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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.

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).

The effects of weak oscillating magnetic fields (OMFs ≤7 mT at 50 Hz) on freezing were studied in three systems of different complexity. To do so, ferric chloride solutions, lactate dehydrogenase (LDH), and minced hake muscle experimentally infected with Anisakis L3 were frozen with and without OMF application. OMFs did not affect freezing kinetics of either ferric chloride solutions or minced hake muscle. LDH activity, Anisakis mortality, and water-holding capacity of the hake muscle after thawing were not affected by OMF either. Further studies are needed to evaluate the effectiveness of stronger OMFs in a wider frequency range., This work has been supported by the Spanish MINECO under the project AGL2012-39756-C02-01 and by the EuropeanUnion Seventh Framework Programme under the project EU PARASITE (GA 312068). A.C. Rodríguez has been supported by the MINECO under predoctoral grant BES-2013-065942.

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.

© 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).

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.

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

© 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.

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).

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.

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.

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

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.

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.

Water status and analyses of free sugars, sugar-alcohols and fructo-oligosaccharides (FOS) were carried out in Fragaria x vesca treated with different high CO 2 concentrations applied to minimise damage caused by storage at 0°C. The thermodynamic parameters such as the amount of unfrozen water (U w), T g, Tg′, and peak position of the O-H stretching vibration were determined in various saccharides including FOS (1-kestose, nystose and kestopentaose) by infrared spectroscopy studies and differential scanning calorimetry. Beneficial high CO 2 treatment (20%) avoided the reduction of unfreezable water fraction and increased endogenous FOS levels, in contrast to that observed in air-stored and in those exposed to higher CO 2 levels (40%). The direct FOS-water interaction, possibly within the hydrogen-bond network of cellular structures, could explain the maintenance of water status, cell integrity and the low water leakage levels in 20% CO 2-treated fruit at values similar to those found in freshly harvested fruit. © 2012 Elsevier Ltd. All rights reserved.

The main organic (acids, amino acids and sugars) and inorganic (mono and divalent cations) solutes associated with changes in water status in response to low temperature and high CO 2 levels were analyzed in untreated and 20% or 40% CO 2 treated strawberries (Fragaria vesca L. cv. Mara de Bois) stored at 0°C. Intercellular water distribution and cellular tissue integrity were visualized using low temperature scanning electron microscopy (LT-SEM). The results indicated that high CO 2 treatments prevented the weight loss and cell structure disorganization observed in untreated strawberries. However, there were differences in water loss regulation dependent on CO 2 levels. In addition to mediating osmotic adjustment, treatment with 20% CO 2 had a protective effect on cellular structure and prevented the movement of water into the intercellular spaces. Specifically, an accumulation in total sugars and proline were detected in 20% CO 2-treated fruit. Moreover, water loss was controlled in these fruit and K +/Na + homeostasis maintained similar to that found in freshly harvested fruit. By contrast, 40% CO 2 controlled water loss, but the intercellular spaces were filled with aqueous solution, possibly as a result of a change in water status. Moreover, these changes were associated with an increase in free soluble Ca 2+. In view of the opposite patterns of accumulation in malic, succinic acid, γ-aminobutyric acid (GABA) and glutamate found when comparing 20% and 40% CO 2 treated strawberries, we suggest that their corresponding metabolic pathways play a regulatory role in CO 2 tolerance. © 2012 Elsevier B.V..

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 CO 2 (20% CO2 for 3 days) 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. © 2011 Elsevier Ltd. All rights reserved.

A cryoprotective chitinase (BChi14) was isolated and purified from the mesocarp of CO2-treated cherimoya fruit (Annona cherimola Mill.) stored at chilling temperature by anion exchange and chromatofocusing chromatography. This hydrolase was characterized as an endochitinase with a Mr of 14.31kDa and a pI of 8.26, belonging to the family 19 of glycosyl hydrolases (GH19). While it was stable over a wide pH range and active in a broad acidic pH range, it had an optimum pH of 7.0. Its optimum temperature was low, 35°C, and it retained about 30% of its maximum activity at 5°C. Moreover, BChi14 was relatively heat unstable and its activity was progressively lost at temperatures above 50°C. Kinetic studies revealed many similarities with other plant endochitinases. However, BChi14 had high kcat (6.93s-1) value for the fluorogenic substrate 4-MU-(GlcNAc)3, reflecting its great catalytic efficiency. Moreover, a thermodynamic characterization revealed that the purified enzyme displayed a high kcat at 37 and 5°C, and a low Ea (11.32kJmol-1). In vitro functional studies indicated that BChi14 had no effect on the inhibition of Botrytis cinerea hyphal growth and no antifreeze activity, as shown by the thermal hysteresis analysis using differential scanning calorimetry. However, the purified endochitinase showed very strong cryoprotective activity against freeze-thaw inactivation of lactate dehydrogenase. The PD50 was 12.5 times higher than that of the cryoprotective protein BSA, and 2 or 3 orders of magnitude greater than sucrose, comparable with that of most cryoactive plant dehydrins. These results, together with the consolidated microstructure and the integrity of CO2-treated mesocarp tissue, indicate that BChi14 is functionally implicated in the mechanisms underlying chilling tolerance activated by high CO2 concentrations. © 2010 Elsevier GmbH., This work was supported by research grants (Projects AGL2002-02308/ALI and AGL2008-02949/ALI) from the CICYT (Spain)

In order to obtain a better understanding of the active defense strategy of cherimoya (Annona cherimola Mill.) fruit, hydrolytic and antifungal activity, as well as expression of proteins functionally and immunogenically related to the pathogenesis-related proteins chitinase (PR-Q) and 1,3-β-glucanase (PR-2), were estimated in fruit at different ripening stages. Increase in expression of the 27 kDa constitutive chitinase and the induction of two new proteins, a 26 kDa chitinase and a 51 kDa 1,3-β-glucanase were associated with enhanced in vitro hydrolytic and antifungal activity of the acidic protein extract in ripe fruit. Ripening modified the expression of constitutive basic isoenzymes, with a sharp decrease in both relative accumulation and hydrolytic activity. Likewise, a new basic 33 kDa chitinase was induced in the over-ripe fruit, concomitant with accumulation of a basic constitutive 76 kDa 1,3-β-glucanase. At this stage, the basic protein extract modified in vitro growth inhibition of Botrytis cinerea. Short-term high CO2 treatment delayed fruit ripening and maintained a similar distribution of activity and isoenzymatic pattern in both protein fractions to that in unripe fruit. These results indicate that the changes in the pattern of defense proteins and hydrolytic activity in cherimoyas appear to be associated with ripening. Moreover, unlike the constitutively expressed isoenzymes, only the transitorily induced chitinases and 1,3-β-glucanases were associated with an active defense-related response. © 2009 Elsevier B.V. All rights reserved., This work was supported by research grants from the CICYT, Spain (Projects AGL2002-02308/ALI and AGL2005-04502/ALI). O.G. was supported by a predoctoral fellowship from the MEC (Spain)

We have analyzed the influence of the stage of ripeness on L-phenylalanine ammonia-lyase (PAL) gene expression, accumulation of anthocyanins and total phenolics, and on antioxidant activity in the skin of table grapes treated with 20% CO2 + 20% O2 + 60 % N2 for 3 or 6 d at low temperature (0 °C). The residual effect of high CO2 treatment after transfer to air was also studied. In early harvested grapes, neither the anthocyanin content nor the accumulation of VcPAL mRNA was affected by any of the CO2 treatments applied. However, in late harvested grapes, the duration of high CO2 treatment determined its effect and a 6 d treatment with CO2 sustained higher levels of total phenolics and anthocyanin accumulation, and VcPAL expression than observed in untreated late harvested grapes. The increased antioxidant capacity was correlated with the total amount of phenolics and anthocyanins. Conversely, in grapes treated for 3 d with CO2 the phenylpropanoid pathway did not appear to be induced and a relationship between antioxidant activity and anthocyanins was not observed. Thus, further studies are needed to identify the most important antioxidants in these treated fruit. © 2009 Elsevier B.V. All rights reserved., This work was supported by research grants AGL2008-02949 from CICYT (Spain

This study focuses on how the length of exposure to chilling temperature and atmosphere storage conditions regulate the hydrolytic activity and expression of chitinase (PR-Q) and 1,3-β-glucanase (PR-2) isoenzymes in cherimoyas (Annona cherimola Mill.). Storage at 6 °C modified the expression of constitutive isoenzymes and induced the appearance of novel acidic chitinases, AChi26 and AChi24, at the onset of the storage period, and of a basic chitinase, BChi33, after prolonged storage. The induction of this basic isoenzyme was concomitant with the accumulation of basic constitutive 1,3-β-glucanases. These low-temperature-induced chitinases modified the growth inhibition in vitro of Botrytis cinerea. Short-term high CO2 treatment activated a coordinated response of acidic chitinases and 1,3-β-glucanases after prolonged storage at chilling temperature. Moreover, the high in vitro cryoprotective activity of CO2-treated protein extracts was associated with the induction of two low molecular mass isoenzymes, AGlu19 and BChi14. Thus, exposure to high concentrations of CO2 modified the response of fruit to low temperature, inducing the synthesis of cryoprotectant proteins such as specific pathogenesis-related isoenzymes that could be functionally associated with an increase in chilling tolerance in vivo. © 2008 Elsevier GmbH. All rights reserved.

In this study, we have analyzed the profiles of individual anthocyanins in the skin of Cardinal table grapes and their contribution to the total antioxidant capacity (TAC) in response to low temperature (0 °C) and high CO2 levels (20% for 3 days). An analysis of the representative colour parameters of this red-violet variety was also determined. The anthocyanin composition was determined using high-performance liquid chromatography coupled to diode array detector and mass spectrometry (HPLC-DAD-MS). The contribution of individual anthocyanins to the TAC value of table grapes was calculated on the basis of their concentration and antioxidant capacity measured as the TEAC value (slope of the anthocyanin/slope of Trolox). Chromatographic analysis identified six anthocyanins, including pelargonidin 3-glucoside (Pg-3-G), in the skin of Cardinal table grapes. Short-term storage at 0 °C in air had an increasing effect on the concentration of each of the identified anthocyanins. After 3 days at 0 °C, untreated grapes had the highest anthocyanin content (27.55 mg/100 g FW) and displayed the largest TAC value (52.45 mM TE/100 g FW). Peonidin 3-glucoside (Pn-3-G) was the predominant anthocyanin, and it was mainly responsible for the rise in the calculated TAC value in untreated grapes. Pn-3-G had a low average TEAC value (1.73 mM), but its contribution could be explained by the sharp increase in the content of this anthocyanin the first days of storage at 0 °C. In contrast, the Pn-3-G content in grapes at the end of the 3-day CO2 treatment did not change, and both the total anthocyanin content and the calculated TAC value remained significantly constant in comparison to the levels in pre-stored grapes. In addition CO2 treatment had a positive effect on the amount of Pg-3-G concomitant with a pronounced decline in delphinidin 3-glucoside (Dp-3-G) and smaller decreases in petunidin 3-glucoside (Pt-3-G) and malvidin 3-glucoside (Mv-3-G). These effects of high CO2 levels on the profile of individual anthocyanins were progressively lost when grapes were transferred to air. Indeed, after prolonged low-temperature storage when the colour of the berries become dark violet, similar calculated TAC values were quantified in untreated grapes and in those exposed to 20% CO2. These data provide new insights about the effect of low temperature and high CO2 levels on the concentration of the individual anthocyanins identified in cardinal table grapes. © 2008 Elsevier B.V. All rights reserved., This work was supported by research grants AGL2005-04502/ALI from CICYT (Spain). M.T.S.-B. and R.M. were supported by “Ramon y Cajal” postdoctoral contracts from the MEC and an I3P fellowship from the CSIC (Spain)

The aim of this work was to determine the physicochemical characteristics of non-conventional starch from cherimoya fruit in order to suggest possible applications of these polymers in various food systems. The influence of the stage of ripening on thermal behaviour of cherimoya fruit starch was also analysed. Starch was visualized by cryo-SEM, and gelatinization and retrogradation properties were studied by differential scanning calorimetry (DSC). DSC was also used for accurate determination of the starch content of cherimoya dry matter. The calorimetric profile for cherimoya fruit starch showed a peak temperature (gelatinization) over a range from 63.7 to 65.2 °C and enthalpy values between 14.8 and 15.9 J g-1 during ripening. Neither temperatures nor melting enthalpy of recrystallized amylopectin differed over the ripening period. The recrystallization rate, as measured by changes in melting enthalpy with time according to the Avrami model, showed a very slight retrograde tendency. It is suggested that these characteristics could be useful in products where good quality and long shelf-life are required. © 2007 Swiss Society of Food Science and Technology., This work was supported by research Grants (Projects AGL2002-02308/ALI and AGL2005-04502/ALI) from the Comisión Interministerial de Ciencia y Tecnología (CICYT), Spain

In this work, we analyzed the kinetic properties of phenylalanine ammonia-lyase (PAL) extracted from >cherimoya> (Annona cherimola Mill.) fruits ripened at ambient temperature (20C) and stored under several environmental conditions, including high CO2 levels (20%) and low temperature (6C). The effect of different ammonia-related compounds on cherimoya PAL activity was also evaluated. PAL exhibited two different Km values for L-phenylalanine (L-Phe) and negative substrate cooperativity, with Hill coefficient (napp) values reaching 0.64 and 0.71 for low temperature and high CO2 levels, respectively. The kinetic analysis revealed that ammonia produced mixed inhibition of PAL enzyme, with inhibition constants (Ki and Ki′ values of 0.57 ± 0.2 mM and 2.54 ± 0.2 mM. We propose that the regulation of PAL by ammonia inhibition and the negative cooperativity may be essential in adjusting the active phenylpropanoid metabolism in Annonas to the requirement of L-Phe and in consequence, to the carbon skeleton demand for other anabolic pathways. © 2007, Blackwell Publishing.

Changes in the state of water of cherimoya (Annona cherimola Mill.) fruit during ripening were monitored using differential scanning calorimetry (DSC) and magnetic resonance imaging (MRI) techniques. The stage of ripening was determined by analyzing the changes in titratable acidity, firmness and total soluble solids contents. Furthermore, quantitative measurements of solutes such as proline, sucrose, glucose and fructose were also studied. The significant increase in the transverse relaxation time (T2) values and the loss of flesh firmness during the initial stage of ripening are consistent with the sustained drop in the unfreezable water weight fraction according to the DSC data. The ripe stage of the fruit was marked by minimum longitudinal relaxation time (T1) values and a rapid upsurge in the unfreezable water weight fraction, greatly influenced by the osmotic adjustments prompted by the significant accumulation of soluble sugars, proline and carboxylates. Changes in structure and solute concentration associated with ripening can be analyzed simultaneously, determining fruit water status by DSC. © 2007 Elsevier B.V. All rights reserved.

The effect of pretreatment with 20% CO2 plus 20% O2 for 3 days was studied with regard to its effectiveness on natural postharvest decay control and its possible induction of specific PR genes in table grapes. Full-length cDNAs encoding a class I chitinase (Vcchit1b) and β-1,3-glucanase (Vcgns1) were isolated from table grapes (Vitis vinifera L. cv. 'Cardinal'). Our results indicate that this short-term high CO2 treatment had a residual effect and significantly reduced decay incidence of table grapes during low temperature storage and upon transfer to 20 °C. Our results indicate that during low temperature storage the expression pattern differed between the two tested PR genes. So, while the abundance of Vcgns1 transcript increased sharply at the beginning of storage at 0 °C, the increase in Vcchit1b mRNA levels was paralleled by the change in total decay. High CO2 pretreatment restrained the up-regulation of Vcgns1 gene expression and delayed the accumulation of Vcchit1b transcript as compared with non-treated grapes. Upon transfer to 20 °C after 33 days of cold storage, when attainment of maximum total decay was observed, there was a sharp increase in the accumulation of Vcchit1b mRNA in both treated and non-treated grapes, which was higher in the non-treated ones. Our results point out that the expression of class I chitinase and β-1,3-glucanase genes is not enhanced in CO2-treated grapes which control total fungal decay. These results suggest, then, that the efficacy of high CO2 pretreatment in reducing total fungal decay is not mediated by induction of the above-mentioned PR genes. © 2006 Elsevier B.V. All rights reserved.

The levels of ammonia in cherimoya (Annona cherimola, Mill) fruit changed in response to different storage conditions. The highest amount of ammonia was observed in fruit stored at a chilling temperature, and the lowest ammonia level was found in CO2-treated fruit at ambient temperature. These fruit presented the highest amount of nitrogen incorporated in polyamines, mainly in spermidine and spermine. Moreover, the fact that the trend in polyamines and ammonia content reverted when treated fruit were transferred to air would appear to confirm preferential reassimilation of ammonia under high CO2 treatment. The increase in phenylalanine ammonia-lyase (PAL) activity observed in ripening fruit at ambient temperature was substantially reduced in fruit stored at a chilling temperature. Our results suggest that PAL activity increases in fruit with a high demand of ammonia for further assimilation processes, and that this activity decreases in fruit where ammonia reassimilation does not take place. Additionally, in the case of CO2-treated fruit, ammonia availability is apparently promoted by the deamination of amino acids, which is reflected in a significant decrease in free amino acid content without a concomitant rise in total protein levels. We observed that the patterns of change in phosphoenolpyruvate carboxylase (PEPC) and PAL activities were opposite. Moreover, coordination between these enzymes was related to the metabolic endogenous levels of ammonia. The role of ammonia levels as a significant metabolic signal for controlling carbon and nitrogen metabolism is discussed. © 2002 Elsevier Science B.V. All rights reserved.

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5.) activity, tanning ability, and polyphenols levels were measured in cherimoya (Annona cherimola Mill.) fruit treated with 20% CO2 + 20% O2 + 60% N2 for 1, 3, or 6 days during chilling temperature (6 cC) storage. The residual effect of CO2 after transfer to air was also studied. These observations were correlated with texture and cellular characteristics, visualized by cryo-SEM. Tanning ability and the early increase in tannin polyphenols induced by chilling temperature were reduced by CO2 treatment. Conversely, high CO2 atmosphere enhanced the nontannin polyphenol fraction as compared with fruit stored in air. Lignin accumulation and PAL activation observed in untreated fruit after prolonged storage at chilling temperature were prevented by high CO2. Moreover, the restraining effect on lignification was less effective when the CO2 treatment was prolonged for 6 days. In addition, fruits held at these conditions had greater firmness and the histological characterization of the separation between cells was similar to that in untreated fruits. We conclude that CO2 treatment modulates the phenolic response that seems to regulate the strength of cell adhesion and so to prevent hardening caused by chilling temperature storage., This work was supported by a CICYT (Spain) grant (ALI99-0954-C03-01). T

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) regulatory properties were studied in non-photosynthetic (mesocarp) and photosynthetic (peel) tissues from cherimoya (Annona cherimola Mill.) fruit stored in air, in order to gain a better understanding of in vivo enzyme regulation. Analyses were also performed with fruit treated with 20% CO2-20% O2 to define the role of PEPC as part of an adaptive mechanism to high external carbon dioxide levels. The results revealed that the special kinetic characteristics of the enzyme from mesocarphigh Vmax and low sensibility to L-malate inhibition - are related to the active acid metabolism of these fruits and point to a high rate of reassimilation of respired C02 into keto-acids. With respect to fruit stored in air, PEPC in crude extracts from CO2-treated cherimoyas gave a similar Vmax (1.12±0.03 μkat-mg-1 protein), a lower apparent Km (68±9 μM for PEP) and a higher I50 of L-malate (5.95±0.3 mM). These kinetic values showed the increase in the affinity of this enzyme toward one of its substrate, PEP, by elevated external CO2 concentrations. The lower Km value and lower sensitivity to L-malate are consistent with higher in vivo carboxylation reaction efficiency in CO2-treated cherimoyas, while pointing to an additional enzyme regulation system via CO2. © 2001 Elsevier Science Ltd. All rights reserved.

Cherimoya fruit (Annona cherimola, Mill.) were kept at 20°C in air or in 20% CO2 for 3 days and then transferred to air, to study the effect of a high CO2 treatment on phenolic metabolism and ripening-related changes. Total polyphenol levels remained constant while a rapid decline in lignin content was observed in cherimoyas stored in air. However, a sharp increase in PAL activity up to the second day at 20°C was observed. The maximum ethylene production was observed 2 days later. At the end of the CO2 treatment, ethylene production was inhibited and PAL activity was similar to that found in air-treated fruit. These data suggest that the increase in PAL activity at 20°C was not affected by high CO 2 and does not relate to ethylene. The CO2 treatment inhibited flesh softening and maintained lignin at levels found in freshly harvested fruit. Exposure to 20% CO2 also improved internal colour and increased the non-tannin polyphenol fraction, but prevented the decline in the tannin fraction otherwise observed upon ripening in air. We concluded that high CO2 treatment at 20°C did not enhance PAL activity and lignin deposition although treated fruits retained more lignin after transfer to air. The possible involvement of PAL activity in the supply of important metabolic compounds for early events of ripening will be discussed. © 2001 Elsevier Science B.V. All rights reserved., This work was supported by a grant from Spain, CICYT (ALI99-0954-C03-01).

In this study we focused on the effect of high CO2 level (20%) on ethylene and polyamine biosynthesis in cherimoya (Annona cherimola Mill.) fruits stored at ripening (20°C) and chilling (6°C) temperatures. At ripening temperature, CO2 inhibited ethylene production, but 1-aminocyclopropane-1-carboxylate (ACC) oxidase activity was similar to that in ripe control fruits. CO2 treatment led to a decline in putrescine (Put) and a major accumulation of spermidine (Spd) and spermine (Spm) without any effect on arginine decarboxylase (ADC) activity. These results confirm the preferential transformation of Put to Spd and Spm in CO2-treated fruits. At chilling temperature, the increase in ACC oxidase activity was inhibited and the Vmax of ADC increased. A combination of chilling temperature storage and high CO2 level led to suppression of basal ethylene production while ACC oxidase activity remained unchanged. In addition, fruits held at these conditions had higher polyamine titres than the untreated control. We propose that, in CO2-treated fruits, the absence of autocatalytic or basal ethylene production, depending on the temperature, may be due to deviation of the S-adenosylmethionine (SAM) pool towards polyamine synthesis, primarily Spd and Spm. © CSIRO 1999.

In this work, we studied the effect of short-term high CO2 treatment (20% CO2 plus 20% O2 for 3 days) on both ethylene and polyamine synthesis in cherimoya fruit stored at 20 and 6°C. The decrease in putrescine content, with no variation in ADC activity, and the accumulation of both spermidine and spermine confirm the preferential transformation of the diamine into polyamines in CO2-treated fruit. The absence of autocatalytic or basal ethylene production with no variation in ACC oxidase activity may be due to deviation to the SAM pool towards polyamine synthesis by high CO2 treatment. These metabolic changes may be responsible for the effect of high CO2 levels on delaying cherimoya fruit ripening and maintaining fruit quality during storage at chilling temperature. Our results are discussed in light of the role attributed to polyamines in cytoplasmic pH regulation.

In this study the effect of treatment with 20% CO2 plus 20% O2 for 3 d on γ-aminobutyric acid (GABA) was analysed and total polyamine titres and the production of some pathogenesis-related proteins (PR-proteins) in cherimoya (Annona cherimola Mill.) fruit stored at low temperature. In immunoassays with anti-PR-Q and PR-2 protein sera, high CO2 levels were found to provoke the co-ordinated accumulation of a chitinase-like protein and 1,3-β-glucanase. Chitinase activity was higher in treated than in untreated fruit. At the end of CO2 treatment, total polyamine and γ-aminobutyric acid content and uptake of O2 were observed to be higher in treated compared to untreated fruit, but the accumulation of these compounds decreased when the fruit was transferred to air. Since this treatment effectively retains the fruit quality (Escribano et al., 1997), high CO2 levels may have a direct effect on the activation of the above-mentioned specific responses that enables cherimoya fruit to overcome chilling temperature. The relationship between the activation of the defence system and the capacity to regulate cytoplasmic pH in CO2-treated fruits was also addressed.

Free polyamine levels were determined in cherimoya (Annona cherimola Mill. cv. ) fruit during ripening at 20 °C. Several parameters including respiration rate, ethylene production and total titratable acidity were analyzed. The polyamine prevailing after harvest was spermidine, although a sharp increase was observed in putrescine content during ripening. The possible relationship between this rise in putrescine level and the ripening process was studied on the basis of the pattern of diamine content in fruit stored over a range of low temperatures (10, 8 and 6 °C). Retardation of the ripening process by cold storage was concurrent with a slower rate of increase in the free putrescine level. Moreover, where ripening was inhibited by storage at the chilling temperature (6 °C), no increase in free putrescine was observed. While no variation was observed in spermidine and spermine content in fruit ripening at 20 °C, levels did undergo change during low temperature storage. The possibility that significant accumulation of the free putrescine titer may be associated with high acidity levels in ripening cherimoya tissues is discussed.

Cellular and Molecular Aspects of the Plant Hormone Ethylene: Proceedings of the international symposium on Cellular and Molecular Aspects of Biosynthesis and Action of the Plant Hormone Ethylene, held in Agen, France, 1992