The molecular mechanisms of Monascus purpureus M9 responses to blue light based on the transcriptome analysis

Light is an important environmental factor that regulates various physiological processes of fungi. To thoroughly study the responses of Monascus to blue light, transcriptome sequencing was performed on mRNAs isolated from samples of Monascus purpureus M9 cultured under three conditions: darkness (D); exposure to blue light for 15 min/d (B15); and exposure to blue light for 60 min/d over 8 days (B60). The number of differentially expressed genes between the three pairs of samples—B15 vs D, B60 vs B15, and B60 vs D—was 1167, 1172, and 220, respectively. KEGG analysis showed the genes involved in primary metabolism including carbon and nitrogen metabolism were downregulated by B15 light treatment, whereas B15 upregulated expression of genes involved with aromatic amino acid metabolism, which associated with development, and branched chain amino acid metabolism, and fatty acid degradation, which can produce the biosynthetic precursors of pigments. When exposed to B60 conditions, genes with roles in carbohydrate metabolism and protein synthesis were upregulated as part of a stress response to blue light. Based on this study, we propose a predicted light-stimulated signal transduction pathway in Monascus. Our work is the first comprehensive investigation concerning the mechanism of Monascus responses to blue light.

activate light-responsive genes transcription 20 . Such genes are involved in various physiological processes constituting a regulatory network in N. crassa 10 . Few papers have reported light regulatory mechanisms in Monascus.
With the rapid development and reduction in cost of next generation sequencing, RNA-seq has been widely used to characterize differentially expressed genes under various conditions 21,22 . Wu et al. measured changes in mRNA levels of light-regulated genes while studying the responses of N. crassa to light 23 . Several candidate genes and signaling pathways associated with light-induced brown film formation in Lentinula edodes have also been analyzed via RNA-seq 24 . In this study, three groups of mRNAs were isolated from the mycelia of M. purpureus M9 samples that were cultivated under one of three conditions: grown in the dark (D); exposed to blue light for 15 min/d (B15); or exposed to blue light for 60 min/d (B60). All samples were grown in their respective conditions for eight days, after which their corresponding transcripts were identified by high throughput sequencing and analyzed using digital gene expression profiles. More than one thousand differentially expressed genes (DEGs) were found to respond to blue light and to exert widespread effects on the metabolism of M9. This work was the first investigation concerning the genome-wide response of Monascus to blue light. Our results will facilitate an understanding of its light-stimulated regulatory mechanisms.

Results
Sequencing and de novo assembly. To better survey the molecular mechanism of blue light-induced transcription in Monascus, two biological replicates of each condition were obtained, with the B15 and B60 treatments labeled the "early" and "late" light response, respectively. Capped and polyadenylated mRNA was purified from mycelia, after which cDNA was produced and sequenced; a total of 24-33 million reads were obtained from the six samples. All error rates were below 0.01%, while GC content accounted for about 52% of sequenced bases, indicating highly accurate sequencing data (Supplementary Data S1). Homology analysis showed that M9 is closely related to M. purpureus YY-1, so we chose to use the published genome of YY-1 as a reference ( Supplementary Fig. S1). The total mapped reads of all six samples possessed greater than 70% sequence identity with respect to the reference genome, again corroborating the validity of our data (Supplementary Data S2) 25 . The strong correlation (R 2 > 0.97) between biological replicates suggested excellent reproducibility of results for cultures grown under the three different conditions ( Supplementary Fig. S2).
Analysis of differentially expressed genes. About 93% of genes were expressed with fragments per kilo base per million reads (FPKM) > 1 at one or more of the time points analyzed; these were considered "expressed genes" (Supplementary Data S3). A Venn diagram illustrates that 1167, 1172, and 220 DEGs were found amongst the three sample groups: "B15 vs D", "B60 vs B15" and "B60 vs D", respectively (Fig. 1). The number of unique The diagram displays the number of unique and shared differentially expressed genes (DEGs) in the three sets of transcripts obtained from mycelia that were grown in the dark (D); exposed to blue light for 15 min/d (B15); and exposed to blue light for 60 min/d (B60). The number in one color represents the unique DEGs of one comparative group. The number in two overlapping colors represents the shared DEGs of two comparative groups. The number in three overlapping colors represents the shared DEGs of three comparative groups.
Scientific RepoRts | 7: 5537 | DOI:10.1038/s41598-017-05990-x DEGs belonging to B15 vs D (338) and B60 vs B15 (281) was much greater than the number belonging to B60 vs D (31), which indicated that the B15 light treatment induced expression of more genes than did B60 when compared to D (Fig. 1). Similar results were observed in a cluster analysis of DEGs: in terms of gene expression, there were relatively large differences between B15 and D, whereas expression in B60 was almost the same as in D. B15 upregulated/downregulated the expression of genes that were downregulated/upregulated under B60 (Fig. 2). It may be that M9 took stress response to short-term blue light stimulus (B15 vs D), so a large number of genes expression were regulated, and then when exposure time was extended to B60, it recovered genes expressions to the D level for adapting blue light (B60 vs B15).
GO functional classification and KEGG pathway enrichment of differentially expressed genes. GO terms significantly enriched in DEGs that were upregulated in B15 vs D group included heme binding, tetrapyrrole binding, oxidoreductase activity, oxidation-reduction process, iron ion binding, acyl-CoA dehydrogenase activity, and transmembrane transport (Fig. 3(A); Supplementary Data S4). Meanwhile, GO terms enriched in DEGs that were downregulated in the B15 set included single-organism metabolic process, single-organism process, oxidation-reduction process, carbohydrate metabolic process, oxoacid metabolic process, cofactor binding, oxidoreductase activity, oxidoreductase activity, and acting on CH-OH group of donors ( Fig. 3(A); Supplementary Data S5). These observations showed that significantly enrichment GO term in B15 vs D group mainly concentrated on oxidoreductase activity and oxidation-reduction process. Moreover, heme and tetrapyrrole are oxidoreductase cofactors and thus involved in redox reactions, supporting a theme in the GO results. Additionally, acyl-CoA dehydrogenase plays a key role in β-oxidation of fatty acids. It is clear that B15 light treatment induced expression of several genes involved in oxidation-reduction reactions in M. prupureus M9.
Comparing the B15 vs D and B60 vs B15 groups, we found that the significantly enriched GO terms downregulated by B15-cofactor binding and single-organism metabolic process-were upregulated by B60 (Fig. 3(B); Supplementary Data S6). Meanwhile, the major GO terms upregulated by B15 were downregulated Unsupervised hierarchical clustering of DEGs representing transcripts obtained from mycelia that were: grown in the dark (D); exposed to blue light for 15 min/d (B15); and exposed to blue light for 60 min/d (B60). The number of reads corresponding to each gene was divided by log 10 (FPKM + 1). Red: upregulated; blue: downregulated.
Scientific RepoRts | 7: 5537 | DOI:10.1038/s41598-017-05990-x by B60 ( Fig. 3(B); Supplementary Data S7). These results indicated that B15 light treatment induced several oxidation-reduction reactions in M9, while the number of these reactions declined to levels typical of the D samples when exposure time is extended to B60.
KEGG pathway analysis revealed that DEGs were significantly enriched in metabolic and signal transduction pathways. Compared with D, B15 light treatment upregulated numerous DEGs, which were significantly enriched in pathways controlling tryptophan metabolism, valine, leucine and isoleucine degradation, ribosome, phenylalanine metabolism, and tyrosine metabolism ( Fig. 4(A); Supplementary Data S8). Tryptophan, phenylalanine and tyrosine are aromatic amino acids and can be converted into several biological compounds by a series of oxidation reactions, including 5-hydroxy tryptamine, heteroauxin, dopamine, melanin involvement in physiology and growth of organism. Valine, leucine and isoleucine are branched chain amino acids; their degradation produces acetyl-CoA and methyl-CoA, which are synthetic precursors of type II polyketides such as Monascus pigments. Genes involved in the branched chain amino acid degradation pathway were upregulated in B15 samples, possibly indicating that Monascus pigment biosynthesis is induced by blue light.
The DEGs that showed significant enrichment within the KEGG pathway were upregulated in B15 vs D group; however, they were all downregulated in B60 vs B15 group ( Fig. 4(B); Supplementary Data S11). Although genes involved in fatty acid and aromatic compound degradation were not considered to be upregulated in B15 vs D group-due to their corrected P-values being greater than or equal to 0.05-they were still downregulated in B60 vs B15 group ( Fig. 4(B); Supplementary Data S11). Conversely, the significantly enrichment pathway which were downregulated in B15 vs D group (Fig. 4(A); Supplementary Data S9), were mostly upregulated in B60 vs B15 group pertained to carbon metabolism, glycolysis/gluconeogenesis, glycine, serine and threonine metabolism, and pyruvate metabolism ( Fig. 4(B); Supplementary Data S10). Even though the P-values of genes involves in the citrate cycle (TCA cycle) and amino acid biosynthesis were not significantly downregulated in B15 vs D group, they were still upregulated in B60 vs B15 group ( Fig. 4(B); Supplementary Data S10). Effects of blue light on primary metabolism of Monascus purpureus M9. According to KEGG pathway analysis, blue light exerted a global influence on the primary metabolism of M. purpureus M9. B15 light treatment reduced carbon and nitrogen metabolism while enhancing aromatic amino acid metabolism, associated with development of Monascus, and branched chain amino acid degradation and fatty acid degradation, with the last two taking part in polyketide biosynthesis. However, during B60 treatment, M9 upregulated carbohydrate metabolism and protein biosynthesis, apparently as part of its stress response to blue light.

Analysis of differentially expressed genes involved in pigment biosynthesis. A majority of
genes involved in Monascus pigment biosynthesis showed differential expression in the B15 vs D and B60 vs B15 comparisons, suggesting that pigment production is regulated by blue light. Expression of gene C5.129-a transcriptional regulator of the pigment biosynthesis gene cluster-as well as the genes comprising that cluster (C6.698, C1.1079, C2.21, C2.23, C2.29, and C2.31) was upregulated by blue light in the B15 set; with the exception of C5.138, those same genes were downregulated to their respective D levels when subjected to B60 treatment. This indicated that B15 light treatment was more favorable to inducing pigment biosynthesis than was B60 (Supplementary Table S1; Supplementary Data S12-17).
In our study, pigment yields were in accordance with gene expression. The color value of intracellular and extracellular Monascus pigments under B15 conditions were slightly higher than those under D conditions, while D and B60 color values were almost identical ( Supplementary Fig. S3(A)). However, in our previous study, we observed significant differences in the concentrations of red, orange, and yellow pigments between the dark and the blue light-exposed samples 14 .
Genes encoding heme-thiolate proteins such as the cytochrome P450 protein family-which are associated with pigment biosynthesis-also undergo light-induced regulation. Expression of the genes encoding cytochrome P450 proteins were upregulated by B15 light treatment and downregulated to their D levels when subjected to B60 conditions. Similarly, the anabolic pathway of heme, a co-factor of polyketide metabolism, was also regulated by blue light in a manner similar to that seen with cytochrome P450. Analysis of differentially expressed genes involved in growth and reproduction. Of 27 DEGs related to development, eight genes linked to asexual sporulation were upregulated by B15 light treatment, while the rest were downregulated compared to their expression levels in the D sample (Supplementary Table S2; Supplementary Data S12-17). The downregulated genes included C7.619, which is involved in initiating mitosis 26 ; C1.187, C1.813, and C3.795, which are associated with spindle formation [27][28][29][30] ; and C1.635, C2.396, and C6.392, which are linked to the spindle formation checkpoint [31][32][33] . These responses suggest that exposure to B15 conditions slows development of M9 (Supplementary Table S2; Supplementary Data S12-17).
However, most of the aforementioned genes recovered to their D-state expression levels after exposure to B60 conditions. It was theorized that M9 slowed growth and reproduction activity as part of an effort to resist blue light-induced stress.
Differential gene expression related to sexual and asexual development in M9 was also observed in response to blue light stimulus. Some genes related to sexual development were downregulated in the B15 set when compared to the D sample, such as C3.689 (which is involved in the initiation of meiosis 34 ), C1.277 (which also has a role in meiosis 35 ), and C5.27, C7.467, C5.108, C4.214, C2.450, which take part in gametogenesis and spore wall assembly (Supplementary Table S2; Supplementary Data 12-17) 36,37 . However, eight genes related to asexual development were upregulated in the B15 set. These included C6.584, which is involved in asexual growth 38 ; C2.339, which contributes to conidium formation; C5.674 and C4.520, which are required for conidium cell wall organization; and C4.715, a gene essential for spore release and hyphal growth in response to stress (Supplementary Table S2; Supplementary . Gene expression measurements were accompanied by phenotypic observation: more conidia were formed under blue light versus the number generated in darkness. Conversely, few cleistothecia appeared when mycelia were exposed to blue light, strongly limiting the formation of ascopores ( Supplementary Fig. S3(B)).  Fig. S4; Supplementary Data S12). IP3 is involved in intracellular Ca 2+ signaling in cells, a mechanism that regulates a host of physiological processes; DAG is a component in the protein kinase C (PKC) signal transduction pathway, which phosphorylates various proteins 39 . The most important signaling pathway in cells, the coupled IP3/Ca 2+ -DAG/PKC, was activated by B15 light treatment. Exposure to B15 conditions also upregulated the genes C3.606 (which encodes the calcium-transporting enzyme ATPase 2), C8.218 and C4.276 (which encode another calcium transport protein) and C1.416 (which codes for a calcium/ calmodulin-dependent protein kinase); each of the aforementioned genes was downregulated to their D-state levels upon exposure to B60 treatment (Supplementary Data S12; Supplementary Data S17).

MAPK signaling pathway is inhibited by blue light.
Mitogen-activated protein kinase (MAPK) cascades are signal transduction pathways that are found throughout eukaryotes, including yeasts, animals and plants. MAP kinase pathways are composed of a MAP kinase, a MAP kinase kinase, and a MAP kinase kinase kinase; the system is regulated by sequential phosphorylation of its components. These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses. Various physiological processes including conidial germination, hyphal fusion and carotenoid biosynthesis are controlled by MAPK cascades in Neurospora crassa 40 , which also play a key role during development and secondary metabolism in A. nidulans, A. niger and A. fumigatus 41,42

Discussion
Light is an important signal for every living cell. In fungi, alterations in sexual development, conidiation, circadian clock resetting, hyperpolarization of the cell membrane, and other metabolic processes have been observed in response to light 9 . More recently, RNA-seq was used to identify components of light perception machinery and their downstream signaling cascades in fungi. Neurospora transcriptome sequencing revealed that light-induced genes can be approximately classified as early response (with peak mRNA levels between 15 and 45 min of light treatment) and late response (with peak mRNA levels between 45 and 90 min of light treatment) 23 . The GATA family transcription factor SUB-1 was required for the activation by light of late response genes 43 . The genes frq (the central clock component frequency) and al-3 (mycelial carotenoid biosynthesis) were both early-and late-response genes 44 . However, the light-stimulated signal transduction pathway in Monascus was still unclear. To better understand the response of Monascus to blue light and its associated light-controlled gene regulatory network, we used transcriptome sequencing to identify M. purpureus M9 transcripts, then compared sequencing data from cells grown in darkness to cells exposed to blue light for 15 min and 60 min periods.
The results showed that blue light functions as a signal to modulate different physiological aspects in M9. Short-term exposure to blue light resulted in oxidative stress, which correlated with upregulation of genes encoding cytochrome C peroxidase (C8.314), peroxidase (C1.1127, C5.557, C8.171), and non-ribosomal peptide synthetase (C1.1079) (Supplementary Data S12). Conversely, there was a reduction in primary metabolic activity, including carbon and nitrogen metabolism; at the same time, M9 upregulated metabolic pathways involving aromatic amino acids, which is affiliated with development of M9, and branched chain amino acid degradation, which produces acetyl-CoA and malonyl-CoA, the biosynthetic precursors of pigments. Moreover, the β-oxidation of fatty acids was enhanced, providing ATP and NADH to fuel increased metabolic activity. It is notable that Monascus pigments play a role in protection against light damage in mycelia. In addition, it was observed that exposure to blue light led to increased conidia growth, possibly indicating that blue light induces a stress response in Monascus that promotes asexual reproduction (via spore formation) ( Supplementary Fig. S3(B)). However, long-term exposure to blue light under B60 conditions induces Monascus to upregulate carbohydrate metabolism and protein biosynthesis, apparently as defensive measures against blue light-induced stress.
Blue light exerted a widespread influence on M. purpureus M9. Based on our transcriptome analysis, we propose a possible blue light-induced signal transduction pathway in Monascus (Fig. 5). Short-term exposure to blue light (the B15 treatment) activated the IP3/Ca 2+ and DAG/PKC signal transduction pathway and inhibited the MAPK pathway, while also regulating the biosynthesis of transcription factors and other proteins that impact metabolic processes. In response to B15 treatment, M9 reduced its primary metabolism and slowed development and reproduction, while upregulating secondary metabolism, especially biosynthesis of photoprotective pigments that absorb blue light. However, no photoreceptors, such as WC-1 and WC-2 in N. crassa, have been identified in M. purpureus M9.
Our study investigated the physiological responses of Monascus to blue light via transcriptome sequencing, revealing that the photoresponse of M. purpureus differs in notable ways from the well-studied system found in N. crassa. Therefore, this work should represent a general advancement in both photobiology and M. purpureus research. We propose a possible light-stimulated signal transduction pathway in Monascus and expect it to provide the foundation for future studies of the molecular mechanisms of Monascus light responses. was reinoculated once a month. Spores suspension of M9 (adjusted to 10 6 spores /ml) was harvested by sterile water and inoculated into rice medium (rice powder, 50 g/L; KH 2 PO 4 , 1.5 g/L; NaNO 3 , 3 g/L and MgSO 4 ·7H 2 O, 1 g/L) in a 12-mm culture dish. The dishes were incubated at 30 °C without shaking in the darkness or exposed to blue light (100 lux) for 15 min/d, 60 min/d for 8 days, respectively. Light source is cool white illuminant of 465 nm without heating.
Fresh mycelia were obtained on the 8th day. Parts of mycelia were placed into 1.5 ml screw-cap tubes and snap-frozen in liquid nitrogen for RNA extraction. The remaining was used to morphology observation and pigments concentration analysis. Dry weight of mycelia was measured and pigment concentration was determined using a UV-visible spectrophotometer. Cleistothecia and conidia formations were observed by Olympus BH2 compound microscope.
RNA extraction, library preparation and sequencing. Total RNA was extracted from frozen mycelia using the Plant RNA Kit (Omega, CA, USA). RNA degradation and contamination was monitored on 1% agarose gels. The purity was checked using the NanoPhotometer ® spectrophotometer (IMPLEN, CA, USA). RNA concentration was measured using Qubit ® RNA Assay Kit in Qubit ® 2.0 Flurometer (Life Technologies, CA, USA) and integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Sequence reads assembly, mapping and annotation. Raw data of fastq format were firstly processed through in-house perl scripts. In this step, clean data were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
The Monascus purpureus YY-1 reference genome and gene model annotation files were downloaded from genome website (http://spxy.tust.edu.cn/duxj/index.html) directly 25 . Index of the reference genome was built using Bowtie v2.2.3 and paired-end clean reads were aligned to the reference genome using TopHat v2.0.12.
Gene function was annotated based on the following databases: the NR (NCBI non-redundant protein sequences), Swiss-Prot (A manually annotated and reviewed protein sequence database), KEGG (KEGG Ortholog database), and GO (Gene Ontology), using BLAST with a cutoff E-value of 10 −5 .
Quantification and differential expression analysis of transcripts. HTSeq v0.6.1 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene.
Differential expression analysis of three conditions (two biological replicates per condition) was performed using the DESeq R package (1.18.0). DESeq provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution 45 . The resulting P-values were adjusted using the Benjamini and Hochberg's approach for controlling the false discovery rate. Genes with an adjusted P-value < 0.05 found by DESeq were assigned as differentially expressed.

GO and KEGG enrichment analysis of differentially expressed genes. GO enrichment analysis of
DEGs was implemented by the GOseq R package, in which gene length bias was corrected. GO terms with corrected P-value < 0.05 were considered significantly enriched by differential expressed genes 46 .
KEGG is a database resource for understanding high-level functions and utilities of the biological system from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-through put experimental technologies 47 . We used KOBAS software to test the statistical enrichment of differential expression genes in KEGG pathways 48 . Confirmation of gene expression level by RT-qPCR. First-strand cDNA was synthesized using the PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa, Japan), with the Oligo dT Primer 15. Gene expression was monitored by RT-qPCR and carried out using the SYBR Premix Ex Taq II (TaKaRa, Japan). RT-qPCR was Scientific RepoRts | 7: 5537 | DOI:10.1038/s41598-017-05990-x performed using the Stratagen Mx3000 P (Agilent) with the following cycling program: hold at 95 °C for 30 s, followed by a three-step PCR (42 cycles of denaturation at 95 °C for 5 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 sec) and dissociation curve analysis (at 95 °C for 15 s, annealing at 60 °C for 30 s, then collecting the dissociation curve from 60 °C to 95 °C, finally at 95 °C for 15 s).

Statistical analysis.
For each sample, three technical replicates of the RT-qPCR assay were used with three biological replicates. Results were expressed as means ± standard deviation (SD) of the number of experiments. T-test for the values was performed at P < 0.05.