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.

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SummaryGrapevine stilbenes, particularly trans‐resveratrol, have a demonstrated pharmacological activity. Other natural stilbenes derived from resveratrol such as pterostilbene or piceatannol, display higher oral bioavailability and bioactivity than the parent compound, but are far less abundant in natural sources. Thus, to efficiently obtain these bioactive resveratrol derivatives, there is a need to develop new bioproduction systems. Grapevine cell cultures are able to produce large amounts of easily recoverable extracellular resveratrol when elicited with methylated cyclodextrins and methyl jasmonate. We devised this system as an interesting starting point of a metabolic engineering‐based strategy to produce resveratrol derivatives using resveratrol‐converting enzymes. Constitutive expression of either Vitis vinifera resveratrol O‐methyltransferase (Vv ROMT) or human cytochrome P450 hydroxylase 1B1 (Hs CYP1B1) led to pterostilbene or piceatannol, respectively, after the engineered cell cultures were treated with the aforementioned elicitors. Functionality of both gene products was first assessed in planta by Nicotiana benthamiana agroinfiltration assays, in which tobacco cells transiently expressed stilbene synthase and Vv ROMT or Hs CYP1B1. Grapevine cell cultures transformed with Vv ROMT produced pterostilbene, which was detected in both intra‐ and extracellular compartments, at a level of micrograms per litre. Grapevine cell cultures transformed with Hs CYP1B1 produced about 20 mg/L culture of piceatannol, displaying a sevenfold increase in relation to wild‐type cultures, and reaching an extracellular distribution of up to 45% of total production. The results obtained demonstrate the feasibility of this novel system for the bioproduction of natural and more bioactive resveratrol derivatives and suggest new ways for the improvement of production yields.

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Quantitation of changes in protein abundance is key to discovering novel biomarkers. Currently, reverse phase liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) can be used to quantify changes in protein expression levels. Nevertheless, quantitative analysis of protein mixtures by HPLC-MS/MS is still hampered by the wide range of protein expression levels, the high dynamic range of protein concentrations and the lack of reliable quantitation algorithms. In this context, we describe two different samples (4-protmix and 8-protmix) suitable for relative protein quantitation using isobaric Tags for Relative and Absolute Quantitation (iTRAQ). Using the 4-protmix, relative protein changes of up to 24-fold were measured. The 8-protmix allowed the quantitation of the relative protein changes in a mixture of proteins within the range of two orders of magnitude in concentration and 10-fold differences in relative abundance.The two reference samples proposed here (4-protmix and 8-protmix) cover a wide range of protein fold changes and protein concentrations, respectively, which can be used to optimize the settings used during acquisition with different mass spectrometers and to test the performance of different quantitation algorithms used for iTRAQ experiments.Three technical replicates corresponding to 800 fmol of 4-protmix and 8-protmix were analyzed by HPLC-MS/MS using two platforms, a ChipLC coupled to a 6530 Q-ToF (Agilent Technologies) and a 1200 nano-HPLC (Agilent Technologies) coupled to -sult, the analysis of the 4-protmix and the 8-protmix all proteins in the samples (Tables 1 and 2 As shown here, we were able to detect up to 24-fold changes in protein ratios. The standard de--sponded to the highest protein fold change in our samples (24-fold).

BackgroundGrapevine (Vitis vinifera L.) is an economically important fruit crop. Quality-determining grape components such as sugars, acids, flavors, anthocyanins, tannins, etc., accumulate in 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 in our understanding of berry development and ripening processes.ResultsWe report the developmental analysis of Vitis vinifera cv. Muscat Hamburg berries at the protein level from fruit set to full ripening. An iTRAQ-based bottom-up proteomic approach followed by tandem mass spectrometry led to the identification and quantitation of 411 and 630 proteins in the green and ripening phases, respectively. Two key points in development relating to changes in protein level were detected: end of the first growth period (7 mm-to-15 mm) and onset of ripening (15 mm-to-V100, V100-to-110). A functional analysis was performed using the Blast2GO software based on the enrichment of GO terms during berry growth.ConclusionsThe study of the proteome contributes to decipher the biological processes and metabolic pathways involved in the development and quality traits of fruit and its derived products. These findings lie mainly in metabolism and storage of sugars and malate, energy-related pathways such as respiration, photosynthesis and fermentation, and the synthesis of polyphenolics as major secondary metabolites in grape berry. In addition, some key steps in carbohydrate and malate metabolism have been identified in this study, i.e., PFP-PFK or SuSy-INV switches among others, which may influence the final sugar and acid balance in ripe fruit. In conclusion, some proteins not reported to date have been detected to be deregulated in specific tissues and developmental stages, leading to formulate new hypotheses on the metabolic processes underlying grape berry development. These results open up new lines to decipher the processes controlling grape berry development and ripening.

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Vitis vinifera cell cultures respond to pathogens and elicitors by synthesizing and extracellularly accumulating stilbenoid phytoalexins. Large amounts of trans-resveratrol (t-R) are produced when a cell culture is elicited with methylated cyclodextrins (MBCD), either alone or combined with methyl jasmonate (MeJA). t-R transport to the extracellular medium, which represents the apoplastic space, would place this antifungal defense right in the battlefield to efficiently fight against pathogen attack. Yet despite their physiological relevance, these transport pathways are mostly unknown. A broad hypothesis-free DIGE-based proteomic experiment of a temporal series of elicited grapevine cell cultures was performed to explore the expression profiles of t-R biosynthetic proteins and other co-expressing proteins potentially involved in such a cell response. A correlation between two tau class glutathione-S-transferases (GSTs) with several stilbene synthase and phenylalanine ammonia-lyase isoforms, and with the t-R metabolite itself, was found and further assessed by a qRT-PCR gene expression analysis. The best candidate, GSTU-2, was cloned from the cDNA of the MBCD + MeJA-elicited grapevine cells and used for Agrobacterium-mediated grapevine cell transformation. The non-elicited lines that overexpressed GSTU-2 displayed an extracellular t-R accumulating phenotype, but stabilization of t-R required the addition to culture medium of adsorbent compounds, e.g., PVP or β-cyclodextrin. The wild-type cell cultures accumulated no t-R, not even in the presence of adsorbents. The transient expression of the GSTU-2-GFP fusion proteins in grapevine cells showed localisation in the plasma membrane, and the immunoprecipitation of HA-tagged GSTU-2 revealed its interaction with HIR, a plasma membrane-bound protein. These findings are consistent with a functional role in transport. This is the first report providing several pieces of experimental evidence for the involvement of a specific tau class GST in t-R transport to the extracellular medium.