A Morphological Study of Lichen-Symbionts, Ascomycetous Fungus Myelochroa leucotyliza and Green Alga Trebouxia sp., with Special Reference to the Mechanism of Lipid (Atranorin) Secretions

Lichens are symbionts of fungi and algae (or cyanobacteria). Lichen forms a unique complex called thallus and produces large amount of lipids of various kinds. These lipids were secreted from the fungal cells and deposited in the thallus to form tremendous amount of crystalline materials. The crystalline materials are considered to have roles to protect the lichen from excess light, bacterial infection, freezing, and etc. This should be the reason that lichens can exhibit a wide ability of adaptations to extreme habitats even as those of alpine, desert, and polar region. From this aspect, we considered the significance of symbiosis of lichen must depend on the unusual lipid production and secretion.<br />　　As mentioned, the fungi produce lipids and secrete them out of cells. On the other hand, the algae were considered to provide precursor sugars for the fungi to produce lipids (Mosbach, 1969, Yamazaki et al., 1965.). Although fungi grown in the wild secrete large amount of lipids to form crystals, those grown in the culture both alone or with algae produce only small amounts and are found unable to form crystals in the culturing medium (Honegger, 1996).<br />　　This study is focused on the mode and the mechanism of the lipid secretion as we considered that the existence of large amount of crystals is signifying the symbiosis of lichens.<br />　　To begin the study we first respectively isolated the fungi and the algae from the thallus of Myelochroa leucotyliza. The isolated samples were cultured both alone or with each other. The cultured samples and the lichen in the wild were examined by means of quick freezing and replicated or substituted electron microscopy. Next, it seemed essential to identify the nature of crystals. X-ray and electron microscope diffraction methods and TLC analysis on the re-crystallized lipid extracted from the wild lichen did the identification. The major component of the re-crystallized material was atranorin.<br />　　According to the observations on the morphology of wild and cultured samples with electron microscopy the following results were obtained; 1) atranorin is the predominant component of the crystalline materials in the thallus, 2) the dynamic morphological change of the plasmamembrane of the fungi occurred in parallel to the activities of lipid secretion, 3) the zymogen granules contained in the fungal cytoplasm exocytose during the lipid secretion, 4) the amount of lipid bodies contained in the fungi inversely changed with those of the crystal deposition outside of the cell.<br />　　It was considered that if we could promote the cultured fungi into actively lipid-secreting cells, and if we could find crystals around the cells, we would have an opportunity to understand the entire scheme of the way the cell secretes lipid and the significance of symbiosis of lichens. Along with this consideration, we cultured fungi in the medium with higher concentration of sugar than usual. According to Hamada's report (1993) that in these condition fungi synthesize large amount of lipid. We detected neither crystal by EM or existence of atranorin by TLC. It was observed, however, that in the fungal cytoplasm cultured in this sugar fortified medium existence of multiple lipid bodies together with numbers of zymogen granules. The appearance of these accumulations of multiple organelles suggested the cellular inhibition of secretions of both lipids and proteins. In order to release the inhibition, we added into the culturing medium various materials that had been reported to promote lipid secretion. Finally, we soaked the fungi growing in this sugar fortified culture with a few drops of the medium in which the algae had been cultured alone. The soaking looked the fungi to release the inhibition and made them actively exocytose zymogen granules. The plasmamembrane changed the shape with many invaginations of omega appearances. The crystals were found outside of the cells.<br />　　The amount and the size of lipid bodies decrease during the active secretion of zymogen granules. However, the lipid-bodies may consist of reservoirs of sterol-derivatives and triacylglycerols. It is considered that they should represent the indirect source of atranorin. Atranorin belongs to polyketides. It has to be produced via acetate-malonate pathway by the interaction of polyketide synthase, in which two phenolic units derived from acetate join to become atranorin. The cytosolic location of polyketide synthase has been reported by showing fluorescent of GFP of the GFP-tagged enzyme. Further, we observed using a fluorescent microscope the possible cytosolic location of atranorin under UV excitation. As atranorin is extremely hydrophobic, the lipid must have being bound with some associating protein in the cytoplasm. However, it is not known how atranorin reaches the inner side of the plasmamembrane.<br />　　The modes of lipid secretion so far survived in the long course of histology are as follows. They are 1) th ...

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