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. 2020 Jan 12;8(1):108.
doi: 10.3390/microorganisms8010108.

Comparative Transcriptome Analyses During the Vegetative Cell Cycle in the Mono-Cellular Organism Pseudokeronopsis erythrina (Alveolata, Ciliophora)

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Free PMC article

Comparative Transcriptome Analyses During the Vegetative Cell Cycle in the Mono-Cellular Organism Pseudokeronopsis erythrina (Alveolata, Ciliophora)

Yiwei Xu et al. Microorganisms. .
Free PMC article

Abstract

Studies focusing on molecular mechanisms of cell cycles have been lagging in unicellular eukaryotes compared to other groups. Ciliates, a group of unicellular eukaryotes, have complex cell division cycles characterized by multiple events. During their vegetative cell cycle, ciliates undergo macronuclear amitosis, micronuclear mitosis, stomatogenesis and somatic cortex morphogenesis, and cytokinesis. Herein, we used the hypotrich ciliate Pseudokeronopsis erythrina, whose morphogenesis has been well studied, to examine molecular mechanisms of ciliate vegetative cell cycles. Single-cell transcriptomes of the growth (G) and cell division (D) stages were compared. The results showed that (i) More than 2051 significantly differentially expressed genes (DEGs) were detected, among which 1545 were up-regulated, while 256 were down-regulated at the D stage. Of these, 11 randomly picked DEGs were validated by reverse transcription quantitative polymerase chain reaction (RT-qPCR); (ii) Enriched DEGs during the D stage of the vegetative cell cycle of P. erythrina were involved in development, cortex modifications, and several organelle-related biological processes, showing correspondence of molecular evidence to morphogenetic changes for the first time; (iii) Several individual components of molecular mechanisms of ciliate vegetative division, the sexual cell cycle and cellular regeneration overlap; and (iv) The P. erythrina cell cycle and division have the same essential components as other eukaryotes, including cyclin-dependent kinases (CDKs), cyclins, and genes closely related to cell proliferation, indicating the conserved nature of this biological process. Further studies are needed focusing on detailed inventory and gene interactions that regulate specific ciliated cell-phase events.

Keywords: RT-qPCR; ciliate; eukaryotes; unicellular transcriptome; vegetative cell cycle.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Comparison of 13 ciliate macronuclear genomes. Blue lines denote the genome size; yellow lines represent the number of chromosomes. Species names are listed on the left, while macronuclear ploidy is shown on the right. Asterisks denote that macronuclear ploidy is unclear. Species names and ploidy are depicted in different colors for different ciliate groups (Spirotrichea: dark blue; Oligohymenophorea: black). All data were obtained from references [20,28,29,30,31,32,33,34,35,36,37].
Figure 2
Figure 2
Statistics of transcriptomic data and cluster analysis of differentially expressed genes (DEGs). (A), Heatmap of the top 100 DEGs between the cell division (D) and growth (G) stages from three replicates. Each column represents a sample, and each row represents a unigene. (B), Relative transcription levels of DEGs in group D in comparison with group G shown by RT-qPCR and RNA-Seq. Blue lines represent the fold change of gene transcription revealed by RNA-Seq using log2. Green bars represent the relative transcription level determined by RT-qPCR using log2 (2−ΔΔct). Error bars represent standard deviations from three independent biological replicates. (C), Length distributions of unigene sequences derived from the transcriptome assembly of Pseudokeronopsis erythrina.
Figure 3
Figure 3
Morphology of Pseudokeronopsis erythrina, analysis, and functional annotation of unigenes. (A,B), P. erythrina from life at growth stage (A) and infraciliature at cell division stages from Chen et al. [15] (B). (C), Venn diagram of DEGs for stage D and stage G. Unigenes shared by stages D and G are in dark purple, while unique ones at stage D and G are in orange and light purple, respectively. (D), Annotation of unigenes according to clusters of orthologous groups (COG). Numbers above bars represent predicted unigenes. Scale bars in (A, B): 60 μm.
Figure 4
Figure 4
Top 30 GO terms and enrichment of the DEGs at the D stage in the biological process category (A), cell components category (B), molecular function category (C), and KEGG pathway enrichment (D). The size of dots indicates the number of DEGs. The color of dots corresponds to the FDR/Q value.
Figure 5
Figure 5
Number of unigenes in Pseudokeronopsis erythrina annotated in KEGG pathways.
Figure 6
Figure 6
RT-qPCR melting curves for five up-regulated (AE) and six down-regulated differentially expressed genes (FK). (A), c37490_g1; (B), c38398_g1; (C), c38773_g1; (D), c52670_g1; (E), c60031_g1; (F), c60031_g1; (G), c56677_g1; (H), c50771_g1; (I), c58897_g1; (J), c47080_g1; (K), c58837_g1. In each figure, “g” and “d” represent stage G and stage D.

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