Regulation of Primary Metabolic Pathways in Oyster Mushroom

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1 Regulation of Primary Metabolic Pathways in Oyster Mushroom Mycelia Induced by Blue Light Stimulation: Accumulation of Shikimic Acid Masanobu Kojima*, Ninako Kimura & Ryuhei Miura Department of Bioscience and Biotechnology, Shinshu University, Kamiina, Nagano , Japan. *Correspondence and requests for materials should be addressed to M.K.

2 Legends: Supplementary Fig. S1. PLC analysis of shikimic acid in oyster mushroom mycelia cultivated in the dark (before irradiation) and under blue light stimulation for h. Supplementary Fig. S. Comprehensive analysis of gene expression in oyster mushroom mycelia after blue light stimulation using a custom microarray: PFD, 150 µmol m - s -1 ; stimulation time, 0, 0.5, 1,,, 1, and h. a h. Altered transcripts were quantified using the comparative method. Genes with greater than or equal to -fold changes in signal intensity were used to identify significant differences in gene expression. Differentially expressed genes were also subjected to K-means analysis and were classified into eight clusters (I VIII). Total number of genes (TNG) categorized in each cluster is shown in a h. Supplementary Fig. S. CA of the time course of gene expression related to aromatic amino acid biosynthesis, glycolysis/gluconeogenesis and the pentose phosphate pathway. Supplementary Fig. S. Glycolysis pathway. EC (PFK) is a rate-determining enzyme.

3 Supplementary Fig. S5. Pentose phosphate pathway. EC (GPD) is a rate-determining enzyme. Supplementary Fig. S. Time course of the expression levels of the rate-determining enzymes as detected by antibody response. The expression levels of the enzymes were analyzed using ImageJ software; n = ; error bar = SD. Supplementary Table S1. CA of the time course induction of 1 primary metabolites by blue light stimulation Supplementary Table S. Probe names of genes represented in the custom microarray, EC number and the KEGG Orthology (KO) ID of the corresponding protein Supplementary Table S. Forward and reverse primers synthesized for quantitative PCR analysis Supplementary Table S. Peptides synthesized for preparation of polyclonal primary antibodies against the target proteins

x 50 mm Detection UV Wavelength: 10 nm Mobile Phase: 1% PO /MeCN = 10/90 Flow Rate: 0.

4 Before irradiation Analysis Conditions: Column: Inertsil ILIC (GL Sciences Ltd.) Particle Size: 5 μm Length: Φ. x 50 mm Detection UV Wavelength: 10 nm Mobile Phase: 1% PO /MeCN = 10/90 Flow Rate: 0. ml/min Oven Temp.: 0 o C Injection Amount: 10 μl Retention Time of Shikimic Acid: 11.5 min After blue light stimulation for h Supplementary Fig. S1

5 a Expression level Cluster I (TNG: 110) b Cluster II (TNG: 75) Stimulation time (h) Expression level Stimulation time (h) c Expression level Cluster III (TNG: 7) Stimulation time (h) Supplementary Fig. Sa-d d Expression level Cluster IV (TNG: 189) Stimulation time (h)

6 e Expression level Cluster V (TNG: 119) Stimulation time (h) f Expression level Cluster VI (TNG: 55) Stimulation time (h) g Expression level Cluster VII (TNG: 5) Stimulation time (h) Supplementary Fig. Se-h h Expression level Cluster VIII (TNG: 115) Stimulation time (h)

7 CA of the Time Course of Gene Expression Related to Phenylalanine, Tyrosine, Tryptophan Biosynthesis (Cf. KEGG map0000) Color range of expression level a ierarchical clustering on genes associated with the enzymes ierarchical clustering on the (time-series) samples EC number a Red and green cells indicate the ratio of each expression level above and below the median, respectively. Supplementary Fig. Sa

8 CA of the Time Course of Gene Expression Related to Glycolysis/Gluconeogenesis (Cf. KEGG map00010) Color range of expression level a ierarchical clustering on genes associated with the enzymes ierarchical clustering on the (time-series) samples a Red and green cells indicate the ratio of each expression level above and below the median, respectively. Supplementary Fig. Sb EC number

9 CA of the Time Course of Gene Expression Related to the Pentose Phosphate Pathway (Cf. KEGG map0000) Color range of expression level a ierarchical clustering on genes associated with the enzymes ierarchical clustering on the (time-series) samples a Red and green cells indicate the ratio of each expression level above and below the median, respectively. EC number Supplementary Fig. Sc

10 C O O O O O O Glucose (Glc) O O C OPO O O O Glucose -phosphate (GP) O C O O O O C OPO Fructose -phosphate (FP) O C OPO O O O C OPO Fructose 1,-bisphosphate (F1,BP) C OPO O C O Dihydroxyacetone phosphate (DAP) O PO COO - Phosphoenolpyruvic acid (PEP) O O - OPO C O -Phosphoglyceric acid (PG) O O - O C OPO -Phosphoglyceric acid (PG) O OPO O C OPO 1,-Bisphosphoglyceric acid (1,BPG) O O C OPO Glyceraldehyde -phosphate (GAP) O CO - Pyruvic acid (Pyr) Supplementary Fig. S

11 C O O O O O O Glucose (Glu) O O C OPO O O O Glucose -phosphate (GP) O O C OPO O O O -Phosphogluconolactone O CO - O O O C OPO - -phosphogluconic acid O CO O O C OPO Erythrose -phosphate (EP) + C O O O O C OPO - Fructose -phosphate (FP) O C O O O O O C OPO - Sedoheptulose 7-phosphate (S7P) + CO O C OPO Glyceraldehyde -phosphate (GAP) O CO O O O C OPO Ribose 5-phosphate (R5P) + C O O O C OPO Xylulose 5-phosphate (Xu5P) C O O O O C OPO Ribulose 5-phosphate (Ru5P) Supplementary Fig. S5

12 a EC : PFK b EC : GPD PFK expression Light stimulation (h) GPD expression Light stimulation (h) EC.5.1.5: DAPS c d e expression Light stimulation (h) 108 expression Light stimulation (h) expression Light stimulation (h) Supplementary Fig. S

13 Supplementary Table S1. CA of the Time Course Induction of 1 Primary Metabolites Induced by Blue Light Stimulation NO. Compound name Blue-Light Stimulation Time vs Standardized Score Dark 0 h h 1 h h h h 1 γ-glu--aminobutyric acid Phosphorylcholine XC001 (Unknown) NADP_divalent N-Acetylglutamic acid Citrulline Isopropylmalic acid Aminobutyric acid XA00 (Unknown) ydroxybutyric acid N-Acetyllysine S-Adenosylmethionine N-Acetylornithine Fructose 1,-diphosphate Cystathionine Adenylosuccinic acid Lys myo-inositol -phosphate Saccharopine ypotaurine exanoic acid N-Acetylglucosamine -phosphate Argininosuccinic acid Uric acid Ornithine Aminovaleric acid Betaine β-ala Ophthalmic acid Choline Glucose 1-phosphate is N-Acetylserine UMP Betaine aldehyde_+o Glucose -phosphate m-ydroxybenzoic acid Butyrylcarnitine Arg ADP-glucoseGDP-fucose Aminobutyric acid O-Phosphoserine S-Adenosylhomocysteine Threonic acid Fructose -phosphate Sucrose '-phosphate Uracil Phosphoglyceric acid N,N-Dimethylhistidine Carboxymethyllysine Aminoisobutyric acid omocitrulline XC0089 (Unknown) Glycerol -phosphate CDP-choline Pro Dihydroxyacetone phosphate UDP-glucoseUDP-galactose Ergothioneine myo-inositol 1-phosphate myo-inositol -phosphate UDP-glucuronic acid Sedoheptulose 7-phosphate Uridine N-Acetylneuraminic acid Phosphoglyceric acid omoserine datp dtdp istidinol Erythrose -phosphate Sebacic acid

14 7 GDP UDP dttp ydroxyproline Cys Adenine GMP ydroxydecanoic acid CoA_divalent UTP Citric acid Ribulose 5-phosphate Glutathione (GS) GABA Aminoadipic acid Phosphogluconic acid N-Formylaspartic acid Pantothenic acid O-Succinylhomoserine Val GTP Creatine Ethanolamine ATP Malic acid N-Acetylaspartic acid Cadaverine FAD_divalent Trp Methylserine ADP Glucuronic acid N-Acetylasparagine Phe Leu Cytidine Carnitine Asp N-Acetylglycine cgmp dtdp-glucose Fumaric acid Methyl--oxovaleric acid -Methyl--oxovaleric acid CTP UDP-N-acetylglucosamine XA000 (Unknown) CDP Trigonelline Ribose 5-phosphate N-Acetylmethionine Terephthalic acid NMN Gln Met Spermidine camp ydroxy--methylglutaric acid Tryptamine Oxypurinol Oxoproline N-Acetylleucine Pipecolic acid Trimethylamine Trehalose -phosphate Guanidinobutyric acid XC000 (Unknown) Methylhistidine Isocitric acid CMP-N-acetylneuraminate β-ala-lys Phenylpyruvic acid ccmp','-ccmp XA000 (Unknown) Glucosaminic acid Galacturonic acid ydroxypropionic acid ,-Diaminobutyric acid

15 19 -Phenylethylamine Lactic acid SDMA Inosine AMP Putrescine Ile Imidazole--acetic acid γ-butyrobetaine Acetamidobutanoic acid Formylanthranilic acid trans-glutaconic acid Adenosine Shikimic acid Glutathione (GSSG)_divalent ADMA Digalacturonic acid Nω-Methylarginine CMP Cysteine glutathione disulfide Ala-Ala Gly Methionine sulfoxide Imidazolelactic acid Ser Isethionic acid Gluconolactone Suberic acid '-Dephospho CoA XC0017 (Unknown) N5-Ethylglutamine Succinic acid ydroxyvaleric acid N,N,N-Trimethyllysine Gluconic acid Guanine Arg-Glu Glycerophosphocholine Allantoic acid Isovalerylcarnitine Isobutyrylcarnitine N-Acetyllysine O-Acetylcarnitine Tyr Sorbitol -phosphate XC005 (Unknown) Serotonin XC01 (Unknown) Guanosine Glycerol Trimethylamine N-oxide N-Acetylglucosamine 1-phosphate Thr Asn Gly-Asp NAD Imidazole--methanol Phthalic acid NADP Glyceric acid N-Carbamylglutamic acid cis-aconitic acid Carnosine Pyruvic acid

16 Supplementary Table S. Probe Names of Genes Represented in the Custom Microarray, EC Number and the KEGG Orthology (KO) ID of the Corresponding Protein Phenylalanine, Tyrosine and Tryptophan Biosynthesis Pathway (KEGG Map0000) KEGG ORTOLOGY EC number Probe Name (KO) ID CUST_178_PI507 K CUST_90_PI507 K007 CUST_81_PI CUST_1055_PI507 K01 CUST_158_PI CUST_71_PI507 K CUST_11589_PI507 K CUST_91_PI507 K CUST_990_PI507 K CUST_7_PI507 K CUST_855_PI507 K01850

17 Glycolysis/Guconeogenesis Pathway (KEGG Map00010) EC number Probe Name CUST_1058_PI507 K0001 CUST_1059_PI K001 CUST_1058_PI507 CUST_10900_PI507 CUST_10901_PI507 CUST_01_PI CUST_5_PI507 K0018 CUST_110_PI507 CUST_10890_PI507 CUST_50_PI CUST_11109_PI507 K CUST_70_PI507 K CUST_719_PI507 CUST_75_PI507 K CUST_970_PI507 K CUST_597_PI507 K CUST_11_PI507 K CUST_8_PI507 K CUST_88_PI507 K CUST PI507 K CUST_889_PI507 K CUST_18_PI507 K CUST_80_PI507 K CUST_50_PI507 CUST_111_PI507 K CUST_11091_PI507 K CUST_01_PI507 K CUST_1_PI507 CUST_79_PI507 K CUST_110_PI507 K01895 KEGG ORTOLOGY (KO) ID

18 Pentose Phosphate Pathway (KEGG map0000) EC number Probe Name CUST_95_PI K000 CUST_110_PI CUST_77_PI507 K CUST_119_PI507 K0015 CUST_817_PI CUST_119_PI507 K001 CUST_105_PI CUST_970_PI507 K CUST_00_PI507 K CUST_55_PI507 K0085 CUST_501_PI K0098 CUST_110_PI507 CUST_1180_PI CUST_11809_PI507 K0105 CUST_710_PI CUST_75_PI507 K CUST_8_PI507 K CUST_595_PI507 K CUST_10980_PI507 K CUST_01_PI507 K01810 CUST_1_PI K0185 CUST_79_PI507 KEGG ORTOLOGY (KO) ID

19 Supplementary Table S. Forward and Reverse Primers Synthesized for Quantitative PCR Analysis EC / Protein ID Forward Primer Reverse Primer EC / 177 5'-TCA GCA ATA ACG TCC CGA TG-' 5'-TTG CTT GAT CGC ATC ACA GG-' EC / '-ACG TTC AAG GAA CCG TTT GG-' 5'-ACC TGC AGC AAA TGG TTC TG-' EC / '-ACA CAT CCA AGA TGC GTT CG-' 5'-AAT TGC CAT GGG AGC AAT CG-' EC / 108 5'-TTC TGA GAT CGA ACG CTT GC-' 5'-TTT CAT GAG TGC AGG CAT CG-' EC / '-ACG CCA AAC TCC TCA AAC AG-' 5'-TTG TTC GGG GCT TTT CGA AG-'

20 Supplementary Table S. Peptides Synthesized for Preparation of Polyclonal Primary Antibodies against the Target Proteins Target protein EC / Protein ID AMW a / kda Synthesized peptide phosphofructokinase (PFK) EC / CDNPGTTNYGEGTGQDTE glucose--phosphate dehydrogenase (GPD) EC / SDSGTIPSMETSDEC -deoxy-d-arabinoheptulosonate 7-phosphate (DAP) synthase -deoxy-d-arabinoheptulosonate 7-phosphate (DAP) synthase -deoxy-d-arabinoheptulosonate 7-phosphate (DAP) synthase a Average molecular weight EC / MGKPLVPDFSDDEKVC EC / CEQNNLLYSELGGRERKP EC / CRQGVRGRRELLKKQARP

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Supplemental file 3. All 306 mapped IDs collected by IPA program. Supplemental file 6. The functions and main focused genes in each network.

LIST OF SUPPLEMENTAL FILES Supplemental file 1. Primer sets used for qrt-pcr. Supplemental file 2. All 1305 differentially expressed genes. Supplemental file 3. All 306 mapped IDs collected by IPA program.