Raquel Rosales

Unraveling the roles of CBF1, CBF4 and dehydrin 1 genes in the response of table grapes to high CO2 levels and low temperature

CBFs (C-repeat binding factors) are transcription factors that are rapidly induced by low temperature and that recognize the CRT/DRE element in the promoter of a set of cold regulated genes, the CBF regulon. Dehydrins are proteins that accumulate in plants under stress conditions, such as low temperature, and some form part of the CBF regulon. To investigate their role in the response of table grape clusters to 0°C long storage as well as to 3-day high CO₂ postharvest treatment, we isolated two partial CBF genes (VvcCBF1 and VvcCBF4) and a full-length dehydrin (VvcDHN1a) from Vitis vinifera cv. Cardinal. Hydrophobic cluster analysis (HCA) identified differences in the secondary and tertiary structure between Vitis CBF4s and CBF1s. Overall, our results showed that, in table grapes, the expression of CBF genes is induced mainly in response to CO₂ treatment, suggesting that the response of DHN1a in this fruit could be attributed to a cold-inducible CBF-independent pathway.

Low Temperature and Short-Term High-CO2 Treatment in Postharvest Storage of Table Grapes at Two Maturity Stages: Effects on Transcriptome Profiling

Table grapes (Vitis vinifera cv. Cardinal) are highly perishable and 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 treatment (20 kPa CO2 + 20 kPa O2 + 60 kPa N2) at 0°C reduced total decay and retained fruit quality in early and late-harvested table grapes during postharvest storage. In order to study the transcriptional responsiveness of table grapes to low temperature and high CO2 levels in the first stage of storage and how the maturity stage affect these changes, we have performed a comparative large-scale transcriptional analysis using the custom-made GrapeGen GeneChip®. In the first stage of storage, low temperature led to a significantly intense change in grape skin transcriptome irrespective of fruit maturity, although there were different changes within each stage. In the case of CO2 treated samples, in comparison to fruit at time zero, only slight differences were observed. Functional enrichment analysis revealed that major modifications in the transcriptome profile of early- and late-harvested grapes stored at 0°C are linked to biotic and abiotic stress-responsive terms. However, in both cases there is a specific reprogramming of the transcriptome during the first stage of storage at 0°C in order to withstand the cold stress. Thus, genes involved in gluconeogenesis, photosynthesis, mRNA translation and lipid transport were up-regulated in the case of early-harvested grapes, and genes related to protein folding stability and intracellular membrane trafficking in late-harvested grapes. The beneficial effect of high CO2 treatment maintaining table grape quality seems to be an active process requiring the induction of several transcription factors and kinases in early-harvested grapes, and the activation of processes associated to the maintenance of energy in late-harvested grapes.

Differential regulation of dehydrin expression and trehalose levels in Cardinal table grape skin by low temperature and high CO2

Dehydrins and trehalose are multifunctional protective biomolecules that play a role in counteracting cellular damage during dehydrative stresses. In this paper, we studied dehydrin isoform patterns, dehydrin gene expression and trehalose levels in the skin of Cardinal (Vitis vinifera L.) table grapes, along with their regulation by different cold postharvest storage conditions. Immunoanalysis with K-segment antibody recognizes four constitutive dehydrins (from 17 to 44 kDa) that are tightly regulated by low temperature and high CO2. Phosphatase treatment showed that DHN44 and DHN22 isoforms are phosphorylated polypeptides, while MALDI-TOF MS and MS/MS analysis suggested that 44 kDa polypeptide may be a dehydrin homodimer. At the transcriptional level, dehydrins are also regulated by low temperature and high CO2, showing a fairly good correlation with their mRNA levels. Trehalose was quantified by high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), revealing a progressive increase of this metabolite throughout storage at 0 °C and the sudden transitory increases in short-term high CO2-treated fruit. We propose that the constitutive presence and up-regulation of dehydrins and trehalose during low temperature postharvest storage could be positively correlated with the relative chilling tolerance of table grapes and the adaptive responses activated by high CO2 levels to preserve cell water status and to counteract the disruption of physiological processes during cold storage.

Effects of High CO2 Levels on Fermentation, Peroxidation, and Cellular Water Stress in Fragaria vesca Stored at Low Temperature in Conditions of Unlimited O2

To better understand the tolerance of strawberries (Fragaria vesca L.) to high CO2 in storage atmospheres, fermentation and cellular damage were investigated. Fruits were stored for 3 and 6 days at 0 °C in the presence of different CO2 levels (0, 20, or 40%) with 20% O2. Changes in pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) gene expression and in fermentative metabolites, as well as in bound water and malondialdehyde (MDA) concentrations, were analyzed. In strawberries stored without added CO2, up-regulation of PDC and ADH was not associated with an increase in fermentative metabolites. By contrast, moderate ethanol fermentation in fruits exposed to 20% CO2 seems to be essential to maintain fruit metabolism, reducing both lipid peroxidation and cellular water stress. However, if the CO2 concentration increases (40%), the excess acetaldehyde and ethanol produced were closely correlated with a decrease in bound water and production of MDA.