DIVISION OF PERIBACTEROID MEMBRANES IN ROOT NODULES OF WHITE CLOVER

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1 J. Cell Set. 69, (1984) 147 Printed in Great Britain The Company of Biologists Limited 1984 DIVISION OF PERIBACTEROID MEMBRANES IN ROOT NODULES OF WHITE CLOVER JOHN G. ROBERTSON Applied Biochemistry Division, Department of Scientific and Industrial Research, Palmerston North, New Zealand AND PAMELA LYTTLETON Department of Chemistry, Biochemistry and Biophysics, Massey University, Palmerston North, New Zealand SUMMARY Division of peribacteroid membranes in the cytoplasm of root nodules of white clover was found, from a study of serial thin sections prepared for electron microscopy, to accompany division of the bacteroids. It was also observed that the peribacteroid membranes appeared to have adhered to various sites on the surface of the bacteroid envelope outer membranes. Wherever peribacteroid membranes were constricted as though undergoing division in the region of the cleft formed by partial division of the bacteroids, these constrictions could be related to the point of adhesion of the peribacteroid membranes to the surface of the bacteroids within the cleft. It was concluded that adhesion of the peribacteroid membranes to the bacteroid envelope outer membranes is likely to be a critical element in the process of division of the peribacteroid membranes. Differences in the degree of adhesion between peribacteroid membranes and the bacteroid envelope outer membranes may explain variations in the number of bacteroids enclosed by peribacteroid membranes in nodules of different legumes. INTRODUCTION Release of rhizobia from infection threads into the cytoplasm of legume nodule meristematic cells is considered to involve an endocytotic process in which the bacteria at the tips of the threads become enclosed by the infection thread membranes (Goodchild & Bergersen, 1966; Newcomb, 1981), which thus form the peribacteroid membranes (Robertson, Lyttleton & Tapper, 1983). In soybean nodules, subsequent division of the bacteroids is accompanied by division of the peribacteroid membranes during the early, but not the later, stages of nodule development (Bergersen & Goodchild, 1973; Bergersen, 1974), so forming peribacteroid membranes enclosing several bacteroids. In other legumes, division of the peribacteroid membranes may accompany division of the bacteroids throughout development of the infected nodule cells, thereby giving rise to peribacteroid membranes enclosing predominantly single bacteroids (Dart, 1977). There appear, however, to be no reports regarding possible mechanisms involved in the process of division of these membranes. Such a process may be an important aspect of legume nodule development, in that it enables the bacteroids to be distributed throughout the plant cell (Robertson et al. 1983). The aim of the present study was to determine whether some indication of the nature of this process might be obtained from an examination of serial sections of tissue taken at various stages of nodule development.

2 148 J. G. Robertson and P. Lyttleton MATERIALS AND METHODS Preparation of nodule tissue and electron microscopy Nodules were obtained from white clover (Trifolium repens L. cv. Grasslands Huia) grown in pumice for 35 days following inoculation of germinated seeds with Rhizobium trifolii NZP566 (Boland, Fordyce & Greenwood, 1978). The nodules were processed for electron microscopy as described previously (Robertson & Lyttleton, 1982), with the exception that, in the preparation of some samples, the immersion of tissue in magnesium/uranyl acetate solution following fixation with osmium was omitted. Alternatively, nodules were obtained from seedlings grown on slopes of Jensen's agar for 7 or 17 days following inoculation with R. trifolii strain 7012 and were processed for electron microscopy as described previously (Ronson, Lyttleton & Robertson, 1981). Thin sections cut with a diamond knife were mounted on copper grids without support films and stained at C with saturated uranyl acetate in 50 % ethanol for 5 min, followed by lead citrate (Venable & Coggeshall, 1965) for 3 min. Sections mounted on gold grids without support films were stained for polysaccharides (Thie'ry, 1967; Robertson, Lyttleton, Williamson & Batt, 1975) by floating grids on 1 % periodic acid for 30 min, 0-2 % thiocarbohydrazide in 20 % acetic acid for 22 h, and 1 % silver proteinate for 30 min, with appropriate washing steps between each reagent. Sections were examined with a Philips EM 200 or 201c microscope at 60 kv. RESULTS Three stages of development of white clover nodules, including the zones of infection, early symbiosis and late symbiosis, are illustrated in Figs 1-3. At each stage of Abbreviations used in Figs 1-15: b, bacteroid; bim, bacteroid envelope inner membrane; bom, bacteroid envelope outer membrane; c, capsule; cw, cell wall; it, infection thread; itm, infection thread membrane; m, mitochondria; ma, infection thread matrix material; pbm, peribacteroid membrane; pbs, peribacteroid space; r, rhizobium; mm, rhizobial envelope outer membrane. Fig. 1. Thin section of the infection zone of a nodule from a 7-day white clover plant inoculated with R. trifolii strain 7012 and grown on an agar slope. The micrograph shows rhizobia on the point of release from the infection thread (arrows) and several bacteroids in the cytoplasm, each enclosed by a peribacteroid membrane. Stained with uranium and lead. X6000. Fig. 2. Thin section of the early symbiotic zone of a nodule from a 17-day white clover plant inoculated with R. trifolii strain 7012 and grown on an agar slope. Bacteroids enclosed singly in peribacteroid membranes are distributed throughout the cytoplasm, interspersed with mitochondria. Stained with uranium and lead. X6000. Figs 3-6. Thin sections of nodules from 35-day white clover plants inoculated with R. trifolii strain NZP566 and grown in pumice culture. Stained with uranium and lead. Fig. 3. Thin section of the late symbiotic zone, showing enlarged bacteroids enclosed singly by peribacteroid membranes and virtually filling the cytoplasm. Mitochondria are evident around the periphery of the cell. X6000. Fig. 4. Dividing bacteroid in the early symbiotic zone. The peribacteroid membranes do not follow the surface of the bacteroid envelope outer membranes in the region of the cleft formed by partial division of the bacteroid. X Fig. 5. Bacteroid in the early symbiotic zone having apparently divided without division of the peribacteroid membrane. X Fig. 6. Portions of two bacteroids in the late symbiotic zone showing close apposition, but not fusion, of the cytoplasmic surfaces of the two peribacteroid membranes at high magnification. X

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5 Division of peribacteroid membranes 151 development, the individual bacteroids, as seen in single thin sections, were always surrounded by peribacteroid membranes. Although these membranes were often closely adjacent to each other in the plant cell cytoplasm (Figs 3, 6), they did not show signs of having fused. Bacteroids that appeared to have divided (Fig. 5), or were in a state of partial division (Fig. 4), were commonly seen in thin sections of the early symbiotic zone (Fig. 2). By contrast, peribacteroid membranes possessing ultrastructural characteristics that indicated that they might be in the process of dividing between bacteroids were rarely observed. It appeared, however, that the peribacteroid membranes must have undergone division during multiplication of the bacteroids, since in the early symbiotic zone (Fig. 2) 98 % of the peribacteroid membranes of 470 counted in thin sections contained only a single organism, and in the late symbiotic Figs 7, 8. Thin sections of the early symbiotic zone of nodules from 35-day white clover plants inoculated with R. trifolii NZP566 and grown in pumice culture. In preparing this tissue for electron microscopy, immersion in magnesium uranyl acetate following osmium fixation was omitted. Stained with uranium and lead. Fig. 7. A-N. Sequence of serial sections through a single bacteroid completely enclosed by a peribacteroid membrane and separated from other bacteroids in the plant cytoplasm. X9000. Fig. 8. A-G. Section numbers 3, 5, 6, 8 11 of a sequence of serial sections through a peribacteroid membrane enclosing several bacteroids. X9000. Figs Thin sections of nodules from 35-day white clover plants inoculated with R. trifolii NZP566 and grown in pumice culture. Fig. 9. Portion of a bacteroid showing several adhesion sites (arrowheads) between the peribacteroid membrane and the bacteroid envelope outer membrane. Stained with uranium and lead. X Fig. 10. Micrograph showing distortion of the peribacteroid membrane and bacteroid envelope outer membrane at an adhesion site (arrowhead) at high magnification. Stained with uranium and lead. X Fig. 11. A-D. Sequence of serial sections showing adhesion of the peribacteroid membrane to the bacteroid envelope outer membrane at irregular sites (arrowheads) over the surface of the bacteroid. Stained with uranium and lead. X Fig. 12. Portion of a bacteroid showing several adhesion sites (arrowheads) between the peribacteroid membrane and the bacteroid envelope outer membrane. The bacteroid envelope inner membrane is also clearly defined. Thin section stained with silver by the Thigry (1967) method for polysaccharides. X Fig. 13. Infection thread showing a rhizobium at the point of release into the cytoplasm of the plant cell. An electron-translucent zone between the infection-thread matrix material and the rhizobium may correspond to rhizobial capsular material that is less pronounced between the rhizobium and the infection thread membrane. Sites of adhesion between the infection thread membrane and the rhizobial envelope outer membrane are evident (arrowheads). Section stained with silver by the Thie'ry (1967) method for polysaccharides. X Fig. 14. Portion of a bacteroid at a stage of division showing adhesion sites (arrowheads) between the peribacteroid membrane and the bacteroid envelope outer membrane in the region of the cleft formed by division of the bacteroid. Stained with uranium and lead. X Fig. 15. A-D. Sequence of serial sections of the early symbiotic zone of a nodule as described for Figs 7,8. An adhesion site (arrowhead) between the peribacteroid membrane and the bacteroid envelope outer membrane in the cleft formed by partial division of the bacteroid is clearly evident in B but is absent in A. X

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8 154 jf. G. Robertson and P. Lyttleton zone (Fig. 3) 96% of 470 peribacteroid membranes counted contained only a single organism. A study of serial sections of nodule tissue confirmed that peribacteroid membranes, apparently enclosing only single organisms when viewed in single thin sections, were in fact not interconnected by junctions of any type (Fig. 7). Very occasionally a study of serial sections of nodule tissue revealed peribacteroid membranes enclosing more than one bacteroid (Fig. 8), although in one section of the series (Fig. 8A) only a single organism was apparent. Where several bacteroids were enclosed they tended to be closely packed together and the peribacteroid membranes were closely associated with large areas of the surfaces of the bacteroids (Fig. 8). A study of the ultrastructure of the peribacteroid membranes enclosing bacteroids at various stages of division revealed that the peribacteroid membranes did not generally follow the contours of the cleft formed at the site of division of the bacteroids (Figs 4, 5). It was observed, however, that at other points over the surface of the bacteroids, the peribacteroid membranes appeared to be closely associated with (Figs 9, 12), and in fact to adhere to (Figs 10, 11) the bacteroid envelope outer membranes. Similar sites of adhesion were observed between the infection thread membrane and the outer surface of the rhizobial envelope in the infection thread at various stages of endocytosis (Fig. 13). Very occasionally, constrictions of the peribacteroid membranes in the region of the clefts formed by the dividing bacteroids were observed in thin sections (Fig. 14). A study of serial sections of bacteroids at various stages of division showed that these constrictions were associated with sites of adhesion of the peribacteroid membranes to the bacteroid envelope outer membranes within the cleft (Fig. 15). DISCUSSION The object of the work reported here was to determine whether any indication could be obtained, from a study of thin sections of legume nodule tissue, of the nature of the process of division of the peribacteroid membranes. It was apparent that adhesion of these membranes to the bacteroid envelope outer membranes is likely to be an important aspect of this process. This conclusion was based on the observation that the peribacteroid membranes appeared to be very closely associated with (Figs 9, 12), and in fact adhering to (Figs 10, 11), the bacteroid envelope outer membrane at various sites over the surfaces of the bacteroids. Where constriction of the peribacteroid membranes was observed within the cleft formed by partial division of the bacteroids, these constrictions were found, in serial thin sections, to be associated with sites of adhesion of the peribacteroid membranes to the bacteroid envelope outer membranes within the clefts (Figs 14, 15). It appears, therefore, that division of the peribacteroid membranes is probably related to the tendency of these membranes to adhere to the bacteroid envelope outer membranes and, as a consequence, to follow the general contour of the surfaces of the individual bacteroids. The mechanism of division of the peribacteroid membranes has not, to our knowledge, been discussed in the literature. However, this process might be regarded as a continuation of the initial process of endocytosis in which the rhizobia at the tips of

9 Division of peribacteroid membranes 155 the infection threads are surrounded by the infection thread membranes and released into the plant cell cytoplasm. During endocytosis, the infection thread membranes appear to be closely associated with the surface of the rhizobial envelope outer membranes (Fig. 13). Previous authors have raised the possibility that this close association may be an important aspect of the endocytotic process (Goodchild & Bergersen, 1966; Kijne, 1975; Newcomb, 1976; Newcomb, Syono & Torrey, 1977; Kijne & Planque\ 1979). It is also evident from published micrographs (Gourret & Fernandez- Arias, 1974; Newcomb, 1976; Newcomb et al. 1977; Bal, Shantharam & Verma, 1980) that peribacteroid membranes occur in close association with, or adhere to, the surface of the bacteroid envelope outer membranes in a variety of legume systems, suggesting that adhesion between these two membranes may be of general importance in the legume-rhizobium symbiosis. It has been widely reported that differences occur in the number of bacteroids enclosed by peribacteroid membranes in nodules of different legumes that have been infected with the same strain of Rhizobium (Dart, 1977; Sutton, Pankhurst & Craig, 1981), and also in particular legumes at different stages of nodule development (Goodchild & Bergersen, 1966). Such differences in the number of enclosed bacteroids might be caused by differences in the degree of adhesion between the peribacteroid membranes and the bacteroid envelope outer membranes in the various systems. Studies of soybean nodules (Goodchild & Bergersen, 1966) do reveal more points of contact between these membranes, where the peribacteroid membranes enclose only single bacteroids at the early stages of nodule development, in comparison to the later stages where several bacteroids are enclosed. Comparison of the soybean (Goodchild & Bergersen, 1966) and white clover (Fig. 8) legume systems highlights the difference in degree of membrane adhesion that occurs where the overall tendency is to enclose several, as opposed to only single, bacteroids. Changes or differences in the number of bacteroids enclosed by peribacteroid membranes in the cytoplasm of legume nodule cells may relate to changes or differences in the composition of the peribacteroid membranes or the bacteroid envelope outer membranes during the process of development of legume root nodules. In this regard it has been reported that the density of the peribacteroid membranes changes during nodule development in lupin (Robertson et al. 1978). Also changes occur in the properties of the rhizobial envelope outer membrane during the transition from the free-living to the bacteroid state in the nodules of several legumes (Bal et al. 1980; Sutton et al. 1981; Bal & Wong, 1982). It is not known whether the observed sites of adhesion between the peribacteroid and infection thread membranes and the rhizobial or bacteroid envelope outer membranes reflect specific interactions between components on the surface of these membranes. Such specificity may, however, be an extremely important aspect of the legume Rhizobium symbiosis. Support for this comes from the observation (Pankhurst, 1974) that the number of bacteroids enclosed by peribacteroid membranes was greater in nodules formed by ineffective mutants of R. trifolli with altered cell wall properties, than in nodules formed by the wild type.

10 156 J. G. Robertson and P. Lyttleton We thank Clive Pankhurst, Alan Craig and Barry Scott for their comments on the manuscript, Douglas Hopcroft and Raymond Bennett for photographic assistance, Dale Rosvall and Pat Phillips for typing, and the Plant Physiology Division, Department of Scientific and Industrial Research, for growth room facilities. REFERENCES BAL, A. K., SHANTHARAM, S. & VERMA, D. P. S. (1980). Changes in the outer cell wall of Rhizobium during development of root nodule symbiosis in soybean. Can. J. Microbiol. 26, BAL, A. K. &WONG, P. P. (1982). Infection process and sloughing off of rhizobial outer membrane in effective nodules of lima bean. Can.J. Microbiol. 28, BERGERSEN, F. J. (1974). Formation and function of bacteroids. In The Biology of Nitrogen Fixation (ed. A. Quispel), pp Amsterdam: North-Holland. BERGERSEN, F. J. & GOODCHILD, D. J. (1973). Cellular location and concentration of leghaemoglobin in soybean root nodules. Aust. J. biol. Sci. 26, BOLAND, M. J., FORDYCE, A. M. & GREENWOOD, R. M. (1978). Enzymes of nitrogen metabolism in legume nodules: a comparative'study. Aust.J. PI. Physiol. 5, DART, P. J. (1977). Infection and development of leguminous nodules. In.A Treatise on Dinitrogen Fixation, section III (ed. R. W. F. Hardy), pp New York: John Wiley & Sons. GOODCHILD, D. J. & BERGERSEN, F. J. (1966). Electron microscopy of the infection and subsequent development of soybean nodule cells. j f. Bad. 92, GOURRET, J.-P. & FERNANDEZ-ARIAS, H. (1974). Etude ultrastructurale et cytochimique de la diffevenciation des bacte'roides de Rhizobium trifolii Dangeard dans les nodules de Trifolium repens L. Can.J. Microbiol. 20, KIJNE, J. W. (1975). The fine structure of pea root nodules. 1. Vacuolar changes after endocytotic host cell infection by Rhizobium leguminosarum. Physiol. PL Path. 5, KIJNE, J. W. & PLANQUE, K. (1979). Ultrastructural study of the endomembrane system in infected cells of pea and soybean root nodules. Physiol. PI. Path. 14, NEWCOMB, W. (1976). A correlated light and electron microscopic study of symbiotic growth and differentiation in Pisttm sativum root nodules. Can.J. Bot. 54, NEWCOMB, W. (1981). Nodule morphogenesis and differentiation. Int. Rev. Cytol. (suppl.) 13, NEWCOMB, W., SYONO, K. & TORREY, J. G. (1977). Development of an ineffective pea root nodule: morphogenesis, fine structure and cytokinin biosynthesis. Can.J. Bot. 55, PANKHURST, C. E. (1974). Ineffective Rhizobium trifolii mutants examined by immune-diffusion, gel-electrophoresis and electron microscopy. J. gen. Microbiol. 82, ROBERTSON, J. G. & LYTTLETON, P. (1982). Coated and smooth vesicles in the biogenesis of cell walls, plasma membranes, infection threads and peribacteroid membranes in root hairs and nodules of white clover. J. Cell Sci. 58, ROBERTSON, J. G., LYTTLETON, P. & TAPPER, B. A. (1983). The role of peribacteroid membrane in legume root nodules. In Advances in Nitrogen Fixation Research (ed. C. Veeger & W. E. Newton), pp The Hague: Nijhoff/Junk. ROBERTSON, J. G., LYTTLETON, P., WILLIAMSON, K. I. & BATT, R. D. (1975). The effect of fixation procedures on the electron density of polysaccharide granules in Nocardia corajiina.j. Ultrastruct. Res. 52, ROBERTSON, J. G., WARBURTON, M. P., LYTTLETON, P., FORDYCE, A. M. & BULLIVANT, S. (1978). Membranes in lupin root nodules. II. Preparation and properties of peribacteroid membranes and bacteroid envelope inner membranes from developing lupin nodules. J. Cell Sci. 30, RONSON, C. W., LYTTLETON, P. & ROBERTSON, J. G. (1981). (Vdicarboxylate transport mutants of Rhizobium trifolii form ineffective nodules on Trifolium repens. Proc. natn. Acad. Sci. U.SA. 78, SUTTON, W. D., PANKHURST, C. E. & CRAIG, A. S. (1981). The Rhizobium bacteroid state. Int. Rev. Cytol. (suppl.) 13,

11 Division of peribacteroid membranes 157 THI RY, J. P. (1967). IVlise en evidence des polysaccharides sur coupes fines en microscopie electronique.j- Microscopie 6, VENABLE, J. H. & COGGESHALL, R. (1965). A simplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25, (Received 4 February 1984-Accepted 21 February 1984)

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