Rachis browning is one of the main factors reducing the quality of table grapes during storage at low temperature. To better understand the effect of a 3-day CO 2 pretreatment (20% CO 2 plus 20% O 2 ) on maintaining the rachis quality of table grapes (Vitis vinifera cv. Cardinal) 5 at 0 ºC, we analyzed the expression of genes codifying enzymes related to the synthesis and oxidation of phenolic compounds (phenylalanine ammonia-lyase, VcPAL; and polyphenol oxidase, GPO) and the detoxification of reactive oxygen species (catalase, GCAT; and ascorbate peroxidase, VcAPX) in rachis of treated and non-treated bunches. Furthermore, due to their role in senescence, the implication of ethylene and abscisic acid (ABA) was also 10 investigated by studying the expression pattern of key regulatory genes for these hormones such as ACC synthase (ACS1) and oxidase (ACO1), VvNCED1 and 2. To determine whether these changes in gene expression were specifically related to rachis deterioration, their expression pattern in pulp and skin of treated and non-treated grapes were evaluated. The appearance of browning in non-treated rachis was associated with an increase in GPO and 15VcPAL mRNA levels, whereas high CO 2 levels arrested this accumulation. In pulp, even though browning was not evident, a slight increase in GPO accumulation in non-treated bunches was detected. Moreover, lipid peroxidation level revealed lower oxidative stress in rachis of CO 2 -treated bunches than in non-treated ones, which seemed to be regulated by VcAPX and GCAT gene expression induction. Interestingly, this regulation was specific to rachis, showing a 20 different pattern in pulp and skin. Regarding phytohormones, our results pointed to the participation of the ethylene biosynthesis pathway in rachis browning. On the other hand, neither VvNCED1 nor VvNCED2 expression levels were altered in rachis, but NCED1 was induced specifically by low temperature in pulp. Overall, our results suggest a specific response of rachis to high levels of CO 2 that could be related to the mitigation of rachis browning. 25

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.

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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.