Project description:Blanc Du bois grapes are gaining popularity in the South eastern US due to its distinctive flavor and disease tolerance characteristics. Berry composition at harvest is a major contributing factor of wine quality. Blanc Du bois grapes are harvested from EL-38 and EL-39 stages depending on the style of wine desired or harvested early to avoid rain nearing harvest. In the current study, gel-free proteome analysis was applied to investigate changes in enzymes, primary and secondary metabolism proteins during ripening and late ripe stage. Grape berries from EL-33, EL-34, EL-36, EL-38 and EL-39 were collected based on brix, acidity and density. Protein extracts from different berry stages were resolved by electrophoresis. Proteins were extracted from the gel as a single band, detained and subjected to proteolysis with sequencing grade trypsin. Trypsin digested peptides from different berry protein extracts were separated on a nano LC and the eluent was sprayed onto to a LTQ Orbitrap Velos mass spectrometer. The raw files were analyzed using Proteome Discoverer with Sequest and Mascot search nodes using Vitis species FASTA database (70,263 entries) and the data were further validated by Scaffold software. A total of 1091, 1131, 1078, 1042 and 1066 proteins were detected in EL-33, EL-34, EL-36, EL-38 and EL-39 of berries respectively. Statistical ANOVA analysis revealed 927 proteins present across the stages that are involved in various biochemical and metabolic pathways. Seventeen proteins including dihydroflavonol reductase, sucrose phosphate synthase, PR proteins increased more than three-fold between ripe and late ripe berry stages. Other proteins that increased during ripe and late ripe stage berries were alcohol dehydrogenase 1, anthocyanidin reductase, phospho-2-dehydro-3-deoxyheptonate aldolase, fatty acid hydroperoxide lyase, cinnamyl alcohol dehydrogenase, -isopiperitenol (-)-carveol and SAM-methyltransferases.
Project description:The emerging roles of endolysosomes (EL) in AF have yet to be discovered, especially how the EL contribute to the development of AF within a slow time frame. To identify the EL role in AF, we applied proteomics, transcriptomics, integrated analysis, electron tomography, western blotting and enzyme assays. To understand the cellular and EL driven dominant pathways, in AF, we applied an organelle protein isolation method, that was previously developed by us (Ayagama et al,.2021) on to an AF goat model. Our findings include upregulation of AMPK pathway, EL-specific proteins that were not identified in the TL, such as GAA, DYNLRB1, CLTB, SIRT3, CCT2, and muscle specific HSPB2. The structural anomalies such as autophago-vacuole formation, irregularly stacked mitochondria, and glycogen deposition provide an insight into of the contribution of EL contribution and the related pathways and molecular mechanisms.
Project description:Background Grapes (Vitis vinifera L.) are economically the most important fruit crop worldwide. However, the complexity of molecular and biochemical events that lead to the onset of ripening of nonclimacteric fruits is not fully understood which is further complicated in grapes due to seasonal and cultivar specific variantion. The Portuguese wine variety Trincadeira gives rise to high quality wines but presents extremely irregular berry ripening among seasons probably due to high susceptibility to abiotic and biotic stresses. Results Ripening of Trincadeira grapes was studied taking into account the transcriptional and metabolic profilings complemented with biochemical data. The mRNA expression profiles of four time points spanning developmental stages from pea size green berries, through veraison and mature berries (EL 32, EL 34, EL 35 and EL 36) and in two seasons (2007 and 2008) were compared using the Affymetrix GrapeGen® genome array containing 23096 probesets corresponding to 18726 unique sequences. Over 50% of these probesets were significantly differentially expressed (1.5 fold) between at least two developmental stages. A common set of modulated transcripts corresponding to 5862 unigenes indicates the activation of common pathways in between years despite the irregular development of Trincadeira grapes. These unigenes were assigned in the functional categories of “metabolism, development”, “cellular process”, “diverse functions”, “regulation overview”, “response to stimulus, stress”, “signaling”, “transport overview”, “xenoprotein, transposable element” and “unknown”. Metabolic profiling using 1H NMR spectroscopy associated to two-dimensional techniques showed the importance of metabolites related to oxidative stress response, amino acid and sugar metabolism as well as secondary metabolism in grape ripening. This information was integrated with transcriptional profiling obtained using genome array and qRT-PCR for five developmental stages (EL 32, EL 34, EL 35, EL 36 and EL 38). Conclusions Altogether the results provide new information regarding the network of events leading to grape ripening as well as highlight features that may be cultivar specific namely in what concerns the role of carbohydrates and growth regulators´ metabolism as well as epigenetic factors and signaling pathways.