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.