Sterol And Galactolipid Composition of the Lichen Photobiont Genus Trebouxia Compared with a Potential Host Lichen in the Genus Usnea

Abstract

Lichens are a diverse group of cosmopolitan fungi that are notable for their close symbiotic relationship with single-celled photosynthetic organisms. These symbionts are typically chlorophyte algae, though some groups host cyanobacteria, which provide the fungal component with photosynthesized nutrients. This relationship is so close that the fungus cannot grow properly without its symbiote, and as a result biochemical analysis of lichens typically looks at both the fungal and algal components together. Understanding these organisms separately and identifying the nature of their symbiosis helps us understand both lichens and symbiosis better, and while the fungus’s reliance on its algal partner complicates this goal, there are ways around it, particularly if we focus on the algal component. Using unialgal cultures of Trebouxia, a common chlorophyte algae symbiote genus which can grow in media without a fungal component, this study identifies the sterols and galactolipids found in isolated Trebouxia erici and Trebouxia excentrica algae and compares them to those of wild Usnea lichens in middle Tennessee, using gas chromatography/mass spectrometry analysis to identify the sterol lipids and positive-ion electrospray/mass spectrometry to identify galactolipids. Trebouxia species are a broadly used lichen symbiont typically found in species of Usnea lichen, which allows us to distinguish which sterol structures in the whole lichen samples are synthesized by the algae and which by the fungus. While there is evidence in the literature that symbiosis changes the metabolism of algae, which could potentially affect sterol synthesis, eliminating the sterols synthesized by the algae from the sterols of the lichen leaves sterols that are either synthesized by the fungus or require its presence to form in the algae. This allows for separate biochemical analysis of these closely paired organisms - for example whole lichen samples contain ergosta-5,8,22E-trien-3β-ol (lichesterol) and 24-methylcholesta-5,7,22E-trien-3β-ol (ergosterol), common lichen sterols, but these are not present in unialgal samples, connecting their synthesis to the fungal symbiont. The greatest percentage of sterols found in algal samples, however, are also found in whole lichen samples in smaller proportions relative to the fungal sterols. These sterols, 24-ethylcholesta-5,22E-dien-3β-ol (stigmasterol) and 24-methylcholest-5en-3β-ol (campesterol), are synthesized by algal symbiont species regardless of the presence of a fungal partner, so are of algal origin. Galactolipids are only found in chloroplasts, which the fungal component of lichens lacks completely, but these lipids are compared between 2 species within an algal genus and between cultured and lichenized algal samples as a lipid type known to belong only to the algal partner. This serves as a baseline for lipid variation between algal sample groups, as well as providing galactolipid data for a group of green algae. This study finds a common monogalactosyldiacylglycerol (MGDG) with an 18:3/16:4 sn-1/sn-2 fatty acyl structure shared by algae across all groups, symbiotic or cultured. T. erici algal groups also shared all their smaller percentage MGDG and digalactosyldiacylglycerol (DGDG) molecules. T. excentrica showed only the 18:3/16:4 MGDG found in both algal groups and a set of 3 DGDG molecules, of which one was not shared by the T. erici lines - an 18:2/16:2 DGDG. Only 2 galactolipids were identified in the Usnea lichen samples, the 18:3/16:4 shared by all algal groups and an 18:3/18:3 DGDG shared by both T. erici lines. This study shows the lipid profiles of T. erici culture lines 911 and 912 are highly similar, with identical galactolipids and only small differences in sterol composition, while T. excentrica has a distinct sterol profile with sitosterol and lacks an unidentified C29 sterol found in both algal groups examined here. T. excentrica also has fewer galactolipids overall compared to T. erici and contains an 18:2/16:2 DGDG not found in any other sample groups. Comparison with whole Usnea lichen samples confirms the presence of lichesterol, ergosterol, and an unidentified C28 sterol in the fungal Usnea that are absent in all algal sample groups.