RESUMO
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.
RESUMO
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.
Assuntos
Dióxido de Carbono/farmacologia , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Plantas/genética , Estresse Fisiológico/fisiologia , Fatores de Transcrição/genética , Vitis/fisiologia , Sequência de Aminoácidos , Clonagem Molecular , Análise por Conglomerados , Temperatura Baixa , DNA de Plantas/genética , Frutas/genética , Frutas/metabolismo , Frutas/fisiologia , Dados de Sequência Molecular , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/fisiologia , Sementes/genética , Sementes/metabolismo , Sementes/fisiologia , Análise de Sequência de DNA , Fatores de Transcrição/química , Ativação Transcricional/genética , Vitis/genética , Vitis/metabolismoRESUMO
Unfreezable water (UFW) content in berry tissues (pulp, skin, seed) and rachis of table grape clusters stored at 0°C has been studied using differential scanning calorimetry. The effect of short exposure to high CO2 (20% CO2 for 3days) and the transfer to air were also studied. Water status of pulp tissues was related to the thawing behaviour and the structural characteristics, using low-temperature scanning electron microscopy (LT-SEM). The UFW content in all tissues increased rapidly in response to high CO2 while it remained stable or decreased in untreated clusters. The strong potential of this beneficial gaseous treatment for increasing the UFW content was also evident after transfer to air. The metabolic adjustment caused by exposure to high CO2, which reduced the amount of water available to be frozen, improved stored fruit quality, thus minimising structural damage and reducing water leakage associated with the freezing-thawing process.
RESUMO
Gene expression of a class I chitinase (Vcchit1b) in the skin of table grapes was analyzed as a molecular marker for changes induced at low temperature and also to study the effect of high CO(2) levels modulating transcript levels at 0 degrees C. An active recombinant VcCHIT1b was overexpressed in Escherichia coli, and as the protein was produced as insoluble inclusion bodies, it was solubilized and refolded. The purified recombinant chitinase showed an optimum pH of 6.0 and a temperature of 50 degrees C, retaining activity at 0 and -10 degrees C. Purified chitinase exerted in vitro antifungal activity against Botrytis cinerea. Furthermore, recombinant chitinase was able to cryoprotect lactate dehydrogenase against freeze/thaw inactivation. However, the recombinant VcCHIT1b did not show any antifreeze activity when the thermal hysteresis activity was measured using differential scanning calorimetry.
