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Keywords:

  • akinete;
  • Azolla;
  • biofilm;
  • cell differentiation;
  • cyanobacteria;
  • membrane vesicle

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    The nitrogen-fixing symbiosis between cyanobacteria and the water fern Azolla microphylla is, in contrast to other cyanobacteria–plant symbioses, the only one of a perpetual nature. The cyanobacterium is vertically transmitted between the plant generations, via vegetative fragmentation of the host or sexually within megasporocarps. In the latter process, subsets of the cyanobacterial population living endophytically in the Azolla leaves function as inocula for the new plant generations.
  • • 
    Using electron microscopy and immunogold-labeling, the fate of the cyanobacterium during colonization and development of the megasporocarp was revealed.
  • • 
    On entering the indusium chamber of the megasporocarps as small-celled motile cyanobacterial filaments (hormogonia), these differentiated into large thick-walled akinetes (spores) in a synchronized manner. This process was accompanied by cytoplasmic reorganizations and the release of numerous membrane vesicles, most of which contained DNA, and the formation of a highly structured biofilm.
  • • 
    Taken together the data revealed complex adaptations in the cyanobacterium during its transition between plant generations.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Among the various cyanobacterial–plant symbioses, the one between cyanobacteria and the water fern Azolla spp. is the only one where the colonizing cyanobacteria are kept through Azolla generations. They are either maintained through the vegetative fragmentation of the plant or kept between Azolla generations via transfer of the cyanobacterium into the reproductive sporocarps of the plants. Sporocarps (mega- and microsporocarps) develop in pairs from each lateral branch point on the ventral side of the Azolla sporophytes (Lumpkin & Plucknett, 1980; Peters & Meeks, 1989; Braun-Howland & Nierzwicki-Bauer, 1990). A small cyanobacterial population is attracted to the apical region of the Azolla sporophyte harbouring the young developing megasporocarp pairs. The cyanobacterium subsequently enters these megasporocarps and gets ‘entrapped’ in a small opening or cavity located below the indusium (upper part of the sporocarp) and on top of the underlying sporangium (Perkins & Peters, 1993; Zheng et al., 1988, 1999; Zheng & Huang, 1994). In comparison to our knowledge of the structure and infection by the cyanobacteria of the developing megasporocarp, our knowledge of the fate of the entrapped cyanobacterial population is limited and improving it is therefore an important overall aim of the present study.

Additionally, the Azolla plant symbiosis is the only obligate three-component symbiosis, composed of heterotrophic bacteria (the bactobionts), cyanobacteria (the cyanobionts) and the eukaryotic host, the pteridophyte Azolla (Zheng et al., 1988; Nierzwicki-Bauer & Aulfinger, 1990, 1991; Carripico, 1991; Lechno-Yossef & Nierzwicki-Bauer, 2002). In natural environments, free-living cyanobacteria are frequently associated with other bacterial populations and often form microbial aggregates, thicker ‘mats’ or highly structured biofilms (Ramsing et al., 2000; Vincent, 2000). Although cyanobacteria form intimate symbioses with many plant taxa (Rai et al., 2002), there is no information available on the occurrence of this phenomenon in relation to plant symbioses, such as during the vertical transfer of cyanobacteria and bacteria between the Azolla generations.

In order to gain a deeper understanding of the mechanisms involved in the unique vertical transfer of cyanobacteria between plant generations, we attempted to clarify aspects related to coordination between plant and cyanobacterial developmental events during the transfer of the symbionts between Azolla microphylla generations. A special focus was on cyanobacterial cell differentiation events, membrane vesicle liberation and cyanobacterial biofilm formation.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plant cultivation and treatment

Azolla microphylla Kaulfus (IRRI No. 4018) was grown in a glasshouse pond at the Department of Botany, Stockholm University (Sweden), and the Biotechnology Institute, Fujian Academy of Agricultural Sciences (China). Twenty pre-sporulating and sporulating Azolla fronds were grown individually at a temperature ranging between 20 and 28°C and examined every day under a stereomicroscope (Nikon SMZ-U, Japan). The development of the sporocarp from the appearance of the sporocarp initial at the shoot apex to the mature megasporocarp was followed and the time recorded for different developmental stages (size and shape). Sporocarps at various developmental stages, from the apex to the fifth branch points further down the main stem of Azolla, were carefully collected with a fine needle and scalpel under the stereomicroscope. The sporocarps were either processed immediately or kept individually in Eppendorf tubes containing distilled water at 4°C for 2 d before subsequent treatments.

Light microscopy

The associated cyanobacteria were squeezed out from three developmental megasporocarps and examined with light and fluorescence microscopy (Olympus BX60, equipped with a 460–490 nm filter). To estimate the number of akinetes in the mature megasporocarp, the lignified upper part of the indusium was removed using needles and the akinetes were dispersed on a glass slide and counted under the microscope. Semithin sections (0.5 µm) of developing sporocarps embedded in Epon 812 (see later) were stained with 0.5–0.7% toluidine blue (Sigma-Aldrich, Steinheim, Germany) and examined using light microscopy.

Specimen preparation for scanning electron microscopy (SEM)

The megasporocarps were fixed in 2.5% glutaraldehyde in phosphate-buffered saline (PBS, pH 7.4) at 4°C for 2 d followed by post-fixation in 1% osmium tetroxide in PBS for 90 min. Fixed samples were rinsed three times with PBS for 10 min each and dehydrated in a graded series of ethanol from 70 to 100% followed by critical point drying using CO2 as transition fluid. The dried specimens, mounted on stubs, were coated with a platinum and palladium layer of c. 20 nm using IB-5 sputter. Examination was made with a JSM-35CF scanning electron microscope (Japan).

Specimen preparation for transmission electron microscopy (TEM)

For structural analysis, the developing sporocarps were fixed and dehydrated as mentioned in the previous section, followed by embedding in Epon 812 (TAAB Laboratories Equipment, Reading, UK). For immunogold labeling, specimens were fixed in 3% paraformaldehyde in PBS for 1 h and washed three times in PBS for 10 min each, dehydrated stepwise in ethanol (70–100%), and embedded in LR White (TAAB, Laboratories Equipment). The samples were sectioned using an ultramicrotome (LKB Ultratome III), and mounted on copper or gold grids. Sections for the ultrastructural analyses were stained for 15 min with uranyl acetate in 50% ethanol followed by 2 min in lead citrate. Examinations were performed with a Zeiss EM-906 transmission electron microscope, operating at 80 kV accelerating voltage.

Immunogold labeling

Grid-mounted ultrathin sections for immunogold labeling were blocked in 10% (w/v) bovine serum albumin (BSA, Sigma A2153) in PBS for 1 h, followed by incubation overnight at 4°C in primary antibody (mouse anti-DNA, Boehinger Mannhein, Germany) diluted 1 : 20 in PBS. Subsequently the grids were washed in PBS (3 × 15 min) and incubated in polyclonal rabbit anti-mouse IgG antibodies (Dako A/S, Glostrup, Denmark) diluted 1 : 100 in PBS for 45 min at room temperature. After washing in PBS (2 × 15 min), the grids were incubated in affinity-purified polyclonal goat anti-rabbit IgG antibodies with 5 nm gold particles attached (GE Healthcare, Uppsala, Sweden) for 45 min, washed in PBS (2 × 15 min) followed by 2 × 15 min washing in distilled water. The grids were air-dried and examined with a transmission electron microscope (Zeiss EM-906) as described earlier. A negative control, omission of the primary antibody, was included to test the specificity and background labeling of the secondary antibodies used.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Megasporocarp development and colonization by cyanobacteria

The time between the appearance of the sporocarp initials at the A. microphylla shoot apex and their maturity, reached at about the fifth branch point (counted from the plant apex) of the A. microphylla fronds, was estimated to approx. 17 d under the growths conditions used. However, the time for their development is strongly influenced by temperature and might also vary between different Azolla species, and can therefore not be generalized. The development of the entrapped cyanobacteria in the megasporocarps of the Azolla fronds located at the second, fourth and fifth branch points, c. 5, 10 and 17 d old, respectively, was examined by bright field and epifluorescence light microscopy and scanning electron microscopy. As seen in Fig. 1(a,c,e), the size of the developing sporocarps increased considerably during maturation along the Azolla fronds. Throughout this developmental sequence, the cyanobacteria were clearly visible as bright blue-green clusters (Fig. 1a,c (left hand arrows) and e); or as red fluorescing clusters, through the megasporocarp wall (Fig. 2a); and, after sectioning the sporocarp (right hand arrows in Fig. 1a,c), already in the youngest sporocarps (second branch point). These cyanobacterial clusters colonized the ‘indusium chamber’ in the upper part of the sporocarps (Figs 1a,c,e, 2a,b). As the enclosed sporangium developed, the relative proportion of the cyanobacterial community became progressively smaller, being pushed up towards the indusium by the growing and maturing sporangium (Fig. 1a,c,e).

image

Figure 1. Development of the Azolla microphylla megasporocarps colonized by cyanobacteria, demonstrated using stereomicroscopy and light microscopy. (a) A young megasporocap (second branch point) of the Azolla frond – the pigmented cyanobacterial colony (arrows) is clearly visible in the indusium chamber above the megaspore. (b) Young nonheterocystous hormogonial filaments still common at this developmental stage. (c) A megasporocarp located at the fourth branch point of the Azolla plant. In the longitudinal semithin section of the sporocarp, the small proportion of the cyanobacterial filaments is apparent (right hand; arrow). (d) At this stage, most of the cyanobacterial cells in the filaments have differentiated into proakinetes, recognized by their oblong shape and larger cell size. (e) A mature megasporocarp located the fifth branch point of the Azolla plant with the cyanobacteria constituting a small proportion of the megasporocarp (arrow). (f) The cyanobacteria now appear as fragmented filaments of mature akinetes with numerous large cyanophycin granules in the cytoplasm (darker granules). The approximate age of the developing megasporocarp is indicated below the figure. Bars, 100 µm (a, c, e); 10 µm (b, d, f).

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image

Figure 2. Colonization of the Azolla microphylla megasporocarp by hormogonia-like cyanobacterial filaments. (a) Fluorescence microscopy of a developing sporocarp (sp) showing the red fluorescing chlorophyll a-containing hormogonium filaments entering the indusium camber through a pore (white arrow). (b) A longitudinal section through the indusium chamber enclosing the large megaspore when viewed by SEM. The entrance pore is still open (black arrow). The cyanobacterial filaments are seen in close contact with the megaspore (sp). Differentiation into akinetes has commenced. (c) Higher magnification of cyanobacteria in (b), showing the oblong proakinetes (pa) and the closely associated, mostly rod-shaped, bacteria (arrows). Bars, 10 µm.

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From hormogonium-like filaments to chains of akinetes

The small-celled cyanobacterial filaments entered the developing sporocarps of A. microphylla through pores at the top of the indusium (Fig. 2a,b). The filaments were, at this stage, already associated with various bacteria (Fig. 2c). As bacteria were also observed at the megaspore initials, the associated bacterial population most likely originates from the apical region (data not shown). A series of major and synchronized developmental changes in the cyanobionts were apparent through the megasporocarp colonization process. In the mature Azolla leaf cavities, the cyanobacterium is typically composed of large-celled (vegetative cells being 3 × 6 µm in size) multi-heterocyst filaments, with akinetes (spores) being occasionally observed in older parts of the plant (data not shown). However, during colonization of the megasporocarp, the cyanobacterium appeared as long motile hormogonium-like filaments, characterized by small and dividing vegetative cells, approx. 2–2.5 µm in width and 2.5–3.5 µm in length, lacking heterocysts (Figs 1b, 3a). On entering through the pore, the size of the hormogonium cells started to increase and could be recognized by a rounder shape (Fig. 3b). Subsequently, these cells (approx. 2.5–3 µm in width and 3–5.5 µm in length) started to differentiate into chains of considerably longer and larger proakinetes (Figs 1d, 2b,c, 3b,c). Along with the akinete development, the filaments also started to fragment. In the mature megasporocarps at the fifth branch point, the majority of the vegetative cells had differentiated into large (5–6 µm in width to 8–12 µm in length), mostly individual, mature akinetes (Fig. 1f). Besides their size, akinetes were recognized by the presense of the numerous larger, electron-dense cyanophycin granules (Figs 1f, 3c,d), functioning as N-reserves (composed of arginine and aspartate). Proakinetes were identified by the formation of a loose fibrillar envelope around the oblong cells (Fig. 3d, insert) and a reorganization of the vegetative cell (thylakoid) membranes and cellular contents. Heterocysts were not observed in cyanobacteria of mature sporocarps. After completion of the sporocarp colonization, the sporocarp pores were closed, hindering further colonization by cyanobacteria and bacteria. The numbers of akinetes present in 25 megasporocarps were counted and ranged from 408 to 595, with an average of 492 akinetes per megasporocarp.

image

Figure 3. Micrographs of cyanobacteria residing in the indusium chamber of differently aged Azolla megasporocarps. (a–c) Light microscopy of cyanobacteria at different developmental stages. (a) A young hormogonium filament squeezed out from a 5-d-old megasporocarp. Note the small cells and the homogenous cellular content. (b) Cyanobacteria in a 10-d-old megasporocarp with rounder and larger cells starting to accumulate dense cyanophycin granules of varying sizes. (c) A filament about to fragment from a 15-d-old megasporocarp. It consists of almost fully mature akinetes, with numerous scattered cyanophycin granules. (d) TEM micrograph of a hormogonium-like filament entrapped in a young megasporocarp. Along the filament a gradual development of akinete initials is apparent, recognized by their cell elongation (as seen also in b) and the electron-dense, large cyanophycin granules (cg). The insert depicts an enlargement of the cell wall showing the initiation of a deposition of extracellular fibrillar material (fm) at this stage. Bars: 10 µm (a–c); 1 µm (d), 0.1 µm (insert in d).

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Membrane vesicles (MVs) liberation

Once the cyanobiont had entered the indusium chamber and the differentiation into proakinetes/akinetes was initiated, numerous membrane vesicles (MVs) were being discharged from the filaments as revealed using TEM analyses (Fig. 4a–c). Approximately 52% of the examined cyanobacterial cells in the indusium chamber released MVs. The likewise enclosed bacteria also released MVs, but these were smaller and fewer (approx. 24% of the bacterial cells examined). The release of MVs was accompanied by deposition of fibrillar material outside the cyanobacterial cell wall outer membrane (Fig. 4c). The latter eventually gave rise to the thick external cell envelope characterizing the akinetes. After the formation of the akinete envelope, MVs of different sizes appeared in the transparent area between the original cell wall and the new envelope (Fig. 4d), indicating that the MVs were produced and secreted from the cyanobacterium. The MVs released often fused to the deposited fibrillar envelope surrounding the cells (Fig. 4c,d). As the outer envelope became wider and multilayered, the MVs were also observed outside the envelope (Fig. 4f). Mature akinetes were surrounded by a multilayered envelope separated from the outer membrane of the cell by a wide electron-transparent area (Fig. 4g). The cyanobacterial MVs were bilayered (likely to be phospholipid membranes), ranging from 20 nm to 1 µm in diameter (see Fig. 4b). Inclusions of different electron densities were also apparent (Fig. 4e). To get additional information on the composition of the MVs, immunogold-TEM labeling, using a mouse-anti-DNA antibody, was performed. The gold particles were, as expected, found in the cyanobacterial cytoplasm (Fig. 5a). In addition, a clear DNA labeling was associated with the interior of the secreted MVs, while when omitting the primary antibody, no label was apparent (Fig. 5b,c). The number of DNA-labeled vesicles were calculated, and out of 98 vesicles counted, 89 were found to be labeled, giving an average of 90% labeling.

image

Figure 4. Transmission electron microscopy micrographs illustrating the release of cyanobacterial membrane vesicles within the indusium chamber. (a) Numerous membrane vesicles (mv) are seen being released into the extracellular space from a cyanobacterial cell (lower part) at the stage when these start to develop into proakinetes. (b) Close-up of several small membrane vesicles and a large membrane vesicle (mv, approx. 1 µm in diameter) being pinched off from the outer cyanobacterial membrane. A flocculent material is apparent in the larger vesicle. (c) A fibrillar material (fm) is starting to be deposited around the cyanobacterial cell wall simultaneously with the release of numerous membrane vesicles (mv). The three-layered Gram-negative cyanobacterial cell wall with its distinct peptidoglycan layer (pg) is apparent. (d) Numerous membrane vesicles in the transparent area between the outer membrane (om) are fusing to the now less transparent envelope (en) of the developing proakinete. The cyanobacterial plasma membrane (pm) and outer membrane (om) are illustrated. (e) Two membrane vesicles containing rounded core structures with higher electron densities. (f) A more mature akinete surrounded by a fully developed outer envelope (en) composed of different density layers. Note the membrane vesicles (mv) now being released from the outer surface of the envelope. (g) The membrane system of a fully mature akinete showing a multiple layered envelope (en) of different electron densities and an electron-transparent area between the envelope and the cyanobacterial outer membrane with membrane vesicles. Bars, 10 nm (a, c–g); 0.5 µm (b).

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image

Figure 5. Immunogold-TEM localization of DNA in cyanobacterial membrane vesicles being released during vertical transmission of the cyanobiont between Azolla plant generations. (a) The localization of the 5 nm-gold particles illustrates the expected occurrence of DNA (arrows) in the cytoplasm of a cyanobacterial proakinete. (b) The localization of the 5 nm-gold particles illustrates a DNA-immuno-label in the membrane vesicles released from a proakinete. (c) Control showing that when the primary anti-DNA antibody was omitted no label was detected. Bars, 30 nm.

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Biofilm formation

Even at an early developmental stage, with the proakinetes still in chains and with bacteria attached, a ‘biofilm’ started to develop around the filaments (Fig. 6a). In nearly mature sporocarps, the biofilm positioned on the solid surface at the inner wall of the lignified indusium developed into a fibrillar matrix as verified by SEM (Fig. 6b), while the space occupied by the prokaryotes, between the indusium and the sporangium, progressively got smaller (Fig. 6d,e). In the upper part, the akinetes were more tightly packed and the abundance of intervening bacteria was higher compared with the lower part of this biofilm. Channels with different widths (0.5–5 µm) developed between the akinete cells (Fig. 6f). At maturation, the biofilm formed a highly ordered structure, ranging from 25 to 30 µm in thickness and 80 µm in diameter, and was characterized by narrow, uniform channels interspersing the cyanobacterial community at the top of the indusium chamber (Fig. 6g).

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Figure 6. Biofilm formation within the Azolla indusium chamber. (a) SEM micrograph showing chains of large, still connected cyanobacterial proakinetes. Bacteria (arrows) are seen attached to the proakinete surface. (b) Larger-magnification SEM micrograph of the biofilm at a later stage, characterized by the cyanobacteria/bacteria community being embedded in a fibrillar matrix (arrows). (c) A more dense flocculent material (f) covering the proakinete community. (d) Akinetes (a) located in the narrow space between the upper cell wall of the sporangium and the indusium (in). (e) TEM of a cross-sectioned, tightly packed biofilm composed of cyanobacteria/bacteria, illustrating its gross architecture at the tip of the mature megasporocarp. (f) TEM micrograph of the cyanobacteria/bacterial biofilm located at the tip of a mature megasporcarp. Numerous bacteria (b) are still seen in the intercellular spaces among the cyanobacterial akinetes with cyanophycin granules (cg) at the upper part of the biofilm. The thin bar (c) illustrates the channel formed between the akinetes at this stage. (g) A higher magnification, illustrating a highly organized biofilm architecture with uniform channels (0.5–1 µm; denoted c) present among the akinetes. Bars, 10 µm.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Throughout its life cycle, the water fern Azolla maintains a unique and intimate association with cyanobacteria and a variety of heterotrophic bacteria. Our data clearly show a hitherto unknown degree of complexity in the organized and intricate developmental processes that ensure the transfer of a cyanobacterial inoculum to the next plant generation. This complexity suggests a long-lasting co-evolution between the symbiotic eukaryotic and prokaryotic partners. A coevolution between the Azolla host and the cyanobacteria has previously been proposed, and recent genetic data support this hypothesis (Papaefthimiou et al., 2008a,b). During colonization of the Azolla sporocarps, the cyanobacterium at the apical meristem appears as long, motile, small-celled, hormogonium-like filaments which enter the young developing sporocarps, a movement apparently guided chemotactically by some unknown attractant. On reaching the inside of the sporocarp, our data unexpectedly revealed that all cells in the hormogonia, in a synchronized fashion, developed into akinetes (spores), a protective life stage. The direct transition from hormogonial cells into thick-walled akinetes has, to our knowledge, not been reported previously for free-living or symbiotic cyanobacteria. In free-living cyanobacteria, differentiation of vegetative cells into akinetes is a phenomenon that is elicited by adverse external conditions (Sutherland et al., 1979; Drews & Weckesser, 1982; Adams & Duggan, 1999). In symbiosis, Azolla is the only cyanobacterial host in which akinetes have been detected, but then only in cavities of old leaves (Becking, 1987; Grilli Caiola et al., 1992). The observed increase in the cyanobacterial cell size, the changed shape, the increased number and distribution of large inclusion bodies in the cytoplasm, such as cyanophycin granules, all support a differentiation into proper akinetes. However, as polyphosphate granules were rarely found at any stage of the cyanobacterial transfer, phosphorus may be in short supply and, indeed, phosphate limitation is known to elicit akinate differentiation in free-living cyanobacteria (van Dok & Hart, 1996; Li et al., 1997; Adams & Duggan, 1999). Another possibility is that the Azolla host produces an ‘akinete-inducing’ factor, comparable to the ‘hormogonium-inducing’ factors released by other symbiotic plant hosts (Rasmussen et al., 1994; see Bergman et al., 2007).

Our data also demonstrate secretion of numerous MVs of varying size from the cyanobacterial cells during sporocarp colonization and akinete differentiation. More than half of the cyanobacterial cells were found to release MVs. Interestingly, the major part (90%) of the MVs counted contained genetic material as shown using immuno-gold-TEM analyses, which may suggest a transfer of DNA from the cyanobacterium to the host. To our knowledge, this is the first demonstration of a release of genetic material via MVs in cyanobacteria. A trans-envelope transporter has recently been suggested for cyanobacteria (Hoiczyk & Baumeister, 1998; Hoiczyk & Hansel, 2000). MVs were also found to be released from the bacteria located in the inducium chamber, although at a lower frequency. The discharge of MVs from Gram-negative bacteria such as Pseudomonas and Escherichia coli has been reported (Kadurgamuwa & Beveridge, 1995; Beveridge, 1999; Yaron et al., 2000). The spherical, bilayered, membranous structures of the MVs found in the cyanobacteria examined here are similar in shape and size to MVs released by these other Gram-negative bacteria (Beveridge, 1999). The discharge of MVs from the cyanobacterial and bacterial cells was also observed in the leaf cavity, but at a low frequency (unpublished data). The vivid secretion of MVs by cyanobacteria in the Azolla sporocarp may have several functions: in the outer envelope development during akinete differentiation, as the MVs were seen fusing to the expanding fibrillar external envelope surrounding the differentiating akinetes; in the formation of the cyanobacterial biofilm, as MVs may simultaneously, or at later stages, deliver material for the generation of the biofilm, as also emphasized for other microbes (Schooling & Beveridge, 2006); and in lateral gene transfer between the symbiotic partners, as many of the MVs contained DNA. We also suggest that transfer (or loss) of DNA from the cyanobacterium to its plant host may explain the documented intimacy in this symbiosis and the difficulty in cultivating the cyanobiont free of its Azolla host (Lechno-Yossef & Nierzwicki-Bauer, 2002).

Biofilms play a role not only in benthic and terrestrial environments but also in plant–microbe associations (Fugua, 2004). Microbial biofilms share common features, such as being attached to abiotic or biotic surfaces; being constituted assemblages of microorganisms; being involved in the development of microcolonies; and in the formation of a highly ordered and elaborate matrixes with channels (Davey & O'toole, 2000; Stoodley et al., 2002). The formation of the cyanobacterial/bacterial biofilm in the megasporocarp of Azolla accompanied the phenotypic changes found, such as development of akinetes. The formation of ‘channels’ interconnecting the akinetes (Fig. 6f,g) may also be signs of a well-organized biofilm (Davey & O'toole, 2000). The bacterium-free channels may function in exchange of nutrients and water, metabolites and gases during the transition of the cyanobiont between generations in the Azolla symbiosis.

In conclusion, our study clearly shows that the cyanobacteria, in order to be transferred to the next generation of Azolla, go through elaborate developmental processes, involving unique cell differentiation events, a DNA release through vesiculation, the latter also potentially involved in biofilm formation. The final outcome of this process is that c. 400–600 cyanobacterial akinetes and numerous bacteria, although highly inferior in their relative biomass to cyanobacteria, are being kept in dormant capture until the sporocarps germinate and better conditions are offered outside the inducium chamber. The cyanobacteria-bacterial community, together with the host plant, act as a highly organized community to ensure survival of this nitrogen-fixing plant symbiosis.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We gratefully acknowledge financial support from the Swedish International Development Cooperation Agency (Sida) through the Swedish Research Links Programme, the Swedish Research Council, the K. and A. Wallenberg Foundation and the Fujian Science and Technology Funds (Minkeji 2003-61, 2001Z026). The authors would like to thank S. Lindwall (at SU) for excellent skills in TEM assistance, J. Klint (at SU) for help in preparing the figures, P. J. Lu for cultivation of Azolla (at FAAS) and L. J. Zhou (at FAAS) for assistance in sampling.

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  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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