The utilization of cellulosic and hemicellulosic polymers in plant biomass for the production of bioethanol or platform chemicals is considered as a possible strategy to reduce carbon dioxide emissions and bypass the current dependence on fossil resources. Renewable lignocellulosic biomass, besides being cheap and abundant, has also the advantage that it does not compete with food production. Currently favored raw materials for this purpose include –among others – wood residues, “energy crops” such as switch grass or Miscanthus, and agricultural byproducts such as wheat straw [1]. The latter is one of the most abundant low-cost sources of lignocellulosic biomass in middle European countries with an annual production of over 130 million tons [2].

For the biotechnological use of these materials, they first need to be pretreated and then hydrolyzed to their monomers. The fungus Trichoderma reesei is currently the major industrial producer of enzymes needed to degrade the above polymers to soluble monosaccharide [1, 3]. Most of these enzymes are not formed during cultivation on monosaccharides such as glucose; the fungus must therefore be grown in the presence of an inducer which is mostly a cellulose and hemicellulose containing waste material [4]. Optimally, this would be the same material for which the produced enzymes are aimed to be applied, because this would ensure the induction of the whole spectrum of enzymes needed. However, this is often not possible because commercial producers prefer the manufacture of an enzyme preparation for a broad range of substrates.

One of the carbon sources that is used for the production of cellulases and hemicellulases by T. reesei is lactose, which is favoured when a soluble and cheap inducing carbon source is preferred, e.g. to facilitate and reduce costs for fermentation control and enzyme recovery [5]. However, cellulase production on lactose occurs at a slower rate and a lower final enzyme yield than on cellulosic materials, and it has also been reported to lead to an enzyme preparation with lower specific activities [6]. Yet an in depth comparison of the enzymes produced on cellulose and lactose has to our knowledge not been published so far.

Here we report a comparison of the transcriptome of T. reesei growing on lactose and cellulose (wheat straw), which not only demonstrates the differences in the enzymes produced but also in the molecular physiology of growth on these two carbon sources.

We have previously described that lactose can induce an almost complete cellulase and hemicellulase enzyme system in T. reesei, and reasoned that this could be due to a preference of the fungus to initiate feeding on lignocellulose by hydrolysing the β-galactoside side chains in the xyloglucans, which are linked to cellulose in the primary cell wall of dicotyledons [19]. In order to test how this induction on lactose would compare to a complex lignocellulosic material – wheat straw, which not only contains cellulose but also xylans but with very little galactose side chains (< 0.1%; [11]) – we compared the transcriptome under both conditions. The results showed that 85 of the 132 genes of the CAZome were expressed both on lactose as well as on wheat straw. However, two thirds of them were significantly stronger expressed on wheat straw than on lactose. One of the major qualitative differences between wheat straw and lactose was a strict dependence of 4 xylanases and 2 β-xylosidases on wheat straw for expression, implying that xylanases are only poorly expressed on lactose. Only xyn2 and xyn3 were also expressed on the latter. These findings suggest that there is indeed no major difference between the induction of cellulolytic enzymes by lactose and cellulose, but that the observed differences are due to the content of xylan. Expression of xyn2 has previously been demonstrated to be triggered by both sophorose (which is considered to be a “cellulose-specific” inducer) as well as xylobiose (considered to be “xylan-specific”) and by lactose [20]. However, xyn1, xyn2, xyn4 and xyn5 have recently also been shown to be differentially induced by D-xylose and L-arabinose [21]. Similarly, Akel et al. [22] have shown that the α-L-arabinofuranosidase genes require the presence of L-arabinose for full induction. We therefore interpret the significantly higher expression of most of the xylanases and hemicelluloses side chain hydrolases to be due to the presence of additional specific inducers for their genes.

These results raise an interesting question: it has been demonstrated that expression of the cellulase and hemicellulase genes in T. reesei is completely dependent on the function of the Zn2Cys6-transcription factor XYR1 [13, 22], and xyr1 itself is induced both on lactose [19], and cellulose [23]. So how could a single transcription factor respond to different inducers in quantitative different ways? The regulation of genes encoding xylanolytic enzymes of the model organism Neurospora crassa has been suggested to involve several regulatory groups: the xylanase regulator XLR-1 (the N. crassa orthologue of XYR1) was suggested to work alone or in combination with other unknown regulators and an XLR-1 independent group of genes was also suggested to exist [24]. Häkkinen et al. [11] have also hypothesized that several regulatory mechanisms, depending on the inducers present, may act on the CAZyme gene promoters simultaneously, and in some cases also in an additive manner. An example of such an additional regulator could be ACE2, which has been demonstrated to assist xylanase gene transcription by enhancing xyr1 transcription and by forming a putative heterodimer with XYR1 [25], whereas it has only a small effect on induction of cellulase gene transcription by solka floc cellulose, and none at all when sophorose is used as an inducer [26]. Another candidate could be the orthologue (Trire2: 26163) of the recently described N. crassa cellulase regulator CLR-2 [27]. In this study, clr2 was found to be induced during growth on lactose or wheat straw. A blastp search with the T. reesei CLR2 protein sequence against the NCBI database revealed that it is an orthologue of the A. nidulans mannanase regulator ManR, for which the DNA-binding motif has been determined as 5’-YAGAAT-3’ [28]. However, a search of the presence of this motif in 1 kb upstream of several CAZome genes that were found to be significantly regulated in this study revealed no consistent picture: 1–2 copies were present in some genes, but completely absent in the majority of them, including the major β-mannanase gene man5 (unpublished data). Whether or not CLR2 or another transcription factor cooperate with XYR1 in the expression of some CAZome genes therefore remains to be determined. We should like to stress that both xyr1 and clr2 belonged to transcript group E, i.e. they were expressed to similar levels on lactose and wheat straw. The different level of expression of various genes on wheat straw and lactose can therefore not be simply the result of an enhanced expression of xyr1.

In this study, we also detected a significant upregulation of genes encoding chitinases, α-galactosidases and mannosidases. These genes were also recently observed to behave different from the major set of cellulase and hemicellulase genes by being moderately or even very low expressed during growth on birch xylan, steam exploded and enzymatically treated bagasse already at the early time points [11]. Interestingly, this upregulation was strongly enhanced in a strain in which the xyr1 gene had been deleted, indicating that XYR1 is a repressor of these genes. While the XYR1 orthologues from A. oryzae, N. crassa and Fusarium graminearum all function in the regulation of xylanase gene expression, XYR1 regulates cellulase gene expression only in T. reesei and A. oryzae. In both N. crassa and A. oryzae – the two fungi in which the effect of xyr1 manipulation has been studied on a genome-wide scale [24, 29] - it has so far been shown only to activate gene expression. However, the variation of occurrence (and in 3 cases even absence) of XYR1 binding sites in the promoters of these genes makes an action of XYR1 as a repressor of these genes unlikely. We consider it rather possible that their strong upregulation in the ∆ xyr1 mutant is due to its inability to grow on wheat straw and the reduced growth on lactose [30], i.e. autophagy. In fact, upregulation of some of the T. reesei chitinases by carbon starvation has been shown [12], but is so far not known for the α-mannanases/mannosidases or α-galactosidases. Since oligosaccharides with these monosaccharide and linkage types are not part of the cell wall polymers of the fungus [31], their induction under starvation requires further studies.

A comparison of the wheat straw and lactose transcriptome did not lead to the detection of major changes in metabolic pathways and the signaling to them with four exceptions: one was a high number of genes associated with phospholipid metabolism and protein secretion, with DNA replication and repair, and finally the massive upregulation of genes for iron homeostasis. As for the first, Schreiber et al. [32] showed that the addition of the phospolipid precursor cholin increased cellulase formation in T. reesei, and at the same time led to an increase in the hyphal content of endoplasmic reticulum. Also, Glenn et al. [33] showed that the hypercellulolytic mutant T. reesei RUT C30 exhibits a proliferated amount of endoplasmic reticulum. It is thus possible that the increased expression of genes associated with phospholipid synthesis is responsible for an enhanced synthesis of endomembrane components required for increased cellulase export from the hyphae.

Another group of upregulated genes was those related to autophagy. To date, more than 30 autophagy-related (ATG) genes have been identified for Saccharomyces cerevisiae and other fungi [34, 35]. BLASTP search of the predicted T. reesei proteins (http://genome.jgi-psf.org/Trire2/Trire2.home.html) against the NCBI database (at a cutoff of E-value < −70 over at least 80% of the sequence length) detected 22 orthologues of ATG genes (see Additional file 7: Table S7) of which 7 were found to be strongly upregulated on wheat straw but not on lactose or glucose. Notably this group included the serine/threonine protein kinase Atg1 [17, 18], which – together with Atg17, Atg29, and Atg31 – forms a protein complex that initiates the formation of autophagosomes [36, 37]. Nitsch et al. [38] have recently shown that autophagy plays important roles in physiological adaptation in submerged cultures under conditions of carbon depletion by organelle turnover and protection against cell death. Our data suggest that the slow growth rate on wheat straw may lead to a carbon uptake rate slow enough to already signal starvation. However, autophagy has also been shown to be necessary for fungal morphogenesis, particularly when attacking other organisms [39, 40], and we can therefore also not rule out that it is crucial for the growth of T. reesei on an insoluble substrate.

The upregulation of genes associated with DNA synthesis and repair appears to indicate the operation of mechanisms leading to damage of DNA during growth on cellulose. While the occurrence of such mechanisms has not been reported so far, we detected a wheat-straw-specific 2.7-fold upregulation of the T. reesei orthologue of the NADPH oxidase NoxA (NOX1; Trire2:79498; see Additional file 2: Table S2). This enzyme generates reactive oxygen species in a regulated manner and is involved in several aspects of fungal biology, including development and plant pathogenesis [41]. We consider it possible that the upregulation of enzymes for DNA repair is a response to the formation of reactive oxygen species by NOX1, although the reason for its upregulation is not clear. Brun et al. [42] reported that Podospora anserina hyphae form specialized structures for cellulose degradation and their formation is differentially regulated by NOX1 and NOX2. Interestingly, Montero-Barrientos et al. [43] reported that an overexpression of nox1 in T. harzianum leads to the increased formation of protease, cellulase, and chitinase activities during mycoparasitic interaction with a fungal host. Nitsche at al. (2013) reported that in A. niger autophagy influences the sensitivity towards oxidative stress. We consider it likely that the detected enhanced autophagic activity on wheat straw (vide supra) also causes the increased expression of genes related to DNA damage repair and oxidative stress.

Finally, a very intriguing finding from this study was the massive upregulation of genes involved in iron homeostasis. This elevated expression of iron-uptake systems on wheat straw can be explained by a lower bioavailable concentration of iron during growth on wheat straw (as shown in this study), which is probably due to the ability of cellulose for sequestering Fe³⁺[44]. In nature, this shortage can be reinforced by the formation of Fe³⁺-oxalate chelates with the oxalic acid secreted by basidiomycetes [45]. It is also possible that this induction of iron assimilating enzymes has the additional benefit of triggering the synthesis of the large number of flavin containing oxidases and monooxygenases that are upregulated during growth on wheat straw and result in an increased demand for iron. As already hypothesized [19], this increased expression of oxidative enzymes could indicate the operation of Fenton chemistry during cellulose degradation by T. reesei. The increased expression of a glucose oxidase (Trire2:22915) and a gluconate kinase (Trire2:71072), as observed in this study, suggests partial degradation of the glucose from cellulose, which could be involved in the provision of hydrogen peroxide for this process.

Our study shows that growth on wheat straw leads in part to an enhanced expression of cellulase and hemicellulase genes by T. reesei, but also to the selective induction of a set of enzymes, notable the majority of the xylanolytic enzymes. This implies that the expression of genes associated with lignocelluloses degradation by T. reesei is subject to as yet unknown regulator proteins which are supposed to cooperate with XYR1. A detailed analysis of transcriptomic changes of genes involved in cellular metabolism and its regulation further identified autophagy, phospholipid biosynthesis, iron homeostasis and DNA repair as processes related to degradation of wheat straw, whose roles warrant further investigations. In addition, manipulation of these genes may be a new tool for strain improvement in T. reesei.