ALBERT

Notes: Yeast cells show an adaptive response to a mild heat shock, resulting in thermotolerance acquisition. This is accompanied by induction of heat-shock protein (hsp) synthesis and rapid accumulation of trehalose. Genetic approaches to determine the specific role of trehalose in heat-induced thermotolerance in Saccharomyces cerevisiae have been hampered by the finding that deletion of TPS1, the gene encoding trehalose-6-phosphate synthase, causes a variety of pleiotropic effects, including inability to grow on glucose-containing media. Here, we have studied a tps1 mutant of the yeast Schizosaccharomyces pombe that reportedly has no such growth defects. We show that tps1 mutants have a serious defect in heat shock-induced acquisition of thermotolerance if conditioned at highly elevated temperatures (40–42.5°C), which, in wild-type cells, prevent hsp but not trehalose synthesis. In contrast, hsp synthesis appears to become particularly important under conditions in which trehalose synthesis is either absent (in tps1 mutant strains) or not fully induced (conditioning at moderately elevated temperatures, i.e. 35°C). In addition, pka1 mutants deficient in cAMP-dependent protein kinase were examined. Unconditioned pka1 cells had low levels of trehalose but a high basal level of thermotolerance. It was found that pka1 mutant cells, contrary to wild-type cells, accumulated large amounts of trehalose, even during a 50°C treatment. pka1 tps1 double mutants lacked this ability and showed reduced intrinsic thermotolerance, indicating a particularly important role for trehalose synthesis, which takes place during the challenging heat shock.

Notes: Synthesis of trehalose in the yeast Saccharomyces cerevisiae is catalysed by the trehalose-6-phosphate (Tre6P) synthase/phosphatase complex, which is composed of at least three different subunits encoded by the genes TPS1, TPS2, and TSL1. Previous studies indicated that Tps1 and Tps2 carry the catalytic activities of trehalose synthesis, namely Tre6P synthase (Tps1) and Tre6P phosphatase (Tps2), while Tsl1 was suggested to have regulatory functions. In this study two different approaches have been used to clarify the molecular composition of the trehalose synthase complex as well as the functional role of its potential subunits. Two-hybrid analyses of the in vivo interactions of Tps1, Tps2, Tsl1, and Tps3, a protein with high homology to Tsl1, revealed that both Tsl1 and Tps3 can interact with Tps1 and Tps2; the latter two proteins also interact with each other. In addition, trehalose metabolism upon heat shock was analysed in a set of 16 isogenic yeast strains carrying deletions of TPS1, TPS2, TSL1, and TPS3 in all possible combinations. These results not only confirm the previously suggested roles for Tps1 and Tps2, but also provide, for the first time, evidence that Tsl1 and Tps3 may share a common function with respect to regulation and/or structural stabilization of the Tre6P synthase/phosphatase complex in exponentially growing, heat-shocked cells.

Notes: The composition of fatty acids and sterols in soil lipid fractions is often used as a global indicator for the status and changes of soil microbial communities. In order to validate such analyses in the context of ectomycorrhizal communities, an experiment was performed in which seedlings of Pinus sylvestris and the fungus Pisolithus tinctorius were grown separately, or combined to form ectomycorrhiza under axenic conditions. Fatty acids of the neutral lipid fraction (NLFAs) and the phospholipid fraction (PLFAs) as well as sterols were identified and quantified by gas chromatography–mass spectrometry. When grown separately, the two organisms differed strongly with respect to the sterol composition. Sterols had a much higher relative abundance in the fungus in comparison with the plant, and the two main fungal sterols, ergosterol and 24-ethyllanosta-8,24(24′)-diene-3beta,22zeta-diol (Et lano 8,24), as well as six minor fungal sterols were not found in the plant. On the other hand, the three sterols found in plant roots were absent from the fungus. With regard to fatty acids, the lipids of both organisms contained the same three major PLFAs, namely n16:0, 18:2–9,12c, and 18:1–9c. However, plant lipids contained, in addition, eight PLFAs and five NLFAs that were not present in the fungus. On the other hand, the fungus contained two PLFAs and two NLFAs that were not present in the plant. When the fungus and the plant were brought together, there was a drastic change in the lipid composition of the root: within a day, all the saturated fatty acids in the NLFA fraction increased very strongly and then slowly decreased but remained at an elevated level throughout the experiment. All these saturated fatty acids also started to appear in the extraradical fungal mycelium; they increased steadily and reached their highest levels at the end of the experiment. These results indicate that in symbiosis, the fungus transports plant lipids from the symbiotic interface to the extraradical mycelium. Concerning sterols, the extraradical mycelium acquired only a small amount of plant-specific sterols. However, its ergosterol content steadily decreased whereas the content of Et lano 8,24 remained high, causing the ratio of these two sterols to decrease from 1 : 7 to 1 : 20, whereas in the ectomycorrhizal root, the opposite phenomenon occurred, so that the ratio increased to a value of almost 1 : 1. The marked changes in the composition of the extraradical mycelium were well reflected in a principal component analysis of all lipid components. The present results show that a given ectomycorrhizal fungus may display markedly different lipid compositions in its intraradical and extraradical parts. In addition, they highlight a potential role of plant lipid transfer from the root to the fungus in the functioning of the ectomycorrhizal symbiosis.