The role of the motile tubular vacuole system in mycorrhizal fungi

Mycorrhizal fungi, to be effective for the plant, must be able to transfer mineral nutrient elements from sites of uptake at hyphal tips across various distances to the exchange region in the mycorrhiza. Vacuoles are likely to be important in this transport, since they contain elements of nutritional significance in abundance. In tip cells of hyphae of most fungi –- known to include three ectomycorrhizal basidiomycetes, an ericoid mycobiont, and two arbuscular mycorrhizal fungi –- the vacuoles form a motile tubular reticulum. The vacuoles are most active in hyphal tips, but non-motile vacuoles at a distance from the tip can be induced to become motile by environmental changes. Neither the tubular vacuolar reticulum nor its contents are properly preserved by conventional fixation and embedding. Vacuolar tubules are readily shown in vivo with fluorescent tracers, throughout the extramatrical mycelium and in outer hyphae of the sheath in eucalypt mycorrhizas synthesised with Pisolithus sp., but they have proved harder to label in field-collected ectomycorrhizas and ericoid mycorrhizas. Freeze-substitution does preserve the structure of vacuoles and vacuolar tubules, and careful anhydrous techniques allow them to be microanalysed, indicating high content of K and P in vacuoles of hyphal tips, and also in sheath and Hartig net of ectomycorrhizas. Vacuoles contain polyphosphate in diffuse, non-granular form. Polyphosphate is present right up to the tip region of hyphae as well as in sheath and Hartig net: thus important mineral nutrient elements are present at both ends of the long hyphal transport pathway. Exactly what happens in between, however, remains to be elucidated.     

Vacuolar reticulum in oomycete hyphal tips: An additional component of the Ca2+Regulatory system?

Cultures of Achlya sp., Phytophthora cinnamomi, Saprolegnia diclina, S. ferax, and S. parasitica, treated with 6-carboxyfluorescein diacetate solution, accumulate 6-carboxyfluorescein in a reticulate system of fine tubules. The network shows longitudinal polarity within the hyphae, tubules being finest toward the hyphal tips. In more mature subapical regions the network is connected with large vacuoles that also accumulate 6-carboxyfluorescein. A morphologically similar system has also been identified in freeze-substituted hyphae of S. ferax. The network is considered to be vacuolar, but differs from the tubular vacuole system of true fungi in that tubules are less motile, more frequently branched, and do not alternate with clusters of spherical vacuoles. The appearance of the network resembles patterns of calcium-sensitive dye staining and it is suggested that the vacuolar reticulum in the tip region of oomycete hyphae may act as a Ca2+ sink. The tubular reticulum in oomycetes is very fragile and can be shown with 6-carboxyfluorescein in only those hyphal tips with a motility and organelle distribution characteristic of growing hyphae with normal morphology. Diverse abnormal hyphae show a range of other fluorochrome localizations. These include large irregular compartments filled with fluorochrome, and fluorescent cytoplasm with organelles and vacuoles standing out in negative contrast. These localizations in abnormal hyphae are correlated with other structural changes indicative of damage. Special care is required in experiments with oomycetes to avoid such artefacts of localization. Copyright 1997 Academic Press. Copyright 1997 Academic Press     

Vacuolar Reticulum in Oomycete Hyphal Tips: An Additional Component of the CaRegulatory System?

Cultures ofAchlyasp.,Phytophthora cinnamomi, Saprolegnia diclina, S. ferax,andS. parasitica,treated with 6-carboxyfluorescein diacetate solution, accumulate 6-carboxyfluorescein in a reticulate system of fine tubules. The network shows longitudinal polarity within the hyphae, tubules being finest toward the hyphal tips. In more mature subapical regions the network is connected with large vacuoles that also accumulate 6-carboxyfluorescein. A morphologically similar system has also been identified in freeze-substituted hyphae ofS. ferax.The network is considered to be vacuolar, but differs from the tubular vacuole system of true fungi in that tubules are less motile, more frequently branched, and do not alternate with clusters of spherical vacuoles. The appearance of the network resembles patterns of calcium-sensitive dye staining and it is suggested that the vacuolar reticulum in the tip region of oomycete hyphae may act as a Ca²⁺sink. The tubular reticulum in oomycetes is very fragile and can be shown with 6-carboxyfluorescein in only those hyphal tips with a motility and organelle distribution characteristic of growing hyphae with normal morphology. Diverse abnormal hyphae show a range of other fluorochrome localizations. These include large irregular compartments filled with fluorochrome, and fluorescent cytoplasm with organelles and vacuoles standing out in negative contrast. These localizations in abnormal hyphae are correlated with other structural changes indicative of damage. Special care is required in experiments with oomycetes to avoid such artefacts of localization.     

Microtubules, but not actin microfilaments, regulate vacuole motility and morphology in hyphae of Pisolithus tinctorius

While it is now recognised that transport within the endomembrane system may occur via membranous tubules, spatial regulation of this process is poorly understood. We have investigated the role of the cytoskeleton in regulating the motility and morphology of the motile vacuole system in hyphae of the fungus Pisolithus tinctorius by studying (1) the effects of anti-microtubule (oryzalin, nocodazole) and anti-actin drugs (cytochalasins, latrunculin) on vacuolar activity, monitored by fluorescence microscopy of living cells; and (2) the ultrastructural relationship of microtubules, actin microfilaments, and vacuoles in hyphae prepared by rapid-freezing and freeze-substitution. Anti-microtubule drugs reduced the tubular component of the vacuole system in a dose-dependent and reversible manner, the extent of which correlated strongly with the degree of disruption of the microtubule network (monitored by immunofluorescence microscopy). The highest doses of anti-microtubule drugs completely eliminated tubular vacuoles, and only spherical vacuoles were observed. In contrast, anti-actin drugs did not reduce the frequency of tubular vacuoles or the motility of these vacuoles, even though immunofluorescence microscopy confirmed perturbation of microfilament organisation. Electron microscopy showed that vacuoles were always accompanied by microtubules. Bundles of microtubules were found running in parallel along the length of tubular vacuoles and individual microtubules were often within one microtubule diameter of a vacuole membrane. Our results strongly support a role for microtubules, but not actin microfilaments, in the spatial regulation of vacuole motility and morphology in fungal hyphae.     

Characterization of a Novel Prevacuolar Compartment in Neurospora crassa

Using confocal microscopy, we observed ring-like organelles, similar in size to nuclei, in the hyphal tip of the filamentous fungus Neurospora crassa. These organelles contained a subset of vacuolar proteins. We hypothesize that they are novel prevacuolar compartments (PVCs). We examined the locations of several vacuolar enzymes and of fluorescent compounds that target the vacuole. Vacuolar membrane proteins, such as the vacuolar ATPase (VMA-1) and the polyphosphate polymerase (VTC-4), were observed in the PVCs. A pigment produced by adenine auxotrophs, used to visualize vacuoles, also accumulated in PVCs. Soluble enzymes of the vacuolar lumen, alkaline phosphatase and carboxypeptidase Y, were not observed in PVCs. The fluorescent molecule Oregon Green 488 carboxylic acid diacetate, succinimidyl ester (carboxy-DFFDA) accumulated in vacuoles and in a subset of PVCs, suggesting maturation of PVCs from the tip to distal regions. Three of the nine Rab GTPases in N. crassa, RAB-2, RAB-4, and RAB-7, localized to the PVCs. RAB-2 and RAB-4, which have similar amino acid sequences, are present in filamentous fungi but not in yeasts, and no function has previously been reported for these Rab GTPases in fungi. PVCs are highly pleomorphic, producing tubular projections that subsequently become detached. Dynein and dynactin formed globular clusters enclosed inside the lumen of PVCs. The size, structure, dynamic behavior, and protein composition of the PVCs appear to be significantly different from those of the well-studied prevacuolar compartment of yeasts.     

Vacuole motility and tubule-forming activity inPisolithus tinctorius hyphae are modified by environmental conditions

Summary Motile tubular vacuole systems have been visualised using DIC optics in living hyphae ofPisolithus tinctorius without loading of any fluorescent tracer. Adding new medium, with or without the tracer CFDA, alters the motility of this system and increases the number of tubules. This response has been shown in individual hyphal tip cells and quantified in populations of tip cells. Vacuoles with motile tubules are also demonstrated in more basal cells of the hyphae, within 600 m of the growing hyphal front. The vacuoles in these cells show more limited motility, but similarly respond to addition of new medium by increased motility and tubular activity. This demonstration that the vacuole system in more mature regions is both motile and interconnected as in the tips, and similarly responds to changes in external conditions, supports the hypothesis that the vacuole system may play a role in long-distance transport. Vacuoles in the most mature cells, more than 600 m behind the hyphal growth zone are not motile. They do not respond to these stimuli and remain spherical and isolated. There are many explanations for this and the present lack of response does not exclude the transport hypothesis. The findings further support the concept that tubular vacuole systems are equivalent to animal endosomal/lysosomal systems and have implications for their motility, especially their plasticity in response to external stimuli, such as fluorescent tracers.Abbreviations CFDA 6-carboxyfluorescein diacetate - DIC differential interference contrast - MMN modified Melin-Norkrans medium - SEM standard error of the mean     

Dynamic organization of vacuolar and microtubule structures during cell cycle progression in synchronized tobacco BY-2 cells

In higher plant cells, vacuoles show considerable diversity in their shapes and functions. The roles of vacuoles in the storage, osmoregulation, digestion and secretory pathway are well established; however, their functions in cell morphogenesis and cell division are still unclear. To observe the dynamic changes of vacuoles in living plant cells, we attempted to visualize the vacuolar membrane (VM) by pulse-labeling tobacco BY-2 cells with a styryl fluorescent dye, FM4-64. By time-sequence observations using confocal laser scanning microscopy (CLSM), we could follow the dynamics of vacuolar structures throughout the cell cycle in living higher plant cells. We also confirmed the dynamic changes of VM structures by the observation using transgenic BY-2 cells expressing GFP-AtVam3p fusion protein (BY-GV). Furthermore, by using transgenic BY-2 cells that stably express a GFP-tubulin fusion protein [BY-GT16, Kumagai et al. (2001) Plant Cell Physiol. 42: 723], we could study the relationship between the dynamics of vacuoles and microtubules. From these observations, we identified, for the first time, some remarkable events: (1) at the late G(2) phase, tubular structures of the vacuolar membrane developed in the central region of the cell, probably in the premitotic cytoplasmic band (phragmosome), surrounding the mitotic apparatus; (2) from anaphase to telophase, these tubular structures invaded the region of the phragmoplast within which the cell plate was being formed; (3) at the early G(1) phase, some of the tubular structures expanded rapidly between the cell plate and daughter nuclei, and subsequently developed into large vacuoles at interphase.     

Spitzenkörper, vacuoles, ring-like structures, and mitochondria of Phanerochaete velutina hyphal tips visualized with carboxy-DFFDA, CMAC and DiOC6(3)

Growth and organelle morphology in the wood rotting basidiomycete fungus Phanerochaete velutina were examined in Petri dishes, on agar-coated slides, and in submerged cultures, using DIC, fluorescence and four-dimensional (4-D; x,y,z,t) confocal microscopy, with several fluorescent probes. Phanerochaete is ideal for this work because of its fast growth, robustness, and use in a wide range of other studies. The probe carboxy-DFFDA, widely used for labelling vacuoles, has no effect either on hyphal tip extension or colony growth at the concentrations usually applied in labelling experiments. Carboxy-DFFDA labels the vacuoles and these form a tubular reticulum in hyphal tip cells. The probe also labels extremely small vesicles (punctate fluorescence) in the apex of tip cells, the Spitzenkörper, and short tubules that undergo sequences of characteristic movements and transformations to produce various morphologies, including ring-like structures. Their location and behaviour suggest that they are a distinct group of structures, possibly a subset of vacuoles, but as yet to be fully identified. Regular incursions of tubules extending from these structures and from the vacuolar reticulum into the apical dome indicate the potential for delivery of material to the apex via tubules as well as vesicles. Such structures are potential candidates for delivering chitin synthases to the apex. Spitzenkörper behaviour has been followed as hyphal tips with linear growth encounter obstacle hyphae and, as the hydrolysis product of carboxy-DFFDA only accumulates in membrane-enclosed compartments, it can be inferred that the labelled structures represent the Spitzenkörper vesicle cloud. Mitochondria also form a reticular continuum of branched tubules in growing hyphal tips, and dual localisation with DiOC₆(3) and CMAC allows this to be distinguished from the vacuolar reticulum. Like vacuolar tubules, mitochondrial tubules also span the septa, indicating that they may also be a conduit for intercellular transport.     

Vacuolar membrane dynamics in the filamentous fungus Aspergillus oryzae

Vacuoles in filamentous fungi are highly pleomorphic and some of them, e.g., tubular vacuoles, are implicated in intra- and intercellular transport. In this report, we isolated Aovam3, the homologue of the Saccharomyces cerevisiae VAM3 gene that encodes the vacuolar syntaxin, from Aspergillus oryzae. In yeast complementation analyses, the expression of Aovam3 restored the phenotypes of both Deltavam3 and Deltapep12 mutants, suggesting that AoVam3p is likely the vacuolar and/or endosomal syntaxin in A. oryzae. FM4-64 [N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl)pyridinium dibromide] and CMAC (7-amino-4-chloromethylcoumarin) staining confirmed that the fusion protein of enhanced green fluorescent protein (EGFP) with AoVam3p (EGFP-AoVam3p) localized on the membrane of the pleomorphic vacuolar networks, including large spherical vacuoles, tubular vacuoles, and putative late endosomes/prevacuolar compartments. EGFP-AoVam3p-expressing strains allowed us to observe the dynamics of vacuoles with high resolutions, and moreover, led to the discovery of several new aspects of fungal vacuoles, which have not been discovered so far with conventional staining methods, during different developmental stages. In old hyphae, EGFP fluorescence was present in the entire lumen of large vacuoles, which occupied most of the cell, indicating that degradation of cytosolic materials had occurred in such hyphae via an autophagic process. In hyphae that were not in contact with nutrients, such as aerial hyphae and hyphae that grew on a glass surface, vacuoles were composed of small punctate structures and tubular elements that often formed reticulum-like networks. These observations imply the presence of so-far-unrecognized roles of vacuoles in the development of filamentous fungi.     

Dispersed polyphosphate in fungal vacuoles in Eucalyptus pilularis/Pisolithus tinctorius ectomycorrhizas.

Ectomycorrhizas produced between Pisolithus tinctorius and Eucalyptus pilularis under axenic conditions were rapidly frozen, freeze-substituted in tetrahydrofuran and embedded anhydrously, and dry-sectioned for X-ray microanalysis. The vacuoles of the sheath and Hartig net hyphae were rich in phosphorus and potassium. They also contained sulfur and variable amounts of chlorine. In anhydrously processed freeze-substituted mycorrhizas, dispersed electron-opaque material filled the fungal vacuoles. X-ray maps indicated that P was distributed evenly throughout the entire vacuole profile and was not concentrated in spherical bodies or subregions of the vacuole. There were no electron-opaque granules surrounded by electron-lucent areas, such as are commonly seen in chemically fixed material. The fungal vacuoles were also rich in K, which similarly gave a signal from the entire vacuolar profile. Such P-rich vacuoles occurred in both the mycorrhizal sheath and Hartig net hyphae. Stained sections of ether-acrolein freeze-substituted mycorrhizas also showed only dispersed material in the fungal vacuoles as, in most cases, did acetone-osmium freeze-substituted material. Precipitation of metachromatic granules by ethanol suggested that large amounts of polyphosphate are stored in these regions under the conditions of our experiments, as well as in the tips of actively growing hyphae of the same fungus. The higher plant vacuoles of ectomycorrhizas gave a much lower signal for K, and P was barely detectable. Much more K was located in the vacuoles of the root exodermal cells than in epidermal cells. The analysis of element distribution between the vacuole and cytoplasm in root cells agrees well with that found for other plant species using other techniques. We conclude that polyphosphate is indeed present in the vacuoles of the fungal cells of these ectomycorrhizas, but that in vivo it is in a dispersed form, not in granules.     

Possible involvement of pleiomorphic vacuolar networks in nutrient recycling in filamentous fungi

Morphological analyses of vacuoles in filamentous fungi in the past decade have led to the remarkable finding that they are highly pleiomorphic organelles. Among them, tubular vacuoles have been implicated in nutrient transport between hyphal tips and the host plant surface in mycorrhizal fungi. However, a series of works suggested the presence of tubular vacuoles in other fungi that are not mycorrhizal, including Aspergillus oryzae, hinting at more general roles of the tubular vacuoles. Recently, we made two key observations by using the fusion protein of enhanced green fluorescent protein (EGFP) with a putative vacuolar t-SNARE in A. oryzae; tubular vacuoles formed more extensively in hyphae that were not in contact with nutrients, and vacuoles that were interconnected by tubules in the mature mycelial region displayed traces of microautophagy-mediated degradation of cytoplasm. The aim of this addendum is to discuss the possible involvement of vacuoles in degrading, transporting, and recycling nutrients from the mature mycelial region to hyphal tips, to support the continuous tip growth.     

Acidic vesicles in living hyphae of an arbuscular mycorrhizal fungus, Gigaspora margarita

Localization and movement of organelles in living hyphae of an arbuscular mycorrhizal fungus, Gigaspora margarita, were observed using a combination of fluorescent probes and laser-scanning confocal microscopy. Dense, evenly distributed acidic vesicles were visible in germ tubes and extraradical hyphae using DIC with the fluorescent acidotropic probe LysoTracker. These vesicles were distinct from both tubular vacuoles stained with DFFDA and lipid bodies stained with BODIPY 493/503 or Nile Red. Tubular vacuole bundles appeared to be influenced by the bidirectional cytoplasmic streaming of acidic vesicles and lipid bodies. Movement of the acidic vesicles occurred bidrectionally at different rates. The size and distribution of lipid bodies were variable. Based on our observations, the function of these organelles is discussed in relation to nutrient translocation in arbuscular mycorrhizas. Abbreviations: AM – arbuscular mycorrhiza; DAPI – 4′,6-diamidino-2-phenylindole; DIC – differential interference contrast; BODIPY 493/503 – 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene; DMSO – dimethyl sulfoxide; FITC – fluorescein isothiocynate; caboxy-DFFDA – Oregon Green 488 carboxylic acid diacetate.     

Possible Involvement of Pleiomorphic Vacuolar Networks in Nutrient Recycling in Filamentous Fungi

Morphological analyses of vacuoles in filamentous fungi in the past decade have led to the remarkable finding that they are highly pleiomorphic organelles. Among them, tubular vacuoles have been implicated in nutrient transport between hyphal tips and the host plant surface in mycorrhizal fungi. However, a series of works suggested the presence of tubular vacuoles in other fungi that are not mycorrhizal, including Aspergillus oryzae, hinting at more general roles of the tubular vacuoles. Recently, we made two key observations by using the fusion protein of enhanced green fluorescent protein (EGFP) with a putative vacuolar t-SNARE in A. oryzae; tubular vacuoles formed more extensively in hyphae that were not in contact with nutrients, and vacuoles that were interconnected by tubules in the mature mycelial region displayed traces of microautophagy-mediated degradation of cytoplasm. The aim of this addendum is to discuss the possible involvement of vacuoles in degrading, transporting, and recycling nutrients from the mature mycelial region to hyphal tips, to support the continuous tip growth.

Addendum to:

Vacuolar Membrane Dynamics in the Filamentous Fungus Aspergillus oryzae

J.Y. Shoji, M. Arioka and K. Kitamoto

Eukaryot Cell 2006; 5: 411-21     

Ni2+ induces changes in the morphology of vacuoles, mitochondria and microtubules in Paxillus involutus cells

Organelles of ectomycorrhizal fungi are known to respond to changes in the extracellular environment. The response of vacuoles, mitochondria and microtubules to short-term nickel (Ni2+) exposure were investigated in hyphal tip cells of a Paxillus involutus from a heavy metal-rich soil. Vacuoles, mitochondria and microtubules were labelled with Oregon Green 488 carboxylic acid diacetate, 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) and anti-alpha-tubulin antibodies, respectively; hyphae were treated with NiSO4 in the range of 0-1 mmol l(-1) and examined microscopically. Untreated hyphal tip cells contained tubular vacuole and mitochondrial networks. Ni2+ caused loss of organelle tubularity and severe microtubule disruption that were exposure-time and concentration dependent. Fine tubular vacuoles thickened and eventually became spherical in some hyphae, tubular mitochondria fragmented and microtubules shortened and aggregated into patches in most hyphae. Tubular vacuoles reformed on NiSO4 removal and tubular mitochondria in the presence of NiSO4 suggesting cellular detoxification. These results demonstrate that Ni2+ induces changes in organelle and microtubule morphology. Recovery of tubular organelles to pretreatment morphology after Ni2+ exposure suggests cellular detoxification of the metal ion.     

Motile tubular vacuoles in extramatrical mycelium and sheath hyphae of ectomycorrhizal systems

Summary Extramatrical mycelium and outer hyphae of the sheath ofEucalyptus pilularis-Pisolithus tinctorius mycorrhizas contain abundant motile tubular vacuoles which accumulate the carboxyfluorescein analogue Oregon Green 488 carboxylic acid. The fluorochrome accumulates in a system of small vacuoles, tubules, and larger vacuoles, which are interlinked, motile, and pleiomorphic, in external hyphae, cords, and hyphae of the outer sheath. There is often a difference in fluorescence between two neighbouring cells, indicating that the dolipore septum exercises control on the movement of material between cells. Generally the motile tubular vacuole system in mycorrhizas resembles that previously found in isolated mycelium. The majority of fungal cells in the sheath contain no fluorochrome even after long exposure of the mycorrhiza to the solution, but with differential interference optics the cells are clearly seen to be alive and to contain vacuoles resembling those in the outer hyphae. In translocation experiments, long-distance transport of the fluorochrome is slow and slight, or even nonexistent in some cases.Abbreviations carboxy-DFF Oregon Green 488 carboxylic acid - carboxy-DFFDA Oregon Green 488 carboxylic acid diacetate - DIC differential interference contrast Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

Laser confocal scanning microscopy of the surface membrane/T-tubular system and the sarcoplasmic reticulum in insect striated muscle stained with DilC18(3)

The structure of the surface membrane/transverse tubular (T-tubular) system and of the sarcoplasmic reticulum (SR) of the labial adductor muscle of the honey bee (Apis mellifera) was examined by laser confocal scanning microscopy, after staining with the fluorescent membrane probe DiIC18(3). The following components of the surface membrane/T-tubular system were visualized: transverse tubular networks that are located in the A-band close to the A-I junction and form dyads with the SR, longitudinal tubules that link the T-tubular networks within the between sarcomeres, and surface invaginations of larger diameter that contain tracheoles. The well developed SR forms a dense network of branching and anastomosing tubules in the A-band. A few tubular elements in the interfibrillar space in the I-band link the SR of adjacent sarcomeres. This study demonstrates the advantages of the laser confocal microscope and lipophilic fluorescent dyes for studying the 3-D structure of cellular membrane systems.     

Laser Confocal scanning microscopy of the surface membrane/T-tubular system and the sarcoplasmic reticulum in insect striated muscle stained with DiIC18(3)

The structure of the surface membrane/transverse tubular (T-tubular) system and of the sarcoplasmic reticular (SR) of the labial adductor muscle of the honey bee (Apis mellifera) was examined by laser confocal scanning microscopy, after staining with the fluorescent membrane probe DiIC₁₈(3). The following components of the surface membrane/T-tubular system were visualized: transverse tubular networks that are located in the A-band close to the A–I junction and form dyads with the SR, longitudinal tubules that link the T-tubular networks within and between sarcomeres, and surface invaginations of larger diameter that contain tracheoles. The well developed SR forms a dense network of branching and anastomosing tubules in the A-band. A few tubular elements in the interfibrillar space in the 1-band link the SR of adjacent sarcomeres. This study demonstrates the advantages of the laser confocal microscope and lipophilic fluorescent dyes for studying the 3-D structure of cellular membrane systems.     

The fluorescent probe HPTS as a phloem-mobile, symplastic tracer: an evaluation using confocal laser scanning microscopy

Confocal laser scanning microscopy (CLSM) has been used to evaluatethe use of the fluorescent probe HPTS (8-hydroxypyrene-1,3,6-trisulphonicacid) as a symplastic tracer. HPTS-acetate was used to loadHPTS into the phloem and its movement was followed in threesystems where symplastic unloading has been proposed. In Arabidopsisroot tips and Abutilon nectaries the intercellular distributionof HPTS differed markedly from that observed with 5-(and 6)-carboxyfluorescein(CF)- HPTS was observed in the nuclei and cytoplasm whilst CFwas rapidly transferred into the vacuoles. In contrast, bothHPTS and CF accumulated in the vacuoles of the vascular parenchymaand nucellus cells following unloading from the phloem of thedeveloping barley caryopsis. The results indicate that HPTShas a number of advantages as a symplastic probe compared withCF. The findings are discussed in relation to the influenceof vacuolar sequestration on dye distribution. Key words: Confocal laser scanning microscopy (CLSM), HPTS, intercellular transport, phloem (unloading)     

Vacuole development in cultured evacuolated oat mesophyll protoplasts

Oat leaf mesophyll protoplasts were evacuolated and shown to develop acidic vacuoles when cultured for 3 d. Vacuole development was followed by cell wall formation. Developing vacuoles, stained with acridine orange, took the form of a tubular network when viewed by confocal laser scanning microscopy. The tubules expanded and fused to form a series of interconnected vacuoles. When thin sectioned material was examined by transmission electron microscopy, the tubular network appeared as a number of small, expanding vesicles. The vacuolar H⁺-ATPase, H⁺-PPase and a membrane integral protein of 23 kDa (VM23) were shown, by Western blotting, to be removed from protoplasts following evacuolation. After 5 d culture the H⁺-ATPase and H⁺-PPase, but not VM23, were detectable in microsomal fractions.This study describes, for the first time, successful vacuole regeneration in a monocotyledenous plant. This regeneration follows a similar pattern to that seen in non-cereal protoplasts.     

Actin microfilaments regulate vacuolar structures and dynamics: dual observation of actin microfilaments and vacuolar membrane in living tobacco BY-2 Cells

Actin microfilaments (MFs) participate in many fundamental processes in plant growth and development. Here, we report the co-localization of the actin MF and vacuolar membrane (VM), as visualized by vital VM staining with FM4-64 in living tobacco BY-2 cells stably expressing green fluorescent protein (GFP)-fimbrin (BY-GF11). The MFs were intensively localized on the VM surface and at the periphery of the cytoplasmic strands rather than at their center. The co-localization of MFs and VMs was confirmed by the observation made using transient expression of red fluorescent protein (RFP)-fimbrin in tobacco BY-2 cells stably expressing GFP-AtVam3p (BY-GV7) and BY-2 cells stably expressing gamma-tonoplast intrinsic protein (gamma-TIP)-GFP fusion protein (BY-GG). Time-lapse imaging revealed dynamic movement of MF structures which was parallel to that of cytoplasmic strands. Disruption of MF structures disorganized cytoplasmic strand structures and produced small spherical vacuoles in the VM-accumulating region. Three-dimensional reconstructions of the vacuolar structures revealed a disconnection of these small spherical vacuoles from the large vacuoles. Real-time observations and quantitative image analyses demonstrated rapid movements of MFs and VMs near the cell cortex, which were inhibited by the general myosin ATPase inhibitor, 2,3-butanedion monoxime (BDM). Moreover, both bistheonellide A (BA) and BDM treatment inhibited the reorganization of the cytoplasmic strands and the migration of daughter cell nuclei at early G1 phase, suggesting a requirement for the acto-myosin system for vacuolar morphogenesis during cell cycle progression. These results suggest that MFs support the vacuolar structures and that the acto-myosin system plays an essential role in vacuolar morphogenesis.