Proteome Profile of American Hybrid Grape cv. Blanc du Bois during Ripening Reveals Proteins Associated with Flavor Volatiles and Ethylene Production

The study of key control points in ripening is essential to improve grape wine quality. Molecular basis of ripening is still far from being understood from the Pierce's disease (PD)‐tolerant grapes predominantly grown in the southeastern United States. To identify proteins expressed during Blanc du Bois grape berry green and ripening stages, proteome analysis from five different stages revealed 1091, 1131, 1078, 1042, and 1066 proteins. Differential expression analysis revealed 551 common proteins across different stages of maturity that are involved in various biochemical and metabolic pathways. The proteins identified were associated with phenylpropanoids, isoquinoline alkaloids, fatty acids, unsaturated fatty acids, and furanones. Our data provide the first step to understand the complex biochemical changes during ripening of PD‐tolerant American hybrid grapes that are popular for their aroma and flavor profile in the southeastern United States. Proteomics data are deposited to the ProteomeXchange PXD004157.     

Grape berry plasma membrane proteome analysis and its differential expression during ripening

High purity berry plasma membranes (PMs) of Vitis vinifera L. cv. Cabernet Sauvignon were isolated by two-phase partitioning of microsome fractions at different stages of berry ripening. PM proteins resolvable by the detergent cocktail of CHAPS and ASB-14 were separated by two-dimensional electrophoresis. A total of 119 protein spots from pre-véraison berry PMs on 2-D gels detected with silver staining were subjected to MALDI-TOF mass spectrometry analysis. Sixty-two spots were identified as putative PM proteins, with 1-6 predicted transmembrane helices, including true PM proteins such as ATP synthase, ABC transporters, and GTP-binding proteins reported in plants. They were then grouped into eight functional categories, mainly involved in transport, metabolism, signal transduction, and protein synthesis. Another 11 spots were identified as proteins of unknown function. The véraison and post-véraison samples stained 98 and 86 spots on the gels, respectively. During the berry ripening process, total PM protein content gradually decreased. Among all identified proteins, 12 showed significant differences in terms of their relative abundance. Increasing ubiquitin proteolysis and cytoskeleton proteins were observed from pre-véraison to post-véraison. Zeatin O-glucosyltransferase peaked at véraison, while ubiquitin-conjugating enzyme E2-21 was down-regulated at this stage. This proteome research provides the first information on PM protein characterization during the grape berry ripening process.     

A Deep Proteomics Perspective Into Grape Berry Quality Traits During Ripening

Discovery‐based proteomics studies have an important role in the understanding of the biochemical processes that occur during grape berry ripening. The ripening process is relevant in determining grape berry quality. For a proteome analysis of grape berry ripening, Kambiranda et al. (2018) applied a label‐free mass spectrometry–based quantitative approach. The authors reported the identification of proteins associated with the production flavor, aroma and ethylene production. Despite the valuable contribution of discovery‐based proteomics studies, the picture is still incomplete. Future efforts in gaining proteome coverage would benefit the identification of proteins associated with grape berry quality traits.     

Differential Expression of Proteins in Red Pear Following Fruit Bagging Treatment

Fruit bagging is a very effective method for study of fruit qualities and anthocyanin synthesis. The characterization of differentially expressed proteins that were isolated from both bagged and normal fruit skin tissue is apparently an essential parameter for understanding the effect of shading on fruit qualities and to understand the mechanism of fruit coloring in Pyrus communis. Proteome maps of both bagged and normal P. communis 'Placer' fruit skin were obtained by performing two-dimensional electrophoresis analysis and compared to assess the extent to which protein distribution differed in pear skin. The comparative analysis showed 38 differentially expressed proteins between the two samples: with three protein spots up-regulated and 35 down-regulated in the bagged fruit. Differentially expressed protein spots were subjected to matrix-assisted laser desorption ionization time of flight (MALDI-TOF) analysis and the data compared to that of known proteins to deduce their possible functions. Of these, 21 protein spots were identified and classified into functional classes. These identified proteins were mainly involved in photosynthesis, signal transduction, energy pathway, protein folding and assembly, and carbohydrate and acidity metabolisms, and were under-expressed in bagged fruit skins. This work provides a first characterization of the proteome changes in response to fruit bagging treatment in red pears.     

Proteomic characterization of the greening process in rice seedlings using the MS spectral intensity-based label free method

Illumination-induced greening in dark-grown plants is one of the most dramatic developmental processes known in plants. In our current study, we characterized the greening process of rice seedlings using comparative proteome analysis. We identified 886 different proteins in both whole cell lysates of illuminated and nonilluminated rice shoots and performed comparative proteome analysis based on the MS spectral intensities obtained for unique peptides from respective proteins. Furthermore, the changes in the levels of individual proteins were then compared with those of the corresponding mRNAs. The results revealed well-coordinated increases in the enzymes involved in the Calvin cycle at both the protein and mRNA levels during greening, and that the changes at the mRNA level precede those at the protein level. Although a much lower effect of illumination was found on the enzymes associated with glycolysis and the TCA cycle, coordinated increases during greening were evident for the enzymes involved in photorespiration and nitrogen assimilation as well as the components of the chloroplastic translational machinery. These results thus define the differential regulation of distinct biological systems during greening in rice and demonstrate the usefulness of comprehensive and comparative proteome analysis for the characterization of biological processes in plant cells.     

Proteins involved in biotic and abiotic stress responses as the most significant biomarkers in the ripening of Pinot Noir skins

We propose an integrated approach, obtained by the combination of multivariate statistics and proteomics, useful to isolate candidate biomarkers for the evaluation of grape ripening. We carried out a comparative 2-DE analysis of grape skins collected in three moments of ripening and analyzed the spot volume dataset through the application of principal component analysis followed by forward stepwise-linear discriminant analysis. This technique allowed to discriminate véraison, quite mature and mature samples, and to sort the matched spots according to their significance. We identified 36 spots showing high discriminating coefficients through liquid chromatography - electrospray ionization - tandem mass spectrometry (LC-ESI-MS/MS). Most of them were involved in biotic and abiotic stress responses indicating these enzymes as good candidate markers of berry ripening. These evidences hint at a likely developmental role of these proteins, in addition to their reported activity in stress events. Restricting the same statistical analysis to the samples belonging to the two last stages, it was indicated that this approach can clearly distinguish these close and similar phases of berry development. Taken all together, these results bear out that the employment of the combination of 2-DE and multivariate statistics is a reliable tool in the identification of new protein markers for describing the ripening phases and to assess the overall quality of the fruit.     

Mapping the proteome of Leishmania Viannia parasites using two-dimensional polyacrylamide gel electrophoresis and associated technologies

In this study we have demonstrated the potential of two-dimensional electrophoresis (2DE)-based technologies as tools for characterization of the Leishmania proteome (the expressed protein complement of the genome). Standardized neutral range (pH 5-7) proteome maps of Leishmania (Viannia) guyanensis and Leishmania (Viannia) panamensis promastigotes were reproducibly generated by 2DE of soluble parasite extracts, which were prepared using lysis buffer containing urea and nonidet P-40 detergent. The Coomassie blue and silver nitrate staining systems both yielded good resolution and representation of protein spots, enabling the detection of approximately 800 and 1,500 distinct proteins, respectively. Several reference protein spots common to the proteomes of all parasite species/strains studied were isolated and identified by peptide mass spectrometry (LC-ES-MS/MS), and bioinformatics approaches as members of the heat shock protein family, ribosomal protein S12, kinetoplast membrane protein 11 and a hypothetical Leishmania-specific 13 kDa protein of unknown function. Immunoblotting of Leishmania protein maps using a monoclonal antibody resulted in the specific detection of the 81.4 kDa and 77.5 kDa subunits of paraflagellar rod proteins 1 and 2, respectively. Moreover, differences in protein expression profiles between distinct parasite clones were reproducibly detected through comparative proteome analyses of paired maps using image analysis software. These data illustrate the resolving power of 2DE-based proteome analysis. The production and basic characterization of good quality Leishmania proteome maps provides an essential first step towards comparative protein expression studies aimed at identifying the molecular determinants of parasite drug resistance and virulence, as well as discovering new drug and vaccine targets.     

Temporal variations of the postnatal rat urinary proteome as a reflection of systemic maturation

The rat kidney matures during the first 2 wk of life, suggesting that temporal variations in the urinary proteome may occur during this period. We describe the urine proteome during postnatal development in the rat and demonstrate specific proteomic changes corresponding to developmental milestones. Urine was collected from 30 rats at five postnatal (P) days of life (P1, P3, P7, P14, and >P30) by bladder aspiration. The proteome was assessed by nano-ESI-LC-MS/MS. For identification, we used stringent criteria to provide a 1% false positive rate at the peptide level. The proteins in common at each time interval decreased during postnatal maturation. When comparing all five developmental times, six proteins were ubiquitously present. We detected 14 proteins involved with cellular adhesion, structure, or proliferation and differentiation only during neonatal development. Additionally, 30 proteins were specific to adults, of which 13 originated from the prostate or seminal vesicle. This is the first MS characterization of the normal urinary proteome in early postnatal rodent development that demonstrates distinct differences correlating with different stages of tissue maturation. Further characterization of the normal urinary proteome may provide the basis for identification of urinary biomarkers of diseases of the urinary tract.     

A DIGE-based quantitative proteomic analysis of grape berry flesh development and ripening reveals key events in sugar and organic acid metabolism

Grapevine (Vitis vinifera L.) is an economically important fruit crop. Quality-determining grape components, such as sugars, acids, flavours, anthocyanins, tannins, etc., are accumulated during the different grape berry development stages. Thus, correlating the proteomic profiles with the biochemical and physiological changes occurring in grape is of paramount importance to advance the understanding of the berry development and ripening processes. Here, the developmental analysis of V. vinifera cv. Muscat Hamburg berries is reported at protein level, from fruit set to full ripening. A top-down proteomic approach based on differential in-gel electrophoresis (DIGE) followed by tandem mass spectrometry led to identification and quantification of 156 and 61 differentially expressed proteins in green and ripening phases, respectively. Two key points in development, with respect to changes in protein level, were detected: end of green development and beginning of ripening. The profiles of carbohydrate metabolism enzymes were consistent with a net conversion of sucrose to malate during green development. Pyrophosphate-dependent phosphofructokinase is likely to play a key role to allow an unrestricted carbon flow. The well-known change of imported sucrose fate at the beginning of ripening from accumulation of organic acid (malate) to hexoses (glucose and fructose) was well correlated with a switch in abundance between sucrose synthase and soluble acid invertase. The role of the identified proteins is discussed in relation to their biological function, grape berry development, and to quality traits. Another DIGE experiment comparing fully ripe berries from two vintages showed very few spots changing, thus indicating that protein changes detected throughout development are specific.     

A multidisciplinary study on the effects of phloem-limited viruses on the agronomical performance and berry quality of Vitis vinifera cv. Nebbiolo

Viral infections are known to have a detrimental effect on grapevine yield and performance, but there is still a lack of knowledge about their effect on the quality and safety of end products. Vines of Vitis vinifera cv. Nebbiolo clone 308, affected simultaneously by Grapevine leafroll-associated virus 1 (GLRaV-1), Grapevine virus A (GVA), and Rupestris stem pitting associated virus (RSPaV), were subjected to integrated analyses of agronomical performance, grape berry characteristics, instrumental texture profile, and proteome profiling. The comparison of performance and grape quality of healthy and infected vines cultivated in a commercial vineyard revealed similar shoot fertility, number of clusters, total yield, with significant differences in titratable acidity, and resveratrol content. Also some texture parameters such as cohesiveness and resilience were altered in berries of infected plants. The proteomic analysis of skin and pulp visualized about 400 spots. The ANOVA analysis on 2D gels revealed significant differences among healthy and virus-infected grape berries for 12 pulp spots and 7 skin spots. Virus infection mainly influenced proteins involved in the response to oxidative stress in the berry skin, and proteins involved in cell structure metabolism in the pulp.     

Proteomic Analysis of Sauvignon Blanc Grape Skin,Pulp and Seed and Relative Quantification of Pathogenesis-Related Proteins

Thaumatin-like proteins (TLPs) and chitinases are the main constituents of so-called protein hazes which can form in finished white wine and which is a great concern of winemakers. These soluble pathogenesis-related (PR) proteins are extracted from grape berries. However, their distribution in different grape tissues is not well documented. In this study, proteins were first separately extracted from the skin, pulp and seed of Sauvignon Blanc grapes, followed by trypsin digestion and analysis by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Proteins identified included 75 proteins from Sauvignon Blanc grape skin, 63 from grape pulp and 35 from grape seed, mostly functionally classified as associated with metabolism and energy. Some were present exclusively in specific grape tissues; for example, proteins involved in photosynthesis were only detected in grape skin and proteins found in alcoholic fermentation were only detected in grape pulp. Moreover, proteins identified in grape seed were less diverse than those identified in grape skin and pulp. TLPs and chitinases were identified in both Sauvignon Blanc grape skin and pulp, but not in the seed. To relatively quantify the PR proteins, the protein extracts of grape tissues were seperated by HPLC first and then analysed by SDS-PAGE. The results showed that the protein fractions eluted at 9.3 min and 19.2 min under the chromatographic conditions of this study confirmed that these corresponded to TLPs and chitinases seperately. Thus, the relative quantification of TLPs and chitinases in protein extracts was carried out by comparing the area of corresponding peaks against the area of a thamautin standard. The results presented in this study clearly demonstrated the distribution of haze-forming PR proteins in grape berries, and the relative quantification of TLPs and chitinases could be applied in fast tracking of changes in PR proteins during grape growth and determination of PR proteins in berries at harvest.     

Rice proteome analysis: a step toward functional analysis of the rice genome

The technique of proteome analysis using 2-DE has the power to monitor global changes that occur in the protein complement of tissues and subcellular compartments. In this review, we describe construction of the rice proteome database, the cataloging of rice proteins, and the functional characterization of some of the proteins identified. Initially, proteins extracted from various tissues and organelles were separated by 2-DE and an image analyzer was used to construct a display or reference map of the proteins. The rice proteome database currently contains 23 reference maps based on 2-DE of proteins from different rice tissues and subcellular compartments. These reference maps comprise 13 129 rice proteins, and the amino acid sequences of 5092 of these proteins are entered in the database. Major proteins involved in growth or stress responses have been identified by using a proteomics approach and some of these proteins have unique functions. Furthermore, initial work has also begun on analyzing the phosphoproteome and protein-protein interactions in rice. The information obtained from the rice proteome database will aid in the molecular cloning of rice genes and in predicting the function of unknown proteins.     

Rice Proteome Database: A Step toward Functional Analysis of the Rice Genome

The technique of proteome analysis using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has the power to monitor global changes that occur in the protein complement of tissues and subcellular compartments. In this study, the proteins of rice were cataloged, a rice proteome database was constructed, and a functional characterization of some of the identified proteins was undertaken. Proteins extracted from various tissues and subcellular compartments in rice were separated by 2D-PAGE and an image analyzer was used to construct a display of the proteins. The Rice Proteome Database contains 23 reference maps based on 2D-PAGE of proteins from various rice tissues and subcellular compartments. These reference maps comprise 13129 identified proteins, and the amino acid sequences of 5092 proteins are entered in the database. Major proteins involved in growth or stress responses were identified using the proteome approach. Some of these proteins, including a β-tubulin, calreticulin, and ribulose-1,5-bisphosphate carboxylase/oxygenase activase in rice, have unexpected functions. The information obtained from the Rice Proteome Database will aid in cloning the genes for and predicting the function of unknown proteins.     

Comparative analyses of the proteomes of leaves and flowers at various stages of development reveal organ‐specific functional differentiation of proteins in soybean

The functional differentiation of protein networks in individual organs and tissues of soybean at various developmental stages was investigated by proteomic approach. Protein extraction by Mg/NP‐40 buffer followed by alkaline phenol‐based method was optimized for proteomic analysis. Proteome analyses of leaves at various developmental stages showed 26 differentially expressed proteins, wherein proteins in translocon at the outer/inner envelope membrane of chloroplast protein‐transport machineries increased significantly at the first trifoliate. Immunoblot analysis showed chaperonin‐60 expressed abundantly in young leaves, whereas HSP 70 and ATP‐synthase β were constitutively expressed in all tissues. The net photosynthesis rate and chlorophyll content showed an age‐dependent correlation in leaves. These results suggest that proteins involved in carbon assimilation, folding and assembly, and energy may work synchronously and show a linear correlation to photosynthesis at developmental stages of leaves. Comparison of flower bud and flower proteome reveals 29 differentially expressed proteins, wherein proteins involved in mitochondrial protein transport and assembly, secondary metabolism, and pollen‐tube growth were up‐regulated during flower development. Together, these results suggest that during developmental stages, each type of tissue is associated with a specific group of proteins; wherein proteins involved in energy, sugar metabolism, and folding, assembly, and destination may play pivotal roles in the maturation process of each organ or tissue.