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.2019 Aug 29;14(8):e0221938.
doi: 10.1371/journal.pone.0221938. eCollection 2019.

De novo transcriptome profile of coccolithophorid alga Emiliania huxleyi CCMP371 at different calcium concentrations with proteome analysis

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De novo transcriptome profile of coccolithophorid alga Emiliania huxleyi CCMP371 at different calcium concentrations with proteome analysis

Onyou Nam et al. PLoS One..

Abstract

The haptophyte alga Emiliania huxleyi is the most abundant coccolithophore in the modern ocean and produces elaborate calcite crystals, called coccolith, in a separate intracellular compartment known as the coccolith vesicle. Despite the importance of biomineralization in coccolithophores, the molecular mechanism underlying it remains unclear. Understanding this precise machinery at the molecular level will provide the knowledge needed to enable further manipulation of biomineralization. In our previous study, altering the calcium concentration modified the calcifying ability of E. huxleyi CCMP371. Therefore in this study, we tested E. huxleyi cells acclimated to three different calcium concentrations (0, 0.1, and 10 mM). To understand the whole transcript profile at different calcium concentrations, RNA-sequencing was performed and used for de novo assembly and annotation. The differentially expressed genes (DEGs) among the three different calcium concentrations were analyzed. The functional classification by gene ontology (GO) revealed that 'intrinsic component of membrane' was the most enriched of the GO terms at the ambient calcium concentration (10 mM) compared with the limited calcium concentrations (0 and 0.1 mM). Moreover, the DEGs in those comparisons were enriched mainly in 'secondary metabolites biosynthesis, transport and catabolism' and 'signal transduction mechanisms' in the KOG clusters and 'processing in endoplasmic reticulum', and 'ABC transporters' in the KEGG pathways. Furthermore, metabolic pathways involved in protein synthesis were enriched among the differentially expressed proteins. The results of this study provide a molecular profile for understanding the expression of transcripts and proteins in E. huxleyi at different calcium concentrations, which will help to identify the detailed mechanism of its calcification.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Differentially expressed genes (DEGs) involved inE.huxleyi at different calcium concentrations.
(A) DEGs from each comparison of different calcium concentrations. Bar charts show up-regulated and down-regulated DEGs in yellow and blue, respectively. (B) Correlation between RNA-seq data and qPCR analysis for validation. (C, D) DEG comparison of the (C) up- and (D) down-regulated groups. The Venn diagram was drawn by Venny2.1 [36].
Fig 2
Fig 2. Putative biomineralization-related genes in DEGs at different calcium concentrations.
The log2 fold changes of the genes are shown as a bar chart (log2FC; ± SE, light gray: [Ca2+] 0.1 mM relative to [Ca2+] 0 mM, dark gray: [Ca2+] 10 mM relative to [Ca2+] 0.1 mM, black: [Ca2+] 10 mM relative to [Ca2+] 0 mM). The gene IDs in the bar chart are in the following order: SLC4-2 (c31857_g11_i1), SLC family (c25647_g1_i1), Na+/H+ exchanger (c31114_g3_i2), delta CA (c24914_g1_i2), calcium-binding GPA (glutamic acid, proline and alanine) protein (c20552_g1_i1), Ca2+/Mg2+ permeable cation channels (LTRPC family) (c30953_g1_i11), fibrillins and related proteins containing a Ca2+-binding epidermal growth factor (EGF)-like domain (JGI ID 118025; c28155_g1_i1), fibrillins and related proteins containing Ca2+-binding EGF-like domain (JGI ID 463266; c28980_g1_i1), hypothetical protein (c34539_g1_i1), eukaryotic initiation factor 4A (eIF4A) (c22513_g1_i1), ABC transporter (c34539_g1_i1), and putative mitochondrial chaperone BCS1 (c23862_g2_i1). Asterisks represent significant expression change (*p < 0.05, **p < 0.005).
Fig 3
Fig 3. Gene Ontology (GO) enrichment analysis of DEGs up-regulated in [Ca2+] 10 mM relative to [Ca2+] 0 mM.
The GO terms were enriched using Blast2GO. The enriched terms were reduced to most specific and shown in the red box (cut-off: FDR<0.05, green: biological process, blue: molecular function, yellow: cellular component).
Fig 4
Fig 4. KEGG pathway enrichment of DEGs at different calcium concentrations.
The pathways enriched in (A) up-regulated and (B) down-regulated DEGs in the comparison between [Ca2+] 10 vs 0 mM and [Ca2+] 10 vs 0.1 mM.
Fig 5
Fig 5. Unigenes differentially expressed in both the transcriptome and proteome.
(A) The differentially expressed genes (DEGs) and proteins (DEPs) in [Ca2+] 10 mM relative to [Ca2+] 0.1 mM (yellow: up-regulated, blue: down-regulated). (B) The putative biomineralization-related DEPs (log2FC; ± SE): AEL1 (c31742_g1_i2), calcium pump (c31573_g1_i1), mitochondrial chaperone BCS1 (c23862_g2_i1), and eIF4A (c22513_g1_i1). (C) Correlation between differentially expressed transcripts and proteins.
Fig 6
Fig 6. KEGG pathways in DEGs and DEPs ofE.huxleyi at different calcium concentrations.
(A) The up- and (B) down-regulated DEGs and DEPs involved in metabolic pathways ofE.huxleyi. The numbers of common and unique DEGs and DEPs are shown in the Venn diagram. The bar chart shows the sum of the pathways commonly involved in DEGs and DEPs.
Fig 7
Fig 7. The hypothetical calcification-related intracellular model shown as a schematic cell diagram.
This calcification-related model is suggested based on previously reported studies [4, 11, 59, 67]. The ABC family transporters (yellow, upper-left) were mostly included in the ‘secondary metabolites biosynthesis, transport, and catabolism’ of the KOG-enriched cluster. The LTRPC family cation channel (JGI ID 460292) and Ca2+-pump (JGI ID 466567) (red, upper-middle) were up- and down-regulated, respectively, at the ambient Ca2+ concentration relative to limited Ca2+ concentrations. Furthermore the HCO3- transporter solute carrier 4 families (JGI ID 436956, 466232) (green, upper-right) are described in this study. The delta CA (JGI ID 195575) (dark red, upper-right) was the only up-regulated carbonic anhydrase in this study. The gene expression of the fibrillins and related proteins containing Ca2+-binding EGF-like domains (JGI ID 118025, 463266) (orange, upper right-hand corner) was also up-regulated at ambient Ca2+ concentration relative to the limited Ca2+ concentrations. The eukaryotic translation initiation factors (eIFs) (eIF4A: JGI ID 312754, eIF2α: JGI ID 366908) (orange, lower-right) were up-regulated at the ambient Ca2+ concentration relative to the limited Ca2+ concentrations as well. The eIFs have the potential to regulate the signaling pathway related to calcification at different calcium concentrations. The molecular chaperones (orange, lower-middle) BiP (JGI ID 442092), BCS1 (JGI ID 369425) and Ero1 (JGI ID 461210) are also possible factors in the calcification process.
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References

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This study was supported by the Basic Core Technology Development Program for the Oceans and the Polar Regions of the National Research Foundation of Korea (NRF,https://www.nrf.re.kr) funded by the Ministry of Science, ICT (2015M1A5A1037053) (E. Jin). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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