Large differences in carbohydrate degradation and transport potential among lichen fungal symbionts.

Lichen symbioses are thought to be stabilized by the transfer of fixed carbon from a photosynthesizing symbiont to a fungus. In other fungal symbioses, carbohydrate subsidies correlate with reductions in plant cell wall-degrading enzymes, but whether this is true of lichen fungal symbionts (LFSs) is unknown. Here, we predict genes encoding carbohydrate-active enzymes (CAZymes) and sugar transporters in 46 genomes from the Lecanoromycetes, the largest extant clade of LFSs. All LFSs possess a robust CAZyme arsenal including enzymes acting on cellulose and hemicellulose, confirmed by experimental assays. However, the number of genes and predicted functions of CAZymes vary widely, with some fungal symbionts possessing arsenals on par with well-known saprotrophic fungi. These results suggest that stable fungal association with a phototroph does not in itself result in fungal CAZyme loss, and lends support to long-standing hypotheses that some lichens may augment fixed CO2 with carbon from external sources.

Novel methods to characterise spatial distribution and enantiomeric composition of usnic acids in four Icelandic lichens.

Usnic acid is an antibiotic metabolite produced by a wide variety of lichenized fungal lineages. The enantiomers of usnic acid have been shown to display contrasting bioactivities, and hence it is important to determine their spatial distribution, amounts and enantiomeric ratios in lichens to understand their roles in nature and grasp their pharmaceutical potential. The overall aim of the study was to characterise the spatial distribution of the predominant usnic acid enantiomer in lichens by combining spatial imaging and chiral chromatography. Specifically, separation and quantification of usnic acid enantiomers in four common lichens in Iceland was performed using a validated chiral chromatographic method. Molecular dynamics simulation was carried out to rationalize the chiral separation mechanism. Spatial distribution of usnic acid in the lichen thallus cross-sections were analysed using Desorption Electrospray Ionization-Imaging Mass Spectrometry (DESI-IMS) and fluorescence microscopy. DESI-IMS confirmed usnic acid as a cortical compound, and revealed that usnic acid can be more concentrated around the algal vicinity. Fluorescence microscopy complemented DESI-IMS by providing more detailed distribution information. By combining results from spatial imaging and chiral separation, we were able to visualize the distribution of the predominant usnic acid enantiomer in lichen cross-sections: (+)-usnic acid in Cladonia arbuscula and Ramalina siliquosa, and (-)-usnic acid in Alectoria ochroleuca and Flavocetraria nivalis. This study provides an analytical foundation for future environmental and functional studies of usnic acid enantiomers in lichens.

Studies on recombination processes in two Chlamydomonas reinhardtii endogenous genes, NIT1 and ARG7.

Integration of exogenous DNA in the unicellular green alga Chlamydomonas reinhardtii is principally carried out by mechanisms involving non-homologous recombination (NHR), rather than homologous recombination (HR). Homologous recombination is, however, the mechanism of choice when it comes to gene targeting. Unfortunately, attempts to establish this method in Chlamydomonas have had limited success. In this study we compared two endogenous genes, NIT1 and ARG7, and their HR/NHR ratios when different types of fragments were used as donors of homologous sequences. Transformation of the auxotrophic strain containing the inactivating point mutation arg7-8 with nonfunctional ARG7 gene fragments overlapping this mutation showed increased HR efficiencies when linearized plasmids were used. Efficiency went down rapidly with decreasing length of ARG7 homology. After identification of the inactivating 6726(G→A) point mutation in nit1-305 strains, an analogous set of experiments was performed. In the case of NIT1, overall efficiency of recombination was 10 to 100 fold lower than with ARG7. In order to better demonstrate HR we introduced three silent mutations close to the position of the point mutations in our transforming plasmids. Sequencing of transformants indicated homologous recombination over a short interval.