Natacha Fontes

Activity of tonoplast proton pumps and Na+/H+ exchange in potato cell cultures is modulated by salt

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.

Grape Berry Vacuole: A Complex and Heterogeneous Membrane System Specialized in the Accumulation of Solutes

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.

Purification and functional characterization of protoplasts and intact vacuoles from grape cells

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.

New Observations on the Integrity, Structure, and Physiology of Flesh Cells from Fully Ripened Grape Berry

The physiological/structural status of the soft ripened berry is still a matter of debate. The isolated mesocarp cells from ripened berries of both wine and table varieties were studied by bright-field, fluorescence, and confocal microscopy and flow cytometry to highlight the organization of berry flesh cell, function, and viability. Flow cytometry analysis confirmed that protoplasting from grape berry mesocarp tissue yields a single heterogenous population of intact and viable cells. Also, the integrity of the plasma membrane and the architecture and complexity of the intracellular membranous system were shown by FM1-43 staining coupled to confocal microscopy imaging. The observed incorporation of the fluorescent glucose analogue 2-NBDG suggests that endocytosis is involved in the transport and intracellular compartmentation of apoplastic sugars. Neutral Red staining confirmed the intricate organization, size, diversity, and integrity of the vacuolar apparatus that is probably related to the multifaceted roles of the vacuoles in the developing fruit.

Isolation and Use of Protoplasts from Grapevine Tissues

Highly pure, intact and functional protoplasts can be obtained from plant tissues, which are readily amenable for challenging with exogenous sugars, acids, analogues, transport inhibitors and drugs. Thus, they may be used as models for both basic research and biotechnological approaches. Some of these studies require the regeneration of plants from protoplasts; however most agronomically important plant species, including grapevine, are recalcitrant to plant regeneration. Oxidative stress has been considered as a crucial factor accounting for the recalcitrance of grapevine protoplasts, as supported by the profiles of generated reactive oxygen species (ROS) and ROS-scavenging enzymes, the modified cell redox state, as well as the altered endogenous titers of polyamine levels. In the present work, methods for the purification of intact and functional protoplasts from grape berry mesocarp tissue and for the isolation and culture of mesophyll protoplasts are described. Methods for the detection of ROS in grapevine protoplasts, together with assays for antioxidant enzyme and antioxidant biomolecules are also detailed.

Flow Cytometry and Fluorescence Microscopy as Tools for Structural and Functional Analysis of Vacuoles Isolated from Yeast and Plant Cells

A series of optimized protocols to isolate vacuoles from both yeast and plant cells, and to characterize the purified organelles at a functional and structural level, are described. For this purpose, we took advantage of the combined use of cell fractionation techniques with different fluorescence-based approaches namely flow cytometry, fluorescence microscopy and spectrofluorimetry. These protocols altogether constitute valuable tools for the study of vacuole structure and function, as well as for the high-throughput screening of drug libraries to identify new molecules that target the vacuole.