Edward C. Cantino

Decay of ? particles in germinating zoospores of Blastocladiella emersonii

SummaryThe breakdown and disappearance of γ particles was followed by electron microscopy on synchronously germinating populations of B. emersonii zoospores. The sequence of morphological changes, unaffected by the method used to induce germination, was divided into four stages. Initially, the γ particles surrounding unit membrane becomes amorphous and buds off vesicles that fuse with the plasma membrane. Simultaneously, ca. 80 mμ vesicles are visible either partially embedded in, or lying free along, the inner surface of the electron dense matrix. These small vesicles seem to originate from a newly detected membrane system in the electron dense γ matrix. In the next stage, γ particles characteristically adhere to various organelles, especially the nuclear cap. In subsequent stages the γ particles continue to decay by releasing 80 mμ vesicles from their matrices and by budding off vesicles from their surrounding membranes. Often, multivesicular bodies are formed when the 80 mμ vesicles become enclosed by the γ particles budding outer membrane. In the final stage, the surrounding membranes of the different particles fuse and produce large vacuoles containing many of the electron dense matrices which, though by now partially fragmented, continue to release 80 mμ vesicles. The relationship of the above events to the formation of the cell wall is discussed.

Utilization of endogenous reserves by swimming zoospores of Blastocladiella emersonii

Summary1. Methods are described for inducing the synchronous release of zoospores from single-generation cultures of Blastocladiella emersonii and preparing washed suspensions of up to 2×1010 non-encysted zoospores for physiological studies. 2. During 5 h incubations of such zoospores in a buffered CaCl2 solution, the rate of oxygen uptake was ca. 10 μl O2 x h-1 x (107 cells)-1, the respiratory quotient was 0.92, the average dry weight (47 picograms) of the spore decreased 1.5 picograms (pg), and other components (per-spore) decreased as follows: nucleic acid, nil; total lipid, 0,95 pg; phospholipid, 0.80 pg; polysaccharide, 0.5–1.0 pg, depending upon initial (1.5–2.3 pg) intracellular levels; protein, 3.0 pg; and total nitrogen 0.4 pg. During this period, 0.38 pg of NH3-nitrogen was released per spore. Correspondingly, the lipid bodies decreased in size and number and the SB-matrix became progressively thinner. 3. It was concluded that during the endogenous metabolism of a non-encysted zoospore of B. emersonii, a significant portion (11.5%) of its protein pool, as well as lipid and polysaccharide components, were degraded.

Chapter 1 The Induction and Early Events of Germination in The Zoospore of Blastocladiella emersonii

Publisher Summary This chapter discusses some aspects of the kinetics of encystment. Spores induced with low temperatures and sulfonic acid azo dyes display similar kinetics, while spores induced with potassium chloride (KCl) show a much greater mean time of encystment. The evidence suggests that the difference in behavior must result from an increase in the number and/or the average rates of encystment processes that ensue after triggering. When the reason for this difference is uncovered, an important aspect of encystment is resolved. But in the meantime, a comparison of the similarities among the induction methods may also be fruitful.

DNA profile of the spore of blastocladiella emersonii: Evidence for ?-particle DNA

SummaryProcedures were developed for isolating DNA from zoospores of Blastocladiella emersonii, critical steps being the use of non-frozen cells, enzymatic elimination of polysaccharide, and a combined treatment with T1RNAse and pancreatic RNAse for complete removal of RNA. Methods for isolating γ-particles from spores were also established. Extraction of DNA directly from purified γ-particles substantiated previous assumptions that they contain DNA. Heat denaturation and preparative CsCl equilibrium sedimentation studies of whole spore DNA revealed the presence of four distinct components, I–IV, with buoyant densities of 1.731, 1.715, 1.705, and 1.687 g/cm3. The major species appeared to be nuclear chromatin. Nucleolar and mitochondrial sites were tentatively assigned to species II and III, respectively. Evidence was presented that species IV resided in γ-particles. Some possible roles for γ-particle DNA were discussed.

Isolation and characterization of microbodies and symphyomicrobodies with different buoyant densities from the fungus Blastocladiella emersonii.

Abstract Microbodies isolated from sporangia of the aquatic fungus Blastocladiella emersonii have a mean buoyant density of 1.222 g/cm3 after centrifugation through a linear sucrose gradient, and contain catalase, isocitrate lyase and malate synthase. Microbodies fuse to produce one symphyomicrobody per zoospore at the time of sporogenesis. An increase in density accompanies this process. The symphyomicrobody has a mean buoyant density of 1.292 g/cm3 while the spores single mitochondrion has a buoyant density of 1.219 g/cm3. Statistical data are also provided for both starting levels and purification of symphyomicrobody and mitochondrial enzyme markers.

The lipid composition of the Blastocladiella emersonii γ particle and the function of γ-particle lipid in chitin formation

Lipid was extracted from isolated γ particles of Blastocladiella emersonii and characterized by means of column and thin-layer chromatography. The major lipid component was a glycolipid. When isolated γ particles were incubated in the presence of the substrate for chitin synthetase, UDP-[1-14C]GlcNAc, labeled glycosyldiglyceride and labeled chitin were formed. When labeled glycolipid was added to a γ-particle preparation along with unlabeled substrate, labeled chitin was again produced. Electron micrographs of γ particles incubated with substrate showed in vitro formation of chitin fibrils. A model is presented correlating the ultrastructural changes in γ particles during zoospore encystment with the biochemical events leading to chitin formation.

Lag-Phase Sporogenesis in Blastocladiella Emersonii: Induced Formation of Unispored Plantlets

A method was perfected for inducing single-spore formation in uninucleate lag-phase germlings of Blastocladiella emersonii. Whether one or more spores was produced was related to the age of the germlings, the number of nuclei present prior to induction, and their apparent position in the mitotic cycle. Amino acids and peptone-yeast extract reversed induction of sporogenesis more effectively than glucose. Changes in cell morphology, polysaccharide, protein, nucleic acids, and dry weight during induction in uninucleate lag-phase germlings were similar to those known to occur in multinucleate log-phase ordinary colorless plants. The viability and general morphology of zoospores produced by lagand logphase plants resembled one another closely.

Concurrent effect of visible light on ?-particles, chitin synthetase, and encystment capacity in zoospores ofBlastocladiella emersonii

SummaryZoospores derived from ordinary colorless plants ofBlastocladiella emersonii grown under 240 μW/cm2 of visible light contain an average of ca. 5 γ-particles and numerous aggregates of cytoplasmic granules which resemble the γ-matrix, while spores from dark-grown plants contain ca. 12 γ-particles but none of the granules. Correspondingly, the amount of chitin synthetase associated with γ-particles is approximately proportional to the number of γ-particles in the two spore types. The foregoing light effects, known to be accompanied by an increased capacity for encystment, probably take place before sporogenesis. Conversely, zoospores derived from dark-grown plants do not encyst appreciably over an 8 h period unless they are illuminated, but the number of γ-particles/spore decreases from 12 to ca. 6 whether or not light is present. Arguments are presented for considering the latter disappearance of γ-particles as an essential “ark reaction” which precedes a photochemical reaction, both being needed for light-induced encystment.

Calcium is a prominent constituent of the Gamma Particle in the zoospore of Blastocladiella emersonii as revealed by X-ray microanalysis

Abstract Energy selective X-ray elemental microanalysis was used to demonstrate the presence of Ca, K and P (relative amounts about 1 : 1 : 4, respectively) in the virus-like Gamma Particles found in the zoospores of the aquatic fungus Blastocladiella emersonii . Some Gamma Particles, however, lack detectable amounts of K. The possible significance of these observations for understanding the triggering of encystment and the initiation of wall synthesis is underscored.

Lipid changes during development of Blastocladiella emersonii

Abstract The lipid composition of swimming spores, cysts and five hour germlings was established. Spores utilized triglycerides first, then phospholipids. Upon encystment all glycolipid components decreased, while in germlings the phospholipids, monoglycerides and sterol esters exhibited a marked increase.