BACKGROUND
trichoderma reesei  represents a model system for investigation of plant cell wall degrading enzymes
<cit> .
especially with the current efforts to increase the efficiency and production of cellulase mixtures for economically competitive second generation biofuels, research towards plant cell wall degrading enzymes has gained increased attention
<cit> .
genetic engineering for strain improvement targets numerous pathways in t. reesei and mainly aims at increased production of cellulolytic enzymes
<cit> .
nutrient availability and utilization are crucial determinants for the survival of t. reesei in a natural habitat.
glycoside hydrolases are the main enzymes for this task as they have roles in degradation of biomass , but they also have functions in defence or pathogenesis and in routine cellular functions such as cell wall remodelling
<cit> .
the genome analysis of t. reesei <cit>  revealed a smaller enzyme set for plant cell wall degradation than expected and recent re-annotation of cazyme  genes updated the number of glycoside hydrolases to 201
<cit> .
nutrient degradation pathways are tightly regulated in order to ensure maximum efficiency with a minimum of resources used.
therefore t. reesei integrates various environmental signals, which result in an adjusted response to the current conditions in its surroundings
<cit> .
in recent years, light response emerged as an important reaction to the environment, which is not only applied in the natural habitats, but is still operative under artificial conditions in the lab or in a fermenter
<cit> .
in t. reesei, almost 3% of all genes are differentially regulated in light and darkness and these genes are enriched in functions of carbohydrate transport and metabolism
<cit> .
t. reesei possesses homologues of the two n. crassa photoreceptors white collar- <dig>  and white collar- <dig>  - two zinc-finger transcription factors, which usually act as a complex
<cit> .
the t. reesei homologues blr <dig> and blr <dig>  are involved in regulation of cellulase gene expression
<cit>  and to some extent in asexual and sexual development
<cit> .
the third n. crassa photoreceptor, vivid  is responsible for gating of light responses
<cit> , acts negatively on the white collar complex  and can act as a universal brake on light response
<cit> .
its orthologue in t. reesei, env <dig>  was found to have a profound effect on light dependent processes and signalling, including regulation of cellulase gene expression
<cit> .
however, despite a number of similar functions of vvd and env <dig>  also in cellulase regulation
<cit> , these factors are not functional homologues
<cit> .
in contrast to n. crassa strains lacking functional vvd
<cit> , deletion strains of env <dig> show a severe growth defect in light
<cit> .
additionally, env <dig> is essential for female fertility of t. reesei in light
<cit> , is assumed to influence camp turnover by a negative effect on phosphodiesterases and impacts regulation of the heterotrimeric g-protein pathway
<cit> .
investigation of the influence of blr <dig>  blr <dig> and env <dig> on efficiency of cellulose degradation under fermentation conditions revealed an influence of all three photoreceptors
<cit> .
the efficiency of the secreted enzyme mixture was enhanced in Δenv <dig>  while the higher cellulolytic activity in Δblr <dig> media was due to increased secretion capacity.
strains lacking blr <dig> did show enhanced biomass accumulation in the presence of cellulose, but production of hydrolytic enzymes was nevertheless weakest in this strain.
however, comparison of transcript levels of cellulases with cellulolytic activity in the culture filtrate or abundance of the respective protein there, revealed discrepancies, especially in light
<cit> .
hence, an influence of components involved in perception and transmission of the light signal could also be responsible for posttranscriptional and/or posttranslational regulation of enzyme production.
utilization of plant cell walls not only involves the uptake of the building blocks of cellulose, but also components released from hemicellulose, such as galactose or arabinose are encountered on this natural substrate and channelled into catabolic pathways
<cit> .
for catabolism of d-galactose, different pathways are used in fungi.
in the leloir pathway conversion of d-galactose in several steps to d-glucose-6-phosphate involves phosphorylation
<cit> .
the alternative oxidoreductive d-galactose pathway has the intermediates d-galactose, galactitol, l-xylo-3-hexulose, d-sorbitol and d-fructose
<cit> .
the enzymes involved in catabolism of d-galactose, aldose reductase xyl <dig>  galactitol dehydrogenase lad <dig>  l-xylo-3-hexulose reductase lxr <dig> and d-sorbitol dehydrogenase xdh <dig> also have functions in degradation of l-arabinose, with additional contribution of the l-xylulose reductase lxr3
<cit> .
in this study, we investigated genome wide transcriptional regulation by blr <dig>  blr <dig> and env <dig> and we aimed to evaluate different regulatory patterns caused by these factors.
we show that the recently discovered imbalance in light dependent gene regulation i. e. an increased number of genes regulated by light
<cit>  is even more pronounced in mutants lacking env <dig> and that around 75% of all genes encoding glycoside hydrolases of t. reesei are differentially regulated in light and darkness in wild-type or mutant strains.
we also found that catabolism of hemicellulase building blocks is subject to regulation by light, which is in part mediated by blr <dig>  blr <dig> and env <dig> with one genomic cluster being regulated by light upon growth on cellulose.
RESULTS
transcriptome analysis of gene regulation by env <dig>  blr <dig> and blr2
we studied differential regulation by env <dig>  blr <dig> and blr <dig> by microarray analysis in light and darkness upon growth on microcrystalline cellulose.
downregulation of the transcription of env <dig> and the photolyase gene phr <dig> in Δblr <dig> and Δblr <dig> in light as well as the regulation pattern of the cellobiohydrolase gene cbh2/cel6a in light and darkness and in Δenv <dig> are in accordance with earlier studies
<cit> .
additionally, qrt-pcr analysis done for previously reported studies with the same experimental setup
<cit>  and data on evaluation of the genes encoding enzymes involved in degradation of pentoses  was in agreement with microarray results and and hence confirm the validity of our results.
in order to gain insight into the impact of components of the light signalling machinery on light responsiveness  of transcript abundance, we compared differential gene regulation between light and darkness in Δenv <dig>  Δblr <dig> and Δblr <dig>  in the wildtype  <dig> % of all genes are regulated in response to light, but this percentage strongly increases upon deletion of the phosducin like protein encoding phlp <dig>  gnb <dig> or gng <dig>  up to 23%
<cit> .
intriguingly, deletion of blr <dig> or blr <dig> also causes the number of light-dependently regulated genes to increase , which indicates that strains lacking these photoreceptors are defective in proper regulation of light responsiveness or adaptation to constant light, but are not blind .
this finding suggests that photoreceptors do not exclusively act as a complex as was already suggested earlier
<cit>  and that additional factors are present in the genome which transmit light signals in the absence of the photoreceptors blr <dig>  blr <dig> and env <dig> as also shown for their orthologues in n. crassa <cit> .
in Δenv <dig> the number of light responsive genes strongly increases to  <dig> % of all genes of t. reesei  .
in all three strains, differential transcription between light and darkness was both positive and negative, which is in agreement with data from t. atroviride <cit> , but in contrast to n. crassa for which only positive regulation by photoreceptors was observed
<cit> .
additionally, we found a remarkably high number of glycoside hydrolase encoding genes and signal transduction components among those downregulated due to illumination, but also numerous transcription factors – particularly in Δenv <dig>  .
interestingly, the number of light responsive genes is higher in the Δenv <dig> mutant compared to the individual Δblr strains and also higher than both Δblr strains together.
this finding confirms earlier results on individual functions of env1
<cit>  and also functions independent of blr <dig> and blr <dig>
functions of genes with differential regulation in Δblr <dig>  Δblr <dig> and Δenv1
due to the considerably increased number of genes differentially regulated between light and darkness in Δblr <dig>  Δblr <dig> and Δenv <dig>  we were interested, which functions were targeted by this regulation.
in Δblr <dig> only  <dig> genes were upregulated by light and most of them only around 2fold, among them a putative sulphate permease  and two putative ß-glucosidases .
also in Δblr <dig> only  <dig> genes are upregulated in light, including an nmt1-like gene  putatively involved in thiamine biosynthesis and the pth <dig> type g-protein coupled receptor gene tr_ <dig>  only tr_ <dig> encoding an unknown hypothetical protein is upregulated in Δblr <dig> and Δblr <dig> in light compared to darkness.
in both photoreceptor mutants the majority of genes was downregulated in light  and exert diverse functions .
detailed analysis revealed that genes significantly enriched in the geneset down regulated in Δblr <dig> in light include metabolism , particularly c-compound and carbohydrate metabolism , secondary metabolism , abc-transporters , oxygen and radical detoxification  including catalase reaction and superoxide metabolism.
env1
, ∆
blr <dig>
and ∆
blr <dig>
genes down regulated in Δblr <dig> in light are significantly enriched for functions in transcription  and protein synthesis , but although numerous metabolic genes are regulated , no significant enrichment in this function was observed.
these obviously different functions of blr <dig> and blr <dig> are in agreement with earlier data in t. atroviride and n. crassa, which indicated independent roles of the two photoreceptors besides their function as complexes
<cit> .
for genes upregulated in light in Δenv <dig> we observed a significant enrichment in metabolic functions , especially in amino acid metabolism , c-compound and carbohydrate metabolism  and lipid, fatty acid and isoprenoid metabolism .
moreover, genes with functions in energy supply, oxidation of fatty acids and cellular transport, particularly c-compound and carbohydrate transport were significantly enriched.
with genes downregulated in Δenv <dig> again metabolic functions are significantly enriched , with strongest enrichment in c-compound and carbohydrate metabolism , but although metabolism of some amino acids is still enriched, the strong enrichment in amino acid metabolism in general as seen for upregulated genes is not obvious in this gene set.
further enrichment occurred with genes involved in sulphur metabolism and sulphate assimilation , cell cycle and dna processing .
from the latter functional group, also genes involved in dna recombination and repair, mitotic cell cycle and cell cycle control, cell division and septum formation/hydrolysis are significantly enriched among those downregulated in Δenv <dig>  which correlates with its strong growth and developmental defect in light
<cit> .
interestingly, also genes involved in translation are significantly enriched among the downregulated genes , which hints at a function of env <dig> in modulation of posttranscriptional regulation of gene expression as suggested for several genes in light in t. reesei <cit> .
additionally also cellular transport, including c-compound and carbohydrate transport, defence mechanisms, stress response, dna damage response, cellular sensing and response, development are enriched in this gene set.
in order to put these results into context, we searched in a hierarchical cluster analysis for genes upregulated in light in the wild-type but not in the mutant strains investigated in this study.
we found a cluster of  <dig> genes which fulfilled this criterion.
genes in this cluster were enriched in functions in metabolism  including nitrogen and sugar metabolism and secondary metabolism, which are also among the functions most elaborately regulated in all three mutants.
the influence of light on transcription of glycoside hydrolase encoding genes is in part mediated by env <dig>  blr <dig> and blr2
previously we showed that in signalling mutant strains of the heterotrimeric g-protein pathway, the number of genes differentially regulated in light and darkness increases and that this effect also concerns glycoside hydrolases
<cit> .
here, we found that deletion of env <dig>  blr <dig> or blr <dig> led to light dependent regulation of  <dig> glycoside hydrolase genes , partially overlapping with gh encoding genes already known to be potentially light regulated
<cit> .
together with those genes, which were shown to be regulated by light in mutants in the heterotrimeric g-protein pathway, in total  <dig> out of  <dig> genes  were found to be differentially regulated in light and darkness in the wildtype and/or in mutant strains.
among these  <dig> genes, all gh families are represented except gh family  <dig>  with its only member beta galactosidase .
however, inspection of transcript levels of the respective gene also differed between light and darkness up to  <dig> fold, which is only slightly below our threshold.
functions of env <dig> in darkness
previous studies showed that env <dig> not only has functions in light, but also in darkness
<cit> , which however remained elusive until now.
the transcriptome data confirmed this assumption and  <dig> genes were found to be specifically regulated more than 2fold  in darkness in Δenv <dig> .
only  <dig> genes were found to be exclusively downregulated in Δenv <dig> in darkness, including two putative fad dependent oxidoreductases , phosphoenolpyruvate carboxykinase tr_ <dig> and one predicted oligopeptide transporter .
those genes showing increased transcript abundance specifically in Δenv <dig> in darkness include  <dig> glycoside hydrolases  and tr_70845), the class ii hydrophobin encoding gene hfb <dig>  one candidate non-ribosomal peptide synthase gene , one catalase , blr <dig> and blr <dig>  moreover, three genes involved in sulphur metabolism  are upregulated.
in summary, the functions influenced by env <dig> in darkness correspond to a subset of functions subject to light response in t. reesei.
these results further reflect that env <dig> exerts individual functions not only in light, but also in darkness.
regulatory targets of blr <dig> and blr <dig> in darkness
in addition to differential regulation between growth in light and growth in darkness, we also studied altered gene regulation in the photoreceptor mutant strains compared to wild-type in light and darkness, which revealed light-dependent regulatory targets of these factors .
previous studies in t. reesei indicated that blr <dig> and blr <dig> not only have functions in light, but that they also regulate gene expression and metabolic functions in darkness
<cit> .
q-rt pcr analysis of the cellobiohydrolase cbh1/cel7a, for which the microarray reached the saturation limit, confirmed a regulatory function of blr <dig> and blr <dig> in darkness .
moreover, individual functions for the two photoreceptors homologues besides their activity as a complex were suggested in n. crassa <cit> .
we therefore analyzed which functions these photoreceptors target in darkness.
we found that in Δblr <dig>  <dig> genes are upregulated in darkness, among them  <dig> glycoside hydrolases including two alpha-glycosidases , two beta-glycosidases , one alpha galactosidase  and one chitinase .
moreover, seven genes involved in sulphur metabolism and  <dig> transporters are among the genes upregulated in Δblr <dig>   <dig> genes were downregulated in Δblr <dig> in darkness.
accordingly, functional category analysis identified c-compound and carbohydrate metabolism in the gene set upregulated in Δblr <dig> and metabolism of several amino acids among the genes down regulated in Δblr <dig> in darkness as significantly enriched .
genes exerting transport functions were significantly enriched in both gene sets in Δblr <dig>
in Δblr <dig>   <dig> genes are upregulated in darkness including  <dig> glycoside hydrolases, among them two beta glucosidases  and one l-arabinofuranosidase  and three transporters.
among the genes down-regulated in Δblr <dig>   <dig> genes involved in sulphur metabolism were found.
funcat analysis revealed significant enrichment in genes involved in polysaccharide and carbohydrate metabolism as well as transport functions among the genes up- or down-regulated in Δblr <dig> in darkness.
hence both photoreceptors play a role in alteration of carbohydrate metabolic functions and transport of compounds with distinct, both positive and negative targets in darkness.
shared regulatory targets of blr <dig>  blr <dig> and env1
light dependent induction of env <dig> transcription requires the presence of blr <dig> and blr2
<cit> .
in order to elucidate which target genes would be regulated by the complex assumed to be formed by blr <dig> and blr <dig> and transmitted involving the function of env <dig>  we screened for genes regulated similarly in Δenv <dig>  Δblr <dig> and Δblr <dig> .
while in darkness no overlap could be detected, we found  <dig> genes to be upregulated in all three mutants in light, which are concluded to represent genes negatively influenced by the blr1/blr <dig> complex via env <dig> in light.
among them were  <dig> glycoside hydrolase family  <dig> genes  as well as one putative zinc binuclear cluster transcription factor .
however, in contrast to the only  <dig> genes negatively influenced by blr <dig>  blr <dig> and env <dig>  we found the majority of common targets of these factors  to be positively regulated in light by the light signalling machinery.
although most of the genes in this group are of unknown function, major targets appear to be the glycoside hydrolases with  <dig> members of diverse families found in this group.
additionally, three putative transcription factors  and two g protein coupled receptors,  were downregulated in all three mutants in light, suggesting altered signal perception as well as output on regulatory targets.
the finding of six genes involved in sulphur metabolism  including the e <dig> ubiquitin ligase lim1
<cit>  supports the hypothesis of a role of this process in light-dependent modulation of gene expression in t. reesei.
additionally, the finding of one hydrophobin gene in this group  is also not without precedent
<cit> .
regulation by a potential negative effect of env <dig> on the blr complex
for n. crassa,  <dig> genes were found to be up-regulated in Δvvd and down-regulated in the white-collar mutants upon growth on cellulose in light, which were significantly enriched in c-compound and carbohydrate metabolism.
hence, carbon metabolism was suggested to be subject to photoadaptation in n. crassa <cit> .
in order to evaluate this hypothesis for t. reesei, we also screened for genes down-regulated due to the lack of a functional photoreceptor complex , but upregulated in Δenv <dig>  lack of env <dig> is in this case assumed to result in increased transcription of genes positively regulated by blr <dig> and blr <dig>  we only found  <dig> genes of those downregulated in Δblr <dig> and Δblr <dig> in light  to be up-regulated in Δenv <dig>  interestingly, these included  <dig> genes involved in pheromone processing and sexual development, two photolyases as well as two transcription factors .
however, no enrichment in carbon metabolic functions as seen for n. crassa was observed.
consequently, in contrast to n. crassa, carbon metabolism appears to be regulated by the influence of blr <dig> and blr <dig> on induction of env <dig> rather than the assumed negative effect of env <dig> on the function of the blr complex in light.
additionally, this discrepancy may reflect the regulatory differences of env <dig> and vvd, which are no functional homologues
<cit> .
distinct targets of the blr1/blr <dig> complex versus env1
despite considerable overlap in their major targets in light with env <dig>  the common targets of blr <dig> and blr <dig>  are distinct from those of env <dig> to a certain extent .
<dig> genes were found to be upregulated in light compared to the wildtype in both Δblr <dig> and Δblr <dig> but not in Δenv <dig>  including one transcription factor ,  <dig> genes involved in sulphur metabolism , one pth11-type gpcr , one polyketide synthase  and one glycoside hydrolase family  <dig> gene .
<dig> genes represent positive targets of the blr1/blr2-complex in light, among them were one polyketide synthase from pks orthologous group 3
<cit>   and one non-ribosomal peptide synthase  as well as  <dig> glycoside hydrolases.
interestingly, we also detected  <dig> transcription factors positively regulated by blr <dig> and blr <dig> but not by env <dig> including one transcription factor  reported to be responsive to light in the absence of major photoreceptors
<cit> .
env <dig> triggers gene regulation also independently of blr1/blr2
the results described above led to the question, which output pathways would be regulated by env <dig> independently of blr1/blr <dig> in light.
more than 55%  of all genes downregulated and even 92%  of those upregulated in a mutant lacking env <dig> are not targets of either blr <dig> or blr <dig> .
moreover, the negative effect of env <dig> in light is much more widespread than that of blr <dig> or blr <dig>  consequently, a function for env <dig> distinct of that of blr <dig> and blr <dig> in light can be assumed.
analysis of negative targets of env <dig> in light revealed an influence on  <dig> putative transcription factors, including two transcription factors  described to be light responsive in the absence of the major photoreceptors in n. crassa <cit> .
moreover,  <dig> photolyases , 5 g protein coupled receptors ,  <dig> genes involved in secretion ,  <dig> genes involved in secondary metabolism  and  <dig> glycoside hydrolase genes were negatively regulated by env <dig>  genes influenced positively by env <dig>  but not by blr <dig> or blr <dig> in light include  <dig> putative transcription factors, among them hap <dig>  encoding an important regulator of cellulase gene expression
<cit> .
additionally,  <dig> genes involved in sulphur metabolism, eight g protein coupled receptors , nine genes involved in secretion, two polyketide synthases , three hydrophobin genes  and most intriguingly, nine glycoside transferase and  <dig> glycoside hydrolase genes are enhanced by env <dig> in light.
we conclude that env <dig> represents a key factor in light-dependent regulation of gene expression, the major effect of which is not exerted concertedly with the blr1/blr <dig> complex.
the considerable number of target genes with functions in carbon utilization and nutrient signal perception suggests a crucial function in interconnecting nutrient with light signalling.
coregulation of genes with the major cellulases
genes regulated consistently under different conditions often participate in the same function.
therefore, investigation of co-regulated genes can help to assign a putative function to unknown genes or reveal processes related to each other
<cit> .
for identification of genes and/or processes involved in the light signalling pathway that modulates the cellulase gene transcription pattern, we examined a cluster of  <dig> genes that are co-regulated with cel6a/cbh <dig> in all of the light affected mutant strains and their parental strain qm <dig>  cel7a/cbh <dig>  the major cellobiohydrolase, is co-regulated with cel6a/cbh <dig>  but due to saturation of the microarray signal for this transcript, we were not able to use the transcript pattern of cel7a/cbh <dig> to evaluate coregulated genes of cellulases.
to confirm that the transcription pattern of cel6a/cbh <dig> in the mutants resembles that of cel7a/cbh <dig>  we performed qrt-pcr, which confirmed coregulation .
we consequently used cel6a/cbh <dig> as representative gene for evaluation of genes coregulated with cellobiohydrolases in the presence or absence of blr <dig>  blr <dig> or env <dig> as described above in light and darkness .
the gene set of the cel6a/cbh <dig> cluster comprised  <dig> more glycoside hydrolase family genes, a swollenin , which was shown to disrupt the structure of cellulosic materials
<cit> , two carbohydrate esterase family genes , cip <dig> and cip <dig> - each containing a carbohydrate-binding module and were previously shown to be co-expressed with cellulases and represent “novel” types of cellulases
<cit> , a flavohemoglobin , a gaba permease  and two potential transcription factors .
in addition, two hypothetical proteins, a wd40-repeat containing protein  and a hypothetical g protein coupled receptor  were also coregulated with cel6a/cbh <dig>  notably, the xylanase regulator  <dig> encoding gene xyr <dig> was also shown to be coregulated with cel7a/cbh <dig> and cel6a/cbh <dig>  which is in agreement with earlier data, because xyr <dig> influences cellulase gene transcription positively and correlates with cellulase transcription levels
<cit> .
however, positive regulation of xyr <dig> by light has not been observed upon growth on lactose
<cit>  and appears to be specific to cellulose, which also suggests differential light dependent regulation of plant cell wall degrading enzymes on these carbon sources.
indeed, previous studies of t. reesei grown on cellulose or lactose showed that the effect of light on cellulase gene expression is positive on cellulose
<cit>  and negative on lactose
<cit> .
light impacts pentose and d-galactose metabolism
the considerable regulation of glycoside hydrolases and hence of mechanisms involved in substrate degradation by env <dig> led us to re-evaluate data on growth patterns for various carbon sources in more detail
<cit> .
the results for growth on  <dig> carbon sources in constant light and constant darkness of the parental strain and the env1-non functional strain env1pas– <cit>  were rearranged and used for hierarchical cluster analysis.
env1pas– showed considerably weaker growth in light than in darkness on most carbon sources, as could be expected from its published growth defect in light
<cit>  .
the growth of the wildtype was enhanced in light in a cluster of  <dig> different carbon sources including d-sorbitol, l-arabinose, d-fructose, d-galactose and xylitol in light, while the env <dig> mutant strain showed a considerably lower growth rate in light on these carbon sources .
hence, the positive influence of light on growth on these carbon sources is likely to be at least in part mediated by env <dig>
<cit> .
hierarchical cluster analysis was performed using hce  <dig>  with default settings.
standard deviations shown result from three biological replicates.
values are represented as percent.
ll means constant light, dd means constant darkness.
d-galactose and pentose catabolism are regulated by light on cellulose
interestingly, this cluster of carbon sources with env <dig> dependent enhanced regulation by light comprises several carbon sources of the d-galactose and pentose catabolism .
the respective enzymes are assumed to be involved in metabolism of hemicellulose degradation products
<cit> .
considering coregulation of many plant cell wall degrading enzymes upon detection of an inducing substrate
<cit> , a regulation of these pathways also on cellulose seemed reasonable and was indeed observed with the microarray data of this study, albeit very low expression levels for lxr <dig>  lxr <dig> and lad <dig> as well as only small differences in transcript levels necessitated independent confirmation of these data.
consequently, we evaluated the microarray data by qrt-pcr for the key components of oxidoreductive pentose and d-galactose pathway with respect to transcriptional regulation by light and/or the photoreceptors .
transcription of all genes involved in catabolism of d-galactose and l-arabinose  except xki <dig> showed a statistically significant increase in light in the wildtype .
this indicates that the cascade starting from d-galactose or l-arabinose and d-xylose, respectively is positively affected by light during growth on cellulose.
besides the leloir pathway, an oxidoreductive pathway was identified in t. reesei and many of the enzymes involved are also part of the l-arabinose and d-xylose pathway
<cit> .
the degradation of the hemicellulosic sugars d-xylose, l-arabinose and d-galactose starts with the same reaction: an nadph-linked reduction by an aldose reductase xyl1
<cit> .
the next step, the oxidation of galactitol or l-arabitol is catalyzed by lad <dig>  the l-arabitol-4-dehydrogenase
<cit> .
recently it was shown that lxr <dig>  encoding an l-xylo-3-hexulose reductase is the missing link in the oxidoreductive d-galactose catabolism in t. reesei <cit> .
the last step of the d-galactose degradation before glycolysis is the mediated by a nad-xylitol dehydrogenase xdh <dig>  which also catalyzes the conversion from xylitol to d-xylulose in the l-arabinose and d-xylose pathways
<cit> .
the last phosphorylation step of the l-arabinose and d-xylose pathway before the substance enters the pentose phosphate pathway is presumably done by the gene product of xki <dig>  a xylulose kinase and a homolog of aspergillus niger xkia <cit> .
differential expression between constant light and constant darkness upon growth on cellulose was abolished upon lack of blr <dig> and blr <dig> for all genes investigated, except xki <dig>  which does not show regulation by light in the wild-type .
transcript abundance predominantly drops to darkness levels in Δblr <dig> and Δblr <dig>  hence, blr <dig> and blr <dig> are concluded to be necessary for positive regulation of this pathway in light.
for env <dig> the situation is more complex.
in the absence of env <dig>  differential expression between light and darkness is abolished for lxr <dig> , lad <dig> and xdh <dig>  in contrast, the difference between transcript levels in light and darkness becomes even more pronounced for lxr <dig> and xyl <dig>  which is due to a strong upregulation of lxr <dig> and of a considerable down-regulation of xyl <dig> in light.
down-regulation of xki <dig> in light upon lack of env <dig> results in differential expression of xki <dig> in this strain, which was not observed in the wild-type.
this effect is also one example, how lack of adaptation as mediated by env <dig>  blr <dig> or blr <dig> could result in an increased number of genes differentially transcribed between light and darkness.
these results are in complete agreement with the results of the biolog analysis, since lack of env <dig> causes decreased transcription of most of the enzymes in the pathway in light .
consequently, while the data for individual genes and growth on individual carbon sources for wild-type and mutant strains only show minor differences in dependence of light , consistent regulation of growth and transcript abundance of the genes encoding enzymes of the oxidoreductive pentose and d-galactose pathway strongly indicates that the response of t. reesei to hemicellulose building blocks is modulated by photoreceptors in response to light.
a meme search using the online version  <dig> .
<dig>  did not reveal a common sequence in these promotors related to known light responsive motifs.
according to the flat hierarchical network as proposed for photoreceptors n. crassa <cit> , the effect seen here is likely to be exerted indirectly.
for xyl <dig>  regulation by the cellulase and hemicellulase regulator xyr <dig> was shown
<cit> .
we therefore screened all gene promoters of the pathway for xyr1-binding sites and only xdh <dig> has a xyr <dig> binding site.
as xyr <dig>  similarly to xdh <dig> and xyl <dig> is positively regulated by env <dig> in light, this regulation might be mediated by xyr <dig>
lxr <dig> is part of a env <dig> regulated cluster in light
while initially assumed to be an l-xylulose reductase as it catalyzed the nadph/nadp + specific reactions for l-xylulose/xylitol and for d-fructose/d-mannitol
<cit> , lxr <dig> later turned out to be a mannitol dehydrogenase
<cit> .
on d-mannitol, our growth analysis showed a slightly slower growth of the wild-type in light.
due to its additional activity on fructose
<cit> , lxr <dig> could also be involved in the increased growth of the wild-type and the decreased growth in the env <dig> mutant on this carbon source in light .
investigation of the genomic region around lxr <dig> revealed that  <dig> genes in its genomic vicinity are regulated negatively  by env <dig> in light upon growth on cellulose and hence form a cluster .
blr <dig> and blr <dig> do not influence the regulation of the cluster.
in contrast to the effect upon growth on mannitol, env <dig> has a strongly negative effect on lxr <dig> and the genes in its cluster upon growth on cellulose .
the cluster is located at scaffold  <dig> between position  <dig> –  <dig> and comprises besides lxr <dig> also a zn-cluster transcription factor , a putative oligopeptide transporter , two probable old yellow enzyme  family flavin oxidoreductases , a putative gtp cyclohydrolase  and a proline oxidase/dehydrogenase involved in conversion of proline for use as a carbon and nitrogen source .
two of these genes, lxr <dig> and tr_ <dig> have homologues in n. crassa , which are responsive to light .
however, neither these nor the other members of this cluster are syntenic in n. crassa.
within the borders of the env1-regulated lxr1-cluster, a light regulated cluster starts , which comprises tr_ <dig>  tr <dig>  as well as a taurine dioxigenase involved in sulphur metabolism  and additionally contains a gatase1-like  domain containing peptidase , which is located outside the lxr <dig> cluster.
consistent targets of photoreceptors in n. crassa and t. reesei
previous studies indicated at least in part comparable regulation of cellulase gene expression by light and photoreceptors in t. reesei and n. crassa <cit> .
we were hence interested, whether the regulatory targets of photoreceptors in these two fungi are similar.
therefore, we re-analyzed the transcriptome data for n. crassa wild-type as well as Δwc- <dig>  Δwc- <dig> and Δvvd upon growth on cellulose in light  for significant 2fold differential regulation in order to be comparable with our data.
<dig> genes were found to be differentially regulated in one or more of the n. crassa photoreceptor mutants and are hence photoreceptor targets in light.
<dig> of these genes have reciprocal best hits  in t. reesei and are therefore likely homologues.
<dig> genes were identified as consistent targets of one or more photoreceptors in n. crassa and t. reesei .
in agreement with previous findings, these genes were enriched in functions of metabolism , particularly c-compound and carbohydrate metabolism  and sugar, glucoside, polyol and carboxylate metabolism .
additionally, functions in c-compound and carbohydrate transport  and glycolysis and gluconeogenesis  were enriched.
among the consistent photoreceptor targets are further five glycoside hydrolases , tr <dig> , tr_ <dig> , tr_ <dig> , tr_ <dig> ), one carbohydrate esterase  and two genes involved in sexual development  including the gene encoding the alpha-type peptide pheromone precursor ppg- <dig> which was shown to impact cellulase regulation in n. crassa <cit> .
two putative sugar transporters  and one putative carboxylic acid transporter  were also among these genes.
although functions in sulphur and amino acid metabolism were not enriched among these  <dig> genes, two putative methionine synthases  as well as one methionine permease  were consistently regulated.
moreover, two transcription factors were found to be consistent targets: tr_ <dig>  which has not yet been characterized and tr_ <dig>  the homologue of tah- <dig>  which is involved in conidiophore development
<cit>  and shows increased transcript levels upon growth on miscanthus
<cit>  in n. crassa.
tr_ <dig> is part of the lxr <dig> cluster described above and intriguingly, also lxr <dig> itself is among the consistently regulated genes.
however, the well characterized cellulase regulators of t. reesei and n. crassa, such as xyr <dig>  cre <dig>  clr- <dig> or clr- <dig> are not among the consistently regulated genes.
discussion
in this study we investigate the light signalling machinery of t. reesei at a genome wide level upon growth on cellulose, which is the closest defined carbon source to what t. reesei encounters in its natural habitat.
we extend previous knowledge on the function of photoreceptors, which was mainly done on glucose and in constant light, with investigating the effect of the photoreceptors in darkness.
this enabled us to study both differential gene regulation between growth in light and darkness  and investigation of regulatory targets  of blr <dig>  blr <dig> and env <dig> in light and darkness.
although only few genes are regulated by the photoreceptors in darkness, a clear role in carbon metabolism and transport functions could be detected in darkness.
these functions resemble also the targets in light, indicating that regulation by photoreceptors is not strictly light dependent.
one of the most intriguing and puzzling findings of this study was the increased number of differentially transcribed genes in the photoreceptor mutants, as we rather expected the opposite effect.
a similar phenomenon was observed for mutants in the g-protein pathway
<cit> .
one explanation for the reason that so many genes are light-regulated in the photoreceptor mutants might be a lack of light adaptation in the photoreceptor mutants: in the comparison between growth in constant darkness and in constant light, many transcripts might appear unchanged due to adaptation to light.
however upon deletion of blr1/blr <dig> or env, both light induction and adaptation are assumed to be lost, which would cause decreased/altered transcript levels in the mutants.
indeed, evaluation of the regulatory targets of blr <dig>  blr <dig> and env <dig> revealed the highest numbers of regulated transcripts to be downregulated in the mutant strains in constant light , which supports this hypothesis.
while we cannot provide a mechanistic explanation yet, our working hypothesis currently involves a higher order regulation to be targeted by the photoreceptors such as chromatin remodelling, which was shown to be impacted by light and the clock
<cit> .
this would explain the wide-spread effect we see and it would be in agreement with a rather tight regulation in the wild-type.
nevertheless, also such a mechanism would require an additional, so far unidentified factor to transmit the light signal in the absence of photoreceptors.
recently, investigation of the effect of photoreceptors on cellulase gene expression in n. crassa revealed that this process is subject to photoadaptation in this fungus.
additionally, the known cellulase repressor gene ace <dig>  the carbon catabolite repressor gene cre <dig> and the cross pathway control protein encoding cpc <dig>  which is involved in response to amino acid starvation response, were found to be regulated by photoreceptors in n. crassa <cit> .
in t. reesei however, the situation appears to be different.
we could identify the cellulase and hemicellulase regulator gene xyr <dig> to be a target of blr <dig> and env <dig> on cellulose, whereas n. crassa xyr- <dig> is not regulated by photoreceptors.
also the t. reesei homologues of clr- <dig> and clr- <dig> <cit> , two recently identified n. crassa cellulase regulators,  are subject to regulation by photoreceptors, although they are not light regulated in the wild-type.
additionally, neither ace <dig> nor cre <dig> or cpc <dig> are among the targets of the light signalling machinery in t. reesei.
hence, despite largely similar global processes to be targeted by blr <dig>  blr <dig> and env <dig>  such as carbon-, amino acid- and sulphur metabolism, the regulation of the involved pathways seems to be achieved differently in n. crassa and t. reesei.
this finding is also in agreement with consistent regulation of metabolic genes but not the respective known regulators between n. crassa and t. reesei in light.
also the effects on glycogen metabolism observed in n. crassa were not obvious for t. reesei.
however, the general influence of photoreceptors on carbon and amino acid metabolism as well as its assumed correlation with cellulase gene expression
<cit>  is clearly conserved in t. reesei and n. crassa.
the results of this study illustrate an important phenotypic characteristic of strains lacking env <dig>  these strains show severely reduced growth rate and conidiation, both in liquid media and on plates in light
<cit> .
this defect is clearly reflected in the strongly altered transcriptome of this strain in light compared to darkness.
in the absence of env <dig>  considerable alterations involving both positive and negative regulation of metabolic genes – especially those in carbon and amino acid metabolism – as well as genes for substrate transport and energy metabolism occurs.
hence env <dig> is of crucial importance for adjustment of metabolism to changing light conditions.
so far we could not identify the reason for the diminished growth rate of Δenv <dig> under the conditions we used and hence effects besides metabolic imbalance may influence the phenotype of this strain.
additionally, it was found that growth rate does not necessarily correlate with production of hydrolytic enzymes in t. reesei .
despite its slow growth, specific cellulase activity secreted into the medium by Δenv <dig> is several fold increased compared to wild-type .
we conclude that env <dig> is an essential regulator/signal transmitter for adjustment of growth rate to environmental conditions in light.
this function, however, is most important on cellulose, as the biolog analysis on numerous carbon sources  showed that the light dependent growth defect of Δenv <dig> does not occur on every carbon source.
therefore, it can be assumed that the function of env <dig> in light is most critical upon growth on cellulose, but less important for growth in the presence of other carbon sources in light.
to complement our transcriptome analysis for growth on cellulose, we compared the growth patterns on different carbon sources in light and darkness for wild-type and an env <dig> non functional strain
<cit> , because of the strong effect of env <dig> on light dependent gene regulation.
earlier analysis had indicated that the degradation of different carbon sources is regulated in a light dependent manner and that env <dig> is involved in the transfer of the positive light signal in many cases
<cit> .
our re-evaluation and cluster analysis showed that especially intermediates of the oxidoreductive d-galactose and l-arabinose pathway , which is also important for utilization of lactose, are among the carbon sources on which growth in the wild-type and the env <dig> mutant were most clearly influenced.
due to the ability of t. reesei to grow and express cellulases on lactose, the degradation of lactose is very well investigated
<cit> .
in eukaryotes lactose is first cleaved to glucose and galactose and galactose is further converted into glucose 6-phosphate by enzymes of the leloir pathway.
together with arabinose, also catabolism of galactose is involved in utilization of hemicellulose building blocks
<cit> .
as for the most part, plant cell wall degrading enzymes are coregulated in t. reesei <cit> , this is in accordance with our finding that the genes encoding the enzymes operative in these pathways
<cit>  are expressed upon growth on cellulose.
these findings are in agreement with the hypothesis that t. reesei senses the presence of plant cell wall material in its environment by detection of building blocks of cellulose and hemicellulose.
interpretation of the respective signals is subsequently adjusted to the requirements in light and darkness by blr <dig>  blr <dig> and env <dig>
CONCLUSIONS
in summary, we showed that blr <dig>  blr <dig> and env <dig> exert important metabolic functions, not only in light, but also in darkness.
lack of components of the light signalling machinery causes considerably increased light responsiveness of transcript levels, likely caused by a light-dependent, positive effect on output pathways.
thereby, the photoreceptors blr <dig> and blr <dig> do not exclusively act as a complex, but additionally have individual targets.
this finding also applies to env <dig>  for which the transcriptome pattern indicates a sizable amount of independent targets in light.
interestingly, the targets of the light response machinery also include the catabolic enzymes necessary for degradation of hemicellulose building blocks i. e. the d-galactose and pentose pathway and lxr <dig>  which adds a new aspect to light dependent gene regulation on cellulose.
comparison with transcript profiles of n. crassa indicates a conserved adjustment of metabolic pathways in light by photoreceptors, but also different regulatory mechanisms applied in order to achieve this effect.

 trichoderma reesei represents a model system for investigation of plant cell wall degrading enzymes. especially with the current efforts to increase the efficiency and production of cellulase mixtures for economically competitive second generation biofuels, research towards plant cell wall degrading enzymes has gained increased attention. genetic engineering for strain improvement targets numerous pathways in t. reesei and mainly aims at increased production of cellulolytic enzymes. nutrient availability and utilization are crucial determinants for the survival of t. reesei in a natural habitat. glycoside hydrolases are the main enzymes for this task as they have roles in degradation of biomass, but they also have functions in defence or pathogenesis and in routine cellular functions such as cell wall remodelling. the genome analysis of t. reesei revealed a smaller enzyme set for plant cell wall degradation than expected and recent re-annotation of cazyme genes updated the number of glycoside hydrolases to 201. nutrient degradation pathways are tightly regulated in order to ensure maximum efficiency with a minimum of resources used. therefore t. reesei integrates various environmental signals, which result in an adjusted response to the current conditions in its surroundings. in recent years, light response emerged as an important reaction to the environment, which is not only applied in the natural habitats, but is still operative under artificial conditions in the lab or in a fermenter. in t. reesei, almost 3% of all genes are differentially regulated in light and darkness and these genes are enriched in functions of carbohydrate transport and metabolism. t. reesei possesses homologues of the two n. crassa photoreceptors white collar- and white collar- - two zinc-finger transcription factors, which usually act as a complex. the t. reesei homologues blr and blr are involved in regulation of cellulase gene expression and to some extent in asexual and sexual development. the third n. crassa photoreceptor, vivid is responsible for gating of light responses, acts negatively on the white collar complex and can act as a universal brake on light response. its orthologue in t. reesei, env was found to have a profound effect on light dependent processes and signalling, including regulation of cellulase gene expression. however, despite a number of similar functions of vvd and env also in cellulase regulation, these factors are not functional homologues. in contrast to n. crassa strains lacking functional vvd, deletion strains of env show a severe growth defect in light. additionally, env is essential for female fertility of t. reesei in light, is assumed to influence camp turnover by a negative effect on phosphodiesterases and impacts regulation of the heterotrimeric g-protein pathway. investigation of the influence of blr blr and env on efficiency of cellulose degradation under fermentation conditions revealed an influence of all three photoreceptors. the efficiency of the secreted enzyme mixture was enhanced in Δenv while the higher cellulolytic activity in Δblr media was due to increased secretion capacity. strains lacking blr did show enhanced biomass accumulation in the presence of cellulose, but production of hydrolytic enzymes was nevertheless weakest in this strain. however, comparison of transcript levels of cellulases with cellulolytic activity in the culture filtrate or abundance of the respective protein there, revealed discrepancies, especially in light. hence, an influence of components involved in perception and transmission of the light signal could also be responsible for posttranscriptional and/or posttranslational regulation of enzyme production. utilization of plant cell walls not only involves the uptake of the building blocks of cellulose, but also components released from hemicellulose, such as galactose or arabinose are encountered on this natural substrate and channelled into catabolic pathways. for catabolism of d-galactose, different pathways are used in fungi. in the leloir pathway conversion of d-galactose in several steps to d-glucose-6-phosphate involves phosphorylation. the alternative oxidoreductive d-galactose pathway has the intermediates d-galactose, galactitol, l-xylo-3-hexulose, d-sorbitol and d-fructose. the enzymes involved in catabolism of d-galactose, aldose reductase xyl galactitol dehydrogenase lad l-xylo-3-hexulose reductase lxr and d-sorbitol dehydrogenase xdh also have functions in degradation of l-arabinose, with additional contribution of the l-xylulose reductase lxr3. in this study, we investigated genome wide transcriptional regulation by blr blr and env and we aimed to evaluate different regulatory patterns caused by these factors. we show that the recently discovered imbalance in light dependent gene regulation i. e. an increased number of genes regulated by light is even more pronounced in mutants lacking env and that around 75% of all genes encoding glycoside hydrolases of t. reesei are differentially regulated in light and darkness in wild-type or mutant strains. we also found that catabolism of hemicellulase building blocks is subject to regulation by light, which is in part mediated by blr blr and env with one genomic cluster being regulated by light upon growth on cellulose.  transcriptome analysis of gene regulation by env blr and blr2 we studied differential regulation by env blr and blr by microarray analysis in light and darkness upon growth on microcrystalline cellulose. downregulation of the transcription of env and the photolyase gene phr in Δblr and Δblr in light as well as the regulation pattern of the cellobiohydrolase gene cbh2/cel6a in light and darkness and in Δenv are in accordance with earlier studies. additionally, qrt-pcr analysis done for previously reported studies with the same experimental setup and data on evaluation of the genes encoding enzymes involved in degradation of pentoses was in agreement with microarray results and and hence confirm the validity of our results. in order to gain insight into the impact of components of the light signalling machinery on light responsiveness of transcript abundance, we compared differential gene regulation between light and darkness in Δenv Δblr and Δblr in the wildtype % of all genes are regulated in response to light, but this percentage strongly increases upon deletion of the phosducin like protein encoding phlp gnb or gng up to 23%. intriguingly, deletion of blr or blr also causes the number of light-dependently regulated genes to increase, which indicates that strains lacking these photoreceptors are defective in proper regulation of light responsiveness or adaptation to constant light, but are not blind. this finding suggests that photoreceptors do not exclusively act as a complex as was already suggested earlier and that additional factors are present in the genome which transmit light signals in the absence of the photoreceptors blr blr and env as also shown for their orthologues in n. crassa. in Δenv the number of light responsive genes strongly increases to % of all genes of t. reesei. in all three strains, differential transcription between light and darkness was both positive and negative, which is in agreement with data from t. atroviride, but in contrast to n. crassa for which only positive regulation by photoreceptors was observed. additionally, we found a remarkably high number of glycoside hydrolase encoding genes and signal transduction components among those downregulated due to illumination, but also numerous transcription factors – particularly in Δenv. interestingly, the number of light responsive genes is higher in the Δenv mutant compared to the individual Δblr strains and also higher than both Δblr strains together. this finding confirms earlier results on individual functions of env1 and also functions independent of blr and blr functions of genes with differential regulation in Δblr Δblr and Δenv1 due to the considerably increased number of genes differentially regulated between light and darkness in Δblr Δblr and Δenv we were interested, which functions were targeted by this regulation. in Δblr only genes were upregulated by light and most of them only around 2fold, among them a putative sulphate permease and two putative ß-glucosidases. also in Δblr only genes are upregulated in light, including an nmt1-like gene putatively involved in thiamine biosynthesis and the pth type g-protein coupled receptor gene tr_ only tr_ encoding an unknown hypothetical protein is upregulated in Δblr and Δblr in light compared to darkness. in both photoreceptor mutants the majority of genes was downregulated in light and exert diverse functions. detailed analysis revealed that genes significantly enriched in the geneset down regulated in Δblr in light include metabolism, particularly c-compound and carbohydrate metabolism, secondary metabolism, abc-transporters, oxygen and radical detoxification including catalase reaction and superoxide metabolism. env1, ∆ blr and ∆ blr genes down regulated in Δblr in light are significantly enriched for functions in transcription and protein synthesis, but although numerous metabolic genes are regulated, no significant enrichment in this function was observed. these obviously different functions of blr and blr are in agreement with earlier data in t. atroviride and n. crassa, which indicated independent roles of the two photoreceptors besides their function as complexes. for genes upregulated in light in Δenv we observed a significant enrichment in metabolic functions, especially in amino acid metabolism, c-compound and carbohydrate metabolism and lipid, fatty acid and isoprenoid metabolism. moreover, genes with functions in energy supply, oxidation of fatty acids and cellular transport, particularly c-compound and carbohydrate transport were significantly enriched. with genes downregulated in Δenv again metabolic functions are significantly enriched, with strongest enrichment in c-compound and carbohydrate metabolism, but although metabolism of some amino acids is still enriched, the strong enrichment in amino acid metabolism in general as seen for upregulated genes is not obvious in this gene set. further enrichment occurred with genes involved in sulphur metabolism and sulphate assimilation, cell cycle and dna processing. from the latter functional group, also genes involved in dna recombination and repair, mitotic cell cycle and cell cycle control, cell division and septum formation/hydrolysis are significantly enriched among those downregulated in Δenv which correlates with its strong growth and developmental defect in light. interestingly, also genes involved in translation are significantly enriched among the downregulated genes, which hints at a function of env in modulation of posttranscriptional regulation of gene expression as suggested for several genes in light in t. reesei. additionally also cellular transport, including c-compound and carbohydrate transport, defence mechanisms, stress response, dna damage response, cellular sensing and response, development are enriched in this gene set. in order to put these results into context, we searched in a hierarchical cluster analysis for genes upregulated in light in the wild-type but not in the mutant strains investigated in this study. we found a cluster of genes which fulfilled this criterion. genes in this cluster were enriched in functions in metabolism including nitrogen and sugar metabolism and secondary metabolism, which are also among the functions most elaborately regulated in all three mutants. the influence of light on transcription of glycoside hydrolase encoding genes is in part mediated by env blr and blr2 previously we showed that in signalling mutant strains of the heterotrimeric g-protein pathway, the number of genes differentially regulated in light and darkness increases and that this effect also concerns glycoside hydrolases. here, we found that deletion of env blr or blr led to light dependent regulation of glycoside hydrolase genes, partially overlapping with gh encoding genes already known to be potentially light regulated. together with those genes, which were shown to be regulated by light in mutants in the heterotrimeric g-protein pathway, in total out of genes were found to be differentially regulated in light and darkness in the wildtype and/or in mutant strains. among these genes, all gh families are represented except gh family with its only member beta galactosidase. however, inspection of transcript levels of the respective gene also differed between light and darkness up to fold, which is only slightly below our threshold. functions of env in darkness previous studies showed that env not only has functions in light, but also in darkness, which however remained elusive until now. the transcriptome data confirmed this assumption and genes were found to be specifically regulated more than 2fold in darkness in Δenv. only genes were found to be exclusively downregulated in Δenv in darkness, including two putative fad dependent oxidoreductases, phosphoenolpyruvate carboxykinase tr_ and one predicted oligopeptide transporter. those genes showing increased transcript abundance specifically in Δenv in darkness include glycoside hydrolases and tr_70845), the class ii hydrophobin encoding gene hfb one candidate non-ribosomal peptide synthase gene, one catalase, blr and blr moreover, three genes involved in sulphur metabolism are upregulated. in summary, the functions influenced by env in darkness correspond to a subset of functions subject to light response in t. reesei. these results further reflect that env exerts individual functions not only in light, but also in darkness. regulatory targets of blr and blr in darkness in addition to differential regulation between growth in light and growth in darkness, we also studied altered gene regulation in the photoreceptor mutant strains compared to wild-type in light and darkness, which revealed light-dependent regulatory targets of these factors. previous studies in t. reesei indicated that blr and blr not only have functions in light, but that they also regulate gene expression and metabolic functions in darkness. q-rt pcr analysis of the cellobiohydrolase cbh1/cel7a, for which the microarray reached the saturation limit, confirmed a regulatory function of blr and blr in darkness. moreover, individual functions for the two photoreceptors homologues besides their activity as a complex were suggested in n. crassa. we therefore analyzed which functions these photoreceptors target in darkness. we found that in Δblr genes are upregulated in darkness, among them glycoside hydrolases including two alpha-glycosidases, two beta-glycosidases, one alpha galactosidase and one chitinase. moreover, seven genes involved in sulphur metabolism and transporters are among the genes upregulated in Δblr genes were downregulated in Δblr in darkness. accordingly, functional category analysis identified c-compound and carbohydrate metabolism in the gene set upregulated in Δblr and metabolism of several amino acids among the genes down regulated in Δblr in darkness as significantly enriched. genes exerting transport functions were significantly enriched in both gene sets in Δblr in Δblr genes are upregulated in darkness including glycoside hydrolases, among them two beta glucosidases and one l-arabinofuranosidase and three transporters. among the genes down-regulated in Δblr genes involved in sulphur metabolism were found. funcat analysis revealed significant enrichment in genes involved in polysaccharide and carbohydrate metabolism as well as transport functions among the genes up- or down-regulated in Δblr in darkness. hence both photoreceptors play a role in alteration of carbohydrate metabolic functions and transport of compounds with distinct, both positive and negative targets in darkness. shared regulatory targets of blr blr and env1 light dependent induction of env transcription requires the presence of blr and blr2. in order to elucidate which target genes would be regulated by the complex assumed to be formed by blr and blr and transmitted involving the function of env we screened for genes regulated similarly in Δenv Δblr and Δblr. while in darkness no overlap could be detected, we found genes to be upregulated in all three mutants in light, which are concluded to represent genes negatively influenced by the blr1/blr complex via env in light. among them were glycoside hydrolase family genes as well as one putative zinc binuclear cluster transcription factor. however, in contrast to the only genes negatively influenced by blr blr and env we found the majority of common targets of these factors to be positively regulated in light by the light signalling machinery. although most of the genes in this group are of unknown function, major targets appear to be the glycoside hydrolases with members of diverse families found in this group. additionally, three putative transcription factors and two g protein coupled receptors, were downregulated in all three mutants in light, suggesting altered signal perception as well as output on regulatory targets. the finding of six genes involved in sulphur metabolism including the e ubiquitin ligase lim1 supports the hypothesis of a role of this process in light-dependent modulation of gene expression in t. reesei. additionally, the finding of one hydrophobin gene in this group is also not without precedent. regulation by a potential negative effect of env on the blr complex for n. crassa, genes were found to be up-regulated in Δvvd and down-regulated in the white-collar mutants upon growth on cellulose in light, which were significantly enriched in c-compound and carbohydrate metabolism. hence, carbon metabolism was suggested to be subject to photoadaptation in n. crassa. in order to evaluate this hypothesis for t. reesei, we also screened for genes down-regulated due to the lack of a functional photoreceptor complex, but upregulated in Δenv lack of env is in this case assumed to result in increased transcription of genes positively regulated by blr and blr we only found genes of those downregulated in Δblr and Δblr in light to be up-regulated in Δenv interestingly, these included genes involved in pheromone processing and sexual development, two photolyases as well as two transcription factors. however, no enrichment in carbon metabolic functions as seen for n. crassa was observed. consequently, in contrast to n. crassa, carbon metabolism appears to be regulated by the influence of blr and blr on induction of env rather than the assumed negative effect of env on the function of the blr complex in light. additionally, this discrepancy may reflect the regulatory differences of env and vvd, which are no functional homologues. distinct targets of the blr1/blr complex versus env1 despite considerable overlap in their major targets in light with env the common targets of blr and blr are distinct from those of env to a certain extent. genes were found to be upregulated in light compared to the wildtype in both Δblr and Δblr but not in Δenv including one transcription factor, genes involved in sulphur metabolism, one pth11-type gpcr, one polyketide synthase and one glycoside hydrolase family gene. genes represent positive targets of the blr1/blr2-complex in light, among them were one polyketide synthase from pks orthologous group 3 and one non-ribosomal peptide synthase as well as glycoside hydrolases. interestingly, we also detected transcription factors positively regulated by blr and blr but not by env including one transcription factor reported to be responsive to light in the absence of major photoreceptors. env triggers gene regulation also independently of blr1/blr2 the results described above led to the question, which output pathways would be regulated by env independently of blr1/blr in light. more than 55% of all genes downregulated and even 92% of those upregulated in a mutant lacking env are not targets of either blr or blr. moreover, the negative effect of env in light is much more widespread than that of blr or blr consequently, a function for env distinct of that of blr and blr in light can be assumed. analysis of negative targets of env in light revealed an influence on putative transcription factors, including two transcription factors described to be light responsive in the absence of the major photoreceptors in n. crassa. moreover, photolyases, 5 g protein coupled receptors, genes involved in secretion, genes involved in secondary metabolism and glycoside hydrolase genes were negatively regulated by env genes influenced positively by env but not by blr or blr in light include putative transcription factors, among them hap encoding an important regulator of cellulase gene expression. additionally, genes involved in sulphur metabolism, eight g protein coupled receptors, nine genes involved in secretion, two polyketide synthases, three hydrophobin genes and most intriguingly, nine glycoside transferase and glycoside hydrolase genes are enhanced by env in light. we conclude that env represents a key factor in light-dependent regulation of gene expression, the major effect of which is not exerted concertedly with the blr1/blr complex. the considerable number of target genes with functions in carbon utilization and nutrient signal perception suggests a crucial function in interconnecting nutrient with light signalling. coregulation of genes with the major cellulases genes regulated consistently under different conditions often participate in the same function. therefore, investigation of co-regulated genes can help to assign a putative function to unknown genes or reveal processes related to each other. for identification of genes and/or processes involved in the light signalling pathway that modulates the cellulase gene transcription pattern, we examined a cluster of genes that are co-regulated with cel6a/cbh in all of the light affected mutant strains and their parental strain qm cel7a/cbh the major cellobiohydrolase, is co-regulated with cel6a/cbh but due to saturation of the microarray signal for this transcript, we were not able to use the transcript pattern of cel7a/cbh to evaluate coregulated genes of cellulases. to confirm that the transcription pattern of cel6a/cbh in the mutants resembles that of cel7a/cbh we performed qrt-pcr, which confirmed coregulation. we consequently used cel6a/cbh as representative gene for evaluation of genes coregulated with cellobiohydrolases in the presence or absence of blr blr or env as described above in light and darkness. the gene set of the cel6a/cbh cluster comprised more glycoside hydrolase family genes, a swollenin, which was shown to disrupt the structure of cellulosic materials, two carbohydrate esterase family genes, cip and cip - each containing a carbohydrate-binding module and were previously shown to be co-expressed with cellulases and represent “novel” types of cellulases, a flavohemoglobin, a gaba permease and two potential transcription factors. in addition, two hypothetical proteins, a wd40-repeat containing protein and a hypothetical g protein coupled receptor were also coregulated with cel6a/cbh notably, the xylanase regulator encoding gene xyr was also shown to be coregulated with cel7a/cbh and cel6a/cbh which is in agreement with earlier data, because xyr influences cellulase gene transcription positively and correlates with cellulase transcription levels. however, positive regulation of xyr by light has not been observed upon growth on lactose and appears to be specific to cellulose, which also suggests differential light dependent regulation of plant cell wall degrading enzymes on these carbon sources. indeed, previous studies of t. reesei grown on cellulose or lactose showed that the effect of light on cellulase gene expression is positive on cellulose and negative on lactose. light impacts pentose and d-galactose metabolism the considerable regulation of glycoside hydrolases and hence of mechanisms involved in substrate degradation by env led us to re-evaluate data on growth patterns for various carbon sources in more detail. the results for growth on carbon sources in constant light and constant darkness of the parental strain and the env1-non functional strain env1pas– were rearranged and used for hierarchical cluster analysis. env1pas– showed considerably weaker growth in light than in darkness on most carbon sources, as could be expected from its published growth defect in light. the growth of the wildtype was enhanced in light in a cluster of different carbon sources including d-sorbitol, l-arabinose, d-fructose, d-galactose and xylitol in light, while the env mutant strain showed a considerably lower growth rate in light on these carbon sources. hence, the positive influence of light on growth on these carbon sources is likely to be at least in part mediated by env. hierarchical cluster analysis was performed using hce with default settings. standard deviations shown result from three biological replicates. values are represented as percent. ll means constant light, dd means constant darkness. d-galactose and pentose catabolism are regulated by light on cellulose interestingly, this cluster of carbon sources with env dependent enhanced regulation by light comprises several carbon sources of the d-galactose and pentose catabolism. the respective enzymes are assumed to be involved in metabolism of hemicellulose degradation products. considering coregulation of many plant cell wall degrading enzymes upon detection of an inducing substrate, a regulation of these pathways also on cellulose seemed reasonable and was indeed observed with the microarray data of this study, albeit very low expression levels for lxr lxr and lad as well as only small differences in transcript levels necessitated independent confirmation of these data. consequently, we evaluated the microarray data by qrt-pcr for the key components of oxidoreductive pentose and d-galactose pathway with respect to transcriptional regulation by light and/or the photoreceptors. transcription of all genes involved in catabolism of d-galactose and l-arabinose except xki showed a statistically significant increase in light in the wildtype. this indicates that the cascade starting from d-galactose or l-arabinose and d-xylose, respectively is positively affected by light during growth on cellulose. besides the leloir pathway, an oxidoreductive pathway was identified in t. reesei and many of the enzymes involved are also part of the l-arabinose and d-xylose pathway. the degradation of the hemicellulosic sugars d-xylose, l-arabinose and d-galactose starts with the same reaction: an nadph-linked reduction by an aldose reductase xyl1. the next step, the oxidation of galactitol or l-arabitol is catalyzed by lad the l-arabitol-4-dehydrogenase. recently it was shown that lxr encoding an l-xylo-3-hexulose reductase is the missing link in the oxidoreductive d-galactose catabolism in t. reesei. the last step of the d-galactose degradation before glycolysis is the mediated by a nad-xylitol dehydrogenase xdh which also catalyzes the conversion from xylitol to d-xylulose in the l-arabinose and d-xylose pathways. the last phosphorylation step of the l-arabinose and d-xylose pathway before the substance enters the pentose phosphate pathway is presumably done by the gene product of xki a xylulose kinase and a homolog of aspergillus niger xkia. differential expression between constant light and constant darkness upon growth on cellulose was abolished upon lack of blr and blr for all genes investigated, except xki which does not show regulation by light in the wild-type. transcript abundance predominantly drops to darkness levels in Δblr and Δblr hence, blr and blr are concluded to be necessary for positive regulation of this pathway in light. for env the situation is more complex. in the absence of env differential expression between light and darkness is abolished for lxr, lad and xdh in contrast, the difference between transcript levels in light and darkness becomes even more pronounced for lxr and xyl which is due to a strong upregulation of lxr and of a considerable down-regulation of xyl in light. down-regulation of xki in light upon lack of env results in differential expression of xki in this strain, which was not observed in the wild-type. this effect is also one example, how lack of adaptation as mediated by env blr or blr could result in an increased number of genes differentially transcribed between light and darkness. these results are in complete agreement with the results of the biolog analysis, since lack of env causes decreased transcription of most of the enzymes in the pathway in light. consequently, while the data for individual genes and growth on individual carbon sources for wild-type and mutant strains only show minor differences in dependence of light, consistent regulation of growth and transcript abundance of the genes encoding enzymes of the oxidoreductive pentose and d-galactose pathway strongly indicates that the response of t. reesei to hemicellulose building blocks is modulated by photoreceptors in response to light. a meme search using the online version. did not reveal a common sequence in these promotors related to known light responsive motifs. according to the flat hierarchical network as proposed for photoreceptors n. crassa, the effect seen here is likely to be exerted indirectly. for xyl regulation by the cellulase and hemicellulase regulator xyr was shown. we therefore screened all gene promoters of the pathway for xyr1-binding sites and only xdh has a xyr binding site. as xyr similarly to xdh and xyl is positively regulated by env in light, this regulation might be mediated by xyr lxr is part of a env regulated cluster in light while initially assumed to be an l-xylulose reductase as it catalyzed the nadph/nadp + specific reactions for l-xylulose/xylitol and for d-fructose/d-mannitol, lxr later turned out to be a mannitol dehydrogenase. on d-mannitol, our growth analysis showed a slightly slower growth of the wild-type in light. due to its additional activity on fructose, lxr could also be involved in the increased growth of the wild-type and the decreased growth in the env mutant on this carbon source in light. investigation of the genomic region around lxr revealed that genes in its genomic vicinity are regulated negatively by env in light upon growth on cellulose and hence form a cluster. blr and blr do not influence the regulation of the cluster. in contrast to the effect upon growth on mannitol, env has a strongly negative effect on lxr and the genes in its cluster upon growth on cellulose. the cluster is located at scaffold between position – and comprises besides lxr also a zn-cluster transcription factor, a putative oligopeptide transporter, two probable old yellow enzyme family flavin oxidoreductases, a putative gtp cyclohydrolase and a proline oxidase/dehydrogenase involved in conversion of proline for use as a carbon and nitrogen source. two of these genes, lxr and tr_ have homologues in n. crassa, which are responsive to light. however, neither these nor the other members of this cluster are syntenic in n. crassa. within the borders of the env1-regulated lxr1-cluster, a light regulated cluster starts, which comprises tr_ tr as well as a taurine dioxigenase involved in sulphur metabolism and additionally contains a gatase1-like domain containing peptidase, which is located outside the lxr cluster. consistent targets of photoreceptors in n. crassa and t. reesei previous studies indicated at least in part comparable regulation of cellulase gene expression by light and photoreceptors in t. reesei and n. crassa. we were hence interested, whether the regulatory targets of photoreceptors in these two fungi are similar. therefore, we re-analyzed the transcriptome data for n. crassa wild-type as well as Δwc- Δwc- and Δvvd upon growth on cellulose in light for significant 2fold differential regulation in order to be comparable with our data. genes were found to be differentially regulated in one or more of the n. crassa photoreceptor mutants and are hence photoreceptor targets in light. of these genes have reciprocal best hits in t. reesei and are therefore likely homologues. genes were identified as consistent targets of one or more photoreceptors in n. crassa and t. reesei. in agreement with previous findings, these genes were enriched in functions of metabolism, particularly c-compound and carbohydrate metabolism and sugar, glucoside, polyol and carboxylate metabolism. additionally, functions in c-compound and carbohydrate transport and glycolysis and gluconeogenesis were enriched. among the consistent photoreceptor targets are further five glycoside hydrolases, tr, tr_, tr_, tr_ ), one carbohydrate esterase and two genes involved in sexual development including the gene encoding the alpha-type peptide pheromone precursor ppg- which was shown to impact cellulase regulation in n. crassa. two putative sugar transporters and one putative carboxylic acid transporter were also among these genes. although functions in sulphur and amino acid metabolism were not enriched among these genes, two putative methionine synthases as well as one methionine permease were consistently regulated. moreover, two transcription factors were found to be consistent targets: tr_ which has not yet been characterized and tr_ the homologue of tah- which is involved in conidiophore development and shows increased transcript levels upon growth on miscanthus in n. crassa. tr_ is part of the lxr cluster described above and intriguingly, also lxr itself is among the consistently regulated genes. however, the well characterized cellulase regulators of t. reesei and n. crassa, such as xyr cre clr- or clr- are not among the consistently regulated genes. discussion in this study we investigate the light signalling machinery of t. reesei at a genome wide level upon growth on cellulose, which is the closest defined carbon source to what t. reesei encounters in its natural habitat. we extend previous knowledge on the function of photoreceptors, which was mainly done on glucose and in constant light, with investigating the effect of the photoreceptors in darkness. this enabled us to study both differential gene regulation between growth in light and darkness and investigation of regulatory targets of blr blr and env in light and darkness. although only few genes are regulated by the photoreceptors in darkness, a clear role in carbon metabolism and transport functions could be detected in darkness. these functions resemble also the targets in light, indicating that regulation by photoreceptors is not strictly light dependent. one of the most intriguing and puzzling findings of this study was the increased number of differentially transcribed genes in the photoreceptor mutants, as we rather expected the opposite effect. a similar phenomenon was observed for mutants in the g-protein pathway. one explanation for the reason that so many genes are light-regulated in the photoreceptor mutants might be a lack of light adaptation in the photoreceptor mutants: in the comparison between growth in constant darkness and in constant light, many transcripts might appear unchanged due to adaptation to light. however upon deletion of blr1/blr or env, both light induction and adaptation are assumed to be lost, which would cause decreased/altered transcript levels in the mutants. indeed, evaluation of the regulatory targets of blr blr and env revealed the highest numbers of regulated transcripts to be downregulated in the mutant strains in constant light, which supports this hypothesis. while we cannot provide a mechanistic explanation yet, our working hypothesis currently involves a higher order regulation to be targeted by the photoreceptors such as chromatin remodelling, which was shown to be impacted by light and the clock. this would explain the wide-spread effect we see and it would be in agreement with a rather tight regulation in the wild-type. nevertheless, also such a mechanism would require an additional, so far unidentified factor to transmit the light signal in the absence of photoreceptors. recently, investigation of the effect of photoreceptors on cellulase gene expression in n. crassa revealed that this process is subject to photoadaptation in this fungus. additionally, the known cellulase repressor gene ace the carbon catabolite repressor gene cre and the cross pathway control protein encoding cpc which is involved in response to amino acid starvation response, were found to be regulated by photoreceptors in n. crassa. in t. reesei however, the situation appears to be different. we could identify the cellulase and hemicellulase regulator gene xyr to be a target of blr and env on cellulose, whereas n. crassa xyr- is not regulated by photoreceptors. also the t. reesei homologues of clr- and clr-, two recently identified n. crassa cellulase regulators, are subject to regulation by photoreceptors, although they are not light regulated in the wild-type. additionally, neither ace nor cre or cpc are among the targets of the light signalling machinery in t. reesei. hence, despite largely similar global processes to be targeted by blr blr and env such as carbon-, amino acid- and sulphur metabolism, the regulation of the involved pathways seems to be achieved differently in n. crassa and t. reesei. this finding is also in agreement with consistent regulation of metabolic genes but not the respective known regulators between n. crassa and t. reesei in light. also the effects on glycogen metabolism observed in n. crassa were not obvious for t. reesei. however, the general influence of photoreceptors on carbon and amino acid metabolism as well as its assumed correlation with cellulase gene expression is clearly conserved in t. reesei and n. crassa. the results of this study illustrate an important phenotypic characteristic of strains lacking env these strains show severely reduced growth rate and conidiation, both in liquid media and on plates in light. this defect is clearly reflected in the strongly altered transcriptome of this strain in light compared to darkness. in the absence of env considerable alterations involving both positive and negative regulation of metabolic genes – especially those in carbon and amino acid metabolism – as well as genes for substrate transport and energy metabolism occurs. hence env is of crucial importance for adjustment of metabolism to changing light conditions. so far we could not identify the reason for the diminished growth rate of Δenv under the conditions we used and hence effects besides metabolic imbalance may influence the phenotype of this strain. additionally, it was found that growth rate does not necessarily correlate with production of hydrolytic enzymes in t. reesei. despite its slow growth, specific cellulase activity secreted into the medium by Δenv is several fold increased compared to wild-type. we conclude that env is an essential regulator/signal transmitter for adjustment of growth rate to environmental conditions in light. this function, however, is most important on cellulose, as the biolog analysis on numerous carbon sources showed that the light dependent growth defect of Δenv does not occur on every carbon source. therefore, it can be assumed that the function of env in light is most critical upon growth on cellulose, but less important for growth in the presence of other carbon sources in light. to complement our transcriptome analysis for growth on cellulose, we compared the growth patterns on different carbon sources in light and darkness for wild-type and an env non functional strain, because of the strong effect of env on light dependent gene regulation. earlier analysis had indicated that the degradation of different carbon sources is regulated in a light dependent manner and that env is involved in the transfer of the positive light signal in many cases. our re-evaluation and cluster analysis showed that especially intermediates of the oxidoreductive d-galactose and l-arabinose pathway, which is also important for utilization of lactose, are among the carbon sources on which growth in the wild-type and the env mutant were most clearly influenced. due to the ability of t. reesei to grow and express cellulases on lactose, the degradation of lactose is very well investigated. in eukaryotes lactose is first cleaved to glucose and galactose and galactose is further converted into glucose 6-phosphate by enzymes of the leloir pathway. together with arabinose, also catabolism of galactose is involved in utilization of hemicellulose building blocks. as for the most part, plant cell wall degrading enzymes are coregulated in t. reesei, this is in accordance with our finding that the genes encoding the enzymes operative in these pathways are expressed upon growth on cellulose. these findings are in agreement with the hypothesis that t. reesei senses the presence of plant cell wall material in its environment by detection of building blocks of cellulose and hemicellulose. interpretation of the respective signals is subsequently adjusted to the requirements in light and darkness by blr blr and env  in summary, we showed that blr blr and env exert important metabolic functions, not only in light, but also in darkness. lack of components of the light signalling machinery causes considerably increased light responsiveness of transcript levels, likely caused by a light-dependent, positive effect on output pathways. thereby, the photoreceptors blr and blr do not exclusively act as a complex, but additionally have individual targets. this finding also applies to env for which the transcriptome pattern indicates a sizable amount of independent targets in light. interestingly, the targets of the light response machinery also include the catabolic enzymes necessary for degradation of hemicellulose building blocks i. e. the d-galactose and pentose pathway and lxr which adds a new aspect to light dependent gene regulation on cellulose. comparison with transcript profiles of n. crassa indicates a conserved adjustment of metabolic pathways in light by photoreceptors, but also different regulatory mechanisms applied in order to achieve this effect. 
in contrast to n. crassa, photoreceptor mediated regulation of carbon metabolism in t. reesei occurs primarily by blr and blr via their positive effect on induction of env transcription, rather than by a presumed negative effect of env on the function of the blr complex. nevertheless, genes consistently regulated by photoreceptors in n. crassa and t. reesei are significantly enriched in carbon metabolic functions. analysis of growth on different carbon sources revealed that the oxidoreductive d-galactose and pentose catabolism is influenced by light and env transcriptional regulation of the target enzymes in these pathways is enhanced by light and influenced by env blr and/or blr additionally we detected an env1-regulated genomic cluster of genes including the d-mannitol dehydrogenase gene lxr with two genes of this cluster showing consistent regulation in n. crassa. the tropical ascomycete trichoderma reesei represents one of the most efficient plant cell wall degraders. we show that one major output pathway of light signalling in trichoderma reesei is regulation of glycoside hydrolase genes and the degradation of hemicellulose building blocks. our transcriptome analysis of strains lacking the photoreceptors blr and blr as well as env revealed a considerable increase in the number of genes showing significantly different transcript levels in light and darkness compared to wild-type. targets of env and blr1/blr are for the most part distinct and indicate individual functions for env and the blr complex besides their postulated regulatory interrelationship. we show that members of all glycoside hydrolase families can be subject to light dependent regulation, hence confirming nutrient utilization including plant cell wall degradation as a major output pathway of light signalling. hence, different regulatory mechanisms are operative in these two fungi, while the light dependent regulation of plant cell wall degradation appears to be conserved.
 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.  our transcriptome analysis of strains lacking the photoreceptors blr and blr as well as env revealed a considerable increase in the number of genes showing significantly different transcript levels in light and darkness compared to wild-type. we show that members of all glycoside hydrolase families can be subject to light dependent regulation, hence confirming nutrient utilization including plant cell wall degradation as a major output pathway of light signalling. in contrast to n. crassa, photoreceptor mediated regulation of carbon metabolism in t. reesei occurs primarily by blr and blr via their positive effect on induction of env transcription, rather than by a presumed negative effect of env on the function of the blr complex. nevertheless, genes consistently regulated by photoreceptors in n. crassa and t. reesei are significantly enriched in carbon metabolic functions. hence, different regulatory mechanisms are operative in these two fungi, while the light dependent regulation of plant cell wall degradation appears to be conserved. analysis of growth on different carbon sources revealed that the oxidoreductive d-galactose and pentose catabolism is influenced by light and env transcriptional regulation of the target enzymes in these pathways is enhanced by light and influenced by env blr and/or blr additionally we detected an env1-regulated genomic cluster of genes including the d-mannitol dehydrogenase gene lxr with two genes of this cluster showing consistent regulation in n. crassa.  we show that one major output pathway of light signalling in trichoderma reesei is regulation of glycoside hydrolase genes and the degradation of hemicellulose building blocks. targets of env and blr1/blr are for the most part distinct and indicate individual functions for env and the blr complex besides their postulated regulatory interrelationship. trichoderma reeseihypocrea jecorinalight responseglycoside hydrolasesd-galactosel-arabinoseenvoyblr1blr2 