Circadian clock- and temperature-associated genes contribute to overall genomic differentiation along elevation in lichenized fungi.

Circadian regulation is linked to local environmental adaptation, and many species with broad climatic niches display variation in circadian genes. Here, we hypothesize that lichenizing fungi occupying different climate zones tune their metabolism to local environmental conditions with the help of their circadian systems. We study two species of the genus Umbilicaria occupying similar climatic niches (Mediterranean and the cold temperate) in different continents. Using homology to Neurospora crassa genes, we identify gene sets associated with circadian rhythms (11 core, 39 peripheral genes) as well as temperature response (37 genes). Nucleotide diversity of these genes is significantly correlated with mean annual temperature, minimum temperature of the coldest month and mean temperature of the coldest quarter. Furthermore, we identify altitudinal clines in allele frequencies in several non-synonymous substitutions in core clock components, for example, white collar-like, frh-like and various ccg-like genes. A dN/dS approach revealed a few significant peripheral clock- and temperature-associated genes (e.g. ras-1-like, gna-1-like) that may play a role in fine-tuning the circadian clock and temperature-response machinery. An analysis of allele frequency changes demonstrated the strongest evidence for differentiation above the genomic background in the clock-associated genes in U. pustulata. These results highlight the likely relevance of the circadian clock in environmental adaptation, particularly frost tolerance, of lichens. Whether or not the fungal clock modulates the symbiotic interaction within the lichen consortium remains to be investigated. We corroborate the finding of genetic variation in clock components along altitude-not only latitude-as has been reported in other species.

Identification and expression of functionally conserved circadian clock genes in lichen-forming fungi.

Lichen-forming fungi establish stable symbioses with green algae or cyanobacteria. Many species have broad distributions, both in geographic and ecological space, making them ideal subjects to study organism-environment interactions. However, little is known about the specific mechanisms that contribute to environmental adaptation in lichen-forming fungi. The circadian clock provides a well-described mechanism that contributes to regional adaptation across a variety of species, including fungi. Here, we identify the putative circadian clock components in phylogenetically divergent lichen-forming fungi. The core circadian genes (frq, wc-1, wc-2, frh) are present across the Fungi, including 31 lichen-forming species, and their evolutionary trajectories mirror overall fungal evolution. Comparative analyses of the clock genes indicate conserved domain architecture among lichen- and non-lichen-forming taxa. We used RT-qPCR to examine the core circadian loop of two unrelated lichen-forming fungi, Umbilicaria pustulata (Lecanoromycetes) and Dermatocarpon miniatum (Eurotiomycetes), to determine that the putative frq gene is activated in a light-dependent manner similar to the model fungus Neurospora crassa. Together, these results demonstrate that lichen-forming fungi retain functional light-responsive mechanisms, including a functioning circadian clock. Our findings provide a stepping stone into investigating the circadian clock in the lichen symbiosis, e.g. its role in adaptation, and in synchronizing the symbiotic interaction.

The Clock Gene TOC1 in Shoots, Not Roots, Determines Fitness of Nicotiana attenuata under Drought.

The highly conserved core circadian clock component TIMING OF CAB EXPRESSION1 (TOC1) contextualizes environmental stress responses in plants, for example by gating abscisic acid signaling and suppressing thermoresponsive growth. Selective interaction of TOC1 with PHYTOCHROME B under far-red-enriched light suggests a connection between circadian gating of light responses and sensitivity to ABA, an important regulator of growth and stress responses, including under drought. However, the fitness consequences of TOC1 function, particularly in the root, are poorly understood. Here, we used the desert annual, Nicotiana attenuata, to investigate the function of TOC1 in shoots and roots for maintaining fitness under drought, in both field and glasshouse experiments. Despite marked decreases in leaf water loss, TOC1-deficient lines failed to maintain fitness in response to drought stress as measured by total seed capsule production. Restoring TOC1 transcript levels in shoots via micrografting was sufficient to restore wild-type drought responses under field conditions. Microarrays identified a coexpression module in leaves strongly linking red and far-red light signaling to drought responses in a TOC1-dependent manner, but experiments with phytochrome-deficient lines revealed that the effects of TOC1 deficiency under drought cannot be attributed to changes in red/far-red light perception alone. Taken together, these results elucidate the sophisticated, tissue-dependent role of the circadian clock in maintaining fitness in the face of long-term abiotic stresses such as drought.