Purification and functional characterization of protoplasts and intact vacuoles from grape cells
© Gerós et al; licensee BioMed Central Ltd. 2010
Received: 7 November 2009
Accepted: 22 January 2010
Published: 22 January 2010
During 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.
Protoplasts 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.
Highly 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.
Enzymatic digestion of grape cells yields highly pure, viable and homogeneous populations of protoplasts
Protoplasts were prepared from Vitis vinifera L. cells (CSB, Cabernet Sauvignon Berry). Cells were cultivated in liquid mineral medium supplemented with 2% (w/v) sucrose. The method of Greuter and Keller  to isolate protoplast from Stachys sieboldii tubers was adapted for grape cells and optimized by introducing several changes, including the composition of the media, enzyme proportion and purification steps. Protoplasting was performed by enzymatic digestion of the cell walls (450 × 106 cells) with 0.007% (w/v) cellulase Y-C and 0.0007% (w/v) pectolyase Y-23 (Kyowa chemical products CO., LTD) in a final volume of 50 ml. Digestion occurred in Gamborg B5 Medium supplemented with 0.4 M sucrose, under shaking (50 rpm), at pH 5.8 and 22°C. Different digestion periods of 4 to 12 h were tested. The resulting protoplasts were gently collected and subsequently purified. Initially, protoplasts were separated by floating, at 150 × g for 8 min, and subsequently washed with the same medium. A discontinuous gradient was prepared by overlaying 1 volume of a solution containing 0.05 M glucose, 154 mM NaCl, 125 mM CaCl2 and 5 mM KCl, pH 5.8, on the protoplast suspension, followed by centrifugation at 150 × g for 8 min. Protoplasts were recovered from the interface of the gradient, resuspended in 3 volumes of the glucose-containing medium and sedimented for 8 min at 150 × g. The pellet was washed in 0.4 M mannitol, 15 mM MgCl2, 5 mM Mes, pH 5.8, resuspended in the same medium and stored at 4°C. Protoplasts were counted in a Malassez chamber under the light microscope. A protoplast yield of 13% was obtained as a result of a 12 h digestion protocol, decreasing approximately to 6% when the digestion lasted 4 h.
Flow cytometry analysis requires that microscopical biological particles be in suspension. It allows the simultaneous quantification of multiple fluorescence emissions in the same cell or biological particle, and scattered light related to morphology . Therefore, individual cells or sub-cellular particles from heterogeneous subpopulations can be physically isolated on the basis of their fluorescence or light scatter properties . In the present work, flow cytometry has been exploited in order to characterize the protoplast and vacuole samples and to individualize the subpopulations, allowing conclusions about the purity of each fraction. Flow cytometric analysis was performed in an Epics® XLTM (Beckman Coulter) flow cytometer equipped with an argon-ion laser emitting a 488 nm beam at 15 mW. Green fluorescence was collected through a 488 nm blocking filter, a 550 nm long-pass dichroic and a 525 nm band-pass filter. For each sample, 20,000 protoplasts and 20,000 vacuoles were analysed at low flow rate. An acquisition protocol was defined to measure forward scatter (FS), side scatter (SS) and green fluorescence (FL1) on a four decades logarithmic scale. Data were analysed by WinMDI 2.8 software. The analysis of the biparametric histograms, plotting log SS against log FS, revealed some heterogeneity in both relative complexity and size of the protoplast population (Figure 1B). However, the subpopulation of protoplasts can be easily identified, since it easily stains well with FDA (gated region P). Above and to the right of this region, there is also a subpopulation that probably consists of protoplast aggregates. The subpopulations with the lowest scatter (below and to the left of region P) correspond mainly to submicroscopic particles - as some cell debris and cell wall residues - of relative low complexity and size, which were co-purified with the protoplasts. Figure 1C depicts the overlay of the green fluorescence and auto-fluorescence histograms of the gated region P. Quantification of the percentage of FDA positive stained cells indicates that the protoplasts population exhibits a high percentage of viability. These findings were previously summarized in the book chapter by Papadakis et al. .
The lysis of grape protoplasts yields highly pure and intact vacuoles
Vacuoles were released upon the protoplast osmotic lysis at a relatively high temperature and were purified by a Ficoll step gradient centrifugation. The methodology was adapted from the protocol used to obtain vacuoles from Arabidopsis protoplasts . The protoplast suspension was added to 2.5 volumes of the pre-warmed (37-45°C) lysis buffer, a solution with reduced osmotic strength containing 0.2 M mannitol, 10% Ficoll (w/v), 15 mM EDTA, 10 mM MOPS, pH 8.0, supplemented with 0.1% BSA and 2 mM DTT, resulting in the release of intact vacuoles. The vacuoles were collected from the vacuole buffer layer after a one-step Ficoll gradient centrifugation of 15 min at 1000 × g. The discontinuous gradient was optimized as follows: one layer of the lysis mixture (10% Ficoll, w/v), one layer of 3.0% Ficoll (w/v) and one layer of vacuole buffer containing 0.5 M mannitol, 10 mM MOPS, pH 7.5 and a protease inhibitor cocktail (Complete, Roche Applied Science, Germany), in the proportion of 7:3:1 volumes. The 3.0% Ficoll solution was prepared by diluting the lysis buffer with vacuole buffer. The vacuoles were counted on a Malassez chamber under the light microscope. In a typical fractionation procedure, an average amount of 4.0 × 106 vacuoles was obtained, corresponding to about 12 % of the total number of protoplasts (purified by a 12-h digestion protocol) subjected to lysis. Since the yield of intact vacuoles was always higher when protoplasts were isolated by a 12-h digestion protocol (not shown), this digestion duration was used in all subsequent experiments. Cytosolic glucose-6-phosphatase was used as a marker enzyme to monitor vacuole purification . The specific activities of the protoplast preparation and vacuole preparation were 715 and 14.6 (nmol min-1 mg prot-1), respectively. Only 2% of the marker enzyme was recovered in the vacuolar fraction, indicating that this sample was strongly depleted in protoplasts and cytosolic contaminations. This conclusion was further supported by microscopic observation and flow cytometry analysis.
Most of the intact vacuoles, ranging in size from 10 to 50 μm, maintained an internal acidic pH and exhibited a red colour after being labelled with Neutral Red, a lipophilic phenazine dye (Sigma-Aldrich), in spite of their resuspension in a buffer at pH 7.5 (Figure 2B). This acidity was relatively stable and was not completely abolished by the incubation with 100 μM CCCP. This may be due to the buffering capacity of organic acids accumulated in the vacuoles, but we must not discard the fact that higher protonophore concentrations could promote red colour dissipation. However, 2.5 mM NH4Cl almost completely abolished the pH gradient across the majority of the vacuoles.
Intact vacuoles from grape cells are physiologically active organelles
Above results show that two distinct primary proton pumps, the vacuolar ATPase and the vacuolar inorganic pyrophosphatase (V-PPase), generate a proton electromotive force, which, in turn, allow the secondary active transport of several compounds that are accumulated in the vacuole, as it has been shown for Ca2+ uptake. However, we must not ignore that the long digestion period used to purify the protoplasts can affect both the vacuolar contents and the activity of the transporter proteins. The predominant activity of the V-PPase compared to that of V-ATPase in intact vacuoles (Figure 4) is in agreement with the earlier results [14, 16–18]. The capacity of the vacuole population to sequester protons strongly suggests the intactness and integrity of these extremely fragile organelles.
Structural studies [19–21] and, more recently, proteomic analysis [22–26] have elucidated some aspects of vacuole function and biogenesis, and have generated an unpredicted interest in the purification of this extremely fragile organelle. During the ripening of fleshy fruits, the vacuoles accumulate water, sugars and secondary metabolites [13, 27, 28]. In spite of its importance for crop yield and quality, the molecular basis of these compartmentation processes is still poorly understood for grapevine. As in many species, the major bottleneck to study these aspects in grapevine is to obtain highly purified vacuoles with a good yield. This work describes the preparation of intact and viable vacuoles from grape cells suspensions, so as to demonstrate their feasibility as a model system to study the mechanisms underlying vacuolar compartmentation. A fast and efficient method has been developed to isolate highly pure and intact protoplast and vacuoles from grape suspension-cultured cells. 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 grapevine breeding programs.
This work was supported in part by the Fundação para a Ciência e a Tecnologia (research projects no. POCI/AGR/56378/2004 and no. PTDC/AGR-ALI/100636/2008; grant no. SFRH/BD/23169/2005 to N.F), the Conférence Française des Présidents d'université (CPU) and Conselho de Reitores das Universidades Portuguesas (CRUP) (Actions Intégrées Luso-Françaises - 2008/2009). The authors would like to thank Filomena Louro of the Scientific Editing Program of Universidade do Minho for revising the English text of the manuscript and Prof. Manuela Côrte-Real for her support and expert assistance on flow cytometry experiments, as well as the COST 858 network on viticulture for facilitating our exchanges.
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