BACKGROUND
the role of light in life has been subject to intensive research with almost all living organisms from animals to bacteria, plants and fungi.
in recent years, interconnections of circadian rhythmicity and its resetting by light with regulation of metabolic pathways became obvious.
proper integration of adaptation to light–dark cycles and adequate adjustment of metabolism is known to be crucial for health, fitness and sometimes even survival of plants  <cit> , mammals  <cit>  and fungi  <cit> .
however, although numerous genes are known to play a role in the integration of nutrient signalling with light dependent adjustment to the environment  <cit> , the molecular basis for the underlying regulatory network remains to be explored in detail.
in fungi, the light status in the environment has a more general effect than only triggering sporulation, defence mechanisms or dna-photolyases in fungi  <cit> .
studies on fungi during the previous century until present time showed that light has an impact on nearly all metabolic processes in fungi like carotenoid metabolism, polysaccharide metabolism, fatty acid metabolism, nitrogen and sulphur metabolism and many more  <cit> .
one of the first model organisms for investigations of light effects on morphology in fungi was trichoderma
<cit> , because of its visible reaction to illumination, which triggers conidiation  <cit> .
in the potent plant cell wall degrader t. reesei
<cit>  signalling events causing regulation of plant cell wall degrading enzymes as well as light response are subject to continued research efforts  <cit> .
one of the most prominent and hence also best studied mechanisms for this purpose is the pathway of heterotrimeric g proteins.
g proteins consist of alpha, beta and gamma subunits.
the heterotrimeric complex receives a signal from the g protein coupled receptors , which is influenced by external stimuli.
in case of activation of the gpcr, a conformational change of the receptor results in an exchange of gdp for gtp at the g alpha subunit.
the active g alpha subunit dissociates from the tightly bound g beta gamma dimer and both are impacting their effectors to affect regulatory pathways of secondary messengers  <cit> .
among these pathways, the camp pathway represents an important output of heterotrimeric g-protein signaling.
in this pathway, the intracellular levels of the secondary messenger camp is regulated by adenylate cyclase, which synthesizes camp and phosphodiesterase , which degrades camp  <cit> .
steady state levels of camp were found to be fine-tuned by a negative feedback loop established by pde and protein kinase a , which is activated by camp .
in t. reesei, transcript levels of the genes encoding the g protein alpha subunits gna <dig> and gna <dig> are regulated by light and the light regulatory protein env <dig> .
env <dig> was found to be essential for elevated camp levels and is assumed to negatively influence phosphodiesterase activity  <cit> .
the camp pathway as a main output pathway of heterotrimeric g-protein signalling was shown to be important for cellulase gene expression in t. reesei
<cit> .
env <dig> is a pas/lov domain containing regulator of light responses and cellulase gene expression  <cit> .
its closest characterized homologue is the neurospora crassa photoreceptor protein vvd, which plays a role in regulation of the circadian rhythm, photoadaptation and sensing of light intensities .
vvd is also known to act as a universal brake in light responses  <cit>  and to modulate activity of the white collar complex  by physical interaction  <cit> .
transcription of env <dig> is strongly induced by light and this induction is dependent on the blue light photoreceptors blr <dig> and blr <dig>  <cit> .
interestingly, envoy also possesses regulatory functions in darkness  <cit>  and represents a crucial signalling component with functions not only in light response, but also in nutrient signal transduction  <cit> .
the investigation of a potential link between light signalling and the pathway of heterotrimeric g proteins revealed the class i phosducin like protein phlp <dig> as a central component.
additionally, the g protein beta and gamma subunits gnb <dig> and gng <dig> were found to be members of this regulatory mechanism, all of which are crucial for tight regulation of light response in t. reesei
<cit> .
transcription of phlp <dig> is responsive to light, with phlp <dig> clearly belonging to the late light responsive genes  as defined by chen  <cit> .
microarray analysis of mutants lacking phlp <dig>  gnb <dig> or gng <dig> showed that their primary function is a positive regulation of target genes in light, with glycoside hydrolases as an important output pathway.
these findings support the idea of a connection between nutrient and light signalling via heterotrimeric g-protein signalling  <cit> .
in agreement with this finding a study in trichoderma atroviride showed that the photoceptors blr <dig> and blr <dig> are crucial for the light stimulated nutrient uptake  <cit> .
based on the extensive evidence for an interconnection between nutrient signalling and light response, we now tackled the issue how this regulatory interaction is established at the molecular level and how the signal is transmitted further.
to this end we investigated the first step of regulation by adjustment of transcript levels, that represents the basis for translation, modification and ultimately signaling output.
we compared genome wide transcriptional regulation by env <dig> with that of the heterotrimeric g-protein components gnb <dig>  gng <dig> and phlp <dig>  which pointed at a mechanism coupling the light signal with the g protein pathway and with glycoside hydrolases as representatives of the nutrient degradation machinery as output pathway.
our subsequent analyses of light response of selected signalling components in numerous mutant strains revealed that mutual regulation of env <dig> and phlp <dig> constitutes one node in the interconnection between nutrient and light signalling, with gnb <dig> as an important factor of signal transmission to downstream targets.
subsequently, we show that the core output functions impacted by env <dig> are regulated via its effect on camp levels.
RESULTS
targets of light- and nutrient signalling show considerable correlation
in order to evaluate the interrelationship between nutrient and light signaling we compared the regulatory targets of these pathways as revealed by transcriptome analysis from strains grown with microcrystalline cellulose as sole carbon source in light and darkness.
thereby, env <dig>  blr <dig> and blr <dig>  <cit>  served as representatives of the light response pathway and phlp <dig>  gnb <dig> and gng <dig>  <cit>  represented the nutrient signaling pathway of heterotrimeric g-proteins.
interestingly, our analysis of the influence of the light response machinery on gene regulation in light and darkness had revealed the strongest effect on positive targets of env <dig>  blr <dig> and blr <dig> in light , the most severe influence being exerted by env <dig>  <cit> .
this condition is similar to the condition most relevant for the function of phlp <dig>  gnb <dig> and gng <dig>  <cit> .
because of the outstanding position of env <dig> in positive regulation of downstream targets in light, we compared the positive phlp1-gnb1-gng <dig> targets  <cit>  with those of env <dig> in light .
intriguingly, we found 77%  of the positive targets of phlp1-gnb1-gng <dig> to overlap with those of env <dig> in light.
in principle, the detected target processes strongly resemble those of the light signalling machinery.
gene set enrichment analysis of these common targets with the p-value threshold for significant enrichment set to ≤ <dig>  revealed enrichment in genes involved in metabolic processes, transport, oxidoreductase activity and regulation.
a specific enrichment of polygalacturonase activity, mainly represented by genes encoding glycoside hydrolases of family  <dig>  suggests that one common target of env <dig>  phlp <dig>  gnb <dig> and gng <dig> could be the enhancement of maceration and soft rotting of plant tissue by weakening the pectin network.
we conclude that the nutrient signals transmitted via phlp1-gnb1-gng <dig> are closely interrelated with light signalling via env <dig>  lack of one of these four components presumably causes the system mediating the respective positive signalling output to shut down.
the data confirm the key function of env <dig> and phlp1-gnb1-gng <dig> in interconnecting nutrient- and light signalling.
light is the most important source of variation in target genes
in order to evaluate the interconnection between light and nutrient signal transmission , we performed a hierarchical cluster analysis and principal component analysis  of genome wide transcript patterns of all strains included in this study .
except for the wildtype, the detected clusters revealed a clear separation between gene regulation in strains grown in light compared to those grown in darkness.
this finding suggests that the tight regulation of light response in the wildtype becomes unbalanced due to the lack of either a central component of the light signalling machinery or a component involved in light dependent transmission of nutrient signals.figure  <dig>
analysis of global genome wide transcript abundance patterns of Δ
blr1
, Δ
blr2
, Δ
env <dig>
Δ
phlp1
, Δ
gnb1
and Δ
gng1
by  principal component analysis  and  hierarchical cluster analysis
.
data on transcript levels of all  <dig> genes of t. reesei <cit>  in the parental strain as well as mutant strains in light and darkness of two biological replicates were used for the analysis.
average standard deviation between two biological replicates was 13%.
however, Δenv <dig> in light appears in this cluster as an outgroup, hence highlighting its distinct function, and Δgnb <dig> in light also occupies a peculiar position.
nevertheless, nutrient and light- signalling strains still appear in separate clusters according to cultivation in light and darkness.
the outstanding position of Δgnb <dig> in light supports the hypothesis that phlp <dig> and gng <dig> are important for appropriate folding of gnb <dig> in light  <cit> , the function of which obviously is more different from that of phlp <dig> and gng <dig> in light than in darkness .
pca analysis confirms this result and shows that light is the major source of variation among the different strains .
we conclude that both env <dig> and gnb <dig> have crucial functions in regulation of downstream output pathways, with env <dig> having an even more widespread effect than gnb <dig>
evaluation of the interconnection between light- and nutrient signalling
because of the considerable overlap between targets of the light and nutrient signalling pathway via env and phlp1-gnb1-gng <dig>  we were interested how the interconnection between the two pathways is established.
since an influence of env <dig> on the genes encoding the g protein alpha subunits gna <dig> and gna <dig> is known  <cit> , we included these g protein alpha subunits in the analysis along with double mutants bearing constitutive alleles and a deletion in env <dig>  qrt-pcr analysis of gene transcription in the parental strain qm <dig> and strains altered in components of the signal transduction pathways of light response and heterotrimeric g protein signalling were applied to reveal the hierarchical order and interrelationships of regulators within the cascade.
in order to minimize influences of growth defects or altered expression of hydrolytic enzymes in light, we chose glycerol as carbon source.
glycerol does not induce cellulase gene expression in t. reesei, but does not prevent induction of cellulase gene expression upon addition of an inducer  <cit> .
additionally, we wanted to gain information on transient processes in addition to constant light and and therefore applied a switch from constant darkness to constant light after 24 hours of growth.
this experimental design also enabled us to distinguish regulatory impacts on early and late light response: strains were incubated in darkness first and illuminated for  <dig>   <dig>   <dig> and 120 minutes.
two biological replicates were evaluated throughout the analysis.
with this analysis we also aimed to identify the central component, from which the integrated signal is channelled for adjustment of the output pathways.
figure  <dig> provides an overview on results of qrt-pcr, which will be discussed in detail in the following.figure  <dig>
overview on qrt pcr analysis of
blr1
,
blr2
,
env1
,
gnb1
,
gng1
,
phlp1
,
gna1
and
gna3
in strains with alterations of these genes.
hierarchical cluster analysis was performed to evaluate similar regulation patterns of genes in the different mutants.
data for the strains gna1qle and gna3qle, which express constitutively activated versions of the respective g-protein alpha subunits and lack the env <dig> open reading frame were taken from  <cit>  in order to provide a complete picture.
env <dig> acts negatively on transcription of photoreceptors
we first tested the regulatory connections of the photoreceptor genes.
env <dig> transcription rapidly reacts to light  <cit>  and hence represents an elrg.
it was already shown that in the absence of either blr <dig> or blr <dig>  transcription of env <dig> is not induced and remains at barely detectable dark-levels  <cit> .
our analysis showed that env <dig> in turn influences photoreceptor transcript levels negatively in darkness and early light response, when env <dig> is strongly induced in the parental strain .
additionally, also blr <dig> and blr <dig> are not transcribed independently under transient conditions of light response, since blr <dig> has a clearly negative effect on blr <dig> transcription .
consequently, we propose a model in which after induction of env <dig> expression, its negative effect on blr <dig> and blr <dig> transcription leads to a steady state level of transcription of these three genes.
the repressing effect of blr <dig> on blr <dig> transcript levels is in accordance with earlier data in t. atroviride showing that blr <dig> is the limiting factor for photoperception and phototransduction  <cit> .figure  <dig>
analysis of
blr1
and
blr2
transcript levels.
transcript abundance is shown in response to light by qrt-pcr in the wildtype and ∆env <dig> strain  as well as in the ∆blr <dig> and ∆blr <dig> strains .
the strains were cultivated on mandels-andreotti minimal media with 1%  glycerol as carbon source.
the strains were kept in darkness for 24 hours and then exposed to light.
samples were taken in darkness, indicated as 24dd, and after  <dig>   <dig>   <dig> and 120 minutes of illumination , respectively.
availability of phlp <dig> has a positive effect on transcript levels of gnb1and gna1
class i phosducin like proteins are supposed to act as co-chaperones for g protein beta and gamma folding  <cit> .
assuming that also in our system a tight regulation of the amount of functional g protein subunits occurs, we investigated whether the availability of phlp <dig> and hence efficiently folded gnb <dig> and gng <dig> would feed back to the respective transcript levels.
we found that presence of phlp <dig> enhances gnb <dig> transcript levels .
transcript levels of gnb <dig> in Δgng <dig> also showed a positive effect of gng <dig> on gnb <dig> transcription , hence suggesting that both phlp <dig> and gng <dig> are important for regulation of gnb <dig>  the amount of the resulting functional g protein beta-gamma complex would consequently be determined by upregulation of gnb <dig> in this three way regulatory interaction.
this result is in agreement with data from constant illumination and on cellulose  <cit> , which indicates the central function of gnb <dig> to be carbon source independent.
moreover, the function of gnb <dig> is concluded to extend beyond a transient effect after illumination.figure  <dig>
regulation by phlp <dig>  gnb <dig> and gng <dig>  influence on gnb <dig>  and gna <dig>  transcript levels in response to light and influence of gnb <dig> on gna <dig> transcription .
transcript levels were measured by qrt-pcr from strains grown on mandels-andreotti minimal media with 1%  glycerol as carbon source.
strains were grown on mandels-andreotti minimal media with 1%  glycerol as carbon source for 24 hours in darkness  and then exposed to light for  <dig>   <dig>   <dig> or 120 minutes , respectively.
while the effect of phlp <dig>  gnb <dig> or gng <dig> on transcription of gna <dig> was negligible , we found a clearly positive influence of phlp <dig> and gng <dig> on transcription of gna <dig> .
interestingly, as seen for the effect of these two factors on gnb <dig>  also their effect on transcription of gna <dig> was similar.
considering that class i phosducin like proteins are assumed to act as co-chaperones, these data can be interpreted in a way that lack of either of these genes causes folding and complex formation with gnb <dig> to fail, which may also be responsible for the effect on gna <dig>  however, no absolute positive effect of gnb <dig> on the gna <dig> transcript pattern was observed .
the g protein alpha subunits act on transcription of phlp1but not gnb1or gng1
in order to establish the position of the g protein alpha subunits gna <dig> and gna <dig> within the network, we investigated transcription patterns of their genes in mutants bearing constitutively activated versions of these genes .
interestingly, we found a positive interconnection of gna <dig> with phlp <dig>  since they showed a mutually positive effect on each other’s transcription in early light response .
similarly, also gna <dig> acts positively on transcription of phlp <dig> , although no effect of phlp <dig> on gna <dig> was observed .
in contrast, no significant impact of gna <dig> or gna <dig> on transcription of the beta- and gamma subunit genes gnb <dig> and gng <dig> was observed, hence placing phlp <dig> in a crucial position in the nutrient signalling cascade, likely as signal transmitter towards the output pathway, but due to its regulatory interaction with gna <dig> and gnb <dig> also as part of a feedback cycle.figure  <dig>
effect of constitutive activation of gna <dig> or gna <dig> and impact of
phlp1
.
transcript ratios of phlp <dig>  and the g protein alpha subunit gene gna3
in response to light upon growth on glycerol as carbon source are shown.
qrt-pcr measurements show that both constitutively activated g alpha subunits  cause enhanced phlp <dig> transcript levels .
phlp <dig> positively influences gna <dig> transcript levels , but not gna <dig> transcript levels .
strains were grown on mandels-andreotti minimal media with 1%  glycerol as carbon source for 24 hours in darkness  and then exposed to light for  <dig>   <dig>   <dig> or 120 minutes , respectively.
phlp <dig> and env <dig> are regulatorily interconnected
with phlp <dig> emerging as a potential node between nutrient signalling and light response, we now analysed at which part of the light response pathway phlp <dig> might intervene.
recently it was shown that the photoreceptors blr <dig> and blr <dig> only have marginal influence on phlp <dig> transcription.
in contrast, env <dig> reduces the differential transcription of phlp <dig> in light and darkness and seems to be more relevant for transcriptional regulation of phlp <dig> than blr <dig> or blr <dig>  <cit> .
therefore, we were interested in the mechanistic roles of phlp <dig> and env <dig> under conditions reflecting early and late light response.
we found that env <dig> has a clearly negative effect on transcription of phlp <dig> and on the other hand phlp <dig> acts positively on early light response of env <dig> transcription .
hence, these two components are likely to establish a steady state level by positive and negative  transcriptional interaction upon illumination.figure  <dig>
regulatory interrelationship of phlp <dig> and env <dig> at the level of transcription.
phlp <dig> transcript levels are enhanced in ∆env <dig> mutant strain compared to the wildtype.
env <dig> transcript levels are decreased in the ∆phlp <dig> mutant strain compared to the wildtype.
strains were cultivated on glycerol as carbon source for 24 hours in darkness  and then exposed to light for  <dig>   <dig>   <dig> or 120 minutes , respectively.
since phlp <dig> impacts transcription of the g protein beta subunit gene gnb <dig>  we were interested whether env <dig> would also influence gnb <dig>  indeed we found that env <dig> negatively regulates transcription of both gnb <dig> and gng <dig>  the transcription patterns of both genes being highly similar in the Δenv <dig> mutant .
notably, the regulatory interaction between env <dig> and phlp <dig> as well as the influence of env <dig> on transcription of gnb <dig> and gng <dig> are most obvious during early stages of light response.
in contrast, the effect of phlp <dig> and gng <dig> on transcription of gnb <dig> can be detected also after early light response.
we conclude that the influence of env <dig> on transcript levels of gnb <dig> and gng <dig> is likely to be mediated via its negative effect on phlp <dig> figure  <dig>
regulatory interrelationship of gnb <dig>  gng <dig> and env <dig> at the level of transcription.
gnb <dig> and gng <dig> show similar transcript patterns in the wildtype strain.
gnb <dig> and gng <dig> transcript patterns in ∆env <dig> show an upregulation at early time points.
strains were cultivated on glycerol as carbon source for 24 hours in darkness  and then exposed to light for  <dig>   <dig>   <dig> or 120 minutes , respectively.
regulation of the output pathways by camp levels
the camp pathway is considered the major output pathway of heterotrimeric g-protein signaling  <cit>  and would hence be likely to act as a coincidence detector between nutrient and light signaling.
for t. reesei, an influence of the two heterotrimeric g-protein alpha subunits gna <dig> and gna <dig> on intracellular camp levels was observed  <cit> .
having established the node between nutrient and light signalling we now wanted to investigate the downstream pathway of this cascade.
deletion of env <dig> is known to result in strongly decreased intracellular camp levels  <cit> , which are likely to represent an output of the regulatory interconnection between light- and nutrient signalling established between env <dig> and phlp <dig>  in the strain lacking acy <dig>  camp production is abolished  <cit>  and consequently the situation resembles that in the strain lacking env <dig>
hence, we performed microarray analysis of a deletion mutant in the gene encoding adenylate cyclase  <dig> , which synthesizes camp.
similar conditions to those used for previous transcriptome analysis of env <dig>  <cit>  i. e. growth on cellulose for 72 hours in light and darkness were applied.
we compared the results with the transcriptome of Δenv <dig> in light and darkness.
indeed, we found a considerable overlap of targets, interestingly only among targets in light and no overlapping targets were found in darkness .
the  <dig> genes up-regulated in light in Δenv <dig> and Δacy <dig> included one polyketide synthase , three putative g-protein coupled receptors  and one transcription factor related to a n. crassa transcription factor responsive to light , as well as two sugar transporters .
<dig> genes were underexpressed in light in Δenv <dig> and Δacy <dig>  among them  <dig> glycoside hydrolases including xyn <dig>  xyn <dig>  bxl <dig>  cel3a, cel3b, egl <dig>  egl <dig>  egl <dig>  cbh <dig>  cel61a and cel61b as well as the genes encoding the auxiliary proteins swollenin, cip <dig> and cip <dig> and the cellulase and hemicellulase regulator gene xyr <dig>  additionally, we found  <dig> genes involved in sulphur metabolism, including the regulator gene lim1
<cit> .
further genes consistently regulated in Δenv <dig> and Δacy <dig> include two g-protein coupled receptors  as well as six putative sugar transporters including the recently characterized lactose permease tr_ <dig>  which is also important for cellulase gene expression on lactose  <cit> .
no contrasting regulation was observed .
functional category analysis revealed that the functions of the consistently regulated genes are enriched in metabolism , especially amino acid metabolism , nitrogen and sulphur metabolism  as well as c-compound and carbohydrate metabolism .
in the latter group particular enrichment in polysaccharide metabolism  was observed.
additionally, transport functions of carbohydrates and amino acids were enriched.
we conclude that a considerable portion of the core functions of env <dig>  are regulated via the effect of env <dig> on camp levels.
discussion
the major influence of light on fungi was studied extensively in the last decades and it could be shown that light affects a broad spectrum of metabolic pathways, morphological changes, growth and secondary metabolism  <cit>  .
since the discovery, that cellulase gene expression is modulated by light  <cit> , studies unravelling the light signalling network influencing the cellulase gene expression were undertaken , which revealed a puzzle of components regulating the model output pathway of cellulase transcription in response to light.
besides the intriguing insights into fungal physiology, these findings also provide perspectives for research towards elucidation of the interplay between light response and metabolism in higher organisms.
the results presented here connect these components by placing them in the background of a signaling network and provide intriguingly new insights into the mechanism how light- and nutrient signalling are connected at the molecular level .figure  <dig>
schematic drawing of the current model for the interrelationship between nutrient and light signaling in
t. reesei
.
the regulatory interplay between nutrient and light signalling involves a limb represented by components of the heterotrimeric g-protein pathway and a limb established by the crucial components of the light response machinery.
central interactors of this connection are env <dig> and phlp <dig>  the positive effect of phlp <dig> on env <dig> transcription in early light regulation  may be responsible for negative regulation of heterotrimeric g-protein signalling by env <dig>  the effect of env <dig> on gnb <dig> and gng <dig> is mediated by phlp <dig>  investigation of the effect of constitutive activation of the g-alpha subunits gna <dig> and gna <dig> on regulation of phlp <dig> transcript levels showed that the nutrient signals transmitted by gna <dig> and gna <dig> impact phlp <dig>  both the light signalling components as well as the factors of heterotrimeric g-protein pathway analyzed in this study were found to dampen light responsiveness of transcript abundance and are likely to constitute a light specific regulatory mechanism sustaining transcript levels of downstream genes relevant during growth in light.
investigation of the camp pathway as depicted in this scheme was described earlier  <cit> .
arrows indicate positive influence while plungers indicate a negative effect.
dashed lines show hypotheses deduced from results in other fungi .
asterisks indicate an effect on light responsiveness of transcript levels.
together with previous studies, the model we propose illustrates the predominantly negative impact of the light signalling machinery and in contrast a largely positive effect of phlp <dig> and other components of heterotrimeric g-protein signalling on regulation of nutrient utilization or enzyme gene transcription, respectively.
hence, the interplay of a positive and a negative limb are proposed to establish a balanced output which integrates both light and nutrient signals.
this mechanism is reminiscent of the positive and negative regulatory cycles triggering light response and circadian rhythmicity in n. crassa
<cit> .
the dissimilarity of the gnb <dig> transcript pattern from those of phlp <dig> and gng <dig> suggests that lack of phlp <dig> and gng <dig> has different consequences than lack of gnb <dig>  hence the alterations seen cannot be exclusively due to perturbed complex formation of gnb <dig> and gng <dig> as supported by phlp <dig>  in mammalian systems individual functions of g-beta subunits independent of the g-gamma subunit were reported  <cit> , which may be one reason for this phenomenon.
this hypothesis is supported by the finding that lack of gnb <dig> causes a slightly different phenotype than that of phlp <dig> or gng <dig>  <cit>  and the characteristic alterations in transcriptome data for a strain lacking gnb <dig>  reflecting partially different targets.
it has to be considered, that the regulatory interconnections revealed in this study must be mediated by transcription factors, which remain to be determined and might include the photoreceptors blr <dig> and blr <dig>  due to the observed function of env <dig> via adjustment of camp levels it appears likely that this regulation also involves posttranslational modification and hence activation/deactivation of these transcription factors by phosphorylation.
these transcription factors could either act directly on the target promotors or alternatively activate/deactivate further regulators.
the latter hypothesis would be supported by the finding that in n. crassa regulation by wc- <dig> is mediated by a flat hierarchical transcription factor network  <cit> .
analysis of interaction partners of the signaling components investigated in this study will provide insight into the mechanisms involved in this regulation and is currently in progress in our lab.
transcriptome analysis revealed a crucial position of env <dig> and gnb <dig> in the interrelationship of light response and nutrient signalling in t. reesei.
interestingly, upon deletion of the respective genes , we saw a considerable increase in genes regulated by light from roughly 3% to more than  <dig> or 30%, respectively  <cit> .
the crucial importance of env <dig> in both nutrient and light signalling is further reflected by the considerable number of regulatory targets distinct from those of the photoreceptor proteins  <cit> .
most of the genes differentially regulated in Δenv <dig> or Δgnb <dig> were downregulated in light, although in Δenv <dig> considerable upregulation also occurs.
the huge impact of light on strains lacking env <dig> was already observed in earlier studies  <cit> , but the extent of the regulatory function of env <dig> in light was still unexpected.
also in strains lacking blr <dig> or blr <dig> considerable downregulation of transcript levels occurred  <cit> .
these findings led us to hypothesize that the light response machinery and particularly env <dig> sustains expression levels of genes responsible for environmental sensing and signal transmission in light, presumably also via phlp1-gnb1-gng <dig>  as these components appear to only have few targets in darkness, this mechanism might not be operative in darkness, which raises the question whether a different pathway is responsible for such an effect in darkness.
correlation of the majority of targets of phlp1-gnb1-gng <dig> with those of env <dig> along with the peculiar position of Δenv <dig> in the cluster analysis of mutant strains and its impact on regulation of g-alpha subunit genes as described earlier  <cit> , places env <dig> upstream of the components of heterotrimeric g protein signalling.
nevertheless, our new transcriptome data on Δacy <dig> also indicate that env <dig> plays an important role in the output pathway of the cascade and that the components of heterotrimeric g-protein signalling analysed here might just be a modulator of the pathway regulated by env <dig>  future research will show if this hypothesis can be substantiated.
evaluation of genome wide transcription patterns and subsequent detailed analysis of light response in several mutant strains enabled us to identify env <dig> and phlp <dig> as a central interlocked regulatory pair connecting the light response pathway with nutrient signalling.
thereby, phlp <dig> acts positively on env <dig> transcription during early light response, which in turn has a negative effect on transcript abundance of blr <dig> and blr <dig> at this time.
considering data on the effect of vvd on the wcc  <cit> , env <dig> likely also supports inhibition of blr complex activity.
env <dig> in turn consistently acts negatively on transcript levels of not only phlp <dig>  but also gnb <dig> and gng <dig>  this result is in accordance with earlier findings in n. crassa
<cit> , showing that early light-responsive genes are mostly involved in primary measures to adjust to the light conditions, including protection from light, photoperception and cell signalling, while functions in c-compound and carbohydrate metabolism predominate among late light responsive genes.
therefore our findings could be interpreted in a way that env <dig> dampens g-protein signalling during early light response by decreasing transcription of phlp <dig>  gnb <dig> and gng <dig> in order to provide resources for protective measures.
the initial positive action of phlp <dig> on env <dig> transcription enhances this effect.
subsequently, the positive action of phlp <dig> on complex formation of the g beta and gamma subunits and hence g-protein signalling – which is likely to transmit nutritional signals - might be important for metabolic adaptation to light.
in the context of the overlap of transcriptomes of deletion mutants in env <dig>  phlp <dig>  gnb <dig> and gng <dig> it becomes clear that this interrelationship is even more profound and extends beyond transient effects in early light response.
as for the downstream pathway of the node between phlp <dig> and env <dig>  we investigated to which extent the regulation of camp levels by env <dig>  which is assumed to be accomplished by a negative effect on phosphodiesterase activity  <cit> , is relevant for gene regulation by env <dig>  as deletion of adenylate cyclase has a similar effect on camp levels as deletion of env <dig> , we performed microarray analysis of Δacy <dig> in light and darkness on cellulose for identification of camp dependent targets of env <dig>  interestingly, we only detected an overlap of regulated genes in light, with most of the consistently regulated genes being upregulated.
the functions of these genes are strongly enriched in metabolic functions – specifically carbon and sulphur/amino acid metabolism.
interestingly, the sulphur source in the medium is crucial for the ability of t. reesei to utilize cellulose.
without sulphate, growth of t. reesei on cellulose in light is severely perturbed and methionine cannot serve to replace sulphate as sulphur source under these conditions  <cit> .
our findings hence indicate that the interrelationship between sulphur and carbon metabolism is likely to involve the function of camp dependent mechanisms.
this overlap in regulated genes in light is further in agreement with the retarded growth phenotype of t. reesei strains lacking env1
<cit> , as retarded growth is also observed upon lack of acy1
<cit> .
however, the mechanism for regulation of growth by camp levels in t. reesei remains to be investigated in detail.
knowledge on the overlap of genes regulated as a consequence of two different factors crucial for normal growth of t. reesei should enable insight in to the mechanism regulating growth in response to camp levels.
in order to narrow down the number of candidate regulators, we checked whether n. crassa homologues of the target genes of env <dig> and acy <dig> in light, for which phenotype analyses of mutants are available, are known to be important for normal growth.
abnormal growth patterns in knock outs were only found for homologues of tr_ <dig>  and tr_ <dig> .
transcript levels of tr_ <dig> in Δacy <dig> and Δenv <dig> are only downregulated about 2fold.
therefore, we consider it unlikely that this putative transporter might contribute to the severe growth defect of Δacy <dig> and Δenv <dig>  n. crassa vib- <dig> is among other functions in cell recognition and programmed cell death, important for response to carbon starvation  <cit> .
the transcript abundance of tr_ <dig> is decreased  <dig> fold in Δacy <dig> and more than 19fold in Δenv <dig>  however, the vib- <dig> deletion mutant only shows a decreased linear growth of about 20% compared to wild-type.
consequently, tr_ <dig> may be in part responsible for the retarded growth in response to reduced camp levels, but additional factors with more significant effects remain to be identified.
CONCLUSIONS
in summary, we found that a sizable amount of independent targets of env <dig> in light shows considerable overlap with targets of the heterotrimeric g-protein components phlp <dig>  gnb <dig> and gng <dig>  complementary investigation of early and late light response revealed that env <dig> represents a crucial node in light signal transduction and exerts its function in part via the regulatory interrelationship with the phosducin like protein phlp <dig>  as well as gnb <dig> and gng <dig>  this interaction between nutrient and light signalling is at least in part mediated by transcriptional interaction of env <dig> and phlp <dig>  downstream targets of the cascade are to a considerable extent regulated via the function of env <dig> in modulation of camp levels.

 the role of light in life has been subject to intensive research with almost all living organisms from animals to bacteria, plants and fungi. in recent years, interconnections of circadian rhythmicity and its resetting by light with regulation of metabolic pathways became obvious. proper integration of adaptation to light–dark cycles and adequate adjustment of metabolism is known to be crucial for health, fitness and sometimes even survival of plants, mammals and fungi. however, although numerous genes are known to play a role in the integration of nutrient signalling with light dependent adjustment to the environment, the molecular basis for the underlying regulatory network remains to be explored in detail. in fungi, the light status in the environment has a more general effect than only triggering sporulation, defence mechanisms or dna-photolyases in fungi. studies on fungi during the previous century until present time showed that light has an impact on nearly all metabolic processes in fungi like carotenoid metabolism, polysaccharide metabolism, fatty acid metabolism, nitrogen and sulphur metabolism and many more. one of the first model organisms for investigations of light effects on morphology in fungi was trichoderma, because of its visible reaction to illumination, which triggers conidiation. in the potent plant cell wall degrader t. reesei signalling events causing regulation of plant cell wall degrading enzymes as well as light response are subject to continued research efforts. one of the most prominent and hence also best studied mechanisms for this purpose is the pathway of heterotrimeric g proteins. g proteins consist of alpha, beta and gamma subunits. the heterotrimeric complex receives a signal from the g protein coupled receptors, which is influenced by external stimuli. in case of activation of the gpcr, a conformational change of the receptor results in an exchange of gdp for gtp at the g alpha subunit. the active g alpha subunit dissociates from the tightly bound g beta gamma dimer and both are impacting their effectors to affect regulatory pathways of secondary messengers. among these pathways, the camp pathway represents an important output of heterotrimeric g-protein signaling. in this pathway, the intracellular levels of the secondary messenger camp is regulated by adenylate cyclase, which synthesizes camp and phosphodiesterase, which degrades camp. steady state levels of camp were found to be fine-tuned by a negative feedback loop established by pde and protein kinase a, which is activated by camp. in t. reesei, transcript levels of the genes encoding the g protein alpha subunits gna and gna are regulated by light and the light regulatory protein env. env was found to be essential for elevated camp levels and is assumed to negatively influence phosphodiesterase activity. the camp pathway as a main output pathway of heterotrimeric g-protein signalling was shown to be important for cellulase gene expression in t. reesei. env is a pas/lov domain containing regulator of light responses and cellulase gene expression. its closest characterized homologue is the neurospora crassa photoreceptor protein vvd, which plays a role in regulation of the circadian rhythm, photoadaptation and sensing of light intensities. vvd is also known to act as a universal brake in light responses and to modulate activity of the white collar complex by physical interaction. transcription of env is strongly induced by light and this induction is dependent on the blue light photoreceptors blr and blr. interestingly, envoy also possesses regulatory functions in darkness and represents a crucial signalling component with functions not only in light response, but also in nutrient signal transduction. the investigation of a potential link between light signalling and the pathway of heterotrimeric g proteins revealed the class i phosducin like protein phlp as a central component. additionally, the g protein beta and gamma subunits gnb and gng were found to be members of this regulatory mechanism, all of which are crucial for tight regulation of light response in t. reesei. transcription of phlp is responsive to light, with phlp clearly belonging to the late light responsive genes as defined by chen. microarray analysis of mutants lacking phlp gnb or gng showed that their primary function is a positive regulation of target genes in light, with glycoside hydrolases as an important output pathway. these findings support the idea of a connection between nutrient and light signalling via heterotrimeric g-protein signalling. in agreement with this finding a study in trichoderma atroviride showed that the photoceptors blr and blr are crucial for the light stimulated nutrient uptake. based on the extensive evidence for an interconnection between nutrient signalling and light response, we now tackled the issue how this regulatory interaction is established at the molecular level and how the signal is transmitted further. to this end we investigated the first step of regulation by adjustment of transcript levels, that represents the basis for translation, modification and ultimately signaling output. we compared genome wide transcriptional regulation by env with that of the heterotrimeric g-protein components gnb gng and phlp which pointed at a mechanism coupling the light signal with the g protein pathway and with glycoside hydrolases as representatives of the nutrient degradation machinery as output pathway. our subsequent analyses of light response of selected signalling components in numerous mutant strains revealed that mutual regulation of env and phlp constitutes one node in the interconnection between nutrient and light signalling, with gnb as an important factor of signal transmission to downstream targets. subsequently, we show that the core output functions impacted by env are regulated via its effect on camp levels.  targets of light- and nutrient signalling show considerable correlation in order to evaluate the interrelationship between nutrient and light signaling we compared the regulatory targets of these pathways as revealed by transcriptome analysis from strains grown with microcrystalline cellulose as sole carbon source in light and darkness. thereby, env blr and blr served as representatives of the light response pathway and phlp gnb and gng represented the nutrient signaling pathway of heterotrimeric g-proteins. interestingly, our analysis of the influence of the light response machinery on gene regulation in light and darkness had revealed the strongest effect on positive targets of env blr and blr in light, the most severe influence being exerted by env. this condition is similar to the condition most relevant for the function of phlp gnb and gng. because of the outstanding position of env in positive regulation of downstream targets in light, we compared the positive phlp1-gnb1-gng targets with those of env in light. intriguingly, we found 77% of the positive targets of phlp1-gnb1-gng to overlap with those of env in light. in principle, the detected target processes strongly resemble those of the light signalling machinery. gene set enrichment analysis of these common targets with the p-value threshold for significant enrichment set to ≤ revealed enrichment in genes involved in metabolic processes, transport, oxidoreductase activity and regulation. a specific enrichment of polygalacturonase activity, mainly represented by genes encoding glycoside hydrolases of family suggests that one common target of env phlp gnb and gng could be the enhancement of maceration and soft rotting of plant tissue by weakening the pectin network. we conclude that the nutrient signals transmitted via phlp1-gnb1-gng are closely interrelated with light signalling via env lack of one of these four components presumably causes the system mediating the respective positive signalling output to shut down. the data confirm the key function of env and phlp1-gnb1-gng in interconnecting nutrient- and light signalling. light is the most important source of variation in target genes in order to evaluate the interconnection between light and nutrient signal transmission, we performed a hierarchical cluster analysis and principal component analysis of genome wide transcript patterns of all strains included in this study. except for the wildtype, the detected clusters revealed a clear separation between gene regulation in strains grown in light compared to those grown in darkness. this finding suggests that the tight regulation of light response in the wildtype becomes unbalanced due to the lack of either a central component of the light signalling machinery or a component involved in light dependent transmission of nutrient signals.figure analysis of global genome wide transcript abundance patterns of Δ blr1, Δ blr2, Δ env Δ phlp1, Δ gnb1 and Δ gng1 by principal component analysis and hierarchical cluster analysis. data on transcript levels of all genes of t. reesei in the parental strain as well as mutant strains in light and darkness of two biological replicates were used for the analysis. average standard deviation between two biological replicates was 13%. however, Δenv in light appears in this cluster as an outgroup, hence highlighting its distinct function, and Δgnb in light also occupies a peculiar position. nevertheless, nutrient and light- signalling strains still appear in separate clusters according to cultivation in light and darkness. the outstanding position of Δgnb in light supports the hypothesis that phlp and gng are important for appropriate folding of gnb in light, the function of which obviously is more different from that of phlp and gng in light than in darkness. pca analysis confirms this result and shows that light is the major source of variation among the different strains. we conclude that both env and gnb have crucial functions in regulation of downstream output pathways, with env having an even more widespread effect than gnb evaluation of the interconnection between light- and nutrient signalling because of the considerable overlap between targets of the light and nutrient signalling pathway via env and phlp1-gnb1-gng we were interested how the interconnection between the two pathways is established. since an influence of env on the genes encoding the g protein alpha subunits gna and gna is known, we included these g protein alpha subunits in the analysis along with double mutants bearing constitutive alleles and a deletion in env qrt-pcr analysis of gene transcription in the parental strain qm and strains altered in components of the signal transduction pathways of light response and heterotrimeric g protein signalling were applied to reveal the hierarchical order and interrelationships of regulators within the cascade. in order to minimize influences of growth defects or altered expression of hydrolytic enzymes in light, we chose glycerol as carbon source. glycerol does not induce cellulase gene expression in t. reesei, but does not prevent induction of cellulase gene expression upon addition of an inducer. additionally, we wanted to gain information on transient processes in addition to constant light and and therefore applied a switch from constant darkness to constant light after 24 hours of growth. this experimental design also enabled us to distinguish regulatory impacts on early and late light response: strains were incubated in darkness first and illuminated for and 120 minutes. two biological replicates were evaluated throughout the analysis. with this analysis we also aimed to identify the central component, from which the integrated signal is channelled for adjustment of the output pathways. figure  provides an overview on results of qrt-pcr, which will be discussed in detail in the following.figure overview on qrt pcr analysis of blr1, blr2, env1, gnb1, gng1, phlp1, gna1 and gna3 in strains with alterations of these genes. hierarchical cluster analysis was performed to evaluate similar regulation patterns of genes in the different mutants. data for the strains gna1qle and gna3qle, which express constitutively activated versions of the respective g-protein alpha subunits and lack the env open reading frame were taken from in order to provide a complete picture. env acts negatively on transcription of photoreceptors we first tested the regulatory connections of the photoreceptor genes. env transcription rapidly reacts to light and hence represents an elrg. it was already shown that in the absence of either blr or blr transcription of env is not induced and remains at barely detectable dark-levels. our analysis showed that env in turn influences photoreceptor transcript levels negatively in darkness and early light response, when env is strongly induced in the parental strain. additionally, also blr and blr are not transcribed independently under transient conditions of light response, since blr has a clearly negative effect on blr transcription. consequently, we propose a model in which after induction of env expression, its negative effect on blr and blr transcription leads to a steady state level of transcription of these three genes. the repressing effect of blr on blr transcript levels is in accordance with earlier data in t. atroviride showing that blr is the limiting factor for photoperception and phototransduction.figure analysis of blr1 and blr2 transcript levels. transcript abundance is shown in response to light by qrt-pcr in the wildtype and ∆env strain as well as in the ∆blr and ∆blr strains. the strains were cultivated on mandels-andreotti minimal media with 1% glycerol as carbon source. the strains were kept in darkness for 24 hours and then exposed to light. samples were taken in darkness, indicated as 24dd, and after and 120 minutes of illumination, respectively. availability of phlp has a positive effect on transcript levels of gnb1and gna1 class i phosducin like proteins are supposed to act as co-chaperones for g protein beta and gamma folding. assuming that also in our system a tight regulation of the amount of functional g protein subunits occurs, we investigated whether the availability of phlp and hence efficiently folded gnb and gng would feed back to the respective transcript levels. we found that presence of phlp enhances gnb transcript levels. transcript levels of gnb in Δgng also showed a positive effect of gng on gnb transcription, hence suggesting that both phlp and gng are important for regulation of gnb the amount of the resulting functional g protein beta-gamma complex would consequently be determined by upregulation of gnb in this three way regulatory interaction. this result is in agreement with data from constant illumination and on cellulose, which indicates the central function of gnb to be carbon source independent. moreover, the function of gnb is concluded to extend beyond a transient effect after illumination.figure regulation by phlp gnb and gng influence on gnb and gna transcript levels in response to light and influence of gnb on gna transcription. transcript levels were measured by qrt-pcr from strains grown on mandels-andreotti minimal media with 1% glycerol as carbon source. strains were grown on mandels-andreotti minimal media with 1% glycerol as carbon source for 24 hours in darkness and then exposed to light for or 120 minutes, respectively. while the effect of phlp gnb or gng on transcription of gna was negligible, we found a clearly positive influence of phlp and gng on transcription of gna. interestingly, as seen for the effect of these two factors on gnb also their effect on transcription of gna was similar. considering that class i phosducin like proteins are assumed to act as co-chaperones, these data can be interpreted in a way that lack of either of these genes causes folding and complex formation with gnb to fail, which may also be responsible for the effect on gna however, no absolute positive effect of gnb on the gna transcript pattern was observed. the g protein alpha subunits act on transcription of phlp1but not gnb1or gng1 in order to establish the position of the g protein alpha subunits gna and gna within the network, we investigated transcription patterns of their genes in mutants bearing constitutively activated versions of these genes. interestingly, we found a positive interconnection of gna with phlp since they showed a mutually positive effect on each other’s transcription in early light response. similarly, also gna acts positively on transcription of phlp, although no effect of phlp on gna was observed. in contrast, no significant impact of gna or gna on transcription of the beta- and gamma subunit genes gnb and gng was observed, hence placing phlp in a crucial position in the nutrient signalling cascade, likely as signal transmitter towards the output pathway, but due to its regulatory interaction with gna and gnb also as part of a feedback cycle.figure effect of constitutive activation of gna or gna and impact of phlp1. transcript ratios of phlp and the g protein alpha subunit gene gna3 in response to light upon growth on glycerol as carbon source are shown. qrt-pcr measurements show that both constitutively activated g alpha subunits cause enhanced phlp transcript levels. phlp positively influences gna transcript levels, but not gna transcript levels. strains were grown on mandels-andreotti minimal media with 1% glycerol as carbon source for 24 hours in darkness and then exposed to light for or 120 minutes, respectively. phlp and env are regulatorily interconnected with phlp emerging as a potential node between nutrient signalling and light response, we now analysed at which part of the light response pathway phlp might intervene. recently it was shown that the photoreceptors blr and blr only have marginal influence on phlp transcription. in contrast, env reduces the differential transcription of phlp in light and darkness and seems to be more relevant for transcriptional regulation of phlp than blr or blr. therefore, we were interested in the mechanistic roles of phlp and env under conditions reflecting early and late light response. we found that env has a clearly negative effect on transcription of phlp and on the other hand phlp acts positively on early light response of env transcription. hence, these two components are likely to establish a steady state level by positive and negative transcriptional interaction upon illumination.figure regulatory interrelationship of phlp and env at the level of transcription. phlp transcript levels are enhanced in ∆env mutant strain compared to the wildtype. env transcript levels are decreased in the ∆phlp mutant strain compared to the wildtype. strains were cultivated on glycerol as carbon source for 24 hours in darkness and then exposed to light for or 120 minutes, respectively. since phlp impacts transcription of the g protein beta subunit gene gnb we were interested whether env would also influence gnb indeed we found that env negatively regulates transcription of both gnb and gng the transcription patterns of both genes being highly similar in the Δenv mutant. notably, the regulatory interaction between env and phlp as well as the influence of env on transcription of gnb and gng are most obvious during early stages of light response. in contrast, the effect of phlp and gng on transcription of gnb can be detected also after early light response. we conclude that the influence of env on transcript levels of gnb and gng is likely to be mediated via its negative effect on phlp figure regulatory interrelationship of gnb gng and env at the level of transcription. gnb and gng show similar transcript patterns in the wildtype strain. gnb and gng transcript patterns in ∆env show an upregulation at early time points. strains were cultivated on glycerol as carbon source for 24 hours in darkness and then exposed to light for or 120 minutes, respectively. regulation of the output pathways by camp levels the camp pathway is considered the major output pathway of heterotrimeric g-protein signaling and would hence be likely to act as a coincidence detector between nutrient and light signaling. for t. reesei, an influence of the two heterotrimeric g-protein alpha subunits gna and gna on intracellular camp levels was observed. having established the node between nutrient and light signalling we now wanted to investigate the downstream pathway of this cascade. deletion of env is known to result in strongly decreased intracellular camp levels, which are likely to represent an output of the regulatory interconnection between light- and nutrient signalling established between env and phlp in the strain lacking acy camp production is abolished and consequently the situation resembles that in the strain lacking env hence, we performed microarray analysis of a deletion mutant in the gene encoding adenylate cyclase, which synthesizes camp. similar conditions to those used for previous transcriptome analysis of env i. e. growth on cellulose for 72 hours in light and darkness were applied. we compared the results with the transcriptome of Δenv in light and darkness. indeed, we found a considerable overlap of targets, interestingly only among targets in light and no overlapping targets were found in darkness. the genes up-regulated in light in Δenv and Δacy included one polyketide synthase, three putative g-protein coupled receptors and one transcription factor related to a n. crassa transcription factor responsive to light, as well as two sugar transporters. genes were underexpressed in light in Δenv and Δacy among them glycoside hydrolases including xyn xyn bxl cel3a, cel3b, egl egl egl cbh cel61a and cel61b as well as the genes encoding the auxiliary proteins swollenin, cip and cip and the cellulase and hemicellulase regulator gene xyr additionally, we found genes involved in sulphur metabolism, including the regulator gene lim1. further genes consistently regulated in Δenv and Δacy include two g-protein coupled receptors as well as six putative sugar transporters including the recently characterized lactose permease tr_ which is also important for cellulase gene expression on lactose. no contrasting regulation was observed. functional category analysis revealed that the functions of the consistently regulated genes are enriched in metabolism, especially amino acid metabolism, nitrogen and sulphur metabolism as well as c-compound and carbohydrate metabolism. in the latter group particular enrichment in polysaccharide metabolism was observed. additionally, transport functions of carbohydrates and amino acids were enriched. we conclude that a considerable portion of the core functions of env are regulated via the effect of env on camp levels. discussion the major influence of light on fungi was studied extensively in the last decades and it could be shown that light affects a broad spectrum of metabolic pathways, morphological changes, growth and secondary metabolism. since the discovery, that cellulase gene expression is modulated by light, studies unravelling the light signalling network influencing the cellulase gene expression were undertaken, which revealed a puzzle of components regulating the model output pathway of cellulase transcription in response to light. besides the intriguing insights into fungal physiology, these findings also provide perspectives for research towards elucidation of the interplay between light response and metabolism in higher organisms. the results presented here connect these components by placing them in the background of a signaling network and provide intriguingly new insights into the mechanism how light- and nutrient signalling are connected at the molecular level.figure schematic drawing of the current model for the interrelationship between nutrient and light signaling in t. reesei. the regulatory interplay between nutrient and light signalling involves a limb represented by components of the heterotrimeric g-protein pathway and a limb established by the crucial components of the light response machinery. central interactors of this connection are env and phlp the positive effect of phlp on env transcription in early light regulation may be responsible for negative regulation of heterotrimeric g-protein signalling by env the effect of env on gnb and gng is mediated by phlp investigation of the effect of constitutive activation of the g-alpha subunits gna and gna on regulation of phlp transcript levels showed that the nutrient signals transmitted by gna and gna impact phlp both the light signalling components as well as the factors of heterotrimeric g-protein pathway analyzed in this study were found to dampen light responsiveness of transcript abundance and are likely to constitute a light specific regulatory mechanism sustaining transcript levels of downstream genes relevant during growth in light. investigation of the camp pathway as depicted in this scheme was described earlier. arrows indicate positive influence while plungers indicate a negative effect. dashed lines show hypotheses deduced from results in other fungi. asterisks indicate an effect on light responsiveness of transcript levels. together with previous studies, the model we propose illustrates the predominantly negative impact of the light signalling machinery and in contrast a largely positive effect of phlp and other components of heterotrimeric g-protein signalling on regulation of nutrient utilization or enzyme gene transcription, respectively. hence, the interplay of a positive and a negative limb are proposed to establish a balanced output which integrates both light and nutrient signals. this mechanism is reminiscent of the positive and negative regulatory cycles triggering light response and circadian rhythmicity in n. crassa. the dissimilarity of the gnb transcript pattern from those of phlp and gng suggests that lack of phlp and gng has different consequences than lack of gnb hence the alterations seen cannot be exclusively due to perturbed complex formation of gnb and gng as supported by phlp in mammalian systems individual functions of g-beta subunits independent of the g-gamma subunit were reported, which may be one reason for this phenomenon. this hypothesis is supported by the finding that lack of gnb causes a slightly different phenotype than that of phlp or gng and the characteristic alterations in transcriptome data for a strain lacking gnb reflecting partially different targets. it has to be considered, that the regulatory interconnections revealed in this study must be mediated by transcription factors, which remain to be determined and might include the photoreceptors blr and blr due to the observed function of env via adjustment of camp levels it appears likely that this regulation also involves posttranslational modification and hence activation/deactivation of these transcription factors by phosphorylation. these transcription factors could either act directly on the target promotors or alternatively activate/deactivate further regulators. the latter hypothesis would be supported by the finding that in n. crassa regulation by wc- is mediated by a flat hierarchical transcription factor network. analysis of interaction partners of the signaling components investigated in this study will provide insight into the mechanisms involved in this regulation and is currently in progress in our lab. transcriptome analysis revealed a crucial position of env and gnb in the interrelationship of light response and nutrient signalling in t. reesei. interestingly, upon deletion of the respective genes, we saw a considerable increase in genes regulated by light from roughly 3% to more than or 30%, respectively. the crucial importance of env in both nutrient and light signalling is further reflected by the considerable number of regulatory targets distinct from those of the photoreceptor proteins. most of the genes differentially regulated in Δenv or Δgnb were downregulated in light, although in Δenv considerable upregulation also occurs. the huge impact of light on strains lacking env was already observed in earlier studies, but the extent of the regulatory function of env in light was still unexpected. also in strains lacking blr or blr considerable downregulation of transcript levels occurred. these findings led us to hypothesize that the light response machinery and particularly env sustains expression levels of genes responsible for environmental sensing and signal transmission in light, presumably also via phlp1-gnb1-gng as these components appear to only have few targets in darkness, this mechanism might not be operative in darkness, which raises the question whether a different pathway is responsible for such an effect in darkness. correlation of the majority of targets of phlp1-gnb1-gng with those of env along with the peculiar position of Δenv in the cluster analysis of mutant strains and its impact on regulation of g-alpha subunit genes as described earlier, places env upstream of the components of heterotrimeric g protein signalling. nevertheless, our new transcriptome data on Δacy also indicate that env plays an important role in the output pathway of the cascade and that the components of heterotrimeric g-protein signalling analysed here might just be a modulator of the pathway regulated by env future research will show if this hypothesis can be substantiated. evaluation of genome wide transcription patterns and subsequent detailed analysis of light response in several mutant strains enabled us to identify env and phlp as a central interlocked regulatory pair connecting the light response pathway with nutrient signalling. thereby, phlp acts positively on env transcription during early light response, which in turn has a negative effect on transcript abundance of blr and blr at this time. considering data on the effect of vvd on the wcc, env likely also supports inhibition of blr complex activity. env in turn consistently acts negatively on transcript levels of not only phlp but also gnb and gng this result is in accordance with earlier findings in n. crassa, showing that early light-responsive genes are mostly involved in primary measures to adjust to the light conditions, including protection from light, photoperception and cell signalling, while functions in c-compound and carbohydrate metabolism predominate among late light responsive genes. therefore our findings could be interpreted in a way that env dampens g-protein signalling during early light response by decreasing transcription of phlp gnb and gng in order to provide resources for protective measures. the initial positive action of phlp on env transcription enhances this effect. subsequently, the positive action of phlp on complex formation of the g beta and gamma subunits and hence g-protein signalling – which is likely to transmit nutritional signals - might be important for metabolic adaptation to light. in the context of the overlap of transcriptomes of deletion mutants in env phlp gnb and gng it becomes clear that this interrelationship is even more profound and extends beyond transient effects in early light response. as for the downstream pathway of the node between phlp and env we investigated to which extent the regulation of camp levels by env which is assumed to be accomplished by a negative effect on phosphodiesterase activity, is relevant for gene regulation by env as deletion of adenylate cyclase has a similar effect on camp levels as deletion of env, we performed microarray analysis of Δacy in light and darkness on cellulose for identification of camp dependent targets of env interestingly, we only detected an overlap of regulated genes in light, with most of the consistently regulated genes being upregulated. the functions of these genes are strongly enriched in metabolic functions – specifically carbon and sulphur/amino acid metabolism. interestingly, the sulphur source in the medium is crucial for the ability of t. reesei to utilize cellulose. without sulphate, growth of t. reesei on cellulose in light is severely perturbed and methionine cannot serve to replace sulphate as sulphur source under these conditions. our findings hence indicate that the interrelationship between sulphur and carbon metabolism is likely to involve the function of camp dependent mechanisms. this overlap in regulated genes in light is further in agreement with the retarded growth phenotype of t. reesei strains lacking env1, as retarded growth is also observed upon lack of acy1. however, the mechanism for regulation of growth by camp levels in t. reesei remains to be investigated in detail. knowledge on the overlap of genes regulated as a consequence of two different factors crucial for normal growth of t. reesei should enable insight in to the mechanism regulating growth in response to camp levels. in order to narrow down the number of candidate regulators, we checked whether n. crassa homologues of the target genes of env and acy in light, for which phenotype analyses of mutants are available, are known to be important for normal growth. abnormal growth patterns in knock outs were only found for homologues of tr_ and tr_. transcript levels of tr_ in Δacy and Δenv are only downregulated about 2fold. therefore, we consider it unlikely that this putative transporter might contribute to the severe growth defect of Δacy and Δenv n. crassa vib- is among other functions in cell recognition and programmed cell death, important for response to carbon starvation. the transcript abundance of tr_ is decreased fold in Δacy and more than 19fold in Δenv however, the vib- deletion mutant only shows a decreased linear growth of about 20% compared to wild-type. consequently, tr_ may be in part responsible for the retarded growth in response to reduced camp levels, but additional factors with more significant effects remain to be identified.  in summary, we found that a sizable amount of independent targets of env in light shows considerable overlap with targets of the heterotrimeric g-protein components phlp gnb and gng complementary investigation of early and late light response revealed that env represents a crucial node in light signal transduction and exerts its function in part via the regulatory interrelationship with the phosducin like protein phlp as well as gnb and gng this interaction between nutrient and light signalling is at least in part mediated by transcriptional interaction of env and phlp downstream targets of the cascade are to a considerable extent regulated via the function of env in modulation of camp levels. 
investigating the downstream part of the cascade we found support for the hypothesis that env is necessary for camp mediated regulation of a considerable part of the core functions of the output pathway of this cascade, including regulation of glycoside hydrolase genes and those involved in nitrogen, sulphur and amino acid metabolism. env and phlp are mutual regulators connecting light signaling with nutrient signaling, with env triggering the output pathway by influencing camp levels. we found that the targets of the light regulatory protein env in light show considerable overlap with those of the heterotrimeric g-protein components phlp gnb and gng detailed investigation of a regulatory interrelationship of these components with env under conditions of early and late light response indicated a transcriptional mutual regulation between phlp and env which appears to dampen nutrient signalling during early light response, presumably to free resources for protective measures prior to adaptation of metabolism to light. therefore we aimed to elucidate the interrelationship between nutrient and light signaling and how the light signal is transmitted to downstream pathways. this interrelationship of light response and nutrient sigalling is crucial for health and fitness. crosstalk between the signalling pathways responding to light–dark cycles and those triggering the adaptation of metabolism to the environment is known to occur in various organisms.
 crosstalk between the signalling pathways responding to light–dark cycles and those triggering the adaptation of metabolism to the environment is known to occur in various organisms. this interrelationship of light response and nutrient sigalling is crucial for health and fitness. the tropical ascomycete trichoderma reesei represents one of the most efficient plant cell wall degraders. regulation of the enzymes required for this process is affected by nutritional signals as well as other environmental signals including light. therefore we aimed to elucidate the interrelationship between nutrient and light signaling and how the light signal is transmitted to downstream pathways.  we found that the targets of the light regulatory protein env in light show considerable overlap with those of the heterotrimeric g-protein components phlp gnb and gng detailed investigation of a regulatory interrelationship of these components with env under conditions of early and late light response indicated a transcriptional mutual regulation between phlp and env which appears to dampen nutrient signalling during early light response, presumably to free resources for protective measures prior to adaptation of metabolism to light. investigating the downstream part of the cascade we found support for the hypothesis that env is necessary for camp mediated regulation of a considerable part of the core functions of the output pathway of this cascade, including regulation of glycoside hydrolase genes and those involved in nitrogen, sulphur and amino acid metabolism.  env and phlp are mutual regulators connecting light signaling with nutrient signaling, with env triggering the output pathway by influencing camp levels. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. issue-copyright-statement© the author 2014 