Plants of Chinese wild V. amurensis accession ‘Heilongjiang Seedling’, which originates in northeast China, were maintained in the grape germplasm repository at the Northwest A & F University, Yangling, Shaanxi, the People’s Republic of China. This accession is highly resistant to cold (Zhang et al., 2012).

In early January 2010, 1-year-old shoots were taken from mature vines for sand-storage at under 4°C. At the end of March, the shoots were retrieved and used for cuttings, soaked for 2 h in Transplantone (500 mg/L), allowed to develop roots, and cultivated in a greenhouse (25°C, light 12000 lux). In July, well-grown and healthy potted plants were selected for further cold stress and total RNA and protein isolation.

‘Heilongjiang seedling’ plants growing in pots were placed in a pre-chilled growth chamber at 4°C. Young leaves, stems and roots were harvested at 0, 2, 6, 12, 24, and 48 h after exposure to 4°C. Samples were frozen in liquid nitrogen prior to extraction of RNA and protein.

Total RNA was extracted using the improved sodium dodecyl sulfate (SDS)/phenol method (Zhang et al., 2003). The first-strand cDNA was synthesized using the Easy Script First-Strand cDNA Synthesis Super Mix (Transgen, China), according to the manufacturer’s protocol. The cDNA templates for 5′- and 3′-Rapid Amplification of cDNA Ends (RACE) were synthesized using the SMARTTM RACE cDNA Amplification Kit (Clontech, Palo Alto, CA, USA). Primers for 5′- and 3′-RACE are listed in Table 1. All amplified RACE fragments were sequenced three times for each sample. Based on the 5′-RACE and 3′-RACE results, a pair of full-length primers VaERD15-F/R (including initiation and termination codons) were designed (Table 1). Amplification and sequencing of the full-length cDNA of VaERD15 were repeated using two replicates.

The cDNA sequence of VaERD15 gene from V. amurensis was translated into amino acid sequences on the NCBI website¹. The protein conserved domain analysis website² was used to predict conserved domains, theoretical molecular weights and isoelectric points. The homologies of ERD15 in V. amurensis and in corn, pepper and Arabidopsis were analyzed by means of DNAMAN analysis software. Finally, phylogenetic analyses were generated using MEGA 5.0 software³.

Total RNA was extracted from various grapevines after exposure to low-temperature stress (4°C) for 0, 2, 6, 12, 24, and 48 h, and first-strand cDNA was synthesized, as previously described. Semi-quantitative PCR was carried out using the primers rtVaERD15-F and rtVaERD15-R (Table 1). The volume of the semi-quantitative PCR amplification was 20 μl, and Actin1 (accession no. AY680701) was used as the internal reference gene. All reactions were repeated for three biological replicates.

Western blotting was carried out to further analyze the expression pattern of VaERD15. Total protein was extracted from various samples according to the method of Méchin et al. (2006). The extracted protein concentration was determined using the method of Bradford (1976). Proteins samples (25 μg) were prepared for SDS-polyacrylamide gel electrophoresis (PAGE), and blotted onto a polyvinylidene fluoride membrane (Roche, product no. 03010040001). Immune antibodies were prepared in our laboratory, as previously reported (Zhang et al., 2014).

The ORF of VaERD15 without a termination codon was obtained by PCR amplification using specific primers VaERD15-XbalI-F and VaERD15-KpnI-R (Table 1). The PCR products digested by XbalI and KpnI were cloned into a pMD18-T vector (TaKaRa, Japan). The fragments were fused into the N-terminus of the GFP expression vector driven by the 35S promoter. The vector carrying 35S::GFP was used as a control. The plasmids of the 35S::VaERD15-GFP fusion construct and 35S::GFP were purified for subsequent experiments. Plasmids were then transformed into onion epidermal cells using the particle bombardment method, as described by Varagona et al. (1992). Transformed onion epidermal cells were cultured on MS media under dark conditions for 24 h at 25°C. Expression of the genes transformed into the onion epidermal cells was observed using confocal laser scanning microscopy (LSM 510 META, ZEISS, Germany).

For the transcriptional activation assay, the ORF of VaERD15 was generated and fused into the frame to the NcoI and BamHI sites of the GAL4 DNA-binding domain in the pGBKT7 vector by recombination reactions (Invitrogen, USA). The expression vector pGBKT7 carrying the GAL4 gene constructed by our laboratory was used as a positive control and the empty pGBKT7 vector was used as a negative control. These constructs were then transformed into the yeast strain AH109. The resulting transformants were streaked onto Synthetic Defined (SD)/-Trp medium. After incubation at 30°C for 3 days, transformed strains on the SD/-Trp plates were selected and streaked onto SD/-Trp/-His/-Ade plates containing x-α-gal; the level of transcriptional activation was evaluated by color reaction.

The full-length cDNA of VaERD15 was amplified by PCR and cloned into the BglII/NcoI site of pCAMBIA3301, generating pCAMBIA3301-VaERD15. The specific primers VaERD15-Ncol-F/R are shown in Table 1. Constructs were verified by sequencing. The constructed plasmid was introduced into Agrobacterium tumefaciens GV3101 cells by electroporation. Transgenic Arabidopsis plants were obtained using the floral dipping method (Clough and Bent, 1998). Putative transgenic Arabidopsis plants harboring the pCAMBIA3301-VaERD15 construct were selected on MS plates containing 10 mg/L Basta.

Homozygous T3 Arabidopsis strains were tested by PCR and genomic DNA was extracted from 3-week-old leaves of the putative transgenic Arabidopsis seedlings using the cetyltrimethyl ammonium bromide (CTAB) method with appropriate modification (Wu et al., 1998). The specific primers VaERD15-NcoI –F/R were used for amplifying the exogenous gene, with pCAMBIA3301-VaERD15 plasmid DNA as a positive control, and DNA from WT Arabidopsis (Col-0) leaves as a negative control. At the same time, leaves of VaERD15-positive Arabidopsis plants were taken for GUS staining, with WT Arabidopsis plants serving as controls.

Southern blot analysis was carried out as previously described Southern (1975). After plant genomic DNA was extracted and purified, 50 μg DNA was digested by the restriction enzyme HindIII in 40 μl for 12 h. Electrophoresis and Southern blot analysis were then carried out according to the standard methods.

Two T3 Arabidopsis lines (L1, L2) were used for the cold tolerance assay. Similarly, robust transgenic and WT 3-week-old Arabidopsis plants were used for the cold treatment. Plants were placed in a pre-chilled chamber at 4°C to cold acclimate for 48 h, and then transferred to a pre-chilled chamber at -6°C for 72 h. Plants suffering from cold stress were transferred to room temperature (approximately 23°C) for 5 days to recover. The phenotypic changes of Arabidopsis plants were observed and photographed during this period. Leaf samples were collected at 0, 2, 4, 8, 12, 24, 48, and 72 h after exposure to cold stress and stored at -80°C for qRT-PCR and Western blot analyses. qRT-PCR was carried out to determine expression changes of VaERD15 and AtERD15 [primers: rtVaERD15-F/R and rtAtERD15- F/R (Table 1)], in vivo, using the Takara SYBR Premix Ex Taq^TM II (Perfect Real Time) on a Bio-Rad IQ5 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). The volume used for qRT-PCR amplification was 20 μl, and AtGAPDH (accession no. 101214) was used as the endogenous reference gene. Western blotting was carried out as previously described. All experiments were carried out for three biological replicates.

Biochemical indices relating to cold stress were determined in 3-week-old Arabidopsis seedlings in both transgenic and control plants. All plants had been subjected to the cold treatment described above. Determinations in this part of the study were carried out for three biological replicates.

Relative electrolyte leakage was assessed according to the previously described method (Weigel et al., 2001). Proline content was determined following the method of Shan et al. (2007). MDA content was measured according to the method of Puckette et al. (2007) with minor modifications. About 200 mg leaves frozen in liquid nitrogen was homogenized in 4 ml 10% trichloracetic acid (TCA), then centrifuged at 10,000 rpm for 10 min. The supernatant (2 ml) was mixed with 2 ml thiobarbituric acid (TBA) and heated at 95°C for 30 min, quickly cooled on ice and then centrifuged at 10,000 rpm for 10 min. The soluble sugar and soluble protein contents were measured according to the methods of Bradford (1976) and Machado et al. (2013), respectively.

The activity of SOD was measured using the nitroblue tetrazolium (NBT) method (Giannopolitis and Ries, 1977; Puyang et al., 2015). POD activity was measured using the method of Pagariya et al. (2012). CAT activity was determined as described by Tseng et al. (2007).

All physiological data was analyzed using the IBM SPSS Statistics 18.0 software. The differences between the transgenic samples and the corresponding wild type samples were calculated by the independent sample t-test. Significant differences were represented by ^∗P < 0.05; ^∗∗P < 0.01.

Specific primers were designed according to the acquired ORF of VaERD15, which had a total length of 423 bp with a 5′-untranslated region (UTR) of 66 bp, a 3′-UTR of 196 bp and an intron size of 88 bp. VaERD15 encoded a predicted polypeptide of 140 amino acids with a molecular weight of 16.2 kD (Zhang et al., 2014), and pI of 4.80. Alignment analysis with the grapevine genome (Jaillon et al., 2007) showed that VaERD15 was initially located on chromosome 13.

Multiple sequence alignment analysis (Figure 1A) showed the VaERD15 shared 36% homology with the ZmERD15 predicted protein (accession no. ACG25626.1), 35% homology with the CaERD15 predicted protein (accession no. ABB89735.1) and 33% homology with the AtERD15 predicted protein (accession no. AAM64638.1). Phylogenetic analyses (Figure 1B) indicated that the relationship of VaERD15 with other ERDs from similar plant species can be divided into two families. The closest relationship with VaERD15 was found in Vitis vinifera and Cucumis sativus.

Semi-quantitative PCR and Western blotting were carried out to determine the expression patterns of VaERD15. The relative expressions at transcription level are shown in Figure 2A; the transcript was detected in all tissues measured. At 0 h, high VaERD15 expression was found in stems, low expression in leaves and zero expression in roots. After 2 h of cold stress, the expression of VaERD15 in stems decreased significantly, but recovered slightly after 12 h. The expression of VaERD15 in leaves and roots showed a rising trend that peaked at 24 h, but then decreased.

The Western blot analysis results are shown in Figure 2B. We observed 16.2 kD bands on the polyvinylidene difluoride, membrane, indicating that the tissue proteins in roots, stems and leaves of V. amurensis accession ‘Heilongjiang seedling’ specifically reacted with VaERD15 antibodies. VaERD15 expression in leaves increased gradually and peaked at 48 h under the cold stress treatment. VaERD15 expression in stems showed a downward trend up to 12 h, which recovered at 24 h, but subsequently decreased. In the roots, VaERD15 expression levels increased, with a maximum at 6 h and a second small peak after 24 h.

The subcellular localization results showed that the 35S: VaERD15-GFP fusion expression vector was transiently expressed in onion epidermal cells. Green fluorescence was only observed in the nuclei, while in the control green fluorescence was visible throughout the entire onion cell (Figure 3). This indicates that VaERD15 is localized to the nuclei.

The AH109 strains with the recombinant plasmid of pGBKT7-GAL4 (positive control) and pGBKT7-VaERD15 or the empty pGBKT7 vector (negative control) were all able to grow well on the SD/-Trp medium (Figure 4A). This demonstrates that the pGBKT7-VaERD15 recombinant plasmid, and the positive and negative controls were all transferred into the yeast. The strains transformed with VaERD15 were able to grow well on the SD/-Trp/-His/-Ade + X-α-gal selective medium, and turned blue on the SD/-Trp/-His/-Ade + X-α-gal medium. Accordingly, the negative control did not grow on the SD/-Trp-His-Ade medium (Figure 4B), indicating that VaERD15 could activate the expression of the reporter gene and synthesize the histidine and adenine required for the normal growth of yeast AH109. Taken together, these results illustrate that VaERD15 could function as a transcriptional activator in yeast.

Arabidopsis plants over-expressing VaERD15 were subjected to PCR. Five T3 Arabidopsis plants that survived on selection medium were used for the PCR experiments, and four expected bands were observed (Figure 5A). We also carried out a GUS staining assay. Results showed that transgenic Arabidopsis leaves were stained blue (Figure 5B), indicating that the GUS was expressed in the plant in vivo. This demonstrates that the plant genome had successfully integrated the target gene VaERD15.

Southern blot analysis was performed using four VaERD15-positive lines to further confirm that VaERD15 had been integrated into the Arabidopsis genome. The Southern blot results showed that specific hybridization bands could be clearly observed in all positive lines (Figure 5C), but not in the WT plants. The four transgenic lines all had a specific hybridization signal, but the band sizes were not all the same. This dissimilarity indicates that the transgenic Arabidopsis plants had different insert sites for VaERD15 in their genomes.

qRT-PCR and Western blotting were carried out to determine expression changes of VaERD15 in vivo. As shown in Figure 6A, VaERD15 transcripts gradually increased in L1 and L2 lines with longer durations of cold stress, but the transcripts were barely detectable in the WT plants. We also detected a change in endogenous AtERD15 after transferring VaERD15 into the Arabidopsis plant. The qRT-PCR results showed that there were no significant differences in the expression of endogenous AtERD15 between wild type and transgenic Arabidopsis, except that the expression in transgenic plants was nearly four times higher than that in WT Arabidopsis at 12 h (Supplementary Figure S1). Western blot analysis showed that the VaERD15 expression in two transgenic Arabidopsis lines both tended to increase with longer durations of cold stress, and the VaERD15 expression level in L1 was higher than that in L2 (Figure 6B). However, only weak bands were observed in the WT plants, indicating that expression levels of VaERD15 in WT and in transgenic Arabidopsis were different.

The cold tolerance assay showed transgenic and WT Arabidopsis were both subjected to freezing injury, but to different extents, with longer durations of cold stress. However, freezing injuries were more serious in WT Arabidopsis. Almost all the transgenic plants suffering from cold stress were able to resume normal growth after being transferred to room-temperature (∼23°C). However, the same recovery did not occur with the WT plants (Figure 7).

Cold tolerance is strongly correlated with a number of physiological parameters in plants (Liu et al., 2010; Li et al., 2014; Xu et al., 2014a). To investigate whether the cold tolerance of Arabidopsis lines over-expressing VaERD15 was improved, we measured several cold-related physiological indices including: relative electrolyte leakage; the contents of proline, MDA; soluble sugars and soluble proteins; and the activities of SOD, POD and CAT.

Results showed that relative electrolyte leakage in both transgenic and WT Arabidopsis plants showed an upward trend with longer durations of low temperature treatment (Figure 8A). Both transgenic and WT Arabidopsis plants showed similar conductivities in the early stages of cold stress but after 2 h, the conductivity in the WT plants was significantly higher than that in the transgenic plants. This indicates that WT plants suffered greater cell membrane damage than the transgenic plants.

We also investigated proline and MDA contents (Figures 8B,C). At the start of the stress period, the proline content of the transgenic plants was slightly higher than that of the WT plants but the difference was not significant. After 4 h of cold stress, proline in the transgenic plants accumulated rapidly, and proline abundance and growth rate were both significantly higher than in the WT plants. Changes in proline content were consistent with the phenotypic changes under cold stress. While the MDA content in the transgenic plants was lower than in the WT plants after 2 h of cold stress, it increased rapidly with longer durations of cold treatment and was consistently higher than in the WT plants.

Similar to conductivity, the soluble sugar contents of both transgenic and WT Arabidopsis plants increased with longer durations of cold treatment (Figure 8D). The soluble sugar content of the transgenic plants was slightly lower than in the WT plants for the first 12 h of cold stress, but then accumulated rapidly to become significantly higher than in the WT plants.

As shown in Figure 8E, the soluble protein content of the transgenic plants was consistently higher than that of the WT plants during cold stress. However, the pattern of change was very similar in both plant, falling to a minimum during the first 12 h, and then gradually increasing.

We also measured the activity of antioxidant enzymes. The results showed that SOD activity in transgenic Arabidopsis was consistently higher than in WT plants (Figure 9A), indicating that plants over-expressing VaERD15 generally had elevated SOD activity. POD and CAT activities showed a late increase. The POD content curve of the transgenic plants was relatively flat, but that of the WT plants decreased over the first 8 h and then increased significantly after 12 h (Figure 9B). The CAT content in the transgenic plants was lower than in the WT plants at first, but rose slightly after 2 h (Figure 9C).