Physiological Plant Pathology (1976) 8, 123-129

Origin of the membrane envelope enclosing the Alnus crispa var. mollis Fern. root nodule endophyte as revealed by freeze-etching microscopy

M. LALoNnEt and I. W. DEVOE

Dcpnrtnunt of Microbiology, Mac&mald Cam@s of McGill Unimxri~, St-c Anne a% Bclkxw, Qulbcc HOA ICO, Canada

(Accepdfor publicafion JVovembcs 1975)

Nitrogen-6xing root nodules of Alnus crispa var. mollis Fem. were studied by freeze-etching microscopy (FEM). Cryo-protection, with dimethylsulfoxide (DMSO), of unfixed nodular tissue, permitted observation of the ultrastructure of the endophyte cells. Replicas of the freeze-etched nodular tissue showed that the host cell wall, host plasmalemma, membrane envelope, endophyte capsule, endophyte cell wall and cell membrane can be differentiated on the basis of their fracture profiles. The pectic capsule surrounding the endophyte cell wall was shown to consist of layers containing randomly distributed granules. It is suggested that these granules represent capsule-bound enzymes. The freeze-etch profile of the mem- brane envelope, which always surrounds the encapsulated hyphal and vesicular forms of the endophyte, was identical to that of the host plasmalemma of the infected cortical cell. These observations confirm the hyphothesis that, after the penetration through the host cell wall, the endophyte hypha enters the cell endocytotically and that its membrane envelope is closely related to the host plasmalemma.

INTRODUCTION The nitrogen-fixing root nodule symbiosis shown by non-leguminous angiosperms permits significant contributions to the nitrogen economy of a number of ecosystems [6]. Unfortunately, isolation and identification of the causal organisms of the root nodule of non-leguminous angiosperms has not been achieved [3, 18, 24, 251. How- ever, based on electron microscope studies of the ultrastructure of non-leguminous root nodules, it is generally agreed that the characters of the endophyte are consistent with the belief that they are prokaryotic actinomycetes [4, 9, 11, 16, 261.

During the initial infection of the host, the free-living form of the actinomycetal endophyte enters the plant root through a root hair [I, 231. Then the hyphal endophyte perforates the host cell wall and enters a cortical cell [13]. As the endophyte emerges from the cell wall, the host cell synthesizes, transports and deposits pectic substances for the encapsulation of the microbial intruder [17]. Surrounding this encapsulation material is a membrane which lies between the endophyte and host cell. Based on transmission electron microscope observations [Z, 16, 301 and scanning electron microscope observations [13], this enclosing membrane envelope appeared to be the invaginated host plasmalemma. This membrane envelope would have a key position during the symbiotic interactions of

t Present address: Unit of Biological Nitrogen Fixation, Botanisch Laboratorium, Nonnensteeg 3, Leiden, The Netherlands.

124 M. Lalonde and I. W. Devoe

the non-leguminous root nodule. We therefore employed the freeze-etching technique to obtain further information on the origin of the enclosing membrane of the A1nu.s crispa var. mollis Fern. root nodule endophyte.

MATERIALS AND METHODS

Green silky alder (Ahus crispa var. mollis Fern.) was grown in glass tubes (20 x 150 mm) each containing 15 cm3 of air-dried sand collected from Tadoussac sand dune (Quebec, Canada). The sand was supplemented to saturation with a nitrogen- deficient nutrient solution [14] used at half strength. After 7 days of germination under diffuse illumination, the alder seedlings were grown in a growth chamber under 30 000 lx at 24 “C for 16 h each day and in darkness at 18 “C for 8 h each night.

Alder roots were examined after 3 months and those nodulated were washed gently in tap water to remove the sand. The root nodules used were removed from the roots by sectioning at the base of the root nodule. This hand sectioning was done under a dissecting microscope with a single edge razor blade. The selected nodules were then immersed in a cryo-protectant solution: glycerol (30% in 0.05 M, pH 7.4 potassium phosphate buffer) or DMSO (30% dimethylsulfoxide in 0.05 M, pH 7.4 potassium phosphate buffer). After an imbibition period of 1 h at room temperature, 1 to 3 nodules were mounted per 3 mm copper disks, and frozen in liquid Freon 22.

Freeze-etching was carried out on a Balzers instrument as described by Moor & Miihlethaler [,?I]. A standard etching time of 90 s at - 100 “C was used in all experiments. Replication was performed by platinum-carbon shadowing followed by carbon reinforcement. Replicas of nodule tissue were cleaned by successive flotations for 2 h on 35% chromic acid, and 30 min on 70% chromic acid, followed by a distilled water rinse and finally by a flotation for 30 min on 6% commercial bleach. Cleaned replicas were picked up on 200 or 300 mesh grids from distilled water. All preparations were examined in an AEI 6B electron microscope operating at 60 kV accelerating voltage.

The terminology described by Miihlethaler [,?.?I for the freeze-etching of cell membranes will be used throughout. Membrane faces revealed by the etching process are called surface faces, the one nearest the interior of the object in question being the inner surface (IS), and the one farthest away, the outer surface (OS). Interior membrane faces, revealed by the fracturing process, are called fracture faces, the one nearest the interior of the object in question being the inner fracture face (IFF), and the one farthest away, the outer fracture face (OFF).

It should be noted that because the membrane envelope surrounding the endo- phyte cells is identified as the invaginated host plasmalemma, in this paper the surface closest to the endophyte capsule will be identified as the outer surface (ME-OS) of the membrane envelope and the surface in contact with the host cell cytoplasm as the inner surface (ME-IS).

RESULTS

Excised root nodules impregnated in 30% glycerol for 1 h showed extensive freezing damage after freeze-etching. Although chemical pre-fixation should have helped glycerol impregnation of the plant tissue [2U], this treatment was avoided because

Freeze-etched endophyte of Alms root nodule 125

pre-fixation of glycerol-protected plant tissue has been shown to alter the fracturing properties of plant membranes [8]. However, the utilization of dimethylsulfoxide (DMSO) as a cryo-protectant, permitted excellent morphological preservation of the fine structures of the unfixed host and endophyte cells of the root nodule. Con- sequently, all the following freeze-etch illustrations were selected from DMSO cryo- protected nodular tissues.

As observed in previous light microscopy [15J, transmission electron microscopy [Iq and scanning electron microscopy studies [13] the freeze-etching technique showed the endophyte of A. crisfia var. mollis Fem. root nodules as hyphal and also as vesicular cells.

In Plate 1 a hyphal endophyte is seen passing through the cell wah of a host cell in the nodular tissue. Numerous hyphae and vesicles can also be seen in thii infected host cortical cell. In Plate 2, which is a higher magnification of the pene- trating hypha shown in Plate 1, it seems evident that the host plasmalemma forms an invagination to enclose the hyphal endophyte. The same Plate 2 also suggests a similarity between the inner fracture face of the exposed membrane envelope, always enclosing the endophyte, and the inner fracture face of the invaginated host plasmalemma. Nevertheless, a further identification and comparison of the other possible fracture planes of the endophyte components must be undertaken before the similarity of the freeze-etch protiles of the membrane envelope and the host plasmalemma can be confirmed.

Replicas of freeze-etched nodular tissues, cryo-protected with DMSO, revealed a variety of fracture profile images. These freeze-etch observations made possible the differentiation of the fracture faces between the host cell wall, host plasma- lemma, membrane envelope, endophyte capsule, endophyte cell wall and endophyte cell membrane. Thus it was possible to compare the fracture characteristics of the host plasmalemma with those of the membrane envelope which always encloses the hyphal or vesicular endophyte.

Host jbm&mma fracture profile

The fracture planes of the host plasmalemma were studied adjacent to the host cell wall of infected cortical cells (Plate 3). The plasmalemma mostly fractured to show a characteristic inner fracture face (P-IFF). The spherical particles on the P-IFF were small and distinct and showed a characteristic distribution pattern (Plate 4). From time to time, the inner surface of the plasmalemma (P-IS) was exposed by the fracturing procedures and was found to be ornamented with vari- ously shaped depressions (Plate 5). Roth fracture planes of the host plasmalemma were easily distinguished from the fibrillar material of the host cell wall (Plates 4 and 5).

Endophyte hypha jkxture jwojiles

Replicas of the freeze-etched hyphal endophyte revealed a variety of fracture plane images. The only face revealed by the fracture of the hyphal cell membrane was covered with densely packed particles which were either spherical or rod-shaped (Plate 6). The capsular material of the hyphal endophyte was composed of granules

10

126 M. Lalonde and I. W. Devoe

randomly distributed in matrix layers (Plates 6, 7 and 8). Surrounding this encap- sulation material, the enclosing membrane envelope was exposed as two freeze- etch profiles : the outer surface and the inner fracture face. The membrane envelope outer surface (ME-OS) was largely covered with particles which were either spherical or rod-shaped (Plate 7). The particles on the membrane envelope inner fracture face (ME-IFF) were small and distinct corresponding in size to the IFF of the host plasmalemma (Plate 8).

In addition, the freeze-etch of the endophyte hypha also permitted to resolve some granules in its cytoplasm. These granules may have been some kind of reserve material. Furthermore, the production of a young vesicle, near the host cell wall, by the terminal swelling of a parental hyphal tip, was easily observed (Plates 9 and 10).

Endophyte vesicles fracture projiles

Replicas of the freeze-etched endophyte vesicles revealed concave and convex frac- ture planes (Plates 10 and 1 l), thus making easier the interpretation of the images. Due to the very characteristic parental hypha attached to it, the endophyte vesicle was easily recognized (Plates 10, 11 and 12).

As observed around the endophyte hypha, the membrane envelope surrounding the endophyte vesicle was revealed as two fracture images: the outer surface (ME- OS) and the inner fracture face (ME-IFF) (Plates 12 and 13). The ME-OS of the vesicle was characterized by a high density of spherical and rod-shaped particles (Plate 12). The ME-IFF of the vesicle was characterized by spherical particles only (Plates 12 andl3), and was similar, if not identical, to the inner fracture face of the host plasmalemma (Plate 4). In a few observations, the ME-IFF of the vesicle was seen to continue around the endophyte vesicle from the host cell cytoplasm (Plate 12). This continuity was also observed at the periphery of the hyphal endophyte (Plate 8). This rare freeze-etch image, of the membrane envelope on the side of the endophyte cells, is explained by the fact that two or more encapsulated endophyte hyphae or vesicles can be enclosed in a common membrane envelope [13, 161.

In other fractured preparations the membrane envelope has been stripped away to expose the capsular material which always surrounds the endophyte vesicle (Plates 14 and 15). This encapsulation material was clearly distinguished as layers containing randomly distributed granules (Plates 14 and 15). However, the poly- saccharidic inner line, situated at the base of the endophyte capsule [17], was never observed in the freeze-etch views of the vesicle. The freeze-etch image of the vesic- ular capsule is identical to that of the hyphal capsule.

The vesicle cell membrane was easily distinguished from the capsular material or the membrane envelope. The vesicle cell membrane was observed as an outer fracture face (M-OFF) which had a characteristic granular appearance with densely distributed spherical and rod-shaped particles (Plates 14 and 15). This M-OFF was also observed as a component of the parental septation separating the parental hypha from the vesicle (Plate 13). An endophyte cell wall fracture view was rarely seen but, when observed, it showed a characteristic fracture face, easily distinguished from the other endophyte fracture planes (Plate 13).

-~-- PLATE 1

PLATES 2, 3,4 and 5

PLATES 6, 7, 8 and 9

PLATES 10 and 11

PLATES 12 and 13

PLATES 14 and 15

All plates were printed to show shadows as white zones. Q: Convex cleavage; IS: concave cleavage; Encircled ) : direction of shadow in the illustration.

PLATE 1. Freeze-etch view of Alms root nodule tissue showing a host cortical cell infected by the hyphal (H) and vesicular (V) forms of the actinomycetal endophyte. Note, in the outlined box, the penetration of an endophyte hypha (H) through the host cell wall (CW). PH: Parental hypha. Bar is 1 urn.

PLATE 2. Plate 2, which is an enlarged detail of Plate 1, shows the penetration of the host cell wall by an endophyte hypha. Notice the invaginated host plasmalemma which is enclosing the encapsulated (C) penetrating hypha. Note also that the freeze-etch image of the plasma- lemma inner fracture face (P-IFF) is similar to that of the membrane envelope inner fracture face (ME-IFF). M: Endophyte cell membrane. Bar is 1 pm.

PLATE 3. Freeze-etch fracture of the cell wall of an infected cortical cell. Notice the plasma- lemma inner fracture face (P-IFF) still adhering to the host cell wall (CW). A concave replica of the inner fracture face of the membrane envelope (ME-IFF) can be recognized surrounding an endophyte vesicle. fm: Frozen medium. Bar is 1 urn.

PLATE 4. Detail of an inner fracture face of the plasmalemma (P-IFF) which is adhering to the fibrillar host cell wall (CW). A frozen medium (fm) can be observed in the plaque well (PW) of this host plasmalemma. Bar is 0.1 pm.

PLATE 5. Detail of the inner surface of the host plasmalemma (P-IS). Note that this surface is characterized by numerous depressions instead of particles. Notice also the characteristic fibrillar structure of the host cell wall (CW). fm: Frozen medium. Bar is 0.1 pm.

PLATE 6. Freeze-etched endophyte hypha showing its cytoplasmic membrane (M) covered by numerous particles. Note the hyphal capsule (C) which is composed of pectic layers with characteristic granules (CG) distributed in them. fm: Frozen medium. Bar is 0.5 urn.

PLATE 7. Endophyte hypha fractured at the level of its membrane envelope outer surface (ME-OS). Note that the hyphal capsule, which is located between the membrane envelope and the endophyte cell wall, is formed of layers (CL). Bar is 0.5 pm.

PLATE 8. Freeze-etch view of the inner fracture face (ME-IFF) of the membrane envelope surrounding an endophyte hypha. Note that the cross-fracture of this hypha has exposed some granules (G) in the hyphal cytoplasm (CY). C: Endophyte capsule; fm: frozen medium. Bar is 0.5 pm.

PLATE 9. This freeze-fracture view illustrates the formation of a young vesicle (YV) at the tip of a parental hypha (PH). hi: Endophyte cell membrane; C: endophyte capsule; fm: frozen medium. Bar is 0.5 urn.

PLATE 10. Note the vesicular form (V) of the endophyte near the host cell wall (CW). A parental hypha (PH), which gave rise to a vesicle, can be easily recognized. The vesicles (V) were fractured at the level of their capsule (C). Bar is 1 urn.

PLATE 11. Concave and convex views of endophyte vesicles fractured at the level of their membrane envelope (ME), capsule (C) or cytoplasm (CY). PH: Parental hypha. Bar is 1 urn.

PLATE 12. Convex view of a parental hypha (PH) with a vesicle attached to it. The freeze- fracture took place at the level of the outer surface (ME-OS) of their common membrane envelope. Some fragments of the inner fracture face (ME-IFF) of the membrane envelope can be seen near the vesicle. Bar is 0.5 pm.

PLATE 13. Freeze-fracture view of the septation separating a parental hypha from its vesicle. The cell membrane outer fracture face (M-OFF) and the cell wall inner surface (W-IS) of the parental septation were exposed during the fracturing process. Note the thick capsule (C), which is formed of layers, below the membrane envelope exposed at the level of its outer surface (ME-OS) or of its inner fracture face (ME-IFF). Bar is 0.5 pm.

PLATE 14. Freeze-etch fracture of a vesicle attached to its parental hypha (PH). The fracture took place at the level of the encapsulation material which is distinguished as layers (CL) with randomly distributed granules (CC) in them. Note that the parental hypha (PH) was cleaved at the level of its septation (PS). Note also that the cell membrane was exposed as an outer fracture face (M-OFF). Bar is 0.5 urn.

PLATE 15. Endophyte vesicle fractured at the level of its capsule which is formed of granules (CG) and of layers (CL). The fracturing also took place at the level of the cell membrane which is exposed as an outer fracture face (M-OFF). s: Vesicle septation; ME: membrane envelope; fm: frozen medium. Bar is 0.5 pm.

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Freeze-etched endophyte of Alnus root nodule 127

DISCUSSION Our interpretation of the images of replicas formed from the freeze-etching of nodular tissues supports the hypothesis that the membrane envelope, always found surrounding the endophyte hyphae and vesicles, originates from the invagination of the host plasmalemma. At the same time, the present freeze-etching microscope (FEM) observations confirm the previous interpretations of the A. tipa var. mollis Fern. root-nodule endophyte structure as observed in light microscopy (LM), trans- mission electron microscopy (TEM) and scanning electron microscopy (SEM) [13, 15, 16-J.

Lalonde & Knowles [17] showed that as the endophyte emerged from the cell wall, the host cell deposited a pectic substance encapsulating the intruder. By means of staining techniques they demonstrated that this pectic capsule was mainly formed of granules and of fibrillar layers. In the present freeze-etch study the endo- phyte capsule was clearly distinguished as numerous layen with randomly distributed granules in them. However, due to their small dimension, the fibrils of the pectic layers were not resolved by the Pt/C replica.

The granules observed in the capsular layer, surrounding both the hyphal and vesicular forms of the endophyte, were similar to previously described membrane- bound particles [7, 221. From these observations, it seems reasonable to speculate that the capsule granules are similar to the globular proteins characteristic of bio- logical membranes [7, 221. This suggestion of capsule-bound proteins, and conse- quently of capsule-bound enzymes, is sustained by the fact that the polysaccharidic capsule of non-leguminous root-nodule endophytes showed electron density after uranyl-lead staining [4, 9-11, 16, Zs]. Bound enzymes, randomly distributed in the capsular pectic layers, could facilitate the uptake or the release of metabolites, or function in the synthesis or the degradation of the capsule peck layers them- selves. Degradation of the pectic capsule could be a means by which the hetero- trophic endophyte obtains carbohydrate (e.g. galacturonic acid) from its auto- trophic host [27J.

The results obtained in the present freeze-etching study are in agreement with the hypothesis that, after the penetration through the host cell wall, the endophyte hypha enters the cell endocytotically and that its membrane envelope must be related to the plasmalemma of the host cortical cell. This endocytotic penetration of the actinomycetal endophyte in the non-leguminous root nodule is similar to that of Rhizobium in the leguminous root nodule [5, 12, 28, 291.

The presence of protein in the host membrane envelope, suggested by numerous spherical and rod-shaped particles observed on the ME-IFF and the ME-OS, might reflect the enzymatic activity which must take place during the symbiotic interactions of the host and endophyte cells. Identification of these enzymes should be attempted by the use of histochemical techniques similar to those employed by Marks & Sprent [19] in their study of the localization of enzymes in soybean root nodules.

Our proposal for the origin of the membrane envelope is based on the assump- tion that the freeze-etch images of a particular membrane type have consistent features mostly due to the globular proteins borne by it [7, 221. One possible way to confirm our interpretation of the freeze-etch images would be the localization

128 M. Lalonde and I. W. Devoe

of a marker enzyme, such as the adenyl cyclase plasmalemma-bound enzyme, to study the relationship between the membrane envelope and the host plasmalemma Km

The authors acknowledge Mr J. E. Gilchrist for his kind assistance with the Balzers equipment. We are indebted to Dr R. Knowles for providing laboratory and growth chamber facilities and for revising the manuscript. This work was supported by a NATO Postdoctoral Fellowship to the first author and a grant from the National Research Council of Canada.

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