Lichens are symbiotic organisms that effectively survive in harsh environments, including arid regions. Maintaining viability with an almost complete loss of water and the rapid restoration of metabolism during rehydration distinguishes lichens from most eukaryotic organisms. The lichen Xanthoria parietina is known to have high stress tolerance, possessing diverse defense mechanisms, including the presence of the bright-orange pigment parietin. While several studies have demonstrated the photoprotective and antioxidant properties of this anthraquinone, the role of parietin in the tolerance of lichens to desiccation is not clear yet. Thalli, which are exposed to solar radiation and become bright orange, may require enhanced desiccation tolerance. Here, we showed differences in the anatomy of naturally pale and bright-orange thalli of X. parietina and visualized parietin crystals on the surface of the upper cortex. Parietin was extracted from bright-orange thalli by acetone rinsing and quantified using HPLC. Although acetone rinsing did not affect PSII activity, thalli without parietin had higher levels of lipid peroxidation and a lower membrane stability index in response to desiccation. Furthermore, highly pigmented thalli possess thicker cell walls and, according to thermogravimetric analysis, higher water-holding capacities than pale thalli. Thus, parietin may play a role in desiccation tolerance by stabilizing mycobiont membranes, providing an antioxidative defense, and changing the morphology of the upper cortex of X. parietina.

Plant dehydration-responsive element binding (DREB) transcription factors (TFs) play important roles during stress tolerance by regulating the expression of numerous genes involved in stresses. DREB TFs have been extensively studied in a variety of angiosperms and bryophytes. To date, no information on the identification and characterization of DREB TFs in Dicranum scoparium has been reported. In this study, a new DBF1 gene from D. scoparium was identified by cloning and sequencing. Analysis of the conserved domain and physicochemical properties revealed that DsDBF1 protein has a classic AP2 domain encoding a 238 amino acid polypeptide with a molecular mass of 26 kDa and a pI of 5.98. Subcellular prediction suggested that DsDBF1 is a nuclear and cytoplasmic protein. Phylogenetic analysis showed that DsDBF1 belongs to group A-5 DREBs. Expression analysis by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) revealed that DsDBF1 was significantly upregulated in response to abiotic stresses such as desiccation/rehydration, exposure to paraquat, CdCl₂, high and freezing temperatures. Taken together, our data suggest that DsDBF1 could be a promising gene candidate to improve stress tolerance in crop plants, and the characterization of TFs of a stress tolerant moss such as D. scoparium provides a better understanding of plant adaptation mechanisms.

Currently, due to the increasing impact of anthropogenic factors and changes in solar activity, the temperature on Earth is rising, posing a threat to biodiversity. Lichens are among the most sensitive organisms to climate change. Elevated ambient temperatures can have a significant impact on lichens, resulting in more frequent and intense drying events that can impede metabolic activity. It has been suggested that the possession of a diverse sterol composition may contribute to the tolerance of lichens to adverse temperatures and other biotic and abiotic stresses. The major sterol found in lichens is ergosterol (ERG); however, the regulation of the ERG biosynthetic pathway, specifically the step of epoxidation of squalene to 2,3-oxidosqualene catalyzed by squalene epoxidase during stress, has not been extensively studied. In this study, we used lichen Lobaria pulmonaria as a model species that is well known to be sensitive to air pollution and habitat loss. Using in silico analysis, we identified cDNAs encoding squalene epoxidase from L. pulmonaria, designating them as LpSQE1 for the mycobiont and SrSQE1 for the photobiont Symbiochloris reticulata. Our results showed that compared with a control kept at room temperature (+20 °C), mild temperatures (+4 °C and +30 °C) did not affect the physiology of L. pulmonaria, assessed by changes in membrane integrity, respiration rates, and PSII activity. An extreme negative temperature (−20 °C) noticeably inhibited respiration but did not affect membrane stability. In contrast, treating lichen with a high positive temperature (+40 °C) significantly reduced all physiological parameters. Quantitative PCR analysis revealed that exposing thalli to −20 °C, +4 °C, +30 °C, and +40 °C stimulated the expression levels of LpSQE1 and SrSQE1 and led to a significant upregulation of Hsps. These data provide new information regarding the roles of sterols and Hsps in the response of lichens to climate change.

Lichens, symbiotic organisms with a high tolerance to harsh environments, possess a greater diversity of sterols than other organisms. Sterols are involved in maintaining membrane integrity, hormone biosynthesis, and signal transduction. (1) Background: A characteristic feature of lichen sterols is a high degree of unsaturation, which influences membrane properties. Desaturases play an important role in the synthesis of unsaturated sterols, in particular, sterol C-5 desaturase (ERG3), which controls the conversion of episterol to ergosterol. Earlier, we demonstrated that the treatment of the lichen Peltigera canina with low and elevated temperatures results in changes in the levels of episterol and ergosterol. (2) Methods: Here, for the first time, we identified ERG3 in P. canina and, using an in silico analysis, we showed that PcERG3 belongs to the superfamily of fatty acid hydrolyases. A phylogenetic analysis was conducted to determine the evolutionary relationships of PcERG3. (3) Results: A phylogenetic analysis showed that PcERG3 clusters with ERG3 from other Peltigeralian and non-Peltigeralian lichens and also with ERG3 from free-living fungi. This suggests that PcERG3 has an ancient evolutionary origin and is related to fungi with lichenized ancestors, e.g., Penicillium. The differential expression of PcERG3 in response to temperature stress, a dehydration/rehydration cycle, and heavy metal exposure suggests that it plays a crucial role in maintaining the balance between more and less saturated sterols and, more generally, in membrane functioning. The multifaceted response of P. canina to abiotic stresses was documented by simultaneously measuring changes in the expression of PcERG3, as well as the genes encoding the heat shock proteins, PcHSP20 and PcHSP98, and PcSOD1, which encodes the antioxidant enzyme superoxide dismutase. (4) Conclusions: These findings suggest that PcERG3 is similar to the sterol C-5 desaturases from related and free-living fungi and plays important roles in the molecular mechanisms underlying the tolerance of lichens to environmental stress.

A promising approach to solve the problem of tolerance of plants in hostile environments is to focus of stress tolerance mechanisms of extremophilic plants, in particular mosses. Along with the universal stress mechanisms, bryophytes exhibit a unique spectrum of secondary metabolites such as carotenoids, a lipophilic metabolite derived from the isoprenoid pathway. The main representatives of carotenoids in mosses are α- and β-carotene, lutein, neo-, viola- and zeaxanthins. Hylocomium splendens is one of the most common and widespread mosses of Northern Hemisphere. The genome of this moss has not been sequenced, and the carotenoid biosynthesis pathway (CBP) genes of this species have not been reported to date. This is the first report to of an attempt to identify and characterize the CBP genes in H. splendens. As a result of cloning, sequencing, and in silico analysis, we identified and characterized ten CBP genes in H. splendens with a full ORF, and prediction of subcellular localization suggests chloroplast localization of CBP proteins. Using multiple alignments and phylogenetic and homology analyses, we demonstrated that the CBP genes of H. splendens share high similarity with the sequences in other bryophytes. Differential expression of CBP transcripts during abiotic stresses was more evident for genes in the middle and downstream steps of CBP. This work provides information on the molecular genetics of CBP in extremophilic bryophytes. Analysis of CBP genes can help to unravel the genetic evolution of carotenoid biosynthesis in plants.