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Abstract: The circadian clock is a fundamental endogenous mechanism of adaptation that coordinates the physiology and behavior of most organisms with diel variations in the external environment to maintain temporal homeostasis. Diatoms are the major primary producers in the ocean. However, little is known about the circadian clock in marine diatoms compared with other organisms. Here, we investigated circadian clock genes, their expression patterns, and responses to environmental stimuli such as light, nitrogen and phosphorus in two marine diatoms, Skeletonema costatum and Phaeodactylum tricornutum, using a combination of qRT-PCR and bioinformatic analysis. We identified 17 and 18 circadian clock genes in P. tricornutum and S. costatum, respectively. Despite significant evolutionary differences, these genes were similar to those of the higher plant Arabidopsis. We also established a molecular model for the marine diatom circadian clock comprising an input pathway, core oscillator, output pathway, and valve effector. Notably, the expression patterns of core clock genes (circadian clock associated 1 (CCA1), late elongated hypocotyl (LHY) and timing of cab 1 (TOC1)) in both species differed from those of terrestrial plants. Furthermore, the expression of these genes was influenced by variations in ambient light, nitrogen and phosphorus availability. Although marine diatoms and higher plants share common circadian clock components, their clock genes have diverged throughout evolution, likely as a result of adapting to contrasting environments.
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Key words:
- circadian clock /
- circadian genes /
- marine diatoms /
- Phaeodactylum tricornutum /
- Skeletonema costatum
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Figure 1. Growth curves of Skeletonema costatum and Phaeodactylum tricornutum under control (a and e), continuous light and dark (b and f), N-deplete and N-resupplied (c and g), P-deplete and P-resupplied (d and h) conditions. The red and blue rectangles indicate the sampling time for molecular analysis while the blue rectangles in c, d, g and h indicate the sampling time in the N- or P-resupplied groups. The red arrow indicates the time point of N or P resupply.
Figure 2. Conservative motif analysis of the core clock genes (CCA1, LHY, and TOC1) in Skeletonema costatum and Phaeodactylum tricornutum based on the databases SMART and Pfam. At is abbreviation of Arabidopsis thaliana; Pt, P. tricornutum; Sc, S. costatum; REC, phosphoacceptor receiver; SANT, switching-defective protein 3 (Swi3), adaptor 2 (Ada2), nuclear receptor co-repressor (N-CoR), transcription factor (TF)IIIB; ANK, ankyrin; Ank2, Ankyrin-B; CCT is timing of cab expression 1.
Figure 3. Expression of CCA1, LHY, and TOC1 in Skeletonema costatum and Phaeodactylum tricornutum within 24 h. Expression of CCA1 in S. costatum and P. tricornutum under control (a-1 and b-1), continuous light (a-2 and b-2), continuous dark (a-3 and b-3), N-deplete (a-4 and b-4), N-resupplied (a-5 and b-5), P-deplete (a-6 and b-6) and P-resupplied (a-7 and b-7) conditions; expression of LHY in S. costatum and P. tricornutum under control (c-1 and d-1), continuous light (c-2 and d-2), continuous dark (c-3 and d-3), N-deplete (c-4 and d-4), N-resupplied (c-5 and d-5), P-deplete (c-6 and d-6) and P-resupplied (c-7 and d-7) conditions; and expression of TOC1 in S. costatum and P. tricornutum under control (e-1 and f-1), continuous light (e-2 and f-2), continuous dark (e-3 and f-3), N-deplete (e-4 and f-4), N-resupplied (e-5 and f-5), P-deplete (e-6 and f-6) and P-resupplied (e-7 and f-7) conditions. Sc represents S. costatum; Pt, P. tricornutum.
Figure 5. The speculative molecular model of the circadian clock in marine diatoms. Genes in the figure are shown in italics, proteins are shown in normal font. Words in red represent the genes that are identified in Skeletonema costatum and Phaeodactylum tricornutum; words in gray represent the genes and proteins that are not identified in S. costatum and P. tricornutum; words in green represent the genes that are identified in S. costatum; words in blue represent the genes that are identified in P. tricornutum. The core feedback loop of the circadian oscillator is highlighted in green square frame; the white area represents daytime, while the shaded area represents nighttime. The environmental signals of the input pathway and the metabolic process of the output pathways showed in a previous study (Zhang et al., 2016).
Table 1. Circadian clock genes identified in the diatoms Skeletonema costatum, Phaeodactylum tricornutum, and other speciesa
Location Gene name Eukaryotic algae Flowering plantsb Skeletonema costatum Phaeodactylum tricornutum Ostreococcus tauri Chlamydomonas reinhardtii Symbiodinium spp. Cyanidioschyzon merolae Arabidopsis thaliana Oryza sativa Input pathway PHYA + nd nd nd − nd + + PYHB + nd nd nd − nd + + PHYC + + nd nd +c nd + + PHYD + + nd nd − nd + + PHYE + + nd nd − nd + + CRY1 + + nd + + + + + ZTL + + nd nd nd nd + + LKP2 + + nd nd nd nd + + FKF1 + + nd nd nd nd + + Core oscillator CCA1 + + + nd nd nd + + LHY + + nd nd nd nd + nd TOC1 + + + + nd nd + nd PRR3 + + nd nd nd nd + nd PRR5 + + nd nd nd nd + nd PRR7 + + nd nd nd nd + nd PRR9 − + nd nd nd nd + nd CK2 + + + + nd nd + + Output pathway CAB2 + + nd nd nd nd + + Valve effector LUX + + nd nd nd nd + nd Note: aProkaryotes are not included in the table because they have completely different clock genes; bonly representative flowering plants are listed; cunknown type. + indicates detected; nd, not detected; −, no data. The content of this table refer to Martínez-García et al. (2000), Noordally and Millar (2015) and Zhang et al. (2016). 
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