Multiple Roles of Soluble Sugars in the Establishment of Gunnera-Nostoc Endosymbiosis

Gunnera plants have the unique ability to form endosymbioses with N₂-fixing cyanobacteria, primarily Nostoc. Cyanobacteria enter Gunnera through transiently active mucilage-secreting glands on stems. We took advantage of the nitrogen (N)-limitation-induced gland development in Gunnera manicata to identify factors that may enable plant tissue to attract and maintain cyanobacteria colonies. Cortical cells in stems of N-stressed Gunnera plants were found to accumulate a copious amount of starch, while starch in the neighboring mature glands was nearly undetectable. Instead, mature glands accumulated millimolar concentrations of glucose (Glc) and fructose (Fru). Successful colonization by Nostoc drastically reduced sugar accumulation in the surrounding tissue. Consistent with the abundance of Glc and Fru in the gland prior to Nostoc colonization, genes encoding key enzymes for sucrose and starch hydrolysis (e.g. cell wall invertase, α-amylase, and starch phosphorylase) were expressed at higher levels in stem segments with glands than those without. In contrast, soluble sugars were barely detectable in mucilage freshly secreted from glands. Different sugars affected Nostoc’s ability to differentiate motile hormogonia in a manner consistent with their locations. Galactose and arabinose, the predominant constituents of polysaccharides in the mucilage, had little or no inhibitory effect on hormogonia differentiation. On the other hand, soluble sugars that accumulated in gland tissue, namely sucrose, Glc, and Fru, inhibited hormogonia differentiation and enhanced vegetative growth. Results from this study suggest that, in an N-limited environment, mature Gunnera stem glands may employ different soluble sugars to attract Nostoc and, once the cyanobacteria are internalized, to maintain them in the N₂-fixing vegetative state.Nitrogen (N) is an essential element for plant growth, but availability of reduced N in the soil is often limiting. Representatives from a wide range of land plants have evolved the ability to form associations with N₂-fixing microbes (Franche et al., 2009). Associations between rhizobia and legume plants are well-characterized examples of plant-bacterial N₂-fixing symbioses. Unlike rhizobia, which generally exhibit narrow host ranges (Kistner and Parniske, 2002), N₂-fixing cyanobacteria are able to form productive associations with a broad range of plants, including bryophytes (hornworts and liverworts), ferns (Azolla), gymnosperms (cycads), and angiosperms (Gunnera; for review, see Rai et al., 2000; Adams et al., 2006). Free-living cyanobacteria within the genus Nostoc can fix N in specialized microoxic cells called heterocysts. The ability of Nostoc to fix N independent of a host environment may facilitate the formation of symbioses with a wide range of plants. Understanding the physiological conditions that enable a plant host to enter into symbiotic associations with cyanobacteria may allow us to extend the benefit of biological N fixation to crops outside the legume family.Nostoc has the ability to differentiate not only into filaments bearing heterocysts but also into transiently motile filaments, known as hormogonia, which enable the cyanobacteria to enter plants (Meeks and Elhai, 2002). Nostoc can be induced to form hormogonia by different environmental stimuli and by a hormogonia-inducing factor released from N-stressed host plants (Meeks and Elhai, 2002; Adams et al., 2006). The attraction of hormogonia to plants is much less specific than that of rhizobia. Hormogonia are attracted to root extracts from either host or nonhost plants and even to certain simple sugars, such as Ara, Glc, and Gal (Nilsson et al., 2006). After entering a plant host, hormogonia revert back to filaments with N₂-fixing heterocysts. Inside the host, further hormogonia formation is suppressed, and heterocysts appear at a frequency of about 30% to 40%, 3- to 4-times higher than that found in free-living Nostoc (Meeks and Elhai, 2002). Although free-living Nostoc species can support N₂ fixation through photosynthesis, under symbiotic conditions they rely on photosynthate from the host plant. In general, the sugars (Suc, Glc, and Fru) known to support heterotrophic growth in the dark by free-living cyanobacteria coincide with those that support nitrogenase activity in Nostoc-plant associations (Meeks and Elhai, 2002). However, the Nostoc-Gunnera association may be exceptional; only Glc and Fru have been shown to sustain nitrogenase activities (Man and Silvester, 1994; Wouters et al., 2000), although Suc anddextrin were able to keep Nostoc alive without light (Wouters et al., 2000). It is evident from cyanobacterial studies that the plant hosts have evolved the ability to regulate cyanobacterial growth and differentiation during symbiotic associations (Meeks and Elhai, 2002).However, because most studies of plant-cyanobacterial associations have focused on the cyanobacterial partner (e.g. Wang et al., 2004; Ekman et al., 2006), the mechanisms through which plant hosts attract, internalize, and maintain cyanobacteria remain to be elucidated (Adams et al., 2006).The Nostoc-Gunnera association is an ideal system with which to study plant-cyanobacteria symbioses, not only because Gunnera is the only genus of angiosperms known to form endosymbioses with N₂-fixing cyanobacteria but also because the association between the two can be readily established in the laboratory (Bergman et al., 1992; Chiu et al., 2005). Nostoc hormogonia enter Gunnera plants through specialized glands located on the stem. As the gland matures, it secretes polysaccharide-rich mucilage that attracts cyanobacteria (Nilsson et al., 2006), supports their growth on the gland surface (Towata, 1985; Chiu et al., 2005), and permits further hormogonia differentiation (Rasmussen et al., 1994). From there, hormogonia enter the gland and penetrate cells near the base of the gland in the stem (Bonnett, 1990; Bergman et al., 1992). Although each gland is only transiently capable of accepting cyanobacteria, new glands continue to form on the stem at the base of each new leaf.In contrast to the development of nodules in legumes, which requires a complex exchange of signals between the two symbiotic partners (Cooper, 2007), stem gland development in Gunnera takes place in the absence of cyanobacteria (Bonnett, 1990). N limitation, however, is a prerequisite for stem gland development (Chiu et al., 2005), as it is for nodulation (Barbulova et al., 2007). We have taken advantage of the N-deficiency-induced gland development in G. manicata to identify factors that enable Gunnera to form endosymbiosis with cyanobacteria. This study investigated changes in the carbohydrate metabolism during Gunnera gland development and discovered that tissue in the mature glands accumulated high levels of soluble sugars prior to the arrival of cyanobacteria. In agreement with this finding, several key genes encoding enzymes for starch/Suc hydrolysis were expressed at higher levels in the gland compared to the stem. Furthermore, we found that various sugars cyanobacteria may encounter as they approach Gunnera glands as opposed to those they would encounter within plant cells differentially affected Nostoc’s ability to form motile hormogonia.     

Phylogeny of symbiotic cyanobacteria within the genus <Emphasis Type="Italic">Nostoc</Emphasis> based on 16S rDNA sequence analyses

A phylogenetic analysis of selected symbiotic Nostoc strain sequences and available database 16S rDNA sequences of both symbiotic and free-living cyanobacteria was carried out using maximum likelihood and Bayesian inference techniques. Most of the symbiotic strains fell into well separated clades. One clade consisted of a mixture of symbiotic and free-living isolates. This clade includes Nostoc sp. strain PCC 73102, the reference strain proposed for Nostoc punctiforme. A separate symbiotic clade with isolates exclusively from Gunnera species was also obtained, suggesting that not all symbiotic Nostoc species can be assigned to N. punctiforme. Moreover, isolates from Azolla filiculoides and one from Gunnera dentata were well nested within a clade comprising most of the Anabaena sequences. This result supports the affiliation of the Azolla isolates with the genus Anabaena and shows that strains within this genus can form symbioses with additional hosts. Furthermore, these symbiotic strains produced hormogonia, thereby verifying that hormogonia formation is not absent in Anabaena and cannot be used as a criterion to distinguish it from Nostoc.The GenBank accession numbers for the cyanobacterial 16S rRNA gene sequences determined in this study are AY742447-AY742454.     

Lichens toGunnera — with emphasis onAzolla

Summary N₂-fixing cyanobacteria occur in symbiotic associations with fungi (ascomycetes) as lichens and with a few green plants. The associated cyanobacterium is always a species ofNostoc orAnabaena. Only a small number of plant genera are involved but there is a remarkable range of host diversity. Associations occur with several bryophytes (e.g.Anthoceros, Blasia, Cavicularia), a pteridophyte (Azolla), cycads (nine genera includingMacrozamia andEncephalartos) and an angiosperm (Gunnera). Except forGunnera, where the cyanobacterium penetrates the plant cells, the cyanobacteria are extracellular with specialized morphological modifications and/or structures of the host plant organs providing an environment which facilitates interaction with the prokaryote.Salient aspects of current knowledge pertaining to the establishment, perpetuation, and functioning of the individual symbioses are summarized. Where possible this includes information concerning recognition and specificity, mode(s) of infection, morphological modifications/adaptations of the host plant and a synopsis of morphological, physiological and biochemical changes common to the symbiotic cyanobacteria. The latter encompasses heterocyst frequencies, enzymes involved in ammonia assimilation, photosynthetic capability and metabolic interaction with the host.TheAzolla-Anabaena symbioses, which have potential agronomic significance as an alternative nitrogen source and maintain continuity with the endophyte through the sexual cycle, are emphasized.     

Characterization of theGeosiphon pyriforme symbiosome by affinity techniques: confocal laser scanning microscopy (CLSM) and electron microscopy

Summary Geosiphon pyriforme represents a photoautotrophic endosymbiosis of aGlomus-like fungus with the cyanobacteriumNostoc punctiforme. The fungus forms unicellular bladders of up to 2 mm in length and 0.5 mm in diameter growing on the soil surface and harboring the endosymbioticNostoc filaments. The cyanobacteria are located in a compartment (the symbiosome) bordered by a host membrane. The space between this symbiosome membrane (SM) and theNostoc cell wall is filled with an about 30–40 nm thick layer of amorphous material, which is present also in the regions of the symbiosome where noNostoc filaments are located. At these sites the amorphous material consists of a 20–30 nm thick layer separating the SM. The region between the SM and the cyanobacterium is defined as symbiosome space (SS). Fungal bladders, hyphae and free livingNostoc were analyzed by affinity techniques as well as the material occurring in the SS. FITC-coupled lectins with sugar specificity to -D-mannosyl/-D-glucosyl (Con A), N-acetyl--D-glucosamine oligomers (WGA), -L-fucosyl (UEA-I), -D-galactosyl (RCA-120), -D-galactosyl (BS-I-B₄), N-acetyl--D-galactosamine (HPA), and sialic acid (EBL) residues were tested. WGA binding and calcofluor white staining demonstrated that the bladder wall as well as the SS contain fibrillar chitin. Of the other lectins only Con A clearly labeled the symbiosome. On the contrary, the lectin binding properties of the slime produced by free livingNostoc-colonies indicate the presence of mannose, fucose, GalNAc, sialic acid, and galactose, while chitin or GlucNAc-oligomers could not be detected. The symbiosome was also investigated electron microscopically. WGA-gold binding confirmed the presence of chitin, while a slight PATAg reaction indicated some polysaccharidic molecules within the SS. Our results show that the amorphous material within the SS contains molecules typical of the fungal cell wall and suggest that the SM is related to the fungal plasma membrane. The applied lectins all bind to the hyphal surface, indicating a high molecular complexity. Mannosyl, -galactosyl, and sialic acid residues are strongly exposed at the outer cell wall layer, whereas GlucNAc, GalNAc, and -galactosyl residues seem to be present in smaller amounts. The symbiotic interface established between the fungus andNostoc inGeosiphon shows many similarities to that occurring between fungi and root cells in arbuscular mycorrhizas.Abbreviations AM arbuscular mycorrhiza - BS-I-B₄ Bandeiraea simplicifolia lectin I isolectin B₄ - CLSM confocal laser scanning microscopy - Con A Concanavalin A - EBL elderberry bark lectin I - FITC fluorescein isothiocyanate - HPA Helix pomatia agglutinin - PATAg periodic acid-thiocarbohydrazide-Ag proteinate - SM symbiosome membrane - SS symbiosome space - RCA-120 Ricinus communis agglutinin 120 - UEA-I Ulex europaeus agglutinin I - WGA wheat germ agglutinin Dedicated to Professor Dr. Peter Sitte at the occasion of his 65th birthday     

The occurrence of fimbriae on a N 2 2 -fixing cyanobacterium which occurs in lichen symbiosis

The Nostoc cyanobiont of the lichen Peltigera canina when grown on N₂ possesses, in the motile stage, discrete unbranched non-flagellar appendages (fimbriae or pili). These arise from the host cell surface in a peritrichous manner, have an axial hole, are 7.0 ±0.3 nm in diameter and are up to 3 m long. They do not haemagglutinate guinea pig red blood corpuscles and differ from the major fimbrial types reported for Gram-negative heterotrophic bacteria and from sex pili. They may be involved in motility and specificity in symbiotic cyanobacteria.     

Developmental patterns related to nitrogen fixation in theNostoc-Gunnera magellanica Lam. symbiosis

Developmental patterns related to nitrogen fixation in the heterocystous cyanobacteriumNostoc harboured in distinct colonies along the stem ofGunnera magellanica Lam. plantlets were examined using successive plant sections. Pronounced morphological, physiological and biochemical alterations in the cyanobacterium were demonstrated. Close to the growing apex the cyanobacterial biomass, contained in smallGunnera cells, was low and consisted mostly of vegetative cells showing a high density of different storage structures except for cyanophycin granules. In contrast, both the total and specific nitrogenase activity and the relative nitrogenase protein level were at maximum within this part; while the frequency of heterocysts increased from zero to 30% within the same area. The nitrogenase protein was localized only in the heterocysts throughout the plant. Further down theGunnera stem there was a progressive increase in both the cyanobacterial biomass and the heterocyst frequency, which finally constituted about 60% of the cyanobacterial cell population. Throughout this part of the stem, cyanophycin granules were frequent in the vegetativeNostoc cells. At the base of the stem, degeneratedNostoc cells dominated and the nitrogenase activity was close to zero, although the nitrogenase protein remained. Degeneration of theNostoc cells and leaf shedding coincided. Both intact plants (approx. 20 mm in height) and plant stem sections (2 mm in length) showed substantial nitrogenase activity, although sectioning caused a 30% reduction in total nitrogenase activity.     

Cytomorphological characters supporting the taxonomic validity ofCyanothece (Cyanoprokaryota)

The fine structure of the type species of the genusCyanothece Komárek 1976,C. aeruginosa, is described and compared with the main cytological characteristics of morphologically related members of the generaCyanobium, Cyanobacterium andSynechococcus. Several morphological features, such as cell walls with thick outer layers containing a special type of vesicles, position of thylakoids, keritomy (net-like appearance of protoplast caused by arrangement of thylakoids, net-like nucleoids and/or by tendency to form intrathylakoidal spaces) and a special structure of mucilaginous envelopes were found to be characteristic of this genus, supporting its separate position among coccal cyanoprokaryotes (cyanobacteria, cyanophytes). The taxonomic significance of ultrastructural features in all mentioned genera is discussed.     

Antialgal activity of a hepatotoxin-producing cyanobacterium,Microcystis aeruginosa

Antimicrobial activity of toxin produced by a freshwater bloom-forming cyanobacterium Microcystis aeruginosa has been studied. When tested against certain green algae, cyanobacteria, heterotrophic bacteria and fungi, the toxin inhibited growth of only green algae and cyanobacteria. The toxin has been partially purified employing Thin layer chromatography (TLC) and High-performance liquid chromatography (HPLC) techniques and appears to be microcystin-LR (leucine–arginine). Both crude and purified toxins showed toxicity to mice, the clinical symptoms in test mice being similar to those produced by hepatotoxin. Purified toxin at a concentration of 50 g ml^–1 caused complete inhibition of growth followed by cell lysis in Nostoc muscorum and Anabaena BT1 after 6 days of toxin addition. Addition of toxin (25 g ml^–1) to the culture suspensions of the Nostoc and Anabaena strains caused instant and drastic loss of O₂ evolution. Furthermore a marked reduction (about 87%) in the ¹⁴CO₂ uptake was also observed at a concentration of 50 g ml^–1. Besides its inhibitory effects on photosynthetic processes, M. aeruginosa toxin (50 g ml^–1) also caused 90% loss of nitrogenase activity after 8 h of its addition. Experiments performed with ¹⁴C-labelled toxin indicate that the toxin uptake by cyanobacterial cells occurs both in light and dark. These results demonstrate that the toxin is strongly algicidal and point to the possibility that it may have an important role in establishment and maintenance of toxic blooms of M. aeruginosa in freshwater ecosystems. The relative significance of the hepatotoxic effect and the algicidal effect of the toxin is discussed with reference both to survival and dominance of M. aeruginosa in nature.     

Cyanobacterial toxicity and migration in a mesotrophic lake in western Washington, USA

In fall 1997, the toxic cyanobacterium Microcystis aeruginosa was documented in Lake Sammamish (western Washington, U.S.A.) for the first time. Cyanobacterial activity and environmental conditions that may promote toxic cyanobacteria were investigated during summer and fall 1999. Development of toxic Microcystis was hypothesized to be due to runoff of nutrients from the watershed (external loading hypothesis) or from vertical migration of dormant cyanobacteria from the nutrient-rich sediments into the water column (cyanobacterial migration hypothesis). Microcystins were detected using an enzyme-linked immunosorbent assay during late August and early September 1999 despite low cyanobacterial abundance. Microcystin concentrations ranged between 0.19–3.8 g l^–1 throughout the lake and at all depths with the exception of the boat launch where concentrations reached 43 g l^–1. Comparison of the conditions associated with the toxic episodes in 1997 and 1999 indicate that Microcystis is associated with a stable water column, increased surface total phosphorus concentrations (> 10 g l^–1), surface temperatures greater than 22°C, high total nitrogen to phosphorus ratios (> 30), and increased water column transparency (up to 5.5 m). Migration of the cyanobacteria, Microcystis and Anabaena, occurred in both the deep and shallow portions of the lake. Microcystis dominated (89–99%) the migrating cyanobacteria with greater migration from the shallow station. External loading of nutrients due to the large rainfall preceding the 1997 toxic episode may have provided the nutrients needed to fuel that bloom. However, toxic Microcystis occurred in 1999 despite the lack of rain and subsequent external runoff. The migration of Microcystis from the nutrient-rich sediments may have been the inoculum for the toxic population detected in 1999.     

Transposition of mobile elements gypsy (mdg4) and hobo in germ-line and somatic cells of a genetically unstable mutator strain of Drosophila melanogaster.

Summary Using the in situ hybridization technique, we have analysed the distribution of mobile elements in the X chromosomes of male offspring of individual mutator strain (MS) males crossed to attached-X females. The experiments demonstrate varying cytological localization of the mobile elements gypsy (mdg4) and hobo among different individuals. The other mobile elements investigated (mdgl, mdg3, 412, 297, copia, 17.6, Doc, H.M.S. Beagle, Springer, FB) display no changes in insertion sites. Such an experiment is equivalent to analysis of separate gametes of an MS individual. Thus, the ability of gypsy and hobo to transpose in germ-line cells is demonstrated directly. Transpositions occur at premeiotic stages of germ cell development, since they appear in clusters. Analysis of gypsy and hobo transposition events shows that they occur independently. The same experiment demonstrates that gypsy localization varies significantly between different salivary gland cells of an MS individual. Two types of gypsy hybridization sites can be distinguished: permanent sites, common to all cells, and additional ones varying between neighbouring salivary gland cells. These additional sites indicate gypsy transposition in somatic cells of the MS. Transposition of the hobo element in somatic cells has also been observed.     

TheGunnera symbiosis: DNA restriction fragment length polymorphism and protein comparisons ofNostoc symbionts

Cyanobacteria separated from symbiosis with several species of the angiospermGunnera were comparatively characterized and correlated with the locales and taxonomy of their host plants. All were identified as strains ofNostoc. Protein profiles and DNA restriction fragment length polymorphisms (from hybridizations with heterologousnifH andglnA probes) determined that three of the four cyanobacteria fromGunnera grown at one site in Sweden, each from a different host species, were very similar or identical. Plants of one species,G. manicata, grown in a second location at the site were infected with a different cyanobiont. Among five isolates from two species ofGunnera, collected in the same locale in New Zealand, three subgroups were documented. Isolates from three differentGunnera species grown in separate locations in the United States were each uniquely different. None of the cyanobacteria differed in the molecular weights of their glutamine synthetase and Fe-nitrogenase proteins. The diversity and accessibility of compatibleNostoc populations present in the soil micro-environment, not a critical selective factor required byGunnera, were concluded to be a major determinant in symbiont selection.     

Glutamine synthetase specific activity and protein concentration in symbiotic Anabaena associated with Azolla caroliniana

Glutamine synthetase (GS) is the primary NH₄ ⁺ assimilating enzyme of cyanobacteria. The specific activities and cellular protein concentration of GS in symbiotic cyanobacteria associated with the water fern Azolla caroliniana were determined and compared to free-living cultures of Nostoc sp. strain 7801, a strain originally isolated from symbiotic association with the bryophyte Anthoceros punctatus. Both the in vitro specific activity and concentration of GS in symbiotic cyanobacteria separated from A. caroliniana were approximately 3-fold lower than the free-living Nostoc sp. strain 7801 culture. These results imply depressed synthesis of GS by the symbiont associated with A. caroliniana.     

Partial sequence of ribulose-1,5-bisphosphate carboxylase/oxygenase and the phylogeny of Prochloron and Prochlorococcus (Prochlorales)

The prochlorophytes, oxygenic photosynthetic prokaryotes having no phycobiliprotein but possessing chlorophylls a and b, have been proposed to have a common ancestry with green chloroplasts, yet this is still controversal. We report here that partial sequence comparisons of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, including sequence data from two prochlorophytes, Prochlorococcus and Prochloron, indicate that Prochlorococcus is more closely related to a photosynthetic bacterium, Chromatium vinosum (-purple bacteria), than to cyanobacteria, while Prochloron is closely related to the prochlorophyte Prochlorothrix and to cyanobacteria. The molecular phylogenetic tree indicates that a common ancestor of Prochlorococcus and -purple bacteria branched off from the land plant lineage earlier than Prochloron, Prochlorothrix, and cyanobacteria.Correspondence to: A. Shimada     

Seed morphology in Southern AfricanOrchidoideae (Orchidaceae)

The seed morphology of 151 species of Southern AfricanOrchidoideae (Orchideae andDiseae; sensuDressler 1981) was studied by means of scanning electron microscopy. Two different seed types were found. (1) In the majority of species the seeds are minute and fusiform. The seed coat is made up of comparatively few concave and elongate testa cells with straight or slightly undulate and generally unthickened anticlinal cell walls. The seed type was here termed Satyrium-type. While most species are very similar in the ornamentation of the periclinal walls of their seed coat, considerable variation was found inHolothrix where two distinct groups can be recognized in this respect. (2) A remarkably different seed type was observed inDisa uniflora and three apparently closely related species (Disa uniflora-type), where large balloon-like seeds occur. Their seed coat consists of convex cells with undulate anticlinal walls. It is suggested that this seed type is a derived condition and has evolved in adaptation to the specialized habitat alongside streams. The possibility of hydrochory in these four species is briefly discussed.     

Occurrence and localization of an uptake hydrogenase in the filamentous heterocystous cyanobacteriumNostoc PCC 73102

Summary Free-living nitrogen-fixingNostoc PCC 73102, a filamentous heterocystous cyanobacterium originally isolated from coralloid roots of the cycadMacrozamia sp., were examined for the presence of an uptake hydrogenase (H₂ase) enzyme. In vivo and in vitro hydrogen uptake measurements were used to study activities and SDS-PAGE and Western immunoblots to reveal occurrence of the hydrogenase protein. Also, transmission electron microscopy and immunocytological labeling were used to study the cellular and subcellular distribution of H₂ase in theNostoc cells. In vivo measurements demonstrated an active uptake of hydrogen in both light and darkness. Light stimulated in vivo hydrogen uptake with approximately 100%, and this was further doubled by increasing the pH₂, from 56 to 208 M H₂. An in vitro hydrogen uptake of 1.1 mol H₂/ mg (protein)/h was observed when using phenazinemethosulphate as e^–-acceptor. Western immunoblots revealed that a polypeptide with a molecular weight of about 55 kDa was immunologically related to uptake H₂ase holoenzyme purified fromAlcaligenes latus. Immunolocalization demonstrated that the H₂ase protein was located both in heterocysts and vegetative cells. A higher specific labeling was associated with the cytoplasmic membranes where the vegetative cells are in contact with each other and where they actually are dividing into two vegetative cells. Using the particle analysis of an image processor, approximately equal H₂ase-gold labeling per cell area was observed in the nitrogen-fixing heterocysts compared to the photosynthetic vegetative cells. This study also shows that there was no correlation between presence of phycoerythrin and uptake H₂ase activity.Abbreviations H₂ase hydrogenase - IgG immunoglobulin G     

Characteristics of Hormogonia Formation by Symbiotic Nostoc spp. in Response to the Presence of Anthoceros punctatus or Its Extracellular Products

Nostocacean cyanobacteria typically produce gliding filaments termed hormogonia at a low frequency as part of their life cycle. We report here that all Nostoc spp. competent in establishing a symbiotic association with the hornwort Anthoceros punctatus formed hormogonial filaments at a high frequency in the presence of A. punctatus. The hormogonia-inducing activity was produced by A. punctatus under nitrogen-limited culture conditions. The hormogonia of the symbiotically competent Nostoc spp. were characterized as motile (gliding) filaments lacking heterocysts and with distinctly smaller cells than those of vegetative filaments; the small cells resulted from a continuation of cell division uncoupled from biomass increase. An essentially complete conversion of vegetative filaments to hormogonia occurred within 12 h of exposure of Nostoc sp. strain 7801 to A. punctatus growth-conditioned medium. Hormogonia formation was accompanied by loss of nitrogen fixation (acetylene reduction) and by decreases in photosynthetic CO₂ fixation and in vivo NH₄⁺ assimilation of 30% and approximately 40%, respectively. The rates of acetylene reduction and CO₂ fixation returned to approximately the control rates within 72 to 96 h after hormogonia induction, as the cultures of Nostoc sp. strain 7801 differentiated heterocysts and reverted to the vegetative growth state. The relationship between hormogonia formation and symbiotic competence is discussed.     

Variability of hydrocarbon and fatty acid components in cultures of the filamentous cyanobacterium Scytonema sp. isolated from microbial community "black cover" of limestone walls in Jerusalem

The hydrocarbon and lipid components of four strains of the filamentous cyanobacterium Scytonema sp. isolated from microbial community Black Cover of limestone walls in Jerusalem were identified by gas chromatography–mass spectrometry using serially coupled capillary columns. The dominant compounds were: 1-heptadecyne (1.5-8%), hexadecanoic acid (14-36%), (Z,Z)-9,12-octadecadienoic acid (12-30%), (Z,Z,Z)-9,12,15-octadecatrienoic acid (6-12%), n-heptadecane (4-16%), and 1-heptadecene (1.5-8%). In addition to unsaturated alkanes and fatty acids, the very long-chain (C₃₀-C₃₂) hydrocarbons, squalene (2.4-3.0%), and branched 4,8,12-trimethyl-C_13:0 acid were also isolated. Two major hydrocarbons were detected in the cyanobacteria species using GC-MS and ¹³C-NMR.     

Embryonic origin and differentiation of the Drosophila heart

We have followed the normal development of the different cell types associated with the Drosophila dorsal vessel, i.e. cardioblasts, pericardial cells, alary muscles, lymph gland and ring gland, by using several tissue-specific markers and transmission electron microscopy. Precursors of pericardial cells and cardioblasts split as two longitudinal rows of cells from the lateral mesoderm of segments T2-A7 (cardiogenic region) during stage 12. The lymph gland and dorsal part of the ring gland (corpus allatum) originate from clusters of lateral mesodermal cells located in T3 and T1/dorsal ridge, respectively. Cardioblast precursors are strictly segmentally organized; each of T2-A6 gives rise to six cardioblasts. While moving dorsally during the stages leading up to dorsal closure, cardioblast precursors become flattened, polarized cells aligned in a regular longitudinal row. At dorsal closure, the leading edges of the cardioblast precursors meet their contralateral counterparts. The lumen of the dorsal vessel is formed when the trailing edges of the cardioblast precursors of either side bend around and contact each other. The amnioserosa invaginates during dorsal closure and is transiently attached to the cardioblasts; however, it does not contribute to the cells associated with the dorsal vessel and degenerates during late embryogenesis. We describe ultrastructural characteristics of cardioblast differentiation and discuss similarities between cardioblast development and capillary differentiation in vertebrates. Correspondence to: V. Hartenstein     

Shifts in the intracellular ATP pools of immobilisedNostoc cells (Cyanobacteria) induced by water stress

Summary Immobilised, desiccated cells ofNostoc commune UTEX 584 have the capacity to increase the size of their extractable intracellular ATP pool upon rewetting. The time taken to recover the pool size depends on the conditions of storage at a particular water potential and the duration of storage. Under the conditions employed, the rewetting of cells induced an increase in ATP pool size at the expense of photophosphorylation or electron transport (oxidative) phosphorylation. The rise in the ATP pool size was instantaneous and was shown to be due to ATP synthesis. This increase did not occur when cells were rewetted in the presence of sodium azide (10 mmol/l), while a partial inhibition was observed with CCCP (carbonyl cyanidem-chlorophenylhydrazone; 2 mol/l). For cells dried at more extreme water potentials, the lag ofc 48 h observed before the ATP pool reached control values is of similar duration to that observed in the recovery of nitrogenase upon rewetting. Chloramphenicol (10 mol/l) stimulated significantly the upshift in the size of the ATP pool ofNostoc cells upon rewetting, yet inhibited completely the rise in nitrogenase activity.     

Genetic variation at theapcAB,cpcAB,gvpA1, andnifH loci and in DNA methylation among N2-fixing cyanobacteria designatedNostoc punctiforme

Genetic similarity among cyanobacteria of a morphological subgroup ofNostoc was evaluated through a comparison of several specific genes and the extent of DNA methylation. Four of six cyanobacteria were originally cultured from facultative symbioses with higher plants (Gunnera andEncephalartos); these and one free-living isolate had been identified or reputed to beN. punctiforme. No consistent correlation to species or symbiotic history was found from DNA hybridizations to genes coding for phycocyanin (cpcAB), allophycocyanin (apcAB), gas vesicle protein (gvpA1), and dinitrogenase reductase (nifH). One gene (gvpC) was not present, andgvpA1 was a single-copy gene in all strains. The gas vesicle genes were concluded to be potentially useful for broadly characterizingNostoc or at least this subgroup. Incubations ofNostoc genomic DNA with 22 restriction endonucleases indicated a high degree of methylation and similarity of its methylated DNA to that of other heterocystous cyanobacteria. The genetic variation of theNostoc isolates was judged to reflect primarily different soil origins.