freeze substitution reveal a nesw model for sporangial...

Freeze substitution reveals a new model for sporangial cleavage in

Phytophthora, a result with implications for cytokinesis in other eukaryotes

G. J. HYDE1*, S. LANCELLE2, P. K. HEPLER2 and A. R. HARDHAM1

1Plant Cell Biology Group, Research School of Biological Sciences, The Australian National University, Canberra ACT, Australia2Botany Department, University of Massachusetts, Amherst MA 01003, USA

•Author for correspondence

Summary

Rapid freezing and freeze substitution (RF-FS) havebeen used to re-examine the process by which themultinucleate sporangium of the Oomycetes, Phy-tophthora cinnamomi and P. palmivora, is sub-divided into uninucleate zoospores. The resultsindicate a new model for sporangial cleavage inPhytophthora and suggest that the currently ac-cepted model is based on interpretation of artefactscaused by chemical fixation. The previous modeldescribes cleavage as a two-stage process in whichspeciah'zed cleavage vesicles first become positionedat the boundaries of each future subdivision andlater fuse to compartmentalize the sporangium. RF-FS, however, indicates that cleavage results from theprogressive extension of paired sheets of membranealong the future subdivision boundaries. Thesesheets finally interconnect and subdivide the sporan-gium. Cleavage vesicles are only evident in prelimi-nary stages of this process and are never aligned

along the future boundaries, contrary to the obser-vations of studies based on chemical fixation. Chemi-cal fixation apparently causes the membranoussheets to vesiculate, even at relatively advancedstages of cleavage, thus giving the misleading im-pression that the resulting network of lined-upvesicles is an intermediate stage in the cleavageprocess. This finding has wide-ranging implicationsfor the understanding of eukaryotic cytokinesis,because all previous studies that describe vesiclealignment and fusion have relied upon chemicalfixation. Other novel features revealed by RF-FSinclude an extensive extracellular matrix within thesporangium that could be involved in zoosporerelease, and a trans-Golgi network.

Key words: cytokinesis, freeze substitution, Phytophthora,Golgi apparatus, extracellular matrix, zoosporogenesis,sporangial discharge.

Introduction

From the great number of ideas that have been putforward to explain the genesis of the additional plasmamembranes required by cells formed during cytokinesis(Pickett-Heaps, 1972a; Rappaport, 1971,1986), two princi-pal models have emerged. The first involves a two-stageprocess in which vesicles are aligned along some part or allof the future plane of cleavage, this part then beingestablished by fusion of the vesicles. The second modelinvolves the progressive extension or stretching of thepartitioning membranes along some part or all of thedeveloping cleavage plane, without any observed prealign-ment of vesicles. Examples in support of both mechanismshave been reported in animal (Thomas, 1968; Rappaport,1986), plant (Gunning, 1982; Volker, 1972), fungal(Hawker and Gooday, 1967; Cole, 1986) and protoctistan(sensu Margulis et al. 1989: Rawlence, 1973; Lokhorst andSegaar, 1989) systems.

In a previous study of chemically fixed sporangia of thenotorious plant pathogen, Phytophthora cinnamomi, weemployed electron microscopy to examine the process bywhich the infectious, motile zoospores of this organism areformed. We concluded that formation of the zoosporicplasma membranes during sporangial cleavage followedthe first of the models described above, that is, fusion ofJournal of Cell Science 100, 735-746 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

prealigned vesicles (Hyde et al. 1991a). This process hasalso been described for P. palmivora (formerly P. parasit-ica, Hohl and Hamamoto, 1967) and all other species ofPhytophthora for which the mechanism of zoosporicplasma membrane genesis has been described (review byHemmes, 1983). In our previous study, however, we notedthat the appearance of elements of the cleavage system ofP. cinnamomi showed some marked variations that did notconform to the vesicle alignment model and that wererelated to the osmolarity of the fixative (Hyde etal. 1991a).These variations led us to test our conclusions byexamining sporangia prepared by rapid freezing, aprocedure which is considered to provide better preser-vation of membrane morphology than chemical fixation.

In this study we re-investigate zoospore formation in P.cinnamomi and P. palmivora using rapid freezing, at bothambient and high pressure, followed by freeze substitution(RF-FS). Our studies have been assisted by the use of amonoclonal antibody that labels cleavage elements in P.cinnamomi and P. palmivora. This is the first time, to ourknowledge, that rapid freezing has been used to studycytokinesis in any eukaryotic organism for which vesiclealignment has been proposed to play a role in partitioningmembrane formation. The results indicate that cleavage ofthe sporangium follows not from the fusion of prealignedvesicles, as suggested by chemical fixation but rather from

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the progressive extension of partitioning membranes. Webelieve that the apparent misinterpretation of the cytoki-netic process that has occurred in Phytophthora mayreflect a much wider problem, involving studies of a greatvariety of eukaryotic organisms. We also report othernovel observations of sporangial structure, and partiallycharacterize an extensive extracellular matrix that de-rives from the contents of the cleavage system and thatmay play a role in zoospore release.

Materials and methods

Organisms and samplingThe cultures of P. cinnamomi and P. palmivora used in this studywere induced to produce sporangia and zoospores by the methodsof Hardham and Suzaki (1986). Briefly, sporangial formation wasinduced by transfer of mycelium to a nutrient-poor medium;sporangial cleavage was induced by treatment with cold distilledwater. Zoospores were released at about 50min (P. cinnamomi)and 20 min (P. palmivora) after a cold shock. Samples were takenbefore induction of cleavage and at intervals between inductionand release.

Freeze fixationHigh pressure freezing. Wet tufts of mycelium with attached

sporangia were placed in gold specimen holders (BalzersBB113142-1) with mineral salts solution (Hardham and Suzaki,1986), (for pre-induction) or distilled water (post-induction) fillingthe remaining space. Pairs of holders were clamped together andfrozen in a Balzers HPM 010 hyperbaric freezer. After freezing,the holders were snapped apart under liquid nitrogen andtransferred to the substitution medium.

Plunge freezing. Tissue was frozen on Formvar-covered loopsfollowing the procedures of Lancelle et al. (1986). To place tissueon the loop, a novel approach was used. The loop was held in placeon the platform of a dissecting microscope by plasticine. About8-10/il of distilled water or mineral salts solution was placed onthe upper face of the loop. A tiny tuft of lightly blotted myceliumwas then placed in this drop and spread out using fine forceps. Thewater was then wicked off and the loop rapidly transferred to theplunging device.

Freeze substitution and preparation for electronmicroscopy

General procedure. Tissue frozen by the above methods wasfreeze substituted using the procedures of Lancelle et al. (1986).The method was modified by the inclusion of 0.05% uranylacetate in the substitution medium, and after 36h at -80°C, thevials were first warmed to — 30 °C for 10 h before being brought toroom temperature. Tissue was then rinsed in acetone severaltimes, and stained en bloc in 5 % uranyl acetate in methanol for2h. After rinsing with acetone, the tissue was infiltrated withEpon resin and polymerized. Sections were stained for 3-5 min inReynold's lead citrate.

Immunolabelling procedure. For material destined for immu-nogold-labelling, the freeze substitution, infiltration and polym-erization procedures of Lancelle and Hepler (1989) were followedwith the inclusion of a 10 h stage at -30°C prior to bringing thesamples to room temperature during freeze substitution. Thematerial was then embedded in LR White resin. Infiltration andpolymerization with UV light were carried out at room tempera-ture. Immunolabelling of sections on gold grids followed themethods of Gubler and Hardham (1988). Monoclonal antibody(mAb) Cpw-1 was used. This mAb has previously been shown tohave a strong affinity for elements of the cleavage system andweaker binding to dictyosomes and peripheral cisternae insporangia of P. cinnamomi (Hyde et al. 1991a). After immuno-labelling, sections were stained with 2 % aqueous uranyl acetatefor 20-30 min, followed by 2 min in lead citrate.

Immunoblot analysisProteins from freeze dried samples were solubilized in SDS

sample buffer (63 mM Tris-HCl, pH6.8, containing 2% SDS,50 mM dithiothreitol, 0.001 % bromphenol blue and 10 % glycerol)for 5 min at 100 °C. After heating, samples were centrifuged andthe supernatant kept on ice until use. Solubilized proteins wereseparated by homogeneous (7 % or 12 % acrylamide) or gradient(5% to 20% acrylamide) SDS-PAGE (Laemmli, 1970) andtransferred to nitrocellulose (Towbin et al. 1979). After transfer,the nitrocellulose sheets were stained with 0.2 % Ponceau S in 3 %trichloroacetic acid, cut into strips and blocked with 5 % skimmedmilk powder in Tris-buffered saline (TBS: 10 mM Tris-HCl,pH7.5 containing 150 mM NaCl). After 1-2 h, the strips werewashed with TBS containing 0.5% Tween 20 (TBST). They werethen incubated with antibodies in ascites fluid (diluted 1/800 inTBST containing 3 % bovine serum albumin) for 1 h, washed fivetimes with TBST for 5 min each, and then incubated for 45 minwith sheep anti-mouse IgG antibodies conjugated to alkalinephosphatase. After washing twice in TBST, twice in TBS and oncein enzyme substrate buffer (ESB: 100 mM Tris-HCl, pH9.5containing 100 mM NaCl and 50 mM MgCl), bound phosphatasewas detected by immersing the strips in enzyme substratecontaining 0.44 % Nitro Blue Tetrazolium (Sigma, St. Louis, USA)and 0.33% 5-brom-4-chloro-3-indolylphosphate (BoehringerMannheim, Germany) in ESB. Prior to antibody incubation, somestrips were treated for l h with either Pronase E (Sigma,1 mgml"1 in 50 mM Tris-HCl, pH 7.5) or sodium periodate (20 mMin sodium acetate buffer, pH4.5).

Results

Sporangia before and during early cleavageThe most notable feature of sporangia rapidly frozenbefore the induction of cleavage and during early post-induction stages was that these were the only sporangia inwhich large discrete cleavage vesicles were observed.These vesicles were most evident in regions of thecytoplasm that were close to the basal body-associated poleof a nucleus and relatively free of other major organelles(Figs 1 and 3). In P. cinnamomi, cleavage vesicles wereespecially numerous in these regions (Fig. 1). In bothspecies the basal body-associated poles of nuclei in thesporangial cortex pointed towards the wall (Figs 1 and 3).During early post-induction, irregularly shaped cleavageelements were often seen around the nuclear pole (Fig. 2).

A significant difference between the two species was thepresence of a single large vacuole in the centre of thesporangium of P. palmivora (Fig. 3). The vacuole wasevident before induction, and was often ringed by amultilayered array of rough endoplasmic reticulum(Fig. 3). Such arrays have not previously been described inPhytophthora and nothing comparable was seen in P.cinnamomi, which has a series of smaller central vacuoles(Hyde et al. 1991a). In these and later stages microtubuleswere seen radiating from the basal bodies of both species

Figs 1-3. Early stages of zoosporogenesis in Phytophthora,seen in freeze substituted sporangia from plunge-frozen (Figs 1,2) and high pressure-frozen material (Fig. 3). In P. cinnamomi(1) and P. palmivora (3), cleavage elements (C) evident asvesicles, and dictyosomes (D) lie near the basal body (B)-associated pole of the nucleus (N) which, for these corticalnuclei, points to the sporangial wall (W). Development of theflagella (F) has begun. In the high pressure-frozen cell (3)cleavage elements do not contain the dense material seen inthe plunge frozen cell and large peripheral vesicles (L) are notwell preserved. The sporangium of P. palmivora contains onelarge central vacuole (V), ringed by layers of roughendoplasmic reticulum. (2) shows irregularly shaped cleavageelements (C) in P. cinnamomi. An elongated cisterna (p) at thetrans face of a dictyosome has a similar appearance to thecleavage elements. X28000 (1), X26000 (2), X21000 (3).

736 G. J. Hyde et al.

Sporangial cleavage in Phytophthora 737

(Fig. 1). In P. palmivora, limited flagellar (Fig. 3) andcleavage plane development was sometimes evident priorto induction.

The conformation of elements of the developing cleavagesystem, at these and later times, was directly comparablein high pressure- and plunge-frozen sporangia. With theexception of the large peripheral vesicles, other sporangialcomponents were equally well preserved by both tech-niques. The large peripheral vesicles were well preservedin plunge-frozen sporangia but not in high pressure-frozenmaterial (Fig. 3; Hyde et al. 19916).

Formation of cortical and internal cleavage planesSubsequent stages of cleavage involved the formation of asingle, cortical cleavage plane parallel to the sporangialwall and a series of internal cleavage planes separatinguninucleate domains. In both species, organization of thecortical plane proceeded more rapidly than that of theinternal planes.

The first indications of the development of the corticalplane were extended cleavage elements which lay mainlyparallel to the sporangial wall (Fig. 4). These cleavageelements were frequently irregularly arranged (Fig. 4),especially at the apex of the sporangium, and wereapparently of greater surface area than would ultimatelybe required. Several sporangial structures appeared to beassociated with the development of the cleavage elements.The contents of elongated cisternae at the trans face of theGolgi apparatus often appeared similar to those ofcleavage elements (Figs 5 and 2). A trans-Golgi networksometimes appeared to interconnect these cisternae withcleavage elements (Fig. 5). Small coated and uncoatedvesicles were often numerous adjacent to cleavage el-ements and dictyosomes, and blebs were frequently seenon the surface of the elements (Figs 5 and 6). Discretecleavage vesicles of the type seen earlier (Fig. 1) were notevident at this or any later stages. Serial section analysisrevealed that occasional circular profiles suggestive ofsuch vesicles (Fig. 4) were continuous with the extendedcleavage elements.

By the time the cortical cleavage plane was fullydeveloped (Fig. 7), most of the irregular and superfluouselements of the cortical plane had disappeared. Thisreduction occurred more rapidly in P. cinnamomi than inP. palmivora. Flagella were evident within the developing(Fig. 4) and completed (Fig. 7) cortical cleavage plane,which is part of the future extracellular space of thecleaved sporangium. The fully developed cortical cleavageplane cut off a shell of cytoplasm between itself and thesporangial plasma membrane (Fig. 7). This shell occasion-ally had connections to the main cytoplasm. Apparentcytoplasmic islands (Figs 7 and 8) which were evident inthe cortical cleavage plane often proved to be projections ofthe shell or of the main cytoplasm when checked by serialsectioning.

The cleavage planes which will surround the internalnuclei of P. cinnamomi appeared to develop initially asmembranous sheets extending back behind the nucleusfrom the narrow pole region (Fig. 9). The distal edges ofthese sheets were commonly dilated (Fig. 9). As in the caseof the developing cortical cleavage plane, there were oftenvery irregular arrangements of cleavage elements aroundthe narrow poles of internal <nuclei. These irregularitiesdisappeared as cleavage progressed. In P. palmivora, therewas no direct evidence of a polar basis to cleavage elementextension in the sporangial interior. This may, however,reflect the difficulty of capturing intermediate stages of

Figs 4-7. Development of the cleavage plane parallel to thesporangial wall in Phytophthora sporangia. (Figs 4 and 5) P.palmivora; (Figs 6 and 7) P. cinnamomi. All cells plunge-frozen. (4) Extended cleavage elements (C), mainly parallel tothe sporangial wall, seen in the sporangial cortex. Cross-sections of tubular portions (T) of the elements sometimes givea misleading impression (as confirmed by serial sectionanalysis) of vesicle alignment. Flagella (F) are seen within thecleavage elements. X9500. (5) An elongated cisterna (P) at thetrans face of a dictyosome has a similar appearance to cleavageelements (C, C). A trans-Golgi network appears tointerconnect this cisterna with the uppermost cleavage element(C). Blebs (boxed area) are seen on the surface of this cleavageelement, and small coated (circled area) and uncoated(arrowhead) vesicles are seen nearby. Peripheral cisternae(arrows) run parallel to the lower cleavage element, x 33 000.(6) Membrane blebs, coated (arrow) and possibly uncoated(arrowhead), seen on the surface of a cleavage element. Acoated vesicle lies nearby (boxed area). X43 000. (7) Flagella(F) and apparent cytoplasmic fragments (stars) are seen withinthe fully developed cortical cleavage plane which cuts off ashell of cytoplasm (s) between itself and the wall, x 10 000.

development in this species due to the rapidity of itscleavage process (Hohl and Hamamoto, 1967). In bothspecies, cleavage progressed as the partitioning mem-branes continued to extend throughout the cytoplasm,interconnecting with each other to delineate the futurezoospores (Figs 10-12). Just prior to release, the zoosporesrounded up and the intercellular spaces became moreobvious (Fig. 13).

The central vacuoles of both species disappeared duringcleavage; there was no indication of their fate. The corticalshell of cytoplasm described earlier (Fig. 7) had alsodisappeared in both species by the completion of cleavage.This occurred at a later stage of internal cleavage in P.palmivora than in P. cinnamomi (compare Figs 10 and 11).Prior to its disappearance the shell became thinner andoften fragmented (Fig. 8). Our evidence suggests that thisfragmentation involved localized fusion of the outermembrane of the cortical cleavage plane with the plasmamembrane of the sporangium (Fig. 8). Serial sectioningshows that the fragments were often interconnected andwere occasionally continuous with the main cytoplasm.The inner membrane of the cortical plane was retainedand formed part of the plasma membrane of the zoospores.

Apart from the cleavage system and an extracellularmatrix described below, two other novel features ofsporangial structure were evident in this study. First,sinuous projections of cytoplasm were observed within thewater expulsion vacuoles of both species and the mem-brane surrounding these vacuoles had numerous vesiclesand blebs associated with it (Figs 11 and 12). Second,peripheral cisternae (flattened organelles previously de-scribed in sporangia of P. cinnamomi at late stages ofcleavage only; Hyde et al. 1991a) were evident in bothspecies from very early cleavage stages (Fig. 5) onwardsand they were much more extensive in the zoosporeinitials (Figs 11 and 12) than previously observed.

The extracellular matrixA notable and previously undescribed feature of sporangiawas the dark, grainy appearance of material within thecleavage system (Figs 1, 2, 4-12). When cleavage wascomplete, this material was external to the zoosporeinitials, forming an extracellular matrix (Figs 11-13).Variability in the apparent density of this material wasmarked in high pressure-frozen material (e.g. Figs 3 and


8). In plunge-frozen material, a consistent decrease indensity was evident in sporangia sampled just prior tozoospore release (Fig. 13).

The extracellular matrix of both species was labelled bymAb Cpw-1 (Figs 14-16). A thick layer of extracellularmatrix was typically seen at the sporangial apex and


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Figs 8-11. Development of the internal cleavage planes ofPhytophthora sporangia. Fig. 8, high pressure-frozen cell;others: plunge-frozen. (8) A fragmented cortical shell ofcytoplasm (s) at the sporangial apex in P. cinnamomi.Fragmentation appears to involve localized fusion with thesporangial plasma membrane (arrows). Large cytoplasmicfragments (stars) are evident within the cortical cleavageplane, x 17 000. (9) Extended cleavage elements run backbehind the basal body-associated pole of an internal nucleus(asterisk). The distal edges (arrows) are dilated (P.cinnamomi). X8500. (10) At a later stage than (9), cleavageelements almost delineate the future zoospore domains. Thecortical cytoplasmic shell has disappeared (P. cinnamomi).X6500. (11) In a sporangium of P. palmivora, in which internalcleavage is more advanced than that shown in P. cinnamomi(10), some of the cortical shell of cytoplasm is still evident (s).Peripheral cisternae (arrows) lie near the future plasmamembranes of the zoospores. A water expulsion vacuole(arrowhead) is evident in one zoospore initial X3000.

showed particularly strong binding of mAb Cpw-1(Fig. 15). The sporangial wall also showed some binding ofmAb Cpw-1, especially near the sporangial apex (Fig. 15),suggesting that the antigen is leaking outwards. As in P.cinnamomi (Hyde et al. 1991a), the central vacuole of P.palmivora was not labelled by mAb Cpw-1 (Fig. 16). Theantigen(s) recognized by Cpw-1 were identified on immu-noblots of sporangial extracts. In P. cinnamomi, Cpw-1bound to several broad bands with apparent molecularweights between 60 and 330 kDa (Fig. 17). Similar resultswere obtained for P. palmivora (data not shown). Thecleavage state of sporangia had no bearing on the numberof bands detected. Pronase treatment of the transferredproteins of P. cinnamomi abolished antibody binding;treatment with periodate did not (Fig. 17).

Discussion

A new model for sporangial cleavage in PhytophthoraThe results of this study describe a significantly differentprocess of partitioning membrane formation to thatpreviously reported during zoosporogenesis in P. palmi-vora and P. cinnamomi (Hohl and Hamamoto, 1967; Hydeet al. 1991a). There was no evidence that cleavage followsfrom the fusion of prealigned vesicles, but rather the dataindicate that subdivision of the sporangium results fromthe progressive extension and eventual interconnection ofmembranous sheets. These new findings are summarizedand compared with the previous model in Fig. 18. Weconsider that the discrepancy between this and previousreports arises from the different techniques used topreserve sporangia, namely chemical fixation and RF-FS.

RF-FS is considered superior to chemical fixation for thepreservation of cell structure generally and membranouscomponents particularly (Lancelle et al. 1985,1986; Gilkeyand Staehelin, 1986; Cresti et al. 1987; Howard andO'Donnell, 1987). Chemical fixation may cause artefactualalterations in the morphology of membranous com-ponents. In a comparative study of wall-destined vesiclesin freeze-substituted and chemically fixed hyphae of theOomycete Saprolegnia, for example, Heath et al. (1985)observed in frozen material densely staining tubularelements that became partially vesiculated and lost theircontents following chemical fixation. Also, studies byMcCully and co-workers have shown that in petiolar hairsof certain plants the membranous canalicular system,which is visible as elongated strands in living cells,becomes highly vesiculated during chemical fixation(O'Brien et al. 1973; Mersey and McCully, 1978) butretains its in vivo morphology when rapidly frozen(McCully and Canny, 1985). We propose that the data fromsporangia preserved by RF-FS also represent more

12

Figs 12-13. Final stages of cleavage in Phytophthora. Plunge-frozen cells. (12) Fully cleaved zoospore initial of P. cinnamomi.Peripheral cisternae (arrows) lie parallel to the zoospore plasma membrane. Sinuous cytoplasmic projections are seen inside thewater expulsion vacuole (WV). Blebs are evident on the vacuolar membranes and small vesicles (boxed area) are seen nearby.X9500. (13) Rounded zoospores seen in the final stages of cleavage in P. cinnamomi. Extracellular matrix material (E) fills thespaces between the zoospores. X8000.


14 15

w

16

v

Figs 14-16. Labelling of contents of cleavage elements and extracellular matrix material with mAb Cpw-1, visualized using asecond antibody, sheep anti-mouse IgG-Au10. All cells high pressure-frozen. (14) Labelling of cleavage elements of the internal (C)and cortical (C) cleavage planes in P. cinnamomi. x 51000. (15) Dense labelling of the thick plug of extracellular matrix material(E) at the apex of a sporangium of P. palmivora. The sporangial wall (w) is also labelled, x 41000. (16) Labelling of part of theinternal cleavage planes (C) of a sporangium of P. palmivora. The large central vacuole (V) is not labelled above background.X40000.

-330-220

1-60

-36

a b e d

Fig. 17. Immunoblotcharacterization ofantigen(s) reacting withmAb Cpw-1. Sporangialproteins of P. cinnamomiwere separated on a SDS-polyacrylamide gradient gel,electrophoretically blotted,stained with Ponceau S(lane a) and probed withCpw-1, followed byperoxidase-conjugatedsecondary antibody (lane b).Lanes c and d show stripspretreated with Pronase andperiodate, respectively,before antibody incubations.Positions of molecular massmarkers (Mrxl0~3) areshown on the right.

faithfully the structure of the living cell, and that theapparent alignment of vesicles seen in previous studies ofcleavage in Phytophthora probably resulted from artefac-tual vesiculation during chemical fixation of membranoussheets similar to those seen in this study. Since thecleavage membranes of chemically fixed sporangia do notvesiculate during the final stages of zoosporogenesis, thenetwork of aligned vesicles seen earlier (Fig. 18) canreadily be misinterpreted as an intermediate stage in thecleavage process. In addition, rather than occurringwithin specialized 'axonemal vacuoles' (Hohl and Hama-moto, 1967; Hyde et al. 1991a; Hemmes, 1983), fiagellardevelopment is shown by RF-FS to occur within thegeneral system of cleavage planes.

Genesis of the cleavage membranesThe present study provides evidence that development ofthe cleavage planes, at least in P. cinnamomi, begins inregions near the basal body-associated nuclear poleswhere dictyosomes are concentrated and cleavage vesiclesare initially clustered. The disappearance of the cleavagevesicles coincides with the formation of the first expansesof cleavage membranes, indicating that cleavage vesiclesmay be incorporated into the developing cleavage planes.


• v • : , / : \

B

D

Fig. 18. A diagrammatic comparison of the process of sporangial cleavage in P. cinnamomi suggested by rapid freezing-freezesubstitution (A, B, C, D) with the previous model (A, B', C, D), based on observations of chemically fixed material. Many featuresof the first sequence are also true for P. palmivora; and many aspects of the second sequence (especially stage C) are shared byother Phytophthora species studied by chemical fixation. In these diagrams, shading represents extracellular matrix material. Inactual sections, the matrix material appeared much denser in sporangia prepared by rapid freezing, as opposed to chemicalfixation. Before induction of cleavage and shortly afterwards (A), specialized cleavage vesicles (•) and dictyosomes (|||) are foundconcentrated at definable poles of nuclei. The poles of cortical nuclei point towards the sporangial wall. Nuclei are evenly spacedwithin the coenocytic sporangium. In the next stage, in rapidly frozen sporangia (B), paired sheets of membrane loop back from thepoles of nuclei in the sporangial interior. In the cortex, one pair of membranous sheets runs parallel to the sporangial wall, andcuts off a shell of cytoplasm between the outer of the two membranes and the sporangial wall. In chemically fixed material (B')similar patterns are seen but these involve planes of discrete vesicles. Next, in frozen sporangia (C), the membranous sheets in thesporangial interior extend and interconnect with each other and the inner of the two cortical membranes, thus subdividing thesporangium into uninucleate, membrane-bound domains. The outer cortical membrane and the cortical shell of cytoplasmdisappear. In chemically fixed material (C), the cortical plane of vesicles and the cytoplasmic shell have disappeared but planes ofvesicles still demarcate domains around the nuclei. In the final stage (D) both rapidly frozen and chemically fixed sporangiacontain fully formed zoospores with a rounded shape and a groove opposite the nuclear pole. Matrix material fills the extracellularspace.

Irregular cleavage elements that are seen in early post-induction stages may be transitional stages in thetransformation of cleavage vesicles into more extendedforms. Further expansion of the partitioning membranesappears to involve the contribution of membrane fromdictyosomes, some of whose cisternae appear intercon-nected with developing cleavage elements through airans-Golgi network. This is the first report of a trans-Golgi network in Phytophthora; the network may bedisrupted during chemical fixation. Coated and uncoatedvesicles, often seen near dictyosomes and cleavageelements, may also be involved in membrane augmen-tation during cleavage element extension. These obser-vations are in agreement with previous proposals of adictyosomal origin for cleavage elements in Phytophthora(Hohl and Hamamoto, 1967; Eisner et al. 1970; Hyde et al.1991a). Vesicles may be transported to the edge of the

expanding system, which is characteristically dilated, orcould be incorporated close to the nuclear pole. Thevesicles and blebs could also play a role in membraneretrieval during development of the cleavage system.

The absence of any structural interconnection betweenthe large vacuoles and the cleavage system and the lack oflabelling of the vacuolar contents with mAb Cpw-1indicate that the large vacuoles do not play a role in thecleavage process.

Implications of this study for cytokinesis in othereukaryotesTo our knowledge, this is the first published study toemploy rapid freezing instead of chemical fixation to studycytokinesis in any system in which partitioning mem-brane formation would be expected to involve the fusion ofprealigned vesicles. As such it represents the fairest test


yet of this model, given the unpredictable preservation ofmembrane form by chemical fixation. The apparent failureof the alignment/fusion model in this study of zoosporoge-nesis in P. cinnamomi and P. palmivora has wide-rangingimplications for our understanding of cytokinesis in othereukaryotes. It has already been proposed (Schroeder, 1970;Rappaport, 1971, 1986) that the small body of studieswhich report vesicle alignment during cytokinesis inanimal cells suffers from the use of a chemical fixationprotocol that leads to vesiculation of the furrowingmembranes. To our knowledge, there has been noconsideration of similar vesiculation during cytokinesis innon-animal systems, although many studies have ex-pressed concern over the difficulty of discerning whetherapparent vesicles, seen in single cross-sections, mightactually be part of a continuous system (Burr and West,1970; Marchant and Pickett-Heaps, 1971; Zaar andKleinig, 1975). Wilson et al. (1990) have recently outlineda theoretical basis for the general case of vesiculation ofmembranes by chemical fixatives. For reasons given belowwe believe that artefactual vesiculation of partitioningmembranes may have occurred in many studies of a widevariety of eukaryotes, especially protoctists.

Absence of loosely arranged vesiclesMany of the studies that report vesicle alignment inprotoctistan systems (e.g. see Goodman and Rusch, 1970;Porter, 1972; Mims, 1973) have one, possibly critical,feature in common with the suspect animal reports andthe chemical fixation studies of zoosporogenesis in Phy-tophthora. In all cases, a stage is described where vesiclesare arranged in a row corresponding to the future plane ofcleavage, but there is, mostly, no prior stage describedwhere the vesicles are loosely arranged in this region. Aloose arrangement of vesicles is, however, typically de-scribed during early cell plate formation in plants (Heplerand Jackson, 1968; Gunning, 1982). It is commonlybelieved that this loose arrangement in plant cells arisesbecause the vesicles are in the process of moving towardsthe future zone of cleavage from either side of it (Heplerand Jackson, 1968; Gunning, 1982). The absence of such astage in some protoctistan systems might be explained byHeath's (1975) proposal that cleavage vesicles in theSaprolegniales move along the future plane of cleavagebefore coming to rest in that plane and fusing. In othercases, insufficient sampling of cells from different stages ofcleavage may have resulted in the loose arrangement ofvesicles having gone undetected. Alternatively, if the

neatly arranged stage is itself an artefact of preparation,then a prior loose stage may not be required for whateveris the true process of membrane genesis. The presentstudy, when viewed in the light of previous studies ofzoosporogenesis in Phytophthora, suggests that the ab-sence of a loosely arranged stage may well be a goodindicator that the neatly arranged stage is an artefact.

Taxonomic heterogeneityThe taxonomic distribution of organisms in which vesiclealignment/fusion has been described is perplexing. Sincethis process is commonly considered to be an evolution-arily advanced feature of cytokinesis (Pickett-Heaps,1972a; Rawlence, 1973), one might expect to trace clearlines of ancestry for this character back through the taxain which it appears towards some primitive organism inwhich it first evolved. The impossibility of doing this isbest illustrated by considering the protoctista. The vesiclealignment model has been proposed for a large number ofphylogenetically distant protoctistan taxa, in many ofwhich there have also been reports of the more primitivemechanism, namely progressive extension of the parti-tioning membranes (Table 1). In a number of cases the twomechanisms have been proposed in reports describingcytokinesis at different (e.g. Labyrinthula sp., Plasmodio-phora brassicae, Oedogonium cardiacum, Table 1) or eventhe same (Physarumpolycephalum, Spirogyra sp., Table 1)stage of the life cycle in the one genus or species.

While taxonomic heterogeneity such as this may have avariety of natural sources (Fowke and Pickett-Heaps,1969; Coss and Pickett-Heaps, 1973; Watson et al. 1985)another possibility is that some variability has arisenfrom methodological complications such as those seen inthis study. It is interesting that, with limited exception(Lucarotti and Federici, 1984), apparently the onlyeukaryotes that exhibit, during cytokinesis, a loosearrangement of vesicles similar to that seen in higherplant cell plate formation are certain green algae (PhylaConjugaphyta and Chlorophyta, sensu Margulis et al.1989), e.g. reports by Fowke and Pickett-Heaps (1969),Pickett-Heaps and Fowke (1970), Floyd et al. (1972),Pickett-Heaps (1973), Marchant and Pickett-Heaps (1973).The green algae and higher plants are commonlyconsidered to be phylogenetically related (Pickett-Heaps,1972a) and these two groups may represent the trueevolutionary lineage of cytokinesis involving vesiclealignment/fusion. Cell plate formation has been proposedto occur in some brown algae (Rawlence, 1973; Markey and

Table 1. Some protoctistan phyla in which both of the main modes of cell cleavage occurPhylum Vesicular alignment/fusion Progressive extension

Oomycota

Labyrinthulomycota

Plasmodiophoromycota

Plasmodial slime moulds

Chlorophyta

Xanthophyta

Conjugaphyta

Phaeophyta

Phytophthora capsiciWilliams and Webster (1970)

Labyrinthula sp.Porter (1972)

Plasmodiophora brassicaeWilliams and McNabola (1967)

Physarum polycephalumGoodman and Rusch (1970)

Oedogonium cardiacumCoss and Pickett-Heaps (1973)

Pseudobumillenopsis pyrenoidosaDeason (1971)

Spirogyra sp.Fowke and Pickett-Heaps (1969)

Ascophyllum nodosumRawlence (1973)

Saprolegnia feraxGay and Greenwood (1966)

Labyrinthula sp.Perkins and Amon (1969)

Plasmodiophora brassicaeGarber and Aist (1979)

Physarum polycephalumZaar and Kleinig (1975)

Oedogonium cardiacumCoss and Pickett-Heaps (1973)

Botrydiopsis alpinaLokhorst and Segaar (1989)

Spirogyra sp.Fowke and Pickett-Heaps (1969)

Chorda tomentosaToth (1974)


Wilce, 1975) but these descriptions do not include theloosely arranged stage and may be cases of artefactualvesiculation.

We believe that descriptions of cell plate formation inplants and certain green algae have features that makethem distinct from, and more credible than, reports ofvesicle alignment/fusion in other eukaryotes. Neverthe-less, the possiblity remains that cell plate membraneswhich have formed in vivo from the fusion of alignedvesicles may exhibit a temporary labile phase, similar tothat shown by cleavage membranes in Phytophthora,during which they are susceptible to vesiculation bychemical fixation.

The extracellular matrix and its possible role in zoosporereleaseThe results describe, for the first time in Phytophthora, thepresence of a dense, grainy material filling elements of thedeveloping cleavage system and forming an extracellularmatrix which surrounds the zoospores in the fully cleavedsporangium (Fig. 18). The passage of this material duringcleavage was traced, in both species, by immunogold-labelling using mAb Cpw-1. In chemically fixed materialof P. cinnamomi, mAb Cpw-1 binds to flocculent materialinside the cleavage system (Hyde et al. 1991a). Thismaterial probably represents all that remains afterchemical fixation of the dense matrix seen in freeze-fixedsporangia. Polyacrylamide gel electrophoresis indicatesthat Cpw-1 binds to one or more proteins with apparentmolecular weights between 60 and 330 kDa. Althoughantibody binding was not abolished by periodate treat-ment, the smearing of the bands is suggestive of extensiveglycosylation of the antigen (Goldkorn et al. 1989).

Extracellular material, often referred to as mucilage,has been described surrounding the spores, sperms andsimilar structures of many protoctists (Moore, 1965;Pickett-Heaps, 19726; Scott and Dixon, 1973; Toth, 1974;Lunney and Bland, 1976; Franke et al. 1977; Duckett andPeel, 1978) and fungi (Ingold, 1968) with several reportsnoting its origin from cleavage elements (Moore, 1965;Scott and Dixon, 1973; Lunney and Bland, 1976; Franke etal. 1977). The major significance of this material is itsproposed capacity to act as a swelling gel (de Bary, 1887;Pickett-Heaps, 19726; Toth, 1974; Lunney and Bland,1976; Duckett and Peel, 1978; Ingold, 1968) or as anosmoticum (Scott and Dixon, 1973; Ingold, 1971) whichmay be involved in events leading to rupture of the storageorgan and release of its contents. Although it has beenconsidered that the swelling gel model is theoreticallyplausible to explain sporangial discharge in Phytophthora(MacDonald and Duniway, 1978; Gisi and Zentmyer, 1980)there has never, until now, been evidence of anyextracellular material extensive enough to be seriouslyconsidered. There is indirect evidence that the extracellu-lar matrix described herein might swell, since the volumeoccupied by it apparently increases just prior to zoosporerelease. If swelling does occur this may account for some orall of the force needed for bursting the sporangium.

Alternatively, the extracellular matrix material mayoperate as an osmoticum. Numerous studies of Phytoph-thora have proposed that sporangial discharge resultsfrom an osmotically driven increase in hydrostaticpressure (reviewed by Gisi, 1983). One deficiency of thehydrostatic model is that it relies on the existence of asemipermeable barrier across which the hypotheticalosmoticum exerts its influence (Gisi and Zentmyer, 1980).The present study indicates that the sporangial membrane

disappears too early (at least in P. cinnamomi) for it tohave any role in this process. A similar dilemma in othersystems has been addressed by examining the possibilitythat the sporangial wall itself may act as a semipermeablebarrier (Money and Webster, 1988, 1989; Money, 1990).While further work is needed to assess the applicability ofthis proposal in Phytophthora, if swelling of an extracellu-lar matrix is responsible for sporangial discharge, then nosemipermeable barrier is required.

In conclusion we point out that this study is anotherdramatic example of the superiority of rapid freezingprocedures over chemical fixation for the preservation ofcell structure. In particular, the maintenance of theapparent true form of the developing cleavage planes andthe trans-Golgi network in Phytophthora demonstratesagain the effectiveness of rapid freezing in bringing tolight extensive membrane systems which have goneundetected in chemically fixed material.

We thank Dale Callaham and Frank Sek for excellent technicalassistance, Hans Hohl, Frank Gubler, Larry Lehnen, Les Watsonand Geoff Wasteneys for helpful suggestions, James Whiteheadfor the illustration and Margaret Wigney for typing themanuscript. This research was supported by a CooperativeResearch Grant from the Australian Department of Industry,Technology and Commerce to G.H. for a visit to Amherst to usethe high-pressure freezer; an Australian Postgraduate ResearchAward (G.H.) and NSF grants DCB-90-04191 (P.K.H.) and BBS-87-14235 (to the University of Massachusetts MicroscopyCenter).

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{Received 5 August 1991 - Accepted 9 September 1991)


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