Figure 1.
Symbiodinium quantification by real-time quantitative PCR (symbiont to host nuclei ratio).
Symbiodinium nuclear EF2, SPS, APX and A. viridis nuclear COP-γ gene copy numbers were assessed by qPCR performed on total genomic DNA extracts from 5 symbiotic anemones (Sy1–Sy5), 6 aposymbiotic anemones (AS1–AS6), in vitro cultured Symbiodinium (CZ) and epidermal tissue fraction (Ep). The histogram represents the relative copy number ratios of EF2, SPS and APX to COP-γ, relative to the Sy1 sea anemone, expressed in fold number.
Figure 2.
Differentially expressed genes (DEG) between symbiotic and aposymbiotic anemones.
A. Heatmap diagram of DEG. Left column (Sy-Apo) represents expression of the 58 up- (SY) and 78 down-regulated (APO) genes (|M|>0.59, B>0) identified from the batch comparison of the 5 pooled symbiotic versus the 6 pooled aposymbiotic anemones. The following columns to the right represent expression of the same genes in each individual symbiotic anemone (Sy1–5) compared with the pooled aposymbiotic (-Apo) anemones and in each individual aposymbiotic anemone (AS1–6) compared with the pooled symbiotic (-Sy) anemones. The dendogram underneath the heatmap represents the cluster array tree result performed on the entire result dataset (with Cluster3). The color intensity code representing M value intervals is shown below. B. A given gene can be differentially expressed in the batch analysis (SY or APO) although only differentially expressed in few individual sea anemones. Histogram shows the number of individual anemones (3–11) each gene is differentially expressed in. Top and bottom panels: gene distribution of SY and APO genes, respectively.
Table 1.
Kern set of symbiotic genes.
Figure 3.
CA2-m, CA2-c, and NPC2-D expression in response to imposed thermal stress.
Expression of CA2-m, CA2-c and NPC2-D after 0, 24 h and 48 h of 8°C heat stress was measured using RT-qPCR. Three anemones (Sy3–5) were assayed. Gene expression, normalized to RCC2 and COP-γ, is given related to t0. Error bars represent standard error.
Figure 4.
Venn diagram of genes expressed in sea anemone tissues.
Distribution of genes (only of cnidarian origin) expressed in the epidermis (Ep) or gastroderm (Ga) compartments (circles), relative to their up-regulation (rectangles) in symbiotic (SY) or aposymbiotic (APO) conditions.
Figure 5.
Tissue-specific expression of targeted genes.
RT-qPCRs were performed on the epidermal (Ep) and gastrodermal (Ga) total RNA extracts from 3 different sea anemones. The histogram shows the relative Ep (gray) versus Ga (green) expression of the genes listed on the histogram (error bars represent standard error).
Table 2.
Examples of A. viridis genes up-regulated in the epidermis (E) or gastroderm (G).
Figure 6.
Gene duplication of NPC2 and Calumenin in anthozoans.
Calumenin (A) and NPC2 (B) homologs alignments (Figure S5 and S6) were subjected to PhyML maximum likelihood phylogenetic analyses. Best-fitted substitution models were: [LG+I+G+F; I = 0.029 F = empirical and gamma = 1.127] for Calumenin and [WAG+I+G; I = 0.036 and gamma = 5.836] for NPC2. Hs: Human, Dr: Danio rerio, Ci: Ciona intestinalis, Sp, Strongylocentrotus purpuratus, Tc: Tribolium castaneum, Dm: Drosophila melanogaster, Ce: Caenorhabditis elegans, Nv: Nematostella vectensis, Av: Anemonia viridis, Ta: Trichoplax adherans, S.cerevisiae and S.pombe: NPC2 homologs in Saccharomyces cerevisiae (Q12408) and Schizosaccharomyces pombe (Q9C0X9), respectively. Protein sequences are given in Table S4. The star points to the A.viridis isoform belonging to the Kern that is up-regulated in the gastrodem of symbiotic anemones.
Figure 7.
Sym32 gene duplication and putative γ-carboxylation regulation model.
A. Color code schematization of the PhyML tree for the FasI domains of the Sym32, Periostin (PN) and BGH3 homologs in sea anemones (Ae, A. elegantissima; Av, A. viridis; Nv, N. vectensis) and human (Hs) (see tree in Figure S7b). Each rectangle represents one FasI domain (small yellow rectangles represent the Signal Sequence). FasI domains 1 and 3 are closely related. The first FasI domains of [AeSym32-1, AvSym32-1 and Nv2Sym-1 and -3] are closely related to each other and to a lesser extent with [AvPN-1 and -3, and NvPN-1 and -3] and finally with [HsPN-1 and -3, and HsBGH3-1 and -3]. A similar relationship exists for the FasI domains 2 and 4. AeSym32, AvSym32 and Nv2Sym are probable orthologs, except that Sym32 appears as the first half of Nv2Sym. The putative ortholog of HsPN is NvPN. B. Heuristic Model. The presence of symbionts activates the expression of calumenin and sym32 genes via an unknown mechanism. The CRS of the Sym32 protein is recognized as substrate by the activated vitamin K cycle (vitamin K is a cofactor produced from the photosynthetic organism) and in turn is γ-carboxylated. Meanwhile, the Calumenin represses the VKOR protein, inhibiting the γ-carboxylase. Two forms of Sym32 are thus expected to be produced from this pathway: the Glu-Sym32 and Gla-Sym32 electrophoretypes (likely corresponding to the two spots PI = 8.2 and PI = 7.9, respectively [51]). Only Sym32 (PI = 8.2) is found to be associated with the symbiosome membrane, underlying a novel functionality for Sym32 and γ-carboxylation.
Figure 8.
Model of pathways highlighted in the aposymbiotic and symbiotic states of sea anemones.
Based on functional homology and protein signature (Table 1), each gene product from the Kern set was assigned a specific sub-cellular compartment or secretion pathway: ExtCell; Extra Cellular milieu; Cyt, Cytosol; Nu, Nucleus; TGN, Trans Golgi Network; ER, Endoplasmic reticulum; LD, Lipid droplets; Mit, Mitochondrion; Per, Peroxisome; Lys, Lysosome; Phag, Phagosome; EE, Early Endosome; RE, Recycling Endosome; LE, Late Endosome; Zoox, Zooxanthellae. The left and right moieties of the diagram show genes with increase transcript abundance in aposymbiotic and symbiotic states, respectively. Circles (aposymbiotic), barrels (solute carrier), and hexagons (symbiotic) represent gene expression specific to epidermis (E, dark grey), gastroderm (G, light grey) or both (E&G, white). Trans-membrane domains are shown by appendages. Each gene number has the corresponding name indicated in the list on the right. Gene products in dashed grey are candidate proteins that could potentially (hypothetic) be present at the perisymbiotic space.