The efficient exclusion of excess Na from the cytoplasm and vacuolar Na(+) accumulation are the main mechanisms for the adaptation of plants to salt stress. This is typically carried out by transmembrane transport proteins that exclude Na(+) from the cytosol in exchange for H(+), a secondary transport process which is energy-dependent and driven by the proton-motive force generated by plasma-membrane and tonoplast proton pumps. Tonoplast enriched-vesicles from control and 150 mM NaCl-tolerant calli lines were used as a model system to study the activity of V-H(+)-PPase and V-H(+)-ATPase and the involvement of Na(+) compartmentalization into the vacuole as a mechanism of salt tolerance in Solanum tuberosum. Both ATP- and pyrophosphate (PP(i))-dependent H(+)-transport were higher in tonoplast vesicles from the salt-tolerant line than in vesicles from control cells. Western blotting of tonoplast proteins confirmed that changes in V-H(+)-PPase activity are correlated with increased protein amount. Conversely, immunodetection of the A-subunit of V-H(+)-ATPase revealed that a mechanism of post-translational regulation is probably involved. Na(+)-dependent dissipation of a pre-established pH gradient was used to measure Na(+)/H(+) exchange in tonoplast vesicles. The initial rates of proton efflux followed Michaelis-Menten kinetics and the V(max) of proton dissipation was 2-fold higher in NaCl-tolerant calli when compared to the control. H(+)-coupled exchange was specific for Na(+) and Li(+) and not for K(+). The increase of both the pH gradient across the tonoplast and the Na(+)/H(+) antiport activity in response to salt strongly suggests that Na(+) sequestration into the vacuole contributes to salt tolerance in potato.

Vacuoles fulfill highly specialized functions depending on cell type and tissue and plant developmental stage. This complex and dynamic organelle is the main reservoir of grape berry cells, playing a major role during fruit development and ripening. Berry development is accompanied by modifications in size, composition, color, texture, flavor, and pathogen susceptibility, primarily because of changes in vacuolar content. Most aroma and flavor compounds are not evenly distributed in the berry, and the number and type of vacuoles may vary depending on the tissue (skin, flesh, and seeds). Together with the lytic and protein storage vacuoles widely distributed in plant cells, phenolic vacuoles are also implicated in cellular storage in grape cells. After veraison, when grape berry growth exclusively results from cell enlargement, tonoplast transporter proteins mediate a massive sugar import and water intake into the vacuole, leading to a large vacuolar expansion. The V-ATPase and V-PPase pumps create a proton electrochemical gradient across the tonoplast, which, in turn, energizes the uptake of charged and uncharged solutes. Several tonoplast proteins mediating the uptake of sugars, organic acids, water, ions, and anthocyanins have been cloned and some have been functionally characterized. The present review focuses on the storage function of vacuoles and on their structure and diversity in relation to development and ripening of the grape berry.

Taking advantage of the low oxidation potential of polyphenolic compounds, voltammetric techniques, such as cyclic voltammetry (CV) and differential pulse voltammetry (DPV) are used rather indiscriminately. In this work, we report Total Polyphenols results (TPP) obtained by these two techniques from a set of nine samples of red and Tawny Port wine. The CV and DPV voltammograms display significant correlations with the physical-chemical parameters used to characterize red and Tawny Port wines, particularly with polyphenols. Although data obtained from CV and DPV for a single polyphenol are directly proportional, important deviations are found between voltammetric results from wines. Results from CV tend to be larger than those from DPV. This difference, that can reach 50 % of the TPP value, was related to the presence of total sulphur dioxide. In view of the present study, the polyphenol quantification in wines should be performed by DPV to minimize the interference of SO2.

The atmospheric conditions are a strong modulator of grape berry composition, but further research is required to better understand this relationship, which is particularly pertinent under the context of climate change. The present study assesses the relationship between interannual variability in atmospheric conditions (mean, maximum and minimum air temperatures and precipitation totals) on grape berry quality attributes in three main Portuguese wine regions—Douro, Dão and Alentejo—and targets two major varieties growing in Portugal (cv. Touriga Nacional and cv. Aragonez/Tempranillo). Berry weight, titratable acidity (TA), pH, potential alcohol (PA), anthocyanins and total phenols index (TPI) data, collected two to three weeks after the end of the veraison until technological maturity, since 1999 in Douro, 2004 in Alentejo and 2008 in Dão, were selected. Meteorological data were obtained from both automatic weather stations and a climatic database defined at a very-high-resolution grid (<1 km) (PTHRES). The influence of daily mean, maximum and minimum air temperatures (November–October) and precipitation totals (April to June and July to September) on the above-mentioned berry quality parameters were first explored to identify the months/periods more influential to grape berry composition. Different statistical approaches were subsequently carried out to explore in greater detail these relationships. At technological maturity, temperature was negatively correlated to berry weight, titratable acidity, anthocyanins and TPI, but was positively correlated to pH and potential alcohol. Moreover, lowest levels of berry weight and TA (and highest levels of pH) were more frequent in warmer regions, while the opposite was seen in the cooler regions. PA, TPI and anthocyanins at maturity did not show a clear trend across regions. In addition, the maturation parameters of each site were grouped into two clusters—years where the maturation parameter is higher (cluster 1) and years where it is lower (cluster 2)—and significant differences in monthly mean temperatures between clusters were found. Overall, temperatures at veraison and maturation periods (June–August) were more influential in determining grape berry composition at harvest. The influence of precipitation was dependent on location and variety. The results also suggested that berry composition in Alentejo is more sensitive to atmospheric variability, while Aragonez seems more resilient than Touriga Nacional. These outcomes are based on a systematized and unprecedentedly large grape berry quality database in Portugal and provided the grounds for the development of grape quality forecast models, either to be used operationally in each vintage or for assessing potential modifications in berry composition in response to changing climates.

BackgroundDuring grape berry ripening, the vacuoles accumulate water, sugars and secondary metabolites, causing great impact in plant productivity and wine quality. However, the molecular basis of these compartmentation processes is still poorly understood. As in many species, the major bottleneck to study these aspects in grapevine is to obtain highly purified vacuoles with a good yield. The present paper describes an isolation method of protoplasts and intact vacuoles from grape berry cells and their functional characterization by transport and cytometric assays.FindingsProtoplasts were prepared by enzymatic digestion of grape cells, and vacuoles were released and purified by a Ficoll step gradient centrifugation. The tonoplast stained strongly with the fluorescent dye FM1-43 and most vacuoles maintained an internal acidic pH, as assessed by Neutral Red. Flow cytometry analysis of vacuole samples incubated with the calcium-sensitive fluorescent probe Fluo-4 AM revealed a well-defined sub-population of intact vacuoles. As assessed by the pH-sensitive probe ACMA, intact vacuoles generated and maintained a pH gradient through the activity of V-ATPase and V-PPase and were able to transport Ca2+ via a proton-dependent transport system.ConclusionsHighly pure, intact and functional protoplast and vacuole populations from grape cells were obtained with the present method, which revealed to be fast and efficient. The capacity of the vacuole population to sequester protons and accumulate Ca2+ strongly suggests the intactness and physiological integrity of these extremely fragile organelles. Grapevine protoplasts and vacuoles may be used as models for both basic research and biotechnological approaches, such as proteomics, solute uptake and compartmentation, toxicological assessments and breeding programs.