Storage temperature is one of the most important factors which affects postharvest fruit quality. Accordingly, low temperature is the most common abiotic stress applied to improve fruit quality during postharvest storage. To cope with low temperature, fruit have developed adaptive mechanisms, involving physiological and biochemical processes together with transcriptomic modifications, including regulatory and functional genes (Maul et al., 2008; Genero et al., 2016; Rosales et al., 2016). Among the regulatory genes, transcription factors play an important role in plant stress responses, acting as coordinators of stress signals and orchestrating the expression of functional genes (Singh et al., 2002; Wang et al., 2016). It is well known that C-repeat/dehydration-responsive element binding factors (CBF/DREB) are transcription factors which are involved in improving cold stress resistance (Stockinger et al., 1997; reviewed by Chew and Halliday, 2011). CBF/DREB proteins, which belong to the subgroup A1 of the DREB proteins and are members of the AP2/ERF (APETALA2/Ethylene-Responsive Factor) transcription factor superfamily, are able to recognize and bind to the DRE/CRT (Dehydration Responsive Element/C-Repeat) DNA regulatory motif in the promoters of many cold-responsive (COR) genes (Yamaguchi-Shinozaki and Shinozaki, 1994; Stockinger et al., 1997). In this respect, dehydrins (DHNs), a subgroup of LEA (Late Embryogenesis Abundant) proteins, are among the most commonly observed proteins which accumulate in plants in response to low temperature and environmental factors leading to cellular dehydration. The presence of DRE/CRT motifs in the promoter of several cold-regulated DHNs suggests that they play a role in the CBF/DREB-mediated signaling pathway (Gilmour et al., 2004; Wisniewski et al., 2006).

CBF/DREB transcription factors were first isolated from Arabidopsis (Stockinger et al., 1997; Medina et al., 1999; Haake et al., 2002) and thereafter they have been isolated from different woody species including almond (Barros et al., 2012), dwarf apple (Yang et al., 2011), sweet cherry (Kitashiba et al., 2004), and trifoliate orange (He et al., 2012). The overexpression of CBF/DREBs increased the tolerance to cold stress in Arabidopsis (Novillo et al., 2007), potato (Pino et al., 2008), and bilberry (Oakenfull et al., 2013). Thus, CBF/DREBs form part of the group of transcription factors identified as the first wave of cold-induced genes (Chinnusamy et al., 2007; Thomashow, 2010; Zhao and Zhu, 2016). The constitutive overexpression of CBFs in Arabidopsis activated the expression of DRE/CRT-containing target genes (including those that encode DHNs proteins such as COR47 and ERD10, or LEA proteins like COR15a) under normal growing conditions and enhanced freezing tolerance in the absence of a cold stimulus (Jaglo-Ottosen et al., 1998; Gilmour et al., 2004).

There is no doubt that CBF/DREB transcription factors are vital for plants to overcome cold stress, but little is known regarding how important they are for fruit. Storage of peach fruit at a chilling injury-delaying temperature (0°C) resulted in a greater accumulation of PpCBF1/5/6 transcripts than in fruit treated with a chilling injury-inducing temperature (5°C) (Liang et al., 2013). Zhang et al. (2017) indicated that several postharvest treatments, including ethylene (Zhao et al., 2009) and nitric oxide (Zhao et al., 2011), as well as hot water (Ma et al., 2014), triggered CBF expression in tomato fruit in the two first cases, and in kiwifruit in the latter, which enhanced cold tolerance during low temperature storage.

Grape (Vitis sp.) is one of the most economically important fruit crops in the world. Most of the studies on Vitis, which have deciphered the effect of CBFs on cold tolerance have been carried out with grapevine seedlings, and there is little information about their role in bunches. Thus, an increase in CBF3 and CBF4 expression was observed in the leaves of Vitis riparia and V. vinifera after 1–2 days at 4°C (Xiao et al., 2006, 2008), contrasting with the quick cold-induction observed in the case of CBF1 and CBF2 (Xiao et al., 2006). Karimi et al. (2015) compared the response of plants of one cultivar of V. riparia and two cultivars of V. vinifera to cold stress, and denoted that V. riparia which is endemic to cold regions, behaved stronger after 10 days of exposure to 4°C and showed higher expression for all the CBFs analyzed. Overexpression of VvCBF4 in V. vinifera cv. Freedom improved freezing survival and reduced freezing-induced electrolyte leakage by up to 2°C in non-cold-acclimated vines (Tillett et al., 2012). Likewise, overexpression of CBF transcription factors from V. vinifera cv. Koshu (Takuhara et al., 2011) or V. riparia (Siddiqua and Nassuth, 2011) in Arabidopsis improved the freezing tolerance of transgenic plants. Furthermore, the results from Siddiqua and Nassuth (2011) indicated that the Arabidopsis lines that overexpressed VrCBF1 or VrCBF4 showed an increase in the accumulation of COR genes, including dehydrin (COR47) and LEA_4 (COR15a), which have at least one DRE/CRT motif in their promoter (Kasuga et al., 1999).

The application of a 3-day gaseous treatment with 20 kPa CO₂ at 0°C maintained the quality of red table grapes cv. Cardinal during postharvest storage at 0°C by reducing total decay and rachis browning (Sanchez-Ballesta et al., 2006; Rosales et al., 2016). Although table grapes are classified as chilling-tolerant, we have pointed out that the gaseous treatment help table grapes to face temperature shifts at 0°C (Sanchez-Ballesta et al., 2006) and thus CO₂-treated berries reached a low ion leakage value during storage at 0°C in comparison to non-treated ones (Rosales et al., 2016). In a previous study, we observed that application of high CO₂ levels at 0°C in table grapes cv. Cardinal for 3 days induced the expression of CBF1 and CBF4 (VviDREBA1-1 in this work) in the pulp and CBF4 in the rachis (Fernandez-Caballero et al., 2012). In a more recent work, we suggested that the beneficial effect of a high CO₂ treatment to maintain Cardinal table grape quality seems to be mediated by the regulation of ERFs (Romero et al., 2016), which belong to the AP2/ERF transcription factor family. In the present study, we have analyzed the effect of a 3-day CO₂ treatment in maintaining the table grape quality of a cultivar which is different from Cardinal, such as Autumn Royal. Unlike red-skinned Cardinal table grape cultivar which matures early (late spring to mid-summer) and presents very few seeds, Autumn Royal is a late-maturing seedless table grape cultivar (from autumn through early winter), with a purple-black to black berry skin. Furthermore, to investigate whether the role played by CBF/DREB transcription factors is cultivar dependent and/or a common feature of other DREBA1s, we have analyzed the transcript accumulation pattern of VviDREBA1-1, VviDREBA1-6, and VviDREBA1-7 in different tissues (skin, pulp, and rachis) of Autumn Royal bunches which were treated and non-treated with 20 kPa CO₂ for 3 days at 0°C and then transferred to air for up to 13 days. Likewise, to study the role of CBF/DREBs in the regulation of DHNs in table grapes we first analyzed the pattern of expression of three dehydrins (VviDHN1a, VviDHN2, and VviDHN4) in different tissues of Autumn Royal bunches which were CO₂-treated and non-treated; and we then analyzed their promoters so as to identify different cis-regulatory elements, including the DRE/CRT motifs. Finally, we examined the DNA-binding specificities of three CBF/DREBs, by using electrophoretic mobility shift assay (EMSA), showing that only VviDREBA1-1 was able to bind in vitro to the CRT element present in the VviDHN2 promoter.

Mature table grapes are non-climacteric fruit with a relatively low respiration rate, but are subject to serious water loss, softening and fungal attack after harvest. Grapes are classified as a non-chilling sensitive fruit and postharvest storage around 0°C is recommended to maintain their quality. However, we have observed that table grapes cv. Cardinal are sensitive to temperature shifts from the field to storage at 0°C with increasing rachis browning, water loss and ion leakage, as well as the activation of phenylpropanoid and pathogenesis related gene expression, whereas a 3-day high CO₂ pretreatment at 0°C avoids and/or modifies these changes (Sanchez-Ballesta et al., 2006; Rosales et al., 2016). In the present study, we have observed that the application of high CO₂ levels for 3 days at 0°C was effective in maintaining the quality of table grapes other than Cardinal, such as Autumn Royal. Previous results in Cardinal berries showed that the CO₂ treatment significantly affected the moisture content of all tissues of the bunch, except for pulp tissues (Goñi et al., 2011). However, similar values of moisture content were quantified in 3-day CO₂-treated and non-treated Autumn Royal berries stored at 0°C, as was observed in strawberries (Blanch et al., 2012). On the other hand, CO₂-treated table grapes showed a low weight loss percentage in comparison to non-treated bunches during storage at 0°C. This effect has been also reported by Martínez-Romero et al. (2003) in table grapes, and in other types of fruit such as nectarines (Retamales et al., 2000), cucumbers (Wang and Qi, 1997), and cherries (Kappel et al., 2002) stored in modified atmosphere packaging at low temperature. Concerning the rachis browning index, it was lower in Autumn Royal bunches treated with CO₂ for 3 days than in non-treated bunches, as has been observed in Cardinal table grapes (Rosales et al., 2013). However, this is not a common response to high CO₂ levels. Thus, rachis appearance was adversely affected in Thompson Seedless and Red Globe bunches stored up to 40 or 45 days at low temperature under controlled atmosphere CO₂ levels ≥15 kPa, respectively, and after 4 days of shelf-life (Retamales et al., 2003). The beneficial effect of the gaseous treatment on the rachis appearance of Autumn Royal bunches could be explained by lower water loss from the rachis under this treatment (Sanchez-Ballesta et al., 2006). Accordingly, in Cardinal bunches the relative water content of the rachis, an indicator of water balance status, decreased considerably throughout storage at 0°C in non-treated bunches whereas the application of 3-day high CO₂ levels reduced this effect (Rosales et al., 2013).

In recent studies, we have shown that maintaining Cardinal table grape quality by the application of a 3-day CO₂ treatment seems to be an active process which requires the activation of transcription factors, such as ERFs, belonging to the AP2/ERF transcription factor family (Romero et al., 2016; Rosales et al., 2016). In addition, we have observed that the gaseous treatment applied to Cardinal bunches induced the expression of other members of this transcription factor family such as CBF/DREB (Fernandez-Caballero et al., 2012). The CBF/DREB proteins play a crucial role in abiotic stress-mediated gene expression, representing one of the most attractive regulons for breeding programs (Zandkarimi et al., 2015) to cope with cold stress. Different studies have indeed shown the relationship between CBF/DREB expression and cold tolerance in different fruit such as tomato, peach, kiwifruit, and mango during low temperature storage in combination with different postharvest treatments (Liang et al., 2013; Ma et al., 2014; Zhang et al., 2017). However, with the exception of the study mentioned above, the relationship of these CBF/DREBs with the different sensitivity to temperature shifts induced in fruit by high CO₂ levels is still unknown. Thus, we have isolated three DREBA1s from Autumn Royal table grapes and characterized their expression pattern in the skin, pulp and rachis during postharvest storage at low temperature with a 3-day high CO₂ treatment. In this work, we observed that gene expression varied according to the VviDREBA1 analyzed and the tissue studied. The storage of Autumn Royal bunches at low temperature under normal atmosphere was enough to activate the expression of VviDREBA1-1 in the pulp and VviDREBA1-7 in the rachis. Likewise, as was observed in Cardinal grapes (Fernandez-Caballero et al., 2012), the 3-day high CO₂ treatment at 0°C modulated the expression of VviDREBA1s by activating VviDREBA1-1 and VviDREBA1-7 in the skin, and VviDREBA1-6 and VviDREBA1-7 in the pulp. On the other hand, it is important to note that the effect of low temperature storage under normal atmosphere modulated VviDREBA1-1 and VviDREBA1-7 after 13 days of storage, while the three VviDREBA1s were induced at the end of the 3-day gaseous treatment at 0°C. This temporal difference could be important to help table grapes face temperature shifts at 0°C. Thus, Santiam tomato fruit showed less sensitivity to chilling than Lichum tomatoes, reflected in the lowest chilling injury index, ion leakage and malondialdehyde content. Meanwhile, LeCBF1 transcript accumulation was higher in Santiam tomatoes, indicating that there was a swift genetic response to chilling stress, which was positively correlated with cold tolerance of the cultivar (Zhao et al., 2009).

It is known that the activation of CBF/DREBs improves the expression of downstream target genes, especially those encoding LEA proteins, including DHNs, as these transcriptional activators can bind to the cis DRE/CRT motif present in the promoter regions of these genes. Overexpression of AtCBF1–AtCBF3 in transgenic Arabidopsis plants resulted in an increase in freeze tolerance and in the activation of different COR genes, including DHNs, at room temperature (Gilmour et al., 2004). A comparative study of two citrus species (Poncirus and Citrus) with different levels of freezing tolerance, showed a correlation between CBF1 expression and the degree of tolerance observed (Champ et al., 2007). These authors further demonstrated that CBF1 specifically recognized the consensus sequence (CCGAC) of the DRE/CRT elements from the dehydrin promoter of Poncirus trifoliata. To study the role of VviDREBA1s in the regulation of VviDHNs, we first studied the gene expression of spliced and unspliced VviDHN1, VviDHN2, and VviDHN4 mRNAs in different tissues of Autumn Royal bunches, both CO₂-treated and non-treated with storage at 0°C. VviDHN1a, VvDHN2, and VviDHN4 are known to undergo alternative splicing (Fernandez-Caballero et al., 2012; Yang et al., 2012). Intron retention during VviDHNs pre-mRNA processing leads to the production of mRNAs with premature stop codons in the intron that give rise to truncated proteins without the K segment, which is characteristic of this protein family. Likewise, Lee et al. (2016) reported that OsCYP19-4 (the primary cyclophilin 19-4 transcript from rice) was able to generate up to eight alternative splice forms via two types of regulated splicing events, intron retention and exon skipping, under low temperatures. Thus, in the case of Arabidopsis, temperature stress (cold and heat) dramatically altered the alternative spliced of pre-mRNAs of several serine/arginine-rich proteins, a conserved family of splicing regulators in eukaryotes (Palusa et al., 2007). Although the functionality of unspliced DHNs is not well known, in a previous work we demonstrated that the unspliced DHN1a variant from V. vinifera cv. Cardinal slightly interacted with DNA (Rosales et al., 2014). In this work, we have observed that unspliced forms showed a higher regulation degree than spliced forms after the CO₂ treatment. Therefore, with the exception of unspliced VviDHN1a and VviDHN4 in rachis, the 3-day CO₂ treatment regulated the transcript levels in the unspliced forms in the different tissues of Autumn Royal.

The study of the VviDHNs promoter regions showed that, whereas the VviDHN2 promoter region showed the presence of one DRE element and one CRT element, VviDHN4 did not show any of them. Likewise, previous results showed the presence of a DRE element in the VviDHN1 promoter (Rosales et al., 2014). Through EMSA assays, our work provides evidence that the recombinant protein VviDREBA1-1 was the only one which showed specific binding to the CRT element (GCCGAC) presented in the VviDHN2 promoter, while no binding was observed to the DRE element (ACCGAC). In a recent work (Carlow et al., 2017), transient transactivation assays showed that all V. riparia CBFs except CBF5 activate via a CRT or DRE promoter element, whereby V. riparia CBF3 (homolog to Vv1DEBA1-6) and CBF4 (homolog to Vv1DEBA1-1) prefer a CRT element. It is known that not all CBF/DREBs have the same affinity and specificity for DRE/CRT elements. Thus, BNCBF17 from Brassica napus showed lower binding specificity than BNCBF5 to the core CCGAC sequence when this was mutated (Gao et al., 2002), while HvCBF1 from barley showed preference for binding to the sequence TTGCCGACAT, which contained the CRT motif instead of DRE (Xue, 2002). Likewise, the analysis of the promoters of COR genes induced in Arabidopsis plants which overexpressed CBFs demonstrated that variations in the sequences around the CRT element could affect the activation of the promoters (Maruyama et al., 2004). In this regard, our results showed that the region flanking the CRT element must have a role in the affinity of the VviDREBA1s, since changes in some nucleotides did not abolish the binding observed with VviDREBA1-1, but its intensity was reduced. Although more studies are needed to be able to understand the binding specificity of CBF/DREBs for target sequences, the results are in concordance with previous results reported by Nassuth et al. (2014) which suggest that CBF/DREBs paralogs in a plant, and possibly orthologs from different species, have a unique preference for DRE/CRT sequences. At this point it is important to remark that the three VviDREBA1s contained the signature sequences, PKKPAGR and DSAWR, flanking the AP2 domain which distinguishes this subgroup of transcription factors from the other AP2/ERF family members (Jaglo et al., 2001). Canella et al. (2010) showed that deletions or mutation in the PKKPAGR sequence greatly impaired the ability of AtCBF1 to induce expression of target COR genes because of its inability to bind to the DRE/CRT element indicating that amino acids beyond the AP2 domain are required. In the specific case of DREBA1s from Autumn Royal, we observed that whereas the PKKPAGR motif was well conserved in VviDREBA1-1 (PKKRAGRxKFxETRHP), there were three (HKRKAGRxKFxETRHP) and five (HKRKTGRxKFxKTRHP) amino acids which were different in VviDREBA1-6 and VviDREBA1-7, respectively. In Vitis, Xiao et al. (2006) observed that despite the three amino acid changes detected in CBF1, targeting and transactivation experiments denoted that CBF1 still functions. Nevertheless, transient activation experiments suggested that V. riparia CBF4 was a more effective activator than VrCBF1 (Xiao et al., 2008), and that Arabidopsis transgenic plants which overexpressed V. riparia CBF1 and CBF4 presented higher amounts of COR genes in the CBF4 lines (Siddiqua and Nassuth, 2011). Likewise, Nassuth et al. (2014) corroborated previous results and showed, with a new system of transactivation, that VrCBF1 and VrCBF4 transcription factors probably activate different overlapping sets of genes, and therefore play unique roles in cold acclimation. Thus, this evidence could be related to the changes observed in the PKKPAGR motif of Vitis CBFs.

Overall, our results demonstrated that the 3-day gaseous treatment was effective in maintaining table grape quality regardless of the cultivar and modulated the expression of VviDREBA1s transcription factors in a tissue dependent way. Furthermore, the application of high levels of CO₂ regulated the retention of introns in the transcripts of the dehydrins in a different manner and in all the tissues analyzed. On the other hand, VviDREBA1-1 was the only transcription factor analyzed that presented in vitro binding capacity to the CRT element of the VviDHN2 promoter region, indicating that the transcriptional regulation of VviDHN1a and VviDHN4 would be carried out by activating other independent routes of these transcription factors (Figure 7). These results, together with the fact that VviDREBA1-1 gene expression was induced by high CO₂ levels in the skin of Autumn Royal fruit and in the pulp and rachis of Cardinal bunches (Fernandez-Caballero et al., 2012), make this transcription factor a good candidate for further research with the aim of improving table grape quality.

MV-H and IR contributed equally to this work. MV-H: Participated in the table grape quality assessments, carried out the RNA extraction, RT-qPCR analysis, semiquantitative RT-PCR, statistical analyses. IR: Carried out the VviDREBA1s isolation and the production of recombinant proteins, the EMSA analysis, edited and collaborated in the first draft of the manuscript. ME: Conceived the postharvest table grapes storage experience, participated in the table grape quality assessments and critically revised the manuscript. CM: Participated in the table grape quality assessments and critically revised the manuscript. MS-B: conceived the VviDREBA1s and DHNs study, supervised and coordinated the experiments, interpreted the results and prepared the first draft of the manuscript. All authors have read and approved this manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.