The Geosiphon symbiosis
A unique symbiosis between a glomeromycotan fungus and cyanobacteria.
Our group worked on the Geosiphon-Nostoc symbiosis for many years, starting from a relatively basic knowledge. Unfortunately, research in our lab had to be stopped, despite the enormous scientific potential of this symbiosis, also to unravel fundamental aspects of the arbuscular mycorrhizal (AM) symbiosis. Reviewers of grant proposals were too much sticking to mainstream approaches (and partly were just incompetent). I mention this so clearly, because this research could have pushed AM research significantly. I am convinced that in future research on this organism will be revived - but it will have to start from scratch, including isolating the organisms from the natural habitat.
We used and proposed the Geosiphon symbiosis as a model for the arbuscular mycorrhiza (AM) symbiosis. However, the Geosiphon symbiosis also is highly interesting in itself, because Geosiphon pyriformis (Kütz.) v. Wettstein is the only known fungus forming endosymbiosis with cyanobacteria (Nostoc punctiforme). Implications of using this symbiosis for AM research are indicated elsewhere. Here, just a mention of the impact of the AM for the entire terrestrial ecosystem as stated by the BEG-Committee (25th May 1993) and many researchers in the field: "The study of plants without their mycorrhizas is the study of artefacts. The majority of plants, strictly speaking, do not have roots; they have mycorrhizas."
Note that some data from some recent publications are not yet discussed here. Also, a genome sequencing project that is driven by a Canadian laboratory is in progress (Krüger and Krüger 2017), but results were not published yet.
The Geosiphon pyriformis - Nostoc symbiosis
Geosiphon pyriformis (Kütz.) v. Wettstein (von Wettstein 1915) forms the only known fungal endocyanosis (endocytobiotic association with a cyanobacterium). The coenocytic fungus forms unicellular, multinucleated cells ('bladders') of up to 2 mm in size (Fig. 1), harboring endosymbiotic, filamentous cyanobacteria of the genus Nostoc. There have been only six reports of this symbiosis being found in nature, at locations ranging from eastern Germany to Austria (Fig. 1b). Probably, the symbiosis is geographically widespread in Central Europe but, due to its small size, rarely reported. Presently, field sites around the small village Bieber in the Spessart Mountains (Germany) are the only known stable natural habitats world-wide (Mollenhauer 1992, Schüßler and Wolf 2005, Krüger and Krüger 2017).
Fig. 1. The Geosiphon-Nostoc symbiosis, isolated from a lab-culture on natural substrate, incubated in liquid medium. The dark appearing bladders are about 1.5 mm in length. The insert shows Geosiphon pyriformis spores, which have a diameter of about 250 µm. All pictures are under copyright.
Fig. 1b. The Geosiphon symbiosis was reported 5 times in the literature, and found close to Munich in the 1970s (Oberwinkler, pers. communication). Presently the only known stable habitat is close to Bieber in the Spessart Mountains. Modified from Schüßler and Wolf (2005). All pictures are under copyright.
The species name 'Geosiphon pyrifome' was sometimes used for the fungus as well as for the symbiosis, because the latter often was regarded as 'phycomycetous lichen'. Nowadays endosymbiotic associations usually are excluded from lichen-definitions (Hawksworth and Honegger 1994). Thus, the species name should be used for the fungus only, also because phylogenetically the Geosiphon fungus belongs to the arbuscular mycorrhiza (AM) forming and related fungi, the Glomeomycota (Fig. 2). Here, the association between the fungus and cyanobacteria is referred to as the Geosiphon-Nostoc symbiosis, or simply the Geosiphon symbiosis, and the species name of the fungus is used in its orthographically correct form, Geosiphon pyriformis (Schüßler 2002).
The individual symbionts
After its original description as Botrydium pyriforme, a siphonal alga (Kützing 1849), Geosiphon pyriformis (as G. pyriforme) was recognized as a phycomycete (fungus with aseptate hyphae; Knapp 1933). Sixty years later, based on suggestions by Walter Gams, it was suggested that Geosiphon could be related to Glomus-like fungi (Mollenhauer 1992). Such fungi form the ecologically and economically extremely important arbuscular mycorrhiza (AM) symbiosis with land plants, so verification would make it conceivable that Geosiphon may also be capable of such association.
Fig. 2. Phylogenetic (maximum likelihood) tree of 'AM fungi' (Glomeromycota), including Geosiphon (modified and updated from Schüßler et al. 2001; Schüßler and Walker 2010; see http://www.amf-phylogeny.com). All pictures are under copyright.
Because the systematics of AM fungi in the last century was based mainly on the characteristics of their spore structure, morphological and ultrastructural criteria of Geosiphon spores were compared with those of some AM fungi (Schüßler et al. 1994). This indeed revealed similarities between G. pyriformis and AM fungi like Diversispora epigaea BEG47 (at that time named Glomus versiforme; see Schüßler et al. 2011). Final evidence showing that Geosiphon is closely related to AM fungi was based on small subunit ribosomal RNA (SSU rRNA) gene sequences (Gehrig et al. 1996). The AM fungi, together with Geosiphon, formed a distinct clade that is not closely related with any other group of the zygomycetes. Further sequence analyses (Sawaki et al. 1999; Schüßler 1999; Schüßler et al. 2001; Redecker et al. 2000b) showed Geosiphon to be most closely related to an AM fungus forming two different spore morphs, at the time named Acaulospora gerdemannii (synanamorph Glomus leptotichum).
Nowadays, the clade containing these lineages is defined as the order Archaeosporales, which represents one of the two basal phylogenetic lineages in the phylum containing the AM fungi (and Geosiphon), the Glomeromycota (Schüßler et al. 2001). In this order, the Geosiphonaceae clusters as sister to the Ambisporaceae, thus appearing to be more derived than the Archaeosporaceae, which branch earlier. This means, that Geosiphon is not representing a sister lineage to the AM fungi, as it sometimes was and is wrongly suggested. It was analyses on the phylogeny of Geosiphon that eventually led to the erection of the Glomeromycota, a widely accepted fungal phylum and, eventually to the phylogenetically based, revised classification of the Glomeromycota (Schüßler and Walker 2010).
The Geosiphon-Nostoc symbiosis attracted interest from the field of AM research. The AM symbiosis is formed by ~80% of all vascular plants studied (Brundrett 2009) and moreover also by lower plants (Read et al. 2000; Schüßler 2000), despite the fact that these plants do not possess roots. By this huge number of plants forming AM it is obvious that the AM is one of the most important factors in land ecosystems (Smith and Read 2008).
The endosymbiont in the Geosiphon symbiosis (Fig. 3) is N. punctiforme, which belongs to a clade of cyanobacteria containing many symbiosis-forming members. In laboratory cultures (Schüßler and Wolf 2005) a strain was used that originally was isolated from the Geosiphon-symbiosis (Mollenhauer 1992). However, various other strains of N. punctiforme from other symbiotic systems (e.g. Anthoceros, Blasia, Gunnera) are also capable of forming symbiosis with G. pyriformis. In the field, G. pyriformis was usually found together with Anthoceros, and the cyanobionts of G. pyriformis associate in symbioses with Anthoceros and Blasia (Mollenhauer 1992).
Fig. 3. Endosymbiotic Nostoc, about 7x6 µm in size, within a Geosiphon bladder. One heterocyst is in focus (arrowhead). All pictures are under copyright.
The 'bacteria like organisms' (BLOs)
It has to be noted, that Geosiphon harbors a further prokaryotic endosymbiont, the so called BLOs (bacteria like organisms; Fig. 4), which are not enclosed by a fungal host membrane but live freely in the cytoplasm (Schüßler et al. 1996, Schüßler 2011). These endosymbiotic bacteria show the typical ultrastructure of those living in most of the AM fungi that were studied for their occurrence. As they are found in very diverse branches of the Glomeromycota they were considered to be widespread glomeromycotan symbionts (Schüßler et al. 1994).
Fig. 4. Electron micrograph of a 'bacteria like endosymbiont' (BLO) in Geosiphon. The BLOs have a diameter of about 0.5 µm and are not enclosed by a host membrane (arrow). The insert shows the plasma membrane of the BLO (arrowhead), as well as its cell wall. Recent studies show them to be Mycoplasma-related, despite the Gram-positive appearance. All pictures are under copyright.
The BLOs are indeed the widespread, ancestral and typical endobacteria in AM fungi. New findings regarding their phylogeny and occurrence in very diverse AM fungal lineages (Naumann et al. 2010) showed that the BLOs are related to the cell wall-lacking Mollicutes. We now know that they are monophyletic and laterally transferred within the AMF since more than 450 million years ago. Their phylogeny and biotrophic lifestyle is shared with the related mycoplasmas, despite the obvious difference of possessing a murein sacculus. It recently turned out that the cell-wall like layer indeed is most likely is not a murein sacculus: in two recent PNAS publications (Torres-Cortés et al. 2015; Naito et al. 2015) describing the genomes of the endobacteria living within AM fungi no cell wall synthesis genes were found. BLOs are Mycoplasma related and evolved within the AM fungi, as diverse communities reflected by plastic genomes.
The Geosiphon symbiosis is facultative for one of the partners (Nostoc can be cultivated without the fungus) and obligate for the other one (Geosiphon is obligatory symbiotic). It is conceivable that the fungus is not restricted to the cyanobacteria as symbiotic partner but also forms symbioses with land plants (see below). However, this assumption is still speculative. In any case, due to its belonging to the Glomeromycota and functional as well as structural similarities to the AM, the Geosiphon symbiosis can play a role as a model symbiosis (Schüßler 2011) for the hard to investigate but extremely important AM, for example for the characterization of symbiosis-related genes (e.g., Schüßler et al. 2006).
Structure, recognition process, and development of the symbiosis
Both symbiosis partners live in the upper layer and on the surface of humid soil, where they get in contact. The interaction is specific by two means: i) only certain Nostoc punctiforme strains are suitable to form this symbiosis, ii) for a successful interaction with the fungus, Nostoc has to be differentiated into a specific stage. This is represented by an early immobile stage of the cyanobacterial developmental cycle, the so called primordium (Mollenhauer et al. 1996). The motile filaments (hormogonia) and late primordial as well as vegetative stages of Nostoc are not recognized by the fungus. When contacting Nostoc, the tip of the fungal hypha bulges out and surrounds part of a cyanobacterial filament, thus incorporating the Nostoc cells (Fig. 5). Usually, 5-15 Nostoc cells are taken up during this process, whereas the heterocysts are never incorporated but ‘cut off' by the fungus (see below). These events are documented in a scientific film available in German and English (Mollenhauer and Mollenhauer 1997).
Fig. 5. CLSM projection of a short hyphae branching from a main hyphae (horizontally oriented, 4-6 µm in diameter) and ‘bulging out’ to enclose a part of a Nostoc filament. The extracellular polysaccharides of Nostoc and the outer layer of the fungal cell wall are labelled by the fluorescence-coupled lectin ConA (green). The Nostoc cells (red autofluorescence, ~4x3 µm in size) that are taken up by the fungal structure show strong deformations and irregular and reduced pigment fluorescence. All pictures are under copyright.
Studies on the development of the Geosiphon-Nostoc symbiosis showed that a successful interaction depends on the appropriate developmental stage of the cyanobacterium (Mollenhauer et al. 1996, Wolf and Schüßler 2005). The life cycle of Nostoc starts from akinetes (spore-like resting stages) leading to vegetative colonies. These colonies release motile trichomes (hormogonia) which are positively phototactic in dim light and negatively in strong light. As a consequence, the hormogonia often congregate just below the soil surface where they spread and meet their symbiotic partners. They eventually undergo a transformation into an aseriate stage called primordium. This then differentiates into so called vegetative cells, which divide and form gelatinous colonies (‘thalli’). Only the very early primordial stage of Nostoc can interact with the fungal partner to give rise to the symbiotic consortium.
The life cycle of the fungal partner starts from resting spores formed in the upper soil layer. The spores (Schüßler et al. 1994) germinate by the outgrowth of a hypha (sometimes more than one), which branches to form a small mycelium of a max. 2-3 centimeters in the soil. When hyphae grow and a hyphal tip contacts a compatible early Nostoc primordium, the fungal hypha bulges out just below the apex. This bulging process is repeated several times so that finally the hyphal tip forms an irregularly shaped structure surrounding a part of a Nostoc primordium. After this incorporation into the fungal hypha, large amounts of cytoplasm streams into this structure. It is swelling and the fungal bladder develops from this Nostoc-containing structure (Fig. 6).
Fig. 6. Young Geosiphon bladders, 100-150 µm in size, formed at the fungal mycelium 7-10 days after initial uptake of the cyanobacteria (left). The irregular structures in the background are vegetatively growing Nostoc colonies. At the right, two mature bladders of about 1 mm length, together with a young bladder, are shown at the right. All pictures are under copyright.
Each individual incorporation event results in the formation of a single pear-shaped above-ground bladder (Knapp 1933). Each bladder represents a polyenergid cell, coenocytic with the fungal mycelium, in which the symbiotic Nostoc cells divide and become physiologically active. Laboratory culturing experiments have shown that, as for AM, phosphate limitation (1-2 µM) of the nutrient solution triggers the stable establishment of the symbiosis. N limitation seems not to be a crucial factor. The same situation is found in the natural habitat, so P-limitation seems to be a driving factor for establishing this symbiosis.
Within the first hours after incorporation into the fungal cytoplasm, the Nostoc filaments become heavily deformed, and some cells may die during this process. The photosynthetic pigments bleach considerably (Fig. 5; Mollenhauer et al. 1996; Schüßler and Wolf 2005). These alterations and significant changes in ultrastructure suggest that during the initial state of endocytotic life the incorporated cyanobacteria suffer severe stress. Within 2-3 days, the enclosed Nostoc cells recover and begin to multiply and grow to reach as much as six times the volume of free-living cells (Schüßler et al. 1996; Mollenhauer and Mollenhauer 1997). Under phosphate limitation the endosymbiotic cyanobacteria divide much faster and form a much higher biomass compared to the free living ones (unpublished). In the symbiosis, the Nostoc cells arrange in filaments in which heterocysts are formed with the same frequency as in the filaments outside the bladders (if cultured under nitrogen limitation). Mature Geosiphon bladders can then reach more than 2 mm in length, and up to six months in lab cultures. They possess a turgor pressure of about 0.6 MPa (=6 bar) (Schüßler et al. 1995).
Structure and compartmentation
The Geosiphon bladder is effectively a multikaryotic cell, coenocytic with the fungal mycelium in the soil. It shows a strong polarity and has a photosynthetic active region in the apical part of the cell exposed to light and air, and a whitish appearing storage region in the basal part embedded in the soil surface, containing many lipid droplets. The center of the bladder is highly vacuolated. Schematic drawings of the compartmentation of Geosiphon are shown in Fig. 7. Ultrastructural observations show the G. pyriformis symbiosis as a system with very close contact between the partners. In fact, it is a symbiotic consortium of three organisms: 1) the fungus, supplying the consortium with inorganic nutrients like phosphate, trace elements, and water, 2) the cyanobacteria, supplying the consortium with carbohydrates by photosynthesis and, at least under some conditions, nitrogen compounds by N2 fixation, 3) the ‘bacteria-like organisms’ (BLOs), which are Mollicutes-related Mycoplasma-like endobacteria (MREs), with yet unknown function.
Fig. 7. Schematic representation of the compartmentation of the Geosiphon-Nostoc symbiosis. At the right, a magnification of the peripheral part of the bladder is shown. Drawings are based on electron microscopical observations. BLO, bacteria-like organism; CW, cell wall; M, mitochondrion; N, nucleus; NC, Nostoc cell; PM, plasma membrane; SM, symbiosome membrane; V, vacuole. All pictures are under copyright.
Within the bladders, the cyanobacteria are located peripherally in a single, cup shaped (often with invaginations) compartment, the symbiosome. The Nostoc cells divide and are physiologically active as endosymbionts in this compartment. Within the cytoplasm of the fungus, glycogen granules exist as storage compounds. No dictyosomes could be found; microtubules can rarely be observed. Fixation of the bladders during preparation for electron microscopy was often inadequate, probably due to the low cell wall permeability, but could be improved by using microwave acceleration.
Preparation of the G. pyriformis spores for electron microscopy (Schüßler et al. 1994) was even more difficult. This problem, caused by the thick spore wall being only slowly permeable to fixatives, also exists with other glomeromycotan species (Maia et al. 1993). Two main storage compounds occur inside the spores: lipid droplets of different size, and ‘structured granules’ that occupy about 25% of the volume. The latter are discussed below with respect to element analysis. They show paracrystalline inclusions, as also found in spores of some other glomeromycotan fungi. Small vacuoles are found in germinating spores and hyphae, often containing dark deposits. These are similar to the deposits in AMF and probably polyphosphate precipitates. The ultrastructure of the Geosiphon-symbiosis was first studied by (Schnepf 1964) and was the crucial investigation leading to the theory of the compartmentation of the eukaryotic cell. The space between the symbiosome membrane and the wall of the enclosed Nostoc cells is only 30-40 nm thick and contains a layer of electron microscopically opaque and amorphous-appearing material which was originally assumed to be slime produced by the endosymbiont (Schnepf 1964). Later ultrastructural and confocal laser scanning microscopical (CLSM) studies by means of affinity techniques revealed that this amorphous layer inside the symbiosome contains chitin (Schüßler et al. 1996). Labelling with wheat germ agglutinin (WGA)-gold conjugates confirmed these results. Thus, the electron opaque layer within the symbiosome represents a ‘rudimentary’ fungal cell wall, showing that the symbiosome membrane surrounding the Nostoc cells is homologous to a fungal plasma membrane.
Clear similarities exist between the fungal cell wall material present in the symbiosome space of the Geosiphon-symbiosis and the thin arbuscular cell wall bordering the symbiotic AM fungus from the colonized plant cell in the AM: both are electron-dense after OsO4 fixation, about 30-40 nm thick, and show the same amorphous structure and appearance. In general, the ultrastructural appearance of G. pyriformis is similar to that of AMF. Considering also the phylogenetic position of G. pyriformis and the known or proposed nutrient flows between the symbiotic partners, it was suggested that the symbiotic interface in the AM and the Geosiphon-symbiosis are homologous (Schüßler et al. 1996). The main difference between the symbioses is the relation of macro- and microbiont. In the Geosiphon-symbiosis the photoautotrophic partner (cyanobacterium) is the microsymbiont, whereas in the AM it is the macrosymbiont (plant) (Figs. 7,8).
Element composition and distribution
It is not yet known why AM fungi cannot be cultured axenically. Also, there is little information available about their trace element requirements and general element composition. Considering the fact that these fungi supply the majority of land plants with inorganic elements, studies on the element composition and transport processes are interesting topics. We have used PIXE (proton induced X-ray emission) measurements to obtain first indications on the macro- and microelement composition of the spores and symbiotic bladders. The element content of some subcellular compartments could be quantitatively measured and, by a differential approach, calculated. PIXE, combined with STIM (scanning transmission ion microscopy) allowed elemental concentrations to be absolutely quantified with a lateral resolution in the 1 µm range and with high accuracy and precision (Maetz et al. 1999a).
Results on the G. pyriformis symbiotic bladders (Maetz et al. 1999b) showed that the fungal partner of the symbiosis, grown on a nutrient poor solution (e.g., containing 1 µM phosphate), accumulate P in the fungus in high concentrations (about 2%), but not in the symbiosome. The P is probably stored as polyphosphate in the vacuoles, as for AM (and many other) fungi. High amounts of Cl (about 2.5%) and K (about 8%), which appear to play major roles in osmoregulation of the fungus, are found (all values given here are related to dry weight, ppm = µg/g DW). The symbiosome (including the cyanobacteria) contains only small amounts of these elements. This is in line with presumed high concentration of monovalent ions in the fungal vacuoles. The macroelements Mg, S, and Ca and the microelements Fe, Mn, Cu, and Zn occur in concentrations comparable to those found in plants. The Se concentration is below 1 ppm. Mo is present within the symbiosome in very low amounts, compared to the rest of the bladder, although Mo is a constituent of nitrogenase, required for N2-fixation of the cyanobacteria. Reasons for this might be that other Mo-enzymes (e.g. nitrate reductase, sulfite oxidase) occur in relevant amounts in the fungal cytoplasm or that Mo is located in the fungal vacuole. Mn and Ni, on the contrary, are present in the symbiosome in much higher amounts than in the rest of the bladder. Much of the Mn (approx. 50 ppm, which is comparable to values found in plant leaves) probably is contained in the water-cleaving Mn protein of photosystem II. Some may be from other enzymes, e.g., Mn-superoxide dismutase (SOD). A likely candidates for enzymes containing the ~50 ppm Ni is cyanobacterial (or secreted fungal) urease; other Ni-containing bacterial enzymes are Ni-SOD and NiFe-hydrogenases.
Unpublished results on the element composition of the Geosiphon spores show that the structured granules (SGs), which are 4-6 µm in diameter, located each within a vesicle, together occupy about 25% of the spore volume and contain most of the total P, K, and S. The S concentration of the spore cell wall is ~0.25%, probably because of high protein content, as shown for an AM fungus (Bonfante and Grippiolo 1984). Compared with the bladders, Cl and K are concentrated within the spores in much lower amounts.
Signal exchange between host and cyanobacterium
It is not known what triggers the recognition process and the morphological changes during the symbiosis establishment. Microscopical studies give no hints for any chemotactic or otherwise directed growth towards the respective symbiosis partner, but the symbiosis compatible Nostoc stage can be synchronized (Schüßler and Wolf 2005) to study this more in detail. Cells of particular strains of N. punctiforme can be incorporated by Geosiphon, resulting in formation of functional symbioses. For other strains, although incorporated, the formation of symbiotic bladders is blocked in an early stage of development. Yet other N. punctiforme strains are not incorporated at all. Further evidence for a specific recognition process is the fact that, among the various developmental stages of Nostoc, only the early primordia are incorporated by the fungus, existing for ~3-12 hours during the life cycle. Not only is the physiological activity of the primordia different from the other stages of the Nostoc life cycle (Bilger et al. 1994) but also the composition of the gelatinous envelope. When differentiating into 'symbiosis compatible' primordia, a mannose containing slime is produced by the cells, whereas other sugars within the extracellular glycoconjugates could be detected only in earlier or later stages of the life cycle (Schüßler et al. 1997). The heterocysts (specialized N2-fixing cells), differentiating at regular spacing along the filaments of the Nostoc primordia when growing under nitrogen limitation always remain outside the fungal hypha during the incorporation process (Mollenhauer et al. 1996). They are not surrounded by a newly appearing mannose-containing glycoconjugate (Schüßler et al. 1997), also indicating a specific recognition of the early primordial cell surface by the fungus. Thus, alterations of extracellular glycoconjugates could be involved in partner recognition. Some unpublished data further indicate a lectin mediated process.
Host-cyanobiont interactions after symbiosis establishment
Morphological changes of the symbionts
The most obvious morphological change taking place after partner recognition is the formation of the Geosiphon bladder. Mature bladders represent big cells, which are coenocytic with the mycelium. They show a clear polarity, with the photosynthetically active symbiotic compartment (symbiosome) located in the apical part of the bladder (Figs. 6,7).
The symbiosome is derived from the invaginated plasma membrane of the fungus and contains the cyanobacteria, which also show an obvious morphological modification, namely the about 6-8 fold volume compared to free living vegetative cells. This is probably caused by the high osmotic pressure inside the bladders. In many plant-symbioses with cyanobacteria are known to increase in size (Bergman et al. 1992; Grilli Caiola 1992), probably as a reaction to the higher osmotic pressure of the surrounding medium. High NaCl concentrations are also known to cause an increase in volume of cyanobacteria (Erdmann and Schiewer 1984). For Geosiphon bladders, the iso-osmolar concentration of sorbitol was measured with oil-filled microcapillaries and determined to be 220-230 mM, corresponding to a turgor pressure (P) of about 0.6 MPa (Schüßler et al. 1995).
However, despite the increase in size, the Nostoc cells inside the Geosiphon bladder have an almost normal ultrastructure. They contain a high number of thylakoids and carboxysomes; one alteration is that the outer membrane is hardly recognizable electron microscopically. Heterocysts are formed with the same frequency as in free living colonies, but their cell wall is thinner in the symbiosis, possibly indicating a lower surrounding O2 concentration.
CO2 fixation and transfer
14C-tracer studies showed that the Geosiphon bladders fix CO2 both in light and in darkness, whereas the rate of CO2 fixation in light is much higher (Kluge et al. 1991). In light, largely phosphate esters, poly-glucans, free sugars (including trehalose and raffinose), some amino acids and organic acids trap 14C. In darkness only malic and fumaric acid together with some amino-acids appear as labeled products. High photosynthetic activity of the endosymbiotic Nostoc cells was also shown by photosystem II chlorophyll-fluorescence kinetics (Bilger et al. 1994). The symbiotic Nostoc cells achieve much higher steady-state quantum yields and electron transport rates when compared to free living Nostoc.
In electrophysiological experiments (unpublished) there were no changes in plasma membrane potential if hexoses and larger amino acids were applied to the nutrient solution. Metabolism of radioactively labelled hexoses by the bladders also could not be detected after usual incubation times. Low cell wall permeability was discussed as the likely reason for the lack of uptake of monosaccharides. This theory is supported by observations showing that the presence of solutes with large molecule radii leads to irreversible cytorrhysis, i.e., collapse of the whole bladder including the cell wall, whereas in presence of small solutes plasmolysis occurred (or cytorrhysis was quickly reversed). This different transport behavior is presumably due to the selective permeability of the bladder wall. By systematically using solutes with known molecular radii, it was shown that the limiting pore radius of the G. pyriformis bladder wall is approx. 0.5 nm, which, compared with other cell wall types, is very small (Schüßler et al. 1995). Such a pore size is too small for an efficient permeation by, e.g., hexose molecules from the outside, but it allows permeation of inorganic hydrated ions like phosphate. Provided that such a small pore size holds true also for the hyphal cell wall, the fungus would not be capable of efficient saprobic acquisition of organic molecules such as glucose, sucrose, larger amino acids, etc. However, cell wall permeability is a complex topic and, e.g., the thin hyphae formed by AMF, known as ‘branching absorbing structures’, might possess different cell wall permeability.
Because AMF obtain up to 20% of the plant-fixed CO2, putatively as monosaccharides and moreover, as recently unraveled, also by plant derived lipids (Keymer et al. 2017), the study of a glomeromycotan sugar transporter that could play a role in the C-transfer from plants to AMF was an important goal. The first glomeromycotan monosaccharide transporter that had been characterized was from the Geosiphon-symbiosis, GpMST1 (Schüßler et al. 2006). This putatively symbiosome-membrane located transporter was demonstrated also to transport sugars potentially deriving from plant cell-wall material (Fig. 8). The GpMST1 sequence moreover provided valuable data for the isolation of orthologues from other AMF and could eventually lead to the understanding of C-flows in the AM.
N2 fixation and transfer
The capability of N2 fixation is indicated by the occurrence of heterocysts, and considerable nitrogenase activity was shown for the bladders (Kluge et al. 1992). In contrast to symbioses of Nostoc with plants, where usually a great enlargement of the heterocyst number indicates N2 fixation as the major role of the cyanobacteria, in Geosiphon the relative heterocyst number does not change. Here, the major role of the endosymbiotic Nostoc is photosynthesis. However, matter exchange between the partners is still poorly investigated, and it is even possible that the second bacterial endosymbiont (BLO; Figs. 4), which is typical for many glomeromycotan fungi, may contribute to N2 fixation.
For the endosymbiotic Nostoc cyanobacteria all inorganic nutrients except N have to be provided by the fungus, as the cyanobacteria live intracellularly. As shown by electrophysiological experiments (unpublished), inorganic ions (nitrate, chloride) and small organic molecules (e.g., glycine, cysteine) lead to rapid, transient depolarization of the plasma-membrane potential of the G. pyriformis bladders, indicating that these substances are actively taken up from the outside.
Regarding N-uptake from the environment and N-transfer in the symbiosis, three ammonium transporters of Geosiphon pyriformis have been characterized in the heterologous yeast system (Ellerbeck et al. 2013), which may play roles in the N-nutrition of the fungus.
Fig. 8. The symbiotic interface and bidirectional nutrient flows in the Geosiphon symbiosis, in comparison to those in the arbuscular mycorrhiza (AM) (from Schüßler et al. 2006). All pictures are under copyright.
Is the symbiosis mutualistic?
The fungus in the Geosiphon symbiosis belongs to the Glomeromycota (Fig. 2) and is, like these, obligatory symbiotic. It is not yet known why glomeromycotan fungi are not capable of non-symbiotic life. Maybe it will be possible in future to develop special culture methods for in vitro growth of AM fungi, including Geosiphon. Generally, in nature glomeromycotan fungi seem not to be capable of saprotrophic life, but are dependent on their symbiosis partners for C-delivery. For Geosiphon bladders it was shown that only molecules can pass the cell wall (Schüßler et al. 1995). The pore size is too small for the uptake of hexoses or disaccharides from the outside, but allows permeation of inorganic ions like phosphate. This might reflect the situation in nature. However, it is also well possible that the fine hyphae growing into the substrate show higher permeability. In any case, by incorporating Nostoc the fungus obtains the needed source for organic compounds.
Nostoc also takes advantage from the co-operation with the fungal host, which probably improves the supply of the endosymbiont with water, phosphate, and also CO2. It is striking that all inorganic nutrients, except N, have to be delivered by the fungus, since the cyanobacteria live intracellular. It should also be kept in mind that the establishment of the Geosiphon symbiosis, like usually also true for the AM, is strongly promoted by P-limitation, which is a severe stress for the photobiont. The endosymbiotic Nostoc cells thus divide and grow faster and bigger than their free living sisters. Preliminary studies moreover show that the intracellular cyanobacteria are protected against heavy metals, which accumulate in the fungus (Scheloske et al. 2001). Therefore, as in the AM, the photobiont seem to be protected against abiotic stress factors, in the Geosiphon symbiosis.
Evolutionary implications with ecological meaning
Most vascular plant species form AM (Smith and Read 2008), including gametophytes and sporophytes of many ferns (Peterson et al. 1981) and Lycopodiaceae (Schmid and Oberwinkler 1993). Also, except for mosses, all groups of bryophytes contain species with AM associations (Ligrone 1988; Ligrone and Lopes 1989; Stahl 1949; Fonseca et al. 2009), indicating an early origin of the AM symbiosis.
In fact, the AM fungi have an ancient fossil record history. Many of the oldest and best preserved ~400 MY old AM fungal fossils in association with plants are known from the Rhynie Chert, radiometrically dated to the early Devonian (e.g., Remy et al. 1994; Dotzler et al. 2009). The oldest known fossils of AM fungal spores and hyphae are from ~460 MY old Ordovician dolomite rock of Wisconsin (Redecker et al. 2000a) and it was concluded that terrestrial AMF already existed at a time when the land flora most likely consisted only of bryophyte-like ‘lower’ plants, 460 MY ago.
From fossil cryptospore assemblages sharing characters with those of extant liverworts (found in what was eastern Gondwana) (Rubinstein et al. 2010), it is estimated that land plants are more than 470 MY old (Early Middle Ordovician). The diversity of these assemblages implies an earlier, perhaps even Cambrian, origin of embryophytes. Early vascular plants already existed ~420 MY ago (Middle Silurian; Cai et al. 1996). A recent molecular clock study (Smith et al. 2010) suggested an origin of land plants around ~477 MY, but this dating in fact refers to the split between bryophytes and the remaining lineages, not the (presumably earlier) origin of the land plant lineage itself. Therefore, a minimum age of 420 MY for the liverwort-vascular plant divergence must be assumed and bryophyte like land plants already were present 510-470 MY ago.
Altogether, these data provide strong support for the hypothesis of Pirozynski and Malloch 1975 that AMF symbioses played a crucial role on the colonization of the land by plants, evolving from a partnership between two aquatic types of organism, algae and ‘oomycetous' fungi (the authors recognized the difference between AM fungi and other ‘phycomycetes’, and thus interpreted them as ‘oomycetes’, which are nowadays known not to be fungi), as the initial step of land plant evolution. A mycotrophic lifestyle could have been essential for an efficient supply of plants with water and nutrients from the soil (Malloch et al. 1980; Marschner and Dell 1994). However, molecular clock estimates always date the origin of the AM fungal lineage as to be at least 50, possibly more than 200 MY earlier than that of land plants. If this holds true, it implies that that there were other types of associations formed by AMF before land plants existed, whether saprobically, parasitically, or already mutualistically. Geosiphon pyriformis, representing a symbiotic association between a glomeromycotan fungus and a photoautotrophic prokaryote, may reflect such an ancestral partnership, and thus, indirectly but substantially, supports the view of Pirozynski and Malloch 1975 regarding pre-Embryophyta associations of AM fungal predecessors. It is very plausible to assume that in the beginning of terrestrial plant life also other associations between glomeromycotan fungi and photoautotrophic organisms (like the ubiquitous cyanobacteria) existed. The present knowledge regarding AM fungi and AM symbiosis evolution was recently discussed and reviewed in Schüßler and Walker 2011.
In summary, glomeromycotan fungi may have adapted to symbiotic life more than 500 MY ago. Without fossil support this is speculative, but G. pyriformis clearly confirms the ability of glomeromycotan fungi to form symbioses with even prokaryotic photoautotrophic organisms. Therefore, cyanobacterial symbioses formed by glomeromycotan fungi could have been an ecologically important step for the colonization of the land habitat.
Arbuscular mycorrhizal fungi form symbioses with most land plants, and AM fungi (e.g., Claroideoglomus claroideum) can be symbiotic with such widely divergent photoautotrophs as hornworts and vascular plants (Schüßler 2000), and the genetic base for this interactions is highly conserved (Wang et al. 2010) and even cyanobacteria in the case of Geosiphon. There are some very fundamental and conserved mechanisms of plant-microorganism interactions present amongst the different AM (-like) associations. When conducting eco-physiological studies involving plants, it is important to consider that in nature the mycorrhizal fungal partners are the main facilitators of nutrient uptake, rather than the plant roots alone. If, as is thought, mechanisms of nutrient acquisition by land plants co-evolved since their origin with the AMF, ecologically and economically important questions might be answered by using the Geosiphon-symbiosis as a model.
A network between fungi, cyanobacteria, and plants?
Against the above described evolutionary background, the interesting question arises as to whether G. pyriformis itself can act as fungal partner to form AM. Unpublished results showed that Geosiphon rDNA can be PCR amplified from plant roots and bryophytes growing in the natural habitat of Geosiphon. However, it cannot be completely ruled out that the sensitive nested PCR approach is detecting tiny amounts of DNA from externally attached hyphae. Future studies at sites where the cyanobacteria symbiosis of Geosiphon never occurs will have to show, whether Geosiphon is indeed forming an AM. If this would be the case, a complex network of ecological importance may be imagined (Fig. 9).
Fig. 9. A putative ecological 'symbiosis network' between cyanobacteria, fungi and plants. Associations or interactions which are not highlighted with blue arrows are hypothetical. Further endosymbionts like the BLOs in glomeromycotan fungi also play an unknown, but probably important role in this network of intimate associations. All pictures are under copyright.
The molecular probes to screen for the occurrence of Geosiphon in the soil and plant roots had been developed, but lack of funds prevented their application. If Geosiphon indeed forms AM with plants, a complex network of biotic interactions would exist in the natural habitat. Within such a network, symbiotic Nostoc could be exchanged between Geosiphon and bryophytes, and Geosiphon could simultaneously form endosymbiosis with Nostoc and AM with plants, thus e.g. transporting and delivering N2 fixed by the cyanobacteria to the plants. I wish future researchers in this field a lot of success and more luck with reviewers, hopefully understanding the potential on this highly interesting symbiosis.
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