Role of transcription factors in the beneficial effect of 3-day high CO2 pretreatment during postharvest storage at low temperature of table grapes

Abstract

[Background and Aims]: Table grape (Vitis vinifera L.) is a non-climacteric fruit, tolerant to low temperature. However, their quality deteriorates during postharvest storage at low temperature mainly because of sensitivity to fungal decay and senescence of rachis. The application of a 3-day CO2 pretreatment (20% CO2 + 20% O2 + 60% N2) at 0ºC reduced total decay and retained bunches quality during postharvest storage. However, the molecular mechanisms involved in this process are not well known. In a recent work, we have shown that the maintenance of Cardinal table grape quality by the 3-day gaseous pretreatment seems to be an active process, activating transcription factors such as WRKYs and ERFs (Ethylene Responsive Factors). In addition, we have observed that the gaseous pretreatment modulated the expression of VviDREBA1-1 in the pulp and rachis of table grapes cv. Cardinal. The fact that DREBA1-1 belong to the same transcription factor family that ERFs seems to indicate that this family could play an important role in the beneficial effect of the gaseous treatment to maintain table grape quality. To better understand the role of transcription factors in the maintenance of table grape quality at low temperature by the application of high CO2 levels we have characterize 5 VviERFs, 3 VviDREBA1s and 15 VviWRKYs in two table grapes cultivars, such as Cardinal and Autumn Royal. Furthermore, to study the role of ERFs/WRKYs in the regulation of pathogenesis related proteins (PRs), and DREBA1s in the regulation of dehydrins (DHNs) in table grapes, we have analyzed the pattern of expression of three PRs (thaumatin (VviTL1), chitinase (Vcchit1b) and β-1,3-glucanase (Vcgns1)) and three DHNs (VviDHN1a, VviDHN2 and VviDHN4) in different tissues of bunches CO2-treated and non-treated. Furthermore, we have identified different cis-regulatory elements, including the GCC/W-box and DRE/CRT motifs, respectively and we have examined the DNA-binding specificities of the mentioned transcription factors by using electro mobility shift (EMSA) assays.

[Experimental Procedure and Main Results]: Table grapes (Vitis vinifera L.) cv. Cardinal were harvested in Camas, Seville, Spain in July 2003 at the early commercial stage (maturity index: 15.24) and table grapes cv. Autumn Royal were picked in Abarán, Murcia, Spain in November 2013 at the late commercial stage (maturity index: 27.97). After random harvesting, bunches were transported in the same day to the laboratory in Madrid (Spain) and treated according to. For each sample, total RNA was extracted three times according to, and treated with DNase I recombinant-RNase free (Roche) for genomic DNA removal. Then, 1 μg of each extraction was used to synthesize cDNA as described by. Relative expression of VviERFs, VviDREBA1s and VviWRKYs, as well as spliced and unspliced transcripts of VviDHN4 was assayed using quantitative RT-PCR (RT-qPCR) as described by. Spliced and unspliced variants of VviDHN1a and VviDHN2 were evaluated by semi-quantitative RT-PCR as described by. The full VviERFs, VviDREBA1s and VviWRKYs open reading frames, including stop codons were amplified by RT-PCR, cloned as N-terminal fusion with an amino acid His tag in pTrcHisA vector (Invitrogen, Carlstad, USA) and transformed into BL21-CodonPlus (DE3)-RIL competent cells as described by. The induction and purification of recombinant proteins was performed according to. Purified recombinant proteins were used to determine DNA binding by EMSA assay. The GCC box, DRE/CRT motifs and W-box were used as probes. Binding reactions were performed in 20 μl final volume containing 2 μg of recombinant proteins, 50 μM unlabeled DNA, 80 nM Biotin-labeled probe, 50 ng μl-1 Poly (dI·dC), 2.5% (v/v) glycerol and 1X binding buffer.

[Main results]: Our results showed that in most of the Cardinal tissues analyzed the VviERFs gene expression was induced after storage under normal atmosphere, although applying high levels of CO2 at 0ºC caused a greater increase in VviERFs transcript accumulation. However, the pattern of expression was different depending on both the tissue and VviERFs. In table grapes cv. Autumn Royal, the 3-day high CO2 treatment applied at 0ºC 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 0ºC. This temporal difference could be important to help table grapes face temperature shifts at 0ºC. The gaseous treatment also activated the expression of WRKYs transcription factors in a tissue-dependent manner in Autumn Royal bunches, inducing levels of 4 VviWRKYs in the skin and 9 in the pulp. The promoter regions of Vcchit1b and Vcgns1 had in common the presence of a single W-box and a GCC box. However, the VviTL1 promoter region included a W-box but not a GCC-box. By other hand, the study of the VviDHNs promoter regions showed that, whereas the VviDHN1 promoter presented a DRE element and the VviDHN2 promoter one DRE element and one CRT element, VviDHN4 did not show any of them. The EMSA assays provided evidences that recombinant VviERF2 and VviERF11 bound specifically to the GCC box presented in the promoter of the above mentioned PRs. However, VviERF069 and VviERF10 can only bind to the GCC box of the Vcgns1 promoter and VviERF6L7 showed no affinity for any of the GCC box studied. However, while VviWRKY14 presented affinity for the W-box of the three PR promoters, VviWRKY42 only had specific affinity to the W-box of the Vcgns1 promoter. In addition, it was showed that VviDREBA1-1 is the only transcription factor analyzed from this family that was able to bind in vitro to the CRT element (GCCGAC) presented in the VviDHN2 promoter.

[Significance of the Study and Conclusions]: To date, there has been no comprehensive analysis regarding the transcriptional responses of transcription factors genes to postharvest storage conditions associated with mechanisms underlying abiotic stress responses, such as low temperature or modified atmosphere. Our work, suggested that the beneficial effect of high CO2 treatment maintaining table grape quality of different cultivars during storage at low temperature seems to be mediated by the regulation of ERFs, DREBA1s and WRKYs transcription factors. In particular, it is interesting to note that VviERF2 gene expression an induction by high CO2 levels in all the tissues analyzed and a significant correlation with the expression of the Vcchit1b and Vcgns1, together with the in vitro binding affinity observed with the GCC box of their promoters. By other hand, the fact that VviDREBA1-1 was the only transcription factor analyzed that presented in vitro binding capacity to the CRT element of the VviDHN2 promoter region, indicated that the transcriptional regulation of VviDHN1a and VviDHN4 would be carried out by activating other independent routes of these transcription factors. These results open interesting.