Colonization of wheat {Triticum vulgare L.) by Ng-fixing cyanobacteria: II. An ultrastructural study

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1 Phytol. (199!), 118, Colonization of wheat {Triticum vulgare L.) by Ng-fixing cyanobacteria: II. An ultrastructural study BY M. GANTAR^ N. W. KERBY* AND P. ROWELL Department of Biological Sciences, University of Dundee, Dundee DDl 4HN, UK ^Institute of Biology, University of Novi Sad, Novi Sad, Yugoslavia {Received 24 October 1990; accepted 26 March 1991) SUMMARY The ultrastructure of a novel association between a N,-fixing cyanobacterium, Nostoc sp. isolated frotn soil, and wheat seedlings grown in liquid culture is described. Cyanobacteria were found either as hormogonia, filaments or aseriate packages. The aseriate packages, which developed from filaments, often formed a thick layer surrounding the root surface. The packages were in intimate contact with adjacent root epidermal cells and could not be removed without damaging the epidermis. Cyanobacteria penetrated both the root epidermis and cortex and formed packages in intercellular spaces. Cyanobacteria] filatnents were occasionally found within plant cells that appeared empty. Additionally, cyanobacteria were observed in association with the stem and on the surfaces of leaves. These findings demonstrate the technical feasibility of forming a novel association between a N^-fixing cyanobacterium and a cereal. Key words: Association, cyanobacteria, Nostoc, ultrastructure, wheat. INTRODUCTION There have been various attempts to create artificial symbioses between non-ieguminous plants and N^fixing microorganisms (for a review see Gusev & Korzhenevskaya, 1990). The Na'^^'^g microorganisms have included Rhizobium, Azotobacter, and Azospirillum and cyanobacteria belonging to the genera Anabaena, Nostoc, Gloeocapsa and Anacystis. The majority of studies use a method that involves the co-culture of plant cells or callus with microorganisms. Other attempts have been made to introduce the unicellular, N^-fixing cyanobacterium Gloeocapsa (Burgoon & Bottino, 1976) and the heterocystous cyanobacterium Anabaena variabilis (Meeks, Malmberg & Wolk, 1978) into protoplasts of tobacco and maize. Protoplasts containing cyanobacteria did not divide and, therefore, stable associations were not obtained (Meeks et al., 1978). However, stable associations were obtained when Anabaena variabilis was co-cultured with tobacco callus (Gusev et al., 1986). Tobacco shoots regenerated from callus, inoculated with cyanobacteria, contained intercellular cyanobacteria that had nitrogenase activity. * To whom correspondence should be addressed. 33 In preliminary studies, we have found that the medium used to culture plant cells (Murashige & Skoog, 1962) inhibits the growth of cyanobacteria. Furthermore, sucrose above a concentration of 2-0 % (w/v) completely inhibited growth of Anabaena variabilis in liquid medium (BG11(,). We have therefore used an alternative approach for forming associations between Ng-fixing cyanobacteria and higher plants which avoids the use of plant tissue culture media. The associations established between species of Nostoc and Anabaena, isolated from soils, and wheat roots appeared to be specific and nitrogenase activity was also demonstrated (Gantar et al., 1991). In this paper we describe the ultrastructure of an association between a Nostoc sp. and wheat seedlings. MATERIALS AND METHODS Nostoc 2S9B was isolated from solonetz soil, from the province of Vojvodina, Yugoslavia and was maintained on BGll medium (Rippka et al., 1979). Colonization of wheat roots was carried out in BGll without nitrate (BGll^) and with nitrate (BGll^-) in liquid culture, at room temperature and a photon flux density of lo/tmol m"^ s~^ Three days after germination, seedlings of wheat {Triticum vutgare L., cv Longbow) were suspended with their roots in

2 486 M. Gantar, N. W. Kerby and P. Rowell a glass vessel containing BGll medium inoculated with Nostoc 2S9B. After 7 d, samples of plant material with associated cyanobacteria were prepared for scanning electron microscopy by fixation in 4"o (v/v) glutaraldehyde in 0-1 M cacodylate buffer ph 7, for 48 h. Samples were washed 3 times in cacodylate buffer prior to postfixation in 1 "i, (w/v) OSO4 in 013 M phosphate buffer (ph 7-3) for 48 h followed by washing in distilled water. Fixed roots were immersed in liquid nitrogen and cut while frozen with a razor blade. Pieces of roots were then dehydrated using an ethanol series ( "o, v/v) and critical point dried. Dried samples were mounted and coated with gold prior to scanning electron microscopy usmg a Jeol JSM-35. Samples for transmission electron microscopy were fixed in the same manner as described above and were embedded in LR-White resin for 72 h. Thin sections were cut using a Reichert 0MU2 microtome and examined witb a Jeol JEM-1200 EX. RESULTS Colonization of wheat roots in co-culture witb Nostoc 2S9B occurred after 3-5 d and a tight association was formed (Gantar et al., 1991). The time required for association was shorter in medium containing nitrate and the cyanobacterial biomass associated with roots (based on chlorophyll a concentration) was higher. Subsequent uitrastructural studies were therefore carried out on associations established in the presence of nitrate. Despite the presence of nitrate, these associations showed high nitrogenase activity (Gantar et al., 1991). Wheat roots were initially colonized by bormogonia, which are sbort motile filaments containing gas vacuoles that may facilitate floatation and contact with roots. Being motile, hormogonia can migrate along the root surfaces which, with the exception of the root cap and an area immediately behind it became colonized (Fig. 1 a). Cyanobacteria were also present on root hairs (Fig. 1 b), which may be the first point of contact with the roots. On the root surface hormogonia developed into long filaments and appeared to be distributed within a mucilaginous layer (Fig. It"), possibly of plant origin, since at this stage of development a cyanobacterial mucilaginous sheath was not evident. The long filaments developed into an aseriate stage, which consisted of filaments tightly packed in a mucilaginous sheath (Figs. It/, 2a, b). Figure \d shows tbe early development of the aseriate stage. Further growth of cyanobacteria was confined to these packages which formed a thick layer around the roots (Fig. 2a); only the outer packages could be removed by washing in a stream of running water. The remaining cyanobacterial packages (Fig. 26) appeared to be very firmly attached to tbe root surface and any further attempts to remove them damaged the root epidermis. The cyanobacterial filaments, inside packages, were compartmentalized and separated from each other by a thin mucilaginous layer. The packages were in intimate association with root epidermal cells and sometimes forined long tubular structures (Fig. 2b). Scanning electron microscopy also indicated the presence of cyanobacteria within tbe root as indicated by light microscopy (Gantar et al., 1991). Cyanobacterial cells were evident in intercellular spaces in the root cortex (Fig. 1 e) and may also be present within some epidermal cells (Fig. 1/). To confirm this, roots were fixed and embedded in resin for transmission electron microscopy. Tbin sections of colonized roots showed the intimate association between cyanobacteria and epidermal cells (Fig. 3 a). Cyanobacterial packages were occasionally found inside apparently degenerating cells, in spaces in the root epidermal layer (Fig. 3 b) and between cortical cells (Fig. 3 c). Individual filaments were occasionally observed within cortical cells (Fig. 3d). Cyanobacterial packages within the cortex were encapsulated in mucilage, as were those on the root surface, and appeared to be embedded in a matrix with other bacteria (Fig. 3 c). Tbe filaments observed in cortical cells did not have a mucilaginous sheath and may be hormogonia (Fig. 3^). After 7 d of co-culturing cyanobacteria with wheat plants, Nostoc filaments had not only extensively colonized the roots but had also migrated to the stems (Fig. 4a) and leaves (Fig. 46). Although not abundant on stems and leaves, hormogonia and beterocystous filaments, but not aseriate packages, were observed. Figures 4c, d show a cross section of a stem with cyanobacteria and bacteria in the spaces between the folded leaves (Fig. 4c, d). The isolate Nostoc 2S9B used in this study was not axenic and plants were not grown under aseptic conditions; therefore, bacteria were frequently observed, sometimes in association with cyanobacteria (Fig. 5 a, 6). Tbe number of bacteria was particularly high on, or in the vicinity of heterocysts (Fig. 5 a). Additionally, bacteria were observed between the aseriate packages and the plant epidermal cells (Fig. 56). Figure 1. Scanning electron micrographs of wheat roots with associated cyanobacteria showing: (a), root cap and adjacent area, which are not colonized by cyanobacteria (bar, ioo /im); (b), a root hair with hormogonia; (f), long cyanobacterial filaments on the root surface and beneath a mucilaginous layer; (rf), initial stage in formation of the aseriate stage; (e), cyanobacteria] filament in an intercellular space between cortical cells; (/), a cyanobacterial filament inside an epidermal cell. Bar, \0/im in (6) to (/).

3 Ultrastructure of a plant-cyanobacterial association 487 Figure 1. For legend see opposite.

4 488 M. Gantar, N. W. Kerby and P. Rowell Figure 2. Scanning electron micrographs of wheat roots showing: (a), a thick layer of cyanobacteria (aseriate stage) on a root surface; (h), packages of cyanobacterial filaments (aseriate stage), forming a tight association with the root surface after removal of the outer packages with water washing. Bar, 10 //m.

5 Ultrastructure of a plant-cyanobacterial association 489 Figure 3. Transmission electron micrographs of wheat roots showing: (a), a thin section through a cyanobacteria! package attached to epidermal cells; (b), cyanobacteria in an epidermal cell and on the surface of the epidermis; (c), an intercellular space in the root cortex, occupied by cyanobacteria in the aseriate stage; {d), a cyanobacterial filament (presumably a hormogonium) in a cortical cell. Bar, 5/<m.

6 490 M. Gantar, N. W. Kerby and P. Rowell Figure 4. Scanning electron micrographs of wheat stem and leaves showing: (a), a hormogonium on the stem surface (bar, lo/^m); (ft), a hormogonium and a heterocystous filament on the leaf surface (bar, 10//m); (f), cross section of the stem (the square indicates the area of higher magnification shown in d, bar, ]000//m); (d), a cyanobacterial filament in the stem (bar, \0/im).

7 Ultrastructure of a plant-cyanobacterial association 491 Figure 5. Scanning electron micrographs of wheat roots showing: (a), a cyanobacterial filament located within a vascular element (note a heterocyst (H) with associated bacteria; bar, 10//m); (ft), bacteria (B) associated with cyanobacterial packages on the surface of the epidermis (bar, 3 DlSCl'SSION In this paper, we describe the ultrastructure of an association between a N^-fixing cyanobacterium, Nostoc 2S9B isolated from soil, and wheat seedlings grown in liquid culture. This cyanobacteriutn has a developmental cycle consisting of hormogonia, heterocystous filaments and an aseriate stage, which may be important in forming a tight association with roots (Gantar et al., 1991). Apart from the well characterized symbiotic associations between higher plants and cyanobacteria, other naturally occurring associations involving cyanobacteria have been described. These include epiphytic growth of Gleotrichia pisum on the aquatic roots and stems of deepwater rice (Whitton et al., 1988) and a range of heterocystous cyanobacteria attached to the lower epidermis of Lemna plants (Duong & Tiedje, 1985). Nitrogenase activity was demonstrated in both of these associations (Duong & Tiedje, 1985; Rother et al., 1988) but the morphology of the associations was not described in detail. In addition to colonizing the surfaces of wheat roots and root hairs, Nostoc 2S9B penetrated the root epidermis and cortex and was present in intercellular spaces and sometimes within plant cells. This is similar to the associations formed when rhizobia were co-cultured with plant callus (Holsten et al., 1971; Rao & Subba-Rao, 1976; Ozawa & Yamaguchi, 1980) and when cyanobacteria were cocultured with tobacco callus. However, in the latter case cyanobacteria were not found w-ithin plant cells (Gusev et al., 1986). The mechanisms by which cyanobacteria penetrate plant cells are as yet unknown. The plant cells containing cyanobacteria appear empty due either to the presence of large vacuoles or to degeneration. Bacteria are found together with cyanobacteria and it is possible that penetration is facilitated by the production of hydrolytic enzymes of bacterial origin (Ozawa & Yamaguchi, 1980). Indeed, a method that has been employed to introduce rhizobia into roots of rice seedlings, without need for plant tissue culture, relies on treatment of the plants with hydrolytic enzymes and polyethyleneglycol (Al-Ma!lah. Davey & Cocking, 1989). In addition to their presence on submerged root surfaces and within roots, cyanobacteria were detected within stems and on the surfaces of leaves, albeit at a much lower frequency than that found in roots. We presume that these cyanobacteria had migrated from the aqueous environment, although their mode of entry into stems is unknown. Aseriate packages were found in stems but were not observed on leaf surfaces (data not show-n). Since Nostoc 2S9B produces heterocysts and shows nitrogenase activity even when the association is grow-n in the presence of nitrate (Gantar et al., 1991) there are several possible advantages over similar novel associations involving rhizobia. Since cyanobacteria can fix Nj aerobically there is no requirement for nodule formation; they are also photosynthetic and may not be reliant on a supply of fixed carbon. Although the phototrophic nature of cyanohacteria could be viewed as a disadvantage for root associations growing in soils, it should be noted that the cyanobacteria in symbiotic association with gymnosperms and angiosperms are located within the plant such that their metabolism is likely to be heterotrophic rather than autotrophic (see Rai, 1990). Indeed, many free-living cyanobacteria can grow heterotrophically (Smith, 1982). Nostoc 2S9B liberates nitrogenous compounds into its growth

8 492 M. Gantar, N. W. Kerby and P. Rowell medium (our unpublished data), as has been reported for other natural iolates of cyanobacteria (see Kerby, Rowell & Stewart, 1989). These nitrogenous compounds may be taken up and assimilated by the plant, tbereby decreasing the reliance of the plant on nitrogen fertilizers. The biochemical and physiological aspects of these novel associations, including benefits to the plant, are currently under investigation. ACKNOWLEDGEMENTS This work was supported by the British Council, The Scientific Fund of the Province of Vojvodina and the University of Dundee. We would also like to thank Heather Bennett and Irene Bennett for their assistance with electron microscopy. REFERENCES AL-MALLAH. M., DA\'EY, M. R. & COCKING. E, C. (1989), Formation of nndular structures on rice seedlings by rhizobia. Journal of Experimental Botany 40, 473^78. BuHGOON, A. C. & BOTTINO, P. J. (1976), Uptake of nitrogen fixing blue-green alga Gloeocapsa into protoplast of tobacco and maize Journal of Heredity , DUONG, T, P, & TIEDJE, J. M, (1985), Nitrogen fixation by naturally occurring duckweed-cyanobacterial associations, Canadian Journal of Microbiology 31, GANTAR. M., KERBV, N, W,, ROWELL, P, & OBREHT. Z, (1991), Colonization of wbeat by N.^-fixing cyanobacteria, I, A survey of soil cyanobacterial isolates forming associations witb roots, Netv Phytologist 118, 477^83, GUSEV, M, V. & KORZHENEVSKAVA, T. (1990), Artificial associations. In: CRC Handbook of Symbiotic Cyanobacteria (Ed. by A. N, Rai). pp , CRC Press, Inc, Boca Raton, GUSEV, M. V., KORZHENBVSKAYA, G., PYVOVAROVA, L, V., BAULINA, O. I. & BuTENKO, R, G, (1986). Introduction of a nitrogen-fixing cyanobacterium into tobacco sboot regenerates. Planta HoLSTEN, R. D.. BURNS, R. C, HARDY, R, W, & HEBERT. R, R. (1971), Establishment of symbiosis between Rhizobium and plant cells in vitro. Nature (London) , KERBV, N, W., ROWELL. P, & STEWART. W. D, P, (1989). The transport assimilation and production of nitrogenous compounds by cyanobacteria and microalagae. In; Algal and Cyanobacterial Biotechnology (Ed, by R, C. Cresswell, T, A. V. Rees & N, Shah), pp, Longman Scientific and Technical, Harlow. MEEKS. J. C. M.'ILMBERG. R. L, & WOLK. C, P- (1978). Uptake of auxotrophic cells of a heterocyst-forming cyanobacterium by tobacco protoplasts, and the fate of their associations. Planta , MURASHIGE, T, & SKOOG, F, (1962), A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15, OzAWA, T. & YAMAGUCHI, M, (1980), Increase in cellulase activity in cultured soybean cells caused by Rhizobium japonicum. Plant Cell Physiology 21, , RAI. A, N, (1990). CRC Handbook of Symbiotic Cyanobacteria. CRC Press, Boca Raton. RAO, V, R. & SURBA-RAO, N, S, (1976), Studies on the interaction of legume root calius with Rhizobium. Zeitschrift fiir Pfianzenphysiologie 80, 14-20, RippKA, R,, DERUELLES, J,, WATERBURV, J, B., HERDMAN, M. & STANIER, R, Y. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Journal of General Microbiology HI, 1-61, RoTHER, J- A,, Aziz. A., KARIM, N, H, & WHITTON. B,.\. (1988). Ecology of deepwater nce-fields in Bangladesh. 4, Nitrogen fixation by blue-green algal communities, Hydrobiologia , SMITH, A. J, (1982). Modes of cyanobacterial carbon metabolism. In; The Biology of Cyanobacteria (Ed, by N. G. Carr & B, A, Whitton), pp, 47-85, Blackweli Scientific Publications. Oxford, WHITTQN, B, A., AZIZ, A,, KAWECKA, B. & ROTHER. J. A. (1988), Ecology of deepwater rice-fields in Bangladesh. 3. Associated algae and macrophytes. Hydrobiologia 169,

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