Day and night regulation of the HO-1/HSP32 synthesis in the harmful dinoflagellate Prorocentrum minimum: response to salinity stress

Abstract

Potentially toxic dinoflagellate species Prorocentrum minimum (Pavillard) Schiller, 1933 colonized the brackish-water Baltic Sea in the early 1980s, where thereafter it has been forming regular harmful blooms. Heat shock proteins (HSP) play an important role in their ability to adapt to environmental stress. In this study, we evaluated how the synthesis of heme oxygenase-1 (HO-1/HSP32), an important part of cellular machinery for cell protection, changes in P. minimum under the influence of salinity stress during the day-night cycle. We identified sequences of the HO-1 homologs in the unannotated P. minimum transcriptomes, represented in the Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP) database. Phylogenetic analysis showed that this protein clustered in a distinct clade and demonstrated an evolutionary relationship with the HO-1 homologs of other taxonomic groups. To identify P. minimum cells that express the HO-1 protein, we used fluorescent flow cytometry. In control conditions (17‰), the number of cells expressing this protein was 2.2 times higher during the day than at night, with the same average fluorescence intensity (1.7 units). During the daytime, the critical salinity conditions of 8‰ did not lead to significant changes in the number of cells expressing HO-1, while in the night culture this salinity shift caused a 2.7-fold increase in the number of cells expressing HO-1, and led to an increase in the intensity of fluorescence up to 2.0 units. The overall DNA and RNA synthesis rates were estimated by the incorporation of 3H-thymidine and 3H-uridine, respectively. Transcription and replication levels were significantly higher under critical salinity conditions (8‰) compared to the control (17‰) and typical sea salinity conditions (35‰). The greatest increase in the DNA synthesis rates (up to 2.7 times) was observed during the daytime in cells exposed to salinity stress at 8‰. In contrast, the greatest increase in the RNA synthesis rates (2.7 times) was registered in cells exposed to salinity of 8‰ at night. Thus, the replication activity of cells in response to salinity stress increased significantly during the day, while transcription levels reached their maximum values at night. These results indicate that a significant increase in the synthesis of both nucleic acids and the HO-1 stress protein can serve as a biomarker of the impact of environmental stress factors, such as salinity. This allowed concluding that the studied parameters demonstrate a high potential for future modeling of harmful algae blooms, particularly those formed by the invasive potentially toxic species under horohaline conditions of the Baltic Sea.