Lipids and ascospore morphology in yeasts
Bareetseng, Andries Sechaba
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Some yeasts produce sexual spores (ascospores) in a variety of shapes and surface ornamentations. These intriguing structures have hitherto been used only in yeast classification. In this study the likely primary function of ascospore shape and ornamentations with associated lipids in water-driven movement as aiding the dispersal of ascospores from enclosed containers (asci), is proposed. This interpretation might find application in nano-, aero- and hydro -technologies with the re-scaling of these structures. Through sexual reproduction, some yeasts produce microscopic containers (asci) that enclose ascospores of many different shapes and various nano-scale surface ornamentations. Some spores are spherical with an equatorial ledge (like the planet Saturn), or resemble hats with a bole and brim, while others look like corkscrews, walnuts, spindles with whip-like appendages, needles, and hairy or warty balls. Until now, these structures were used to classify yeasts and little thought was apparently given to their possible functional role. From literature and microscopic observations, it was found that the yeasts Dipodascopsis uninucleata and Dipodascus have evolved sophisticated means that enable the dispersal of oxylipin-coated spherical and bean-shaped spores from bottle-shaped containers (asci) without inverting or shaking them. Here, spores are pushed by turgor pressure towards the narrow opening and then ejected. Studies of Dipodascopsis suggest that oxylipin-coated, interlocked hooked ridges on the surfaces and stretching across the length of bean- to ellipsoidal-shaped spores are responsible for the alignment of the latter. Here, spores inside the container are positioned side-by-side in a column of linked clusters with elongated sides attached by interlocking hooked ridges in gear-like fashion and orientated mainly with one end towards the opening. It was concluded that hooked ridges form turbine-like structures at both ends, causing propeller-like rotation when the spores are pushed by water pressure towards the ascus opening. This rotational movement loosens the spores (by the unlocking the hooked-ridges) near the container neck, which is necessary for sliding past each other for eventual release. Eventually, spores are released individually from the bottle-shaped ascus while rotating at about 1200 rpm at approximately 110 length replacements per second. With some species of the genus Dipodascus, compressible oxylipin-coated sheathed surface structures and not gears are used to separate and loosen spherical spores in a similar bottle -shaped container before individual release under turgor pressure. These spores simply slide past each other when pressed towards the opening. It is presumed that more complex mechanics are needed to allow the effective release of bean- to ellipsoidal-shaped ascospores compared to spherical sheathed ascospores, for which alignment and rotation are unnecessary. Using gas chromatography-mass spectrometry, it was discovered that a saturated 3-OH 14:0 (mass fragments:175 [CH3O(CO)-CH2-CHO-TMSi]; 330 [M + ]; 315 [M + -15]) is produced by the yeast Eremothecium ashbyii. In order to map the oxylipin’s location in the yeast, antibodies (against these oxylipins) and immunofluorescence microscopy on cells in sexual mode was employed. The oxylipin was present as part of a V-shaped structure on sickle-shaped spores. With the aid of confocal laser scanning microscopy to observe cells treated with antibody and fluorescine (FITC anti-rabbit IgG), it was concluded that the hydrophobic V-shaped structure was present as a mirror image on both sides at the blunt end of an otherwise hydrophilic spore as indicated by differential ascospore staining. Scanning electron microscopy showed this structure to be fin-like protuberances. Next, the function of these fin-like structures and ascospore shape was addressed. Using microscopy, it was discovered that spores are sometimes forced through the ascus with the spiked tip rupturing the ascus wall. Water pressure caused a boomerang movement when the blunt end is pushed forward with the spike leading the way in a circular motion. This happens only when micron streams of water move across the fins from the blunt end towards the tip of the spore. It is believed that this part of the study has only scratched the surface of water-driven ascospore movement in yeasts on a micrometer scale and that the mechanical implications of many spore shapes with a large number of different hydroxy oxylipin-lubricated, nano-scale surface ornamentations await similar explanation and elaboration. Why did some yeasts evolve peculiar spore movement with the beneficial consequence, so far as we can see, to escape from closed or partially closed containers? Of course, this should be important from a survival point of view since without this ability, yeasts will probably not be able to disperse properly. It is believed that if appropriate ultrastructural studies (using glutadialdehyde and osmium tetroxide as fixatives) are conduc ted on yeasts aimed at exposing ascospore surface ornamentations and not merely membrane structure, conducted in the past, clues can be gained to reveal the mechanics behind the motion of nano-sized particles in fluids. Consequently a further aim of this study became to assess ascospore structure (using above ultrastructural method) especially nano-scale ornamentations with associated lipids especially oxylipins in various unrelated yeasts. These were obtained for the yeasts Eremothecium sinecaudum (ascospores corkscrew-shaped and coated with oxylipins), Dipodascopsis uninucleata var. wickerhamii (smooth surfaces without oxylipins), Lipomyces kononenkoae (smooth ascospore surface with lipid sacs), L. tetrasporus (ridged ascospore surface with lipid sacs), Saturnispora saitoi (Saturn-shaped ascospores covered with oxylipins), Ascoidea africana (hat-shaped ascospores covered with oxylipins), Ambrosiozyma platypodis (double brimmed hat-shaped ascospores), Nadsonia commutata (ascospore surface warty; cells contain oxylipins) and N. fulvescens (ascospore surface hairy-like; cells contain oxylipins). Interesting patterns regarding lipid turnover (i.e. total-, neutral-, phospho-, glycolipids and associated fatty acids) were found when asexual and sexual stages of above yeasts are compared.