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.     

Protein expression profiles in an endosymbiotic cyanobacterium revealed by a proteomic approach

Molecular mechanisms behind adaptations in the cyanobacterium (Nostoc sp.) to a life in endosymbiosis with plants are still not clarified, nor are the interactions between the partners. To get further insights, the proteome of a Nostoc strain, freshly isolated from the symbiotic gland tissue of the angiosperm Gunnera manicata Linden, was analyzed and compared with the proteome of the same strain when free-living. Extracted proteins were separated by two-dimensional gel electrophoresis and were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry combined with tandem mass spectrometry. Even when the higher percentage of differentiated cells (heterocysts) in symbiosis was compensated for, the majority of the proteins detected in the symbiotic cyanobacteria were present in the free-living counterpart, indicating that most cellular processes were common for both stages. However, differential expression profiling revealed a significant number of proteins to be down-regulated or missing in the symbiotic stage, while others were more abundant or only expressed in symbiosis. The differential protein expression was primarily connected to i) cell envelope-associated processes, including proteins involved in exopolysaccharide synthesis and surface and membrane associated proteins, ii) to changes in growth and metabolic activities (C and N), including upregulation of nitrogenase and proteins involved in the oxidative pentose phosphate pathway and downregulation of Calvin cycle enzymes, and iii) to the dark, microaerobic conditions offered inside the Gunnera gland cells, including changes in relative phycobiliprotein concentrations. This is the first comprehensive analysis of proteins in the symbiotic state.     

Arabinogalactan proteins occur in the free-living cyanobacterium genus Nostoc and in plant-Nostoc symbioses

Arabinogalactan proteins (AGP) are a diverse family of proteoglycans associated with the cell surfaces of plants. AGP have been implicated in a wide variety of plant cell processes, including signaling in symbioses. This study investigates the existence of putative AGP in free-living cyanobacterial cultures of the nitrogen-fixing, filamentous cyanobacteria Nostoc punctiforme and Nostoc sp. strain LBG1 and at the symbiotic interface in the symbioses between Nostoc spp. and two host plants, the angiosperm Gunnera manicata (in which the cyanobacterium is intracellular) and the liverwort Blasia pusilla (in which the cyanobacterium is extracellular). Enzyme-linked immunosorbent assay, immunoblotting, and immunofluorescence analyses demonstrated that three AGP glycan epitopes (recognized by monoclonal antibodies LM14, MAC207, and LM2) are present in free-living Nostoc cyanobacterial species. The same three AGP glycan epitopes are present at the Gunnera-Nostoc symbiotic interface and the LM2 epitope is detected during the establishment of the Blasia-Nostoc symbiosis. Bioinformatic analysis of the N. punctiforme genome identified five putative AGP core proteins that are representative of AGP classes found in plants. These results suggest a possible involvement of AGP in cyanobacterial-plant symbioses and are also suggestive of a cyanobacterial origin of AGP.     

The role of papillae during the infection process in the Gunnera-Nostoc symbiosis

Gunnera manicata L. glands consist of up to nine separate papillae. Surgical removal of papillae showed that more than two papillae were needed for successful infection with Nostoc. Infection occurs only in the enclosed space between adjacent papillae. Dividing Gunnera cells in the enclosed space are the sites of infection.     

The α-amylase inhibitor of bean seed: two-step proteolytic maturation in the protein storage vacuoles of the developing cotyledon

In the nitrogen fixing symbiosis between Nostoc and the angiosperm Gunnera , the cyanobiont is found in stem glands and is thought to have a heterotrophic mode of nutrition. To investigate whether the photosynthetic machinery in the cyanobiont is down-regulated in the symbiosis, the presence of the phycobiliproteins, phycoerythrin and phycocyanin, and ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco, EC 4.1.1.39) in cyanobionts of Gunnera magellanica Lam. and in a free-living (cultured) isolate of the cyanobacterium was studied by immunoelectron microscopy. Carboxysomes were numerous in all vegetative cells (ca 3.5 per cell section), and on an area basis they showed a high Rubisco label compared to the cytoplasm; but recalculation on a volume basis demonstrated that the carboxysomal fraction of Rubisco decreased in the cyanobiont along the plant stem. Along the whole Gunnera stem both types of phycobiliproteins were present in the symbiotic Nostoc and in amounts equivalent to or above those detected in the free-living isolate. As the symbiotic Nostoc is located intracellularly, out of reach of light in the plant stem, the findings indicate a lack of regulation of the photosynthetic protein synthesis in the symbiotic state.     

Photoregulated or Energy Dependent Process of Hormogonia Differentiation in Nostoc sphaeroides Kutzing （Cyanobacterium）

Abstract: Hormogonium, which was thought to play an important role in the dispersal and survival of these microorganisms in their natural habitats, is a distinguishable developmental stage of heterocystous cyanobacteria. The present study examined the effects of different light conditions and sugars on the differentiation of Nostoc sphaeroides Kützing to the hormogonia stage. Results showed that differentiation of hormogonia was light dependent in the absence of sugar, but that close to 100% of cyanobacteria differentiated to hormogonia in the presence of glucose or sucrose, irrespective of the light conditions. This differentiation was inhibited, even in the presence of sugars, upon application of an inhibitor of respiration. Following the testing of different sugars, the effects of different lights were examined. It was found that 5–10 umol‐nT2‐s‐1 photon flux density was optimal for hormogonia differentiation. One hundred percent differentiation was obtained with white light irradiation, in contrast with irradiation with green light (80% differentiation) and red light (0–10% differentiation). Although they showed different efficiencies in inducing hormogonia differentiation in N. sphaeroides, the green and red radiation did not display antagonistic effects. When the additional aspect of time dependence was investigated through the application of different light radiations and an inhibitor of protein synthesis, it was found that the initial 6 h of the differentiation process was crucial for hormogonia differentiation. Taken together, these results show that hormogonia differentiation in N. sphaeroides is either a photoregulated or an energy dependent process. (Managing editor: Ping HE)     

Genetic diversity of Nostoc microsymbionts from Gunnera tinctoria revealed by PCR-STRR fingerprinting

The cyanobacteria belonging to the genus Nostoc fix atmospheric nitrogen, both as free-living organisms and in symbiotic associations with a wide range of hosts, including bryophytes, gymnosperms (cycads), the small water fern Azolla (Pteridophyte), the angiosperm genus Gunnera, and fungi (lichens). The Gunnera–Nostoc symbiosis is the only one that involves a flowering plant. In Chile, 12 species of Gunnera have been described with a broad distribution in the temperate region. We examined the genetic diversity of Nostoc symbionts from three populations of Gunnera tinctoria from Abtao, Chiloé Island, southern Chile, and microsymbionts from other two species of Gunnera from southern Chile, using PCR amplification of STRR (short tandemly repeated repetitive) sequences of the Nostoc infected tissue. To our knowledge, this is the first report of PCR fingerprinting obtained directly from symbiotic tissue of Gunnera. Genetic analyses revealed that Nostoc symbionts exhibit important genetic diversity among host plants, both within and between Gunnera populations. It was also found that only one Nostoc strain, or closely related strains, established symbiosis with an individual plant host.     

Early events during the establishment of the Gunnera/Nostoc symbiosis

The symbiosis between Gunnera and Nostoc was reconstituted using G. chilensis Lam. and G. manicata Linden, respectively, and three different Nostoc strains. Six stages characterised by specific modifications in both the cyanobiont and the host were recognised during the infection process. Mucilage-secreting stem glands developed on the Gunnera stems independent of the presence of cyanobacteria (Stage I). Soon after addition of the Nostoc isolates to the plant apices, an abundant differentiation of motile hormogonia commenced. The cyanobacteria accumulated in the mucilage on the surface of the gland (Stage II), and the hormogonia then proceeded into the stem tissue through intercellular channels (Stage III). At the channel bases, Nostoc was detected between the cell walls of small, densely cytoplasmic Gunnera cells and also in elaborate folds of these (Stage IV). The Gunnera cell walls subsequently dissolved adjacent to the cyanobacteria and Nostoc entered the host cells (Stage V). Once the intracellular association was formed, a high proportion of the vegetative Nostoc cells differentiated into heterocysts (Stage VI). Nostoc changed from being rich in inclusions (particularly cyanophycin) while on the gland surface into a comparatively non-storing form during penetration and the early intracellular stages. Bacteria were numerous on the gland surface, fewer in the channels, and were never detected within the Gunnera cells, indicating the existence of specific recognition mechanisms discriminating between conceivable microsymbionts. Mechanisms behind mutual adaptations and interactions between the two symbionts are discussed.The technical assistance of Anette Axen and Gary Wife is gratefully acknowledged. Financial support was provided by the Swedish Natural Science Research Council and the Hierta-Retzius foundations.     

Cyanobacteria-bryophyte symbioses

Cyanobacteria are a large group of photosynthetic prokaryotesof enormous environmental importance, being responsible fora large proportion of global CO₂ and N₂ fixation. They formsymbiotic associations with a wide range of eukaryotic hostsincluding plants, fungi, sponges, and protists. The cyanobacterialsymbionts are often filamentous and fix N₂ in specialized cellsknown as heterocysts, enabling them to provide the host withfixed nitrogen and, in the case of non-photosynthetic hosts,with fixed carbon. The best studied cyanobacterial symbiosesare those with plants, in which the cyanobacteria can infectthe roots, stems, leaves, and, in the case of the liverwortsand hornworts, the subject of this review, the thallus. Thesymbionts are usually Nostoc spp. that gain entry to the hostby means of specialized motile filaments known as hormogonia.The host plant releases chemical signals that stimulate hormogoniaformation and, by chemoattraction, guide the hormogonia to thepoint of entry into the plant tissue. Inside the symbiotic cavity,host signals inhibit further hormogonia formation and stimulateheterocyst development and dinitrogen fixation. The cyanobiontsundergo morphological and physiological changes, including reducedgrowth rate and CO₂ fixation, and enhanced N₂ fixation, andrelease to the plant much of the dinitrogen fixed. This shortreview summarizes knowledge of the cyanobacterial symbioseswith liverworts and hornworts, with particular emphasis on theimportance of pili and gliding motility for the symbiotic competenceof hormogonia. Key words: Bryophyte, cyanobacteria, gliding motility, pili, symbiosis Received 1 August 2007; Revised 23 December 2007 Accepted 7 January 2008     

Cellular differentiation in the cyanobacterium Nostoc punctiforme

Nostoc punctiforme is a phenotypically complex, filamentous, nitrogen-fixing cyanobacterium, whose vegetative cells can mature in four developmental directions. The particular developmental direction is determined by environmental signals. The vegetative cell cycle is maintained when nutrients are sufficient. Limitation for combined nitrogen induces the terminal differentiation of heterocysts, cells specialized for nitrogen fixation in an oxic environment. A number of unique regulatory events and genes have been identified and integrated into a working model of heterocyst differentiation. Phosphate limitation induces the transient differentiation of akinetes, spore-like cells resistant to cold and desiccation. A variety of environmental changes, both positive and negative for growth, induce the transient differentiation of hormogonia, motile filaments that function in dispersal. Initiation of the differentiation of heterocysts, akinetes and hormogonia are hypothesized to depart from the vegetative cell cycle, following separate and distinct events. N. punctiforme also forms nitrogen-fixing symbiotic associations; its plant partners influence the differentiation and behavior of hormogonia and heterocysts. N. punctiforme is genetically tractable and its genome sequence is nearly complete. Thus, the regulatory circuits of three cellular differentiation events and symbiotic interactions of N. punctiforme can be experimentally analyzed by functional genomics.     

Photoregulated or Energy Dependent Process of Hormogonia Differentiation in Nostoc sphaeroides Kützing (Cyanobacterium)

Hormogonium, which was thought to play an important role in the dispersal and survival of these microorganisms in their natural habitats, is a distinguishable developmental stage of heterocystous cyanobacteria. The present study examined the effects of different light conditions and sugars on the of hormogonia was light dependent in the absence of sugar, but that close to 100% of cyanobacteria differentiated to hormogonia in the presence of glucose or sucrose, irrespective of the light conditions. This differentiation was inhibited, even in the presence of sugars, upon application of an inhibitor of respiration.Following the testing of different sugars, the effects of different lights were examined. It was found that 5-10 μmol.m-2.s-1 photon flux density was optimal for hormogonia differentiation. One hundred percent differentiation was obtained with white light irradiation, in contrast with irradiation with green light (80%differentiation) and red light (0-10% differentiation). Although they showed different efficiencies in induc ing hormogonia differentiation in N. sphaeroides, the green and red radiation did not display antagonistic effects. When the additional aspect of time dependence was investigated through the application of different light radiations and an inhibitor of protein synthesis, it was found that the initial 6 h of the differentiation process was crucial for hormogonia differentiation. Taken together, these results show that hormogonia differentiation in N. sphaeroides is either a photoregulated or an energy dependent process.     

Characterization of the nitrate-nitrite transporter of the major facilitator superfamily (the nrtP gene product) from the cyanobacterium Nostoc punctiforme strain ATCC 29133

The products of the NpR1527 and NpR1526 genes of the filamentous, diazotrophic, fresh-water cyanobacterium Nostoc punctiforme strain ATCC 29133 were identified as a nitrate transporter (NRT) and nitrate reductase (NR) respectively, by complementation of nitrate assimilation mutants of the cyanobacterium Synechococcus elongatus strain PCC 7942. While other fresh-water cyanobacteria, including S. elongatus, have an ATP-binding cassette (ABC)-type NRT, the NRT of N. punctiforme belongs to the major facilitator superfamily, being orthologous to the one found in marine cyanobacteria (NrtP). Unlike the ABC-type NRT, which transports both nitrate and nitrite with high affinity, Nostoc NrtP transported nitrate preferentially over nitrite. NrtP was distinct from ABC-type NRT also in its insensitivity to ammonium-promoted regulation at the post-translational level. The nitrate reductase of N. punctiforme was, on the other hand, inhibited upon addition of ammonium to medium, lending ammonium sensitivity to nitrate assimilation.     

Evolution of sucrose synthesis

Cyanobacteria and proteobacteria (purple bacteria) are the only prokaryotes known to synthesize sucrose (Suc). Suc-P synthase, Suc-phosphatase (SPP), and Suc synthase activities have previously been detected in several cyanobacteria, and genes coding for Suc-P synthase (sps) and Suc synthase (sus) have been cloned from Synechocystis sp. PCC 6803 and Anabaena (Nostoc) spp., respectively. An open reading frame in the Synechocystis genome encodes a predicted 27-kD polypeptide that shows homology to the maize (Zea mays) SPP. Heterologous expression of this putative spp gene in Escherichia coli, reported here, confirmed that this open reading frame encodes a functional SPP enzyme. The Synechocystis SPP is highly specific for Suc-6(F)-P (K(m) = 7.5 microM) and is Mg(2+) dependent (K(a) = 70 microM), with a specific activity of 46 micromol min(-1) mg(-1) protein. Like the maize SPP, the Synechocystis SPP belongs to the haloacid dehalogenase superfamily of phosphatases/hydrolases. Searches of sequenced microbial genomes revealed homologs of the Synechocystis sps gene in several other cyanobacteria (Nostoc punctiforme, Prochlorococcus marinus strains MED4 and MIT9313, and Synechococcus sp. WH8012), and in three proteobacteria (Acidithiobacillus ferrooxidans, Magnetococcus sp. MC1, and Nitrosomonas europaea). Homologs of the Synechocystis spp gene were found in Magnetococcus sp. MC1 and N. punctiforme, and of the Anabaena sus gene in N. punctiforme and N. europaea. From analysis of these sequences, it is suggested that Suc synthesis originated in the proteobacteria or a common ancestor of the proteobacteria and cyanobacteria.