Transcriptomic Analysis of Cold-Induced Temporary Cysts in Marine Dinoflagellate Prorocentrum cordatum

Abstract

Dinoflagellates are unicellular organisms that are crucial components of aquatic ecosystems, known as important primary producers and causes of harmful blooms. They have complex life cycles, including immotile stages, which contribute to their distribution and survival in unfavorable conditions. Temperature changes, primarily cold stress, significantly impact dinoflagellate physiology, influencing metabolic processes, growth rates, and encystment/excystment cycles. This study investigates the transcriptome of temporary cold-induced cysts in the marine planktonic dinoflagellate Prorocentrum cordatum. We compared gene expression in cysts subjected to a 7-h cold incubation with those returned to standard cultivation conditions and motile vegetative cells. Our results showed a marked predominance of downregulated genes in cold-induced cysts. Encystment affected signaling pathways, including calcium and protein kinase signaling, as well as RNA and protein metabolism. Upon returning to standard conditions, RNA metabolism was reactivated; upregulation of genes encoding some calcium-binding proteins and kinases was observed. Additionally, we analyzed RNA-binding pentatricopeptide repeat-containing proteins, the genes encoding which changed their expression in P. cordatum cysts, for similarities to plant MRL1 proteins. Finally, we focused on MEI2-like proteins to confirm their role in non-sexual cyst formation and position them within the diversity of MEI2 homologs in dinoflagellates.

Keywords: MEI2; cold stress; cyst; dinoflagellate; transcriptome.

Figure 1

Light microscopy of cold-induced cysts of Prorocentrum cordatum at 40× (a) and 100× (b) magnification. Cellulose thecal plates are stained blue with Calcofluor White M2R (a′,b′). The red signal represents chloroplast autofluorescence.

Figure 2

(a) Hierarchical clustering heatmap of DEGs in Cold 7 h, Cold 7 h + ST 3 h, and Control (with 3 replicates for each condition). A total of 384 genes with padj < 0.05 and |log₂ fold change| > 1 were analyzed. The color scale represents row z-scores of log₂-transformed TPM + 1 values. The distance matrix was calculated using Pearson correlation, and clusters were generated using the complete linkage (furthest neighbor) method. (b) Volcano plots depicting differential gene expression between cells subjected to cooling for 7 h and cells from the control group (Cold 7 h vs. Control), between cells cooled for 7 h and then incubated under standard cultivation conditions for 3 h, and cells from the control group (Cold 7 h + ST 3 h vs. Control), and between the two experimental groups (Cold 7 h + ST 3 h vs. Cold 7 h). Genes with padj < 0.05 and |log₂ fold change| > 1 are marked in color: blue dots indicate downregulated genes, while red dots indicate upregulated genes. Black dots indicate genes whose expression did not change significantly. The dotted lines mark the threshold values.

Figure 3

(a) Rooted maximum likelihood phylogenetic tree of MEI2 homologs from dinoflagellates, inferred using Q.pfam+R8 evolutionary model. Bootstrap values (based on 10.000 ultrafast replicates) and Bayesian posterior probabilities are indicated at the nodes (not shown when below 60/0.90). Black circles indicate nodes with full support (bootstrap value/posterior probability =100/1.00). MEI2-like sequences from ciliates were used as an outgroup. The reconstruction is based on the dataset from Palii et al. [23], supplemented with eight sequences of P. cordatum encoded by genes significantly differentially expressed in cold-induced cysts and in cells returned to standard cultivation conditions. Only the names of these sequences and those comprising the outgroup are shown. Clade color coding corresponds to Figure 2 in Palii et al. [23]. P. cordatum sequences corresponding to DEGs downregulated in the comparison Cold 7 h vs. Control are highlighted in blue, sequences also corresponding to DEGs upregulated in the comparison Cold 7 h + ST 3 h vs. Cold 7 h are labeled with purple asterisks. The sequence CAK0884715.1, highlighted in gray, is duplicated in accordance with the position inferred by Bayesian analysis. Sequences corresponding to DEGs upregulated and downregulated in the comparison Cold 7 h + ST 3 h vs. Control are highlighted in orange and cyan, respectively. (b) Fragment of the multiple sequence alignment of the eight MEI2-like sequences from P. cordatum corresponding to DEGs and the MEI2 sequence from the fission yeast Schizosaccharomyces pombe. This fragment corresponds to the RRM3 domain. Key conserved structural and functional sites in the β-strand regions, potentially involved in RNA binding are underlined. A modification in the second site of the sequence CAK0884715.1 is indicated with a blue border. Highlighting and labels follow the same format as in (a).