Ultrastructural changes during sporangium formation and zoospore differentiation in Blastocladiella Emersonii.

TLDR: Samples from synchronized cultures of Rlastorlacliella e m e ~ s o n i i were examined by electron microscopy from the late log phase to the completioli of zoospore differentiation, which covered general details of fungal fine structure lower aquatic fungi.

Abstract: Samples from synchronized cultures of Rlastorlacliella e m e ~ s o n i i were examined by electron microscopy from t,he late log phase to the completioli of zoospore differentiation, Log-phase plants contain t,he usual cytoplasmic organelles but also have an unusual system of large tubules ca. 45 mp 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. ext,ranuclear centrioles at each pole. The papilla is formed by a process of localized cell wall breakdown and deposit,ion of the papilla material by secretory granules. Differentiation of zoospores begins when one of the t,wo cent,rioles associated with each nucleus elongates to form a basal body. The flagella fibers arise from the basal body and elongate into an espanding 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. THEGENERAL details of fungal fine structure lower aquatic fungi, which, in contrast to the have been well covered in recent reviews (Hawker, higher groups, can usually be fixed well with 1965; Rloore, 1965; Braclier, 1967). Relatively osmium tetroxide alone, or in combination with little work has been reported, however, on the glutaraldehyde. TTTo published research papers on aquatics have represented systematic studies Reeeived for publication 12 hIay 1967. of development; the excellent reports on gamete This research was supported by Grant No. AI-04783 from the National Institute of Allergy and Infectiou.. differentiation in Allomyces .macrogpnus by Diseases, National Institutei of Health Blonde1 and Turian (1960), and flagella developThe second authol expreises his slncere gratitude to ment in gametangia of A . ar.buscula by Renaud Dr. A. E. 17atter, University of Colorado Jledical School, and Ssvift (1964). Zoospore differentiation in A . Denver, for introduc~ng him to the techniques of electron micwscopy The authols are also indebted lo Dr. T7atter ,gavanicus has been reported in summary form by for assistance in the preparation of Fig 50, 51, 53, and 54 i\\loore (1964b, c; 1965), but the micrographs February, 19681 LESSIE AND LOVETT-ULTRASTRUCTURE IN BLASTOCLADIELLA 221 have not yet been published. So far as we are conducted a t 2-4 C. Sections were cut with glass aware, the initial fine structural events through knives on a Cambridge-Huxley ultramicrotome. which log-phase plants are converted to zooGold and silver sections were mounted on collosporaagia (or gametangia) have not been dion-covered grids and the sections stained for described. 10-15 min in an alkaline solution of lead citrate The basic ultrastructural organization of the (Reynolds, 1963). The sections were examined zoospore produced by Blastocladiella enzersonl'~ and photographed in a Philips EhI-200 electron Cantirio and Hyatt 11~as originally described b>microscope. Figures 50, 51, 53, 54 were prepared Cantino et al. (1963), and this 117ork was extended from material embedded in Vestopal and were riot by the isolatio~l and characterization of the stained. distinctive nuclear cap by Lovett (1963). Some additional structural details have recently beer1 ~ I E S U L T S T ~ ~ differentiati011 induced by the reported by Reichle and Fuller (1967). As part change in medium takes place in two distinct of a continuing study of the biochemical basis phases, the presporulation modification of the for development in this fungus, we have also plant to form a multinucleate zoosporangium examined the ultrastructural changes that acand the subsequent formation of the individually compaiijthe forniation and germination of B. organized uninucleate zoospores. The results -rvill enze~sonii zoospores in synchronized cultures. deal with three separate stages: the structure of In this paper we discuss the process of sporangium the cytoplasm in log-phase plants before 15% formation and zoospore differentiation in posthr, sporangium formation between 16 and 18 exponential phase plants. hr, and zoospore differentiation from 18 to 19 hr. I\\~ATERIALSAND METHODS-The procedures Log-phase plants-The cytoplasm of growing used for growing B. enzersonii, and for the inplants contains many nuclei with large granular duction of synchronous zoospore differentiation nucleoli; numerous mitochondria; occasional, have been described elsewhere (Murphy arid dense and amorphous bodies which appear to be Lovett, 1966; Lovett, in press). Nost of the microlipid globules; more numerous, smaller, irregularly graphs to folio\\\\were prepared from a single shaped granules with moderately dense granular culture with an estimated synchrony of 90%. A contents surrounded by a single-layered memsecond culture with 95 % synchrony was used to brane (sb, Fig. I ) ; large numbers of polysaccharide obtain material during the exponential gro~vth granules; and densely packed ribosomes (Fig. 1). phase. With the latter, samples were removed A moderate amount of irregularly organized for fixation at 30-min intervals between 12 and endoplasmic reticulum occurs and appears mairlly 14 hr. For the differentiation sequence in the to be of the smooth type with little conspicuous first culture, similar sanlpIes were removed every association with ribosomes. Many vacuoles are 15 mi11 from 16 to 17 hr, and then every 12 min present but vary in number and size from plant until zoospores mere released. Beginning at 16 to plant. The cytoplasm in the basal area near the hr, I in1 of culture was also removed and fixed rhizoids usually has a rather ragged appearance every 30 min for the estimation of culture because of a higher concentration of small vacuoles synchrony. and a paucity of ribosomes (Fig. 12). The latter For electroll microscopy 5-7 ml-samples were condition, plus unusually long mitochondria, imnlediately pipetted into a test tube containing is also characteristic of the anucleate rhizoids. 1 ml of cold 2 % glutaraldehyde in pH 7.8 veronal The ~olysaccharide granules occur both as acetate buffer (0.018 A[ sodium veronal, 0.018 M smaller P-particles (Revel, 1964) of 250-300 A sodium acetate) to prefix. After a 3-4-min centrifdiam and as aggregates of these in rosettes or ugation, the supernatant was decanted and the \"-particles (Fig. 6). The polysaccharide granules pellet of plants fixed for 1 hr in the 2% are frequently clustered around mitochondria buffered glutaraldehyde (Chrispeels and Vatter, and occasionally occur in very large aggregates; 1963). The glutaraldehyde was followed by a particles which resemble the smaller P-particle 1-hr post-fixation in 1 % osmiunl tetroxide in are also found in some mitochondria (Fig. 6). the same pH 7.8 buffer containing 0.001 A[ CaC12. A striking feature of the cytoplasm in growing After fixation the pellets were dehydrated by plants is the presence of large numbers of tubular passage through a graded series of ethanol structures. Nost of these are found in organized solutions containing 1% MgC12 and finally bundles that wind and branch throughout the absolute ethanol. The ethanol \\\\-as replaced by cytoplasm (Fig. 1, 2). The individual tubules propylene oxide, and the dehydrated material ' have a diam of approximately 45 mp and appear was embedded in a plastic mixture consisting of to be membranous structures with some addi15 ml Araldite 6005, 25 ml Epon 812, 55 ml tional fine material attached to the outer surface. dodecyl succinic anhydride, 3 ml dibutyl phthaIn some longitudinal sections the lumen of the late, and 1.5 ml 2,4,5-tri (dimethylaminomethyl) tubules has a distinctly higher density than the phenol (DATP-30) (AIollenhauer, 1964). All steps immediately surrounding area within the bundle. except the infiltration and embedding were Because of their large size relative to microtubdw, 222 [Vol. 55 hl \\ IERIChN JOUENAL O F BOTANY 223 February, 19681 LESSIE A N D LOVETT-ULTRASTRUCTURE IN BLASTOCLADIELLA we will refer to them as \"macrotubules.\" The well-organized bundles of macrotubules are not surrounded by a limiting membrane but are nearly devoid of ribosomes or other particles between the constituent tubules. Cross sections of bundles (Fig. 2) show a regular (ca. 115 m,u center to center) spacing with each inner macrotubule lying in the center of a hexagonal array. The number of tubules per group ranges from as few as 2 or 3 to 30 or more. The bundles of macrotubules show considerable irregular branching as they ramify through the cytoplasm and give the impression of forming a more or less continuous networli. Occasional sections have shown macrotubules terminating in irregular vesicles or cisternae (Fig. 3, 4, 5). The bundles gradually lose their organization and the macrotubules disappear during the subsequent diff erentiation of the sporangium and spores. Sporanyiuin formation-Immediately after the change of medium at 16 hr, the cytoplasm of the plants appears similar to that during the growth phase. At approximately 16% hr a round of nuclear division starts, and the formation of a cross wall to separate the sporangium from the rhizoids begins. Nuclear division-Somatic nuclear division takes place xvithin the intact nuclear membrane and the granu