifera] 1-Cys peroxiredoxin [Vitis vinifera] caffeic acid 3-O-methyltransferase [Vitis vinifera] phenylalanine ammonia-lyase [Vitis vinifera] 4-coumarate–CoA ligase-like 1 [Vitis vinifera] peroxidase 25-like [Vitis vinifera] trans-cinnamate 4-monooxygenase [Vitis vinifera] peroxidase 4-like [Vitis vinifera] peroxidase 4 [Vitis vinifera] peroxidase 42 [Vitis vinifera] caffeoyl-CoA O-methyltransferase [Vitis vinifera] phenylalanine ammonia-lyase G1 [Vitis vinifera] beta-glucosidase BoGH3B [Theobroma cacao] caffeic acid O-methyltransferase isoform X1 [Vitis vinifera] berberine bridge enzyme-like 15 [Vitis vinifera] beta-glucosidase BoGH3B-like [Prunus mume]

2

Va

Vv

1.97 1.49 1.40 1.38 1.30 1.27 1.22 0.79 0.74 0.69 0.66 0.59 – – – – – – – – – – – – – –

1.99 1.24 – 0.69 – 0.72 0.52 0.83 0.72 0.69 0.74 – 1.84 0.32 0.66 0.37 0.83 0.77 0.75 0.26 0.46 0.58 0.64 0.57 0.66 0.71

The italic or bold colours of fold change values indicate statistically significant increased or decreased of proteins in each grapevine. Other proteins identified in this pathway but didn’t change in both grapevines were shown in Supplementary Table S6.

DAPs in V. amurensis were associated with phenylpropanoid biosynthesis, including the enzymes phenylalanine ammonia-lyase, shikimate Ohydroxycinnamoyl transferase, cinnamoyl-CoA reductase 1, 4-coumarate–CoA ligase 2, and caffeic acid 3-O-methyltransferase (Table 3). Only three phenylpropanoid biosynthesis proteins (mannitol dehydrogenase¸ cytochrome P450 84A1, and caffeic acid 3-O-methyltransferase) became more abundant in V. vinifera during CA. While 20 DAPs in this pathway decreased in V. vinifera, only five of them did so in V. amurensis (Table 3). These results strongly indicate that the phenylpropanoid biosynthesis pathway is differently regulated in V. amurensis and V. vinifera during CA.

of their tissue-specific cold responses, and we present candidate proteins and pathways that could be explored in future studies to further elucidate the excellent cold hardiness of V. amurensis. In general, CArelated treatments in model plants and crops are mainly performed under highly controlled conditions; however, the samples in this experiment were collected from a vineyard under the natural CA conditions during late fall and early winter. In addition to a gradual reduction in temperature, several environmental factors, such as photoperiod, the daily temperature range, wind, and rainfall, also affect the dormancy of the buds; therefore, the iTRAQ-determined data presented here reflect the real proteomic modifications that occur under the naturally complex CA conditions.

3.6. A qRT-PCR analysis revealed the correlation between expression and proteomic levels

4.1. Carbohydrate and energy metabolism

To detect the transcriptional-level changes in the genes encoding the identified DAPs and their correlation with the iTRAQ-determined protein abundance, 12 DAPs were randomly selected and subjected to a qRT-PCR analysis. The Pearson correlation coefficient between the iTRAQ and qRT-PCR data was high (R2 > 0.5) across all selected genes (Fig. 4). All detected genes, including those encoding eight upregulated and four downregulated DAPs, had similar expression patterns to the iTRAQ data, although the expression levels of the detected genes changed more dramatically than the protein-level differences. Several genes, such as those encoding remorin and ubiquinol, showed an obvious accumulation at the transcription level in V. vinifera, but not at the protein level (Fig. 4). Post-transcriptional modification and translational regulation may explain the inconsistent results between the transcriptional and translational levels.

Some of the DAPs involved in carbohydrate metabolism were downregulated in both species (Fig. 2), consistent with former results in grafted watermelon (Citrullus lanatus) (Shi et al., 2019) and Carex rigescens (Li et al., 2017). Transketolase (VIT_215s0048g00370.1), a key enzyme of carbohydrate metabolism (Kosová et al., 2018; Rocha et al., 2014), was downregulated in both grapevine species (Supplementary Table S4), as were the majority of DAPs related to amino sugar and nucleotide sugar metabolism (Fig. 2 and 3), suggesting that cold stress inhibited the interconversion and biosynthesis of carbohydrates in the buds. As the main carbohydrate reserve in plants, starch accumulates in the leaves during the day and is degraded throughout the night to be exported to the cytosol in the form of soluble sugars such as glucose, maltose, and sucrose (Gibon et al., 2004). In woody plants such as grapevines, starch is stored in the heterotrophic tissues for use during spring and winter (Hiraki et al., 2019). Sugars not only store energy and carbon, but also act as membrane stabilizers and antioxidants in response to cold stress (Yue et al., 2015). In the present study, the starch and sucrose metabolic pathways were elevated in response to CA, especially in V. vinifera, indicating that the degradation of starch into

4. Discussion In this study, we identified DAPs in V. amurensis and V. vinifera buds during CA. We explored the differences between the patterns of protein accumulation in these two species, which increased our understanding 6

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Fig. 4. Relative expression of 12 candidate DAPs for qRT-PCR analysis and protein abundance correlation. Error bars indicate the standard error of the mean and the asterisks (*, ** and ***) represent significant differences at P < 0.05, 0.01 and 0.001, respectively.

sucrose and/or raffinose plays a crucial role in the response to cold stress (Figs. 2 and 3C). Similar findings have been reported in tea (Camelia sinensis) (Yue et al., 2015) and blueberry (Vaccinium corymbosum) (Lee et al., 2012). Also, starch biosynthesis-related enzymes, such as granule-bound starch synthase 1 (VIT_202s0025g02790.2 and VIT_215s0048g02600.1), were upregulated in both species during CA (Supplementary Table S4). These results suggest that the accumulation of carbohydrates through the induction of starch and sucrose metabolism might improve cold hardiness. The proteins related to energy metabolism became more abundant during CA, and most of them accumulated to higher levels in V. amurensis than in V. vinifera. The upregulation of the ATP-dependent zinc metalloproteases FTSH 2 and 6 (VIT_214s0108g00590.1 and VIT_212s0028g01600.1) and ATPase (VIT_211s0052g00970.1) (Supplementary Table S3) suggests that energy metabolism increased during CA, which is consistent with the increased energy demand reported in Hibiscus sp. plants during this process (Paredes and Quiles, 2015). In contrast, the inhibition of V-ATPase activity was previously reported to be one of the primary events in chilling injuries (Yan et al., 2006). The V-type proton ATPase subunit E (VIT_202s0025g01000.1) was downregulated in both grapevine species, consistent with observations in rice (Oryza sativa) (Rocha et al., 2014) and sunflower (Helianthus annuum) (Kosová et al., 2018) under cold stress. Photosynthesis-related pathways and the corresponding DAPs were more strongly downregulated in cold-sensitive V. vinifera than in V. amurensis (Fig. 3 and Supplementary Table S5). Similar findings have been reported in winter turnip rape (Brassica rapa spp. oleifera), grafted watermelon seedlings, and dormant grapevine plants (George et al., 2018; Shi et al., 2019; Xu et al., 2018a,b). Paredes and Quiles (2015) reported that photosystem II (PSII) was inhibited under cold stress in chilling-sensitive Chinese hibiscus (Hibiscus rosa-sinensis). Oxygenevolving enhancer (OEE), a chloroplast protein, was downregulated to

enhance the stability of PSII under cold stress (Xu et al., 2018a,b), which was consistent with its downregulation in the two grapevines during CA, suggesting a reduced capacity for light absorption in these plants. Furthermore, two drivers of the photosynthetic electron transport of PSII (Shi et al., 2019), the chlorophyll a/b-binding proteins VIT_212s0055g01110.1 and VIT_208s0007g02190.1, were decreased in V. amurensis and V. vinifera during CA. The reduced levels of these photosynthetic proteins indicate a reduction in their light absorption, energy capture, and partitioning of photosynthate assimilates in the buds during CA. 4.2. Stress response and defense-related proteins In plants, the stress-induced accumulation of excessive reactive oxygen species (ROS) causes detrimental oxidative damage to the cellular proteins and membrane lipids, and even cell death. The major ROS scavengers, such as glutathione, ascorbates, and secondary metabolites (flavonoids, carotenoids), therefore function to maintain the balance of ROS under stress conditions (Sharma et al., 2012). Glutathione Stransferase (GST) is involved in the detoxification of ROS by their conjugation to glutathione (Gharechahi et al., 2016). In the present study, the upregulation of GST3 (VIT_212s0028g00920.2) in both species suggested that its antioxidative function was involved in CA. NADH dehydrogenase, a large protein complex in the respiratory chain, was upregulated in both species during CA. This enzyme impairs the accumulation of excess ROS levels during CA (Podgorska et al., 2013), which suggests that it may play a similar role in these grapevine species. Cell dehydration during CA is necessary for reducing cellular susceptibility to freezing injury (Ouyang et al., 2019). The LEA protein family, known to be responsive to abiotic stresses in many plant species (Gao and Lan, 2016), includes the dehydrins, which can prevent 7

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freezing injury through their retention of water molecules (Liu et al., 2017). The increased abundance of dehydrin proteins in plant buds in response to CA and dormancy has been previously noted (Rubio et al., 2018; Wisniewski et al., 2014). In the present study, the elevation of two LEA proteins (VIT_209s0002g06070.1 and VIT_205s0020g00840.1) and the dehydrin ERD14 (Early Response to Dehydration 14, VIT_218s0001g00360.1) in both V. amurensis and V. vinifera are consistent with former studies (Salzman et al., 1996; Navarro et al., 2015), suggesting that the accumulation of LEA and ERD proteins is closely correlated with CA and freezing tolerance in buds.

prevents freezing damage and cell collapse (Le Gall et al., 2015). The biosynthesis of secondary metabolites such as phenolics, flavonoids, and lignin is also important for plant defense mechanisms. We therefore hypothesize that the excellent cold hardiness of V. amurensis can be at least partially attributed to its increased phenylpropanoid biosynthesis during CA. CRediT authorship contribution statement Valerie Farai Masocha: Writing - original draft, Formal analysis. Qingyun Li: Formal analysis, Methodology, Software. Zhenfei Zhu: Visualization, Investigation, Formal analysis. Fengmei Chai: Data curation, Formal analysis. Xiaoming Sun: Investigation, Formal analysis. Zemin Wang: Data curation, Formal analysis. Long Yang: Project administration. Qingfeng Wang: Resources, Project administration. Haiping Xin: Conceptualization, Project administration, Resources, Validation, Writing - review & editing.

4.3. Protein biosynthesis and metabolism Low temperatures cause the unfolding, oxidation, misfolding, and degradation of proteins; therefore, it is necessary to eliminate these damaged proteins and replace them with new proteins, which is often achieved with the help of protective chaperone proteins (Gharechahi et al., 2016). The high abundance of the DAPs was found to associate with the chaperone and folding catalysis pathway and the pathway of protein processing in the endoplasmic reticulum (Figs. 2 and 3). Ribosomal biogenesis-related proteins showed increased accumulation levels in the cold-sensitive V. vinifera, but did not increase during CA in V. amurensis (Fig. 3C). Ribosomal biogenesis has been linked to stress signaling and responses (Palm et al., 2018); therefore, we speculate that proteins in V. vinifera suffer more damage and require more extensive repair processes than those in V. amurensis during CA. Molecular chaperone proteins such as the heat shock proteins (HSPs) play a significant role in protecting plants against extreme temperatures. They are known for regulating protein folding to prevent further cellular damage (Wang et al., 2004). HSP accumulation has been suggested to be a possible stimulus for bud dormancy in grapes (Ophir et al., 2009). Here, we observed that two HSP proteins (VIT_211s0037g00510.1 and VIT_209s0002g06790.1) and the chaperone protein ClpB4 (Caseinolytic peptidase B4, VIT_204s0008g05870.1) were significantly upregulated in both grapevine species during CA (Supplementary Table S4). In the present study, an increased abundance of cysteine protease (VIT_204s0023g02450.1) and ATP-dependent zinc metalloprotease FTSH 6 (VIT_214s0108g00590.1) in both species suggests that many proteins were commonly involved in protein degradation processes during CA (Supplementary Table S3).

Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgements This work was supported by the National Key Research and Development Program of China (2018YFD1000300), the National Natural Science Foundation of China (NSFC Accession No. 31672132) and the Grape Breeding Project of Ningxia (NXNYYZ201502). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.scienta.2019.109143. References Alvarez, S., Choudhury, S.R., Pandey, S., 2014. Comparative quantitative proteomics analysis of the ABA response of roots of drought-sensitive and drought-tolerant wheat varieties identifies proteomic signatures of drought adaptability. J. Proteome Res. 13 (3), 1688–1701. Amme, S., Matros, A., Schlesier, B., Mock, H.P., 2006. Proteome analysis of cold stress response in Arabidopsis thaliana using DIGE-technology. J. Exp. Bot. 57 (7), 1537–1546. Bardou, P., Mariette, J., Escudie, F., Djemiel, C., Klopp, C., 2014. Jvenn: an interactive Venn diagram viewer. BMC Bioinformatics 15, 293. Berkman, S.J., Roscoe, E.M., Bourret, J.C., 2019. Comparing self-directed methods for training staff to create graphs using Graphpad Prism. J. Appl. Behav. Anal. 52 (1), 188–204. Chai, F., Liu, W., Xiang, Y., Meng, X., Sun, X., Cheng, C., Liu, G., Duan, L., Xin, H., Li, S., 2019. Comparative metabolic profiling of Vitis amurensis and Vitis vinifera during cold acclimation. Hortic. Res. 6, 8. Chen, T., Zhang, L., Shang, H., Liu, S., Peng, J., Gong, W., Shi, Y., Zhang, S., Li, J., Gong, J., Ge, Q., Liu, A., Ma, H., Zhao, X., Yuan, Y., 2016. iTRAQ-based quantitative proteomic analysis of cotton roots and leaves reveals pathways associated with salt stress. PLoS One 11 (2), 0148487. Chinnusamy, V., Zhu, J.K., 2007. Cold stress regulation of gene expression in plants. Trends Plant Sci. 12 (10), 444–451. Deng, J., Yin, X., Xiang, Y., Xin, H.P., Li, S.H., Yang, P.F., 2017. iTRAQ-based comparative proteomic analyses of two grapevine cultivars in response to cold stress. Curr. Proteomics 14 (1), 42–52. Díaz-Riquelme, J., Grimplet, J., Martinez-Zapater, J.M., Carmona, M.J., 2012. Transcriptome variation along bud development in grapevine (Vitis vinifera L.). BMC Plant Biol. 12 (1), 181. Fennell, A., 2004. Freezing tolerance and injury in grapevines. J. Crop. Improv. 10 (1-2), 201–235. Fennell, A., Hoover, E., 1991. Photoperiod influences growth, Bud Dormancy, and coldacclimation in Vitis-Labruscana and V-Riparia. J. Am. Soc. Hortic. Sci. 116 (2), 270–273. Fennell, A.Y., Schlauch, K.A., Gouthu, S., Deluc, L.G., Khadka, V., Sreekantan, L., Grimplet, J., Cramer, G.R., Mathiason, K.L., 2015. Short day transcriptomic programming during induction of dormancy in grapevine. Front. Plant Sci. 6, 834. Gao, J., Lan, T., 2016. Functional characterization of the late embryogenesis abundant (LEA) protein gene family from Pinus tabuliformis (Pinaceae) in Escherichia coli. Sci. Rep. 6, 19467. George, I.S., Fennell, A.Y., Haynes, P.A., 2018. Shotgun proteomic analysis of

4.4. Phenylpropanoid biosynthesis pathway Environmental stresses are known to induce the biosynthesis of secondary metabolites such as phenylpropanoids, flavonoids, and anthocyanins (Leyva et al., 1995; Liu et al., 2018; Sun et al., 2018a,b). Previous proteomics studies of Cabernet Sauvignon grape cell suspensions exposed to thermal stresses revealed that low temperatures enhanced the phenylpropanoid pathways (George et al., 2015). In this study, several DAPs involved in phenylpropanoid biosynthesis, such as cytochrome P450 84A1 (VIT_204s0023g02900.1), accumulated in the buds of both V. amurensis and V. vinifera during CA, confirming that low temperatures induce phenylpropanoid biosynthesis in these species. We revealed that the phenylpropanoid biosynthesis pathway of V. vinifera and V. amurensis was enriched, but in contrasting species-specific patterns (Fig. 3A and D); for example, several DAPs, such as phenylalanine ammonia-lyase (VIT_208s0040g01710.1), were upregulated in V. amurensis but downregulated in V. vinifera (Table 3). As the catalyst of the first committed step of phenylpropanoid biosynthesis, phenylalanine ammonia-lyase has been extensively studied in both the abiotic and biotic stress responses of plants (George et al., 2015; Leyva et al., 1995), and also plays a significant role in the production of phenolics and their subsequent incorporation into the cell wall as suberin or lignin (Solecka, 1997). The lignification of plant tissues is a stress-sensing and signal-transduction mechanism that 8

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V.F. Masocha, et al. photoperiod regulated dormancy induction in grapevine. J. Proteomics 187, 13–24. George, I.S., Haynes, P.A., 2014. Current perspectives in proteomic analysis of abiotic stress in Grapevines. Front. Plant Sci. 5, 686. George, I.S., Pascovici, D., Mirzaei, M., Haynes, P.A., 2015. Quantitative proteomic analysis of cabernet sauvignon grape cells exposed to thermal stresses reveals alterations in sugar and phenylpropanoid metabolism. Proteomics 15 (17), 3048–3060. Gharechahi, J., Sharifi, G., Komatsu, S., Salekdeh, G.H., 2016. Proteomic analysis of crop plants under low temperature: a review of cold responsive proteins. In: Salekdeh, G. (Ed.), Agricultural Proteomics Volume 2. Springer, Cham, pp. 97–127. Gibon, Y., Blasing, O.E., Palacios-Rojas, N., Pankovic, D., Hendriks, J.H., Fisahn, J., Höhne, M., Günther, M., Stitt, M., 2004. Adjustment of diurnal starch turnover to short days: depletion of sugar during the night leads to a temporary inhibition of carbohydrate utilization, accumulation of sugars and post-translational activation of ADP-glucose pyrophosphorylase in the following light period. Plant J. 39 (6), 847–862. Guy, C.L., 1990. Cold acclimation and freezing stress tolerance: role of protein metabolism. Annu. Rev. Plant Physiol. 41 (41), 187–223. Haider, S., Pal, R., 2013. Integrated analysis of transcriptomic and proteomic data. Curr. Genomics 14 (2), 91–110. Hincha, D.K., Zuther, E., 2014. Plant cold acclimation and freezing tolerance. Methods Mol. Biol. 1166, 1–6. Hiraki, H., Uemura, M., Kawamura, Y., 2019. Calcium signaling-linked CBF/DREB1 gene expression was induced depending on the temperature fluctuation in the field: views from the natural condition of cold acclimation. Plant Cell Physiol. 60 (2), 303–317. Ji, W., Cong, R., Li, S., Li, R., Qin, Z., Li, Y., Zhou, X., Chen, S., Li, J., 2016. Comparative proteomic analysis of soybean leaves and roots by iTRAQ provides insights into response mechanisms to short-term salt stress. Front. Plant Sci. 7, 573. Kanehisa, M., Furumichi, M., Tanabe, M., Sato, Y., Morishima, K., 2017. KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Res. 45 (D1), D353–D361. Khalil-Ur-Rehman, M., Sun, L., Li, C.X., Faheem, M., Wang, W., Tao, J.M., 2017. Comparative RNA-seq based transcriptomic analysis of bud dormancy in grape. BMC Plant Biol. 17 (1), 18. Kosová, K., Vítámvás, P., Prášila, I.T., Renaut, J., 2011. Plant proteome changes under abiotic stress — contribution of proteomics studies to understanding plant stress response. J. Proteomics 74 (8), 1301–1322. Kosová, K., Vítámvás, P., Urban, M.O., Prášil, I.T., Renaut, J., 2018. Plant abiotic stress proteomics: the major factors determining alterations in cellular proteome. Front. Plant Sci. 9, 122. Le Gall, H., Philippe, F., Domon, J.M., Gillet, F., Pelloux, J., Rayon, C., 2015. Cell wall metabolism in response to abiotic stress. Plants (Basel) 4 (1), 112–166. Lee, J.H., Yu, D.J., Kim, S.J., Choi, D., Lee, H.J., 2012. Intraspecies differences in cold hardiness, carbohydrate content and β-amylase gene expression of Vaccinium corymbosum during cold acclimation and deacclimation. Tree Physiol. 32 (12), 1533–1540. Levitt, J., 1980. Responses of plants to environmental stress, 2nd ed. Chilling, Freezing, and High Temperature Stresses Vol. 1 Academic Press, Orlando. Leyva, A., Jarillo, J.A., Salinas, J., Martinez-Zapater, J.M., 1995. Low temperature induces the accumulation of phenylalanine ammonia-lyase and chalcone synthase mRNAs of Arabidopsis thaliana in a light-dependent manner. Plant Physiol. 108 (1), 39–46. Li, M., Zhang, K., Long, R., Sun, Y., Kang, J., Zhang, T., Cao, S., 2017. iTRAQ-based comparative proteomic analysis reveals tissue-specific and novel early-stage molecular mechanisms of salt stress response in Carex rigescens. Environ. Exp. Bot. 143, 99–114. Liu, G.T., Ma, L., Duan, W., Wang, B.C., Li, J.H., Xu, H.G., Yan, X.Q., Yan, B.F., Li, S.H., Wang, L.J., 2014. Differential proteomic analysis of grapevine leaves by iTRAQ reveals responses to heat stress and subsequent recovery. BMC Plant Biol. 14 (1), 110. Liu, Q., Luo, L., Zheng, L., 2018. Lignins: biosynthesis and biological functions in plants. Int. J. Mol. Sci. 19 (2), 335. Liu, Y., Wang, L., Zhang, T., Yang, X., Li, D., 2017. Functional characterization of KS-type dehydrin ZmDHN13 and its related conserved domains under oxidative stress. Sci. Rep. 7 (1), 7361. Mathiason, K., He, D., Grimplet, J., Venkateswari, J., Galbraith, D., Or, E., Fennell, A., 2009. Transcript profiling in Vitis riparia during chilling requirement fulfillment reveals coordination of gene expression patterns with optimized bud break. Funct. Integr. Genomics 9 (1), 81–96. Moriya, Y., Itoh, M., Okuda, S., Yoshizawa, A.C., Kanehisa, M., 2007. KAAS: an automatic genome annotation and pathway reconstruction server. Nucleic Acids Res. 35 (Suppl._2), W182–185. Navarro, S., Vazquez-Hernandez, M., Rosales, R., Sanchez-Ballesta, M.T., Merodio, C., Escribano, M.I., 2015. Differential regulation of dehydrin expression and trehalose levels in Cardinal table grape skin by low temperature and high CO2. J. Plant Physiol. 179, 1–11. Ophir, R., Pang, X., Halaly, T., Venkateswari, J., Lavee, S., Galbraith, D., Or, E., 2009. Gene-expression profiling of grape bud response to two alternative dormancy-release stimuli expose possible links between impaired mitochondrial activity, hypoxia, ethylene-ABA interplay and cell enlargement. Plant Mol. Biol. 71 (4-5), 403. Ouyang, L., Leus, L., De Keyser, E., Van Labeke, M.C., 2019. Seasonal changes in cold hardiness and carbohydrate metabolism in four garden rose cultivars. J. Plant Physiol. 232, 188–199. Palm, D., Streit, D., Shanmugam, T., Weis, B.L., Ruprecht, M., Simm, S., Schleiff, E., 2018. Plant-specific ribosome biogenesis factors in Arabidopsis thaliana with essential function in rRNA processing. Nucleic Acids Res. 47 (4), 1880–1895. Parada, F., Noriega, X., Dantas, D., Bressan-Smith, R., Perez, F.J., 2016. Differences in respiration between dormant and non-dormant buds suggest the involvement of ABA

in the development of endodormancy in grapevines. J. Plant Physiol. 201, 71–78. Paredes, M., Quiles, M.J., 2015. The effects of cold stress on photosynthesis in Hibiscus plants. PLoS One 10 (9), 0137472. Podgorska, A., Gieczewska, K., ŁUKAWSKA-KUŹMA, K., Rasmusson, A.G., Gardeström, P., SZAL, B., 2013. Long-term ammonium nutrition of Arabidopsis increases the extrachloroplastic NAD(P)H/NAD(P)+ ratio and mitochondrial reactive oxygen species level in leaves but does not impair photosynthetic capacity. Plant Cell Environ. 36 (11), 2034–2045. Rocha, A.G., Mehlmer, N., Stael, S., Mair, A., Parvin, N., Chigri, F., Teige, M., Vothknecht, U.C., 2014. Phosphorylation of Arabidopsis transketolase at Ser428 provides a potential paradigm for the metabolic control of chloroplast carbon metabolism. Biochem. J. 458 (2), 313–322. Rubio, S., Noriega, X., Pérez, F.J., 2018. Abscisic acid (ABA) and low temperatures synergistically increase the expression of CBF/DREB1 transcription factors and coldhardiness in grapevine dormant buds. Ann. Bot. 123 (4), 681–689. Rubio, S., Perez, F.J., 2019. ABA and its signaling pathway are involved in the cold acclimation and deacclimation of grapevine buds. Sci. Hortic. 256, 108565. Salzman, R.A., Bressan, R.A., Hasegawa, P.M., Ashworth, E.N., Bordelon, B.P., 1996. Programmed accumulation of LEA-like proteins during desiccation and cold acclimation of overwintering grape buds. Plant Cell Environ. 19 (6), 713–720. Sharma, P., Jha, A.B., Dubey, R.S., Pessarakli, M., 2012. Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. J. Bot. 2012, 26. Shi, X., Wang, X., Cheng, F., Cao, H., Liang, H., Lu, J., Kong, Q., Bie, Z., 2019. iTRAQbased quantitative proteomics analysis of cold stress-induced mechanisms in grafted watermelon seedlings. J. Proteomics 192, 311–320. Solecka, D., 1997. Role of phenylpropanoid compounds in plant responses to different stress factors. Acta Physiol. Plant. 19 (3), 257–268. Steponkus, P.L., 1984. Role of the plasma membrane in freezing injury and cold acclimation. Annu. Rev. Plant Physiol. 35 (1), 543–584. Sun, C.H., Yang, C.Y., Tzen, J.T.C., 2018a. Molecular identification and characterization of hydroxycinnamoyl transferase in tea plants (Camellia sinensis L.). Int. J. Mol. Sci. 19 (12), 3938. Sun, X., Matus, J.T., Wong, D.C.J., Wang, Z., Chai, F., Zhang, L., Fang, T., Zhao, L., Wang, Y., Hai, Y., Wang, Q., Li, S., Liang, Z., Xin, H., 2018b. The GARP/MYB-related grape transcription factor AQUILO improves cold tolerance and promotes the accumulation of raffinose family oligosaccharides. J. Exp. Bot. 69 (7), 1749–1764. Sun, X., Zhang, L., Wong, D.C.J., Wang, Y., Zhu, Z., Xu, G., Wang, Q., Li, S., Liang, Z., Xin, H., 2019. The ethylene response factor VaERF092 from Amur grape regulates the transcription factor VaWRKY33, improving cold tolerance. Plant J. 99 (5), 988–1002. Sun, X., Zhao, T., Gan, S., Ren, X., Fang, L., Karungo, S.K., Wang, Y., Chen, L., Li, S., Xin, H., 2016. Ethylene positively regulates cold tolerance in grapevine by modulating the expression of ETHYLENE RESPONSE FACTOR 057. Sci. Rep. 6, 24066. Thalmann, M., Santelia, D., 2017. Starch as a determinant of plant fitness under abiotic stress. New Phytol. 214 (3), 943–951. Thomashow, M.F., 1999. PLANT COLD ACCLIMATION: freezing tolerance genes and regulatory mechanisms. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 571–599. Thomashow, M.F., 2010. Molecular basis of plant cold acclimation: insights gained from studying the CBF cold response pathway. Plant Physiol. 154 (2), 571–577. Tian, T., Liu, Y., Yan, H., You, Q., Yi, X., Du, Z., Xu, W., Su, Z., 2017. agriGO v2.0: a GO analysis toolkit for the agricultural community, 2017 update. Nucleic Acids Res. 45 (W1), W122–W129. Vanderschuren, H., Lentz, E., Zainuddin, I., Gruissem, W., 2013. Proteomics of model and crop plant species: status, current limitations and strategic advances for crop improvement. J. Proteomics 93, 5–19. Vitulo, N., Forcato, C., Carpinelli, E.C., Telatin, A., Campagna, D., D’Angelo, M., Zimbello, R., Corso, M., Vannozzi, A., Bonghi, C., Lucchin, M., Valle, G., 2014. A deep survey of alternative splicing in grape reveals changes in the splicing machinery related to tissue, stress condition and genotype. BMC Plant Biol. 14 (1), 99. Wang, W., Vinocur, B., Shoseyov, O., Altman, A., 2004. Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends Plant Sci. 9 (5), 244–252. Wisniewski, M., Nassuth, A., Teulières, C., Marque, C., Rowland, J., Cao, P.B., Brown, A., 2014. Genomics of cold hardiness in woody plants. Crit. Rev. Plant Sci. 33 (2-3), 92–124. Xiao, H., Siddiqua, M., Braybrook, S., Nassuth, A., 2006. Three grape CBF/DREB1 genes respond to low temperature, drought and abscisic acid. Plant Cell Environ. 29 (7), 1410–1421. Xiao, H., Tattersall, E.A., Siddiqua, M.K., Cramer, G.R., Nassuth, A., 2008. CBF4 is a unique member of the CBF transcription factor family of Vitis vinifera and Vitis riparia. Plant Cell Environ. 31 (1), 1–10. Xin, H., Zhu, W., Wang, L., Xiang, Y., Fang, L., Li, J., Sun, X., Wang, N., Londo, J.P., Li, S., 2013. Genome wide transcriptional profile analysis of Vitis amurensis and Vitis vinifera in response to cold stress. PLoS One 8 (3), 58740. Xu, W., Li, R., Zhang, N., Ma, F., Jiao, Y., Wang, Z., 2014. Transcriptome profiling of Vitis amurensis, an extremely cold-tolerant Chinese wild Vitis species, reveals candidate genes and events that potentially connected to cold stress. Plant Mol. Biol. 86 (4-5), 527–541. Xu, Y., Yuan, Y., Du, N., Wang, Y., Shu, S., Sun, J., Guo, S., 2018a. Proteomic analysis of heat stress resistance of cucumber leaves when grafted onto Momordica rootstock. Hortic. Res. 5 (1), 53. Xu, Y., Zeng, X., Wu, J., Zhang, F., Li, C., Jiang, J., Wang, Y., Sun, W., 2018b. iTRAQbased quantitative proteome revealed metabolic changes in winter turnip rape (Brassica rapa L.) under cold stress. Int. J. Mol. Sci. 19 (11), 3346. Yan, S.-P., Zhang, Q.-Y., Tang, Z.-C., Su, W.-A., Sun, W.-N., 2006. Comparative proteomic analysis provides new insights into chilling stress responses in rice. Mol. Cell.

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Scientia Horticulturae 263 (2020) 109143

V.F. Masocha, et al. Proteom. 5 (3), 484–496. Yue, C., Cao, H.L., Wang, L., Zhou, Y.H., Huang, Y.T., Hao, X.Y., Wang, X.C., Wang, B., Yang, Y.J., Wang, X.C., 2015. Effects of cold acclimation on sugar metabolism and sugar-related gene expression in tea plant during the winter season. Plant Mol. Biol. 88 (6), 591–608.

Zhang, N., Zhang, L., Zhao, L., Ren, Y., Cui, D., Chen, J., Wang, Y., Yu, P., Chen, F., 2017. iTRAQ and virus-induced gene silencing revealed three proteins involved in cold response in bread wheat. Sci. Rep. 7 (1), 7524. Zieske, L.R., 2006. A perspective on the use of iTRAQ™ reagent technology for protein complex and profiling studies. J. Exp. Bot. 57 (7), 1501–1508.

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