3.2 | DESeq2 analysis
Overall, the number of differentially expressed transcripts per taxon was high, with 9,069 transcripts being assigned to ascomycetes, 25,109 transcripts to chlorophytes (green algae) and 2,476 transcripts to cyanobacteria (comprising only the longest isoform per ’gene’). A large number of genes was differentially expressed between tripartite morphs and cyanomorphs (hereafter referred to as photomorph-mediated), however, only ascomycete genes were considered for further analyses (312 genes differentially expressed; adjusted Benjamini-Hochberg p -value < 0.05 and a log2-fold change > |2|). Additionally, many genes were differentially expressed when comparing 25 °C with the control temperature of 4 °C_1; in this case, genes from all three lichen symbionts were analyzed. At this temperature setting, 2,862 ascomycete genes, 9,275 green algal genes and 663 cyanobacterial genes were differentially expressed. DESeq2 analysis produced fewer differentially expressed genes when comparing 4 °C_1 with 15 °C (860 ascomycete, 3,258 algal and 148 cyanobacterial genes) as well as with 4 °C_2 (198 ascomycete, 4,095 algal and 53 cyanobacterial genes) (Fig. S1). Principal Component Analysis was used to assess the effects of temperature and photomorph on the overall expression pattern of the symbionts (Fig. 3, S2). On the one hand, the mycobiont (Fig. 3) clearly showed temperature-dependent expression, with clusters for low, medium and high temperatures. The photomorph-effect on mycobiont differential gene expression was less pronounced (2,862 temperature-mediated vs 312 photomorph-mediated differentially expressed ascomycete genes). On the other hand, the temperature-effect on green algal and cyanobacterial gene expression was low (Fig. S2).
3.3 | Differentially expressed genes
When comparing photomorphs, 123 of the 200 most significantly differentially expressed ascomycete genes were upregulated in the cyanomorph and 77 in the tripartite morph (Fig. 4A). Regarding gene expression at different temperatures, 103 of the 200 most significantly differentially expressed ascomycete genes were downregulated at 25 °C when compared to 4 °C_1, whereas only one cyanobacterial and one green algal gene were downregulated (Fig. 4B). We also checked the expression patterns of the 200 temperature-mediated DEGs at the other temperatures (Figs. S3, S4). Overall, of the top 200 DEGs, a somewhat higher proportion of photobiont genes than of ascomycete genes could be functionally annotated (cyanobacteria: 92.5%; green algae: 89%; ascomycetes: 81.5% (temperature-mediated) and 74% (photomorph-mediated)). Table 1 shows the top five significantly differentially expressed genes of each organism for the parameters in question (gene lists with the 200 top DEGs: Tables S3-S6).
3.3.1 | Ascomycete genes / photomorph
GO annotations of the ascomycete DEGs illustrate a variety of distinct biological processes in the cyano- and the tripartite morph (Fig. 5). In both morphs, the majority of ascomycete DEGs were annotated to oxidation-reduction processes. In the tripartite morph, another substantial process was transmembrane transport, whereas the processes tricarboxylic acid cycle and phospholipid biosynthesis comprised a smaller number of DEGs. In the cyanomorph, the remaining ascomycete DEGs were annotated to carbohydrate metabolic processes and to protein phosphorylation. As these biological processes are relatively unspecific, the individual genes with the greatest significance were scrutinized with UniProt BLAST to obtain a more detailed picture of their putative functions.
About a quarter of the top 200 ascomycete DEGs could not be functionally annotated and 50.5% of the top 200 photomorph-mediated DEGs were also temperature-dependent (adjusted Benjamini-Hochberg p -value < 0.05). In addition, the most significantly differentially expressed ascomycete genes were blasted to a local filtered metagenomic database we built using sequences of three species of Peltigeraincluding P. britannica . Of the 200 most significantly differentially expressed ascomycete genes, only three could not be matched with our Peltigera database. These three genes were removed and substituted with the next three genes – which could be matched successfully – from the gene list (Tables S3-S6).
The highest level of differential expression of ascomycete genes was found for a transcript encoding an isopenicillin N synthetase which was expressed in the cyanomorph. The expression of this gene also showed a temperature response, being downregulated at 15°C. Other ascomycete genes upregulated in the cyanomorph encoded cell wall synthesis proteins, e.g., SUN domain proteins and an alpha-1,3-glucan synthase; but proteins that seem to be responsible for cell wall synthesis were expressed in the tripartite morph as well, e.g., chitin synthase. Furthermore, there were indications of morph-dependent differential ascomycete gene expression regarding stress-responsive genes. For example, in the tripartite morph, a transcript encoding glutathione-S-transferase (GST) was upregulated, while in the cyanomorph, the upregulation of para -aminobenzoic acid synthase indicated stress response. We also found evidence of photobiont-mediated differential carbohydrate metabolism in the lichenized fungus. Various genes of carbohydrate pathways were found upregulated in either the cyanomorph (carbohydrate esterase family 4, α-1,2-mannosidase, various transporters) or the tripartite morph (galactonate dehydratase, D-xylose reductase). The results imply that processing of carbon compounds and provision of carbon differs among photomorphs.
3.3.2 | Ascomycete genes / temperature
We found temperature-mediated differential expression of various ascomycete genes. Of the top 200 temperature-mediated ascomycete DEGs, 16% were photomorph-mediated as well. Many of the genes upregulated at 15 °C and 25 °C were stress-related, like genes encoding proteins directly responsible for heat stress responses such as heat shock proteins (HSP) and chaperonins (Fig. 6). On the other hand, some of the DEGs had an indirect role in stress responses. The latter included, among others, a small ubiquitin-related modifier (Rad60-SLD domain-containing protein) and ARPC5 (Actin-related protein 2/3 complex subunit 5). Furthermore, two hours of exposure to 25 °C led to the activation of transposons; in both morphs, various ascomycete genes encoding for proteins from transposon TNT 1-94 were upregulated as well as one gene that was identified as a retrotransposable element.
In addition to upregulation of genes involved in stress responses, downregulation of a large number of genes was observed at 25 °C. These genes could often only be annotated roughly, e.g., to enzyme classes like oxidases and hydrolases or transporter proteins like those of the major facilitator superfamily. Genes that could be annotated more thoroughly were part of various pathways, including translation and transcription as well as some genes encoding mitochondrial proteins. GTPase activity and GTP-binding, ATPase activity as well as NAD(P)-binding were major functions downregulated at 25 °C.
3.3.3 | Cyanobacterial genes / temperature
Functional annotation of cyanobacterial genes exposed to the temperature treatments revealed a number of upregulated genes that had two main functions: stress responses and photosynthesis. The former comprises a group of genes encoding HSPs and chaperonins as well as other genes involved in stress response mechanisms, including modulators (e.g. Dps, lysine–tRNA ligase, Bax inhibitor-1), various response regulators of signaling cascades, genes involved in DNA repair (e.g. recA ) and an antibiotic (bleomycin) resistance protein; these were upregulated at 15 °C and 25 °C (Fig. 6). Photosynthesis genes, which were upregulated at 15 and 25 °C, performed various photosynthetic functions of both the photosystem I and II as well as the cytochrome complex and the ATP synthase.
3.3.4 | Green algal genes / temperature
The temperature-stress induced DGE results for the green algae were similar to those of the cyanobacteria. Most of the green algal genes upregulated at increased temperatures could be functionally annotated to genes encoding various photosynthetic proteins and to stress-response proteins. Genes encoding stress-response proteins, including HSPs, chaperonins as well as proteins for DNA repair mechanisms and signal transduction were upregulated mainly at 25 °C (Fig. 6). Furthermore, at 25 °C, increased expression of proteins associated with lipid metabolism (e.g., sterol 14 desaturase, 3-hydroxy-3-methylglutaryl coenzyme A synthase, prolycopene isomerase) was observed. The main biological process attributed to lipid metabolism by topGO analysis was “lipid metabolic process”; other lipid-metabolism related biological processes included lipid transport and carotenoid biosynthetic process.