High Viscosity and Anisotropy Characterize the Cytoplasm of Fungal Dormant Stress-Resistant Spores

Abstract

Ascospores of the fungus Talaromyces macrosporus are dormant and extremely stress resistant, whereas fungal conidia-the main airborne vehicles of distribution-are not. Here, physical parameters of the cytoplasm of these types of spores were compared. Cytoplasmic viscosity and level of anisotropy as judged by spin probe studies (electron spin resonance) were extremely high in dormant ascospores and during early germination and decreased only partly after trehalose degradation and glucose efflux. Upon prosilition (ejection of the spore), these parameters fell sharply to values characteristic of vegetative cells. These changes occurred without major volume changes that suggest dramatic changes in cytoplasmic organization. Azide reversibly inhibited prosilition as well as the decline in cytoplasmic parameters. No organelle structures were observed in etched, cryoplaned specimens of ascospores by low-temperature scanning electron microscopy (LTSEM), confirming the high cytoplasmic viscosity. However, cell structures became visible upon prosilition, indicating reduced viscosity. The viscosity of fresh conidia of different Penicillium species was lower, namely, 3.5 to 4.8 cP, than that of ascospores, near 15 cP. In addition the level of anisotropic motion was markedly lower in these cells (h 0 /h ؉1 ‫؍‬ 1.16 versus 1.4). This was confirmed by LTSEM images showing cell structures. The decline of cytoplasmic viscosity in conidia during germination was linked with a gradual increase in cell volume. These data show that mechanisms of cytoplasm conservation during germination differ markedly between ascospores and conidia.Ascospores of the fungus Talaromyces macrosporus are sexual structures that exhibit extreme resistance to heat, high pressure, drought, and freezing (14, 15). They contain an extremely high level of trehalose (13) and have relatively low amounts of water in an aqueous environment. The spores show constitutive dormancy in rich media, and germination is triggered and synchronized by a short heat treatment at 85°C. Ascospores of T. macrosporus can germinate after 17 years of storage (40) and belong to the most resilient eucaryotic structures described hitherto. They can survive a heat treatment at 85°C for 100 min or high pressurization at 1,000 MPa for 5 min (14). Upon heat activation, the spores degrade their trehalose within 100 min, followed by a rapid release of glucose into the bathing medium up to 10% of the cell wet weight. After 2.5 h, the outer cell wall opens, and the protoplast encompassed by the inner cell wall is ejected in a fast process (seconds) termed prosilition. The ejected cell then swells and forms a germ tube, resembling events that occur in other fungal spores. Conner et al. (10) studied the cellular basis of heat resistance in relatively young (11-day) and older (25-day) ascospores of Neosartorya fischeri exhibiting different heat resistance levels (D 82 of approximately 23 and Ͼ60 min, respectively). The ascospores showed differences in the inner cell wall region at the lateral ridge...

Invited Review: Cryo-scanning electron microscopy (CSEM) in the advancement of functional plant biology. Morphological and anatomical applications

“…Scale bar = 1.0 mm. Reproduced from Dijksterhuis et al (2007) with permission from Eukaryotic Cell. germinating seeds of lettuce (Lactuca sativa L.) were studied by Nijsse et al (1998Nijsse et al ( , 2004 with CSEM.…”

“…Timing of these events could be modified by changing the composition of the agar. The fine details of the ejection process are examined in beautiful CSEM images of cryo-planed spores in a subsequent paper (Dijksterhuis et al 2007). The thick spiny cell wall develops a longitudinal slit through which the thin smooth cell wall is ejected with its contained protoplast.…”

Germination of conidia of Aspergillus niger is accompanied by major changes in RNA profiles

“…The environmental conditions are signaled by receptor(s) via Ras/MAPK and cAMP/PKA signal-transduction pathways (Osherov & May 2000, Liebmann et al 2004, Reyes et al 2006, Zhao et al 2006). Upon activation of germination, the disaccharide trehalose and the polyol mannitol are degraded (Witteveen & Visser 1995, Thevelein 1996, d’Enfert et al 1999, Fillinger et al 2001, Ruijter et al 2003, Dijksterhuis et al 2007). As a consequence, glycerol is formed, which is indicative for an active glycolysis (d’Enfert 1997).…”

Water- and Air-Distributed Conidia Differ in Sterol Content and Cytoplasmic Microviscosity

“…A strong decrease in cytoplasmic viscosity is also observed during the germination of stress-resistant ascospores of Talaromyces macrosporus (1). Furthermore, the onset of germ tube formation (Fig.…”

“…The viscosity of the interior of the cell has been correlated with the dormancy and stress resistance of fungal spores (1). Spin label electron spin resonance (ESR) spectroscopy was utilized to measure the cytoplasmic microviscosity of conidia using the spin label perdeuterated TEMPONE (4-oxo-2,2,6,6-tetramethylpiperidine- N-oxy; Sigma, St. Louis, MO).…”