Diurnal-Rhythmic Relationships between Physiological Parameters and Photosynthesis- and Antioxidant-Enzyme Genes Expression in the Raphidophyte Chattonella marina Complex

Abstract

Diurnal rhythms in physiological functions contribute to homeostasis in many organisms. Although relationships between molecular biology and diurnal rhythms have been well studied in model organisms like higher plants, those in harmful algal bloom species are poorly understood. Here we measured several physiological parameters and the expression patterns of photosynthesis-related and antioxidant-enzyme genes in the Chattonella marina complex to understand the biological meaning of diurnal rhythm. Under a light-dark cycle, Fv/Fm and expression of psbA, psbD, and 2-Cys prx showed significant increases in the light and decreases during the dark. These rhythms remained even under continuous dark conditions. DCMU suppressed the induction of psbA, psbD, and 2-Cys prx expression under both light regimes. Oxidative stress levels and H₂O₂ scavenging activities were relatively stable, and there was no significant correlation between H₂O₂ scavenging activities and antioxidant-enzyme gene expression. These results indicate that the Chattonella marina complex has developed mechanisms for efficient photosynthetic energy production in the light. Our results showed that this species has a diurnal rhythm and a biological clock. These phenomena are thought to contribute to the efficiency of physiological activities centered on photosynthesis and cell growth related to the diurnal vertical movement of this species.

Keywords: Chattonella marina complex; antioxidant enzyme; diurnal rhythm; photosynthesis.

Figure 1

Diagram showing the sampling times of cultures of Chattonella under light conditions of 14 h light:10 h dark (LD) (A) and continuous dark (24D) (B) after initial 7 h irradiation. Samples were collected at 12 time points throughout the experiment as indicated by arrows: at 12:00 (noon), 3:00, 6:00, and 9:00 p.m. on day 1, and at 12:00 (midnight), 3:00, 6:00, and 9:00 a.m. and 12:00 (noon), and 3:00, 6:00, and 9:00 p.m. on day 2. The white background shows periods of irradiation, and the gray background indicates the dark periods.

Figure 2

Cell densities (A) and Fv/Fm ratios (B) in cultures of Chattonella under two light regimes: 14 h light:10 h dark (LD), and continuous dark (24D). The top half of each panel shows LD conditions, and the bottom half is 24D. A white background indicates a light period, and a gray background indicates a dark period. The light period under LD was from 5:00 a.m. until 7:00 p.m. For 24D, the dark period started at 7:00 p.m. Values are mean ± SD (n = 4).

Figure 3

Superoxide production (A), H₂O₂ concentrations (B), and H₂O₂ scavenging activity (C) in cultures of Chattonella under different light regimes. H₂O₂ scavenging activity is expressed in units of catalase activity (units/cell). A white background indicates a light period, and a gray background indicates a dark period. The light period under LD was from 5:00 a.m. to 7:00 p.m. For 24D, the continuous dark period started at 7:00 p.m. Values are mean ± SD (n = 4).

Figure 4

Relative gene expression levels of psbA (A) and psbD (B) in cultures of Chattonella under different light regimes, as determined by qPCR analysis. White background shows light periods, and gray background shows dark periods. The light period under LD was from 5:00 a.m. to 7:00 p.m. For 24D, the continuous dark period started at 7:00 p.m. Values are mean ± SD (n = 4).

Figure 5

Relative gene expression levels of Cu/Zn sod (A), gpx (B), cat (C), apx (D), trx (E), and 2-Cys prx (F) in cultures of Chattonella under different light regimes, as determined by qPCR analysis. A white background indicates light periods, and a gray background indicates dark periods. The light period under LD was from 5:00 a.m. to 7:00 p.m. For 24D, the continuous dark period started at 7:00 p.m. Values are mean ± SD (n = 4).

Figure 6

Gene expression response of psbA, psbD, and 2-Cys prx in Chattonella treated with DCMU. A white background indicates light periods, and a gray background indicates dark periods. The left and right panels represent LD and 24D conditions, respectively. White circles, solvent control + light conditions; white triangles, DCMU + light; black circles, solvent control + dark; black triangles, DCMU + dark. DCMU, or ethanol (as a solvent control), was added at midnight. Values are mean ± SD (n = 4). * p < 0.05 (Student’s t-test was used to compare solvent control and DCMU treatment groups under LD and 24D conditions).

Figure 7

Summary of results obtained from this study.