Multilamellar Surface Layer of the Cell Wall of Albugo candida and ...
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Vol. 142, No. 2 JOURNAL OF BACTERIOLOGY, May 1980, p. 689-693 0021-9193/80/05-0689/05$02.00/0 Multilamellar Surface Layer of the Cell Wall of Albugo candida and Phycomyces blakesleeanus JALPA P. TEWARI,'* WILLIAM P. SKOROPAD,2 AND SUDARSHAN K. MALHOTRA' Biological Sciences Electron Microscopy Laboratory, University of Alberta, Edmonton, Alberta T6G 2E9,1 and Department of Plant Science, University of Alberta, Edmonton, Alberta T6G 2E32 The surface layer of the cell wall of the sporangia of Albugo candida and of the sporangiophores of Phycomyces blakesleeanus was composed of a series of lamellae. The evidence from freeze-fracture, freeze-etch, and single-stage replicas indicated that the lamellae are organized as bilayers, an organization associated with the presence of lipids. The role of these lamellae in dispersibility and resistance is discussed. Surface characteristics of the propagules of fungi may play a role(s) in their dispersibility and resistance to desiccation. In many fungi these roles have been attributed to the presence of a layer consisting of rodlets on the surface of spores (2). The rodlets are not unique to spores since they are also present on the surface of the hyphae of wild-type Neurospora crassa (Tewari and Malhotra, unpublished data) where they may serve to protect the hyphae against desic- cation. During an investigation of phycomycetous fungi (Albugo candida and Phycomyces blak- esleeanus), it was observed that the surface layer of these fungi had characteristic lamellar orga- nization, usually associated with the presence of lipids. Such a structure, although suited for pro- tection against desiccation, does not appear to have been reported in the available literature. Consequently, the structure of this surface layer in the two fungi is reported in the present paper with a view toward understanding the structure and chemical nature of their surface layer. A. candida is an obligate parasite of the cruciferous plants and causes heavy economic loss all over the world. The sporangia of this fungus repre- sent the repeating progapule stage (1). P. blak- esleeanus is a saprophytic fungus and has been extensively studied to elucidate the mechanism of photoreception and other behavioral re- sponses (3, 8, 10, 20). MATERLALS AND METHODS Mature leaves of the Polish rapeseed cultivar Torch (Brassica campestris) infected with A. candida were collected from the University of Alberta Farm in Ed- monton, Alberta. Cultures of P. blakesleeanus were grown in glucose-asparagine agar (22) in petri dishes kept in moist chambers under constant fluorescent light at room temperature (-22°C). The sporangia of A. candida and the sporangio- phores of P. blakesleeanus (stage IV; see reference 3) were fixed overnight in a 1:1 mixture of 3% glutaral- dehyde and 3% formaldehyde in 0.1 M phosphate buffer (pH 7). They were then soaked in 25% glycerol in water for 30 min, placed on Au-Ni specimen holders, frozen in Freon 22, and stored in liquid nitrogen. Unidirectionally shadowed platinum-carbon replicas of the materials fractured at -100°C were made in a Balzers BA 360M high-vacuum freeze-etch unit, using standard techniques (11, 18). For making replicas of the freeze-etched specimens, materials fixed in the aldehyde mixture were washed in distilled water for 2 h and processed as described above except that the fractured material was etched for 1.5 min at -100°C. Pt-C replicas of the surface of the two materials were prepared as follows. A thin layer of Tissue-Tek (an embedding medium for frozen tissue specimens; Ames Co., Elkhart, Ind.) was spread on a microscope slide, and the sporangia of A. candida were dusted directly on it by teasing the pustules on rapeseed leaves with a needle. Replicas were then prepared in a Balzers BA 360M unit as described above except that the whole operation was conducted at room tem- perature. Replicas of the sporangiophores of P. blak- esleeanus prepared as above revealed extensive shrinkage and had to be discarded. Therefore, surface replicas of the sporangiophores were made by the following freeze-drying procedure. The sporangio- phores were fixed in the aldehyde mixture as described above and washed in distilled water for 2 h. Small pieces of the sporangiophores were positioned in a single layer on Au-Ni specimen holders and frozen in Freon 22 after blotting as much free water as possible. They were transferred to the stage of a Balzers BA 360M unit and freeze-dried for about 5 h at -100°C in a vacuum of _ 10-6 torr. During this period, the knife at liquid nitrogen temperature was positioned above the specimens and acted as a cold trap. The specimens were then replicated as described earlier. The sections and replicas were examined in Philips EM 200 or EM 201 electron microscopes. In each electron micrograph of the replicas, the direction of shadowing is indicated by an arrow at the lower-right corner. The freeze-fractured and freeze-etched faces are labeled according to the terminology of Branton et al. (4). 689 on February 12, 2018 by guest http://jb.asm.org/ Downloaded from
Transcript of Multilamellar Surface Layer of the Cell Wall of Albugo candida and ...
Vol. 142, No. 2JOURNAL OF BACTERIOLOGY, May 1980, p. 689-6930021-9193/80/05-0689/05$02.00/0
Multilamellar Surface Layer of the Cell Wall of Albugocandida and Phycomyces blakesleeanus
JALPA P. TEWARI,'* WILLIAM P. SKOROPAD,2 AND SUDARSHAN K. MALHOTRA'
Biological Sciences Electron Microscopy Laboratory, University ofAlberta, Edmonton, Alberta T6G 2E9,1and Department ofPlant Science, University ofAlberta, Edmonton, Alberta T6G 2E32
The surface layer of the cell wall of the sporangia of Albugo candida and ofthe sporangiophores of Phycomyces blakesleeanus was composed of a series oflamellae. The evidence from freeze-fracture, freeze-etch, and single-stage replicasindicated that the lamellae are organized as bilayers, an organization associatedwith the presence of lipids. The role of these lamellae in dispersibility andresistance is discussed.
Surface characteristics of the propagules offungi may play a role(s) in their dispersibilityand resistance to desiccation. In many fungithese roles have been attributed to the presenceof a layer consisting of rodlets on the surface ofspores (2). The rodlets are not unique to sporessince they are also present on the surface of thehyphae ofwild-type Neurospora crassa (Tewariand Malhotra, unpublished data) where theymay serve to protect the hyphae against desic-cation.During an investigation of phycomycetous
fungi (Albugo candida and Phycomyces blak-esleeanus), it was observed that the surface layerof these fungi had characteristic lamellar orga-nization, usually associated with the presence oflipids. Such a structure, although suited for pro-tection against desiccation, does not appear tohave been reported in the available literature.Consequently, the structure of this surface layerin the two fungi is reported in the present paperwith a view toward understanding the structureand chemical nature of their surface layer. A.candida is an obligate parasite ofthe cruciferousplants and causes heavy economic loss all overthe world. The sporangia of this fungus repre-sent the repeating progapule stage (1). P. blak-esleeanus is a saprophytic fungus and has beenextensively studied to elucidate the mechanismof photoreception and other behavioral re-sponses (3, 8, 10, 20).
MATERLALS AND METHODS
Mature leaves of the Polish rapeseed cultivar Torch(Brassica campestris) infected with A. candida werecollected from the University of Alberta Farm in Ed-monton, Alberta. Cultures of P. blakesleeanus weregrown in glucose-asparagine agar (22) in petri disheskept in moist chambers under constant fluorescentlight at room temperature (-22°C).The sporangia of A. candida and the sporangio-
phores of P. blakesleeanus (stage IV; see reference 3)
were fixed overnight in a 1:1 mixture of 3% glutaral-dehyde and 3% formaldehyde in 0.1 M phosphatebuffer (pH 7). They were then soaked in 25% glycerolin water for 30 min, placed on Au-Ni specimen holders,frozen in Freon 22, and stored in liquid nitrogen.Unidirectionally shadowed platinum-carbon replicasof the materials fractured at -100°C were made in aBalzers BA 360M high-vacuum freeze-etch unit, usingstandard techniques (11, 18).
For making replicas of the freeze-etched specimens,materials fixed in the aldehyde mixture were washedin distilled water for 2 h and processed as describedabove except that the fractured material was etchedfor 1.5 min at -100°C.
Pt-C replicas of the surface of the two materialswere prepared as follows. A thin layer of Tissue-Tek(an embedding medium for frozen tissue specimens;Ames Co., Elkhart, Ind.) was spread on a microscopeslide, and the sporangia of A. candida were dusteddirectly on it by teasing the pustules on rapeseedleaves with a needle. Replicas were then prepared ina Balzers BA 360M unit as described above exceptthat the whole operation was conducted at room tem-perature. Replicas of the sporangiophores of P. blak-esleeanus prepared as above revealed extensiveshrinkage and had to be discarded. Therefore, surfacereplicas of the sporangiophores were made by thefollowing freeze-drying procedure. The sporangio-phores were fixed in the aldehyde mixture as describedabove and washed in distilled water for 2 h. Smallpieces of the sporangiophores were positioned in asingle layer on Au-Ni specimen holders and frozen inFreon 22 after blotting as much free water as possible.They were transferred to the stage of a Balzers BA360M unit and freeze-dried for about 5 h at -100°C ina vacuum of _ 10-6 torr. During this period, the knifeat liquid nitrogen temperature was positioned abovethe specimens and acted as a cold trap. The specimenswere then replicated as described earlier.The sections and replicas were examined in Philips
EM 200 or EM 201 electron microscopes. In eachelectron micrograph of the replicas, the direction ofshadowing is indicated by an arrow at the lower-rightcorner. The freeze-fractured and freeze-etched facesare labeled according to the terminology of Brantonet al. (4).
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690 TEWARI, SKOROPAD, AND MALHOTRA
RESULTS AND DISCUSSIONThe overall structure of the cell wall in the
sporangia of A. candida and the sporangio-phores of P. blakesleeanus as seen in routinelyfixed and sectioned material is well-known (7,12) and is therefore not described in this paper.In both these organisms, an electron-dense cu-ticle (or pellicle) is reported to be present at thesurface of the cell wall in sectioned material, andthe surface layer described in this paper corre-sponds to the position of the cuticle in thinsections. The structure of this layer is very sim-ilar in the two organisms studied. Consequently,it is described in detail in A. candida and differ-ences, if any, are indicated for P. blakesleeanus.
Freeze-fractures of the surface layer in A.candida revealed a series of lamellae (Fig. 1).This multilamellar layer always fractured tan-gentially, even though the underlying cell wallwas cross-fractured. This indicated a path ofleast resistance in that zone in the frozen mate-rial. Also, based on studies on biological mem-branes, it is now generally agreed that fracturingin frozen material takes place through the hy-drophobic molecular core of membrane (13, 19).Furthermore, the fracturing pattern of the sur-face layer in the form of lamellae resembles thefreeze-fractured artificial membranes made fromlipid (17). It is therefore conjectured that thefracturing pattem of the surface layer in A.candida and P. blakesleeanus is due to thepresence of hydrophobic zones in the variouslamellae.The tangential fractures of the inner lamellae
had particles measuring -7 nm (range 4.5 to 9nm), whereas those of the outer lamellae werestudded with plaques measuring '15 nm (range11 to 23 nm; Fig. 2). These estimates were ar-rived at after allowing a correction factor of 5nm for thickness of the Pt-C film (6, 16). Thisdifferentiation into particle- and plaque-bearinglamellae was not apparent in P. blakesleeanuswhere fractured faces of all the lamellae hadplaques (Fig. 3 and 4). Although the nature orthe significance of these particles and plaques isnot known, it is relevant that the particles seenon many freeze-fractured biological membranesare believed to be proteinaceous in nature (9).Also, it has been shown that artificial lipid bilay-ers lacking protein also have particles on theirfractured faces (21).The freeze-etched replicas of the sporangia of
A. candida revealed the etched face (or thesurface) in addition to the fractured faces (Fig.5). The etched face revealed abundant particu-late material. It should be remarked that per-haps this particulate material represents debristhat abounds in the field situation from where
FIG. 1 and 2. Replicas of freeze-fractured spo-rangia of A. candida. Note that the fractured facesEF andPF are composed ofa series oflamellae. Thesuperficial lamellae (arrows) have plaques, whereasthe deep-seated ones (arrowheads) have particles.Asterisk, Background ice; CW, cell wall; E, echinu-lation. Bar = 0.5 Inm.
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CELL WALL OF A. CANDIDA AND P. BLAKESLEEANUS 691
PF . r
_7w-PF
- - E -- f s'%- 5
FIG. 3 and 4. Replicas offreeze-fractured sporan-giophore of P. blakesleeanus showing the multila-mellar organization of the surface layer (arrows).Fractured faces (PF) of the lamellae show plaques.CW, Cell wall; C, cytoplasm. Bar = I ,m (Fig. 3); 0.5un (Fig. 4).
this material was collected. It appears that thedebris dispersed in the liquid medium in whichthe material was suspended for processing and
bound to the surface ofthe sporangia. The debriszv on the etched face differed in dimension (Fig. 5)X from the particles and plaques seen on the frac-
tured face and served as a useful marker for the;L etched face. Disregarding the debris, the etched
face showed a series of smooth lamellae. Thefact that one sees a multilamellar structure onthe etched face indicates that these lamellae arediscontinuous, otherwise one would see only asingle lamella. Furthermore, since the etchedface differs from the fractured face in its appear-ance, it would appear that the latter is not asurface fracture; instead it is a fracture some-where through the middle of a lamella, as in thecase of biological membranes by virtue of theirbeing organized as bilayers.
It has been reported that the etched face maybe covered with a sheet of nonsublimable water(16) and, hence, may not reveal the true surface.In this respect, the direct replicas of the surfaceproved to be useful. In both the materials inves-tigated, these replicas revealed a few smoothlamellae on the surface (Fig. 6 and 7). These
FIG. 5. Replica of freeze-etched sporangium of A.candida. The fractured face (PF) has lamellae withparticles (arrowheads) and plaques (large arrow),whereas the etched face (ES) has lamellae (smallarrows) with smooth faces. Double arrows indicatethe debris on the etched face. E, Echinulation. Bar= 0.5 um.
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692 TEWARI, SKOROPAD, AND MALHOTRA
-' layer of the cefl wall consists of a series of-.__ bilayers, an organiation usually associated with
the presence of lipids. Such a structure is notdiscernible in routinely prepared thin sections
t__ ~~~~~~~(7,11).Both the sporangia ofA. candida (14) and the
sporangiophores of P. blakesleeanus (Tewari_= W and Malhotra, unpublished data) are hydropho-
bic. It could be that the outermost lamellae (orparts thereof) exposed to the natural environ-ment are organized as monolayers with theirhydrophobic aspects facing the outside. Thiswould be consistent with the orientation of am-phipathic molecules at the air-water interface(15). Such a structure could be responsible for
- imparting hydrophobicity to these fungi. Never-theless, it should be pointed out that most of thematerial we studied was prepared after suspen-sion in an aqueous medium. Under these condi-tions, the monolayer arrangement of moleculesis likely to undergo a change to a configuration__ that would be stable in an aqueous environment.As such, some of our observations may not re-flect the structure of fungi in their natural en-vironment. Although the multilamellar layer has
A so far not been chemically characterized, on theM, ^ 3 basis of evidence presented in this paper, it_ appears to consist largely of lipids. The available
data are inadequate to conclude the identity ofother chemicals, if any, associated with it. In thisconnection, it is instructive to note that therodlets present on the surface of the spores ofascomycetes, basidiomycetes, and deuteromy-cetes are thought to be largely composed ofprotein and polysaccharide (2). Also, the hydro-phobicity ofthe rodlet-bearing structures cannotbe related to any known chemical componentother than to the surface relief (5).The sporangia of A. candida are formed in
dry masses and dispersed primarily by air cur-rents. The function of the multilamellate layeron the surface of the cell wall could be to facili-tate air dispersal and protect the sporangia fromdesiccation. In the sporangiophore of P. blak-esleeanus, it would serve the latter functionsince these structures are not dispersed.
ACKNOWLEDGMENTSThis work was financed through grants from Alberta Wheat
' -̂¢ Pool, Alberta Agricultural Research Trust (to W.P.S.), andFIG. 6 and 7. Single-stage replicas of the sporan- Natural Sciences and Engineering Research Council of Can-
gium ofA. candida (Fig. 6) and sporangiophore ofP. ada (to S.K.M.).blakesleeanus (Fig. 7). Note the discontinuous lamel- LITERATURE CITEDlae (arrows) with smooth faces. E, Echinulation. Bar 1T6TR ItEd= 0.5 tmn (Fig. 6); 1 Snm (Fig. 7) 1. Alesopoulos, C. J. 1962. Introductory mycology. Second
edition, John Wiley & Sons, Inc., New York.views were basically similar to those of the 2. Beever, R. E., and G. P. Dempsey. 1978. Function ofetched face. rodlets on the surface of fungal spores. Nature (London)
etcnclusion. On the basis of the evidence 272:608-610.Conclusions. On the basis of the evidence 3. Bergman, K., P. V. Burke, E. Cerdi-O1medo, C. N.
presented above, it appears that the surface David, M. Delbriick, K. W. Foster, E. W. Goodell,
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5. Fisher, D. J., and D. V. Richmond. 1970. The electro-phoretic properties and some surface components ofPenicillium conidia. J. Gen. Microbiol. 64:205-214.
6. Garber, M. P., and P. L Steponkus. 1974. Identificationof chloroplast coupling factor by freeze-etching andnegative-staining techniques. J. Cell Biol. 63:24-34.
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