Mn2+/DMF stimulation produces cAMP in an ACY1-dependent manner
We previously demonstrated that a biologically relevant level of extracellular Mn2+ or DMF markedly stimulates cellulase overexpression in T. reesei [19, 20]. As a secondary messenger, cAMP is involved in responses to extracellular signals. To examine whether Mn2+/DMF stimulation had any effect on cAMP concentration in T. reesei, precultured mycelia of T. reesei QM6a was inoculated into fresh liquid MM containing 1% Avicel (cellulase-inducing conditions) as the sole carbon source with no further supplementation, or with the addition of 10 mM Mn2+ or 1% DMF, as previously described [19].
The intracellular cAMP concentrations of T. reesei QM6a after the addition of Mn2+ and DMF are presented in Fig. 1. Stimulation with 10 mM Mn2+ produced a 76.5% increase in the intracellular cAMP concentration compared with that of the control (no addition) under the same growth conditions. Similarly, stimulation with 1% DMF resulted in a 74% increase in intracellular cAMP concentration compared with the control. These results showed that Mn2+/DMF stimulation could induce cAMP accumulation in T. reesei. Supplementation with forskolin, a direct adenylate cyclase activator [25], also increased the formation of intracellular cAMP by 50% compared with that of the control (Fig. 1). These results indicate that Mn2+/DMF stimulation can result in cAMP accumulation.
To further clarify whether the increased cAMP was mediated by the adenylate cyclase under Mn2+/DMF stimulation, we constructed an adenylate cyclase gene acy1 deletion strain (Δacy1) to detect its function during cAMP accumulation following Mn2+/DMF stimulation. The growth rate of the Δacy1 strains was clearly slower than that of the wild-type strain QM6a (Additional File 1: Figure S1), which is consistent with data from Schuster et al. [14]. We then compared the intracellular cAMP concentration in the Δacy1 strain under different conditions. Mn2+/DMF or Forskolin supplementation did not induce cAMP accumulation in the Δacy1 strains, which differed from the wild-type strain QM6a (Fig. 1). However, exogenous application of dbcAMP increased the cAMP concentration in both Δacy1 and wild-type QM6a (Fig. 1). The results indicated that Mn2+/DMF stimulation can result in cAMP accumulation in an ACY1-dependent manner.
cAMP signalling mediates Mn2+/DMF-stimulated cellulase overexpression in T. reesei
We found that the concentration of cytosolic cAMP was increased by Mn2+/DMF stimulation (Fig. 1). We thus examined whether cAMP signalling was responsible for Mn2+/DMF-stimulated cellulase overexpression.
To explore the role of cAMP signalling in Mn2+/DMF stimulation, cellulase production in the Δacy1 strains in response to Mn2+ and DMF supplementation was detected. As shown in Fig. 2a and b, supplementation with 10 mM Mn2+ or 1% DMF led to an almost 2.5-fold improvement in cellulase production (CMCase and pNPCase activities) in wild-type QM6a, but did not result in increased cellulase production in the Δacy1 strains. The pNPCase activity in the acy1 re-complementation strain Racy1 was similar to that in QM6a (Additional File 2: Figure S2). There was no obvious difference in CMCase or pNPCase activities with and without Mn2+/DMF addition in the Δacy1 strains (Fig. 2a, b).
RT-qPCR was performed to determine the transcription levels of the main cellulase genes cbh1 and egl1 in the QM6a and Δacy1 strains in response to Mn2+/DMF addition. In agreement with the levels of CMCase and pNPCase activities, deletion of acy1 abolished the Mn2+/DMF-stimulated overexpression of cellulase genes compared with the wild-type strain QM6a at all time points (Fig. 2c, d). Cellulase production was detected in the Δacy1 strains in response to exogenous dbcAMP or dbcAMP in combination with Mn2+ and DMF. As shown in Additional File 3: Figure S3, supplementation with 5 mM dbcAMP led to a significant improvement in cellulase production (CMCase and pNPCase activities) in both Δacy1 strains and wild-type QM6a.
These results indicate that cAMP signalling mediates Mn2+/DMF-stimulated cellulase overexpression in T. reesei.
cAMP elevation can induce cytosolic Ca2+ burst and Ca2+ signalling
Our previous results suggested that calcium signalling is the key reason for the Mn2+/DMF-stimulated cellulase overexpression in T. reesei [19, 20]. Presently, cAMP signalling also appeared to mediate Mn2+/DMF-stimulated cellulase overexpression (Fig. 2). To gain insight into the relationship between cAMP and calcium signalling, the effect of cAMP on the cytosolic Ca2+ was examined by the use of Fluo-4 AM, a Ca2+-specific fluorescent probe [26].
As illustrated in Fig. 3a and b, the addition of Mn2+, DMF, Forskolin, and dbcAMP induced a Ca2+ burst in wild-type QM6a by increasing cytosolic Ca2+ to concentrations from 70 to 100% higher than that in cells not cultured with Mn2+, DMF, Forskolin, or dbcAMP. As noted above, Mn2+, DMF, forskolin, and dbcAMP significantly increased the concentration of cytosolic cAMP in QM6a (Fig. 1). These data suggested that a high concentration of cytosolic cAMP is associated with increased cytosolic Ca2+ content (Figs. 1 and 3).
The effect of cAMP on cytosolic Ca2+ was also examined in Δacy1 strains. As shown in Fig. 3a and b, Mn2+, DMF, and forskolin did not increase the cytosolic Ca2+ concentration in the Δacy1 strains. However, dbcAMP resulted in an approximately 80% increase in cytosolic Ca2+ concentration in Δacy1 strains compared with the concentration in the absence of any added compound (Fig. 3a, b). Thus, when cAMP synthesis is blocked in Δacy1 strains, Mn2+, DMF, and forskolin addition cannot induce cytosolic Ca2+ burst, while exogenous dbcAMP addition can. This is because dbcAMP can still increase the cytosolic cAMP concentration in Δacy1 strains, while Mn2+, DMF, and Forskolin cannot (Fig. 1). These data suggested that Mn2+/DMF stimulation can increase cytosolic cAMP content, which induces cytosolic Ca2+ burst in T. reesei QM6a.
The effect of cAMP on Ca2+ signalling was further examined in the QM6a and Δacy1 strains. As shown in Additional File 4: Figure S4, the addition of Mn2+, DMF, and forskolin significantly upregulated the expression levels of three Ca2+ signalling genes (cam, cna, and crz1) in wild-type QM6a compared to untreated cells. However, the expression levels of cam, cna1, and crz1 remained stable in acy1 deletion strains, irrespective of whether Mn2+, DMF, and forskolin were added to the cells. The addition of dbcAMP resulted in an increase in the expression levels of cam, cna1, and crz1 in both wild-type QM6a and Δacy1 strains compared to untreated cells. These data suggest that an increase in cAMP levels induces a cytosolic Ca2+ burst that activates the Ca2+ signal transduction pathway in T. reesei.
To further investigate the link between cAMP and Ca2+ signalling, we measured cellulase production in response to exogenous dbcAMP and dbcAMP in combination with LaCl3. LaCl3 is a plasma membrane Ca2+ channel blocker that inhibits the cytoplasmic Ca2+ burst. As shown in Additional File 5: Figure S5, supplementation with 5 mM dbcAMP led to a significant increase in cellulase production (CMCase and pNPCase activities) in wild-type QM6a. However, cellulase activities decreased significantly when LaCl3 supplements were added compared to the no-LaCl3 control. These results suggest that cAMP signalling causes Ca2+ signalling to regulate cellulase production in T. reesei.
PLC-E mediates both Mn2+- and DMF-stimulated cellulase overexpression
To gain further insight into the mechanism by which Mn2+/DMF stimulates cellulase overexpression, we compared the transcriptomes of three T. reesei QM6a cultures, with no addition, with the addition of 10 mM Mn2+, or with the addition of 1% DMF (liquid MM containing 1% Avicel as the sole carbon source) incubated at 28 °C and 200 rpm for 36 h [20]. This resulted in the retrieval of 846 genes that were differentially expressed following 10 mM Mn2+ addition compared to the control (no addition). Of these 846 genes, 580 were up-regulated and 266 were down-regulated (Additional File 6: Figure S6, Additional File 7: Table S1). In our previous study, we compared the transcriptomes of T. reesei QM6a cultured with no additions and the addition of 1% DMF. We observed that 81 genes were upregulated and 21 were downregulated following 1% DMF addition, compared to the control [20]. In the current study, we analysed the overlap between the differentially expressed genes regulated by Mn2+ and those in DMF. Sixty-three genes were differentially expressed in the presence of both Mn2+ and DMF (Additional File 8: Table S2). Of these genes, 56 were upregulated and 7 genes were downregulated by both Mn2+ and DMF compared with no addition. The same genes were up- or down-regulated under Mn2+/DMF stimulation, implying a similar putative mechanism of cellular overexpression by Mn2+/DMF.
Of the genes that were consistently differentially expressed by both Mn2+ and DMF, 11 cellulose degradation-related genes were significantly upregulated in the presence of 1% DMF or 10 mM Mn2+ (Additional File 9: Table S3). Two main cellobiohydrolase-encoding genes (cbh1 and cbh2; IDs: 123,989 and 72,567), three endoglucanase-encoding genes (IDs: 122,081, 120,312, and 49,976), including two main endoglucanase genes (egl1 and egl2), one beta-glucosidase-encoding gene (cel3b; ID: 121,735), a xylanase-encoding gene (xyn3; ID: 120,229), and transcription of accessory protein-encoding genes, including those of swollenin gene swo1 (ID: 123,992) and cip2 (ID: 123,940) [7], were consistently upregulated in response to 10 mM Mn2+ and DMF addition. The transcriptome data agreed with our previous cellulase activity and qPCR results [19, 20].
Of the genes that were consistently differentially expressed by both Mn2+ and DMF, the transcriptional levels of plc-e, which encodes a phospholipase C protein, were significantly upregulated in response to 10 mM Mn2+ and DMF addition (Additional File 10: Table S4 and [20]). Phospholipase C activity is related to calcium release from intracellular pools, which increases the concentration of cytosolic Ca2+ resulting in a cytosolic Ca2+ burst [11]. We previously reported that the transcriptional level of plc-e was remarkably upregulated in DMF-stimulated strains and suggested that PLC-E is involved in DMF-stimulated cellulase overexpression [20]. The latest results implied that PLC-E might also participate in Mn2+-stimulated cellulase overexpression. Thus, we further examined the role of PLC-E in cellulase overexpression under Mn2+ addition. As shown in Fig. 4, the effect of Mn2+ stimulation on cellulase production was remarkably reduced in the Δplc-e mutant, which was constructed in our previous study [20]. A marked increase in cellulase production (Fig. 4a, b) and the transcription levels of the main cellulase genes (Fig. 4c, d) stimulated by Mn2+ were observed in the wild-type QM6a, while the effect of Mn2+ stimulation on cellulase expression was remarkably reduced in the plc-e deletion strain at all time points (Fig. 4). The growth rate related to cellulase activity and transcription levels of cbh1 and egl1 after Mn2+ stimulation in the Δplc-e mutant were significantly decreased compared with those in wild-type QM6a (Additional File 11: Figure S7). The pNPCase activity in the plc-e re-complementation strain Rplc-e was similar to that in QM6a (Additional File 2: Figure S2). These results indicate that PLC-E is vital for cellulase overexpression in response to both Mn2+ and DMF stimulation.
PLC-E exerts an important link between cAMP and Ca2+ signalling in the expression of cellulase
Earlier studies revealed that extracellular signals can activate phospholipase C (PLC), which is correlated with calcium signalling [11, 27]. Presently, the Ca2+ burst depended on the accumulation of cAMP, and PLC-E was a mediator in Mn2+/DMF-stimulated cellulase overexpression (Figs. 3, 4) and [20]. Additionally, the cytosolic cAMP concentration in the Δplc-e strains showed no obvious change compared with that of QM6a under Mn2+/DMF stimulation, forskolin supplementation, or exogenous dbcAMP addition (Fig. 1). Thus, we hypothesised that PLC-E is an important link between cAMP and Ca2+.
To clarify whether PLC-E is involved in Ca2+ bursts induced by cAMP, the Ca2+ concentration was compared between the QM6a and Δplc-e strains treated with Mn2+, DMF, forskolin, and dbcAMP. The cytosolic Ca2+ levels remained almost stable or were only slightly enhanced in the Δplc-e strains in the presence of Mn2+/DMF stimulation, forskolin supplementation, or exogenous dbcAMP addition. A significant Ca2+ burst was observed in wild-type QM6a under all these conditions compared with that of the untreated control (Fig. 5a, b). The cytosolic Ca2+ burst induced by cAMP was significantly weakened in the Δplc-e strains. These results suggest that PLC-E mediates the Ca2+ burst induced by cAMP. Additionally, the cytosolic cAMP concentration in the Δplc-e strains was similar to that of QM6a under conditions of Mn2+/DMF stimulation, forskolin supplementation, or exogenous dbcAMP addition (Fig. 1). These results suggest that cAMP induces Ca2+ bursts through PLC-E. Furthermore, the significantly upregulated expression levels of three Ca2+ signalling genes (cam, cna and crz1) in wild-type QM6a induced by cAMP were significantly weakened in the Δplc-e strains (Additional File 12: Figure S8). The collective results implicated PLC-E as an important link between cAMP and Ca2+ signalling in cellulase expression.