Germination of the resting sporangia ofPhysoderma maydis,the causal agent ofPhysoderma disease of maize

Summary The structural and developmental characteristics of the resting sporangium in uniflagellate phycomycetes, together with the type of zoospore, are of high taxonomic value. Among these fungi, however, only a few electron microscopic investigations have been published on this topic, mainly due to technical problems. In the present study ofPhysoderma maydis (Blastocladiales) these problems were overcome as the resting sporangia in this species are formed synchronously, in large numbers, the germination is readily induced and the impermeability of the resting sporangium wall can be circumvented by shaking the prefixed sporangia with glass beads.The germination of the resting sporangia ofP. maydis is described by correlative light and electron microscopic studies and discussed in relation to related investigations on sporogenesis: The germination process starts by a breakdown of large electron-dense accretions found in the resting stage. Simultaneously, the peripheral location of the lipid bodies is lost. The large operculum is pushed open by a protrusion of the inner sporangial wall; an additional wall layer is formed during this process. Synaptonemal complexes are found in the nuclei at this stage, as are nuclear division figures which suggests anEuallomyces type of life cycle for this fungus. Cleavage vesicles, formed from dictyosomes or endoplasmic reticulum, ultimately separate the sporangial content into meiospores. The sequential assembly of organelles into the side body complex is described. Sequestering of the ribosomes into a nuclear cap is interpreted as taking place immediately prior to zoospore discharge.     

Ultrastructural changes during sporangium formation and zoospore differentiation in Blastocladiella Emersonii

Samples from synchronized cultures of Blastocladiella emersonii were examined by electron microscopy from the late log phase to the completion of zoospore differentiation. Log-phase plants contain the usual cytoplasmic organelles but also have an unusual system of large tubules ca. 45 mμ diam that ramify in organized bundles throughout the protoplast. After induction, zoosporangium differentiation requires a 2-hr period in which the nuclei divide, a cross wall forms to separate the basal rhizoid region, and an apical papilla is produced. Nuclear division in B. emersonii is intranuclear with a typical microtubular spindle apparatus and paired, unequal, extranuclear centrioles at each pole. The papilla is formed by a process of localized cell wall breakdown and deposition of the papilla material by secretory granules. Differentiation of zoospores begins when one of the two centrioles associated with each nucleus elongates to form a basal body. The flagella fibers arise from the basal body and elongate into an expanding vesicle formed by the fusion of small secondary vesicles. The cleavage planes are formed by fusion of vesicles similar to those associated with flagellum initiation. When cleavage is complete, each sporangium contains ca. 250–260 uninucleate spore units with their flagella lying in the cleavage planes. Probable fusion of mitochondria to produce the single mitochondrion of the zoospore occurs after cleavage; the mitochondrion does not take its position around the basal body and rootlets until just before zoospore release. The ribosomal nuclear cap is organized and enclosed by a membrane formed through fusion of many small vesicles during a short period near the end of differentiation.     

Characterisation of the water expulsion vacuole inPhytophthora nicotianae zoospores

Summary The water expulsion vacuole (WEV) in zoospores ofPhytophthora nicotianae and other members of the Oomycetes is believed to function in cell osmoregulation. We have used videomicroscopy to analyse the behaviour of the WEV during zoospore development, motility and encystment inP. nicotianae. After cleavage of multinucleate sporangia, the WEV begins to pulse slowly but soon attains a rate similar to that seen in motile zoospores. In zoospores, the WEV has a mean cycle time of 5.7 ± 0.71 s. The WEV continues to pulse at this rate until approximately 4 min after the onset of encystment. At this stage, pulsing slows progressively until it becomes undetectable. The commencement of WEV operation in sporangia coincides with the reduction of zoospore volume prior to release from the sporangium. Disappearance of the WEV during encystment occurs as formation of a cell wall allows the generation of turgor pressure in the cyst. As in other organisms, the WEV inP. nicotianae zoospores consists of a central bladder surrounded by a vesicular and tubular spongiome. Immunolabelling with a monoclonal antibody directed towards vacuolar H⁺-ATPase reveals that this enzyme is confined to membranes of the spongiome and is absent from the bladder membrane or zoospore plasma membrane. An antibody directed towards plasma membrane H⁺-ATPase shows the presence of this ATPase in both the bladder membrane and the plasma membrane over the cell body but not the flagella. Analysis of ATPase activity in microsomal fractions fromP. nicotianae zoospores has provided information on the biochemical properties of the ATPases in these cells and has shown that they are similar to those in true fungi. Inhibition of the vacuolar H⁺-ATPase by potassium nitrate causes a reduction in the pulse rate of the WEV in zoospores and leads to premature encystment. These results give support to the idea that the vacuolar H⁺-ATPase plays an important role in water accumulation by the spongiome in oomycete zoospores, as it does in other protists.Abbreviations BMM butyl methylmethacrylate - F fix 4% formaldehyde fixation - GF fix 4% formaldehyde and 0.2% glutaraldehyde fixation - V-ATPase vacuolar H⁺-ATPase - WEV water expulsion vacuole     

Ultrastructural observations of mature and encysting zoospores ofPythium proliferum de Bary

Summary The process of zoospore maturation and encystment inP. proliferum was studied by electron microscopy. General ultrastructural features of the mature, swimming zoospore were found to be similar to those previously described for other oomycetes in both the attachment and ultrastructure of the flagella as well as the type and distribution of cellular organelles. Associated with extensive areas of RER in the mature zoospores were unusual, electrondense, bar-like structures. These structures were found in the groove region of young zoospores and at the periphery of encysting zoospores. Their possible function is discussed. The five main types of vesicles observed during encystment, as seen grouped in this study, along with the vesicles described in previous studies of oomycete encystment, were in table form and individually discussed. Interesting correlations appear to exist in the types of vesicles that are present within the oomycetes studied thusfar.     

Zoosporulation of a new Perkinsus species isolated from the gills of the softshell clam Mya arenaria

A gill-associated Perkinsus sp. isolated from the softshell clam (Mya arenaria) is described as a new species, P. chesapeaki sp. nov. Examination of the parasite in seawater cultures revealed life cycle stages and zoosporulation processes similar to those described for other species of the genus Perkinsus. Prezoosporangia developed thickened cell walls upon contraction of the cytoplasm and development of a distinctive clear area between the cell wall and the protoplast. Successive bipartition of the protoplast led to the formation of hundred's of zoospores within mature sporangia. Zoospores were released into seawater through one or more discharge tubes. Ultrastructural studies revealed an oblong zoospore possessing two flagella that arose from a concave side located in the upper third of the zoospore body. The anterior flagellum possessed a unilateral array of hair-like structures. A large anterior vacuole and basolateral nucleus dominated the cytoplasm of the zoospore body. The presence of a rudimentary apical complex including an open-sided conoid, rhoptries, micronemes, and subpellicular microtubules were also discerned. Differences in zoospore morphology, and sequence analyses of two genes previously reported, support the designation of the gill-associated Perkinsus from the softshell clam as a new species.     

Ultrastructural Changes Associated with Spore Formation in Sporangia and Sporangiola of Thamnidium elegans Link

Fully formed pre-cleavage sporangia and sporangiola of Thamnidiumelegans Link were bounded by a primary wall plus a thick, internalsecondary wall layer. In sporangia in late pre-cleavage, Golgi-likecisternae were associated with groups of cytoplasmic vesiclesof characteristic size and appearance which were not found insporangia containing large cleavage vesicles. In both sporangia and sporangiola, protoplast cleavage was effectedby enlargement of endogenous cleavage vesicles each containinga lining layer of variable appearance, mutual fusion of cleavagevesicle membranes and fusion of cleavage vesicle membranes withthe plasmalemma. Golgi-like cisternae and small vesicular profileswere present in sporangium protoplasts at all stages of cleavagevesicle enlargement. In sporangia, the columella zone was delimitedby cleavage vesicles and separated from the sporogenous zoneby a fibrillar wall. A similar wall, which sometimes protrudedto form a small columella, was formed in sporangiola. Recently delimited spore protoplasts were bounded by plasmalemmamembrane derived from cleavage vesicle bounding membrane andsporangium or sporangiolum plasmalemma and surrounded by aninvesting layer derived from cleavage vesicle lining material.The investing layer at first appeared single, but later twoelectron opaque profiles were discernible. The spore wall wasformed between the investing layer and the plasmalemma. Wallsof sporangia and sporangiola which contained fully formed sporesconsisted of the primary layers only.     

The ultrastructure of zoospores ofAphanomyces euteiches and of their encystment and subsequent germination

Summary The ultrastructure ofAphanomyces euteiches during the periods of zoospore motility, encystment, and germination has been studied. The motile spore has two heterokont flagella inserted laterally into the groove of the zoospore body where each is attached to a kinetosome. The kinetosomes and flagella are anchored into the zoospore body by rootlets comprised of two rows of microtubules with up to 12 microtubules in the outer row and are attached by fine threads to a striate fiber bundle. Secondary microtubules are attached at right angles at regular intervals along the rootlets. An unidentified body, 1.25m in diameter, containing helical fibers 16 nm in diameter is present in each zoospore. This body is situated near the two kinetosomes on the side of the pyriform nucleus opposite the contractile vacuole. The Golgi complex is between the nucleus and the contractile vacuole. The latter is surrounded by a 0.5–1.0m wide zone of Golgi proliferated vesicles. Ribosomes are generally absent from this region. Endoplasmic reticulum containing tubules within the expanded cisternae are also present. Vesicles with striated electron opaque inclusions and vesicles containing a granular cortex and center that developed in previous stages of zoosporogenesis were also present. During encystment of the zoospore the latter vesicles disappear. The two flagella are shed at this time leaving a membrane-bounded granular knob protruding from each of the kinetosome terminal plates. The contractile vacuole becomes disorganized and the zoospore assumes a spherical shape. Cyst wall deposition begins immediately and is completed in 30 minutes. The spore begins to germinate 1 hour following initiation of encystment with the appearance of a bulge in the cyst wall which elongates into a germ tube. Mitotic nuclear division follows.Research supported by the College of Agricultural and Life Sciences Station Project No. 1281.Research assistant and Professor. The advice and assistance of G. A. deZoeten, G. R.Gaard, and S.Vicen are most gratefully acknowledged.     

FINE STRUCTURE OF THE ZOOSPORE OF OEDOCLADIUM CAROLINIANUM (CHLOROPHYTA) WITH SPECIAL REFERENCE TO THE FLAGELLAR APPARATUS1, 2

Ultrastructure of the motile zoospore has been investigated in Oedocladium catolinianum & Hoffman. An unwalled zoospore is usually produced from the contents of a terminal vegetative cell and consists of two principal regions: a small anterior dome and a larger body region; a ring of flagella marks the juncture of these two areas. Chloroplast inclusions consist of thylakoids, mature and incipient pyrenoids, starch and striated microtubules; no eyespot has been observed. Zoospores appear to possess permanent contractile vacuoles with numerous accessory vacuoles, coated vesicles and occasionally coated tubules. The cytoplasm of the dome contains numerous mitochondria ER and golgi bodies, as well as two distinct types of vesicles. The first contains an electron-dense; granular core and is surrounded by a loose, sinuate membrane. The second vesicle is electron-opaque and is found at the apex of the dome: it contains mucopolysaccharides employed during zoospore adhesion. A complex flagellar apparatus encircles the lower region of the dome. It consists of ca. 30–65 flagella, a ring-shaped fibrous band, flagella roots and additional supporting material. The flagella and roots alternate with one another beneath the fibrous band. The compound flagellar roots consist of two superimposed components: an outer ribbon-like unit composed of three microtubular elements and a single striated inner component. A band of support material lies beneath the proximal end of the basal bodies. It is a continuous fibrous band, although it often appears as three distinct, repetitive units.     

A putative DEAD-box RNA-helicase is required for normal zoospore development in the late blight pathogen Phytophthora infestans

The asexual multinucleated sporangia of Phytophthora infestans can germinate directly through a germ tube or indirectly by releasing zoospores. The molecular mechanisms controlling sporangial cytokinesis or sporangial cleavage, and zoospore release are largely unknown. Sporangial cleavage is initiated by a cold shock that eventually compartmentalizes single nuclei within each zoospore. Comparison of EST representation in different cDNA libraries revealed a putative ATP-dependent DEAD-box RNA-helicase gene in P. infestans, Pi-RNH1, which has a 140-fold increased expression level in young zoospores compared to uncleaved sporangia. RNA interference was employed to determine the role of Pi-RNH1 in zoospore development. Silencing efficiencies of up to 99% were achieved in some transiently-silenced lines. These Pi-RNH1-silenced lines produced large aberrant zoospores that had undergone partial cleavage and often had multiple flagella on their surface. Transmission electron microscopy revealed that cytoplasmic vesicles fused in the silenced lines, resulting in the formation of large vesicles. The Pi-RNH1-silenced zoospores were also sensitive to osmotic pressure and often ruptured upon release from the sporangia. These findings indicate that Pi-RNH1 has a major function in zoospore development and its potential role in cytokinesis is discussed.     

The ultrastructure of cell wall formation and of gamma particles during encystment of Allomyces macrogynus zoospores

Structural changes during cell wall formation by populations of semisynchronously germinating zoospores were studied in the water mold Allomyces macrogynus. Fluorescence microscopy using Calcofluor white ST (which binds to -1,4-linked glycans) demonstrated that Calcofluor-specific material was deposited around most cells between 2–10 min after the induction of encystment (beginning when a wall-less zoospore retracts its flagellum and rounds up). During the first 15 min of encystment there was a progressive increase in fluorescence intensity. Ultrastructural analysis of encysting cells showed that within 2–10 min after the induction of encystment small vesicles 35–70 nm diameter were present near the spore surface, and some were in the process of fusing with the plasma membrane. The fusion of vesicles with the zoospore membrane was concomitant with the appearance of electron-opaque fibrillar material outside the plasma membrane. Vesicles similar to those near the spore surface were found within the gamma () particles of encysting cells. These particles had a crystalline inclusion within the electron-opaque matrix. During the period of initial cyst cell wall formation numerous vesicles appeared to arise at the crystal-matrix interface. Approximately 15–20 min was required for the cell wall to be formed. We suggest that the initial response of the zoospore to induction of encystment is the formation of a cell wall mediated by the fusion of cytoplasmic vesicles with the plasma membrane.Non-Standard Abbreviations GlcNac N-Acetylglucosamine - DS sterile dilute salts solution - PYG peptone-yeast extract-glucose broth     

Golgi apparatus activity and membrane flow during scale biogenesis in the green flagellateScherffelia dubia (Prasinophyceae). I: Flagellar regeneration

Summary Cells ofScherffelia dubia regenerate flagella with a complete scale covering after experimental flagellar amputation. Flagellar regeneration was used to study Golgi apparatus (GA) activity during flagellar scale production. By comparing the number of scales present on mature flagella with the flagellar regeneration kinetics, it is calculated that each cell produces ca. 260 scales per minute during flagellar regeneration. Flagellar scales are assembled exclusively in the GA and abstricted from the rims of thetrans-most GA cisternae into vesicles. Exocytosis of scales occurs at the base of the anterior flagellar groove. The central portion of thetrans-most cisterna, containing no scales, detaches from the stack of cisternae and develops a coat to become a coated polygonal vesicle. Scale biogenesis involves continuous turnover of GA cisternae, and scale production rates indicate maturation of four cisternae per minute from each of the cells two dictyosomes. A possible model of membrane flow routes during flagellar regeneration, which involves a membrane recycling loop via the coated polygonal vesicles, is presented.     

THE DEVELOPMENT AND CYTOLOGY OF THE EPIBIOTIC PHASE OF PHYSODERMA PULPOSUM

Lingappa , Yamuna . (U. Michigan, Ann Arbor.) The development and cytology of the epibiotic phase of Physoderma pulposum. Amer. Jour. Bot. 46(3) : 145-150. Illus. 1959.—Physoderma pulposum, a chytrid parasite on Chenopodium album L. and Atriplex patula L., has a zoosporangial epibiotic phase. The latter consists of extramatrical sporangia and intramatrical bushy rhizoids, both enclosed in large protruding galls. The sporangia are subspherical, up to 350μ in diameter, and may produce hundreds of planospores. If planospores settle on the host surface, they develop narrow germ tubes which penetrate the epidermal cells and develop into rhizoids. The planospore body, however, remains on the host surface and develops into a mature epibiotic sporangium in about 20-25 days at 16°C., 12-15 days at 20-25°C., or 6-8 days at 30°C. During development, its nucleus and daughter nuclei divide mitotically with intranuclear spindles until the sporangium contains several hundred nuclei. This is followed by progressive cleavage which delimits the planospore rudiments. When mature sporangia are placed in fresh water, the planospores are quickly formed within 1 hr. at 25°C. and begin to swarm within the sporangia. They escape in large numbers through an opening formed by the deliquescence of a papillum in the sporangial wall. The planospores are subspherical or elongate, 3-5 × 4-6 μ, and each has an eccentric orange-yellow refractive globule and a flagellum 18-22 μ in length. The electron micrographs of the flagella indicate that the flagella are absorbed from tip backward during encystment of the planospores. By periodic inoculation of the host plants with planospores from epibiotic sporangia, as well as from germinating resting sporangia, generation after generation of epibiotic sporangia have been obtained for 4 years. This proves the existence of a eucarpic, epibiotic, ephemeral zoosporangial phase in P. pulposum. Field observations on the duration and sequence of development of the fungus indicate that the endobiotic resting sporangial phase always follows the epibiotic phase. The results of infection experiments also indicate that the epi- and endobiotic phases belong to one and the same fungus, P. pulposum.     

Zoospore development in the oomycetes

Oomycetes cause destructive diseases on both animals and plants. The epidemic spread of oomycete diseases is primarily based on rapid dispersal from host to host by free swimming zoospores. These single-nucleated spores are formed in sporangia and are only released in aqueous environments. Oomycetes are classified in the Kingdom of the Stramenopiles or Chromista, which is comprised of several organisms, including the golden brown algae. The unique shared attribute found in most Stramenopiles is the morphology of the zoospores and especially the structure of their two flagella. They have one tinsel flagellum, and one whiplash flagellum. Only the tinsel flagellum has distinctive flagellar hairs. Zoospore formation can occur within minutes and it is considered one of the fastest developmental processes in any biological system. Once released from the sporangium they are able to exhibit chemotactic responses, electrotaxis, and autotaxis or autoaggregation to target new hosts for infection. Here we discuss the latest discoveries in the development and biology of the oomycete zoospore.     

Ultrastructural analysis of flagellar development in plurilocular sporangia of Ectocarpus siliculosus (Phaeophyceae)

Flagellar development in the plurilocular zoidangia of sporophytes of the brown alga Ectocarpus siliculosus was analyzed in detail using transmission electron microscopy and electron tomography. A series of cell divisions in the plurilocular zoidangia produced the spore-mother cells. In these cells, the centrioles differentiated into flagellar basal bodies with basal plates at their distal ends and attached to the plasma membrane. The plasma membrane formed a depression (flagellar pocket) into where the flagella elongated and in which variously sized vesicles and cytoplasmic fragments accumulated. The anterior and posterior flagella started elongating simultaneously, and the vesicles and cytoplasmic fragments in the flagellar pocket fused to the flagellar membranes. The two flagella (anterior and posterior) could be clearly distinguished from each other at the initial stage of their development by differences in length, diameter and the appendage flagellar rootlets. Flagella continued to elongate in the flagellar pocket and maintained their mutually parallel arrangement as the flagellar pocket gradually changed position. In mature zoids, the basal part of the posterior flagellum (paraflagellar body) characteristically became swollen and faced the eyespot region. Electron dense materials accumulated between the axoneme and the flagellar membrane, and crystallized materials could also be observed in the swollen region. Before liberation of the zoospores from the plurilocular zoidangia, mastigoneme attachment was restricted to the distal region of the anterior flagellum. Structures just below the flagellar membrane that connected to the mastigonemes were clearly visible by electron tomography.     

In vitro studies on the mechanisms of endospore release by Rhinosporidium seeberi

Studies of Rhinosporidium seeberi have demonstrated that this organism has a complex life cycle in infected tissues. Its in vivo life cycle is initiated with the release of endospores into a host's tissues from its spherical sporangia. However, little is known about the mechanisms of sporangium formation and endospore release since this pathogen is intractable to culture. We have studied the in vitro mechanisms of endospore release from viable R. seeberi's sporangia. It was found that watery substances visibly stimulates the mature sporangia of R. seeberi to the point of endospore discharge. The internal rearrangement of the endospores within the mature sporangia, the opening of an apical pore in R. seeberi's cell wall, and the active release of the endospores were the main features of this process. Only one pore per sporangium was observed. The finding of early stages of pore development in juvenile and intermediate sporangia suggested that its formation is genetically programed and that it is not a random process. The stimulation of R. seeberi's sporangia by water supports the epidemiological studies that had linked this pathogen with wet environments. It also explains, in part, its affinities for mucous membranes in infected hosts. The microscopic features of endospore discharge suggest a connection with organisms classified in the Kingdom Protoctista. This study strongly supports a recent finding that placed R. seeberi with organisms in the protoctistan Mesomycetozoa clade. This revised version was published online in June 2006 with corrections to the Cover Date.     

Brassica napus pollen development during generative cell and sperm cell formation

Summary Brassica napus pollen development during the formation of the generative cell and sperm cells is analysed with light and electron microscopy. The generative cell is formed as a small lenticular cell attached to the intine, as a result of the unequal first mitosis. After detaching itself from the intine, the generative cell becomes spherical, and its wall morphology changes. Simultaneously, the vegetative nucleus enlarges, becomes euchromatic and forms a large nucleolus. In addition, the cytoplasm of the vegetative cell develops a complex ultrastructure that is characterized by an extensive RER organized in stacks, numerous dictyosomes and Golgi vesicles and a large quantity of lipid bodies. Microbodies, which are present at the mature stage, are not yet formed. The generative cell undergoes an equal division which results in two spindle-shaped sperm cells. This cell division occurs through the concerted action of cell constriction and cell plate formation. The two sperm cells remain enveloped within one continuous vegetative plasma membrane. One sperm cell becomes anchored onto the vegetative nucleus by a long extension enclosed within a deep invagination of the vegetative nucleus. Plastid inheritance appears to be strictly maternal since the sperm cells do not contain plastids; plastids are excluded from the generative cell even in the first mitosis.     

A spectrophotometric approach for determining sporangium and zoospore viability of Plasmopara viticola

A spectrophotometric method for determining the viability of sporangia and zoospores of the oomycete Plasmopara viticola (causal agent of grapevine downy mildew) is described and evaluated to overcome the limitations of currently available methods for assessing propagule viability. Sporangia produced on leaf discs in the laboratory were harvested at different days after the initiation of sporulation (DAS) to yield differences in sporangium viability. Sporangia were suspended in sterile water, the suspensions were placed in a cuvette, and sporangium germination was monitored in a spectrophotometer (λ = 600 nm) at 2- to 3-min intervals for 5 hr. Absorbance started to increase after sporangia were suspended in water for ~30–60 min followed by major peak(s) for younger sporangia (1–3 DAS), whereas low to no increase in absorbance was observed for senescent sporangia (>7 DAS). Microscopic observation confirmed that the increase in absorbance corresponded to the release and active swimming of zoospores, whereas absorbance decreased when zoospores encysted and settled. A positive correlation (r = .839, p = .0365) was observed when the time to the initial increase in absorbance was plotted against the age of sporangia. The time to the absorbance peak (marking the time of maximum zoospore movement) was shortest for immature sporangia (0 DAS), longest for young sporangia (2 DAS) and decreased for mature and senescent sporangia. A similar pattern was observed for the standardized area under the absorbance curve (indicating the overall quantity of zoospores released), for which values were lowest for immature and senescent sporangia, highest for young sporangia and intermediate for mature sporangia. Consistent patterns obtained across two independent experiments suggest that the method is reproducible and may be further developed for other zoospore-releasing pathogens.     

Allomyces neo-moniliformis gametogenesis

Summary InAllomyces neo-moniliformis meiosis takes place during resting sporangium germination. The meiospores are characteristically binucleate and biflagellate as described byEmerson (1938) andTeter (1944). A variation in the number of nuclei and flagella per meiospore from two is correlated with germination of the resting sporangia under reduced oxygen tension. The meiospores are extremely poor swimmers and are typically amoeboid. At encystment the gamma bodies of the cell are mobilized and appear involved in cyst wall synthesis. A single mitotic division of each nucleus gives rise to four nuclei. Gamete cleavage is as described for spore cleavage inBlastocladiella (Lessie andLovett 1968). The assembly of the nuclear cap and side body complex of the spore are extremely late processes in gametogenesis. The gametes are released when the single papilla dissolves. The gametes fuse in pairs and after zygote formation the cell is uninucleate with two flagella. The biflagellate zygote is an active swimming cell. The presence of homothallism or hetero-thallism inA. neo-moniliformis is discussed.     

Changes in wall and internal structure of allomyces-resistant sporangia during germination

The electron microscope was used to examine changes which take place in wall, as well as in internal, structure during germination of mature resistant sporangia of Allomyces neo-moniliformis. When the resistant sporangia are first placed in water to initiate germination, nuclei, mitochondria, and endoplasmic reticulum are not evident, though after the sporangia have been in water for more than 30 min all of these structures become visible. At this time no cracks are evident in the resistant sporangial wall and the cell membrane appears highly convoluted. Within the next 30 min the outer wall splits and the inner wall expands considerably as the protoplast increases in volume. At the same time the cell membrane straightens out, apparently in response to the protoplasmic expansion. The “cementing substances” begin to dissolve about this time so that 1 1/2 hr after placement in water the outer wall is completely separated from the inner wall which now acts as the cell wall. Cleavage appears to be initiated by the invagination of the cell membrane and by the appearance of segments of endoplasmic reticulum with filled vesicles at one end. Between 2 1/2 and 3 hr after placement in water zoospores are released.     

A Role for the Membrane in Regulating Chlamydomonas Flagellar Length

Flagellar assembly requires coordination between the assembly of axonemal proteins and the assembly of the flagellar membrane and membrane proteins. Fully grown steady-state Chlamydomonas flagella release flagellar vesicles from their tips and failure to resupply membrane should affect flagellar length. To study vesicle release, plasma and flagellar membrane surface proteins were vectorially pulse-labeled and flagella and vesicles were analyzed for biotinylated proteins. Based on the quantity of biotinylated proteins in purified vesicles, steady-state flagella appeared to shed a minimum of 16% of their surface membrane per hour, equivalent to a complete flagellar membrane being released every 6 hrs or less. Brefeldin-A destroyed Chlamydomonas Golgi, inhibited the secretory pathway, inhibited flagellar regeneration, and induced full-length flagella to disassemble within 6 hrs, consistent with flagellar disassembly being induced by a failure to resupply membrane. In contrast to membrane lipids, a pool of biotinylatable membrane proteins was identified that was sufficient to resupply flagella as they released vesicles for 6 hrs in the absence of protein synthesis and to support one and nearly two regenerations of flagella following amputation. These studies reveal the importance of the secretory pathway to assemble and maintain full-length flagella.