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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.

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).

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.

Biocontrol is one of the most promising alternatives to synthetic fungicides for food preservation. Botrytis cinerea, Alternaria alternata, and Aspergillus section Nigri are the most concerning pathogens for grape development. However, frequently, other species, such as Penicillium glabrum in this study, are predominant in spoiled bunches. In this work, 54 native yeasts from vineyards were screened by direct confrontation in potato dextrose agar plates as antagonists against P. glabrum. Isolates of Pichia terricola, Aureobasidium pullulans, and Zygoascus meyerae were selected for their antagonist activity in vitro, plus isolates of Pichia kudriavzevii, Hormonema viticola, and Hanseniaspora uvarum were used as negative controls. However, in vivo, confrontations in wounded grapes showed disagreement with direct confrontation in vitro. P. terricola, P. kudriavzevii, H. viticola, Z. meyerae, and H. uvarum significantly reduced the incidence of P. glabrum on grapes. Nevertheless, P. terricola, H. viticola, and H. uvarum themselves spoiled the wounded grapes. Inhibitions were associated with different mechanisms such as the production of volatile organic compounds (VOCs), lytic enzymes, biofilm formation, and competition for nutrients. The isolates of P. kudriavzevii L18 (a producer of antifungal VOCs which completely inhibited the incidence of P. glabrum) and Z. meyerae L29 (with pectinase, chitinase and &beta, glucanase activity and biofilm formation which reduced 70% of the incidence of P. glabrum) are proposed as suitable biocontrol agents against P. glabrum.

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.

© 2015 Elsevier B.V.. Ca2+-ATPases are plasma membrane and intracellular membrane transporters that use the energy of ATP hydrolysis to pump cytosolic Ca2+ out of the cell (PMCA) or into internal stores. These pumps are the main high-affinity Ca2+ systems involved in the maintenance of intracellular free Ca2+ at the properly low level in eukaryotic cells. The failure of neurons to keep optimal intracellular Ca2+ concentrations is a common feature of neurodegeneration by aging and aging-linked neuropathologies, such as Alzheimer's disease (AD). This disease is characterized by the accumulation of β-amyloid senile plaques and neurofibrillary tangles of tau, a protein that plays a key role in axonal transport. Here we show a novel inhibition of PMCA activity by tau which is concentration-dependent. The extent of inhibition significantly decreases with aging in mice and control human brain membranes, but inhibition profiles were similar in AD-affected brain membrane preparations, independently of age. No significant changes in PMCA expression and localization with aging or neuropathology were found. These results point out a link between Ca2+-transporters, aging and neurodegeneration mediated by tau protein., Ministerio de Economía y Competitividad and Fundación Marcelino Botín (to A.M.M.); Junta de Extremadura and FEDER

© 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).

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

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

The synaptosomal plasma membrane Ca 2+ -ATPase (PMCA) plays an essential role in regulating intracellular Ca 2+ concentration in brain. We have recently found that PMCA is the only Ca 2+ pump in brain which is inhibited by amyloid-β peptide (Aβ), a neurotoxic peptide implicated in the pathology of Alzheimer's disease (AD) [1], but the mechanism of inhibition is lacking. In the present study we have characterized the inhibition of PMCA by Aβ. Results from kinetic assays indicate that Aβ aggregates are more potent inhibitors of PMCA activity than monomers. The inhibitory effect of Aβ could be blocked by pretreating the purified protein with Ca 2+ -calmodulin, the main endogenous activator of PMCA, and the activity of truncated PMCA lacking the calmodulin binding domain was not affected by Aβ. Dot-overlay experiments indicated a physical association of Aβ with PMCA and also with calmodulin. Thus, calmodulin could protect PMCA from inhibition by Aβ by burying exposed sites on PMCA, making them inaccessible to Aβ, and also by direct binding to the peptide. These results suggest a protective role of calmodulin against neuronal Ca 2+ dysregulation by PMCA inhibition induced by Aβ.

Dysregulation of cellular Ca2+ homeostasis and β-amyloid peptide (Aβ) have been linked to cell dysfunction and death in neurodegenerative disorders, such as Alzheimer's disease (AD). We have recently reported that Ca2+-dependence of plasma membrane Ca2+-ATPase (PMCA) activity is altered in AD-affected brains, and in pig brain in the presence of Aβ fragment (25-35) and full-length Aβ1-40 or 1-42 peptides, being inhibited by those peptides. In the present work, performed with Aβ1-42, we found that Ca2+-calmodulin, the main endogenous activator of PMCA, was able to block the inhibitory effect of Aβ on PMCA activity. Besides, the activity of truncated PMCA mutant lacking the C-terminal domain involved in calmodulin binding was not inhibited by Aβ. Dot blot overlay assays further confirmed an interaction of Aβ peptide with both PMCA and calmodulin. Thus, calmodulin may prevent the inhibitory effect of Aβ on PMCA activity by a mechanism that involves its direct binding to the protein, changing PMCA conformation and thus occluding peptide binding to PMCA, and/or its direct binding to the peptide. These finding may reveal a protection role of calmodulin against the Aβ-induced neurotoxicity in AD.This work was supported by Ministerio de Ciencia e Innovacion (MICINN, project BFU2008-00182), Fundacion Marcelino Botin, Junta de Extremadura and FEDER.