Seasonal pattern of apoplastic solute accumulation and loss of cell turgor during ripening of Vitis vinifera fruit under field conditions

Using a novel pressure membrane (PM) apparatus for the extraction of apoplastic fluid from field-grown grape (Vitis vinifera L.) berries, our hypothesis that significant apoplast solutes accumulate at the beginning of the ripening process (i.e. veraison), and that this accumulation might contribute to progressive berry softening due to a progressive loss of mesocarp cell turgor pressure (P) was tested. It was necessary to correct the solute potential (Ψs) of fluid collected with the PM for dilution due to the presence of a dead volume in the apparatus, but after correction, the Ψs obtained with the PM agreed with that obtained by low speed centrifugation. A clear decline in fruit apoplastic solute potential (ψSA) began approximately 10 d prior to fruit coloration, and it was found to be coincident with a decline in mesocarp cell P and fruit elasticity (E). By late in fruit development when berry growth ceased (90 d after anthesis), both apoplast and fruit Ψs reached almost –4 MPa. These results support the hypothesis that a decrease in ψSA is responsible for the observed loss in mesocarp cell P, and is the mechanistic cause of berry softening

Transcriptome profiling of grapevine seedless segregants during berry development reveals candidate genes associated with berry weight

Indexación: Web of Science; PubMedBackground Berry size is considered as one of the main selection criteria in table grape breeding programs. However, this is a quantitative and polygenic trait, and its genetic determination is still poorly understood. Considering its economic importance, it is relevant to determine its genetic architecture and elucidate the mechanisms involved in its expression. To approach this issue, an RNA-Seq experiment based on Illumina platform was performed (14 libraries), including seedless segregants with contrasting phenotypes for berry weight at fruit setting (FST) and 6–8 mm berries (B68) phenological stages. Results A group of 526 differentially expressed (DE) genes were identified, by comparing seedless segregants with contrasting phenotypes for berry weight: 101 genes from the FST stage and 463 from the B68 stage. Also, we integrated differential expression, principal components analysis (PCA), correlations and network co-expression analyses to characterize the transcriptome profiling observed in segregants with contrasting phenotypes for berry weight. After this, 68 DE genes were selected as candidate genes, and seven candidate genes were validated by real time-PCR, confirming their expression profiles. Conclusions We have carried out the first transcriptome analysis focused on table grape seedless segregants with contrasting phenotypes for berry weight. Our findings contributed to the understanding of the mechanisms involved in berry weight determination. Also, this comparative transcriptome profiling revealed candidate genes for berry weight which could be evaluated as selection tools in table grape breeding programs.http://bmcplantbiol.biomedcentral.com/articles/10.1186/s12870-016-0789-

Proteomic analysis of the effects of ABA treatments on ripening Vitis vinifera berries

The control of ripening of the non-climacteric grapevine fruit is still a matter of debate, but several lines of evidence point to an important role for the hormone abscisic acid (ABA). The effects of ABA treatments on Cabernet Sauvignon berries before and at véraison were studied using a 2-DE proteomic approach. Proteins from whole deseeded berries (before véraison) and berry flesh and skin (at véraison) treated with 0.76 mM ABA and collected 24 h after treatment were separated and analysed. A total of 60 protein spots showed significant variations between treated and control berries, and 40 proteins, mainly related to general metabolism and cell defence, were identified by LC MS/MS. Our results show that ABA acts mainly through the regulation of mostly the same proteins which are involved in the ripening process, and that several of these changes share common elements with the ABA-induced responses in vegetative tissues

Berry Flesh and Skin Ripening Features in Vitis vinifera as Assessed by Transcriptional Profiling

Background Ripening of fleshy fruit is a complex developmental process involving the differentiation of tissues with separate functions. During grapevine berry ripening important processes contributing to table and wine grape quality take place, some of them flesh- or skin-specific. In this study, transcriptional profiles throughout flesh and skin ripening were followed during two different seasons in a table grape cultivar ‘Muscat Hamburg’ to determine tissue-specific as well as common developmental programs. Methodology/Principal Findings Using an updated GrapeGen Affymetrix GeneChip® annotation based on grapevine 12×v1 gene predictions, 2188 differentially accumulated transcripts between flesh and skin and 2839 transcripts differentially accumulated throughout ripening in the same manner in both tissues were identified. Transcriptional profiles were dominated by changes at the beginning of veraison which affect both pericarp tissues, although frequently delayed or with lower intensity in the skin than in the flesh. Functional enrichment analysis identified the decay on biosynthetic processes, photosynthesis and transport as a major part of the program delayed in the skin. In addition, a higher number of functional categories, including several related to macromolecule transport and phenylpropanoid and lipid biosynthesis, were over-represented in transcripts accumulated to higher levels in the skin. Functional enrichment also indicated auxin, gibberellins and bHLH transcription factors to take part in the regulation of pre-veraison processes in the pericarp, whereas WRKY and C2H2 family transcription factors seems to more specifically participate in the regulation of skin and flesh ripening, respectively. Conclusions/Significance A transcriptomic analysis indicates that a large part of the ripening program is shared by both pericarp tissues despite some components are delayed in the skin. In addition, important tissue differences are present from early stages prior to the ripening onset including tissue-specific regulators. Altogether, these findings provide key elements to understand berry ripening and its differential regulation in flesh and skin.This study was financially supported by GrapeGen Project funded by Genoma España within a collaborative agreement with Genome Canada. The authors also thank The Ministerio de Ciencia e Innovacion for project BIO2008-03892 and a bilateral collaborative grant with Argentina (AR2009-0021). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

NMR methods to monitor the enzymatic depolymerization of heparin

Heparin and the related glycosaminoglycan, heparan sulfate, are polydisperse linear polysaccharides that mediate numerous biological processes due to their interaction with proteins. Because of the structural complexity and heterogeneity of heparin and heparan sulfate, digestion to produce smaller oligosaccharides is commonly performed prior to separation and analysis. Current techniques used to monitor the extent of heparin depolymerization include UV absorption to follow product formation and size exclusion or strong anion exchange chromatography to monitor the size distribution of the components in the digest solution. In this study, we used 1H nuclear magnetic resonance (NMR) survey spectra and NMR diffusion experiments in conjunction with UV absorption measurements to monitor heparin depolymerization using the enzyme heparinase I. Diffusion NMR does not require the physical separation of the components in the reaction mixture and instead can be used to monitor the reaction solution directly in the NMR tube. Using diffusion NMR, the enzymatic reaction can be stopped at the desired time point, maximizing the abundance of larger oligosaccharides for protein-binding studies or completion of the reaction if the goal of the study is exhaustive digestion for characterization of the disaccharide composition. In this study, porcine intestinal mucosa heparin was depolymerized using the enzyme heparinase I. The unsaturated bond formed by enzymatic cleavage serves as a UV chromophore that can be used to monitor the progress of the depolymerization and for the detection and quantification of oligosaccharides in subsequent separations. The double bond also introduces a unique multiplet with peaks at 5.973, 5.981, 5.990, and 5.998 ppm in the 1H-NMR spectrum downfield of the anomeric region. This multiplet is produced by the proton of the C-4 double bond of the non-reducing end uronic acid at the cleavage site. Changes in this resonance were used to monitor the progression of the enzymatic digestion and compared to the profile obtained from UV absorbance measurements. In addition, in situ NMR diffusion measurements were explored for their ability to profile the different-sized components generated over the course of the digestion

Collaborative Enhancement of Antibody Binding to Distinct PECAM-1 Epitopes Modulates Endothelial Targeting

Antibodies to platelet endothelial cell adhesion molecule-1 (PECAM-1) facilitate targeted drug delivery to endothelial cells by “vascular immunotargeting.” To define the targeting quantitatively, we investigated the endothelial binding of monoclonal antibodies (mAbs) to extracellular epitopes of PECAM-1. Surprisingly, we have found in human and mouse cell culture models that the endothelial binding of PECAM-directed mAbs and scFv therapeutic fusion protein is increased by co-administration of a paired mAb directed to an adjacent, yet distinct PECAM-1 epitope. This results in significant enhancement of functional activity of a PECAM-1-targeted scFv-thrombomodulin fusion protein generating therapeutic activated Protein C. The “collaborative enhancement” of mAb binding is affirmed in vivo, as manifested by enhanced pulmonary accumulation of intravenously administered radiolabeled PECAM-1 mAb when co-injected with an unlabeled paired mAb in mice. This is the first demonstration of a positive modulatory effect of endothelial binding and vascular immunotargeting provided by the simultaneous binding a paired mAb to adjacent distinct epitopes. The “collaborative enhancement” phenomenon provides a novel paradigm for optimizing the endothelial-targeted delivery of therapeutic agents

Applications of Wine Pomace in the Food Industry: Approaches and Functions

Winemaking generates large amounts ofwine pomace, also called grape pomace. This by-product has attracted the attention of food scientists and the food industry, due to its high content in nutrients and bioactive compounds. This review mainly focuses on the different published approaches to the use of wine pomace and its functions in the food industry. Traditionally, wine pomace has been used to obtain wine alcohol, food colorings, and grape seed oil. More recently, research has focused in the production of other value-added products, such as extracts of bioactive compounds, mainly phenols, recovery of tartaric acid, and the making of flours. The most common functions associated with wine pomace products are their use as antioxidants, followed by their use as fortifying, coloring, and antimicrobial agents. These products have mainly been applied to the preparation of meat and fish products and to, a lesser extent, cereal products.Autonomous Government of Castilla y León, Spain, through the research project BU282U13