Timing of perialgal vacuole membrane differentiation from digestive vacuole membrane in infection of symbiotic algae Chlorella vulgaris of the ciliate Paramecium bursaria

Each symbiotic Chlorella of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole derived from the host digestive vacuole to protect from lysosomal fusion. To understand the timing of differentiation of the perialgal vacuole from the host digestive vacuole, algae-free P. bursaria cells were fed symbiotic C. vulgaris cells for 1.5min, washed, chased and fixed at various times after mixing. Acid phosphatase activity in the vacuoles enclosing the algae was detected by Gomori's staining. This activity appeared in 3-min-old vacuoles, and all algae-containing vacuoles demonstrated activity at 30min. Algal escape from these digestive vacuoles began at 30min by budding of the digestive vacuole membrane into the cytoplasm. In the budded membrane, each alga was surrounded by a Gomori's thin positive staining layer. The vacuoles containing a single algal cell moved quickly to and attached just beneath the host cell surface. Such vacuoles were Gomori's staining negative, indicating that the perialgal vacuole membrane differentiates soon after the algal escape from the host digestive vacuole. This is the first report demonstrating the timing of differentiation of the perialgal vacuole membrane during infection of P. bursaria with symbiotic Chlorella.     

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.     

Endosymbiosis of Chlorella species to the ciliate Paramecium bursaria alters the distribution of the host’s trichocysts beneath the host cell cortex

Each symbiotic Chlorella of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole membrane derived from the host digestive vacuole membrane. Alga-free paramecia and symbiotic algae can grow independently. Mixing them experimentally can cause reinfection. Earlier, we reported that the symbiotic algae appear to push the host trichocysts aside to become fixed beneath the host cell cortex during the algal reinfection process. Indirect immunofluorescence microscopy with a monoclonal antibody against the trichocysts demonstrates that the trichocysts change their locality to form algal attachment sites and decrease their density beneath the host cell cortex through algal reinfection. Transmission electron microscopy to detect acid phosphatase activity showed that some trichocysts near the host cell cortex are digested by the host lysosomal fusion during algal reinfection. Removal of algae from the host cell using cycloheximide recovers the trichocyst's arrangement and number near the host cell cortex. These results indicate that symbiotic algae compete for their attachment sites with preexisting trichocysts and that the algae have the ability to ensure algal attachment sites beneath the host cell cortex.     

Genomic reduction and evolution of novel genetic membranes and protein-targeting machinery in eukaryote-eukaryote chimaeras (meta-algae)

Chloroplasts originated just once, from cyanobacteria enslaved by a biciliate protozoan to form the plant kingdom (green plants, red and glaucophyte algae), but subsequently, were laterally transferred to other lineages to form eukaryote-eukaryote chimaeras or meta-algae. This process of secondary symbiogenesis (permanent merger of two phylogenetically distinct eukaryote cells) has left remarkable traces of its evolutionary role in the more complex topology of the membranes surrounding all non-plant (meta-algal) chloroplasts. It took place twice, soon after green and red algae diverged over 550 Myr ago to form two independent major branches of the eukaryotic tree (chromalveolates and cabozoa), comprising both meta-algae and numerous secondarily non-photosynthetic lineages. In both cases, enslavement probably began by evolving a novel targeting of endomembrane vesicles to the perialgal vacuole to implant host porter proteins for extracting photosynthate. Chromalveolates arose by such enslavement of a unicellular red alga and evolution of chlorophyll c to form the kingdom Chromista and protozoan infrakingdom Alveolata, which diverged from the ancestral chromalveolate chimaera. Cabozoa arose when the common ancestor of euglenoids and cercozoan chlorarachnean algae enslaved a tetraphyte green alga with chlorophyll a and b. I suggest that in cabozoa the endomembrane vesicles originally budded from the Golgi, whereas in chromalveolates they budded from the endoplasmic reticulum (ER) independently of Golgi-targeted vesicles, presenting a potentially novel target for drugs against alveolate Sporozoa such as malaria parasites and Toxoplasma. These hypothetical ER-derived vesicles mediated fusion of the perialgal vacuole and rough ER (RER) in the ancestral chromist, placing the former red alga within the RER lumen. Subsequently, this chimaera diverged to form cryptomonads, which retained the red algal nucleus as a nucleomorph (NM) with approximately 464 protein-coding genes (30 encoding plastid proteins) and a red or blue phycobiliprotein antenna pigment, and the chromobiotes (heterokonts and haptophytes), which lost phycobilins and evolved the brown carotenoid fucoxanthin that colours brown seaweeds, diatoms and haptophytes. Chromobiotes transferred the 30 genes to the nucleus and lost the NM genome and nuclear-pore complexes, but retained its membrane as the periplastid reticulum (PPR), putatively the phospholipid factory of the periplastid space (former algal cytoplasm), as did the ancestral alveolate independently. The chlorarachnean NM has three minute chromosomes bearing approximately 300 genes riddled with pygmy introns. I propose that the periplastid membrane (PPM, the former algal plasma membrane) of chromalveolates, and possibly chlorarachneans, grows by fusion of vesicles emanating from the NM envelope or PPR. Dinoflagellates and euglenoids independently lost the PPM and PPR (after diverging from Sporozoa and chlorarachneans, respectively) and evolved triple chloroplast envelopes comprising the original plant double envelope and an extra outermost membrane, the EM, derived from the perialgal vacuole. In all metaalgae most chloroplast proteins are coded by nuclear genes and enter the chloroplast by using bipartite targeting sequences--an upstream signal sequence for entering the ER and a downstream chloroplast transit sequence. I present a new theory for the four-fold diversification of the chloroplast OM protein translocon following its insertion into the PPM to facilitate protein translocation across it (of both periplastid and plastid proteins). I discuss evidence from genome sequencing and other sources on the contrasting modes of protein targeting, cellular integration, and evolution of these two major lineages of eukaryote "cells within cells". They also provide powerful evidence for natural selection's effectiveness in eliminating most functionless DNA and therefore of a universally useful non-genic function for nuclear non-coding DNA, i.e. most DNA in the biosphere, and dramatic examples of genomic reduction. I briefly argue that chloroplast replacement in dinoflagellates, which happened at least twice, may have been evolutionarily easier than secondary symbiogenesis because parts of the chromalveolate protein-targeting machinery could have helped enslave the foreign plastids.     

Infection of algae-free Climacostomum virens with symbiotic Chlorella sp. isolated from algae-containing C. virens

The ciliate Climacostomum virens normally contains algae as symbionts in its cytoplasm and retains them over many generations. An aposymbiotic strain of C. virens which cannot re-establish a new symbiotic association by ingestion of algae derived from green Climacostomum was recently isolated in our laboratory. Results of infection experiments showed that all newly ingested, potentially symbiotic algae were digested in food vacuoles. To clarify whether these ciliates have completely lost their ability to sustain symbiosis with algae or whether this ability can eventually be re-established, infection experiments were performed using a microinjection technique. We have achieved successful infection of algae-free Climacostomum using this method. The endosymbiont population was established in ciliates from as few as 3-5 injected algae, which have retained an intact perialgal vacuole membrane around them. To our knowledge, this is the first evidence of successful infection of aposymbiotic ciliates with algae by microinjection.     

Electron Microscopic Observations on the Symbiosis of Paramecium bursaria and its Intracellular Algae*

SYNOPSIS. Observations were made on the fine structure of Paramecium bursaria and its intracellular Chlorella symbionts. Emphasis was placed on the structure of the algae and structural aspects of the relationship between the organisms. The algae are surrounded by a prominent cell wall and contain a cup-shaped chloroplast which lies just beneath the plasma membrane. Within the cavity formed by the chloroplast are a large nucleus, a mitochondrion, one or more dictyosomes, and numerous ribosomes. The chloroplast itself is made up of a series of lamellar stacks each containing 2–6 or more thylakoids with a granular stroma and starch grains intercalated between the stacks. The thylakoid stacks of mature algae are frequently more compact than those of recently divided algae. A large pyrenoid is located within the base of the chloroplast. It is made up of a granular or fibrillar matrix surrounded by a shell of starch. The matrix is bisected by a stack of 2 thylakoids. Prior to the division of the chloroplast the pyrenoid regresses; pyrenoids subsequently form in the daughter chloroplasts thru condensation of the matrix material and the reappearance of a starch shell. This shell appears to be formed by the hollowing-out of starch grains already present in the chloroplast stroma. Accordingly, in this case, starch moves from the stroma to the pyrenoid. The algae are located thruout the peripheral cytoplasm of the Paramecium. Each alga is located in an individual vacuole except immediately following division of the algae when the daughter cells are temporarily located in the vacuole which harbored the parental cell. Shortly thereafter the vacuole membrane invaginates, thereby isolating the daughter algae into individual vacuoles. Degenerating symbiotic algae are seen; because these are frequently found in vacuoles with bacteria, they are presumed to be undergoing digestion. Due to the conditions of culture these algae could have been either of intracellular or extracellular origin.     

Brandtia ciliaticola gen. et sp. nov. (Chlorellaceae,Trebouxiophyceae) a common symbiotic green coccoid of various ciliate species

Many freshwater protists harbor unicellular green algae within their cells and these host‐symbiont relationships slowly are becoming better understood. Recently, we reported that several ciliate species shared a single species of symbiotic algae. Nonetheless, the algae from different host ciliates were each distinguishable by their different genotypes, and these host‐algal genotype combinations remained unchanged throughout a 15‐month period of sampling from natural populations. The same algal species had been reported as the shared symbiont of several ciliates from a remote lake. Consequently, this alga appears to play a key role in ciliate‐algae symbioses. In the present study, we successfully isolated the algae from ciliate cells and established unialgal cultures. This species is herein named Brandtia ciliaticola gen. et sp. nov. and has typical ‘Chlorella‐like’ morphology, being a spherical autosporic coccoid with a single chloroplast containing a pyrenoid. The alga belongs to the Chlorella‐clade in Chlorellaceae (Trebouxiophyceae), but it is not strongly connected to any of the other genera in this group. In addition to this phylogenetic distinctiveness, a unique compensatory base change in the SSU rRNA gene is decisive in distinguishing this genus. Sequences of SSU‐ITS (internal transcribed spacer) rDNA for each isolate were compared to those obtained previously from the same host ciliate. Consistent algal genotypes were recovered from each host, which strongly suggests that B. ciliaticola has established a persistent symbiosis in each ciliate species.     

Cycloheximide induces synchronous swelling of perialgal vacuoles enclosing symbiotic Chlorella vulgaris and digestion of the algae in the ciliate Paramecium bursaria

Cycloheximide is known to inhibit preferentially protein synthesis of symbiotic Chlorella of the ciliate Paramecium bursaria, but to hardly host protein synthesis. Treatment of algae-bearing Paramecium cells with cycloheximide induces synchronous swelling of all perialgal vacuoles that are localized immediately beneath the host's cell membrane. In this study, the space between the symbiotic algal cell wall and the perialgal vacuole membrane widened to about 25 times its normal width 24 h after treatment with cycloheximide. Then, the vacuoles detached from beneath the host's cell membrane, were condensed and stained with Gomori's solution, and the algae in the vacuoles were digested. Although this phenomenon is induced only under a fluorescent light condition, and not under a constant dark condition, this phenomenon was not induced in paramecia treated with cycloheximide in the light in the presence of the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. These results indicate that algal proteins synthesized in the presence of algal photosynthesis serve some important function to prevent expansion of the perialgal vacuole and to maintain the ability of the perialgal vacuole membrane to protect itself from host lysosomal fusion.     

Symbiotic Chlorella vulgaris of the ciliate Paramecium bursaria plays an important role in maintaining perialgal vacuole membrane functions

Treatment of symbiotic alga-bearing Paramecium bursaria cells with a protein synthesis inhibitor, cycloheximide, induces synchronous swelling of all perialgal vacuoles at about 24h after treatment under a constant light condition. Subsequently, the vacuoles detach from the host cell cortex. The algae in the vacuoles are digested by the host's lysosomal fusion to the vacuoles. To elucidate the timing of algal degeneration, P. bursaria cells were treated with cycloheximide under a constant light condition. Then the cells were observed using transmission electron microscopy. Results show that algal chloroplasts and nuclei degenerated within 9h after treatment, but before the synchronous swelling of the perialgal vacuole and appearance of acid phosphatase activity in the perialgal vacuole by lysosomal fusion. Treatment with cycloheximide under a constant dark condition and treatment with chloramphenicol under a constant light condition induced neither synchronous swelling of the vacuoles nor digestion of the algae inside the vacuoles. These results demonstrate that algal proteins synthesized during photosynthesis are necessary to maintain chloroplastic and nuclear structures, and that inhibition of protein synthesis induces rapid lysis of these organelles, after which synchronous swelling of the perialgal vacuole and fusion occur with the host lysosomes.     

Expression of the nucleus-encoded chloroplast division genes and proteins regulated by the algal cell cycle

Chloroplasts have evolved from a cyanobacterial endosymbiont and their continuity has been maintained by chloroplast division, which is performed by the constriction of a ring-like division complex at the division site. It is believed that the synchronization of the endosymbiotic and host cell division events was a critical step in establishing a permanent endosymbiotic relationship, such as is commonly seen in existing algae. In the majority of algal species, chloroplasts divide once per specific period of the host cell division cycle. In order to understand both the regulation of the timing of chloroplast division in algal cells and how the system evolved, we examined the expression of chloroplast division genes and proteins in the cell cycle of algae containing chloroplasts of cyanobacterial primary endosymbiotic origin (glaucophyte, red, green, and streptophyte algae). The results show that the nucleus-encoded chloroplast division genes and proteins of both cyanobacterial and eukaryotic host origin are expressed specifically during the S phase, except for FtsZ in one graucophyte alga. In this glaucophyte alga, FtsZ is persistently expressed throughout the cell cycle, whereas the expression of the nucleus-encoded MinD and MinE as well as FtsZ ring formation are regulated by the phases of the cell cycle. In contrast to the nucleus-encoded division genes, it has been shown that the expression of chloroplast-encoded division genes is not regulated by the host cell cycle. The endosymbiotic gene transfer of minE and minD from the chloroplast to the nuclear genome occurred independently on multiple occasions in distinct lineages, whereas the expression of nucleus-encoded MIND and MINE is regulated by the cell cycle in all lineages examined in this study. These results suggest that the timing of chloroplast division in algal cell cycle is restricted by the cell cycle-regulated expression of some but not all of the chloroplast division genes. In addition, it is suggested that the regulation of each division-related gene was established shortly after the endosymbiotic gene transfer, and this event occurred multiple times independently in distinct genes and in distinct lineages.     

Cytological,genetic, and biochemical characteristics of an unusual non‐Chlorella photobiont of Stentor polymorphus collected from an artificial pond close to the shore of Lake Biwa,Japan

Stentor polymorphus (Müller) Ehrenberg (Heterotrichea, Ciliophora) is a trumpet‐form ciliate with endosymbiotic green algae (photobionts). Previous reports have indicated that this photobiont has a form typical of Chlorella Beijerinck (Trebouxiophyceae), with one chloroplast and a distinct pyrenoid. We collected S. polymorphus from an artificial pond on the shore of Lake Biwa in Japan. Microscopic examination demonstrated that there was no pyrenoid in the photobiont, and thus we analyzed the cytological, genetic, and biochemical characteristics of these algal cells. Cultured photobionts were smaller than those in hospite. Phylogenetic analyses placed the photobiont within the genus Mychonastes Simpson and Van Valkenburg, a chlorophycean alga occurring in fresh/brackish waters that lacks pyrenoids. Mychonastes provided little photosynthate to its host; the quantity of carbohydrate amounted to only 2% of that provided to Paramecium bursaria (Ehrenberg) Focker by its photobiont.     

基于核及叶绿体基因序列探讨中国绿水螅共生单细胞绿藻系统发生地位

研究对中国绿水螅共生绿藻的核18S rRNA基因全长序列及其叶绿体9个基因(atpA、chlB、chlN、petA、psaB、psbA、psbC、psbD及rbcL)片段序列进行了克隆和测序, 并基于18S rRNA基因序列及叶绿体9个基因序列的整合数据分别通过最大似然法(Maximum-likelihood)和贝叶斯分析(Bayesian inference)对中国绿水螅(Hydra sinensis)共生单细胞绿藻的系统发生地位进行了探讨。系统发生表明: (1)中国绿水螅共生绿藻属于共球藻纲(Trebouxiophyceae)小球藻目(Chlorellales), 但不属于其中的小球藻属(Chlorella); (2)来源于草履虫、水螅、地衣及银杏的共生绿藻均在共球藻纲支系, 而来源于蛙类和蝾螈的共生绿藻属于绿藻纲(Chlorophyceae)支系。无论在共球藻纲支系还是在绿藻纲支系, 不同来源的共生藻并没有排他性地聚为单系群而在系统树中与其他自由生活的绿藻混杂排列, 来自不同宿主的共生绿藻没有共同起源。     

The endosymbiotic unit ofClimacostomum virens andChlorella sp. I. Morphological and physiological studies on the algal partner and its localization in the host cell

Summary The ciliateClimacostomum virens forms an endosymbiotic association with coccoid chlorophycean algae which can be isolated and grown as sterile mass cultures with an inorganic medium. According to both morphological and physiological properties, the algae probably belong to the genusChlorella and have some features in common with symbiotic chlorellae isolated from the ciliatesParamecium bursaria andStentor polymorphus. These and other endosymbiotic chlorellae studied so far excrete maltose or glucose, but algae fromClimacostomum virens show a different excretion pattern by releasing glucose, fructose and xylose. Possible biosynthetic pathways are discussed. Algae inClimacostomum virens are either located individually in special perialgal vacuoles where they are probably protected against attack by host lytic enzymes or to a lesser extent in food vacuoles in different states of digestion. The endosymbiotic character of theClimacostomum virens-Chlorella sp.-association is discussed.Dedicated to Prof. H. A.von Stosch on the occasion of his 75th birthday.     

Physiological changes of a green alga (Micractinium sp.) involved in an early-stage of association with Tetrahymena thermophila during 5-year microcosm culture

Endosymbioses between phototrophic algae and heterotrophic organisms are an important symbiotic association in that this association connects photo- and heterotrophic metabolism, and therefore, affects energy/matter pathways and cycling in the ecosystem. However, little is known about the early processes of evolution of an endosymbiotic association between previously non-associated organisms. In previous studies, we analyzed an early process of the evolution of an endosymbiotic association between an alga and a ciliate by using a long-term culture of an experimental model ecosystem (CET microcosm) composed of a green alga (Micractinium sp.), a bacterium (Escherichia coli), and a ciliate (Tetrahymena thermophila). The results revealed that an algal type, isolated from 5-year cultures of the microcosm, prolonged the longevity of the ancestral and derived clones of T. thermophila in the absence of bacteria, suggesting that a cooperative algal phenotype that benefited the ciliate had evolved in the microcosm. Here, we investigated the physiological changes of the derived Micractinium clones that benefited Tetrahymena, focusing on the release of carbohydrates by and abundance of photopigments in the ancestral and 2 derived algal clones (SC10-2 and SC9-1) isolated from inside Tetrahymena cells. Analyses using HPLC revealed that the algal isolates released glycerol and sucrose at higher concentrations per cell and also contained higher levels of photopigments per cell at pH 7.2, in comparison with the ancestral strain. These phenotypic characters were considered responsible for the increased longevity of Tetrahymena cells, and thus supported the cooperator alga hypothesis.     

Arrest of cytoplasmic streaming induces algal proliferation in green paramecia

A green ciliate Paramecium bursaria, bearing several hundreds of endosymbiotic algae, demonstrates rotational microtubule-based cytoplasmic streaming, in which cytoplasmic granules and endosymbiotic algae flow in a constant direction. However, its physiological significance is still unknown. We investigated physiological roles of cytoplasmic streaming in P. bursaria through host cell cycle using video-microscopy. Here, we found that cytoplasmic streaming was arrested in dividing green paramecia and the endosymbiotic algae proliferated only during the arrest of cytoplasmic streaming. Interestingly, arrest of cytoplasmic streaming with pressure or a microtubule drug also induced proliferation of endosymbiotic algae independently of host cell cycle. Thus, cytoplasmic streaming may control the algal proliferation in P. bursaria. Furthermore, confocal microscopic observation revealed that a division septum was formed in the constricted area of a dividing paramecium, producing arrest of cytoplasmic streaming. This is a first report to suggest that cytoplasmic streaming controls proliferation of eukaryotic cells.     

Evolutionary History of the Enzymes Involved in the Calvin–Benson Cycle in Euglenids

Euglenids are an ancient lineage that may have existed as early as 2 billion years ago. A mere 65 years ago, Melvin Calvin and Andrew A. Benson performed experiments on Euglena gracilis and elucidated the series of reactions by which carbon was fixed and reduced during photosynthesis. However, the evolutionary history of this pathway (Calvin–Benson cycle) in euglenids was more complex than Calvin and Benson could have imagined. The chloroplast present today in euglenophytes arose from a secondary endosymbiosis between a phagotrophic euglenid and a prasinophyte green alga. A long period of evolutionary time existed before this secondary endosymbiotic event took place, which allowed for other endosymbiotic events or gene transfers to occur prior to the establishment of the green chloroplast. This research revealed the evolutionary history of the major enzymes of the Calvin–Benson cycle throughout the euglenid lineage and showed that the majority of genes for Calvin–Benson cycle enzymes shared an ancestry with red algae and/or chromophytes suggesting they may have been transferred to the nucleus prior to the acquisition of the green chloroplast.     

Aurearenophyceae classis nova, a new class of Heterokontophyta based on a new marine unicellular alga Aurearena cruciata gen. et sp. nov. inhabiting sandy beaches

A new heterokontophyte alga, Aurearena cruciata gen. et sp. nov., was isolated from sandy beaches in Japan. Isolates were characterized by light and electron microscopy, spectroscopy of pigment composition, and molecular phylogenetic analyses using 18S rDNA and rbcL. The alga usually possessed a cell wall but also retained two heterokont flagella beneath the cell wall. Each walled cell first produced only a single flagellate cell that subsequently divided into two flagellate cells. Electron-opaque vesicles, possibly associated with cell wall formation, were observed beneath the cell membrane. The chloroplast consisted of two compartments, each enclosed by a chloroplast envelope and the inner membrane of the chloroplast endoplasmic reticulum; these two compartments were surrounded by a common outer membrane of chloroplast endoplasmic reticulum. Molecular phylogenetic trees suggested that this alga was a new and independent member of the clade that included the Phaeophyceae and Xanthophyceae (PX clade). A new class, Aurearenophyceae classis nova was proposed for A. cruciata.     

PHOSPHORUS STATUS AND CYTOPLASMIC STRUCTURES IN SCENEDESMUS (CHLOROPHYCEAE) UNDER DIFFERENT METABOLIC REGIMES1

Phosphorus deficiency affects the anatomy of the unicellular green alga Scenedesmus obtusiusculus Chad. and influences the distribution of other inorganic elements in the cell in addition to phosphorus. Scenedesmus was grown under standard conditions with or without phosphorus. Cells were then cultured with phosphorus under conditions favouring glycolysis, respiration, or photophosphorylation for 2 h or photosynthesis for up to 8 h. The dominating features of phosphorus starvation, were loss of phosphorus and coions from polyphosphate bodies, accumulation of starch, decrease in the volume density of ribosomes both in the chloroplast and cytoplasm, and an increase in wall thickness. Under conditions favoring photosynthesis the mass fraction for phosphorus is low after 1 h, exceptionally high by 2 h, and diminishes by 8 h. High amounts of phosphorus are also regained under conditions favoring glycolysis and photophosphorylation but not respiration. After 2 h under photosynthetic conditions the volume densities of the chloroplast, cytoplasmic ribosomes, the vacuole, and the mitochondrion increased over controls. By 8 h the relative volume of the single ramified mitochondrion had decreased slightly and recognizable segments of it were sequestered within the vacuome. The autophagic nature of the vacuole was further evidenced by the presence of ribosomes and whorls of lamellae within it. Serial sections showed that all polyphosphate granules and sequestered materials were located within a continuous vacuolar cisterna.     

Impact of hypoosmotic challenges on spongy architecture of the cytoplasm of the giant marine alga Valonia utricularis

Summary. The ultrastructure of the several micrometers thick cytoplasmic layer of the giant marine alga Valonia utricularis displays characteristics which are apparently linked with the capability of this alga to regulate turgor pressure. Transmission and scanning electron microscopy of cells prefixed in different ways, including a protocol that allows prefixation of the alga in a turgescent state, revealed a highly dendritic network of cytoplasmic strands connecting and enveloping the chloroplasts and the nuclei. Innumerable vacuolar entities are embedded in the network, giving the cytoplasm a spongy appearance. Vacuolar perfusion of turgor-pressure-clamped cells with prefixation solution containing tannic acid presented evidence that these vacuolar entities together with the huge central vacuole form a large unstirred continuum. In contrast to the tonoplast, the plasmalemma followed smoothly the lining of the cell wall, even at the numerous cell wall ingrowths. Sucrose, but not polyethylene glycol 6000, induced chloroplast clustering. Acute hypoosmotic treatment (established by reduction of external NaCl or by replacement of part of the external NaCl by equivalent osmotic concentrations of sucrose or polyethylene glycol 6000) resulted in a local relocation of the chloroplasts and cytoplasm towards the central vacuole. This effect did not occur when the relatively low reflection coefficients of these two osmolytes were taken into account. The increase in spacing between the spongy cytoplasm and the plasmalemma by chloroplast relocation (viewed by confocal laser scanning microscopy) was associated with a speckled appearance of the affected surface area under the light microscope. As indicated by electron microscopy, hypoosmotically induced chloroplast relocation resulted from disproportionate swelling of the vacuolar entities located close to the plasmalemma. The cytoskeleton in the cytoplasm and the mucopolysaccharide network in the central vacuole apparently resisted swelling of these compartments. This finding has the important consequence that relevant hydrostatic pressure gradients can be built up throughout the entire multifolded vacuolar space. This gradient could represent the trigger for turgor pressure regulation which is manifested electrically first in the tonoplast.Correspondence and reprints: Lehrstuhl für Biotechnologie, Biozentrum, Am Hubland, 97074 Würzburg, Federal Republic of Germany.     

The formation of vacuole membranes in the presence and absence of cell nucleus in amoebae

The formation of new vacuoles around injected algae was studied in amoebae at intervals after enucleation. Algae initially lay free for 3 h within the amoeba cytoplasm, after which they gradually became individually enclosed within single-vacuole membranes. During the initial stage of vacuole formation (4–24 h), discontinuous pieces of non-trilaminar membrane-like structures appeared around the algae, which were not associated with any preexisting membranes. Over 85 and 50% of injected algae became enclosed in vacuoles in amoebae that had been without nuclei between 0–16 and 18–36 h, respectively. Thus, it appeared that vacuole-forming activity was not under the immediate control of cell nuclei. This mode of vacuole formation suggested that the initial vacuole membranes might arise de novo.