Search

Blueberries (Vaccinium corymbosum L.) are becoming increasingly popular for their nutritional and health benefits, and their economic value is therefore increasing. The loss of quality that can occur due to softening and fungal attack is an important consideration when marketing blueberries. Despite the added value of blueberries, no studies have been carried out on how the fruit arrives at the outlets just before purchase by the consumer in terms of firmness, physico-chemical parameters, phenolic compounds, and fungal growth. The aim of this work has been, therefore, to investigate possible differences in quality parameters between blueberries purchased from ten different outlets, regardless of the supplier. The results showed that all the samples were of acceptable quality, although they all had a low maturity index at the point of sale. None of the samples studied showed clear signs of fungal decay at the time of purchase, although we were able to grow and identify some pathogen specimens after cultivation. In terms of total phenolic and anthocyanin content, as well as antioxidant activity, all the samples showed low values, possibly due to their postharvest storage, but they were within the expected range for this fruit. On the other hand, differences in the measured parameters were observed between samples of the same cultivar while no differences were found between conventionally and organically grown blueberries. This suggests that preharvest (such as edaphoclimatic conditions, agricultural practices, and cultivars) and postharvest factors (such as treatments used, storage, and transport temperatures) could influence the berry quality when they reach the consumer.

Soft fruits are appreciated for their taste qualities and for being a source of health-promoting compounds. However, their postharvest is affected by their high respiratory rates and susceptibility to fungal decay. Our aim here is to provide a perspective on the application of short-term high-CO2 treatments at a low temperature to maintain the postharvest quality of soft fruits. This work also suggests using a multi-omics approach to better understand the role of the cell wall and phenolic compounds in maintaining quality. Finally, the contribution of high-throughput transcriptomic technologies to understand the mechanisms modulated by the short-term gaseous treatments is also highlighted., This work was supported by CICYT Project PID2020-113965RB-I00/AEI/10.13039/501100011033.

This article belongs to the Special Issue Biochemical and Molecular Approaches to Postharvest Research., Short-term gaseous treatments improve rachis quality during table grape postharvest, but little is known about the mechanisms involved. In this work, we observed that the application of a 3-day CO2 treatment at 0 °C improved rachis browning of Superior Seedless and Red Globe bunches, affecting the non-enzymatic antioxidant system by reducing the total phenolic content, the antioxidant activity and the expression of different stilbene synthase genes. Lipid peroxidation levels revealed lower oxidative stress in CO2-treated rachis of both cultivars linked to the activation of the enzymatic antioxidant system. Furthermore, whereas a positive correlation was denoted between rachis browning and the accumulation of key ABA regulatory genes in Red Globe bunches, this effect was restricted to ACS1, a key synthetic ethylene gene, in Superior Seedless clusters. This work also corroborated the important role of ethylene-responsive factors in the beneficial effect of the gaseous treatment, not only in the berries but also in the rachis. Finally, the application of the gaseous treatment avoided the induction of cell wall-degrading enzyme-related genes in both cultivars, which could favor the maintenance of rachis quality. This work provides new insight into specific responses modulated by the gaseous treatment focused on mitigating rachis browning independently of the cultivar., This work was supported by CICYT projects AGL2017-85291-R (MINECO/AEI/FEDER, UE) and PID2020-113965RB-I00/AEI/10.13039/501100011033.

Soft fruits are appreciated for their taste qualities and for being a source of health-promoting compounds. However, their postharvest is affected by their high respiratory rates and susceptibility to fungal decay. Our aim here is to provide a perspective on the application of short-term high-CO

Dehydrins, a family of hydrophilic and intrinsically disordered proteins, are a subgroup of late embryogenesis abundant proteins that perform different protective roles in plants. Although the transition from a disordered to an ordered state has been associated with dehydrin function or interactions with specific partner molecules, the question of how the primary and secondary dehydrin protein structure is related to specific functions or target molecule preferences remains unresolved. This work addresses the in silico sequencing analysis and in vitro functional characterization of two dehydrin isoforms, VviDHN2 and VviDHN4, from Vitis vinifera. Conformational changes suggest potential interactions with a broad range of molecules and could point to more than one function. The in silico analysis showed differences in conserved segments, specific amino acid binding sequences, heterogeneity of structural properties and predicted sites accessible for various post-translational modifications between the sequence of both dehydrins. Moreover, in vitro functional analysis revealed that although they both showed slight antifungal activity, only VviDHN4 acts as a molecular shield that protects proteins from freezing and dehydration. VviDHN4 also demonstrated high potential as a chaperone and reactive oxygen species scavenger, in addition to presenting antifreeze activity, all of which confirms its multifunctional nature. Our findings highlight the significant role of Y-segments and the differential and specific amino acid composition of less conserved segments that are rich in polar/charged residues between S- and K-segments, coupled with post-translational modifications, in modulating and switching dehydrin biological function., This research was financed by the CICYT Spanish Projects AGL 2014-53081-R (MINECO) and AGL 2017-85291-R (MINECO/AEI/FEDER, UE). M. Vazquez-Hernandez acknowledges the support of the FPI program financed by the MICINN.

The application of one or two short-term treatments with high levels of CO2 during 3 days at 0 °C maintained the quality of Autumn Royal table grapes (Vitis vinifera) during storage at 0 °C. We have analyzed how the application of a 3-day gaseous treatment, for one or two times at 0 °C, influences on common (VviPAL, VviCHS, VviCHI, VviF3′H, VviF3′5′H, VviF3H and VviLDOX) and branch-specific (VviFLS1, VviLAR1, VviLAR2, VviANR and VviUFGT) flavonoid gene expression in the skin of Autumn Royal table grapes. Likewise, the content of flavonols, flavan-3-ols and anthocyanins were identified with Q-TOF equipment and quantified by HPLC-quadrupole together with the total phenolic content and the antioxidant capacity by the ABTS and FRAP methods. Moreover, we have also used a solid-state voltammetry methodology to compare the effect of the application of one or two gaseous treatments in the skin of table grapes stored at 0 °C. Results revealed that the application of one or two gaseous treatments modulated the expression of flavonoid gene expression and the levels of catechin, in the case of one application, or quercetin-3-glucoside and five anthocyanins in fruit treated twice, maintaining their levels after 28 days of storage at 0 °C similar to those recorded in freshly harvested fruit. Satisfactorily, the electrochemical approach was useful to distinguish between treated and non-treated samples not only in the first stage of storage but also after 16 days., This work was supported by the spanish CICYT projects AGL2014-53081-R and AGL2017-85291-R (MINECO/AEI/FEDER, UE). I. Domínguez thanks the University of Almeria for the Hipatia postdoctoral contract.

This article belongs to the Special Issue Gene Transcriptional Regulation in Crops during Postharvest., Despite the fact that many studies have examined the effectiveness of different gaseous postharvest treatments applied at low temperature to maintain table grape quality, the use of ethanol vapor has hardly been investigated. Thus, this work has studied the effectiveness of ethanol vapor-generating sachets in the maintenance of It 681–30 table grape quality, a new cultivar, during storage at low temperature and after the shelf-life period at 20 °C. To this end, various quality assessments have been carried out and the effect of the ethanol treatment on the expression of different genes (phenylpropanoids, transcription factors, PRs, and aquaporins) was determined. The results indicated that the application of ethanol vapor reduced the total decay incidence, weight loss, and the rachis browning index in It 681–30 grapes stored at 0 °C and after the shelf-life period at 20 °C, as compared to non-treated samples. Moreover, the modulation of STS7 and the different PR genes analyzed seems to play a part in the molecular mechanisms activated to cope with fungal attacks during the postharvest of It 681–30 grapes, and particularly during the shelf-life period at 20 °C. Furthermore, the expression of aquaporin transcripts was activated in samples showing higher weight loss. Although further work is needed to elucidate the role of ethanol in table grape quality, the results obtained in this work provide new insight into the transcriptional regulation triggered by ethanol treatment., This work was supported by CICYT projects AGL2014-53081-R and AGL2017-85291-R (MINECO/AEI/FEDER, UE).

Fatty acids (FAs) play important roles in membrane fluidity, energy status and the production of specific FA-dependent esters. However, little is known about the modifications in FAs induced by high CO2 that can improve fruit quality and prevent the disorders associated with storage at low temperature. For this purpose, total, free and esterified FAs in neutral and polar lipid fractions were characterized and quantified and their relationship with straight-chain esters was examined in strawberries treated with high doses of CO2 and different times of exposure during early storage at 0 °C. In strawberries stored without added CO2 (T1), storage at 0 °C led to an imbalance in the saturated/unsaturated ratio of polar lipids, mainly due to the decrease in the amount of saturated fatty acids (SFA). In addition, a decrease in the amount of free polyunsaturated fatty acids (PUFA) linked to a prevalence of butanoates and hexanoates esterified to long alcohols was observed. A strong negative correlation (P ≤ 0.01; r ≥ 0.7) between butanoate esters and free PUFA was quantified. In contrast, the application of 2 d 20 kPa CO2 treatment (T2), increased the amount of PUFA from the neutral and polar fractions, with a preference for α-linolenic acid (18:3n3) which results in a rise in the 18:3/18:2 ratio, and which can confer membrane stability and fluidity. Furthermore, our results also showed a strong positive correlation (P ≤ 0.01; r ≥ 0.75) between α-linolenic acid (18:3n3) of polar lipids and some ethyl esters that contribute mostly to the aroma in strawberries. However, 3 d 40 kPa CO2 treatment (T4) depleted the content of FAs from all the lipid fractions in association with deficit ATP levels and an increased lipid peroxidation. These results suggest an active lipid breakdown which could play a causal role in the reported increased leakage of cellular water into intercellular air spaces in stressed-CO2 fruit. Taken together, our results provide new insights into the beneficial effect of 2 d 20 kPa CO2 treatment at 0 °C on the enhancement of PUFA in the polar lipid fraction and on the emission of esters other than ethyl acetate, which could improve membrane fluidity and could contribute to enriching aroma in strawberries., This work was financed by Spanish Project AGL2017-85291-R (MINECO/AEI/FEDER, UE).

Cell membranes provide a link between postharvest disorders and their integrity is essential for reducing table grape deterioration due to dehydration and susceptibility to fungal infection. We examined the relationship between ultrastructural membrane integrity at the subcellular level (transmission electron microscopy), cell (H2O2 generation) and membrane (malondialdehyde accumulation) oxidative status, membrane fatty acid composition (polar lipid fraction) and berry quality in order to determine the critical membrane events associated with long-term storage disorders at 0 °C in table grapes (Vitis vinifera L.) cv. Autumn Royal, and evaluate their response to the application of a single or dual short-term 20 kPa CO2 treatment. The results revealed a progressive disorganization of the cytoplasm and the breakdown of the cell organelle ultrastructure, associated with elevated hydrogen peroxide generation and lipid peroxidation rates and an imbalance in the saturated/unsaturated ratio of polar lipids, mainly due to the decrease in the degree of unsaturation of 18-carbon fatty acids. Conversely, high CO2 levels maintained the integrity of the microstructure of cell and energy-related organelles that are essential for metabolic damage repair and cell membrane restoration, reduced oxidative damage in cells and membranes, and increased the unsaturation of 18-carbon fatty acids, lipid unsaturation ratio and index of unsaturated fatty acids (IUFA) in the membrane polar lipids. The results also revealed that the more short-term high CO2 treatment is applied to Autumn Royal grapes, the more cytological evidence there is of enhanced tolerance to long-term cold and prevention of storage disorders in terms of cluster weight loss, rachis browning, berry water loss and decay incidence., This research was financed by the CICYT Spanish Projects AGL2014-53081-R (MINECO) and AGL2017-85291-R (MINECO/AEI/FEDER, UE). M. Vazquez-Hernandez acknowledges the support of the FPI program financed by the MICINN.

Sucrose is a key player in strawberry metabolism. However, the molecular mechanisms underlying sucrose status in strawberries during later ripening stages and postharvest storage remains quite limited. Therefore, we identified members of various gene families encoding key enzymes involved in sucrose metabolism, analyzed their expression profiles and determined the sugar content in strawberries at different ripening stages and postharvest treatments. Three different ripening stages around harvest time, almost red (AR), full red (FR) and dark red (DR) were analyzed, as well as after pre-treatment with high CO2 levels (18 % CO2, 2 d) during low temperature (LT) storage and further shelf-life at 20 °C. The accumulation of sucrose, hexoses, trehalose, myo-inositol, fructo-oligosaccharides (FOS) and raffinose family oligosaccharides (RFOs) was analyzed. Our results indicate that the transition from FR to DR stages was characterized by a prominent accumulation of hexoses, sucrose and a sharp decrease in the levels of FOS. We therefore suggest that FOS, in addition to their protective role in stressful conditions, are also important metabolic signals of the end of ripening process. The highest levels of RFOs was detected in strawberries at LT in air, making them a possible marker of damage, an idea reinforced too by the low RFOs and high myo-inositol quantities found in high CO2 protective treatment. Interestingly, the sucrose accumulation in fully ripe strawberries and in high CO2-treated ones was linked to a significant decrease in the expression of FvVIN2 (vacuolar invertase), an opposite trend to that observed in fruit stored in air. Furthermore, high levels of CO2 prevented both the upregulation of FvSS1 and the sharp increase in the expression of FvCWINV1 observed at low temperature in air. The efficiency of high CO2 pre-treatment on sucrose accumulation was particularly evident during the shelf-life period at 20 °C (SL) after LT storage. Our study provides new insights into how strawberries become sweet by retaining sucrose content. Additionally, our results clearly highlight the need to specify the sugar status at harvest to know the effectiveness of a treatment in maintaining quality attributes., This work was supported by the spanish CICYT project AGL2017-85291-R (MINECO/AEI/FEDER, UE).

Phenolic compounds, such as phytoalexin resveratrol, can be induced in grapes in response to biotic and abiotic stresses and have been related in many healthy effects. Stilbene synthases (STSs) are the key enzyme responsible for resveratrol biosynthesis. They have been already isolated and characterized from several plant species, however, VviSTS is a multigene family and little is known about their modulation in response to the application of gaseous treatments that maintain table grapes quality during postharvest. In this work, we have analyzed the effect of a 3-day CO

In this study, we analyzed the effect of the application of high levels of COOur results indicate that the application of a 3-day gaseous treatment was effective in maintaining the quality of black and white table grapes for up to 28 days of storage at 0 °C. The electrochemical methodology applied was useful for differentiating between skin and pulp samples, and between black and white table grape cultivars. Some molecular mechanisms were modulated in the skin of both cultivars to overcome oxidative stress, which was not manifested with the same intensity in grapes treated with COThe 3-day gaseous treatment was useful to maintain black and white table grape quality at 0 °C. The voltammetry analysis indicated that CO

WRKY transcription factors are regulated by biotic and abiotic stress in Vitis vinifera, although little is known about their role in grape berries. The latest V. vinifera reference genome (PN40024 12 × .v2, VCOST.v3 annotation) gave us a good opportunity to perform an analysis of VviWRKYs, identifying 61 genes. Our study examines whether the application of high CO2 levels for 3 d to maintain table grape quality activated the gene expression of 15 WRKYs in the skin and pulp of grapes (cv. Autumn Royal) stored at 0 °C. Results showed that the induction of VviWRKYs gene expression by CO2 was tissue dependent, being this effect mainly observed in the pulp where 9 out of the 15 genes analyzed were activated by the gaseous treatment. The expression analysis of genes associated with ethylene (ACC synthase1 (ACS1) and ACC oxidase1 (ACO1)) and ABA (9-cis-epoxycarotenoid dioxygenase1 (NCED1)) biosynthesis indicated that the expression of ACO1 in the skin of non-treated samples and in the pulp of CO2-treated ones correlated with the changes observed in VviWRKYs. In addition, we studied the role of WRKYs in the modulation of genes encoding pathogenesis-related proteins (PRs) in table grapes. Results showed there was a significant positive correlation between WRKYs modulated by CO2 in the skin and pulp with a β-1,3-glucanase (Vcgns1) and a thaumatin (VvTL1), respectively. Through electrophoretic mobility shift assay (EMSA) we observed that VviWRKYIIa_3 recombinant protein was able to in vitro bind to the W-box present in the promoter of the three PRs analyzed while VviWRKYIII_3 was only able to specifically bind to the Vcgns1 probe. Our results support the fact that high CO2 treatment is an active process that requires the activation of transcription factors, such as those of the WRKY family, which could participate in the molecular events involved in maintaining table grape quality during postharvest., This work was supported by CICYT projects AGL2014-53081-R and AGL2017-85291-R (MINECO/AEI/FEDER, UE). MV-H was supported by a predoctoral contract from the MEC.

This article belongs to the Special Issue Gene Transcriptional Regulation in Crops during Postharvest., Table grape is a fruit with increasing interest due to its attributes and nutritional compounds. During recent years, new cultivars such as those without seeds and with new flavors have reached countries around the world. For this reason, postharvest treatments that retain fruit quality need to be improved. However, little is known to date about the biochemical and molecular mechanisms related with observed quality improvements. This review aims to examine existing literature on the different mechanisms. Special attention will be placed on molecular mechanisms which activate and regulate the different postharvest treatments applied in order to improve table grape quality., This research was funded by the Spanish Ministerio de Economía y Competitividad and Fondo Europeo de Desarrollo Regional with the AGL2014-53081-R and AGL2017-85291-R (MINECO/AEI/FEDER, UE) projects.

Shelf-life quality was improved when Cardinal table grapes (Vitis vinifera L.) were pretreated with 20 kPa of CO for three days at the beginning of a long-term cold storage. This pretreatment was effective in avoiding postharvest losses of cv. Cardinal grapes in terms of water loss, oxidative damage and disease prevention. To elucidate those physiological and biochemical factors involved in preserving the postharvest shelf-life quality of table grapes, we studied the expression pattern of defense proteins such as pathogenesis-related proteins (PRs) and dehydrins, as well as the profile of protective osmolytes. The efficacy of a short-term high CO pretreatment in reducing fungal disease could be mediated by the increase in the low molecular mass chitinase isoform of 16 kDa, which is up-regulated in the skin of CO-treated grapes in parallel with the shelf-life fungal decay control. In addition, the increasing accumulation of a 22 kDa dehydrin isoform and the endogenous levels of organic osmolytes proline and glycine betaine in fruit stored at 20 °C (mainly in CO-treated fruit) revealed that these protective biomolecules might play a more effective role in maintaining the structural and cellular homeostasis of table grapes after the shelf-life period, helping to reduce water loss and membrane oxidative damage (malondialdehyde accumulation) associated with the senescence-related disorders of postharvest table grapes., This research was supported by the CICYT Projects AGL2014-53081-R (MINECO) and AGL2017-85291-R (MINECO/AEI/FEDER, UE). M. Vazquez-Hernandez acknowledges the support of the FPI program financed by the MICINN.

Strawberries are characterized by an intense flowery odor, which is associated with specific ester species. In this study, the effect of high CO2 atmospheres (0, 20 or 40 kPa) at 0°C on alcohol dehydrogenase (ADH) gene expression and on selected volatile esters of 'Mara des Bois' strawberries was analyzed. Acetaldehyde and ethanol concentrations, and those of hexanal and 1-hexanol were compared in strawberries stored under different high CO2 atmospheres. The abundance of each detected volatile esters varied as a function of CO2 concentration during storage. When compared with fruit at harvest, those stored at low temperature without added CO2 had a marked induction of ADH expression that was associated with the accumulation of 1-hexanol and esters typically related to fatty acid breakdown. Moreover, these fruit had the lowest concentrations of ethanol and some ethyl esters, such as ethyl butanoate. In contrast, a moderate accumulation of ethanol and specific esters associated with strawberry flavor were observed in fruit stored in a 20 kPa CO2. The shift towards ethanolic fermentation in fruit treated with 20 kPa CO2 for 3 days was associated with 1-hexanol accumulation. However, 40 kPa-treated fruit accumulated the lowest levels of octyl 3-methyl butanoate, the volatile ester with the strongest flavor in this cultivar. This reduction in conjunction with the accumulation of higher levels of acetaldehyde, ethanol and ethyl acetate could explain the negative impact on such fruits maintained in an excessively high CO2 atmosphere.

The role played by heat shock proteins (HSPs) in improving fruit quality during postharvest treatments has mainly been studied regarding heat treatments, while little is known about the effect of CO2 treatments. In this study, we have analyzed the gene expression of five heat shock proteins (HSPs) and one heat shock factor (HSF) in the skin of red table grapes (Vitis vinifera cv. Cardinal) to determine whether a pretreatment with high CO2 levels (20 kPa) modulated their expression and how the length of the treatment (1 or 3 d) could influence this change. The 3-d high CO2-treatment was effective in reducing total decay and induced the accumulation of three small HSPs (VviHSP18.1, VviHSP18.2 and VviHSP22.0), whereas VviHSP70.0 and VviHSF4-a gene expression were induced by both treatments. To shed light on the putative physiological role of a small HSP (VviHSP18.1) acting as a chaperone, the recombinant protein was overexpressed in Escherichia coli. It was then purified and mass spectrometry confirmed that the isolated protein was VviHSP18.1, belonging to class I sHSP. Although the purified protein was stable at different high temperatures, when temperature was above 70 °C, a weaker and smaller protein band appeared which was identified by mass spectrometry as VviHSP18.1 with a C-terminal truncation. The recombinant VviHSP18.1 protein displayed chaperone activity which protects citrate synthase (CS) and malate dehydrogenase (MDH) from thermal aggregation at 45 °C, and also displayed the protection of alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH) activities at 55 °C and 65 °C, respectively. By contrast, VviHSP18.1 did not protect LDH from freezing-induced inactivation. Taken together, these results support the hypothesis that a high CO2 treatment is an active process where HSPs could participate in preventing the denaturation and dysfunction of different proteins., This work was supported by CICYT projects AGL2014-53081-R and AGL2017-85291-R (MINECO/AEI/FEDER, UE). The proteomic analysis was performed in the Proteomics Facility of UCM that belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001.

© 2015 Elsevier GmbH. 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 44kDa) 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 44kDa 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., This research was supported by research Grant (Project AGL2011-26742/ALI) from CICYT (Spain).

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.

C-repeat/dehydration-responsive element binding factors (CBF/DREB) are transcription factors which play a role in improving plant cold stress resistance and recognize the DRE/CRT element in the promoter of a set of cold regulated genes. Dehydrins (DHNs) are proteins that accumulate in plants in response to cold stress, which present, in some cases, CBF/DREB recognition sequences in their promoters and are activated by members of this transcription factor family. The application of a 3-day gaseous treatment with 20 kPa CO2 at 0∘C to table grapes cv. Autumn Royal maintained the quality of the bunches during postharvest storage at 0∘C, reducing weight loss and rachis browning. In order to determine the role of CBF/DREB genes in the beneficial effect of the gaseous treatment by regulating DHNs, we have analyzed the gene expression pattern of three VviDREBA1s (VviDREBA1-1, VviDREBA1-6, and VviDREBA1-7) as well as three VviDHNs (VviDHN1a, VviDHN2, and VviDHN4), in both alternative splicing forms. Results showed that the differences in VviDREBA1s expression were tissue and atmosphere composition dependent, although the application of high levels of CO2 caused a greater increase of VviDREBA1-1 in the skin, VviDREBA1-6 in the pulp and VviDREBA1-7 in the skin and pulp. Likewise, the application of high levels of CO2 regulated the retention of introns in the transcripts of the dehydrins studied in the different tissues analyzed. The DHNs promoter analysis showed that VviDHN2 presented the cis-acting DRE and CRT elements, whereas VviDHN1a presented only the DRE motif. Our electrophoretic mobility shift assays (EMSA) showed that VviDREBA1-1 was the only transcription factor that had 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. Our results suggest that the application of high CO2 levels to maintain table grape quality during storage at 0∘C, leads to an activation of CBF/DREBs transcription factors. Among these factors, VviDREBA1-1 seems to participate in the transcriptional activation of VviDHN2 via CRT binding, with the unspliced form of this DHN being activated by high CO2 levels in all the tissues analyzed., This work was supported by the European Union under the 7th Framework Programme FP7-PEOPLE-2012-CIG no. 321694 and by CICYT projects AGL2011-26742 and AGL2014-53081-R. MV-H was supported by a predoctoral contract from the MEC., USD 1,822.53 APC fee funded by the EC FP7 Post-Grant Open Access Pilot

Reducing water consumption and increasing the quality of vegetables is a particularly high priority in agricultural production. Thus, we set out to analyze the effect of restricting the water supply on water relations, rheological properties, and the accumulation of solutes and protective fructooligosaccharides (FOS) and raffinose family compounds (RFOs) in lettuces grown under controlled greenhouse conditions. In addition, we analyzed whether water restriction can prevent senescence-related changes and overcome the stress imposed by subsequent exposure to low temperature. Lettuces var. Maravilla de Verano were grown under three different water supply regimes, well-watered (WW), moderate water deficit (MWD) and severe water deficit (SWD). Our results indicate that accumulated transpiration (AT) was higher in WW plants than in lettuces subjected to water deficit. The relative water content (RWC) was significantly influenced by a restricted water supply but not by additional low temperature stress. Water deficit caused a significant decrease in the amount of unfreezable water (Uw), determined calorimetrically, in association with a significant decrease in total water content (T). After the additional low temperature stress, there was no further drop in Tw, although a significant decrease in Uw was evident, mainly in SWD lettuces. A moderate water deficit enhanced nystose and kestopentaose accumulation. After imposing low temperature stress, MWD lettuces had a lower apparent viscosity, concomitant with an increase in firmness, fewer senescence-related changes and a sharp increase in raffinose. We conclude that moderate water limitation, improving the endogenous levels of FOS and reducing the cleavage of wall polysaccharides backbones, thus reducing viscosity and increasing firmness, could be useful to retain water inside cells, avoiding quality loss and increasing the capacity of the lettuce to resist low non-freezing temperatures., The authors gratefully acknowledge the financial support by the CICYT Project (AGL2014–53081-R) from the MICINN (Spain).

Treatment with high CO atmospheres is effective in preventing strawberry decay and increasing firmness. Nevertheless, CO stress generates energy disturbances associated with a high rate of fermentation. This study was designed to measure the impact of high CO stress (3 d, 40 kPa CO) and its subsequent removal on fruit quality, osmotic balance and water relations. Fructo-trisaccharides isomers and raffinose were characterized and quantified by mass spectrometry (MS and MS). CO stress removal was marked by both a rapid upsurge in the ratio of unfreezable water to total water and an osmotic adjustment prompted by the accumulation of soluble sugars, 1-kestose, raffinose and galactinol. Due to strong increase in galactinol concentrations, we propose it as a suitable biomarker for fruit having undergone stresses associated with osmotic imbalance., This work was financed by a CICYT Project (AGL2014–53081-R) from the MICINN (Spain).

© 2014, The Author(s). It has been recognized that high CO2 concentrations maintain the freshness of strawberries, reducing weight loss and the incidence of decay. Since strawberries have high tolerance threshold to CO2, it is of interest to know what changes occur in cellular water distribution when the threshold is reached or exceeded. Moreover, as strawberries are prone to producing exudate during storage at low temperatures, application of technologies improving water retention is needed. Changes in bound water content were analyzed in relation to simple sugars, polyols, and fructooligosaccharides (FOS) in Fragaria vesca stored at low temperature (0 °C) in the presence of 0, 20, or 40 % CO2. Fruits were analyzed at the end of a 3-day treatment and after transfer to air for one additional day. Unfavorable storage conditions were associated with a decrease in the levels of bound water and in that of 1-kestose and myo-inositol. However, the 1-kestose and myo-inositol content was enhanced when 40 % CO2-treated strawberries were transferred to air at the same time as an increase in bound water content and a restoration of intracellular water took place. The ability of the fruit to modulate the accumulation of these compounds, and by extension their water binding strength, in conjunction with the pool of the bound water fraction, appears to be a mechanism associated with the maintenance of cellular structural integrity. Variation in the percentage of air space and in the weight loss rate produced by enhancing the CO2 treatment during storage at 0 °C was also analyzed., This work was financed by the CICYT Project AGL2011-26742. We thank the Hugo Vela nursery for providing certified organic strawberries with requisites in compliance with CE Regulations 834/2007 and 889/2008.

Ethylene response factors (ERFs) play an important role in plants by regulating defense response through interaction with various stress pathways. After harvest, table grapes (Vitis vinifera L.) are subject to a range of problems associated with postharvest storage at 0°C, such as fungal attack, water loss and rachis browning. The application of a 3-day high CO2 treatment maintained fruit quality and activated the induction of transcription factors belonging to different families such as ERF. In this paper, we have isolated five VviERFs from table grapes cv. Cardinal, whose deduced amino acid sequence contained the conserved apetalous (AP2)/ERF domain. The phylogeny and putative conserved motifs in VviERFs were analyzed and compared with those previously reported in Vitis. VviERFs-c gene expression was studied by quantitative real-time RT-PCR in the different tissues of bunches stored at low temperature and treated with high levels of CO2. The results showed that in most of the tissues analyzed, VviERFs-c gene expression was induced by the storage under normal atmosphere although the application of high levels of CO2 caused a greater increase in the VviERFs-c transcript accumulation. The promoter regions of two PRs (pathogenesis related proteins), Vcchit1b and Vcgns1, were obtained and the in silico analysis revealed the presence of a cis-acting ethylene response element (GCC box). In addition, expression of these two PR genes was analyzed in the pulp and rachis of CO2-treated and non-treated table grapes stored at 0°C and results showed significant correlations with VviERF2-c and VviERF6L7-c gene expression in rachis, and between VviERF11-c and Vcchit1b in pulp. Finally by using electro mobility shift assays, we denoted differences in binding of VviERFs to the GCC sequences present in the promoters of both PRs, with VviERF6L7-c being the only member which did not bind to any tested probe. Overall, our results suggest that the beneficial effect of high CO2 treatment maintaining table grape quality seems to be mediated by the regulation of ERFs and in particular VviERF2-c might play an important role by modulating the expression of PR genes., This work was supported by the European Union under the 7th Framework Programme FP7-PEOPLE-2012-CIG no. 321694 and by CICYT projects AGL2011-26742 and AGL2014-53081-R. IR and MV-H were supported by a postdoctoral Juan de la Cierva contract from the MICINN and a predoctoral contract from the MEC, respectively., USD 2,116.5 APC fee funded by the EC FP7 Post-Grant Open Access Pilot

Fructo-oligosaccharides (FOS) have been recognized as health food ingredients with a protective effect against environmental stresses in plants. We have analyzed the profiles of individual FOS in Cardinal table grape pulp, until now undetected, and quantified their changes in response to low temperature and high CO2 levels. FOS separation and quantification was carried out using anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), and the glucose, fructose and sucrose content of the grapes was also determined. Five FOS were identified and quantified: 1-kestose, neokestose, nystose, nystose b and kestopentaose. While in non-treated table grapes the endogenous FOS remained at steady state levels during storage at 0 °C, exposure to 20% CO2 for 3 days significant increases the levels of 1-kestose and kestopentaose, members of the inulin series. Considering the competitive advantage afforded by CO2-treated grapes, this transitory FOS accumulation could provide protection against damage caused by low temperature storage. © 2011 Elsevier Ltd. All rights reserved., The authors wish to thank the Spanish Ministry of Science and Innovation (Project AGL2008-02949/ALI) for financial support

11páginas, 6 figuras, 4 tablas., A 1,3-β-glucanase with potent cryoprotective activity was purified to homogeneity from the mesocarp of CO subindice 2-treated cherimoya fruit (Annona cherimola Mill.) stored at low temperature using anion exchange and chromatofocusing chromatography. This protein was characterized as a glycosylated endo-1,3-β-glucanase with a Mr of 22.07 kDa and a pI of 5.25. The hydrolase was active and stable in a broad acidic pH range and it exhibited maximum activity at pH 5.0. It had a low optimum temperature of 35 °C and it retained 40% maximum activity at 5 °C. The purified 1,3-β-glucanase was relatively heat unstable and its activity declined progressively at temperatures above 50 °C. Kinetic studies revealed low k subindice cat (3.10 ± 0.04 s−1) and K subindice m (0.32 ± 0.03 mg ml−1) values, reflecting the intermediate efficiency of the protein in hydrolyzing laminarin. Moreover, a thermodynamic characterization revealed that the purified enzyme displayed a high k subindice cat at both 37 and 5 °C, and a low E subindice a (6.99 kJ mol−1) within this range of temperatures. In vitro functional studies indicated that the purified 1,3-β-glucanase had no inhibitory effects on Botrytis cinerea hyphal growth and no antifreeze activity, as determined by thermal hysteresis analysis using differential scanning calorimetry. However, a strong cryoprotective activity was observed against freeze–thaw inactivation of lactate dehydrogenase. Indeed, the PD subondice 50 was 8.7 μg ml−1 (394 nM), 9.2-fold higher (3.1 on a molar basis) than that of the cryoprotective protein BSA. Together with the observed accumulation of glycine-betaine in CO subindice 2-treated cherimoya tissues, these results suggest that 1,3-β-glucanase could be functionally implicated in low temperature-defense mechanism activated by CO subindice 2., Proyects AGL2002-02308/ALI and AGL2008-02949/ALI) from CICYT (Spain).

Anthocyanins and sugars are involved in the responses of fruit to environmental stress. The effect of low temperature (0 °C) in 'Camarosa' strawberries and also the influence of high CO2 levels (20% CO2 for 3 days) on the water-soluble sugars and on the levels of individual anthocyanins were assessed, as well as their association with antioxidant activity, total phenolics and colour. Anthocyanins were assessed by HPLC-DAD-MS, myo-inositol by IC-PAD and the antioxidant capacity using the ABTS{radical dot} scavenging assay. When compared with untreated strawberries, exposure to high CO2 prevented the increases in total phenolics and each of the five major anthocyanins quantified, while it triggered the consumption of soluble sugars, mainly sucrose and myo-inositol. The decrease of these soluble solutes could represent an adaptive strategy of CO2-treated strawberries to cope with storage at low temperature. © 2010 Elsevier Ltd. All rights reserved.

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.

© 2015 Elsevier B.V.. Short postharvest exposure of strawberries to high CO2 levels provides significant benefits in reducing decay and controlling physiological disorders during storage at 0°C. To define the different strategies employed by strawberries to tolerate high CO2 concentrations, the impact of different CO2 concentrations on energy and fermentative metabolism was studied under the same conditions of O2 availability. Our data indicate that metabolic depression represents a strategy to effectively adapt to beneficial high CO2 concentrations, with a decrease in ATP levels and in the energy charge, along with moderate ethanolic fermentation. Moreover, the induction of fermentative genes does not appear to be essential for the accumulation of fermentative metabolites. By contrast, when fruit is stored in air without added CO2, the metabolism is not directed towards fermentation and is accompanied by a high ATP/ADP ratio and energy charge. However, when exposed to 40kPa CO2, the excessively low energy charge and excessive decrease in ATP could not match the ATP requirements, in a process that ultimately causes significant perturbations including a high lipid peroxidation., This work was financed by CICYT grant AGL2014-53081-R from the MICINN of the Spanish government. R.R. was supported by a postdoctoral JAE contract from the CSIC

Annona fruit ripening is associated with a high rate of reassimilation of respired CO 2 , and a large increase in titrable acidity mainly due to malic acid accumulation. However, little is known about the mechanisms involved in acidity changes. In this work, the kinetic properties and activities of phosphoenolpyruvate carboxylase, malate dehydrogenase and malic enzyme, implicated in the regulation of malate metabolism and by extension in cytoplasmic pH, have been analysed. Kinetic studies revealed that the Km values of NADP-malic enzyme for malate were higher than those of malate dehydrogenase for oxaloacetate (1.44 ′ 0.03 mM v 0.78 ′ 0.02 mM). These results together with the higher V m a x value of malate dehydrogenase (more than 26-fold) indicated more efficiency in the reaction of malate synthesis v decarboxylation.The in vitro properties of the enzymes and the fact that the activity of malate dehydrogenase is much higher than the activity of NADP-malic enzyme account for the particular high titratable acidity in cherimoyas during ripening. Our results further indicate that the balance between malic acid synthesis and consumption seems to be effectively regulated and consistent with a controlled cytoplasmic pH value, measured by 3 1 Phosphorus nuclear magnetic resonance spectroscopy.

Dehydrins (DHNs), group II LEA (Late Embryogenesis Abundant) proteins, are among the most commonly observed proteins which accumulate in plants in response to cold and any other environmental factors, causing the dehydration of cells. In previous studies, we isolated a YSK2-type VvcDHN1a gene from table grapes (Vitis vinifera cv. Cardinal) which presented two spliced variants (the spliced, DHN1a_s and the unspliced, DHN1a_u). Their expression was induced by low temperature storage and CO2, although with different accumulation patterns. DHN1a_u codifies for a truncated YS protein lacking ¿- and K-segments, which might affect its functionality. In this work, we expressed both DHN1a_s and DHN1a_u recombinant proteins in Escherichia coli. We carried out a number of in vitro assays to analyze the implications that ¿- and K-segments have in the protective role of VvcDHN1 against different abiotic stresses and their antifungal activity against the fungal pathogen Botrytis cinerea. Our results showed that unlike DHN1a_u, DHN1a_s has a potent cryoprotective effect on lactate dehydrogenase activity, protects malate dehydrogenase against dehydration and partially inhibits B. cinerea growth. Moreover, the DHN1a promoter presented cis-regulatory elements related to cold and drought, as well as biotic stress-related elements. We also observed that both spliced variants interact weakly with DNA, suggesting that K-segments are not involved in DNA binding. Overall, this work highlights the crucial role of ¿- and K-segments in DHNs function in plant response to abiotic stress showing for the first time, the potential role of the V. vinifera DHN1a_s in the protection against freezing and dehydration as well as inhibiting B. cinerea growth., This work was supported by CICYT project AGL2011-26742 and by the European Union under the 7th Framework Programme FP7-PEOPLE-2012-CIG No. 321694. R.R. and I.R. were supported by a postdoctoral JAE contract from the CSIC and a postdoctoral Juan de la Cierva contract from the MICINN, respectively. The work benefited from the networking activities within the European COST Action FA1106-QualityFruit.

Although potassium participates in distinct mechanisms that influence grape growth and development, including osmoregulation, little is known about the association between water and potassium in grape during storage at low temperature. We analyzed the relationship between potassium and the bound water fraction in the skin of early-harvested Cardinal table grapes (Vitis vinifera L.) from two different harvest years, both of which were stored at 0°C for 3 days in air (20% O2+0.03% CO2) or in air+CO2 (20% O2+20% CO2). The relative K+ content and distribution in the skin cells was determined by energy dispersive X-ray microanalysis, revealing a non-uniform accumulation of K+ in grape skin cells. Storage at 0°C in air causes a significant decrease in bound water levels and greater soluble-water K+ accumulation, irrespective of the harvest year. Furthermore, low temperature-scanning electron microscopy images revealed that the epidermal and the first hypodermal layers of the cells were compressed in the skin of fruit stored in air. However, when exposed to air plus 20% CO2, there was no decrease in the bound water content or in the associated K+ accumulation, nor were the outer skin cells compressed. © 2014 Elsevier B.V.

The effect of short-term high CO2 on maintaining postharvest cherimoya (Annona cherimola Mill.) fruit quality during storage at low temperature was analyzed. Pretreatment with an atmosphere of 20% CO2 and 20% O2 for 3 days retained fruit firmness and prevented an increase in the immunodetected levels of polygalacturonase-related protein during storage at 6 °C. Color scale values, chlorophyll content, and levels of the immunoreactive large subunit of ribulose 1,5-biphosphate carboxylase protein were higher in treated than in air-stored fruit. Short-term high CO2 treatment had a long residual effect at low temperature and improved storage life by preventing fruit softening and delaying senescence-like responses in green peel of cherimoya fruit tissues.

SummaryCherimoya fruits (Annona cherimola, Mill.) were kept in 20% O2 + 20% CO2 for up to 3 d at 20°C and then transferred to air to study the effect of high CO2 levels on fermentation enzymes, ethanol and acethaldehyde content and ripening evolution. Ethanol and acetaldehyde content increased during ripening in air mainly associated with the first respiration peak. At the end of the short-term high CO2 treatment, cherimoya fruit had a lower aerobic respiration rate while concentrations of acethaldehyde and ethanol were greatly increased compared with those of air-control fruit. The activation of fermentation pathway by high CO2 atmosphere is mainly due to an enhancement in pyruvate decarboxylase (PDC) activity. High CO2 treatment prevented the ripening process but, after transfer to air a decrease in fermentation metabolism was recorded and fruit was able to ripen, showing a typical decrease in tissue pH.

Treatment of harvested strawberries (Fragaria vesca L. Mara des Bois) with high concentrations of CO2 is an effective means of limiting fungal decay and avoiding disorders caused by low temperature storage. In the present study, we investigated the role of NADP-ME gene expression and activity in lowering malic acid contents and in the provision of NADPH required for the regeneration of the reduced form of glutathione (GSH). We also measured glutathione reductase (GR: EC 1.6.4.2) activity in strawberries treated with different high CO2 concentrations (0, 20 and 40% for 3 days) during storage at 0 °C. A decrease in malic acid content in fruit exposed to 20% CO2 was primarily mediated by the stimulation of NADP-ME activity, rather than associated with changes in the expression of cytosolic NADP-ME genes. Moreover, malic acid decarboxylation was associated with a marked increase in GR activity, which may account for the increased levels of glutathione in fruit following exposure to 20% CO2. This chain of events was not observed in untreated fruit stored in air or in fruit treated with 40% CO2, suggesting that the unique cellular redox status of 20% CO2-treated fruit plays an important role in detoxification and protection from damage during storage. Based on these findings, we propose that NADP-ME activation in fruit exposed to 20% CO2 provides NADPH for glutathione regeneration by GR, thereby conferring protection against the cellular damage caused by low temperatures or excessive high CO2 levels., This work was supported by the CICYT Projects AGL2008-02949 and AGL 2011-26742

The peel of `Fino de Jete' cherimoya (Annona cherimola Mill.) stored at 20 °C in air and in an atmosphere with high levels of CO2 was analyzed for changes in protein levels, color, chlorophyll content, and firmness. The accumulation of immunoreactive proteins was studied using Rubisco and polygalacturonase antibodies. During storage in air, cherimoya peel tissues exhibited a decrease in chlorophyll content and an immunodetected drop in the abundance of the large subunit of ribulose 1,5-biphosphate carboxylase (LSR). An immunodetected rise in polygalacturonase (PG)-related protein was quantified. High CO2 levels delayed the softening of cherimoya fruit, retarded the maximum accumulation of PG-related protein, and maintained LSR levels, greenness, and chlorophyll content of peel tissues.

Storage at 6 °C inhibited the ripening process and caused severe damage in cherimoya fruit (Annona cherimola Mill. cv. ‘Fino de Jete’). In the present study, we analyzed the modifications in protein pattern, free amino acid content and protease activity of cherimoyas during storage at this chilling temperature. SDS-PAGE analysis revealed non-accumulation of some polypeptides related to the ripening process due to storage at 6 °C, and two-dimensional electrophoresis confirmed the appearance of specific low-temperature polypeptides. While many polypeptides observed in freshly harvested fruit persisted during storage, several acid polypeptides were detected only during the first few days of storage at 6 °C. A substrate-dependent change in protease activity was also found in fruit under chilling temperature storage, as compared to ripening fruit. After a decrease to barely detectable levels during the early phase of cold storage, the proteolytic activity then increased, mainly hydrolizing endogenous proteins to free amino acid components.

This paper deals with the impact of low temperature and high CO 2 levels on flavonols, proanthocyanidins, and anthocyanins, synthesized via branched pathways from common precursors, in strawberries (Fragaria vesca L.). Flavonoids were identified with Q-TOF equipment and quantified by HPLC-quadrupole. Proanthocyanins B1 and B3 accumulated in CO 2-treated strawberries, whereas in untreated (air) fruit, flavonoid production was redirected toward anthocyanin accumulation with a sharp decrease in catechin and procyanidin B3 levels. Moreover, in CO 2-treated fruit, mainly in those with 20% CO 2, anthocyanin accumulation did not decline. Due to its antifungal activity, catechin induction in CO 2-treated strawberries could explain the capacity of high CO 2 treatments to reduce fungal decay. Ascorbic acid content increased in 40% CO 2-treated fruits, whereas in those treated with 20% CO 2 an increase in flavonol content was observed. Despite these differences, similar antioxidant capacities were found in untreated and CO 2-treated Mara de Bois strawberries. © 2012 American Chemical Society.

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 CO 2 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 CO 2 treatment, suggesting that the response of DHN1a in this fruit could be attributed to a cold-inducible CBF-independent pathway. © 2012 Elsevier GmbH.