4 | Discussion
This study reveals functional characteristics of lichen symbioses by
contrasting gene expression patterns of the fungal partner growing in
associations with different photobionts, i.e., thallus sectors
containing cyanobacteria or green algae as predominant photosynthetic
partners. We focused specifically on the fungal genes differentially
expressed between morphs as well as three different temperature
treatments, and on temperature-related differential expression of green
algal and cyanobacterial genes. Our analyses reveal fungal gene
expression differences mediated by different photobionts and
temperatures. In the threesome partnership of P. britannica ,
stress responses are triggered at markedly different temperatures in
cyanobacteria, lichen-forming fungi and green algal symbionts.
4.1 | Ascomycete genes / photomorph
Differential gene expression in lichen-forming fungi is mediated by
interactions with different photosynthetic partners. For example, the
upregulation of an ascomycetous isopenicillin N synthetase in the
cyanomorph could be attributable to mycobiont-photobiont interactions.
Metabolic interactions between lichenized fungi and their photosynthetic
partners have been shown to be able to affect the production of lichen
substances (Shrestha & St. Clair, 2013), some of which have antibiotic
(Gazzano et al., 2013; Shrestha & St. Clair, 2013) or growth-inhibitory
properties (Ocampo-Friedmann & Friedmann, 1993; Ranković & Mišić,
2008). Penicillin is a known fungal β-lactam antibiotic which mainly
controls gram positive bacteria (Holtman, 1947) and isopenicillin N
synthetase is essential for the production of penicillin (Müller et al.,
1991). By producing this antibiotic, the lichenized fungus in our study
might be able to control growth and population size of the gram-positive
part of its bacterial microbiome and potentially also of its
intrathalline cyanobionts. This is further indicated by the upregulation
of fungal genes encoding velvet domain-containing proteins in the
cyanomorph, as these genes also play a role in secondary metabolism and
antibiotic biosynthesis in model fungi Penicillium andAspergillus (Kopke et al., 2013; Kato et al., 2003). Excessive
growth of Nostoc photobionts may be detrimental to the symbiosis,
as Nostoc produces toxins in various lichen species, especially
in those growing in humid climates (Kaasalainen et al., 2012), such asP. britannica . The mycobiont could be able to avoid
cyanotoxin-induced damage by expressing isopenicillin N synthetase and
velvet domain proteins in the cyanomorph. Subsequent studies are
necessary to test this hypothesis. Currently it remains unclear if otherPeltigera species living in symbiosis with only cyanobacterial
photobionts also express genes involved in the production of
antibiotics. We hypothesize that the regulation of cyanobacterial growth
could be important for maintaining long-term cohesion of largePeltigera thalli under varying environmental conditions. The
upregulation of the antibiotic proteins depended on temperature as well,
but the pattern was not uniform. This suggests upregulation results from
a combination of factors, like the presence of Nostoc and
temperature changes.
In either photomorph, different genes responsible for cell wall
synthesis or cell wall modification were upregulated, e.g. SUN domain
proteins in the cyanomorph and chitin synthase in the tripartite morph
(downregulated at 25 °C) (Gastebois et al., 2013; Garcia-Rubio et al.,
2020; Bowman & Free, 2006). Fungal hyphal structures and their cell
wall play a vital part in the infection of host organisms such as plants
(Hopke et al., 2018) and in the process of host interaction (Geoghegan
et al., 2017) – as is the case in lichen symbioses (Kono et al., 2020;
Honegger, 1986). In addition, various environmental factors influence
the remodeling of fungal cell walls (Patel & Free, 2019). The
lichenized fungus might interact differently with its two photosynthetic
partners and, mediated by its respective partner, different aspects of
cell wall (trans)formation may be required to establish contact sites.
The green algal partner Coccomyxa contains resilient
sporopollenin biopolymers in its cell walls, which the fungus cannot
penetrate or degrade (Honegger & Brunner, 1981). Instead of haustoria,
the mycobiont forms wall-to-wall appositions (Honegger, 1984). The
cyanobacterial partner Nostoc usually isn’t penetrated either
(Honegger, 1984, 1985), but the hyphae encircle the Nostoc cells
tightly and sometimes even invaginate them (Pawlowski & Bergman, 2007).
Also, in Peltigera species with Nostoc , intrawall
haustoria have been described, which penetrate the membrane of the
cyanobacterial cell wall (Koriem & Ahmadjian, 1986). The establishment
of these contact sites has an effect on the symbiotic relationship as
they allow nutrient transfer between the symbionts (Kono et al., 2020)
which is often considered the functional core of lichen symbioses.
However, differential gene expression of cell wall modifying genes
induced by photosynthetic partners may impact lichens on a much broader
scale. They could be involved in the formation of the strikingly
different phenotypes which P. britannica (alongside other
lichens) develops with the goal to optimize different aspects of the
symbiotic relationship. In foliose lichens (such as Peltigera )
the mycobiont actively positions the photobionts within the thallus to
ensure optimal acclimatization to its environment (Honegger, 2012). The
mycobiont might also control growth and proliferation of the photobionts
(Honegger, 2012; Hyvärinen et al., 2002). However, co-development of the
symbionts is needed for the successful establishment of a lichen thallus
(Hill, 1985). Conclusively, the differential expression of cell wall
(trans)formation genes between morphs could cause the different
phenotypes or at least contribute to their formation.
The interaction between mycobiont and photobiont can also be seen in the
upregulation of ascomycete stress response genes, e.g., that of
glutathione-S-transferase (GST) in the tripartite morph and that ofpara -aminobenzoic acid (PABA) synthase in the cyanomorph; the
expression of both of these enzymes was temperature-dependent as well,
as they were downregulated at 25 °C. GSTs are, inter alia, active during
oxidative stress (Morel et al., 2009). As desiccation leads to an
accumulation of reactive oxygen species (ROS), oxidative stress occurs
in desiccating organisms, including lichens (Kranner et al., 2008;
Holzinger & Karsten, 2013). A major player providing tolerance towards
oxidative stress is glutathione (GSH), an antioxidant which reduces ROS,
whereby GSH itself is being oxidized into glutathione disulfide (GSSG)
(Kranner et al., 2008). The enzyme GST is a major driver of this
detoxification process as it is responsible for the conjugation of GSH
onto ROS (Kammerscheit et al., 2019). Lichens that tolerate desiccation
to a higher degree usually contain an increased GSH pool in their
hydrated state, enabling a rapid oxidation to GSSG during desiccation
and causing an increased GSSG pool (Kranner, 2002). The upregulation of
GST in our samples could therefore be a result of initial desiccation
and desiccation-induced accumulation of ROS, even though the thalli were
watered regularly and were sampled while fully hydrated. The
upregulation of GST occurred in the tripartite morph only – although
both morphs received identical treatment. This pattern might be due to
an increased desiccation tolerance of lichens with green algal
photobionts, relative to those with cyanobacteria (Kranner, 2002), or it
might reflect inherent differences in physiological properties among
photomorphs, e.g. water holding capacities of the gelatinous sheath ofNostoc sp. (Liang et al., 2014). Future studies should conduct
specific experiments to characterize these putative physiological
differences. Furthermore, the expression of PABA synthase in the
cyanomorph indicated stress response, as this enzyme has been shown to
improve tolerance to thermal stress in Agaricus bisporus (Lu et
al., 2014) and to enhance UV-C resistance in Arabidopsis thaliana(Hu et al., 2019). As cyanobacteria are more susceptible to heat stress
(see below) as well as high light when hydrated than green algae
(Gauslaa et al., 2012), the expression of PABA synthase could be a
response of the lichenized fungus to environmental stress in which the
mycobiont supports the photobiont in keeping the holobiont vital.
Photomorph-dependent differential expression of genes involved in fungal
carbon metabolism were expected as both partners produce distinctive
carbon-based secondary compounds. Green algae like Coccomyxaproduce polyols such as ribitol (Richardson & Smith, 1968), whereas
cyanobionts produce glucans and glucose (Hill, 1972), all of which are
translocated and taken up by the mycobiont. In the mycobiont, these
carbohydrates are converted into the energy-storing compound mannitol
(Palmqvist et al., 2008; Grzesiak et al., 2021). The obtained sugars
either serve a nutritional purpose (i.e. growth and respiration)
(Palmqvist et al., 2008; Smith, 1963) or they are conducive to stress
tolerance (e.g. protection during desiccation) (Farrar, 1976; Spribille
et al., 2022). As the carbon-based substrates are distinct, the
mycobiont requires different enzymes for substrate transport and
transformation. Although various genes responsible for carbohydrate
metabolism were found upregulated in both morphs, none of these could be
assigned directly to glucan or ribitol metabolism. Yet, a number of
studies have shown the complex nature of carbohydrate movement within
lichens and have emphasized photobiont-induced distinctions (Smith et
al., 1969; Hill, 1972; Richardson et al., 1967; Palmqvist et al., 2008;
Hill & Ahmadjian, 1972; Kono et al., 2020). In our study, the
expression of ascomycete genes involved in carbon metabolism was
temperature-dependent in some cases, and some genes (e.g., carbohydrate
esterase family 4 and galactonate dehydratase) were upregulated at 4
°C_1 and 4 °C_2, which might have to do with cold tolerance
mechanisms, as carbon metabolism plays a role in tolerance and
acclimation to cold. In photosynthetic organisms, primary as well as
secondary carbon metabolites have been proven to be essential to
withstand cold temperatures (Fürtauer et al., 2019; Calzadilla et al.,
2019; Tarkowski & Van den Ende, 2015). The lichenized fungus might
respond similarly to cold temperatures by metabolizing the carbohydrates
it obtains from its photosynthetic partners; indeed, previous studies
have shown that polyols, such as ribitol and mannitol, serve as
cryoprotectants (Fontaniella et al., 2000; Hájek et al., 2009).
We found over 300
photomorph-mediated differentially expressed genes in the lichenized
fungus. More than half of these genes were also differentially expressed
at different temperatures. Therefore, the differential expression of
these ascomycete genes appears to be the result of a combination of
factors – photobiont type plus specific stimulus. However, the results
clearly indicate photobiont-mediated differential fungal gene
expression.
4.2 | Ascomycete genes / temperature
A stepwise temperature increase from 4 to 25 °C resulted in the
upregulation of various stress-response genes in the lichenized fungus,
with upregulation beginning already at 15 °C. Heat shock proteins and
chaperonins are proteins directly involved in stress responses, giving a
clear indication that the organism is stressed at elevated temperatures
when it has been pre-acclimated to cold; as does the upregulation of
proteins which are only indirectly involved in stress response
mechanisms, such as the Rad60-SLD domain and ARPC5. SLDs are SUMO-like
domains and – as SUMO (small ubiquitin-like modifier) proteins – SLDs
are responsible for the SUMOylation of a range of other proteins
(Ghimire et al., 2020; Prudden et al., 2009). Protein SUMOylation is of
great relevance as it renders targeted proteins useful for various vital
biological processes. Heat stress has been described as one of the
factors leading to increased SUMOylation activity (Zhou et al., 2004).
Shortly after a rise in temperature, SUMO conjugates accumulate,
indicating that SUMOylation might be an early stress response system
(Kurepa et al., 2003). SUMOylation is the starting point of a cascade of
cellular processes in reaction to stress as it activates target
proteins, such as heat shock factors, which in turn activate specific
proteins, such as heat shock proteins (Kurepa et al., 2003). Activation
of HSPs as a consequence of SUMOylation has been described for various
organisms, including A. thaliana (Kurepa et al., 2003) andCandida albicans (Leach et al., 2011). The upregulation of
Rad60-SLD in P. britannica photomorphs at 15 and 25 °C therefore
indicates fungal responses to thermal stress. Similarly, the expression
of ARPC5 at 25 °C reflects stress response processes. ARPC5 is a member
of the multiprotein complex Arp2/3; in the nucleus, the Arp2/3 complex
contributes to DNA repair mechanisms as it promotes migration of DNA
double-strand breaks which are to be repaired (Schrank et al., 2018). An
upregulation of DNA repair mechanisms at high temperatures is expected
because elevated temperatures can cause heat-induced DNA damage (Oei et
al., 2015; Steinhäuser et al., 2016).
Furthermore, in both photomorphs, an upregulation of ascomycete
transposon genes was detected at 25 °C. Transposons, or transposable
elements (TE), are DNA sequences which can change their position in the
genome (Muñoz-López & García-Pérez, 2010). Transposon translocations
can affect gene functioning, especially when they are inserted into a
gene’s coding region. The movements of TEs are subject to prior
activation; stress conditions can serve as stimuli for TE activation
(Dubin et al., 2018). Increased TE transcription has been described for
other organisms experiencing heat stress, such as A. thaliana(Huang et al., 2018). In the pathogenic ascomycete fungusMagnaporthe grisea, heat stress, copper sulfate and oxidative
stress cause activation of retrotransposons (Ikeda et al., 2001).
Therefore, the upregulation of TEs in our P. britannica specimens
at 25 °C might result from thermal stress. The biological consequences
of stress-related TE translocations (Negi et al., 2016) would be an
interesting area of future studies.
In addition to upregulation of ascomycete stress response genes,
downregulation of a large number of genes was observed at 25 °C.
Functional annotation of these genes proved difficult, as many could
only be annotated roughly (e.g., to enzyme classes). Genes that could be
annotated more precisely were mostly part of regular metabolic pathways.
GTPase and ATPase activity as well as NAD(P)-binding were major
functions downregulated at 25 °C. The same is true for various genes
responsible for translation and transcription and for some genes
encoding mitochondrial proteins. The latter suggests a reduction of
mitochondrial function; similar results have been described for stressedSaccharomyces cerevisiae cells (Sakaki et al., 2003). Curbed
metabolism in stress situations might be beneficial to allocate the
available energy resources to stress response pathways, allowing
organisms to survive under suboptimal conditions (Peredo & Cardon,
2020).
The results illustrate that an organism exposed to heat stress does not
solely react by means of expression of stress genes but also by
downregulation of other genes such as those involved in metabolic
pathways under normal conditions. In the lichenized fungus, heat stress
responses such as upregulation of HSPs were already induced at 15 °C; at
25 °C, the fungus appeared to be highly stressed. Therefore, long-term
exposure to high temperatures could result in severe damage, especially
when the lichen’s respiration rate increases, causing a negative carbon
balance over extended time periods (Sundberg et al., 1999; Lange &
Green, 2005).
Interestingly, of the 200 ascomycete genes most significantly
differentially expressed between temperatures, 103 were downregulated at
25 °C, and 27 of these downregulated genes were also differentially
expressed between photomorphs. Of the 97 fungal genes upregulated at 25
°C, only seven were photomorph-mediated. Stress-related proteins are
highly conserved (Elliott, 1998) and stress responses are vital for
survival, which could explain why their upregulation at 25 °C occurs
largely independent of associations with a specific partner.
4.3 | Cyanobacterial genes / temperature
The relatively low number of just below 2,500 cyanobacterial transcripts
detected in our samples results from the method of library construction
involving selection of poly-A mRNA. Cyanobacterial transcripts were
found nonetheless due to carry-over, but as a result the number of
cyanobacterial genes with significantly different expression was
relatively small. Provided that this carry-over is a random process,
cyanobacterial transcripts would be sampled depending on their frequency
in the RNA pool; nevertheless, the results have to be interpreted with
due caution.
Increased temperature led to significant differential expression of
(heat) stress and photosynthesis-related cyanobacterial genes. The
former category comprises HSPs, chaperonins and Dps (DNA-binding protein
from starved cells), as well as genes indirectly involved in stress
response mechanisms, e.g. lysine-tRNA ligase. Dps is a highly conserved
protein which is part of various stress response pathways (Karas et al.,
2015). Its two main functions in stress responses are DNA binding –
i.e. shielding the DNA from reactive chemicals – and ferroxidase
activity. Ferroxidase oxidizes Fe2+ to
Fe3+, and thereby prevents the formation of hydroxyl
radicals via the Fenton reaction (Fe2+ +
H2O23+ +
OH– + •OH) (Calhoun & Kwon, 2011;
Nair & Finkel, 2004). The lysine-tRNA ligase is responsible for the
formation of lysyl-tRNA which is of relevance in protein synthesis,
transferring lysine into ribosomes (Wu et al., 2007). Besides, the
enzyme has another function – as has been assessed forEscherichia coli – as it synthesizes various adenyl
dinucleotides, particularly Ap4A. This function of
lysine-tRNA ligase is active only under stress and leads to an
accumulation of Ap4A. Ap4A serves as a
modulator of heat shock response, binding to and modifying stress
proteins (Onesti et al., 1995).
The second category of genes upregulated at higher temperatures in our
samples are photosynthetic genes. These genes were annotated to various
photosynthetic functions of photosystems I and II as well as the
cytochrome complex and ATP synthase. Expectedly, higher temperatures led
to an increase in photosynthetic activity (Lommen et al., 1971), but the
expression of photosynthetic genes might as well be caused by
heat-induced structural changes of the photosynthetic machinery, such as
protein complexes (Allakhverdiev et al., 2012; Ivanov et al., 2017).
A cyanobacterial gene encoding a bleomycin resistance protein was highly
significantly upregulated at 15 °C and 25 °C, but the reason for its
upregulation is difficult to determine. This protein confers resistance
to the antibiotic bleomycin (Dumas et al., 1994). In E. coli it
has been shown that the presence of antibiotic resistance genes likely
has been induced by adaptation to stress (like thermal stress) as
tolerance towards antibiotics underlies similar mechanisms as tolerance
towards heat (Cruz-Loya et al., 2019) This might potentially explain why
a gene encoding a bleomycin resistance protein was upregulated at
elevated temperatures in the Nostoc cyanobiont of P.
britannica . Besides, Keszenman et al. (2000 & 2005) have demonstrated
that the upregulation of bleomycin resistance genes is a side effect of
heat stress in S. cerevisiae , as the yeast cells proved to be
resistant to bleomycin treatment after having been exposed to heat
stress. This correlation between heat stress and bleomycin resistance is
probably the result of cross-linking of DNA repair mechanisms (Keszenman
et al., 2000, 2005). A potential role of bleomycin-resistance genes in
DNA repair has already been proposed in previous studies on E.
coli (Blot et al., 1991).
A range of cyanobacterial genes of the P. britannicaphotosymbiodemes show temperature-mediated differential gene expression.
Most DEGs are responsible for either heat stress responses or
photosynthesis. In general, our results demonstrate that in
cyanobacteria, heat stress is induced at a temperature of 15 °C already,
similar to what we found in the lichen-forming fungus. This coincides
with personal observations that P. britannica cyanomorphs and
compound thalli only grow in shady and moist habitats where
environmental fluctuations are minimal. The preference of these sites
might simply reflect the cyanobionts’ limited tolerance towards high
temperatures and desiccation. The upregulation of genes of the
photosynthetic apparatus at higher temperatures suggests an increase in
the cyanobacteria’s photosynthetic activity. Hence, elevated
temperatures are not merely a stressor but are beneficial for
cyanobacteria to a certain extent. It seems, however, unlikely that the
favorable conditions for photosynthesis counterbalance the detrimental
effects caused by heat stress, otherwise the cyanomorphs and compound
thalli would not be habitat-restricted to the coolest, moistest sites
available.
4.4 | Green algal genes / temperature
Similar to the cyanobacterial genes, most of the green algal genes
upregulated at higher temperatures were either part of photosynthetic or
stress response pathways. HSPs, chaperonins as well as proteins for DNA
repair and signal transduction were expressed primarily at 25 °C. This
suggests that the green algal partner tolerates heat to a greater extent
than the cyanobacterial and fungal partners. This result is consistent
with findings of Green et al. (2002) as well as personal observations
that P. britannica tripartite morphs grow in relatively open
habitats exposed to more fluctuations in temperature, light and water
availability. Furthermore, photosynthetic activity of the algae was
enhanced at 15 °C and 25 °C, so tripartite morphs may partly benefit
from a rise in temperature. However, the actual optimal photosynthetic
temperature is difficult to determine, as it is species-dependent
(Wagner et al., 2014) as well as dependent on various other
environmental factors (Alam et al., 2015; Green et al., 2002). Given the
upregulation of photosynthetic genes at 15 and 25 °C, one can conclude
that both temperatures are within the range of optimal temperature for
net photosynthesis in P. britannica photosymbiodemes. However, as
25 °C leads to an expression of genes relevant for heat shock responses,
a long-term exposure to higher temperatures could – at least partly –
inactivate the photosynthetic apparatus (Ivanov et al., 2017). A
prolonged increase in temperature could also negatively impact carbon
balance if the lichen’s respiration rate outweighs its photobiont’s
photosynthesis rate. Elevated respiration after temperature increases
has been described for lichens and their photobionts (Palmqvist et al.,
2008; Sundberg et al., 1999), however, the respiration rate usually
normalized after one to three hours (Sundberg et al., 1999). There is no
evidence in our dataset that a rise in temperature led to an elevated
algal respiration rate; based on this the carbon balance in the lichen
is most likely still positive. Gas-exchange measurements would be useful
to settle this issue, but were beyond the scope of the current study.
We also observed upregulation of proteins associated with lipid
metabolism at 25 °C. This metabolic activity could result from lipid
remodeling induced by heat stress, especially in regard to membrane
lipids. Heat can compromise the structural integrity of membranes and
consequently an organism’s vitality. In order to counteract membrane
disintegration, a variety of lipids are synthesized and accumulated in
the cell (Zhang et al., 2020), such as saturated fatty acids (Barati et
al., 2019), whereas other lipids, mostly polyunsaturated fatty acids,
undergo selective degradation or are converted to storage lipids
(Légeret et al., 2016). These metabolic conversions of lipids seem to
allow the algae to cope with an increase in temperature (Zhang et al.,
2020; Song et al., 2018). Therefore, the expression of lipid metabolism
proteins at 25 °C might be an indirect response to stress induced by
elevated temperatures.
These results help us in understanding the ecological conditions under
which lichen symbioses grow in nature. Compound thalli of
photosymbiodemes represent an attractive study system as both
photomorphs grow under the same environmental conditions, so both
photobionts do not only have to tolerate these conditions but must also
benefit from them to establish a successful symbiosis. Therefore,
photosymbiodemes are restricted in their distribution to certain
ecological niches (Green et al., 1993; Lange et al., 1988; Purvis,
2000), a circumstance we were also able to observe in our study, as theP. britannica specimens only grew in damp, relatively hidden
spots such as small crevices of lava rocks or under branches of birch
shrubs growing on a small slope. Solitary tripartite morphs, on the
other hand, grew in open areas that are more exposed to environmental
fluctuations, especially regarding light, temperature and humidity.
Solitary cyanobacterial thalli were missing in the collection area at
Heiðmörk. These distribution patterns of P. britannica seem to be
indicative of the respective photobiont’s stress tolerance. More studies
of gene expression in lichen photomorphs are needed to understand to
which extent the patterns reported here hold true for other taxa as
well.