- Research article
- Open Access
New insights into the tonoplast architecture of plant vacuoles and vacuolar dynamics during osmotic stress
© Reisen et al; licensee BioMed Central Ltd. 2005
Received: 08 April 2005
Accepted: 04 August 2005
Published: 04 August 2005
The vegetative plant vacuole occupies >90% of the volume in mature plant cells. Vacuoles play fundamental roles in adjusting cellular homeostasis and allowing cell growth. The composition of the vacuole and the regulation of its volume depend on the coordinated activities of the transporters and channels localized in the membrane (named tonoplast) surrounding the vacuole. While the tonoplast protein complexes are well studied, the tonoplast itself is less well described. To extend our knowledge of how the vacuole folds inside the plant cell, we present three-dimensional reconstructions of vacuoles from tobacco suspension cells expressing the tonoplast aquaporin fusion gene BobTIP26-1::gfp.
3-D reconstruction of the cell vacuole made possible an accurate analysis of large spanning folds of the vacuolar membrane under both normal and stressed conditions, and suggested interactions between surrounding plastids. Dynamic, high resolution 3-D pictures of the vacuole in tobacco suspension cells monitored under different growth conditions provide additional details about vacuolar architecture. The GFP-decorated vacuole is a single continuous compartment transected by tubular-like transvacuolar strands and large membrane surfaces. Cell culture under osmotic stress led to a complex vacuolar network with an increased tonoplast surface area. In-depth 3-D realistic inspections showed that the unity of the vacuole is maintained during acclimation to osmotic stress. Vacuolar unity exhibited during stress adaptation, coupled with the intimate associations of vacuoles with other organelles, suggests a physiological role for the vacuole in metabolism, and communication between the vacuole and organelles, respectively, in plant cells. Desiccation stress ensuing from PEG treatment generates "double" membrane structures closely linked to the tonoplast within the vacuole. These membrane structures may serve as membrane reservoirs for membrane reversion when cells are reintroduced to normal growth conditions.
3-D processing of a GFP-labeled tonoplast provides compelling visual constructions of the plant cell vacuole and elaborates on the nature of tonoplast folding and architecture. Furthermore, these methods allow real-time determination of membrane rearrangements during stresses.
Space-filling, turgor-driving vacuoles must have originated at a very early stage of biological evolution and have subsequently evolved to undertake various functions, well-known in algae, fungi (including yeast), and plants . Many of the advanced, complex functions operate on or are closely associated with the vacuolar sap-bounding membrane, i.e. the tonoplast. A detailed structural study of dynamic events mediated by the tonoplast should extend our knowledge of its cellular functions. In the past, in vivo observations of the vacuolar membrane were restricted by the resolution of light microscopy. Like other cell components of a size below the limit of resolution of the light microscope, the tonoplast has been studied mostly by electron microscopy. However, this permits only post-mortem observations of thin sections from rapidly frozen freeze-substituted or chemically fixed cells , or replicas of fracture faces from fast-frozen cells [3, 4]. Although these studies have provided invaluable insights into the organization and biogenesis of the tonoplast, analysis of thin specimens provides only limited information about its spatial architecture. High voltage electron microscopes have the ability to retrieve large amounts of information from thick sections through fixed cells, but depend on the use of a restricted number of selective (non-vital) "staining" techniques to overcome the decrease in image contrast typically seen at high accelerating voltages [4–6]. As such, the use of these different techniques makes it difficult to study and understand the dynamic changes of the vacuolar membrane in living cells.
The confocal fluorescence microscope can eliminate out-of-focus blur. Three-dimensional (3-D) data from intact biological specimens can therefore be obtained by non-invasive optical sectioning . A new revolution in microscopy came over a decade ago with the use of "green fluorescent protein" (GFP) from jellyfish in vivo . GFP and its variants are now frequently used to generate a fluorescent organelle, e.g. mitochondria , chloroplasts  or components of the secretory system , allowing one to study their dynamics in living cells. Furthermore, several studies present data of GFP-labeled tonoplasts [12–21], of which only a few followed tonoplast dynamics [20, 21].
Since the best resolution of living cells is obtained with laser scanning confocal microscopy images, plant-compatible GFP cDNA  was fused, in frame, to the 3' end of the cauliflower BobTIP26-1 tonoplast-specific aquaporin cDNA cloned in our laboratory . The resultant chimeric gene was expressed in tobacco cells (var. Wisconsin 38) grown in suspension, which display a GFP-decorated tonoplast . This method of in vivo labeling was used to 3-dimensionally reconstruct the tonoplast of cells at different stages of growth and under various osmotic stresses. Our data show that the GFP-decorated vacuole in tobacco suspension cells is a single continuous compartment. Furthermore, osmotic stress conditions yield a greater tonoplast surface area while maintaining vacuole unity. Moreover, PEG treatment generates spherical structures that are associated with the inner side of the vacuolar membrane. We propose that these structures serve as membrane reservoirs necessary for membrane reorganization after cells are returned to normal growth conditions.
Three-dimensional reconstruction of the vacuole
We have recently expressed a tonoplast aquaporin fusion gene BobTIP26-1::gfp under the CaMV35S promoter in tobacco cells . The proper targeting of the resultant fusion protein to the tonoplast was confirmed by laser scanning confocal microscopy (Additional file 1). Tobacco cells expressing the chimeric protein were used for 3-D reconstructions of the cell vacuoles.
Cellular architecture details that are evident as a result of 3-D reconstructions include the demonstration of a vacuole with numerous transvacuolar strands, outwardly radiating from the nuclear region to the cell periphery (Fig. 3e, 3f). 3-D reconstructions also showed that chloroplasts do not merely surround the nucleus; their distribution also parallels that of the transvacuolar strands.
Tonoplast behavior during plasmolysis
During the plasmolysis step, a peculiar folding of the GFP-labeled tonoplast characterized by complex curling surrounding the nucleus developed within the vacuole (Fig. 4a, 4b). Closer examination of the curled structures revealed that they were slightly more fluorescent than the tonoplast to which they were tethered, an observation that might be due to the joining of two adjacent labeled membranes. Furthermore, the vacuole and the tonoplast remained intact and no vesicle formation was detected. A cellular tomogram through a plasmolysed cell clearly demonstrates the uninterrupted integrity of the tonoplast, its folds and curves being readily visible (Additional File 4). The absence of tonoplast inside the Hechtian strands was confirmed by the absence of labeling (Additional File 4). However, transvacuolar cytoplasmic strands could still be observed inside the vacuoles of plasmolysed (Fig. 4a, arrowhead; Additional File 5) and deplasmolysed cells (Additional file 5). These intravacuolar structures appear to be less flexible than the peripheral tonoplast. Cells were kept plasmolysed for 10 to 15 min before they were bathed again in normal MS culture medium. Plasmolysed cells returned to their normal shape after about 10 to 20 min (Additional File 5), and the curled structures unwound. The position of the nucleus within the cell was nearly constant throughout the plasmolysis-deplasmolysis process.
Effects of osmotic stress on tonoplast architecture
The vacuolization phenomenon could be monitored under Nomarski contrast interference optics (Fig. 5c). Furthermore, this phenomenon was more easily observed with fluorescent labeling of the tonoplast (Fig. 5d). In these cultures, only ~25% of the cells were completely plasmolysed (Fig. 5c arrows), and did not show any fluorescence, indicating that they are non-acclimated, dead cells (Fig. 5d arrows). Additionally, vital staining with neutral red showed that only acclimated cells contained the dye in their vacuoles (data not shown).
To determine if the apparently numerous, independent, small vacuoles of acclimated cells are part of a continuous vacuolar compartment, 3-D reconstructions of the entire vacuole were achieved. The images indicated the existence of a complex vacuolar network. A depth inspection with stereo-viewers showed interconnections between the vacuolar cavities in completely reconstructed vacuoles. Halves of such reconstructed vacuoles are presented in Figure 5e. The nuclear pouch can be seen at the cell center, surrounded by small, interconnected vacuolar cavities. Despite strong evidence for the existence of a solitary vacuolar space, we cannot rule out the possibility that acclimated cells contain small, discrete cavities. However, no small structures of this nature could be discerned using the complicated reconstructions. Since vacuolar cavities are interconnected, we suggest that the tonoplast surface area increases significantly while the continuity of the vacuolar lumen remains unaltered. The software (Imaris 2.7) used for the 3-D reconstructions did not allow quantification of the tonoplast surface area, nor comparison of the tonoplast surfaces of acclimated cells to those of normal cultured cells.
Tonoplast architecture modifications during dehydrative stress
3-D reconstruction of the vacuole under native conditions
The plant vacuole is a multi-functional organelle  which serves as a true milieu intérieur , playing key roles during cell growth , and possibly in the osmoregulation of water during osmotic stress of the cytoplasm . For the purpose of redefining the vacuole architecture, we present the use of 3-D reconstructions of the vacuolar apparatus from cells containing a GFP-labeled tonoplast under "native" conditions. The tonoplast marker system  was developed to gain new insights of the tobacco cell vacuole morphology during both normal and osmotic stress growth conditions. A 3-D representation of the vacuole using confocal serial pictures offers the most accurate illustration of how a vacuole is folded within a plant cell. Indeed, previous examination of mitotic BY-2 cells expressing a GFP-labeled syntaxin yielded comprehensive images of vacuolar architecture by reconstructing 3-D surfaces obtained from sequential confocal sections . A side effect of the morphology of the tonoplast due to the over-expression of a membrane protein such as BobTIP26-1::GFP can, a priori, not be excluded, but results obtained for both 35S-GFP-AtVam3p and the lipophilic probe FM4-64 in BY-2 cells revealed that vacuolar morphology was not artificially affected in transgenic BY-2 cells . The fluorescent tonoplast in BobTIP26-1::gfp expressing cells appears to be similar to the FM4-64 stained one in BY-2 cells, assuming that vacuolar morphology in BobTIP26-1::gfp expressing cells is not altered.
The software used for the representation of the laser confocal microscope data offered two forms of volume visualization: isosurface rendering and direct volume rendering . We chose the isosurface rendering method, in that it allowed us to represent a shaded surface as an easily interpretable 3-D object. However, the two techniques are complementary and the conclusions offered by the final 3-D pictures are equivalent.
A 3-D vacuole structure of BY-2 tobacco cells expressing TIP::gfp was first established by Mitsuhashi and co-workers , but their representation was mainly a projection view of sequential confocal images that revealed the presence of several large vacuoles folded within the cell. However, as demonstrated in the present study, a projection view may not be as informative as 3-D reconstruction (Fig. 1, Fig. 3). Whereas projection views showed multiple vacuoles folded within a single cell, 3-D reconstructions accurately portray a single vacuolar continuum within cells cultivated under normal growth conditions (Fig. 3). Such continuity of the vacuolar lumen has been described by Palevitz and co-workers , who analyzed the vacuole during cell differentiation of Allium stomata cells. Although thin sections of these fixed cells observed by electron microscopy revealed individual small vacuoles, a 3-D reconstruction using 0.25 to 0.50 μm thick serial sections, viewed at 100 kV, clearly showed the sections originated from a continuous reticulate network.
The concept of a continuous vacuole is important for the plant cell because the content of the vacuolar sap can thereby flow between all regions of the cell when homeostatic measures are undertaken by stressed cells.
To obtain enhanced resolution of the vacuole, reconstructions based on electron tomograms should be used as exemplified in the data obtained for both Arabidopsis thaliana mitotic cell plate formation [35–37] and the Golgi apparatus of animal  and yeast  cells.
Tonoplast surface architecture
With 3-D reconstructions of the vacuole, we were able to analyze its surface architecture from a unique perspective. Earlier 3-D renderings of the vacuole-cytoplasm interface of tobacco cell vacuoles showed ripples on the acridine orange labeled vacuole surface , which differ from surface furrows we have described. Indeed the vacuole ripples dip into the cytoplasm, but not into the vacuole . We observed the furrows just after isosurface reconstruction and they seem to result from the pressure exerted by some organelles onto the turgid vacuole. The organelles were thus squeezed between the plasma membrane and the tonoplast creating hence the furrows on the cytoplasmic interface with the vacuole. We show here that the 3-D rendering is also useful to visualize interactions between organelles.
The tonoplast surface is enlarged during osmotic stress
Although membranes are targets of stress-induced cellular damage, and vacuoles are thought to have a central role during stress, few studies of the tonoplast during stress conditions have been reported. We reasoned that scrutinizing the pattern of the tonoplast during different osmotic stresses would build upon our current knowledge of how the cell organizes this membrane during environmental changes. Glycophytic cells, such as tobacco cells (Nicotiana tabacum var. Wisconsin 38), were previously analyzed for their ability to acclimate to NaCl  and determined to have an increased vacuolization as well as an extensive network of transvacuolar membrane strands . Transvacuolar strands are easily visualized with Normarski optics under a light microscope; however, their composition is more difficult to deduce. Our use of the tonoplast intrinsic GFP-tagged protein that we developed  offers in situ evidence that transvacuolar strands transecting the vacuole lumen also contain tonoplast associated molecules (Fig. 1, Fig. 3f).
Tobacco cells growing in the presence of diverse osmotica developed an extensive membrane network that appears to transect the vacuole, thereby creating multiple compartments. Observations of similar phenomena were previously described for cells grown during NaCl acclimation . With a 3-D representation, we found that growth in media supplemented with salt, mannitol or sorbitol resulted in transvacuolar strands completely changing their organizational pattern, shifting from a thin shape during normal conditions to larger surface areas. Concomitant with strand pattern alterations was the appearance of several small cavities. These phenomena were observed regardless of the osmoticum used for acclimation. The 3-D picture obtained from acclimated cells clearly demonstrates that the number of vacuoles remains constant while the total tonoplast surface area increases, thereby creating a more complex vacuolar pattern (Fig. 5e). Thus, the ratio of tonoplast surface area to volume of cytoplasm is optimized for exchanges (transport of water and ions) between the cytosol and the vacuole. As the total surface area of tonoplast membrane increases, it follows that the total mass of membrane compounds also increases. It is notable that similar vacuolization changes were observed to occur after hyper-osmotic treatment of wild type tobacco cells; therefore, the increased membrane surface is linked neither to the presence of the aquaporin nor the greater cell size of BobTIP26-1::gfp expressing cells . Measurements of the surface areas of the tonoplasts in acclimated cells are complicated by the absence of vacuole shape uniformity. In contrast, the prolate spheroid shape of Vicia faba guard cells allows for the determination of both their surface areas and volumes after 3-D reconstruction efforts . In our studies such standardization of conditions was not possible.
Folding of the plasmolytic vacuole
Notwithstanding the massive changes in vacuolar surface area which accompany plasmolysis, only a few observations on the fate of the vacuole during osmotic contraction have been described in the relevant literature. Plasmolysis-deplasmolysis cycles were realized in a perfusion chamber, allowing observation of fluorescent tonoplast labeled with GFP. During plasmolysis, it was observed that the tonoplast undergoes folding and that the vacuole does not vesiculate into discrete multilamellar vesicles severed from the tonoplast as "sac-like, rod-like or doughnut-shaped structures" as previously described . Furthermore, complete tomography of a plasmolysed cell (Additional File 4) demonstrated that the tonoplast folds in a peculiar way inside the protoplast, rather than being broken into small vesicles, as reported earlier . Such a process seems better suited than vesicle formation for a faster reestablishment of the vacuole during deplasmolysis. No membrane fusion is necessary.
Transvacuolar strands, thin tubular structures that traverse the vacuole, were observed not to break down both during plasmolysis and after deplasmolysis (Additional File 5). Earlier studies showed that transvacuolar strands were stabilized by actin filaments [44, 45] and rearranged by myosin motors through their interactions with actin filaments . Despite these observation of strand stability, they have also been described as dynamic and delicate . Our data support the notion that transvacuolar strands exhibit both "strength" and stability, in that their positions remaining static inside the vacuole during abrupt environmental changes (Additional File 5).
PEG treatment results in spherical structures composed of tonoplast
Surprisingly, spherical structures with twice the fluorescent intensity as the tonoplast were observed inside the vacuole lumen of tobacco suspension cells cultured in MS medium supplemented with 10% PEG8000. These structures were similar to those frequently observed in either tonoplast GFP-labeled tobacco leaves [47, 48] or in Arabidopsis cotyledons . Additionally, such structures have been described in cotyledons, hypocotyls and roots of the Arabidopsis vacuolar biogenesis bub (bubble-bath) mutants , as well as in transiently transformed Nicotiana benthamiana plants expressing a GFP fusion protein homologous to a high-affinity tonoplast phosphate transporter . The structures we observed during PEG treatment were independent of the water regulation or over-expression of an aquaporin gene because we, as well as other authors, found similar bulbs in Arabidopsis transformed cells with native TIP1;1 promoter (Bouhidel K., personal communication) and in cells expressing other tonoplast proteins under the control of the 35S promoter . The structures observed in Arabidopsis cotyledons  were called "bulbs", a term which reflects a spherical structure that is not entirely closed. The open section of these bulbs may be the result of incomplete vesicle formation . The bulbs appear to be attached to the actin cytoskeleton, as actin inhibitor treatment allowed their immobilization and 3-D reconstructions . The 3-D images of the spherical structures we observed in tobacco suspension cells cultured under dehydration stress are similar to the 3-D reconstructed cylindrical structures observed in GFP-AtVam3p expressing root protoplasts of A. thaliana . Recently, fluorescent circular structures were seen in germinating pollen tubes of δ -TIP::GFP expressing Arabidopsis plants . These mobile cytoplasmic invaginations may be a widespread characteristic of actively growing tissues.
Why do vacuoles of PEG acclimated suspension cells contain "bulbs"? PEG mimics dehydration when applied to the culture medium, resulting in a decrease of turgor. Similarly, cotyledons undergo a desiccation process. In Spirodela intermedia upper mesophyll cells, a breakdown of the tonoplast into small vesicles was observed after PEG inclusion . Correlation of observed membrane fluorescence intensity with protein quantity suggests that the membrane of the spherical vacuolar structures is likely to contain twice the amount of aquaporins per surface area as the peripheral tonoplast, suggesting a higher level of water exchange. The spherical structures could reflect the presence of lipid domains in the tonoplast where aquaporins are concentrated and vesicle formation occurs. Indeed, some studies showed a high tonoplast fluidity , as well as a specific feature of fatty acid composition that may be responsible for the tonoplasts' unique fluidity and high elasticity  required for osmotic processes in the cell. Our results support the invagination model, postulated by Uemura and co-workers , where parts of the tonoplast form a double-layered membrane structure inside the vacuolar lumen. The spherical structures could serve as a reservoir, not only for membrane expansion, but also to allow for quicker homeostasis adjustments. A higher tonoplast surface area to cell volume ratio would greatly enhance the cells' capacity to maintain large ion pools during growth .
The data presented in this study demonstrates the utility of the aquaporin BobTIP26-1::GFP as a powerful tool for visualizing 3-D membrane rearrangements within stressed tobacco cells. The technique used here provides highly resolved pictures and support of the notion that tobacco suspension cells contain a single major vacuole with a lytic function. Further investigations are required to establish the exact origin and function of the membrane enclosed, intra-vacuolar circular structures exhibited by tobacco cells under PEG mediated stress.
Tobacco (Nicotiana tabacum L. var. Wisconsin 38) suspension cells expressing BobTIP26-1::gfp  were grown in MS medium , with regular shaking, at 24°C under constant photosynthetic illumination (200 μE·m-2·sec-1).
Three to seven day-old cells were analyzed under a Leica TCS 4D laser confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with an argon-krypton laser (488/515 BP-FITC). The laser was focused on individual cells through a 40x NA1 oil-immersion objective. For each cell, a stack of between 100 and 200 images was collected (resolution 256 × 256 with 0.50 to 0.65 μm of z-step). Merged individual confocal images (red and green channels) were composed using Corel Photo-Paint 7 software (Corel, Ottawa, Canada). To obtain 3-D reconstructions, confocal image stacks were imported into the three-dimensional visualization software Imaris 2.7 (Bitplane AG, Switzerland) running on a Silicon Graphics® Octane2™ workstation (SGI, Paris, France). After baseline subtraction, a subregion was defined. Then, the isosurface module of Imaris was used to reconstitute the 3-D pictures. An adequate isovalue was defined for each channel prior to viewing of the computed surface using IvView. Isosurface rendered pictures were then stored as tiff files using the MediaRecorder media tool. Realistic on-screen representations of 3-D reconstructions were analyzed in depth with stereoscopic viewing devices (CrystalEyes®, StereoGraphics®).
Cell perfusion was performed in a homemade perfusion chamber linked to a peristaltic pump (flux = 20 mL·h-1) (IBMP, Strasbourg, France). BobTIP26-1::gfp expressing cells were first adhered to poly-lysinized glass cover slips. The following solutions were used for cell perfusions: MS supplemented with either 0.17 M NaCl or 0.6 M mannitol or 0.5 M sorbitol. Plasmolysis and deplasmolysis were monitored using an inverted laser confocal microscope LSM510 (Zeiss Axiovert 100 M, Jena, Germany) fitted with a Zeiss 63X water-immersion objective. Confocal time lapse series were then collected. Osmolarities were measured with a Vapro® vapor pressure osmometer (Model 5520; Wescor, Logan, UT, USA).
Acclimation stress conditions
Five mL of BobTIP26-1::gfp expressing cells at exponential growth phase, were subcultured in MS medium supplemented with any of the following osmotica: 170 mM NaCl, 0.6 M mannitol, 0.5 M sorbitol or 10% PEG8000. The stressed and control (MS without an osmoticum) cells were analyzed over 7 days and 3-D reconstructed as described above. Fluorescent intensities were measured using IPLab software (Scanalytics, Fairfax, VA, USA).
The authors would like to thank J.C. Robbins for English corrections and meaningful discussions. D.R. was supported by the Ministère de l'Education Nationale de la Recherche et de la Technologie (N° 99-5-12166). The Octane2™ workstation and the Imaris software were used at the CMAB-SERCOBIO (Université de Bourgogne). We thank C. Ritzenthaler at The Inter-Institute Confocal Microscopy Plate-Form in the Institut de Biologie Moléculaire des Plantes (Strasbourg) for his assistance with the perfusion experiments. The Conseil Régional de Bourgogne financed this work.
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