BY LILIAN E. HAWKER AND MARGARET A. GOODAY. Department of Botany, University of Bristol. {Received l"] June 1968) SUMMARY

Transcription

1 . (1969) 68, FUSION, SUBSEQUENT SWELLING AND FINAL DISSOLUTION OF THE APICAL WALLS OF THE PROGAMETANGIA OF RHIZOPUS SEXUALIS (SMITH) CALLEN: AN ELECTRON MICROSCOPE STUDY BY LILIAN E. HAWKER AND MARGARET A. GOODAY Department of Botany, University of Bristol {Received l"] June 1968) SUMMARY Conjugating progametangia of Rhizopus sexualis rapidly enlarge and become flattened at the apical zone of contact. The walls over this zone fuse immediately, lose their separate identity and form a single plate. The fusion wall then thickens by gelatinization and swelling: there is no evidence of deposition of secondary wall material. The plasmalemmas on either side of the fusion wall become wrinkled and enclose pockets of electron transparent material along the sides adjacent to the wall. Vesicles and later lomasome-like masses are seen in a few of these pockets. Numerous small vesicles are present in the adjacent zones of cytoplasm on either side of the wall; some may be seen attached to the plasmalemmas. The wall then thins from the centre of the plate outwards but remains in being until after the gametangia have been delimited by new septa. Cell fusion then takes place by the development at the centre of the fusion wall of a hole which enlarges rapidly until the whole of the plate is dissolved. The evidence from electron micrographs suggests that the dissolution process is entirely chemical and that both matrix and microfibrillar skeleton are dissolved enzymatically while still enclosed in the plasmalemma. INTRODUCTION The formation of the zygospores of members of the Mucoraceae is a familiar phenomenon but details of many stages in this process are either not known or have been wrongly interpreted. Previous papers by the present writers (Hawker and Gooday, 1967, 1968) have given details of the formation of the septa delimiting the gametangia of Rhizopus sexualis (Smith) Callen and of the development of the ornamentation of the zygospore wall. The present paper is concerned with the fusion of the apical parts of the walls of the conjugating progametangia to form a fusion plate and with the subsequent dissolution of this plate allowing cell fusion of the gametangia. Vuillemin (1903) described the events following contact between the conjugating branches of Sporodinia grandis (syn. Syzygites megahcarpus). He noted that the tips of these branches became flattened by reciprocal conapression so that the conjugated progametangia were spindle-shaped, with the fused apical walls forming a partition across the zone of contact. He also observed that this 'primary median partition' increased in thickness after the gametangia had become delimited by the formation of new septa. He refers to the formation of a 'secondary median wall' but it is not clear whether he considered this to be the result of the temporary addition of new wall material to the primary partition or merely the swelling of the latter. 133

2 134 LILIAN E. HAWKER AND MARGARET A, GOODAY The present writers have found no reference to the actual process of fusion of the progametangial walls in any recent studies by optical or electron microscopy. Sassen (1962), however, examined the breakdown of the fusion wall in the young zygospore of Phycomyces blakesleeanus and considered this to be entirely due to enzyme activity. Later, in 1964, he compared the process to cell wall dissolution in other examples of cell fusion. METHODS The methods used were described in a previous paper (Hawker and Gooday, 1967). The development sequence was built up from evidence from some twenty sectioned zygospores. RESULTS Early contact stage The progametangia at the time of contact are surrounded by uniform walls which are relatively thin (c. 0,1 ^ thick) and which are less electron dense than the cytoplasm and, therefore, show as a light grey layer in electron micrographs. As soon as contact is established the size of the progametangia increases rapidly and their shape alters. The apices become flattened by compression and the greatest diameters of both progametangia are just behind their flattened apices so that each is at flrst carrot-shaped and later turnipshaped. At an early stage after initial contact the separate identities of the original apical walls of the conjugating progametangia can no longer be distinguished and the groove (g in Plate i, Nos. i and 2) at the edge of the zone of contact is seen to be partially filled with apparently gelatinized or mucilaginous material. Shrinkage during fixation tends to tear the newly fused walls from the progametangial cytoplasts, but it is significant that the plate remains as a single entity and does not split into its original components (Plate I, No. i). Median sections of newly fused progametangia show that the regions adjoining the fusion wall are rich in nuclei and mitochondria but poor in discernible food reserve material. Nuclei and mitochondria are also present in the peripheral parts of the progametangia further back from the zone of fusion, but the central core here contains numerous lipid droplets and storage vacuoles containing unidentified material. Histochemical staining (Hawker, Abbot and Gooday, 1968) shows that glycogen is also present in this area. Glycogen can be detected in electron micrographs of zygospores at a slightly later stage (Plate i. No. 3). Swelling of fusion wall After the apical parts of the progametangial walls have fused together, the resulting fusion plate rapidly increases in thickness, but the appearance in both electron micrographs and photomicrographs is consistent with this increase in thickness being a result of gelatinization rather than being due to the deposition of new material. Plate i. No. 3 shows the thickened wall soon after fusion has occurred and before the initiation of the gametangial septa. It suggests that gelatinization is proceeding centrifugally from the originally inner layer of each of the component walls towards the plane of fusion, since the central part of the fusion wall is more electron dense and is less amorphous than the parts adjoining the plasmalemmas of the two progametangial cytoplasts. The general appearance is consistent with a progressive dissolution of the microfibrillar

3 Wall fusion and dissolution in Rhizopus 135 skeleton by the action ot enzymes passing through the plasmalemmas towards the wall. The plasmalemmas along the fusion wall show a slight wrinkling instead of having a smooth outline in section and there is a narrow electron-transparent zone between plasmalemma and wall. At a slightly later stage when the gametangial septa have begun to cut off the gametangia (Plate 2, No. 5), the fusion wall has thickened further. The most striking change, however, is a great increase in the degree of wrinkling of the plasmalemmas on either side of the fusion wall (Plate 2, No. 4; Plate 3, No. 7). These wrinkles surround pockets of electron transparent material between the plasmalemma and the wall, which itself no longer has an entire outline. In some of these pockets one or more darker bodies can be seen (Plate 2, No. 4), which at higher magnification (Plate 2, No. 6) are apparently membrane-bounded vesicles. Numerous vesicles can be seen in the cytoplasm near the plasmalemma (Plate 2, No. 4; Plate 3, No. 7) or attached to it. These are of two kinds: (i) very small vesicles with electron dense contents and (ii) slightly larger ones with much less dense contents. The observations suggest the passage of material, enclosed in vesicles, across the plasmalemma, but it has not been possible to determine the direction of movement. However, since the available electron micrographs of several spores at this stage give no indication of breakdown and absorption of the vesicles in the cytoplasm it is unlikely that they contain wall material passing into the cytoplast and being absorbed there. It is more likely that the vesicles contain wall-dissolving enzymes formed in the cytoplast and passing through the plasmalemma into the peripheral layers of the fusion wall. Plate 2, No. 4 and Plate 3, No. 7 show vesicles of both types, either fusing with the apex of one of the plasmalemma wrinkles or being given off by it as a bubble. It is not, of course, impossible that there is transport both ways, or that the two types of vesicle behave differently. Thinning of central area of fusion plate At the stage shown in Plate 2, No. 4 the fusion wall forms a plate of approximately equal thickness extending across the whole width of the spore initial and separating the two progametangia. By the time that the septa delimiting the gametangia are fully formed the fusion plate has reached and passed its maximum thickness and has become progressively thinner from the peripheral parts towards the central area (Fig. ia). The plasmalemmas are still wrinkled; the pockets referred to above have coalesced to give a continuous electron transparent zone and some now contain systems of vesicles or tubules of sufficient complexity to be described as lomasomes (Fig. ig). It is clear that the process of dissolution of the fusion plate has progressed but the plate is still continuous and cell fusion has not yet taken place. Breakdown of the fusion wall Soon after the completion of the gametangial septa, fusion of the gametangia is completed. Communication between them is first established by the final dissolution of a patch of fusion wall to form an irregular central hole. Sassen (1962) claims that with Fhycomyces other holes develop rapidly in the peripheral parts of the fusion wall. Sections of Rhizopus zygospores at this stage (Plate 3, No. 8; Fig. ib) show isolated pieces of the fusion wall embedded in the endoplasm. This could be the result of the formation of additional holes in the disintegrating wall but could equally well be interpreted as due to the irregular shape of the enlarging central hole (Fig. id and e).

4 136 LILIAN E. HAWKER AND MARGARET A. GOODAY The remaining parts of the fusion wall become irregular in thickness, probably owing to release of tension (Fig. ib), but are still surrounded by the plasmalemma. Frequently (g) Fig, I, Three stages in dissolution of fusion wall of zygospore of Rhizopus sexualis, drawn from electron micrographs. Walls shown by dense stippling, (a) Thinning of wall has begun at centre but wall has not yet been breached (cf, Plate 2, No, 5), (b) Wall has been completely dissolved away in at least one place (cf, and Plate 3, No, 9). (c) Only peripheral parts of fusion wall left, zygospore by now beginning to enlarge, (d) and (e) Diagrammatic, illustrating the way a section such as that of (b) eould result from the development of either a single hole at the centre of the fusion wall becoming of irregular outline or the development of several holes (line indicates plane of section), (f) Drawn from electron micrographs of sections of the same zygospore as that shown in Plate 3, No, 9 and shown diagrammatically in (b) above, illustrating the swollen rim of one of the isolated patches of dissolving fusion wall (shading indicates electron density of wall), (g) Lomasome-like bodies seen along dissolving fusion wall in some but not all specimens, drawn from an electron micrograph. PI, plasmalemma; w, wall; 1, lomasome, the part of the wall bordering a hole is swollen and distorted (Fig. if). The hole(s) enlarge and soon coalesce until a peripheral fringe is all that remains of the fusion plate (Fig. ic). This fringe may persist for some time and the exact stage at which it is finally

5 Wall fusion and dissolution in Rhizopus 137 absorbed may vary, but in normal zygosporcs no trace of tbe fusion wall is left by tbe time tbe ornamented mesospore (Hawker and Gooday, 1968) is fully developed. Tbe breakdown of tbe fusion wall is accompanied by an increase in the number of vesicles and an increase in the proportion of type (i) (p. 135) in the vicinity of the disintegrating wall fragments. Again it is not clear whether these organelles contain wall material being translocated into the cytoplasm or whether they contain further supplies of wall-dissolving enzymes approaching the rapidly disappearing wall or whether both processes are taking place simultaneously. DISCUSSION Vuillemin's (1903) description of conjugation in Syzygites megalocarpa does not entirely agree with the present account of the process in Rhizopus sexualis since he claimed that the fusion wall continued to thicken after the delimitation of the gametangia. Details of the dissolution of the fusion wall are, however, difficult to determine with the optical microscope alone. The process of breakdown of the fusion plate in R. sexualis is essentially similar to that described by Sassen (1962) for Phycomyces blakesleeanus in that dissolution begins by thinning at the centre followed by the development of a central hole and then proceeds irregularly. Sassen refers to a layer of cytoplasm free of nuclei and mitochondria but does not specify the exact stage to which this observation applies. In Rhizopus (p. 134) mitochondria and nuclei are numerous in the apical parts of the conjugating progametangia and near the newly fused wall but, later, when dissolution of the latter commences there is a narrow zone largely free of these organelles adjacent to it. At no stage does the zygospore of R. sexualis show the large central vacuole described and figured by Sassen for Phycomyces, although many smaller vacuoles containing unidentified material develop in the interior of tbe maturing spore. Perhaps the most interesting features of the dissolution of the fusion wall in Rhizopus are (i) the wrinkling of the plasmalemmas bordering this wall and (2) the association of the small endoplasmic vesicles with the plasmalemmas. Sassen's material of Phycomyces was fixed with osmium tetroxide and his published electron micrographs do not show clear details of either plasmalemmas or vesicles, but he refers to the presence of the latter and considers them to be part of the endoplasmic reticulum, without, however, postulating any particular function for them. Several authors have reported in a number of fungi, vesicles similar in appearance to those described in the present paper, but associated with areas of active wall formation, in contrast to their association with wall dissolution in Rhizopus. A zone staining deeply with iron-haemotoxylin at the apex of hyphae of two species of Coprinus was observed by Brunswik (1924) who termed it a 'Spitzenkorper'. Similar effects were seen by Girbardt (1955, 1957) in other Basidiomycetes and in species of Aspergillus and Penicillium. McClure, Park and Robinson (1968) report tbe presence of 'Spitzenk5rper'-like structures in the hyphae of several higher (septate) fungi. They claimed that electron microscopy showed these bodies to contain numerous small vesicles which McClure et al. (1968) suggest 'fuse with the plasmalemma liberating their contents as part of the process of wall formation'. The individual vesicles resemble the larger (type (ii), p. 135) of those associated with the disintegrating fusion wall of the Rhizopus zygospore. McClure et al. (1968) were unable to detect a well-defined Spitzenkorper in the hyphal apex of R. stolonifer (syn. nigricans) although they observed a number of small moving particles which could have been vesicles. They give no report

6 138 LILIAN E. HAWKER AND MARGARET A. GOODAY of electron microscope observations with this fungus. Marchant, Peat and Banbury (1967) in a study of hyphal growth and wall synthesis in Fhycomyces blakesleeanus, Fiisarium culmorum, Coprinus lagopus and Fythium ultimum and Peat and Banbury (1967) in a study of the growth of sporangiophores of Fhycomyces report the presence of numerous vesicles in all areas of active wall formation. They conclude that these vesicles are produced by the endoplasmic reticulum, travel towards the periphery of the hyphal apex and fuse with the plasmalemma, giving it a crenellated appearance (similar to that seen in the plasmalemmas bordering the disintegrating fusion wall of the Rhizopus zygospore). The striking similarity in appearance of areas of active wall formation described by the above authors with that of a dissolving wall described in the present paper is of considerable significance. No information is available concerning the nature of the contents of the endoplasmic vesicles, but it might be supposed that these are either enzymes associated with the synthesis or degradation of wall materials, or are the basic cell wall materials themselves. The former is the more likely supposition, since the basic cell wall components are probably of a sufficiently simple nature (e.g. hexose sugars) to permit direct passage through the plasmalemma without an elaborate mechanism of vesicle conveyance. It then becomes likely that the enzymes are in both instances conveyed through the plasmalemma towards the wall. The activity of the enzymes would then be directed towards wall synthesis or wall breakdown according to a localized regulating factor. Marchant, Peat and Banbury (1967) report also the presence of multivesicular bodies in the three chitin-producing species examined by them and postulate that these function in the formation of the chitin microfibrils of the wall. Similar bodies have been seen hy the present writers in Rhizopus (Plate 2, No. 5) but not with sufficient frequency or regularity to warrant any interpretation of their role in wall formation or breakdown. Lomasomes were also reported by Marchant et al. (1967) and are considered to have been formed from the multivesicular bodies. They described additional structures having sonne features in common with lomasomes but thought to have arisen by elaboration of the plasmalemma independently of the multivesicular bodies. Lomasomelike bodies have been seen also in association with the dissolving fusion wall of the Rhizopus zygospore (p. 135) but in view of the widely different interpretations of the nature and function of lomasomes put forward by various authors (summarized by Bracker, 1967 and by Marchant et al., 1967) attempts to interpret their role would be premature. The evidence presented for Rhizopus supports Sassen's conclusion that chemical dissolution of the fusion wall is complete and that there is no microfibrillar residue left free in the cytoplasm after cell fusion. Thus Frey-Wyssling's (1959) suggestion that with cellulose plant cell walls (in the formation of sieve plates) the matrix may be dissolved enzymatically without the destruction of the cellulose microfibrils is not paralleled in known examples of dissolution of chitinous walls (viz. the fusion walls of zygospores of Rhizopits and Fhycomyces and the fusion wall of conjugating cells of the yeast Hansenuk wingei (Brock, 1961)). ACKNOWLEDGMENTS Thanks are due to the Science Research Council for a grant in aid of this investigation, to Dr M. F. Madelin and other colleagues for the stimulus of helpful discussions of the results, to Miss B. Reynolds for making the tracing for Plate i. No. i and to Mr G. H. Rogers for technical assistance in preparing prints for the plates.

7 Wall fusion and dissolution in Rhizopus 139 REFERENCES BRACKER, C. E. (1967). Ultrastructure of fungi. A. Rev. Phytopath., 5, 343. BROCK, T. D. (1961). Physiology of the conjugation process in the yeast Hansenula wingei.j. gen. Microbiol., 26, 487, BRUNSWIK, H, (1924), Untersuchungen liber die Geslechts- und Kernverhaltnisse bei der Hymenomyceten- Gattung Copriniis. Bot. Abb. K. Goebel, 5. FREY-WYSSLING, A. (1959), Die Pflanzliche Zellwand. Springer, Berlin, GiRBARDT, M, (1955). Lebendbeobachtungen an Polystictus versicolor (L.), Flora, yena, 142, 540. GiRBARDT, M, (1957), Der Spitzenkorper von Polystictus versicolor (L,), Planta, 50, 47. HAWKER, L. E., ABBOTT, P. McV, & GOODAY, M, A. (1968). Internal changes in hyphae of Rhizopus sexualis (Smith) Callen and Mucor hiemalis Wehm. associated with zygospore formation. Ann. Bot. N.S., 32, 137. HAWKER, L. E. & GOODAY, M. A. (1967). Delimitation of the gametangia of Rhizopus sexualis (Smith) Callen: an electron microscope study of septum formation, J. gen. Microbiol., 49, 371. HAWKER, L, E. & GOODAY, M. A. (1968). Development of the zygospore wall in Rhizopus sexualis (Smith) Callen, J. gen. Microbiol. (In press.) MCCLURE, K, W,, PARK, D, & ROBINSON, P, M. (1968), Apical organization in the somatic hyphae of fungi, J. gen. Microbiol., 50, 177. MARCHANT, R., PEAT, A. & BANBURY, G. H. (1967). The ultrastructural basis of hyphal growth. New Phytol, 66, 623. PEAT, A. & BANBURY, G. H. (1967). Ultrastructure, protoplasmic streaming, growth and tropisms of Phycomyces sporangiophores. II. The ultrastructure of the growing zone. New Phytol., 66, 475. SASSEN, M. M. A. (1962). Breakdown of cell wall in zygote formation of Phycomyces blakesleeaniis. Kon. Akad. V. Wet. Amsterdam, C65, 447. SASSEN, M. M. A. (1964). Growth and breakdown of cell walls. Rep. ^rd Eur. Reg. Conf. Elec. Microsc, Prague. VuiLLEMiN, C. R. (1903). La membrane des zygospores de Mucorinees. C. r. hebd. Seanc. Acad. Sci., Paris, 137, 869.

8 140 LILIAN E. HAWKER AND MARGARET A. GOODAY EXPLANATION OF PLATES Key to general lettering: fw, fusion wall; g, groove between two recently conjugated progametangia; pi, plasmalemma; er, endoplasmic reticulum; m, mitochondrion; n, nucleus; gl, glycogen; fv, food vacuole; v, small vesicle (vi, type (i); vii, type (ii) see p. 135). No. I. A tracing from an electron micrograph, No. 5 was taken with a Zeiss photomicroscope, all others are electron micrographs. Lines = 1 n except in No. 6 where line = o.i //. PLATE I No. I. Tracing from an electron micrograph of an L.S. of newly conjugated progametangia. Apical walls have fused and do not separate from each other even when shrinkage during processing causes the fusion wall to tear away from the cytoplasts. The groove between the progametangia is already filling. Mitochondria are shown solid black, nuclei by coarse stippling, food vacuoles by lighter stippling, lipid droplets left unshaded. Note zonation: narrow zone (zi) next to fusion wall contains numerous mitochondria; behind this (zii) is a wider zone containing both mitochondria and nuclei plus a few lipid droplets, next (zih) is a zone containing a central mass of food vacuoles (contents undetermined, but thought to be phospholipids) of irregular size and shape and a few lipid droplets, the whole surrounded by a peripheral layer containing mitochondria and nuclei; next a zone (ziv) in which lipid droplets are more numerous than the irregular food vacuoles; and finally (zv) the tapering base of the progametangium which resembles the parent zygophore in structure. Note the preponderance throughout the progametangium of paired nuclei (suggesting recent division). Glycogen can be seen in patches throughout the progametangium in electron micrographs of larger magnification but is not shown in the tracing. No. 2. Section through area of the groove (g in No. i) enlarged to show the loose, probably mucilaginous material filling this groove. No. 3. Central part of fusion wall of a zygospore slightly older than that shown in Nos. i and 2, showing wrinkled plasmalemma and adjacent zones containing small vesicles. PLATE 2 No. 4. Fusion wall showing dissolution commencing. Plasmalemma more deeply wrinkled than in No. 3 and numerous pockets (p) of electron-transparent material developed between it and fusion wall. Dark bodies are present in a few of these pockets (d). The cytoplasm adjacent to the plasmalemmas contains numerous small ovoid vesicles of two main types, very small ones with electron-dense contents and larger ones with less-dense contents. Both types may be seen in contact with small bits of er (x), free in the cytoplasm, and in contact with plasmalemma (y). A single multivesicular body (mv) is shown. No. 5. Photomicrograph of zygospore from which sections shown in Nos. 4, 6 and 7 were later cut, to show stage of progametangial fusion, i.e. fusion wall still not breached, gametangia completely delimited by septa(s). No. 6. Small part of same section as that of No. 4 (but not shown in No. 4) at high magnification to show membrane surrounding a dark body in one of the pockets between plasmalemma dissolving fusion wall. PLATE 3 No. 7. Part of the section shown in No. 4 enlarged to show relationship between vesicles and plasmalemma. No. 8. Section cut from older zygospore showing isolated piece of fusion wall, eroded and irregular in shape. Note vesicles surrounding it.

9 THE NEW PHYTOLOGIST, 68, i PLATE I 1/ Kf^S'/ LILIAN E HAWKER AND MARGARET A. GOODAY I^.<4LL FUSION AND DISSOLUTION IN RHIZOPUS (facing page 140)

10 THE NEW PHYTOLOGIST, 68, i PLATE 2 J LILIAN E. HAWKER AND MARGARET A. GOODAY WALL FUSION AND DISSOLUTION IN RHIZOPUS

11 THE NEW PHYTOLOGIST, 68, i PLATE 3 LILIAN E. HAWKER AND MARGARET A. GOODAY W'^Li FUSION AND DISSOLUTION IN RHIZOPUS

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