Fig 1.
Cultured Saccharina latissima gametophytes used in this study.
Vegetative filaments of A) males and B) females under RL vegetative growth conditions. The same male C) and female D) cultures after 8 d in WL gametogenic conditions. Solid and open arrows indicate sites of antheridial and oogonial development, respectively. Scale bar = 50 μm.
Fig 2.
Cluster analysis of differential gene expression.
Heatmap of 512 KEGG-annotated genes showing differential expression between gametophyte transcriptomes, either between sexes (F = female; M = male gametophytes) or timepoints (time = 0, 1, 6, 8 d). Expression values for each KEGG gene (row) are normalized across all samples (columns) by Z-score. Both column and row clustering were applied, and distinct gene clusters identified by the Gap statistic method are shown to illustrate the major expression patterns observed in the data.
Fig 3.
Female gametophyte expression.
MA plots (log expression ratio vs. mean average expression) comparing female gametophyte gene expression (as KEGG-annotated genes) under vegetative growth (d0) with gametogenic conditions after a) 1 d, b) 6 d, and c) 8 d in WL. Each point on the plots represents a unique KEGG gene, with differentially expressed genes shown as larger orange points (edgeR, FDR < 0.05). The plots show expression on the x-axes as average Log2 counts per million (CPM), and the ratio of RL/WL expression as Log2(RL/WL) is shown on the y-axes. Venn diagrams summarise the expression changes across timepoints for genes up-regulated (upper Venn) and down-regulated (lower Venn) in gametogenic (WL) compared with vegetative (RL) conditions. A full list of KEGG gene annotations can be found in S2 Table.
Fig 4.
MA plots (log expression ratio vs. mean average expression) comparing male gametophyte gene expression (as KEGG-annotated genes) under vegetative growth (d0) with gametogenic conditions after a) 1 d, b) 6 d, and c) 8 d in WL. Each point on the plots represents a unique KEGG gene, with differentially expressed genes shown as larger orange points (edgeR, FDR < 0.05). The plots show expression on the x-axes as average Log2 counts per million (CPM), and the ratio of RL/WL expression as Log2(RL/WL) is shown on the y-axes. Venn diagrams summarise the expression changes across timepoints for genes up-regulated (upper Venn) and down-regulated (lower Venn) in gametogenic (WL) compared with vegetative (RL) conditions. A full list of KEGG gene annotations can be found in S2 Table.
Table 1.
Core “early responsive” KEGG genes in female and male gametophytes.
Fig 5.
Comparative expression in males and females during gametogenesis.
Venn diagrams showing numbers of differentially expressed genes (KEGG-annotated genes) upregulated at each experimental timepoint in females a) and males b). Differentially expressed genes under vegetative conditions only are highlighted in red, “core” genes overexpressed in all conditions in one of the sexes are highlighted in bold, while WL-responsive genes are highlighted in purple (females) or blue (males). MA plots (log expression ratio vs. mean average expression) of female vs. male gametophyte expression of KEGG-annotated genes in vegetative growth conditions c) 0 days, and after d) 1 day, e) 6 days and f) 8 days of culture under WL gametogenic conditions. Each point represents a unique KEGG gene, with differentially expressed shown as larger green points (edgeR; FDR < 0.05) and gene labels are colour-coded according to the Venn diagrams to the left (vegetative = red; “core” = black; WL-responsive = purple or blue for females and males, respectively). The plots show expression on the x-axes as average Log2 counts per million (CPM), and the ratio of female/male expression as Log2(F/M) is shown on the y-axes. KEGG gene information: 1) Female vegetative (RL): lplA: lipoate-protein ligase; ATG101: autophagy-related protein 101; ATP23: mitochondrial inner membrane protease; NOP15: nucleolar protein 15; TOM40: mitochondrial import receptor subunit; SEC13: protein transport protein; fabI: enoyl-[acyl-carrier protein] reductase I; RRP41: exosome complex component; PSMB5: 20S proteasome subunit beta 5; EIF2S1: translation initiation factor 2 subunit 1. 2) Female “core” up-regulated: HERC3: E3 ubiquitin-protein ligase; phoB1: two-component system, OmpR family, alkaline phosphatase synthesis response regulator; pepM: phosphoenolpyruvate phosphomutase; CTSE: cathepsin E; NOX2: NADPH oxidase 2; NAGLT1: MFS transporter, FHS family, Na+ dependent glucose transporter 1; SLC24A5: solute carrier family 24 (sodium/potassium/calcium exchanger), member 5; SYF1: pre-mRNA-splicing factor; ECT2: protein ECT2; KCTD9: BTB/POZ domain-containing protein; LSM3: U6 snRNA-associated Sm-like protein; INO1: myo-inositol-1-phosphate synthase; ispE: 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; MSH2: DNA mismatch repair protein; HPGDS: prostaglandin-H2 D-isomerase / glutathione transferase. 3) Male “core” up-regulated: HMGB2: high mobility group protein B2; SHPRH: E3 ubiquitin-protein ligase; GAL3ST3: galactose-3-O-sulfotransferase 3. 4) Female WL-responsive: ABHD11: abhydrolase domain-containing protein 11; ABHD17: abhydrolase domain-containing protein 17; ACACA: acetyl-CoA carboxylase / biotin carboxylase 1; accC: acetyl-CoA carboxylase, biotin carboxylase subunit; AGPHD1: hydroxylysine kinase; APTX: aprataxin; ASPM: abnormal spindle-like microcephaly-associated protein; CEP41: centrosomal protein; CHMP4: charged multivesicular body protein 4; D2HGDH: D-2-hydroxyglutarate dehydrogenase; DECR2: peroxisomal 2,4-dienoyl-CoA reductase; DEPDC5: DEP domain-containing protein 5; DPM1: dolichol-phosphate mannosyltransferase; dxr: 1-deoxy-D-xylulose-5-phosphate reductoisomerase; EIF2B2: translation initiation factor eIF-2B subunit beta; gdhA: glutamate dehydrogenase (NADP+); groES: chaperonin GroES; GSTK1: glutathione S-transferase kappa 1; HDAC11: histone deacetylase 11; ilvH: acetolactate synthase I/III small subunit; ITGB1: integrin beta 1; LYRM4: LYR motif-containing protein 4; MCM7: DNA replication licensing factor; MINDY3_4: ubiquitin carboxyl-terminal hydrolase; msrB: peptide-methionine (R)-S-oxide reductase; mtnD: 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase; NDUFAB1: NADH dehydrogenase (ubiquinone) 1 alpha/beta subcomplex 1; NIT1: deaminated glutathione amidase; NTO1: NuA3 HAT complex component; nuoB: NADH-quinone oxidoreductase subunit B; nusG: transcriptional antiterminator; PDCD5: programmed cell death protein 5; pepD: dipeptidase D; petF: ferredoxin; PEX4: peroxin-4; PGK: phosphoglycerate kinase; POLB: DNA polymerase beta; PPIH: peptidyl-prolyl isomerase H (cyclophilin H); RP-L15: large subunit ribosomal protein L15; SLC25A4S: solute carrier family 25 (mitochondrial adenine nucleotide translocator); thiG: thiazole synthase; trxA: thioredoxin 1; USP34: ubiquitin carboxyl-terminal hydrolase 34; XDH: xanthine dehydrogenase/oxidase; XRCC2: DNA-repair protein; yggS: PLP dependent protein; yhbH: putative sigma-54 modulation protein. 5) Male WL-responsive: ALG5: dolichyl-phosphate beta-glucosyltransferase; ANO7: anoctamin-7; APC7: anaphase-promoting complex subunit 7; argC: N-acetyl-gamma-glutamyl-phosphate reductase; ARL2BP: ADP-ribosylation factor-like protein 2-binding protein; ARL3: ADP-ribosylation factor-like protein 3; ARL8: ADP-ribosylation factor-like protein 8; AURKX: aurora kinase; AVIL: advillin; BBS9: Bardet-Biedl syndrome 9 protein; CCNT: cyclin T; CCT6: T-complex protein 1 subunit zeta; CETN3: centrin-3; CK: creatine kinase; CNOT1: CCR4-NOT transcription complex subunit 1; DEK: protein DEK; DNAAF2: dynein assembly factor 2, axonemal; DNAI1: dynein intermediate chain 1, axonemal; DNAJB1: DnaJ homolog subfamily B member 1; DNAL1: dynein light chain 1, axonemal; DNALI: dynein light intermediate chain, axonemal; DUR3: urea-proton symporter; DYNC2H: dynein heavy chain 2, cytosolic; DYNC2LI: dynein light intermediate chain 2, cytosolic; DYX1C1: dyslexia susceptibility 1 candidate gene 1 protein; ELF2C: eukaryotic translation initiation factor 2C; ERN1: serine/threonine-protein kinase/endoribonuclease IRE1; GATM: glycine amidinotransferase; GINS4: GINS complex subunit 4; HECTD3: E3 ubiquitin-protein ligase; HNRNPR: heterogeneous nuclear ribonucleoprotein R; HS3ST5: [heparan sulfate]-glucosamine 3-sulfotransferase 5; IFT22: intraflagellar transport protein 22; IFT43: intraflagellar transport protein 43; IFT46: intraflagellar transport protein 46; IFT80: intraflagellar transport protein 80; IFT81: intraflagellar transport protein 81; IFT172: intraflagellar transport protein 172; ILK: integrin-linked kinase; ITPR1: inositol 1,4,5-triphosphate receptor type 1; KIF4_21_27: kinesin family member; KIF13: kinesin family member 13; lysC: aspartate kinase; MBD4: methyl-CpG-binding domain protein 4; MCM3: DNA replication licensing factor; MYO7A: myosin VIIa; ORC1: origin recognition complex subunit 1; PELI: pellino; PIN1: peptidyl-prolyl cis-trans isomerase NIMA-interacting 1; POLA1: DNA polymerase alpha subunit A; PPP1R42: protein phosphatase 1 regulatory subunit 42; PRKX: protein kinase X; PRPF19: pre-mRNA-processing factor 19; RAB28: Ras-related protein; RDH12: retinol dehydrogenase 12; RENT2: regulator of nonsense transcripts 2; REV1: DNA repair protein; RNF115_126: E3 ubiquitin-protein ligase; RVB2: RuvB-like protein 2; SLC25A28_37: solute carrier family 25 (mitochondrial iron transporter); SMC4: structural maintenance of chromosome 4; TRAF3IP1: TRAF3-interacting protein 1; TUBB: tubulin beta; U2AF1: splicing factor U2AF 35 kDa subunit; UBE2D: ubiquitin-conjugating enzyme E2 D.
Table 2.
Over-expressed genes in female gametophytes.
Table 3.
Flagella-related genes over-expressed in male gametophytes.