Comparative Proteomics Reveals Differential Mechanism of Root Cold-resistance between Vitis. Riparia × V. Labrusca and Cabernet Sauvignon

Grapevines, containing large amounts of bioactive metabolites that offer health benets, are widely cultivated around the world. The cold damage of growing outside with extreme low temperature during overwintering stage limits the expansion of production. Although the levels of morphological, biochemical and molecular in different Vitis species exposure to different temperatures have been investigated, differential expression of proteins in roots is still limited. Here, the roots of cold-resistant (Vitis. riparia × V. labrusca, T1) and cold-sensitive varieties (Cabernet Sauvignon, T3) at −4°C as well as of the former at −15°C (T2) were measured by iTRAQ-based proteomic analysis, expression levels of genes encoding candidate proteins were validated by qRT-PCR. The results showed that the root activity of cold-resistant variety was stronger than that of cold-sensitive variety, and it declined with the decrease of temperature. A total of 25 proteins were differentially co-expressed at T2 versus (vs) T1 and T1 vs T3, and these proteins were involved in stress response (e.g. DHN1, SHSPCP and USPCP), bio-signaling (e.g. PKCP, S/TPP and nsS/TP), metabolism (e.g. GluP, GluBE and PE), energy (e.g. AAC, AAACP and NADCP), and translation (e.g. rpL14, rpS21 and PPI). The relative expression levels of the candidate 13 genes were consistent with their fold-change values of proteins. The signature translation pattern for the roots at spatio-temporal treatments of varieties and temperatures provides insight into the differential mechanism of cold resistance of grapevines.


Introduction
Grapevines (Vitis vinifera L.) are rich in phenolics, avonoids and resveratrol that have many biological activities such as antioxidant, inhibition of plasma platelet aggregation, and cyclooxygenase [1,2].
Nowadays grapevines are cultivated in many countries around the world, principally distributed in Europe [3]. In China, the optimum regions for cultivation are mainly distributed in Gansu, Inner Mongolia, Ningxia, Shaanxi, Shandong and Xinjiang [4]. Currently, thirteen varieties of Vitis that are large-scale cultivated ( 100,000 hm 2 ) in the world mainly include: Cabernet Sauvignon, Merlot, Chardonnay, Syrah, Sauvignon Blanc and Pinot Noir [5].
In the commercial large-scale cultivation, grapevines are frequently exposed to environmental stresses such as drought, salinity and extreme temperatures [6]. Most cultivated grapevines are suited to grow in temperate and subtropical regions with mild winter conditions [7]. However, grapevines are often grown outside with severe winter conditions characterized by low temperature, which limits the current and future expansion of production [8]. In order to diminish the freeze damage, several efforts including evaluating the cold-resistant species or varieties as well as revealing the mechanism of cold-resistance have been conducted. For the evaluating the cold-resistant species or varieties, V. riparia is found to be the most cold-resistant species and V. labrusca belongs to medium resistance among of 7 wild Vitis species native to America [9]; the cold resistance of V. riparia × V. labrusca (Beta) and V. berlandieri × V. riparia (5BB) is stronger than that of Cabernet Sauvignon and Merlot [10]. For the revealing the mechanism of cold-resistance, physiological and biochemical metabolites (e.g. soluble sugars, proteins and hormones) in buds, branches and roots were determined [11][12][13]; key genes (e.g. CBF/DREB, ICE and AP2/ERF) enhancing freezing tolerance were identi ed [14][15][16]; and differentially expressed genes among different species and temperatures were analyzed by transcriptomics [8, 17,18].
Previous literatures have reported that grape branches and buds can survive low temperature of −13°C or lower, but roots have weaker cold resistance than the above-ground parts [19,20]. To date, although the levels of osmoregulatory metabolites, the activities of antioxidant enzymes, and the expression levels of cold resistance genes between different Vitis species or low temperature have been investigated in extensive experiments [11][12][13][14][15][16][17][18], differential expression of proteins in roots between cold-resistant and cold-sensitive species or varieties as well as different low temperatures has not been determined and identi ed. In this study, the roots of cold-resistant (V. riparia × V. labrusca, T1) and cold-sensitive varieties (Cabernet Sauvignon, T3) at −4°C as well as the former at −15°C (T2) were spatio-temporally measured by quantitative iTRAQ-based proteomic analysis. We found that 25 proteins were differentially coexpressed at the three treatments and their biological functions were involved in stress response, biosignaling, metabolism, energy and translation; the expression levels of related genes were validated by qRT-PCR.

Difference in root activity
As shown in Fig. 1, there was a 1.68-fold decrease of root activity at T2 compared to T1, and a 2.54-fold greater at T1 than T3. Moreover, the root activities presenting at the different treatments suggest that the roots can be used for proteomic analysis.

Analysis of differentially expressed proteins (DEPs)
To reveal the cold resistance of root in grapevines, the DEPs in roots of V. riparia × V. labrusca and Cabernet Sauvignon at different treatments were analyzed by iTRAQ. A total of 56 and 143 DEPs were obtained at T2 vs T1 and T1 vs T3, respectively (Fig. 2). The cluster heat maps of the DEPs at T1, T2 and T3 were shown in Fig. 3.

Discussion
Plants are frequently exposed to environmental stresses such as low temperature, which plays a major role in the distribution of plant species. Adaptation and acclimation to cold stress result from integrated events occurring at all levels of organization, from the anatomical and morphological level to the cellular (e.g. changes in cell cycle, division and wall architecture), biochemical (e.g. producing osmoregulatory compounds such as proline and glycine betaine), and molecular level (e.g. linking the perception of a stress signal with the genomic responses) [21]. In this study, we found that there was a stronger root activity in V. riparia × V. labrusca than Cabernet Sauvignon, and the root activity declined with the decrease of temperature; a total of 25 proteins were differentially co-expressed in V. riparia × V.
labrusca and Cabernet Sauvignon at −4 o C and/or −15 o C treatments, and these 25 DEPs were classi ed into 5 categories including: stress response, bio-signaling, metabolism, energy, and translation.
The root system is an important organ absorbing water and minerals from the soil, storing foods (e.g. starch, polysaccharides and secondary metabolites), and synthesizing the vital substances (e.g. amino acids, hormones and vitamins) for plant growth and development [22,23]. Here, a stronger root activity at −4 o C was observed in V. riparia × V. labrusca than Cabernet Sauvignon, authenticating that the cold-resistance of V. riparia × V. labrusca is higher than that of Cabernet Sauvignon in eld [10].
For the DEPs involved in stress response, previous literatures have reported that dehydrin (DHN) are highly hydrophilic proteins that are involved in cold acclimation processes [24]. Small heat shock proteins (sHSPs) are ubiquitous stress proteins proposed to act as chaperones and have been ascribed an unusual diversity of functions in the cellular response to environmental stress [25]. Universal stress proteins (USPs) are stress-responsive proteins that may contain a single USP domain or two tandem repeats of USP domains [26,27]. Ferritins (FERs) are a broad superfamily of iron storage proteins, exert a ne tuning of the quantity of metal required for metabolic purposes and help plants to protect against oxidative stress [28]. The GluDP plays a role in cell protection against oxidative stress by detoxifying peroxides [29]. Glutathione peroxidases (GPXs) are key enzymes of the cell antioxidant defense system and involved in scavenging oxyradicals [30]. Investigations have found that the genes DHN in tomato [31], shsp16.9 in rice [32], USPs in Arabidopsis [33], TaFER-5B in Triticum aestivum [34], and GluPX in Taxus chinensis [35] were overexpressed in response to cold stress. In this study, the overexpression of the proteins (DHN1, SHSPCP, USPCP, FER, GluDP and GPX) that enhance cold stress are consistent with higher cold-resistance of V. riparia × V. labrusca than Cabernet Sauvignon.
For the DEPs involved in bio-signaling, both protein kinases (PKs) and protein phosphatases (PPs) play important roles in determining the magnitude and duration of a signaling event, with PKs catalyzing the transfer of a phosphate moiety from ATP to proteins and PPs acting to remove this phosphate group by hydrolysis [36]. Protein serine/threonine phosphatases family from plants constitute PP1, PP2A, PP2B, and novel phosphatases, which have multiple biological functions by regulating a wide variety of cellular signal transduction pathways in response to stresses [37]. The RAD23 is involved in cell cycle regulation, protein quality control, DNA damage response and cellular metabolism [38]. Investigations have found that the genes GsLRPK in Glycine soja [39], S/TPP in rice [40], and RAD23 in apple [41] were overexpressed in response to cold stress. In this study, the overexpression of the proteins (PKCP, S/TPP, nsS/TPK and RAD23) was also observed in higher cold-resistance of V. riparia × V. labrusca compared to Cabernet Sauvignon as well as at lower temperature −15 o C compared to −4 o C.
For the DEPs involved in metabolism, the GluP is an important allosteric enzyme in carbohydrate metabolism [42]. The GluBE is involved in the pathway starch biosynthesis, which is part of glycan biosynthesis [43]. The PE is involved in the pathway pectin degradation and in glycan metabolism [44]. The ABHD family of proteins in plants in uences the lipid biosynthesis more towards leaf lipids such as galactolipids and less towards storage lipids [45]. The ProIP catalyzes speci cally hydrolysis of Nterminal proline from peptides [46]. The MT is involved in the sterol and steroid biosynthesis [47]. Extensive experiments have demonstrated that there are overexpression and activity of enzymes that participate in soluble sugars biosynthesis and starch degradation, which produce proper metabolites to adjust the metabolism and physiology of the plant to cold stress [48]. In this study, the overexpression of the proteins (GluP, GluBE, PE, ABHD3CP, ProIP and MT) was also observed in V. riparia × V. labrusca freezing temperature (−15 o C) might be a part of the mechanism associated with the delay of senescence and death [49].
For the DEPs involved in energy, the AAC plays a key role in the energetic cell metabolism because it exchanges ATP and ADP, product, and substrate of the mitochondrial ATP synthase, respectively [50]. The AAA protein family is a group of ATPases that are associated with various cellular activities [51]. The NAD(P) plays crucial roles in pro-oxidant and antioxidant metabolism and the NAD contents are both exible and potentially important in determining cell fate [52]. The NDUFB7 is an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I), which functions in the transfer of electrons from NADH to the respiratory chain [53]. The phytocyanins (PCs) are a class of plant-speci c blue copper proteins and play critical roles in plant growth and development [54]. The SDHFS is involved in complex II of the mitochondrial electron transport chain and is responsible for transferring electrons from succinate to ubiquinone [55]. Extensive experiments have demonstrated that the membranes become less uid and the proteins components can no longer function normally in chilling-sensitive plants, the result is inhibition of H + -ATPase activity, energy transduction and enzymedependent metabolism [39]. In this study, the overexpression of the proteins (AAC, AAACP, NADCP, NDUFB7, PCP and SDHFS) was observed in the V. riparia × V. labrusca variety.
For the DEPs involved in translation, the large and small subunits of ribosomal proteins are structural constituents of ribosomes, which perform the essential task of protein synthesis in the cell [56]. The PPI functions in the folding of membranal proteins [57]. Investigations have found that the genes SOL34 in Glycine max [58], RPS5 in Arabidopsis [59], and OsCYP19-4 that has the PPI activity in rice [60] were overexpressed in response to cold stress. In this study, the overexpression of the proteins (rpL14, rpS21 and PPI) could be required for growth acclimation to cold stress during overwintering stage.

Conclusion
From the above observations, the root activity of cold-resistant Vitis species is stronger than that of coldsensitive species, and it declines with the decrease of temperature. The DEPs observed in roots between the cold-resistant and cold-sensitive species as well as different temperatures suggest translation-based regulation of roots in response to low temperature. The biological function of the 25 DEPs involved in stress response, bio-signaling, metabolism, energy, and translation should be further investigated using transgenic assays.

Plant material
The one-year-old seedlings (cutting-propagated) of V. riparia × V. labrusca (cold-resistant variety) and Cabernet Sauvignon (cold-sensitive variety) were planted and grown in eld with glass separated (depth 50 cm, width 150 cm) to avoid the roots interfering with each other. Complex fertilizer (N+P 2 O 5 +K 2 O≧500 g/L, Cu+Fe+Mn+Zn+B: 3-30g/L; 100 ml per plant) was applied each year in the soil at the depth from 10 to 30 cm, the soil water content was controlled from 45% to 55%. After three years, the lateral roots of V. riparia × V. labrusca were collected (n = 9 plants) at the depth of 20 cm when the average temperature of soil surface was at −4°C (T1; December 12) and −15°C (T2; January 12), respectively; and the lateral roots of V. vinifera were collected (n = 9 plants) at −4°C (T3; December 12). The collected samples were rapidly frozen in liquid nitrogen for measurement of root activity and analysis of proteomics. The two varieties V. riparia × V. labrusca and Cabernet Sauvignon were identi ed by Prof. Delong Yang (Gansu Agricultural University, China). The samples were collected from the germplasm resources nursery of wine grapes built by our own laboratory, and the voucher specimens were deposited in the herbarium of Gansu Agricultural University, China.

Measurement of root activity
Root activity was measured according to a triphenyl tetrazolium chloride (TTC) method [61]. Brie y, roottip samples (100 mg) were cut into pieces and then placed into a glass tube (10 mL). The TTC solution (0.4% w/v, 3 mL) and Na 2 HPO 4 -KH 2 PO 4 buffer (0.1 mol/L, 3 mL, pH 7.0) were sequentially added in the tube. After incubated at 37°C for 1 h, H 2 SO 4 (1 mol/L, 1.5 mL) was added into the mixture to stop the reaction. The colored samples were transferred into a sealed tube, methanol (15 mL) was added and then the mixture was incubated at 37°C for 4 h to decolor. Absorbance readers were taken at 485 nm and root activity was evaluated based on μg of triphenylformazan (TTF).

Protein extraction, quanti cation and digestion
Total protein samples were extracted according to a previous protocol [62] with some modi cations.
Brie y, root samples (0.5 g) were ground into powder in liquid nitrogen and then dissolved in pre-chilled (−20°C) trichloroacetic acid/acetone (10% w/v, 5 mL). After precipitated at −20°C for 12 h, the homogenate was centrifuged at 14,000 g at 4°C for 15 min. After the supernatant removed and the precipitate suspended in acetone at −20°C for 2 h, the suspension was centrifuged at 14,000 g at 4°C for 10 min. Following exhaustive suspension in acetone (× 3), the precipitate was dissolved in triethylammonium bicarbonate (0.5 mol/L, 0.5 mL) at 4°C for 1 h and then centrifuged at 14,000g at 4°C for 10 min. Finally, the supernatant was transferred to a new tube. The quality of the extracted protein was quanti ed by SDS-PAGE. The protein samples were digested using a Filter aided sample preparation method . Brie y, the iTRAQ-labeled peptides (5 μg) were separated by a Thermo Scienti c Easy C 18 column (75 μm × 100 mm, 3 μm) with gradient elution from 2% B to 45% B in 120 min (A: 0.1% formic acid in H 2 O, B: 0.1% formic acid in acetonitrile) at a ow rate of 300 nl/min. All tandem MS were produced following higher collision energy dissociation method.
Protein identi cation and function annotation Protein identi cation was performed using a decoy database search with the false discovery rate < 1.0% and more than one identi ed peptide. Protein quantitation was analyzed using an iTRAQ 8-plex combined with Mann-Whitney Test. Differentially expressed proteins (DEPs) were selected with a fold-change (FC) value 1.2 or 0.82 (The ratio of T2 vs T1 or T1 vs T3) and P-value 0.05 [69]. Protein functions were annotated against the databases including Swiss-Prot and Gene Ontology (GO) [70][71][72].

RNA extraction and quantitative real-time PCR (qRT-PCR)
Western blotting was used to identify protein expression due to high resolution and sensitivity [73,74], while the decrease of immune reactivity and nonspeci c bands exist in plants [75]. qRT-PCR was alternatively selected to indirectly validate the protein expression [67,76,77]. In this study, the qRT-PCR was used to identify the expression level for genes encoding the candidate proteins. Brie y, RNA samples were extracted from roots using a plant RNA kit. Primer sequences ( Table 2) were designed in primer-blast of NCBI. First-strand cDNA was synthesized using a FastKing RT Kit. PCR ampli cation was carried out using a SuperReal PreMix. Actin was used as an internal reference and the relative expression level (REL) was calculated using a 2 −△△Ct method [78]. Table 2 Sequences of primer used in qRT-PCR analysis.      Cluster heat maps of the differentially expressed proteins (DEPs) at T1, T2 and T3 treatments.   The relative expression level of genes involved in stress response (A), bio-signaling (B), metabolism (C), energy (D), and translation (E) at different treatments, as determined by qRT-PCR.

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