THE NATURE AND FUNCTION OF THE MITOCHONDRION- LIPID-SYMPHYOMICROBDDY COMPLEX IN THE ZDOSPORE 0F BLASTOCLADIELLA EMERSONII Dissertation for the Degree of Ph. D. MICHIGAN STATE'UNIVERSITY GARY LYNN MILLS 1976 I“; iv F2“? .2 “5 v .31.;33 “iii-Jt a. This is to certify that the thesis entitled The Nature and Function of the Mitochondrion- Lipid-Symphyomicrobody Complex in the Zoospore of Blastocladiella emersonii presented by Gary Lunn Mills has been accepted towards fulfillment of the requirements for Ph.D. degree in Botany git/ml C 634%. Major professor DateNoyi 9/ (q 7Q 0-7639 ABSTRACT THE NATURE AND FUNCTION OF THE MITOCHONDRION-LIPID-SYMPHYOMICROBODY COMPLEX IN THE ZODSPORE OF BLASTOCLADIELLA EMERSONII By Gary Lynn Mills The zoospores of Blastocladiella emersonii possess an elaborate, tightly organized, membrane bound assemblage of organelles. One especially prominent component is the side body complex. This complex consists of a single mitochondrion, a number of discrete lipid globules and a single-membrane-bound structure, once named the SB matrix. A study was made of the lipid composition of the zoospores, the lipid changes during development and the chemistry or enzymology of the indivi- dual components of the side body complex. The specific purpose was to try to obtain a better understanding of the functional nature of this group of organelles. The zoospores of a, emersonii, when derived from cultures grown on solid media, contained about 11% total lipid. This lipid was separated chromatographically on silicic acid into neutral lipid (46.6%), glycolipid (15.8%), and phospholipid (37.6%). Each class was fractionated further on columns of silicic acid, Florisil, or diethylaminoethyl- cellulose, and monitored by thin-layer chromatography. Triglycerides were the major neutral lipids, mono- and diglycosyldiglycerides the major glycolipids, and phosphatidylcholine and phosphatidylethanolamine the major phospholipids. Other neutral lipids and phospholipids detected Gary Lynn Mills were: hydrocarbons, free fatty acids, free sterols, sterol esters, diglycerides, monoglycerides, lysophosphatidylcholine, lysophosphatidyl- ethanolamine, phosphatidic acid, phosphatidylserine and phosphatidylino- sitol. Palmitic, palmitoleic, stearic, oleic, y-linolenic and arachi- donic acids were the most frequently occurring fatty acids. When a. emersonii was grown in [l,2-14CJ-acetate-labeled liquid media, lipid again accounted for ll% of both the mature plants and the zoospores released from them. The composition of the lipid extracted from such plants and spores was also the same. However, it differed markedly from that of the lipid in spores harvested from solid media, consisting of 28.3% neutral lipid, l2.0% glycolipid, and 59.0% phospholipid. The major lipids were the same as those derived from plate grown cultures. The lipid composition of swimming spores, cysts and five hour germlings was also established. Spores utilize triglycerides first, then phospholipids. Upon encystment all glycolipids decreased, while in germlings the phospholipids, monoglycerides and sterol esters exhibited a marked increase. Lipid globules were isolated and characterized both chemically and morphologically. They were composed mainly of triglycerides and free sterols, the combination accounting for over 90.0% of the total weight of the globules. Smaller amounts of diglycerides, carotenoids, fatty free acids, phospholipids and protein were found. No sterol esters or mono- glycerides were detected. Morphologically, the isolated lipid globules resembled the lipid globules jn_§jtu, They were spherical, 0.4 pm to l.5 um in diameter and lacked a trilaminar membrane. Gary Lynn Mills Since the SB matrix lies in close proximity to the lipid globules which are primarily triglycerides, and since the triglycerides decrease as the spores swim, the SB matrix might be functioning as a microbody. Microbodies can be identified ultracytochemically with the diamino- benzidine (DAB) catalase test. When zoospores and sporangia of B, emersonii were incubated in the DAB reaction mixture both the SB matrix in the zoospores and the sb granules in the sporangia exhibited a catalase positive reaction. The two types of organelles are ontogene- tically interrelated; the sb granules are microbodies, and these give rise to the symphyomicrobody (formerly the SB matrix) by symphyogenesis. Microbodies were isolated from sporangia of B. emersonii. They had a mean buoyant density of 1.222 g/cm3 after centrifugation through a linear sucrose gradient, and contained catalase, isocitrate lyase and malate synthase activities. Symphyomicrobodies were also isolated from zoospores. They had a mean buoyant density of 1.292 g/cm3, hence an increase in density accompanied the formation of symphyomicrobodies by symphyogenesis. The spores single mitochondria had a buoyant density of l.2l9 g/cm3. Statistical data are provided for starting levels and purification of symphyomicrobody and mitochondrial enzyme markers. THE NATURE AND FUNCTION OF THE MITOCHONDRION-LIPID-SYMPHYOMICROBODY COMPLEX IN THE ZOOSPORE OF BLASTOCLADIELLA EMERSONII By Gary Lynn Mills A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1976 [(Iil Ill. I II‘III In memory of Wiliam G. Fields ii ACKNOWLEDGMENTS The author would like to express sincere thanks to Dr. E. C. Cantino for his enthusiastic support and excellent guidance during the course of this study. I would also like to thank the other members of my guidance committee, Dr. N. E. Good, Dr. H. A. Imshaug, and Dr. C. J. Pollard for their assistance. I would also like to acknowledge the National Institute of Health and the National Science Foundation as the sources of funds for my research assistantship. TABLE OF CONTENTS Page LIST OF TABLES ................................................. v LIST OF FIGURES ................................................ vi LIST OF NON-STANDARD ABBREVIATIONS ............................. viii INTRODUCTION ................................................... 2 EXPERIMENTAL I. Lipid composition of the zoospores of Blastocladiella emersonii ............................. 7 II. Lipid changes during development of Blastocladiella emersonii ............................. 33 III. Isolation and characterization of lipid globules from the zoospores of Blastocladiella emersonii ....... 45 IV. The single microbody in the zoospore of Blastocladiella emersonii is a 'symphyomicrobody' ..... 51 V. Isolation and characterization of microbodies and symphyomicrobodies with different buoyant densities from the fungus Blastocladiella emersonii ............ 74 DISCUSSION .................................................... 87 BIBLIOGRAPHY .................................................. 97 APPENDIX A Cytochemical localization of acid phosphatase in zoospores of B, emersonii ................................ 11] APPENDIX B Mitochondrial and microbody enzyme activity during development of B, emersonii .............................. 114 iv Table osmAwN TO. ll. 12. I3. LIST OF TABLES Lipid composition of zoospores produced on solid media .................................................. Composition of zoospore phospholipid ................... Molar ratios for zoospore phospholipids ................ Composition of zoospore neutral lipid .................. Principal fatty acids in zoospore lipid ................ Composition of lipid in plants and zoospores pro- duced in liquid media .................................. The percent composition of lipid during swimming and development ........................................ Composition of lipid globule ........................... Composition of the separated neutral lipid associated with the lipid globules after silicic acid column chromatography ......................................... Comparison of isolated lipid globules from B, emersonii with other isolated lipids ................... Specific activities of enzymes associated with organelles, including their initial activities in the zoospore homogenates ............................ The buoyant densities of isolated organelles ........... Mitochondrial and microbody enzyme activities during different developmental stages of B, emersonii .............................................. Page I3 l7 I8 23 24 26 38 52 54 57 77 83 Figure I0. . (a) (b) (C) LIST OF FIGURES Representative elution patterns for dry weight of lipid and total phosphorus obtained by column chromatography of total phospholipid on DEAE- cellulose .............................................. Glycolipid fractions collected by Florisil column chromatography and resolved further by TLC ............. Components in the neutral lipid fraction separated by TLC and visualized by H2304 charring ................ Separable components in the 14C-labeled neutral, glycolipid, and phospholipid extracted from post- cleavage plants of B, emersonii and resolved by TLC, and the profiles for distribution of radio- activity in these components ........................... Changes in total and labeled lipid/1010 cells during swimming and development ............................... Changes in profiles of labeled lipids in zoospores during swimming ........................................ Changes in profiles of labeled lipids in zoospores, cysts and germlings .................................... Electron microgram of a thin section through B emersonii zoospore showing the in situ appearance of the lipid globules and their—relationship to the spores single mitochondrion and symphyomicrobody ... Thin section of the isolated lipid layer ............... High magnification of lipid globule .................... The relationship of the symphyomicrobody-lipid com- plex to other organelles in the side body of the B, emersonii zoospore .................................. The response of the SB matrix in zoospores ole. emersonii to the Beard and Novikoff DAB test for catalase activity ...................................... vi Page I6 20 22 28 36 39 42 SI SI SI 63 66 Figure Page 11. Sections through sporangia at the time of sporo- genesis showing the DAB test for catalase activity ..... 68 12. Formation of the symphyomicrobody (by symphyogenesis) and the symphyomicrobody-lipid complex in the zoospore of_B. emersonii ........................................ 69 13. Distribution of organelle protein, and activities of mitochondrial enzymes and microbody enzymes after sucrose density gradient separation of components of the 300 xg supernatant from zoospores ............... 79 14. Electron micrograph of isolated mitochondria, fixed in 0.5% glutaraldehyde and 0.5% 0504, and postfixed in 1% 0504, each for 1 h at 4°C, dehydrated, and embedded in Spurr's medium ............................. 80 15. Distribution of protein and isocitrate lyase activity after sucrose density gradient separation of compounds of the l0,000 xg pellet from sporangia ................ 82 16. The response of zoospores to the Gomori lead nitrate cytochemical procedure for localization of acid 113 phosphate activity ..................................... vii Phospholipids LPC LPE PA PC PE PI PS Neutral Lipids DG FFA FS HC MG SE TG Glycolipids DGDG MGDG PGDG LIST OF NON-STANDARD ABBREVIATIONS Lysophosphatidylcholine Lysophosphatidylethanolamine Phosphatidic acid Phosphatidylcholine Phosphatidylethanolamine Phosphatidylinositol Phosphatidylserine Diglyceride Free fatty acid Free sterol Hydrocarbon Monoglyceride Sterol ester Triglyceride Diglycosyldiglyceride Monoglycosyldiglyceride Polyglycosyldiglyceride viii INTRODUCTION In 1949, a new species of the Chytridiomycete, Blastocladiella, was discovered in a fresh water pond on the campus of the University of Pennsylvania and subsequently described (Cantino, 1951). Two years later it was designated a new species, Blastocladiella emersonii (Cantino and Hyatt, 1953), and since that time this fungus has become an important experimental organism for studying morphological, physiological and developmental interrelationships (see reviews: Cantino and Lovett, 1964; Cantino, 1966; Cantino et al., 1968; Truesdell and Cantino, 1971; Lovett, l975; Cantino and Mills, 1976). The life cycle of B, emersonii is rather simple although variations do occur (Hennessy and Cantino, 1972; Cantino and Myers, 1974). The fungus produces posteriorly uniflagellated zoospores. During their existence, these zoospores undergo mainly catabolic activities, very few synthetic processes having been detected ($011 and Sonneborn, 1971; Suberkropp and Cantino, 1973; Lovett, 1975). The zoospores round up, retract their flagella and encyst, a process involving dedifferentiation accompanied by striking ultrastructural changes (3011 et al., 1969; Truesdell and Cantino, 1971) and the production of a cell wall (Myers and Cantino, 1974). After a short time the cysts produce rhizoids and become germlings. The germlings undergo nuclear division and exponential growth and generate plants bearing multinucleate sporangia. Exponential growth culminates at the time of papillae formation, after which the sporangia undergo cytodifferentiation with formation of zoospores. The zoospores are released ng_the exit papillae and the cycle is repeated. No sexual stage is known. I. I [III III I It I l I I {III [N I I III I. The organelle arrangement in the unflagellated zoospore is highly ordered (Cantino and Mills, 1976). Each cell contains: a nuclear apparatus consisting of the nucleus, nucleolus and the nuclear cap, which houses most of the spore's ribosomes; a kinetosome-rootlet- centriole complex situated at the posterior end of the nucleus, and continuous with the flagellar axoneme; cytoplasmic gamma particles which are involved in cell wall synthesis; and the side body complex which is made up of several organelles. It is this latter group of organelles to which this thesis is directed -- the aim being to try to understand the function of this side body complex. The history of the side body complex is lengthy and there has been much confusion about the terminology. StUben (1939) was the first to observe, by means of light microscopy, what he called the "seitenkbrper", i.e. the side body, in zoospores of other species of the Blastocladiaceae. Couch and Niffen (1942) identified one of the components of the side body as a group of fat bodies. It was not, however, until 1963 that the true nature of the side body was determined. Cantino et al. (1963) and Lovett (1963) showed ng_electron microscopy that the side body complex consisted of the mitochondrion, lipid granules and particles of unknown material. These unidentified particles were later labeled "sb granules" (Lessie and Lovett, 1968). The sb granules were at first treated as separate entities; however, Cantino and Truesdell (1970) using serial sections showed that the sb granules were segments of a larger continuous structure; the latter was labeled the "SB matrix". The side body complex is located in the posterior portion of the zoospore. The lipid granules are dispersed along the outer surface of the long "arm" of the mitochondrion. Molded against them is the SB matrix, an organelle bound by a unit membrane that has a granular to amorphous texture of moderate electron density. A sheet of double mem- brane, the backing membrane, covers the whole complex of organelles and is continuous with the outer unit membrane of the nuclear apparatus. Within the past few years, structural aggregates resembling either the whole side body complex or just the SB matrix-lipid grouping have been seen in zoospores of Blastocladiella brittanica (Cantino and Truesdell, 1971) and other Chytridiomycetes. Especially noteworthy is the side body complex described by Martin (1971) for the zoospores of Coelomomyces punctatu , wherein lipid globules are positioned between the mitochondrion on one side and a unit membrane bound SB matrix on the other. A somewhat comparable arrangement occurs in the zoospores of Coelomomyces psorophorae (Hhisler et al., 1972) and Coelomomyces indicus (Madelin and Beckett, 1972). Similarly, something resembling a side body complex was observed in the zoospores of Allomyces (Fuller and Olson; 1971), where they called the membrane bound body a "StUben body", a term which was thought to be more general. Olson (1973) concluded later, using serial sections, that the side body complex in the meiospores of Allomyces had a three dimensional organization resembling the one in B, emersonii. Stfiben bodies were also reported to occur in Phlyctochytrium arcticum (Chang and Barr, 1973), and zoospores of Harpochytrium hedinii (see Travland and Whisler, 1971) possess a tight association of lipid-like globules, a single "rumposome", and profiles of electron dense granular material resembling the SB matrix in B, emersonii. The important question is: what is the function of these various bodies which are enclosed by a single membrane, composed of a moderately I I. I ...II I III I II I III I I I III A III [III E ‘I‘ (III Ill! [1 l liI‘ I electron opaque substance, and closely associated with lipid bodies and mitochondria? Recently a number of people studying Chytridiomycetes have concluded, based strictly on morphological criteria, that these organelles are microbodies. McNitt (1974) described a rumposome-lipid-microbody complex in Phlyctochytrium irregulare. Chong and Barr (1974) observed microbodies in Entophlyctis conferrae-glomeratae, microbody-lipid complexes in Rhizophydium patellarium and microbody-1ipid-mitochondrial complexes in Catenaria anguillulae. Held (1975) observed a tight association of lipid globules and microbodies which were surrounded by a backing membrane in Rozella allomycis. In each of the above cases, however, identification of these unit membrane—bound organelles as microbodies was entirely based on morphology. Because these organelles were not studied chemically or enzymologically, their functional nature remained unknown. Over the past few years, a large literature about microbodies has accumulated. These organelles are bound by a unit membrane and have been found in animals, plants, protozoa, algae and fungi (DeDuve, 1969; Hruban and Rechcigl, 1969; Tolbert, 1971; Vigil, 1973; Richardson, 1974; Frederick et al., 1975). Microbodies are 0.3 um to 1.5 um in diameter, generally are associated with lipid bodies and/or endoplasmic reticulum and have a fine granular matrix of varying electron density. Physiologically, microbodies have been classified as peroxisomes or glyoxysomes (DeDuve, 1969). Peroxisomes play a role in glycolate metabolism and photorespiration (DeDuve, 1969; Tolbert, 1971). Glyoxysomes function in the conversion of lipids to carbohydrates (Beevers, 1969; Richardson, 1974). Morphologically, the SB matrix in the B, emersonii zoospore resembles a microbody, and it is always found in close association with lipid I I IIIlI III' I‘lllllll HfIIll‘fll [III I'll. ll ‘1 E‘I‘I‘fiil {fl bodies---a common characteristic of glyoxysomes. However, the SB matrix is some 3-4 times larger than most microbodies, and the zoospores do not possess an endoplasmic reticulum (Cantino et al., 1963), although an association between sb granules and endoplasmic reticulum may occur during zoosporogenesis (Lessie and Lovett, 1968). If the SB matrix is a microbody, it might be functioning as a glyoxy- some. Glyoxysomes play a central role in gluconeogenesis by virtue of their capacity to utilize lipids yig_B-oxidation, and to mediate the forma- tion of succinate ng_isocitrate lyase and malate synthase by way of the glyoxylate cycle. Succinate then finds its way to mitochondria where it is further metabolized. The net synthesis of carbohydrate from lipid is therefore dependent on the metabolic interplay between glyoxysomes and mitochondria. The association of components in the side body complex, i.e. the lipid SB matrix and the single mitochondrion, seems to be an ideal arrangement for the metabolism of lipid by a glyoxysomal-type particle. It has been known for some time that the zoospores of B, emersonii contain at least some of the glyoxylate cycle enzymes. Isocitrate lyase was detected in zoospore homogenates and purified some 52 fold from 200- sporangial preparations (McCurdy and Cantino, 1960). Suberkropp and Cantino (1973) also found that the total lipid decreased as the zoospores swam. These data, along with the morphological characteristics, suggest that the SB matrix may be a glyoxysome. If the 58 matrix is a glyoxysome. what is its function and what is its relationship to the other organelles in the side body complex? In this study, I have attempted to elucidate some of the answers to these questions in two ways. First, the lipid composition of the zoospores was determined, I I I II II I I II [fl-ll" till" I III.‘ IIHIIEIILIIII (r and changes in these lipids were followed during different developmental stages. Second, the individual components of the side body complex were isolated and partially characterized. I Lipid Composition of the Zoospores of Blastocladiella emersonii Except for a reference to Katsura (1970), cited by Gay et al. (1971), which we have not seen, apparently the only available data on the lipid content of fungal zoospores, as estimated by direct chemical analysis, are to be found in two recent reports on the water mold Blastocladiella emersonii (Cantino and Hyatt, 1953). The first of these (Suberkropp and Cantino, 1973) established changes in the quantity of lipid/cell during endogenous metabolism of swimming zoospores; the second (Smith and Silverman, 1973) provided a pre- liminary description of changes in lipid composition after zoospore germination. We have been investigating the lipids in certain organelles in these zoospores (for a review of their structure, see Truesdell and Cantino, 1971), for which the composition of total cell lipid constitutes an essential reference point. In this communication, we characterize the whole spore lipid of B, emersonii, and provide some comparative information about the lipid content of the plants from which such spores are derived. MATERIALS AND METHODS Production of zoospores on a solid medium. The original strain of B, emersonii (Cantino and Hyatt, 1953) was grown on peptone-yeast extract-glucose (Difco; PYG) agar by inoculating with 4 x 105 spores per standard Petri plate and culturing at 22°C in the dark. Zoospores were obtained about 24 h later from first generation plants by flooding each plate with 5 m1 of water and filtering 15 min later. After population densities were established with a model B Coulter counter, the zoospores were sedimented at 1,000 x g for 5 9 min. Under these conditions, the yield was approximately 4 x 10 zoospores per 100 plates. Production of plants and zoospores in a liquid medium. PYG broth cultures were prepared, inoculated, and induced to release zoospores at 22°C, by the method of Myers and Cantino (1971). [1,2-14C] sodium acetate (New England Nuclear Corp.: 50 uCi) was added 9 h after 4M. For studies inoculation, the final concentration being 5 x 10' of zoosporangial lipid, thalli were harvested either just before zoospore cleavage or after zoospores had been cleaved but not released (ca. 23 h after inoculation); for studies of zoospore lipid, spores were collected about 1 h later. Extraction of lipid. Spore pellets were washed with water, sedi- mented, sonically treated (30 s, 80H), and extracted at room temperature with chloroform-methanol (2:1, v/v) overnight. Additional extractions did not increase yields. The spore homogenate was filtered through a coarse, fritted-glass Buchner funnel, and the filtrate was evaporated to dryness under N2. Nonlipid contaminants were removed with Sephadex 6-25 by the method of Rouser and Fleischer (1965). Column chromatography. Lipids were fractionated into neutral, glyco-, and phospholipids on activated silicic acid (100 mesh, Mallinckrodt Chemical Co., St. Louis, Mo.). Neutral lipids were separated further on silicic acid after removal of fatty acids (Dittmer and Wells, 1969), or on 7% hydrated Florisil (Carroll and Serdarevich, 1967). Glycolipids were separated into individual components with Florisil (Radin, 1969), and phospholipids were fractionated on diethylamino- ethyl (DEAE)-cellulose (Sigma Chemical Co., St. Louis, M0.) by the method of Rouser et a1. (1969). Thin-layer and paper chromatography. Thin-layer chromatography (TLC) was used to check the purity of column fractions. Neutral lipids were separated with petroleum ether-diethyl ether-acetic acid (80:20:1, by volume) by using ITLC-SG chromatography media (Gelman Instru- ment Co., Ann Arbor, Mich.). Phospholipids were chromatographed on the same media with isopropanol-ammonium hydroxide (100:7, v/v); glycolipids were separated with the same solvent on ITLC-SA media. Phospholipids were also chromatographed two-dimensionally on Redi- Coats (Supelco Inc., Bellefonte, Pa.) by using chloroform-methanol- ammonium hydroxide (60:25:5, v/v) in the first direction and chloroform-acetone-methanol-acetic acid-water (3:4:l:l:0.5, v/v) in the second direction. IO Lipid components were visualized with ultraviolet light, I2 vapor, or a saturated solution of KZCrO4 in 70% (v/v) H2504 (Skipski and Barclay, 1969). Specific sprays included SbCl3 for sterols and sterol esters (Skipski and Barclay, 1969), 0.2% ninhydrin in butanol for free amino groups. Dragendorff reagent for the detection of choline-containing compounds (Skipski and Barclay, 1969), and the reagent of Dittmer and Lester (1964) for P. Glycolipids were detected with orcinol (Skipski and Barclay, 1969), phenol-H2504 (Gray, 1965), or diphenylamine (Skipski and Barclay, 1969). Descending chromatography of water-soluble hydrolysis products was carried out on Whatman no. 1 paper. Glycerol phosphate esters were resolved with phenol-water (100:38, v/v) (White and Frerman, 1967), and the phosphate groups were detected by the salicylsulfonic acid-FeCl3 procedure (Yorbeck and Marinetti, 1965) or with acid molyb- date (Hanes and Isherwood, 1949). Glycolipid hydrolysis products were chromatograhed with prropanol-ammonium hydroxide-water (6:3:1, v/v) or ethyl acetate-pyridine-water (12:5:4, by volume; Isherwood and Jermyn, 1951). Ammoniacal AgNO3 was used to detect carbohydrates. The hydrolysis products were also studied by TLC. Phosphate-impregnated Chromagram sheets (Eastman; 6061 silica gel) were prepared, spotted, and, after multiple development, sugars were located thereon, all by the method of Welch and Martin (1972). Hydrolysis procedures. Glycerol phosphate esters were prepared by deacylating the phospholipids in 0.2 N methanolic NaOH for 15 min at room temperature (Kates, 1972). The solution was partitioned against II CHCl3 and the aqueous portion was neutralized with Dowex-50 (H+). The water-soluble hydrolysis products were concentrated almost to dryness under a stream of N2 at 40°C, and used for chromatography. Phospholipids were also deacylated by mild alkaline hydrolysis at 0°C (White and Frerman, 1967). Glycolipids were hydrolyzed in 2 N HCl for 2 h at 100°C. The hydrolysate was extracted three times with petroleum ether; the HCl was removed under a stream of N2 or by drying the samples over KOH pellets. Analytical procedures. Lipid-P was determined after digestion of samples with 10 N H2504 by a modification of Bartlett's method (Bartlett, 1959). Total N was assayed with the micrOprocedure of Sloane-Stanley (1967) or by direct nesslerization with commercial (Harleco dry pack) Folin-Wu (Folin and Wu, 1919) reagent. Acyl esters were estimated by the ferric hydroxamate method (Rapport and Alonzo, 1955) with tripalmitin as a standard. Glycerol analyses (Hanahan and Olley, 1958) were based on the determination of formaldehyde produced by oxidation of glycerol with periodate by using a-glycerol phosphate as a standard. Total hexoses were determined with anthrone (Wells and Dittmer, 1963) or the phenol-sulfuric acid method (Dubois et al., 1956). Glucose was also analyzed enzymatically (Glucostat, Worthington Biochemical Corp., Freehold, N.J.), and total hexosamines were estimated by a modification (Dittmer and Wells, 1969) of the Elson- Morgan reaction. 12 Gas chromatography, Fatty acid methyl esters of the total lipid and the neutral, glyco-, and phospholipid fractions were prepared by saponi- fication and extraction of fatty acids (Dittmer and Wells, 1969); the latter were methylated with BF3 (Metcalfe and Schmitz, 1961). Methyl esters were examined with a Packard gas chromatograph model 7300 equipped with a flame ionization detector. The methyl esters were separated on a column (0.32 by 200 cm) packed with 15% Lac-2R-446 on Chromosorb W (80 to 100 mesh) operated at 187°C. The carrier gas was He; the injector port temperature was 193°C; and the detector temperature was 225°C. The esters were identified by their retention times relative to methyl ester standards. Materials. Phospholipid standards were prepared from egg yolks. The phospholipids were extracted with chloroform-methanol (1:1, v/v), separated from neutral lipids by silicic acid chromatography, and then fractionated on DEAE-cellulose. The phospholipids were purified further by TLC and compared with published data on egg yolk phospho- lipids (Rhodes and Lea, 1957). Some organic reagents and most solvents were redistilled before use. Methyl esters were obtained from Supelco Inc., Bellefonte, Pa. RESULTS Characterization of total lipid in spores produced on solid media. Lipid extracts were fractionated on silic acid columns (Table 1). Neutral, glyco-, and phospholipid accounted for 46.6, 15.8, and 37.6%, I3 TABLE 1. Lipid composition of zoospores produced on solid mediaa Fractionb PercentageC Neutral lipid 46.6 :_2.2 Glycolipid 15.8 :_2.4 Phospholipid 37.6 :_2.5 aPercentages were established gravimetrically after the lipid had been fractionated on silicic acid columns (2 by 8 cm). bFractions were eluted successively with 150 ml of chloroform, 100 ml of acetone, and 150 ml of methanol. CMeans and standard deviations for four experiments. I4 respectively, of the total lipid. The latter constituted 11% of the dry weight of the zoospore. Phospholipid. The phospholipid was fractionated further on DEAE- cellulose, and the purity of each fraction was verified by TLC (Fig. 1). Peak I contained phosphatidylcholine (PC; Rf 0.26; all Rf values listed in this report are average values for many runs) and lysophos— phatidylcholine (LPC; Rf 0.12). Both spots were molybdate positive and gave reactions for choline. Phosphatidylethanolamine (PE; Rf 0.72) and lysophosphatidylethanolamine (LPE; Rf 0.45) were found in peaks II and III, respectively. Both spots were ninhydrin and molyb- date positive, as was phosphatidylserine (PS; Rf 0.00), the only compound in peak V. Peak IV was due to oxidation products of PE. Peak VI contained two components, phosphatidic acid (PA; Rf 0.78) and phosphatidylinositol (PI; Rf 0.25); both spots for peak VI were molybdate positive and ninhydrin negative. The quantitative composi- tion of the total phospholipid is shown in Table 2. The phospholipids were also characterized by determining their molar ratios of Pzacyl esters:glycerol:N (Table 3). Theoretical and actual values agreed closely. The high acyl ester content of LPC was due to contamination with PC. Glycerol phosphate esters obtained by deacylation of the phos- pholipids were examined by paper chromatography. Seven compounds were detected, each one reacting positively to the acid molybdate spray for P. The Rf values of the glycerylphosphoryl derivatives corresponded I5 Fig. 1. Representative elution patterns for milligrams dry weight of lipid (right axis) and micrograms of total P (left axis) obtained by column chromatography of total phospholipid on DEAE-cellulose. The column (2 by 20 cm) was loaded with 1,190 ug of phospholipid-P (obtained by silicic acid chromatography). and 50-m1 fractions (horizontal axis) were collected at a rate of 3 ml/min; 1,163 pg of phospholipid-P (97.7%) were recovered. The elution sequence 1 to 6 (top) was as follows: chloroform-methanol (9:1, vol/vol), chloroform-methanol-acetic acid (7:3:0.002, vol/vol), methanol, chloroform—acetic acid (3:1, vol/vol), acetic acid, chloroform-methanol-ammonium hydroxide (32:8:1, vol/v01). The insert shows the results of TLC of compounds in peaks I to V1, phospholipid being visualized by H2504 charring. Both peaks I and VI contained two phospholipid components. Phosphatidyl choline (PC) and phosphatidic acid (PA) were in the peak fractions for I and VI, re- spectively, whereas lysophosphatidyl choline (LPC) and phosphatidyl inositol (PI) occurred in the corresponding shoulders. 17 TABLE 2. Composition of zoospore phospholipid Component Percentagea Phosphatidylcholine 55.0 :_3.0 Lysophosphatidylcholineb 6.3 :_2.6 Phosphatidylethanolamine 22.1 :_2.6 Lysophosphatidylethanolamine 6.3 :_1.7 Phosphatidylserine 3.0 :_1.0 Phosphatidic acid 3.0 :_1.0 Phosphatidylinositol 4.3 :_1.7 aMeans and standard deviations for three experiments based on the amount recovered after column chromatography. bDetermination by P analysis after preparative TLC of column fractions containing lysophosphatidylcholine and phosphatidylcholine (see insert, Fig. l). 18 TABLE 3. Molar ratios for zoospore phospholipidsa Pzacyl esters:glycerol:N Compound Actual Theoretical PC l.00:l.93:0.96:0.85 l:2:l:l LPC l.00:l.47:0.95:0.97 l:1:1:l PE l.00:2.13:l.09:0.97 l:2:l:l LPE l.00:l.08:l.00:l.13 I:l:l:l PS 1.00:2.00:O.83:0.90 1:2:1:l PA 1.00:1.96:1.0l 1:2:1 PI l.00:2.11:1.00 1:2:1 aThe phospholipids used to establish molar ratios were taken either from column fractions (where purity was verified by TLC) or directly from TLC plates. 19 with published data (Kates, 1972) and those for our standards prepared from egg yolks. Two compounds (glycerylphosphorylserine and glyceryl- phosphorylethanolamine) were ninhydrin positive; two inositol-containing glycerylphosphoryl esters were detected. Glycolipid. This lipid class was separated into five fractions on Florisil; seven different spots were derived therefrom by TLC (Fig. 2). Fraction I contained one component (Rf 0.86) which represented 10% of the total glycolipid as determined gravimetrically. Fractions II and III also contained one component each, both having an Rf of 0.55; they represented 38 and 32% of the total glycolipid, respectively. Another 12% of the glycolipid was present in fraction IV in the form of three components with Rf values of 0.31, 0.20, and 0.08. Fraction V had one component (Rf 0.88); it represented 8% of the glycolipid. Results obtained with spray reagents suggested that all the fore- going substances were orcinol and diphenylamine positive except IVa and IVb; the latter were weakly orcinol and molybdate positive. The components in fractions II, III, and IVa were also ninhydrin positive. The glycolipid fraction (15.8% of the total lipid [Table 1]) contained 6.8% of the P in the total lipid extract. Analyses by phenol- sulfuric acid and anthrone methods, with glucose as a standard, suggested that 23% of the glycolipid was carbohydrate. However, judging by enzymatic assays with glucose oxidase, only 6% of this carbohydrate was actually glucose. Thin-layer and paper chromatography of the acid hydrolysis products indicated that, in addition to glycerol, several substances that behaved 20 .I.‘ II III IV V 2...: 0c: ‘4‘“ Fig. 2. Glycolipid fractions collected by Florisil column chromato- graphy (1 by 15 cm) and resolved further by TLC. Fractions I to V were obtained by successive elutions with 50 m1 of chloroform-acetone (1:1, vol/vol), acetone, 95% acetone, 90% acetone, and methanol. The chromato- grams were visualized by H2804 charring. 21 like carbohydrates were present in the glycolipid. Fractions I and V appeared to contain monoglycosyldiglycerides judging from Rf values (Fig. 2) and the fact that each released only one carbohydrate after hydrolysis and TLC. Fractions II and III behaved like diglycosyl- diglycerides chromatographically (Fig. 2), and each released two components, one of them being ninhydrin positive. Three carbohydrates were produced by Fraction IV, one of which was ninhydrin positive. Neutral lipid. This group of lipids was resolved by TLC into 13 spots (Fig. 3). Neutral lipid was also column fractionated through two different media (Table 4), both yielding comparable results when assayed gravimetrically and monitored by TLC. The FFA, whether removed before fractionation on silicic acid or fractionated directly on Florisil, constituted 8% of the total neutral lipid. They migrated to or near the solvent front, as did the hydrocarbons, which accounted for another 13 to 14%. Triglycerides (TG; Rf 0.89) made up the major class (28 to 30%), whereas free sterols (FS; Rf 0.84) and sterol esters (SE; Rf 0.79 and 0.73) constituted 12 to 13% and 15 to 18%, respectively. Both FS and SE reacted positively to the SbCl3 spray. Diglycerides (DG) represented 8 to 9% of the neutral lipid and contained four components (Rf 0.65, 0.58, 0.45, and 0.38), whereas monoglycerides (MG; Rf 0.30, 0.22, 0.09, and 0.00) represented 12%. Fattypacid composition. Zoospores contain at least 20 fatty acids. The principal ones (Table 5) were palmitic, palmitoleic, stearic, oleic, y-linolenic, and arachidonic acids. Nineteen fatty acids were 22 FFA, HC *TG FS SE Fig. 3. Components in the neutral lipid fraction separated by TLC and visualized by H2504 charring. Neutral lipids are, from top to bottom: free fatty acids (FFA), hydrocarbons (HC), triglycerides (TG), free sterols, (FS), sterol esters (SE), diglycerides (0G), and monoglycerides (HG). 23 TABLE 4. Composition of zoospores neutral lipida Percentage Fraction By silicic By Florisil acid Hydrocarbons 14.0 _+__2.0b 13 Free fatty acids 8.3 :_1.1 8 Triglycerides 29.8 :_2.1 28 Free sterols 12.3 :_l.7 l3 Sterol esters 15.0 :_1.0 18 Diglycerides 9.0 :_l.4 8 Monoglycerides 11.6 i 1.0 12 aDetermined gravimetrically after fractionations on either silicic acid or 7% hydrated Florisil. bMeans and standard deviations based on three determinations. 24 TABLE 5. Principal fatty acids in zoospore lipid Percentage of fatty acid in Fatty acida Total Neutral Glyco- Phos- 1ipid lipid lipid pholipid 16:0 (palmitic) 28 19 38 39 16:1 (palmitoleic) 4 4 5 3 18:0 (stearic) 9 9 3 2 18:1 (oleic) 32 35 23 14 18:3 (y-linolenic) 12 6 18 23 20:4 (arachidonic) 7 5 0 13 aThe first and second numbers represent length of carbon chain and number of double bonds, respectively. 25 detected in the neutral lipid fraction, palmitic and oleic acids account- ing for over 50% of them. Eight fatty acids were found in the glycolipid fraction, palmitic, oleic, and y-linolenic being the major ones, and arachidonic being conspicuously absent. The phospholipid fraction con- tained 12 fatty acids of which the major ones were palmitic, oleic, y—linolenic, and arachidonic. Lipid composition of sporangia and zoosppres produced in liquid media. The proportions of neutral, glyco-, and phospholipid in zoospores de- rived from liquid cultures were very different than those for zoospores from agar media (Table 6, column 3 versus Table 1), even though the total lipid in the two kinds of zoospores was the same, i.e., about 11%. On the other hand, the proportions of these three lipid classes were about the same in the mature (i.e., post cleavage) zoospore-producing parent plants (column 2, Table 6) as they were in the zoospores themselves (column 3, Table 6); however, the lipid composition characteristic for both zoospores and postcleavage plants was different than that of pre- cleavage plants (column 1, Table 6). These conclusions were substantiated by the distribution of radioactivity in the three lipid classes (columns 4, 5, and 6, Table 6) derived from precleavage plants, postcleavage plants, and zoospores from liquid cultures containing (140) acetate. The three lipid classes derived from zoospores also resembled those from postcleavage plants when characterized further by TLC. The results obtained by similarly separating the labeled neutral, glyco-, and phospholipid via TLC and then scanning the chromatograms 26 TABLE 6. Composition of lipid in plants and zoospores produced in liquid mediaa Composition Distribution of by weight (%) 14c (%) Fraction Pre- Post- Pre- Post- cleav- cleav- Spores cleav- cleav- Spores age age age age plants plants plants plants Neutral lipid 17.7 25.2 27.2 16.0 23.2 29.4 Glycolipid 13.1 12.0 12.4 15.3 11.7 11.6 Phospholipid 69.2 62.8 60.4 68.7 65.1 59.0 aTotal lipid was extracted and separated as before silicic acid columns. b Counted with a Tracerlab Versa/matic scaler. (see Table 1) on 27 for radioactivity are also delineated (Fig. 4). Most of the radio- activity in the neutral lipid fraction was associated with TG (A-I). FS (A-II) and SE (A-III) were also major components, whereas small amounts of label were associated with the MG and DG. Monoglycosyl- diglyceride (B-I) and diglycosyldiglycerides (B-II) were the major components labeled in the glycolipid fractions; peaks B-III and B—IV are mixtures of glycolipids that were not resolved completely in this solvent system. PC (C-III) and PE (C-I) were the major labeled com- pounds in the phospholipid fraction, LPE (C-II) and LPC (C-IV) also being present. Minor phospholipids were PA, PI, and PS. DISCUSSION The wall-less, motile zoospores of B, emersonii possess an elaborate, tightly organized, membrane—bound corps of organelles; one especially prominent component is a cluster of discrete lipid globules partially wedged into a structure which, until its function and chemical composi- tion has been at least partially characterized, is being called "SB matrix" (Truesdell and Cantino, 1971). These zoospores can develop along four different macrocylic pathways (Cantino, 1966), as well as a microcyclic pathway (Hennessy and Cantino, 1972), thereby producing plants which give rise to new generations of swarm cells. Against this background, we wish to comment briefly about the extractable lipid produced by this fungus. Both zoospores and sporulating cells contain neutral lipids, glyco- lipids, and phospholipids, which vary in amount and relative proportions 9 a. CO I! ugfl Fig. 4. Separable components in the 14C-labeled neutral lipid (A), glycolipid (B), and phospholipid (C) extracted from postcleavage plants of B, emersonii and resolved by TLC, and the profiles for distribution of radioactivity in these components. The arrow (in A) indicates a shift to a 2.5-fold reduction in amplification of the signal; 0 represents the origin. Tracings were made with a Tracerlab 4-pi scanner. 29 depending on the developmental stage. Additionally, we have shown in this report that the proportions of these three lipid classes in 200- spores derived from plate-grown cultures of ordinary colorless (0C) plants differed from the corresponding amounts in zoospores derived from liquid cultures; this observation is consistent with accounts showing that other microorganisms also change in lipid composition as they adjust to different environmental conditions (Bowman and Mumma, 1967; Kostiw et al., 1972). With B, emersonii, the temperature, pH, and composition of the media used for the two kinds of cultures were similar; however, population densities-especially when considered in relation to the amounts of oxygen apparently available-were quite different. We think this difference may account for the corresponding dissimilarities in lipid composition of the zoospores; our reasoning is as follows. We observed (Table 6), as did Smith and Silverman (1973), that there was about 45 to 50% more neutral lipid in zoospores than in sporulating plants when both were derived from liquid cultures. The transition (pre- cleavage plants + postcleavage plants + free swimming zoospores) was associated (Table 6) with a shift in neutral lipid from 16.8% through 24% to 28.3%, respectively. When grown in well-aerated liquid cultures, 00 plants increase exponentially in weight up to about the time of sporogenesis (Goldstein and Cantino, 1962; Khouw and McCurdy, 1969); the corresponding increases in respiration and other evidence for oxidative activity (Khouw and McCurdy, 1969; McCurdy and Cantino, 1960), including the reutilization of lactic acid (Cantino, 1965), suggest 30 that there was a continuously increasing demand for 02 up to the end of their generation time. In plate cultures, on the other hand, neither forced aeration nor agitation was employed; the demand for oxygen may have existed, but its availability was undoubtedly restrict— ed by various diffusion processes and perhaps other factors; hence, the average 02 uptake per plant was probably very much reduced, whereas lactic acid simultaneously accumulated in the medium. This information and the fact that the population density of the plants in plate cultures (ca. 2 x 105 thalli/m1 after submersion in flooding water) was about five times greater than that in liquid cultures (ca. 0.4 x 105 plants/ml) lead us to suspect that exogenous oxygen deficiencies and corresponding shifts toward fermentative metabolism prior to and during sporogenesis may have been responsible, at least in part, for the increased proportion of neutral lipids in B, emersonii grown on solid media. The composition of the extractable lipid in spores derived from plate cultures was also different than that in spores derived from liquid cultures. In the former, the major lipids were PC (20.3%), TG (13.5%), and diglycosyldiglycerides (DGDG; 13.3%), whereas PE, FS plus SE, and lysophosphatides (LP) accounted for 6.7, 12.2, and 4.9% of the lipid, respectively. In the latter, PC and PE were the most abundant components, and lesser amounts of TG, DGDG, LP, FS, and SE were also present, the phosphatides constituting 62% of the total lipid. These results are consistent with the phospholipid contents of zoospores de- rived from liquid cultures as reported by Smith and Silverman (1973) and Suberkropp and Cantino (1973), i.e., 55% and up to 85% of the 31 extractable lipid, respectively. The latter value is not out of line because it was unquestionably inflated by its inclusion of glycolipids; the fractionation techniques used at that time separated the total lipid into only two classes, neutral lipids and polar lipids. Although the quantity of sterols and SE in the zoospores of B, emersonii seems to exceed the amount in most other Phycomycetes (Weete, 1973), the fatty acid composition of the B, emersonii zoospores does resemble that of other phycomycetes (Bowman and Mumma, 1967; Chenouda, 1970; Gordon et al. 1971; Shaw, 1966; Sumner and Morgan, 1969). In addition, y-linolenic acid---once thought (Shaw, 1966) to be both characteristic of and limited to the phycomycetes, but recently found (Safe and Brewer, 1973) to occur in other fungi---was associated predominantly with polar lipids in the zoospores we analyzed, as it was in the mixture of variously aged thalli harvested from multiple generation of B, emersonii cultures by Sumner (1970). However, the average degree of saturation among the fatty acids we extracted from zoospores was greater (an approximate estimate can be derived from the data in Table 5) than the average value obtained by Sumner (1970) for plants. This apparent difference between spores and plants of B, emersonii could, of course, be due simply to differences in culture conditions rather than stages in ontogeny; on the other hand, our results do agree with Sumner's (1970) observations in that more polyunsaturated fatty acids were associated with polar lipids than with neutral lipids. The glycolipid in B, emersonii seems to be of an unusual nature. It is also the most homeostatic of the three lipid classes, apparently 32 being stabilized at a level of about 12 to 15% of the total lipid what- ever the developmental stage -- whether the glycolipid is derived from zoospores or sporangia, mature or immature, or extracted from plate-grown or liquid-grown cultures. This conclusion contrasts sharply with the results of Smith and Silverman (1973), who concluded that glycolipid accumulated during sporulation. Although they did not specify how many spores were released by their sporulating plants, the information provided suggests that they may have been microcyclic plants similar to the uni- spored plantlets studied by Hennessy and Cantino (1972). The physiologi- cal changes occurring after induction of sporogenesis in lag-phage (microcyclic) germlings and log-phase (macrocyclic) plants are known to show similarities but also some differences (Hennessy and Cantino, 1972). Perhaps the latter includes lipid composition. Our unpublished data lead us to believe that the glycolipids are associated with specific organelles in B, emersonii, which could account for the stability of this class of lipids. A more detailed study is under- way to identify the nature of these glycolipids and their possible rela- tionships to certain functional aspects of the zoospore of B, emersonii. II Lipid Changes During Development of Blastocladiella emersonii Fungi accumulate lipids (Cochrane, 1958) in the form of globules that probably serve as reserves (Walker and Throneberry, 1971). The zoospores and plants of the aquatic fungus Blastocladiella emersonii Cantino and Hyatt also contain lipid bodies (Cantino and Truesdell, 1970; Lessie and Lovett, 1968; Lovett and Cantino, 1960). Under conditions of starvation these zoospores utilize stored polysaccharides and lipid (Suberkropp and Cantino, 1973). Recently the lipid components have been identified and quantified (Mills and Cantio, 1974). The present report documents changes in lipid classes and their individual components during different develop- ment stages of B, emersonii. MATERIALS AND METHODS Culture techniques. Blastocladiella emersonii was grown at 22°C in 9 l. modified PGY broth (Myers and Cantino, 1971). The culture, inoculated with 2.5 x 108 - 4.5 x 108 spores obtained from PYG agar plates, was aerated (10 1./min) and illuminated with "cool white" fluorescent lighting, 555-712 uW/cmz. After 9 h of growth 50 uCi of NaOAc-[U-14C], (54 mCi/— 4 M. Growth was mM) and NaOAc were added to give a final concn of 5 x 10- continued for an additional 9 h at which time aeration was stopped, the plants were allowed to settle, and the spent medium was removed by suction. The plants were washed once with 4 l. of sporulation inducing medium (0.5 mM MOPS [Calbiochem], pH 6.8, containing 0.1 mM CaClz) and resuspended in 33 34 IO 1 1. of the same solution; aeration was resumed. From 10 to 3.5 x 1010 spores were synchronously released 6-7 h later. Suspensions of zoospores were passed through filter paper and con- centrated by centrifugation (0 h spores) or they were treated in one of two ways: (a) the spores were allowed to swim in the aerated buffered medium for 5 to 10 h or (b) they were induced to encyst by chilling to 4-6°C (avg. time required to reach temp., 45 min). Then PYG broth was added immediately to full strength and equilibrated to 22°C. Approximately 95% encystment occurred within 15 min, this being faster than in most non- nutrient systems (Truesdell and Cantino, 1971). The cells were then ex- tracted immediately or after 5 h of growth (germlings with mainly two nuclei). Lipid extraction. Zoospores, cysts and germlings were concentrated by cen- trifugation (100 xg for 7 min), washed with H20 and then recentrifuged. The pellets were suspended in 2:1 CHCl3-Me0H (19 vol. solvent/vol. material), sonicated at 80 W for 30 sec, and extracted overnight at room temp. The extract was filtered through a coarse fritted glass BUchner funnel, evaporat— ed to dryness under N2, and suspended in 5 ml CHCl3-Me0H (19:1). Non-lipid contaminants were removed with Sephadex G-25 (Rouser and Fleischer, 1965). Chromatography. The lipid was then applied to a 2 x 8 cm silicic acid column and separated into classes. Neutral lipids were eluted with 100 m1 CHCl glycolipids with 100 m1 MeZCO and phospholipids with 100 m1 MeOH. 39 Each class was taken to dryness under a stream of N2 at 40° and resuspended in 2 m1 CHCl -Me0H (2:1). The fractions were then used for dry wt 3 35 determination, TLC, and the measurement of total 14C incorporated. Neutral lipids were separated on Gelman ITLC-SG chromatography media, using light petrol-EtZO-HOAC (80:20zl). Glycolipids and phospholipids were chromato- graphed on ITLC-SA and ITLC-SG media, respectively, using iso-PrOH-NH40H (100:7). Lipids were located by using H2304 and heat and identified by comparison with previously identified B, emersonii spore lipids (Mills and Cantino, 1974). Quantitative data for individual components were determined from total peak areas obtained from scans (Tracerlab 4 Pi scanner) for radio- activity in uncharred TLC's. RESULTS Radioactivity from NaOAc-[U-14C] was incorporated into the lipids of synchronously produced zoospores (Fig. 5, 0 h). Following fractionation into neutral lipids, glycolipids and phospholipids, the percentage of 14C incorporated each class was determined by both dry weight and total (Table 7, 0 h). Individual classes were separated by TLC (Fig. 6, 0 h) and the major lipid components were identified by techniques used earlier (Mills and Cantino, 1974) and quantified by measuring their total radio- activity (Fig. 6, 0 h). The T6 and FS were the major neutral lipids, SE and MG being minor components with undectable label. The major glycolipid was DGDG, while the minor glycolipids were MGDG and PGDG. The phospholipid fraction contained two major peaks; one consisted of PC and LPC, the other contained PE and LPE. Minor phospholipids were PS and PI. These data were then compared with the results obtained from zoospores which had been swimming for 5 and 10 h, from encysted spores, and from 5 h germlings. 36 O u .. -II II - -1 SI .... 5. '= :' 0 e p- - p l I II it II "81 “I'll“ Fig. 5. Changes in total (o---o) and labeled (o---o) lipid/1010 cells during swimming and development. Lower curves, swimming spores; upper curves, encystment and growth. 38 N.mn o._m o.m~ m.m m.¢_ m.mp mewpscmm 0.0“ m._u ¢.w m.m o.—N «.mP pmAU “.mm o.om m.¢m N.¢N o.mm m.mm ; o_ m.mm m.mo _.m_ o.mF m.__ m.m~ ; m m.Pm m.~m N.n_ m.NF m.P~ m.m_ ; o Ego p3 xeo Ego p3 xgo Ego p3 ago wmmpm eeee_eeeme;a eeawpeoxew eeaep Feeoeoz mg» we #3 Ace >5 vmmmmmmm mm pcmsQoFm>wu new mcwssezm mzwczu anwF mo copuwmonsou acousmn och .moepisuiowuumowumc eo cowumcoacoucp use mFqu .m m4mmgmu m.o m.o we 1 u . Ne mco: o._-m.o uwawb mmuxEoLmzuumm mnmp umFQoLu ..Fm pm mcwumzmsmp um; wpznopm m.N mung» mco: ¢.N mcoc mmumcp m.¢m mcoc m.P-¢.o cwawb .wcomgmsm am 4a qua mm mm a: we we mcmgnswz Aggy w~_m mEmz .wmm w mugaom cowpwmoquu & muwaWF ummeomv cmcpo saw: wwcomgmsm am Eocw mm—znoFm vwawF ummeomm mo comwgmaeou .op m4m<~ 58 _.nm N.NN u m.~ m.— w.o o.m o.m N.N m.~ m.o o.F o.F money «.0 m.o P.F ¢.N m.mm 8mm m.mm om acmmmgq mm.o-m_.o mmsomoawb mu?gmuxpmwgu - o.N-m.o m:_bmopa ucmmmca o.m-o._ meomogmcam mFQPmeQ cowguuwawp pcmmmga m.F-m.o owEmmpaopxU mom, .wvgmnEOA w scarcum em>._ “mm anP ..Pm pm colgmm tm>wp 8mm Romp ..Po um mxumn mcoumpapoo “gamma comp ..Fm pm mw~cmxomz Lm>w_ panama A.u.b=ouv o_ m_nm» 59 m.~m N.N m.m m.m m+ o.m n.¢ o.~ m.m m.¢m «.mm mm mmPUWmm> avg?— ucmmmca _.o mpwmoaeou mmpuwmm> “commas o.m-m.o ULQLP m_aswm mpznopm “Camden o.N-o.F __o _Nmp .cmHBOH w gmamscmppoz FNm_ ..Pm pm cmpp< mcoumpxuou coma swam Fump .cmpuop w gmzmgcwppoz _Nmp ..Fm um cmp_< mcoumquou camp swam mnmp ..Fm pm mxz~3 Fmemem mmuxgomwb A.v.u:oov op mpnwh 60 .mmo mm nwaw_o;amo;a mo pgmwmz cmrzmmFOE mmmgm>m cm mam :wmpoca Em P mewE=mmm cm>wm mowpmg sogm umpm_:m_mo$ .mem_ uwamp mo _E\mn op op u:m_m>*:cm ucm ucmmmga mm: cwmpogmm .xuv>wummowumg mo covpsawgpmrv :o mmmmmu umcwneou mgmz meowpmmgm we mam who .mmvemmemwgp »_:wms “an .uPQPF Fmpom mm umumoamgn .:m>wm mm=Fm> o: pan pcmmmgam A.u.p=ouv o, m_amp IV The Single Microbody in the Zoospore of Blastocladiella emersonii is a 'Symphyomicrobody' 'Microbodies' which have been found in most major groups of organisms (DeDuve, 1969), display some or all of the following features (Mollenhauer et al., 1966; Frederick et al., 1968; Hruban and Rechcigl, 1969): a round to elongate structure 0.3 u to 1.5 u in diameter and delimited by a ca. 60 A - 80 A membrane; a close association with endoplasmic reticulum or lipid bodies; a matrix of moderate electron density in which electron opaque inclusions; crystalloid structures, or both may be embedded. Micro- bodies have also been categorized physiologically as either peroxisomes or glyoxysomes (DeDuve, 1969; Tolbert, 1971). Peroxisomes play a role in glycolate metabolism and photorespiration, and produce and degrade H202 (DeDuve, 1969; Tolbert, 1971). Glyoxysomes, frequently associated with lipid bodies (Vigil, 1970, 1973; Mendgen, 1973; Silverberg and Sawa, 1973; Trelease et al., 1974), are involved in converting fats to carbohydrates (Beevers, 1969; Tolbert, 1971). In recent years, workers have labeled various particles in the zoospores and sporangia of aquatic fungi as microbodies. However, none of them has published evidence for the origin, fate, or enzyme content of such organelles, nor any other significant supporting evidence that they are truly microbodies. In this report, we provide ultracytochemical evidence for the occurrence of microbodies in zoospores and sporangia of the fungus Blastocladiella emersonii Cantino and Hyatt. We also present arguments for the method of origin of a novel microbody, here named 61 62 the 'symphyomicrobody', and discuss its possible function in the zoospore and its fate after encystment. MATERIALS AND METHODS B, emersonii was cultured at 22°C in the dark on Difco PYG agar in standard Petri plates after inoculation with ca. 4 x 105 spores. The subsequent first generation of zoospores was pre-fixed on the plates for 10 min with 0.5% glutaraldehyde (GTA) in 0.1 M sodium cacodylate (SC) at pH 7.2, filtered, centrifuged (500 x g, 5 min), and fixed (2 h, 4°C) with 2% GTA in SC. Zoosporangial plants were grown at 22°C in PYG broth (Myers and Cantino, 1971), collected (500 xg, 3 min) at the time of papilla formation (ca. 22 h after inoculation), and fixed (1 h, 22°C) with 2% GTA in SC. Zoospores and sporangia were rinsed thrice with SC, pre- incubated in 0.05 M 2-amino-2-ethyl-l,3 propanediol buffer (AEP), pH 10, for 30 min, and then incubated at 37°C for 60 and 90 min, respectively, in the complete reaction mixture: 5 ml 0.05 M AEP, 0.1 ml 3% H202, and 10 mg 3,3'-diaminobenzidine 4 H01 (DAB). The final pH was adjusted to 9. Cells were also incubated in complete reaction mixtures containing 0.01 M KCN, and in control mixtures without H202. After incubation, cells were rinsed for 30 min in 0.05 M AEP (pH 9), rinsed thrice in SC, postfixed in 2% 0504 (l and 2 h, respectively; 22°C), again rinsed thrice in SC, dehydrated through a graded series of alcohol followed by propylene oxide, and then embedded in Spurr's low viscosity resin (Spurr, 1969). Thin sections were examined with a Philips 300 electron microscope. RESULTS AND DISCUSSION The posteriorly uniflagellated zoospores of B, emersonii contain a 63 Fig. 9. The relationship of the symphyomicrobody---lipid complex to other organelles in the side body of the Blastocladiella emersonii zoospore. Lipid granules (L) are molded against the symphyomicrobody (SB), a spoon-bowl shaped organelle with an irregularly wavy rim. The $8 lies next to the long 'arm' of the mitochondrion (H), usually in intimate contact with it. A sheet of double membrane (the backing membrane; BM) covers the SB-L-M complex, and is 'attached' at several places (e.g., as at X), i.e., is continuous with, the outer unit membrane around the nucleus and its nuclear cap. All of this is surrounded, in turn, by the plasma membrane (PM) of the zoospore. The single flagellum is also shown. 64 nucleus, a nuclear cap, gamma particles, and a side body complex (Cantino et al., 1963; Cantino and Truesdell, 1970); the latter consists of a single mitochondrion, lipid granules, and the SB matrix (Fig. 9). The SB matrix resembles a microbody in some respects; it is unit membrane limited, somewhat electron opaque, and always closely associated with lipid bodies---a common characteristic of glyoxysomes. But, in comparison with a typical microbody, the SB matrix is gigantic (although this could conceivably be misleading in that its size is based on a three dimensional model derived from studies of serial sections [Cantino and Truesdell, 1970] whereas that of most other microbodies has been based on two dimen- sional measurements of random thin sections). Another difference stems from the fact that microbodies are frequently associated with endoplasmic reticulum (Vigil, 1973), while the zoospores of B, emersonii do not possess an endoplasmic reticulum (Cantino et al., 1963). Microbodies have been identified by the use of ultracytochemical techniques. Although many enzymatic activities have been used as 'markers' for microbodies, only catalase apparently occurs in almost all of them (DeDuve, 1969). It can be demonstrated ultracytochemically (Novikoff and Goldfischer, 1968; Frederick and Newcomb, 1969), and its presence has been used to establish the presence of microbodies in various organisms (Beard and Novikoff, 1969; Frederick and Newcomb, 1969; Vigil, 1970; Matsushima, 1972; Stewart et al., 1972). We have applied the procedure of Beard and Novikoff (1969) to B_emersonii zoospores. Incubation in the DAB/H202 medium produced a very dense deposit over the SB matrix (Fig. 10), but not elsewhere. The reaction did not occur when H202 was absent, and it was partially inhibited by 0.01 M KCN. It appears, 65 Fig. 10. The response of the SB matrix in zoospores of B, emersonii to the Beard and Novikoff DAB test for catalase activity. The reaction product is localized in the SB matrix (solid arrows, top and bottom left) of the test spores, but not in the control spores (broken arrows, top and bottom right). Bar = 1.0 u. 66 67 therefore, as if the SB matrix contains catalase, and that it may be a sort of microbody. If the SB matrix is a microbody, what is its origin? The genesis of microbodies has been associated with endoplasmic reticulum (Matsushima, 1972; Vigil, 1973; Silverberg and Sawa, 1973; Richardson, 1974). The zoospores of B, emersonii do not have endoplasmic reticulum; it is present, however, in developing sporangia, and therein it is closely associated with numerous, small, irregularly shaped, single membrane bound particles of moderate electron density labeled 'sb granules' (Lessie and Lovett, 1968). When we tested 8, emersonii sporangia for catalase ultracyto- chemically in an attempt to locate possible precursors of the SB matrix, only the sb granules gave a positive reaction (Fig. 11). Lessie and Lovett (1968) showed that these granules, along with lipid bodies, surround the nucleus during sporogenesis, and that both sb and lipid then become aligned alongside the mitochondrion in the finished zoospore while they are still inside the sporangium. Since it has also been shown (Cantino and Truesdell, 1970) that, in free swimming zoospores, such sb granules are interconnected into a single, large, unit membrane bound 58 matrix (Fig. 9), it is now reasonable to conclude that the SB matrix is formed by 'symphyogenesis'---i.e., that it is produced by the union of formerly separate elements (Fig. 12). And, since we herewith provide evidence that the SB matrix possesses catalase activity, and that it is the only organelle in the zoospore that does so, the SB matrix can properly be called a 'symphyomicrbody'. The building blocks from 68 ~. .3:- -..~ at 'H- e. I 1. u‘ . _ "t . a I " ',‘~ 3}}? ' ... ' k . . . ,, _, _ » I It ‘ . \J I 54. " p Hzflé ' A .0 ' l-l‘. . , . ’5': .;4. - _ . .. ‘ '3 “:Vk‘ " . . v v’ .. ., . 4 J 5. C -. 5 ; . , l . 'n Fig. 11. Sections through sporangia at sporogenesis (ca. 22 h. after inoculation). Arrows point to the 'sb granules'. Catalase positive reactions are seen at top and bottom left, and negative reactions in controls at right top and bottom. Bars = 0.5 u and 0.1 u. top and bottom respectively. 69 LIPID BODIES MICROBODIES 000 .5 formerly ‘sb C granules' 900 .5: SYMPHYOMICRO BODY I/formerly '88 matrix' T SYMPHYOMICROBODY- Ll PID c. ‘.P Gill? Fig. 12. The genesis of the symphyomicrobody-~1ipid complex in Blastocladiella emersonii. See text for details. 70 which it is constructed, i.e., the catalase positive sb granules of Lessie and Lovett (1968), are therefore microbodies. What is the role and fate of the symphyomicrobody? The symphyomicrobody is closely associated with lipid particles and the mitochondrion; perhaps, therefore, it functions as a glyoxysome. Glyoxysomes play a central role in gluconeogenesis by virtue of their capacity to burn up fat and mediate the glyoxylate cycle ng_isocitrate lyase and malate synthetase, thus bringing about net synthesis of succinate which presumably finds its way to mitochondria where it is further metabolized (Beevers, 1969; Tolbert, 1971; Richardson, 1974). A key point in this operation is the fact that continuous activity of glyoxysomes requires the cooperation of outside enzymes (MUller et al., 1968; Beevers, 1969); therefore a metabolic inter- play ng_movement of C4 and C6 molecules back and forth is thought to occur between glyoxysomes and mitochondria. Isocitrate lyase activity was detected long ago (McCurdy and Cantino, 1960) in B, emersonii zoospore extracts; our work in progress has verified its presence, as well as that of malate synthetase, in spore populations. Preliminary data suggest that isocitrate lyase activity is associated with a particulate fraction of buoyant density 1.28 g/cm3 on a sucrose gradient. However, there is not yet enough information available about other enzyme activities and metabolic properties of the symphyomicrobody of the zoospores of B, emersonii to establish if this giant organelle should be considered a glyoxysome or peroxisome or both---or, neither one. 71 If the symphyomicrobody behaves like a conventional glyoxysome, it presumably functions while a zoospore is swimming rather than during its encystment. Zoospores lose 64% of their triglycerides after 5 h of swim- ming, but there is no significant decrease in lipid when they encyst (Mills et al., 1974). Also, during swimming the symphyomicrobody decreases in size (Suberkropp and Cantino, 1973) whereas upon encystment it becomes somewhat branched (established from serial sections of zoospore populations induced to encyst synchronously; Cantino et al., unpublished data). Little is known directly about the fate of the symphyomicrobody after germination. However, if isocitrate lyase were associated exclusively with the symphyomicrobody in the B, emersonii zoospore and with microbodies at other stages of its life history, then the available analytical data (McCurdy and Cantino, 1960) for total isocitrate lyase per plant could mean that new generations of microbodies are not produced until some 3-6 h after a zoospore has germinated. However, electron microscopic evidence to support or refute this possibility is not yet available. The occurrence of microbodies and side-body complexes in other aquatic fyggj, The three-dimensional shape of the symphyomicrobody in B, emersonii was established (Cantino and Truesdell, 1970) from studies of serial sections. Such serial section evidence has not been published, to our knowledge, for the shape of microbodies in other aquatic fungi (however, in this connection see Olson, 1973, below)---nor, for that matter, in other organisms. Furthermore, the size of the B, emersonii symphyo- microbody, as already emphasized (Suberkropp and Cantino, 1973), depends upon how long a zoospore has been swimming; similarly, this has not been established for microbodies in other aquatic fungi. 72 Within the past few years structural aggregates resembling either the whole side body complex in B, emersonii (i.e. a symphyomicrobody [SB]-1ipid-mitochondrial complex bound by a backing membrane) or just the SB-lipid combination, have been seen in the zoospore of Blastocladiella britannica (Cantino and Truesdell, 1971) and other Chytridiomycetes. Especially noteworthy among the latter is the side body complex in the zoospores of Coelomomyces punctatus (Martin, 1971) which greatly resembles that in Blastocladiella emersonii. A somewhat comparable arrangement apparently occurs in two other species, Coelomomyces psorophorae (Whisler et al., 1972) and B, indicus (Madelin and Beckett, 1972). The latter authors also report a sequence of events in the formation of the side body complex in the sporangia of Coelomomyces similar to that which occurs during sporogenesis in Blastocladiella emersonii. A side body complex is also said to occur in the zoospores of Catenaria anguillulae (Chong and Barr, 1974). Similarly, an arrangement (made up of profiles termed 'StUben bodies') resembling a side body complex has been observed in zoospores of Allomyces (Fuller and Olson, 1971); 'StUben bodies' also reportedly occur in Phlyctochytrium arcticum (Chong and Barr, 1973). Recently, Olson (1973) concluded that the side body complex in the meiospores of Allomyces has a three dimensional organization resembling the one in Blastocladiella emersonii. Zoospores of Rozella allomycis also contain aggregates of "several lipid globules, a folded backing membrane, and a microbody" (Held, 1973), and microbody lipid complexes are said to occur in species of Entophlyctis and Rhizophydium (Chong and Barr, 1974). Finally, Harpochytrium hedinii zoospores contain 73 (Travland and Whisler, 1971) a tight association of lipid globules, a single rumposome, and profiles of electron dense granular material re- sembling sections through the SB-matrix in Blastocladiella emersonii. Although the various profiles seen thus far in thin sections through fungal zoospores, as outlined above, resembles microbodies on structural grounds, supplementary evidence of a functional nature is now highly desirable. Furthermore, it will be most interesting to find out how many of them are formed by the process of symphyogenesis, and whether or not any of them decrease in size while zoospores are swimming as does the symphyomicrobody of B, emersonii, for these are characteristics that we have not encountered in the literature on microbodies. From these latter two points of view in particular, the B, emersonii symphyomicrobody apparently encompasses something more than that circumscribed by today's stereotype of the glyoxysome. However, if the symphyomicrobody is a gly- oxysome, then the manner in which it is placed in the side body complex - in intimate contact with both the single mitochondrion, on the one hand, and the spore's total complement of lipid globules, on the other---illus- trates what appears to be an exceptionally favorable arrangement for carrying out the supposed mission of a glyoxysome. V Isolation and Characterization of Microbodies and Symphyomicrobodies with Different Buoyant Densities from the Fungus Blastocladiella emersonii Since the discovery of the functional nature of mammalian and leaf peroxi- somes (DeDuve, 1966; Tolbert and Yamazaki, 1969) and glyoxysomes (Brei- denbach and Beevers, 1967), there have been many publications on micro- bodies; they are summarized in a symposium (Hogg, 1969), a book (Hruban and Rechcigl, 1969), and reviews (Tolbert, 1971; Vigil, 1973; Richardson, 1974; Frederick et al., 1975). Plant microbodies, extensively charac- terized morphologically and biochemically (Vigil, 1973; Frederick et al., 1975), have been classified as glyoxysomes, peroxisomes and "unspecialized microbodies" (Huang and Beevers, 1971; Huang and Beevers, 1973; Huang, 1975). In contrast, descriptions of fungal microbodies have been far more limited (Frederick et al., 1975), identifications being based mainly on fine structural criteria. Analyses of isolated microbodies involving more than one enzyme have been limited to studies with Saccharomyces (Szabo and Avers, 1969), Neurospgra (Kobr et al., 1969) and Coprinus (O'Sullivan and Casselton, 1973). The zoospores and zoosporangia of the water mold Blastocladiella emersonii (Cantino and Hyatt, 1953) contain two kinds of catalase posi- tive organelles, symphyomicrobodies and microbodies (Mills and Cantino, 1975). They are ontogenetically related in that several microbodies 74 75 give rise to a single large microbody by symphyogenesis. The purpose of this report is to provide statistically circumscribed data about the enzymatic activities of isolated symphyomicrobodies, and biochemical and physical supporting evidence for the method of origin of this unusual organelle. MATERIALS AND METHODS Synchronous cultures of B, emersonii were grown essentially as described (Lovett, 1967) except that synchronous sporogenesis was induced after 18 h at 22°C. Zoosporangia were collected by filtration at the time of papilla formation; spores were collected after filtration by centrifuga- tion. Zoosporangia and spore pellets were suspended in a homogenizing medium consisting of 50 mM Na cacodylate, pH 7.5, containing 0.5 M sucrose, 2.5% Ficoll, 10 mM MgClZ, 10 mM KCl, and 5 mM EDTA. Sporangia were broken by sonication (X3, 30 sec, 60 W, 4°C), with one min intervals between each treatment; spores were disrupted by passing through a 27 gauge hypodermic needle until they were broken as observed microscopically. Sporangial homogenates were passed through four layers of 8-ply cellulose gauze, and both the spore and sporangial homogenates were centrifuged at 300 x g for 7 min. The sporanigal supernatant was centrifuged at 10,000 x g for 15 min. The pellet was resuspended in homogenizing medium and applied to a continuous sucrose gradient, as was the 300 x g spore super- natant. The sucrose gradient (BO-65%, w/w) was formed over a cushion of 65% (w/w) sucrose. All sucrose solutions contained 5 mM EDTA. After centrifugation for 4 h at 24,000 rpm (61,000 x g to 110,000 x g) in a swinging bucket rotor #969, model B02 International ultracentrifuge, 76 fractions were collected by drops. All steps were done at 0-4°C. Sucrose concentrations were measured refractometrically; protein was determined colorimetrically (Lowry et al., 1951). Enzyme activities were followed spectrophotometrically at a ca 23°C using a Gilford model 222 photometer and model 2453 linear potentiometer recorder (Honeywell). The enzymes assayed and methods employed were: succinic dehydrogenase, EC 1.3.99.1 (Hiatt, 1961), fumarase, EC 4.2.1.2 (Racker, 1950), catalase, EC 1.11.1.6 (Beers and Sizer, 1952), isocitrate lyase, EC 4.1.3.1 (Dixon and Kornberg, 1959), and malate synthase, EC 4.1.3.2. (Ornston and Ornston, 1969). Specific activities are designated as umoles of substrate used or product formed x min.1 x mg protein". RESULTS Of many techniques used to try to rupture spores with minimal organelle damage, passage through a syringe needle yielded the best results, with reproducible specific activities (Table 11). Most of the microbody and mitochondrial enzyme activities (ca 65% and 80%, respectively) were associated with particulates. Homogenates freed of large debris (300 x g) and centrifuged through sucrose density gradients yielded four protein peaks (Fig. 13, top). Fractions 1-5 contained soluble protein. Fractions 7—10 contained mitochondria-lipid-symphyomicrobody complexes (Mills and Cantino, 1975; Cantino and Mills, 1976) as determined both enzymatically and electron microscopically; they had a buoyant density of 1.19 g/cm3. Fractions 16-19 (1.23 g/cm3) were enriched for mitochon- dria (Fig. 14). The densest (1.29 g/cm3) fractions, 25-28, contained symphyomicrobody material. 77 .mpmxomcn cw .mpcmswgmaxm mpmcmmmm mo cmasac m.o.m.H :mmz Amy _.e m.N m.e_ o.o_ e.oF ufiom mmacm>< covpmmwmwczq o.m N.m N.mfi N.m~ o.mN u_oc Essexmz Amy Ampv Amy Amy Ampv mm__. “ONO. emm.e eomo. mmmo. mp_mcmmeo .H m_oe. .H mmm_. .H omm.mfi .H m_mo. .H memo. umbm_0mw ..<.m AN_V Am_v Ao_v Am_v Ammv emmo. mmmo. mom. “moo. mmoo. muacmmoeo; .H Nmmo. .H Femo. .H mom._ .H page. A«V.H mmoo. meoam ..<.m mmmcmmocuxsmo mmmguc»m mmmzm mmmcmssm mwcwumzm mmmFmamo mumpmz mpmcuvmomH mmsz~cm mecmco;00pwz mmez~cm amonocmwsozgaszm mmpmcmmoso: mcoamooN m2» cw mmwpw>wumm mepwcv mecu mcwu:_ucw .mmFchmmco 59w; mmpmwmommm mmsxucm mo mmppw>wumm owmwomam .FF m4mmm8 mm.~m No.0m mm.¢ m.mv ¢.mm m.m Fm.mppm pcmpa ; w_ mo.om mp.mm mm.m m.mm n.5F m.F om.FN¢P ucmpa ; mF NF.m m¢.¢ mqm. N.m_ m.op m.~ 5N.mom pcm_q ; NF No.m mm._ mofi. m.nm N.mp —.~ om.mm pcmpa ; m mm; 2; N8. 7mm 0.3 m; 2.: 22m 5 0 0mm. mom. mmo. m.om m.o~ a.m mm.em meePeemm e m FmF. mmm. NFC. m.oP m.mm m.p cm.m~ «mac _mm. Nem. mmo. e.mm m.a~ m.m me._N meaamaON mmmcm53m mmmcmmocuxcmm mmmxp mmmem23m mmmcmmoemxzmc mmmz_ “cape mmmpm mwcwmmzm mmmcu_UOmH mwcwmuzm mpmgpwmomH \cwmpocm an _mucmsmo_m>mo Am_o_xv mea_a\»me>wpd< Amo_xv mmwow>_md< aweeamam wwcomcmem am mo mmmmum FmpcmanFm>mm pcmsmwmwm mcwczu mmwuw>wumm mechm xmonocm_s use meeucocmouwz .m_ mmm