Three-dimensional analysis of the senescence program in rice (Oryza sativa L.) coleoptiles

The cytological sequence of senescence-related changes in coleoptiles of rice (Oryza sativa L. cv. Nippon-bare) was studied using fluorescence and electron microscopy. The coleoptiles reach full size 3 d after sowing, then rapidly senesce and wither completely by day 7. The interveinal region in cross-sections taken 1 mm from the tip of the coleoptile was selected for this analysis. Fluorescence microscopy using samples embedded in Technovit 7100 resin, electron microscopy and immunoelectron microscopy using DNA-specific antibodies were used to elucidate the sequence of senescence-related events. These occur in the following order: (i) degradation of the chloroplast DNA (cpDNA); (ii) condensation of the nucleus in conjunction with a decrease in the size of the dense-chromatin region, shrinkage of the chloroplast, degradation of ribulose-1, 5-bisphosphate carboxylase/oxygenase, dilation of the thylakoid membranes, increase in size and number of osmiophilic globules, condensation of the cytoplasm; (iii) disorganization of the nucleus, degeneration of the tonoplast; (iv) complete loss of the cytoplasmic components, distortion of the cell wall, invasion of microorganisms into the intercellular spaces and ultimately into the cell itself. The mitochondria maintain their ultrastructural integrity and a constant level of mitochondrial DNA throughout senescence. In young mesophyll cells, invagination of the tonoplast into the vacuole frequently occurs. This occasionally includes cytoplasmic material, which is digested in the vacuole as senescence proceeds. Immunoelectron microscopy suggests that cpDNA degradation involves rough digestion first, rather than rapid, direct decomposition of the DNA into nucleotides. The fragmented cpDNA is then dispersed throughout the chloroplast and cytoplasm. Received: 9 April 1998 / Accepted: 11 June 1998     

DEVELOPMENT OF PERIPHERAL VACUOLES IN PLANT CELLS

The vacuolar apparatus of various plant cells consists of two distinct features: the large central vacuole and peripheral vacuoles which are derived from invaginations of the plasma membrane. Peripheral vacuoles are conspicuous structures in both living and fixed hair or filament cells of Tradescantia virginiana. They occur as spherical structures along the inner boundary of the peripheral cytoplasm and can be recognized as projections into the central vacuole. These structures are variable in size and number within a cell and can represent a significant proportion of the volume of the vacuole. Peripheral vacuoles most frequently are observed in motion with the streaming cytoplasm although their velocity is usually somewhat slower that that of the cytoplasmic organelles. Ultrastructural studies show two closely approximated membranes, one for each vacuole, in areas where a peripheral vacuole projects into the central vacuole. These are separated by an intermembrane zone continuous with the peripheral cytoplasm. The movement of organelles over the perimeter of the peripheral vacuole is presumed to occur along this intermembrane zone. The internal area of the peripheral vacuoles may appear empty although some contain a vesicular content of unknown origin and function.     

FINE STRUCTURE OF CAPITULAR FILAMENTS IN THE COENOCYTIC GREEN ALGA PENICILLUS1,2,3

Capitular filaments of Penicillus capitatus contain a large central vacuole. The parietal cytoplasm is densely packed, devoid of chloroplasts in the growing tip, and becomes convoluted and sponge-like as extensions of the vacuole penetrate the cytoplasm in mature portions of the filament. Structure of organelles and their distribution in the filament are described. The vacuole contains a variety of inclusions, such as membranous configurations, spherical bodies, electron dense bodies, and calcium oxalate monohydrate crystals, each of the latter surrounded by a chamber associated with microtubules. Endophytic bacteria are present throughout the vacuole and occasionally in the tip cytoplasm. Some vacuolar components of P. pyriformis are described for comparison.     

ULTRASTRUCTURE OF DEVELOPING AND MATURE NONARTICULATED LATICIFERS IN THE MILKWEED ASCLEPIAS SYRIACA L. (ASCLEPIADACEAE)

The ultrastructure of developing and mature nonarticulated laticifers in Asclepias syriaca L. (the common milkweed) was studied by conventional fixation and staining techniques and by osmium impregnation techniques. The mature laticifer protoplast in A. syriaca possesses a large central vacuole with an intact vacuolar membrane. Formation of this vacuole apparently results from dilation and subsequent enlargement of endoplasmic reticulum and possibly in part by fusion of smaller vacuoles and limited cellular-lytic autophagy. Widespread digestion or autophagy of cytoplasm within vacuoles is not evident. Nuclei, mitochondria, dictyosomes, and small vesicles are the most prominent components distributed in the peripheral cytoplasm. Plastids appear to degenerate as the laticifer matures. The specialized cellular component, latex, which is the vacuolar content of the laticifer, is interpreted to be produced in the cytoplasm and subsequently incorporated into the large central vacuole. Rubber globules, the most prominent latex component, are surrounded by a membrane that does not have a trilaminate structure. Globules are associated with an electron-dense fibrillar component in the vacuole.     

Development of endosperm in Arabidopsis thaliana

 The process of endosperm development in Arabidopsis was studied using immunohistochemistry of tubulin/microtubules coupled with light and confocal laser scanning microscopy. Arabidopsis undergoes the nuclear type of development in which the primary endosperm nucleus resulting from double fertilization divides repeatedly without cytokinesis resulting in a syncytium lining the central cell. Development occurs as waves originating in the micropylar chamber and moving through the central chamber toward the chalazal tip. Prior to cellularization, the syncytium is organized into nuclear cytoplasmic domains (NCDs) defined by nuclear-based radial systems of microtubules. The NCDs become polarized in axes perpendicular to the central cell wall, and anticlinal walls deposited among adjacent NCDs compartmentalize the syncytium into open-ended alveoli overtopped by a crown of syncytial cytoplasm. Continued centripetal growth of the anticlinal walls is guided by adventitious phragmoplasts that form at interfaces of microtubules emanating from adjacent interphase nuclei. Polarity of the elongating alveoli is reflected in a subsequent wave of periclinal divisions that cuts off a peripheral layer of cells and displaces the alveoli centripetally into the central vacuole. This pattern of development via alveolation appears to be highly conserved; it is characteristic of nuclear endosperm development in angiosperms and is similar to ancient patterns of gametophyte development in gymnosperms. Received: 21 September 1998 / Revision accepted: 17 November 1998     

The Contractile Vacuole of Amoeba proteus. III. Vacuolar Response to Phagocytosis1

The reaction of the contractile vacuole of Amoeba proteus to single and multiple phagocytosis under controlled conditions has been studied. Fluid intake into the cytoplasm from the phagosomes induces secretion by the contractile vacuole of equivalent excess volumes:. Vacuolar response is rapid (200 sec) and may be initiated by increases of protoplasmic hydration of as little as 1%. Cytoplasmic uptake of fluid from the phagosome can occur against an osmotic gradient; thus some form of active transport is implied.     

Immunohistochemical identification of the cytoskeletal elements in the notochord cells of bony fishes

The medulla of the unconstricted notochords of the shortnose sturgeon, Acipenser brevirostratus, and African lungfish, Protopterus annectens, and the cellular component of the intervertebral joint tissue of the teleost fish, Perca flavescens, are comprised of cells with a large central vacuole. Previous studies on the fine structure of this tissue revealed that the cytoplasm surrounding these vacuoles consists of 10-nm-diameter intermediate filaments. Since in mammals there are a large number of tissue-specific types of intermediate filaments, this study uses antibodies to mammalian intermediate filaments to determine the type of filaments present in the notochord cells of bony fishes. Positive labeling using a polyclonal antibody to human skin keratins is observed in the cytoplasm of the notochord cells in the intervertebral tissues of Perca. These tissues are also probed with the AE series antibodies that label keratins found in mammalian epithelial cells. In both Protopterus and Acipenser the peripheral cytoplasm of the notochord cells is labeled with all three AE antibodies. In Perca only the AE3 antibody probe produces positive staining. These staining patterns are consistent with previous studies on the localization of cytokeratins in fish tissues and indicate that the intermediate filaments in the notochord cells of bony fishes are immunologically similar to the mammalian keratins. J. Morphol. 236:105–116, 1998. © 1998 Wiley-Liss, Inc.     

Floral meristems as a source of enhanced yield and quality of DNA in grasses

Floral meristems of Lolium and Festuca grasses give a 5- to 19-fold enhancement in yield of extracted DNA in comparison with leaves. Meristems also provide highly pure DNA samples. The method could be useful for applications in molecular genetics in many species of the Gramineae. Received: 14 April 1998 / Revision received: 8 June 1998 / Accepted: 10 July 1998     

Desiccation and osmotic stress increase the abundance of mRNA of the tonoplast aquaporin BobTIP26-1 in cauliflower cells

Changes in vacuolar structure and the expression at the RNA level of a tonoplast aquaporin (BobTIP26-1) were examined in cauliflower (Brassicaoleracea L. var. botrytis) under water-stress conditions. Gradual drying out of slices of cauliflower floret tissue caused its collapse, with a shrinkage in tissue and cell volumes and an apparent vesiculation of the central vacuole, whereas osmotic stress resulted in plasmolysis with a collapse of the cytoplasm and the central vacuole within. Osmotic stress caused a rapid and substantial increase in BobTIP26 mRNA in slices of floret tissue. Exposure of tissue slices to a regime of desiccation showed a slower but equally large rise in BobTIP26 mRNA followed by a rapid decline upon rehydration. In situ hybridization showed that BobTIP26-2 mRNA is expressed most highly in meristematic and expanding cells of the cauliflower florets and that desiccation strongly increased the expression in those cells and in differentiated cells near the xylem vessels. These data indicate that under water-deficit conditions, expression of the tonoplast aquaporin gene in cauliflower is subject to a precise regulation that can be correlated with important cytological changes in the cells. Received: 21 October 1998 / Accepted: 10 February 1999     

Glucose repression and autolysis ofSaccharomyces cerevisiae cells: alterations in the cytochemical localization of acid phosphatase

Summary Allerations in the localization of acid phosphatase inSaccharomyces cerevisiae during glucose repression and during autolysis have been studied. Cell morphology becomes distinctly changed after only 2 h in the presence of high glucose concentration while after 3 h of glucose repression the majority of the mitochondirial structures resemble promitochondria. Yeast cells repressed for 6 h contain almost completely degraded mitochondrial structures and numerous lipid droplets in the central vacuole and cytoplasm. Destruction of mitochondria is accompanied by the accumulation of acid phosphatase in these organelles and in the cytoplasm whereas its activity in the central vacuole is lowered, most probably because of the leakage of the enzyme into the cytoplasm.No preferential breakdown of mitochondria is observed during autolysis. On the contrary, mitochondria are apparently the last to be degraded. Digestion of cytoplasmic regions and membranous elements occurs intravacuolarly after sequestration by protrusions of the central vacuole which are formed at the initial stages of autolysis. Acid phosphatase is not released from the central vacuole, suggesting indirectly that vacuole enzymes do not migrate into the cytoplasm during autolysis.     

Components of the endocytic and recycling trafficking pathways interfere with the integrity of the Legionella‐containing vacuole

Legionella pneumophila requires the Dot/Icm translocation system to replicate in a vacuolar compartment within host cells. Strains lacking the translocated substrate SdhA form a permeable vacuole during residence in the host cell, exposing bacteria to the host cytoplasm. In primary macrophages, mutants are defective for intracellular growth, with a pyroptotic cell death response mounted due to bacterial exposure to the cytosol. To understand how SdhA maintains vacuole integrity during intracellular growth, we performed high‐throughput RNAi screens against host membrane trafficking genes to identify factors that antagonise vacuole integrity in the absence of SdhA. Depletion of host proteins involved in endocytic uptake and recycling resulted in enhanced intracellular growth and lower levels of permeable vacuoles surrounding the ΔsdhA mutant. Of interest were three different Rab GTPases involved in these processes: Rab11b, Rab8b and Rab5 isoforms, that when depleted resulted in enhanced vacuole integrity surrounding the sdhA mutant. Proteins regulated by these Rabs are responsible for interfering with proper vacuole membrane maintenance, as depletion of the downstream effectors EEA1, Rab11FIP1, or VAMP3 rescued vacuole integrity and intracellular growth of the sdhA mutant. To test the model that specific vesicular components associated with these effectors could act to destabilise the replication vacuole, EEA1 and Rab11FIP1 showed increased density about the sdhA mutant vacuole compared with the wild type (WT) vacuole. Depletion of Rab5 isoforms or Rab11b reduced this aberrant redistribution. These findings are consistent with SdhA interfering with both endocytic and recycling membrane trafficking events that act to destabilise vacuole integrity during infection.     

Sodium fluxes in roots of Eleocharis uniglumis, a brackish water species

Abstract Fluxes of sodium across the plasmalemma and tonoplast of the roots of Eleocharis uniglumis have been measured using ²²Na. E. uniglumis (one glumed spike rush) was collected from an estuarine habitat where it was growing in a wide range of salinities (1 mM-50 mM Na). Compartmental analysis was used to determine sodium concentrations in the cytoplasm and the vacuole. Application of the Ussing-Teorell equation revealed the presence of sodium pumps in the plasmalemma and the tonoplast. Active sodium transport into the cytoplasm from the bathing medium was found to occur in most of the external sodium concentrations investigated. There also appeared to be active transport of sodium into the cytoplasm from the vacuole. In contrast to halophytes, high levels of sodium appeared to be accumulated in the cytoplasm of E. uniglumis roots.     

Compartmentation and fluxes of inorganic phosphate in photosynthetic cells

An analysis of the compartmentation and fluxes of inorganic phosphate in isolated cladophyll cells from Asparagus officinalis was made in parallel with an ultrastructural study. The elution pattern of labelled inorganic phosphate (which indicates that the asparagus cells are behaving as a system of three compartments in series) was used to quantify the fluxes between the vacuole, cytoplasm and free space. A relaxation time of 198 min was calculated for inorganic phosphate exchange between the vacuole and cytoplasm. It is, therefore, suggested that the vacuole serves to buffer the cytoplasmic inorganic phosphate concentration in the long term. However, in the short term, exchange with the vacuole will not appreciably affect the cytoplasmic inorganic phosphate concentration and thus the partitioning of photosynthetically fixed carbon.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - P_i inorganic phosphate     

Ultrastructural Study of an Endosymbiotic Alga and Its Host Ciliate Stentor niger

Stentor niger collected in the suburbs of Hiroshima contained in its cytoplasm several hundreds of endosymbiotic algae and innumerable brownish pigment granules. The body of the ciliate was dark due to a mixture of the green endosymbiotic algae and brown pigment granules. The algae belonged to the genus Chlorella; each was enclosed in a perialgal vacuole and dispersed uniformly in the host cytoplasm from the myoneme layer inward to the center of the ciliate. The cell wall and plasma membrane of the alga enclosed a nucleus, chloroplast, mitochondrion, Golgi complex, accumulation bodies, myelinated vesicles, and many ribosomes. The chloroplast occupied more than half of the volume of the alga and contained a conspicuous pyrenoid. Algal multiplication occurred by two successive divisions of an alga, leading to four autospores within a perialgal vacuole; the walls of the vacuole invaginated to separate the autospores each into its own vacuole. Three types of pigment granules were scattered uniformly throughout the cytoplasm of the ciliate. The ultrastructure of the membranellar region, somatic cortex, and macro- and micronucleus of the ciliate are also described.     

Uptake and compartmentalisation of fluorescent probes byPisolithus tinctorius hyphae: evidence for an anion transport mechanism at the tonoplast but not for fluid-phase endocytosis

Summary Membrane-impermeant fluorescent probes, such as Lucifer Yellow carbohydrazide, 6-carboxyfluorescein, and high-molecular-mass fluorescent dextrans (10 and 70 kDa) are not internalised by actively-growing hyphal tip-cells ofPisolithus tinctorius even after prolonged exposure to the probe. These findings suggest that fluid-phase endocytosis may not occur in these fully turgid tip-growing hyphae. In contrast, a number of membrane-permeant fluorescent probes, including 6-carboxfluorescein diacetate, the novel fluorescein-substitute Oregon Green 488 carboxylic acid diacetate, and the thiol-reactive Cell Tracker reagents 7-amino-4-chloro-methylcoumarin and 5-chloromethylfluorescein diacetate, are taken up by these hyphae and their fluorescent products accumulate in the vacuole system. Accumulation of the fluorescent products of both 6-carboxyfluorescein diacetate and Oregon Green 488 carboxylic acid diacetate in the vacuole system is inhibited by the anion transport inhibitor probenecid and instead these fluorochromes remain in the cytoplasm. These results suggest that the membrane-permeant esters 6-carboxyfluorescein diacetate and Oregon Green 488 carboxylic acid diacetate are first hydrolysed in the cytoplasm and that their fluorescent products are subsequently sequestered across the tonoplast via an anion transport mechanism. Such an anion transport mechanism has been hitherto unrecognised in fungi and may serve to detoxify the fungal cytoplasm by the removal of naturally-occurring unwanted anions. Probenecid-inhibitable organic anion transporters are also located at the limiting membrane of the animal endosomal/lysosomal system and at the tonoplast of higher plants. Our results further support the idea that the tubular vacuole system inP. tinctorius is similar to animal endosomal/lysosomal and plant vacuole systems.     

Nuclear centering in Spirogyra: force integration by microfilaments along microtubules

The contribution of microtubules and microfilaments to the cytomechanics of transverse nuclear centering were investigated in the charophycean green alga Spirogyracrassa (Zygnematales). Cytoplasmic strands of enhanced rigidity and fasciate appearance radiate from the rim of the lenticular nucleus through the vacuole, frequently split once or twice and are attached to the helical chloroplast bands in the peripheral cytoplasm. The nucleus is encased in tubulin and a web of F-actin. Bundles of microtubules, emerging from the nuclear rim, are organized into dividing fascicles within the strands and reach to the inner surface of the chloroplast envelope. Organelles are translocated in both directions along similarly arranged fascicles of microfilament bundles which extend from the nucleus to the peripheral actin cytoskeleton. Application of microtubule- and/or microfilament-depolymerizing drugs affected the position of the nucleus only slowly, but in distinct ways. The differential effects suggest that nuclear centering depends on the tensional integrity of the perinuclear scaffold, with microfilaments conveying tension along stabilized microtubules and the actin cytoskeleton integrating the translocation forces generated within the scaffold. Received: 9 December 1996 / Accepted: 29 April 1997     

CYTOPLASMIC ORGANIZATION AND RHYTHMIC STREAMING IN GROWING BLADES OF CAULERPA PROLIFERA

Light- and electron-microscopic studies of the growing blades and their meristematic tips in Caulerpa prolifera have been correlated with time-lapse photographic studies. The growing blade may be divided into three zones based on the level of maturation. A “meristemplasm” is present at the tip of the growing blade and at sites of wounding. The cytoplasm of these growing regions has an abundant endoplasmic reticulum, Golgi bodies, amyloplasts, and nuclei, but lacks chloroplasts and a central vacuole. An intermediate zone lies between the white meristemplasm and the green, mature basal zone. The basal zone contains a parietal cytoplasm with organelles typical of the Chlorophyta and a dominant central vacuole. Two distinct systems of cytoplasmic streams occur in the basal zone and both contain packets of microtubules orientated parallel to the axis of the streams. As the blade matures and growth ceases, the dominant central vacuole forms up to the tip. In developing blades the tips become white in the absence of light as a result of basipetal movement of the chloroplasts. In plants kept on a 12-hr L: 12-hr D cycle, blade growth and chloroplast migration at the blade tip are rhythmic, and the peak occurs about 2-4 hr after initiation of the dark phase. Experiments are reported using continuous light or darkness after three 24-hr periods of 12-hr L: 12-hr D phasing.     

Symbiotic Chlorella sp. of the ciliate Paramecium bursaria do not prevent acidification and lysosomal fusion of host digestive vacuoles during infection

Summary. Each symbiotic Chlorella sp. of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole derived from the host digestive vacuole, and thereby the alga is protected from digestion by lysosomal fusion. Algae-free cells can be reinfected with algae isolated from algae-bearing cells by ingestion into digestive vacuoles. To examine the timing of acidification and lysosomal fusion of the digestive vacuoles and of algal escape from the digestive vacuole, algae-free cells were mixed with isolated algae or yeast cells stained with pH indicator dyes at 25 ± 1 °C for 1.5 min, washed, chased, and fixed at various time points. Acidification of the vacuoles and digestion of Chlorella sp. began at 0.5 and 2 min after mixing, respectively. All single green Chlorella sp. that had been present in the host cytoplasm before 0.5 h after mixing were digested by 0.5 h. At 1 h after mixing, however, single green algae reappeared in the host cytoplasm, arising from those digestive vacuoles containing both nondigested and partially digested algae, and the percentage of such cells increased to about 40% at 3 h. At 48 h, the single green algae began to multiply by cell division, indicating that these algae had succeeded in establishing endosymbiosis. In contrast to previously published studies, our data show that an alga can successfully escape from the host’s digestive vacuole after acidosomal and lysosomal fusion with the vacuole has occurred, in order to produce endosymbiosis. Correspondence and reprints: Biological Institute, Faculty of Science, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753-8512, Japan.     

Phosphate uptake and polyphosphate metabolism of mycorrhizal and nonmycorrhizal roots of pine and of Suillus bovinus at varying external pH measured by in vivo 31P-NMR

 Comparative in vivo ³¹P-NMR analyses of mycorrhizal and nonmycorrhizal roots of Pinus sylvestris and the fungus of Suillus bovinus in pure culture were used to investigate alterations in phosphate metabolism due to changes in external pH in the range 3.5–8.5. All control samples maintained a constant pH in both cytoplasm and vacuole. Mycorrhizal roots and pure fungus, but not nonmycorrhizal roots, transformed accumulated inorganic phosphate into mobile polyphosphate with a medium chain length. Phosphate uptake rates and polyphosphate accumulation responded differently to external pH. In all cases, maximal phosphate uptake occurred at an external pH close to 5.5. At an external pH of 8.5, both roots and fungus showed a distinct lag in phosphate uptake, which was abolished when the external pH was lowered to 7.5. An irreversible effect on phosphate uptake as a consequence of variation in external pH was also observed. The central role of the fungus in regulating mycorrhizal phosphate metabolism is discussed. Accepted: 15 April 1997