Fine structure of bacteroids in root nodules of Vigna sinensis, Acacia longifolia, Viminaria juncea, and Lupinus angustifolius

Vol. 91, No. 3 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, Mar., 1966

Copyright © 1966 American Society for Microbiology

Fine Structure of Bacteroids in Root Nodules of Vigna sinensis, Acacia longifolia, Viminaria juncea, and Lupinus angustifolius P. J. DART' AND F. V. MERCER Joint Plant Physiology Un2it, Botany Building, Untiversity of Sydney, Sydntey, anid Divisioni of Food Preservation, Commonzwealth Scienitific and Industrial Research Organlizationl, Ryde, Australia Received for publication 22 November 1965

ABSTRACT DART, P. J. (University of Sydney, Sydney, Australia), AND F. V. MERCER. Fine structure of bacteroids in root nodules of Vigna sinensis, Acacia longifolia, Viminaria juncea, and Lupinus angustifolius. J. Bacteriol. 91:1314-1319.-In nodules of Vigna sinensis, Acacia longijolia, and Viminariajuncea, membrane envelopes enclose groups of bacteroids. The bacteroids often contain inclusion granules and electron-dense bodies, expand little during development, and retain their rod form with a compact, central nucleoid area. The membrane envelope may persist around bacteroids after host cytoplasm breakdown. In nodules of Lupinus angustifolius, the membrane envelopes enclose only one or two bacteroids, which expand noticeably during development and change from their initial rod structure.

Legume-Rhizobium associations which form nodules fall into six, fairly discrete, cross-inoculation groups. This paper describes some details of bacteroid fine structure in the cowpea-soybean and in lupin cross-inoculation groups. Earlier observations of soybean (1) and, later, of Lupinus luteus (8) dealt with the fine structure of two

RESULTS

The fine structural organization of host cells from the bacteroid zones of effective nodules of V. sinensis, A. longifolia, and V. juncea is very similar. Early in bacteroid development, the Rhizobium cells are dispersed through the cytoplasm of a vacuolated cell along with the host nodules from these two groups. cell organelles. The bacteroids are rod-shaped, have a central nucleoid area with compact fibrils, AND METHODS MATERIALS are bounded by a plasma membrane and cell of sinensis Savi var. Nodules from plants Vigna Poona (cowpea), Acacia longifolia (Andrews) WilId. wall membrane, and contain prominent, electron(acacia), Viminiaria j]uncea (Schrad) Hoffmgg., and dense bodies and occasionally electron-transLupinus angustifolius L. (New Zealand blue lupin) parent regions presumably corresponding to were examined. The lupin, acacia, and cowpea plants inclusion granules (poly-f3-hydroxybutyrate?). A were effectively nodulated, respectively, by Rhizobium narrow, electron-empty space surrounds each strains W72, SU318, and a strain collected by one of bacteroid; at the interface with the host cytous (P. J. D.) from Northern Australia. The plants plasm, a membrane envelope develops, apwere grown (without nitrogen) in sand culture in a parently de novo, involving synthesis of a new glass house. Viminaria nodules were obtained from membrane rather than organization of existing plants growing in sandy soil in the bush near Sydney; membranes (see also 2). Often, the electron-empty the nodules were presumably effective, since the plants were vigorous, with dark green leaves, and the large spaces around two or more rhizobia are connodules contained an extensive, pink-red (presumably tinuous, and here membrane envelope developleghemoglobin) pigmented area. Slices from the pink ment again foUows the interface of this space to red bacteroid-filled zones of the nodules were fixed with the ground cytoplasm. The development of in KMnO4 or OS04 , embedded in Araldite, sectioned, the membrane envelopes does not appear to and examined in a Siemens Mk 1 electron microscope involve any specific alignment of the host cell as described previously (2). endoplasmic reticulum that would suggest a 1 Present address: Soil Microbiology Department, special involvement of the reticulum in the enveRothamsted Experimental Station, Harpenden, Herts., lope formation. Division of the bacteroids apparently occurs England. 1314

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during, and possibly also after, membrane envelope formation, for in the mature Rhizobiumfilled host cell, several (in some sections of cowpea, 20) bacteria are enclosed in each envelope (Fig. 1, 4, 6). The number of bacteroids enclosed in each membrane envelope is fewer in V. juncea and A. longifolia nodules, although up to eight have been counted. Serial sections are needed to determine more precisely the distributions of bacteroids per envelope. Each bacteroid expands during development in the nodule, but retains the same general rod structure with a well-organized, central nucleoid area. Inclusion granules and electron-dense bodies are still present in some of the bacteroids, and are particularly prominent in cowpea nodules. In nodules of V. juncea and A. longifolia, the envelope-enclosed bacteroids are often very elongate. Occasionally, a Y form of bacteroid is found in cowpea nodules. The bacteroids in the envelopes are surrounded immediately by the electron-empty space and then by a sparsely distributed, granular material. The envelope itself has a typical unit membrane structure and is slightly wider than the bacteroid cell wall and plasma membranes (Fig. 3). The latter membrane is rarely resolved in the rhizobia in these nodules. At the completion of bacteroid enclosure by the membrane envelope, most host cell organelles are displaced to the cell periphery. A greater volume of host cytoplasm, however, appears to separate individual membrane envelopes in the mature bacteroid zone of the cowpea, V. juncea, and A. longifolia nodules than in the corresponding zone in Trijfolium subterraneum and Medicago tribuloides nodules (2, 4). The close packing of the bacteroids in these latter nodules gives an impression of specific orientation that is not apparent in the cowpea, V. juncea, or A. longifolia nodules. In older zones of the nodules, rhizobia can be found still enclosed by their membrane envelopes, even though the host cell cytoplasm has broken down (Fig. 2, 5). The rhizobia appear normal, although occasional empty vesicles resembling lysed bacteria are found (Fig. 2). The organization of bacteroids in L. angustiJolius nodules is markedly different from those of cowpea, acacia, and V. juncea. A membrane envelope usually surrounds each bacteroid (Fig. 7). Often, two Rhizobium cells are apparently enclosed by a single envelope, but without serial sections it is not possible to determine whether the two images represent separate cells or are "branches" of a single cell. Y- and club-shaped bacteroids do occur (Fig. 7, inset). The bacteroids themselves are much swollen, compared with the

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normal rod-shaped Rhizobium cells, and occasionally contain intracytoplasmic membranes. The mature bacteroid nucleoid is usually dispersed throughout the cell and is not organized in a compact central unit. The space between membrane envelope and bacteroid cell wall membrane, in both KMnO4 and OS04 fixation, is generally electron-lucent and contains no organized granular material, as in the other three kinds of nodules. OS04 fixation shows that the host cell cytoplasm surrounding each bacteroid contains numerous ribosomes (mainly free) and a background matrix of medium electron density (Fig. 7, inset). DISCUSSION

The finding of membrane envelopes around the bacteroids in legume nodules of different niiorphology, and from host species of different cross-inoculation groups, suggests that the envelope may be invariably present around Rhizobium cells in a nitrogen-fixing nodule. The structure of ineffective nodules is currently being examined by us. The possible function of the membrane in regulating bacteroid development has been dis-

cussed previously (2). Two main types of development of Rhizobium cells into bacteroids in the nodule can presently be distinguished. In cowpea, V. juncea, A. longijolia, and soybean (1), all members of the cowpea-soybean cross-inoculation miscellany, the Rhizobium cells expand very little during nodule development, retaining a rod form with a central compact nucleoid area. Several bacteroids are enclosed by a single membrane envelope. Development is similar in nodules of Phaseolus vulgaris (7). The other pattern of development occurs in the medic (2, 3, 4, 9), clover (2, 3, 4, 10), and pea (5, 6) inoculation groups, in which the bacteroids expand greatly during development and lose their previous rod structure. The nucleoid region in the bacteroids is only sparsely filled with fibrillar material, and each bacteroid is usually enclosed singly in a membrane envelope. In the latter type of development, multiplication of rhizobia in the host cell apparently precedes membrane envelope synthesis; in the cowpea group, division of rhizobia apparently can also take place within the membrane envelope. The fine structure of bacteroids in the blue lupin nodules resembles the clover-medic-pea type. Each bacteroid is usually enclosed in its individual membrane envelope and expands noticeably during development, losing its former rod shape and compact nucleoid area. A similar development occurs with the bacteroids of L. luteus (8).

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FIG. 1. Mature bacteroid zonie of Acacia longifolia nodule, showing membrane envelopes (e.g., arrows) eniclosinig uniits of several bacteroids; i = inclusioni grantule. X 23,000. KMnO4 fixation.

FIG. 2. Older regioni of nodule where host cytoplasm has collapsed, leaving bacteroids still entclosed by membrane enivelopes. Two of the bacteroids (1) have lysed. X 15,000. FIG. 3. Bacteroid zone of the nodule, showing the "unit membrane" nature of the envelopes; me = membrane enzvelope. X 100,000. 1316

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FIG. 4. Mature bacteroid zone of Viminaria juncea nodule, with bacteroids enclosed by membrane envelopes (e.g., arrows). Symbols: n = bacteroid nucleoid; i = inclusion granule. X 20,000. KMnO4fixation. FIG. 5. Region of nodule where host cytoplasm has collapsed, leaving bacteroids enclosed by membrane envelopes. X 17,000. 1317

FIG. 6. Vignia sinienisis ntodule, mature bacteroid zone, showing several bacteroids eniclosed by a membranie enlvelope (arrow). Intclllsio11 graniules (i) antd electr-on-denise bodies (d) are promin1en1t. Other symbols: n = bhcteroid tiucleoid; w = host cell wall. KMn104fixation. X 22,000. FIG. 7. Lupinius aligustifolius niodules, showinzg swolleni bacteroids enlclosed, usually sinzgly, by a membrane enivelope. Part of this figure is enilarged (iniset), showinig the membranie enivelope (me) distinlct from the bacteroid cell wall membrane anid plasma membranie. The host cell ribosomes lie fiee in a matr-ix of medium electroln-denisity su-rrounidinig the enivelope. The otheer iniset shows a "Y-shaped" bacteroidl; w = host cell wall. OS04 fixation. X 17,000. Imisets. X 50,000 amid X 10,000.

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It is tempting to suggest that, within a particular cross-inoculation group, bacteroid development follows a similar pattern. This hypothesis is currently being examined by us in detail. ACKNOWLEDGMENTS We are grateful to D. G. Drummond for the use of the facilities of the Electron Microscope Unit, University of Sydney. One of us (P. J. D.) was supported by a Research Fellowship of the Linnean Society of New South Wales.

LITERATURE CITED 1. BERGERSEN, F. J., AND M. J. BRIGGS. 1958. Studies on the bacterial component of soybean root nodules: cytology and organisation in the host tissue. J. Gen. Microbiol. 19:482-490. 2. DART, P. J., AND F. V. MERCER. 1963. Development of the bacteroid in the root nodule of barrel medic (Medicago tribuloides Desr.) and

subterranean clover (Trifolium subterranieum L.). Arch. Mikrobiol. 46:382-401. 3. DART, P. J., AND F. V. MERCER. 1963. Membrane envelopes of legume nodule bacteroids. J. Bacteriol. 85:951-952.

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4. DART, P. J., AND F. V. MERCER. 1964. Fine structure changes in the development of the nodules of Trifolium subterranieum L. and Medicago tribuloides Desr. Arch. Mikrobiol. 49:209-235. 5. DIXON, R. 0. D. 1964. The structure of infection threads, bacteria and bacteroids in pea and clover root nodules. Arch. Mikrobiol. 48: 166-178. 6. GRILLI, M. 1963. Osservazioni sui rapporti tra cellule ospiti e rizobi nei tubercoli radicali di Pisello (Pisum sativum). Caryologia 16:561594. 7. GRILLI, M. 1964. Richerche al microscopio ellet-

tronico sui tubercoli radicali di alcune leguminose. Giorn. Bot. Ital. 71:62-67. 8. JORDAN, D. C., AND I. GRINYER. 1965. Electron microscopy of the bacteroids and root nodules of Lupinus luteus. Can. J. Microbiol. 11:721725. 9. JORDAN, D. G., I. GRINYER, AND W. H. COULTER. 1963. Electron microscopy of infection threads and bacteria in young root nodules of Medicago sativa. J. Bacteriol. 86:125-137. 10. MOSSE, B. 1964. Electron-microscope studies of nodule development in some clover species. J. Gen. Microbiol. 36:49-66.