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Molecular insights into the circadian clock in marine diatoms
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Shufeng Zhang1, 2, T, Yue Wu1, 2, T, Lin Lin1, 2, Dazhi Wang1, 2, 3, *
Acta Oceanologica Sinica | 2022, 41(8) : 87 - 98
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Acta Oceanologica Sinica | 2022, 41(8): 87-98
• Marine Biology •
Molecular insights into the circadian clock in marine diatoms
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Shufeng Zhang1, 2, T, Yue Wu1, 2, T, Lin Lin1, 2, Dazhi Wang1, 2, 3, *
Affiliations
  • 1 State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen 361102, China
  • 2 College of the Environment and Ecology, Xiamen University, Xiamen 361102, China
  • 3 Key Laboratory of Marine Ecology & Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
Published: 2022-08-25 doi: 10.1007/s13131-021-1962-4
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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.

circadian clock  /  circadian genes  /  marine diatoms  /  Phaeodactylum tricornutum  /  Skeletonema costatum
Shufeng Zhang, Yue Wu, Lin Lin, Dazhi Wang. Molecular insights into the circadian clock in marine diatoms[J]. Acta Oceanologica Sinica, 2022 , 41 (8) : 87 -98 . DOI: 10.1007/s13131-021-1962-4
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1 Introduction
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The circadian clock is an endogenous regulatory mechanism that allows acclimation to cyclical changes in the external environment, thereby enabling organisms to predict temporal changes, adjust physiological homeostasis, improve their ability to compete, and maximize their chances of survival (Harmer, 2009). It takes about 24 h for the Earth to rotate, and the circadian clock creates a physiological and metabolic rhythm, referred to as the circadian rhythm, that mimics the cycle of the surrounding environment (Harmer, 2009). Circadian rhythms are ubiquitous and have been identified in numerous organisms ranging from prokaryotic cells to multicellular eukaryotes (Bruce, 1970; Konopka and Benzer, 1971; Feldman and Hoyle, 1973; Staiger, 2002).
The rhythm-generating oscillator is a complex network of interlocking feedback loops (Troein et al., 2009). Previous studies have shown that the circadian clock has three distinctive features: (1) as an endogenous regulatory mechanism, the clock produces a circadian rhythm in a manner that is independent signals from the external environment (Harmer, 2009); (2) the circadian clock can be reset by changes in the external environment (Dodd et al., 2005); and (3) the circadian clock can compensate for changes in temperature (Salomé et al., 2010). To synchronize time and space with the external environment, organisms use the circadian clock to regulate gene expression, as well as physiological and biochemical reactions; by synchronizing these processes, an organism can gain significant survival benefit from a changing environment (Barak et al., 2000; Harmer, 2009). The circadian clock controls protein abundance and activity in response to environmental conditions by regulating transcription, translation, and posttranslational modifications, resulting in an oscillatory period that is almost 24 h long (Greenham and McClung, 2015).
Although the mechanisms that regulate the circadian clock are complex, they are known to comprise an input path, a core oscillator, an output path, and a valve effector in higher plants (Harmer, 2009). Environmental signals, such as light and nutrient which are drivers of circadian rhythms, are transmitted to the core oscillator through the input pathway, while the output pathway amplifies the rhythmic signal generated by the core oscillator and transmits it to appropriate downstream genes (McClung, 2001, 2011). The core oscillator is the main component of the circadian clock, and is described as a transcriptional regulatory feedback loop consisting of positive and negative interactions among three components, namely, two MYB domain-containing transcription factors (circadian clock associated 1 (CCA1) and late elongated hypocotyl (LHY)), and a member of the pseudo-response regulator (PRR) protein family (timing of cab 1 (TOC1)) (Alabadí et al., 2001). The expression levels of CCA1 and LHY generally peak just before or after the onset of illumination. Their products bind to the evening element (AAATATCT) on the TOC1 promoter and inhibit its expression. The expression level of TOC1 is known to peak in the evening owing to the reduced expression of CCA1 and LHY, thereby releasing the inhibitory effect on TOC1 (Alabadí et al., 2001; McClung, 2001, 2011). Loss of functional activity in any of the core clock genes results in a shortened clock period, whereas their overexpression confers arrhythmicity to multiple outputs (Schaffer et al., 1998; Wang and Tobin, 1998; Strayer et al., 2000; Alabadí et al., 2001). Signals generated by the core oscillator result in physiological and biochemical responses via the output of downstream circadian clock genes (McClung, 2001, 2011).
Numerous studies have investigated the circadian clocks of several model species, including cyanobacteria (Kondo et al., 1994), Chlamydomonas (Bruce, 1970), Drosophila melanogaster (Konopka and Benzer, 1971) Arabidopsis thaliana (Harmer et al., 2000), the mouse (Mus musculus) (Turek et al., 2005), and humans (Homo sapiens) (Wright et al., 2013). These studies have helped elucidate the structural and regulatory mechanisms of the circadian clock. Several studies have also shown that marine phytoplankton exhibit 24 h period circadian rhythms in a range of processes, including physiological and biochemical pathways, gene expression patterns, and gathering behavior (Schweiger et al., 1964; Ragni and d'Alcalà, 2007; Wada et al., 2011; Filonova et al., 2013; Noordally and Millar, 2015). Transcription of the core cell cycle genes, including cyclins and cyclin-dependent kinases, is under circadian control in the unicellular green alga Ostreococcus, independent of the photosynthetic capacity (Moulager et al., 2007). Furthermore, the circadian clock genes CCA1 and TOC1 have been identified in Ostreococcus tauri (Noordally and Millar, 2015), and have also been characterized in A. thaliana. Annunziata et al. (2019) demonstrated that bHLH-PAS nuclear protein RITMO1 regulates diel rhythm in the marine diatom P. tricornutum and widely represents in algal genomes. Collectively, these data suggest that marine phytoplankton possess a circadian clock system similar to that of higher plants. However, at present, little is known of the circadian clock system in marine phytoplankton.
Diatoms are among the most important phytoplankton in the ocean, accounting for 20% of global carbon fixation and 40% of marine primary production (Goldman, 1993; Falkowski et al., 1998; Field et al., 1998). Consequently, diatoms play an essential role in regulating global biogeochemical cycles and climate (Goldman, 1993; Falkowski et al., 1998; Field et al., 1998). Circadian clock is important for regulating phytoplankton cell growth, gene expression, pigment synthesis, phototactic movements, and carbon fixation (Noordally and Millar, 2015). In our previous study, we identified several homologs of circadian clock genes, including CCA1 and PRR5, in the transcriptome of the marine diatom Skeletonema costatum (Zhang et al., 2016), which suggested the likely existence of a circadian clock in marine diatoms. In the present study, we investigated the expression patterns of circadian clock genes and their responses to environmental stimuli in two marine diatoms, S. costatum and Phaeodactylum tricornutum, and constructed a molecular model for the marine diatom circadian clock. Our results demonstrated that marine diatoms possess the same genetic clock components as higher plants, but the clock genes have diverged, likely due to their contrasting living environments.
2 Materials and methods
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2.1 Organisms and culture conditions
Strains of Skeletonema costatum (Greville) Cleve, 1873 and Phaeodactylum tricornutum Bohlin, 1897 were kindly provided by the Collection Center of Marine Algae, Xiamen University, China. Cells were routinely maintained in f/2 medium at 20°C under a 14 h:10 h light: dark cycle at a light intensity of 100 μmol/(m2·s) provided by a cold fluorescent lamp, as previously described (Zhang et al., 2016).
2.2 Cell synchronization
Before the experiments, cultures of S. costatum and P. tricornutum were synchronized for 48 h using a dark-induction method (Wang et al., 2013). Synchronized cells were then transferred to fresh f/2 medium and cultured for three generations (Zhang et al., 2016). Cells were used for experiments when they were in the exponential growth phase. An initial cell density of 1×104 cells/mL was used for each diatom species and experiments were performed in triplicate.
2.3 Responses of core clock genes to environment stimuli
To elucidate the sensitivity of S. costatum and P. tricornutum core clock genes to environmental changes, the two diatom species were grown under different light regimes and nitrogen (N, NaNO3) and phosphorus (P, NaH2PO4) conditions. Synchronized cells were transferred to fresh f/2 medium at 20°C at an initial cell density of 1×104 cells/mL. Light: dark cycles of 14 h:10 h, 24 h:0 h, and 0 h:24 h were set as the control group, continuous light group, and continuous dark group, respectively, each with three replicates. For the N experiment, synchronized cells were transferred to N-free f/2 medium as the N-deplete group, while N was added to the N-deplete group at a final concentration of 880 μmol/L as the N-resupplied group. For the P experiment, synchronized cells were transferred to f/2 medium with P concentration of 0.5 μmol/L as the P-deplete group, while P was added to the P-deplete group at a final concentration of 30 μmol/L as the P-resupplied group.
2.4 Physiological analysis
Three 1 mL and three 15 mL aliquots of culture media were collected daily at 11:00 for cell counting and the photochemical efficiency of photosystem II (Fv/Fm) analysis, respectively (Zhang et al., 2016). Culture medium (25 mL) from each nutrient treatment was filtered through a GF/F membrane, and dissolved N and P concentrations in the filtrate were measured using continuous flow analysis (CFA-SAN Plus/Skalar Analytik, Erkelenz, Germany).
2.5 Identification of circadian clock genes in S. costatum and P. tricornutum
In this study, a reference list of 26 circadian clock genes that had been previously identified in eukaryotic plants, including A. thaliana and rice (Oryza sativa) (Table S1), were used to identify circadian clock genes in S. costatum and P. tricornutum. The protein sequences of circadian clock genes from higher plants, such as A. thaliana, were first downloaded from the UniProt (http://www.uniprot.org/ ) and NCBI databases as query sequences. A local BLAST search (e-value<10−5) was then carried out using the transcriptome of S. costatum and the genome of P. tricornutum as databases. Candidate clock genes were then screened and verified using the online BLASTP tool in the NCBI database. The core clock genes (CCA1, LHY, and TOC1) in S. costatum and P. tricornutum were searched for conservative motif based on the databases SMART and Pfam, and compared with the Arabidopsis clock core genes to further confirm the authenticity of the core clock genes in diatoms.
2.6 Phylogenetic analysis of core clock genes
The deduced amino acid sequences of the core clock genes (CCA1, LHY, and TOC1) in S. costatum and P. tricornutum were aligned to those in the NCBI database. Phylogenetic analysis of all aligned sequences was carried out using Molecular Evolutionary Genetics Analysis (MEGA) 5.0 software. The length of CCA1, LHY, and TOC1 are 496, 749 and 496 amino acids, respectively. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1 000 replicates) are shown next to the branches. Based on the defined haplotype sequences of the corresponding gene coding regions, molecular evolutionary trees were constructed using the neighbor-joining method.
2.7 RNA extraction and cDNA synthesis
Samples for gene expression analysis were collected every 3 h from 9:00 on the third day for a period of 48 h (Figs 1a−h), while samples were collected from the N- and P-resupplied media after N and P addition (Figs 1c, d, g and h). From each sample, 50 mL of culture medium was filtered through a polycarbonate membrane (pore-size 3 μm; Millipore Corporation, USA) and resuspended in 1 mL of Trizol reagent (Invitrogen, USA). The sample was then immediately frozen in liquid nitrogen and stored at −80°C until RNA extraction (Zhang et al., 2016). Total RNA was extracted using TRI-Reagent (MRC, USA) and the RNeasy Mini Kit (Qiagen, Hilden, Germany) (Zhang et al., 2016). Then, RNA was reverse transcribed into cDNA for qPCR using the FastKing RT Kit with gDNase (Qiagen, Germany).
2.8 Quantitative PCR analysis
Quantitative PCR was performed using the Bio-Rad CFX96 Touch real-time PCR detection system (Applied Biosystems, USA) with the SuperReal PreMix Plus (SYBR Green) Kit (Tiangen, China). The primers for the circadian clock genes were designed using Primer Premier 5 software and are listed in Tables S2 and S3. The 18S rRNA (Kang et al., 2012) and TATA box binding protein genes (Seo et al., 2020) were used as internal reference genes for S. costatum and P. tricornutum, respectively. Amplification was performed in a 20 μL reaction volume, as follows: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 30 s and 60°C for 60 s. Gene expression profiles were subsequently calculated using the 2−△Ct method.
2.9 Statistical analysis
Significant differences (p≤0.05) among gene expression patterns were identified by one-way ANOVA followed by Duncan’s multiple range test. Results are presented as mean±standard deviation of relative mRNA expression.
3 Results
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3.1 Identification of circadian clock genes in S. costatum and P. tricornutum
In this study, 17 and 18 circadian clock genes were identified in P. tricornutum and S. costatum, respectively (Table 1). Three phytochrome genes (PHYC, PHYD, and PHYE), a cryptochrome circadian regulator 1 gene (CRY1), zeitlupe (ZTL), flarin binding, kelck repeat, f-box 1 (FKF1), and LOV kelch protein 2 (LKP2), which are all located in the input pathway of the circadian clock, were identified in both S. costatum and P. tricornutum. However, the phytochrome genes PHYA and PHYB were only detected in S. costatum (Table 1). Three key genes of the circadian clock core oscillator (CCA1, LHY, and TOC1) were identified in both diatom species. Based on the analysis results of databases SMART and Pfam found that CCA1 and LHY of the two diatoms contained the DNA binding domains from MYB proteins, as well as the switching-defective protein 3 (Swi3), adaptor 2 (Ada2), nuclear receptor co-repressor (N-CoR), transcription factor (TF)IIIB (SANT) domain family, both diatom TOC1 and Arabidopsis TOC1 had characteristic Co, constanslike, and timing of cab expression 1 (CCT) motif (Fig. 2). The PRR5 and PRR7 genes, known to play a role in the morning loop of the circadian clock, were identified in S. costatum, whereas the PRR9 gene was only identified in P. tricornutum. The TOC1 and LUX ARRHYTHMO (LUX) genes, known to play a role in the evening loop, identified in both diatom species, as were PRR3 and casein kinase 2 (CK2), two core oscillator genes, and one output pathway gene, chlorophyll a/b binding protein (CAB2) (Table 1).
3.2 Expression patterns of core clock genes in S. costatum and P. tricornutum
The expression profiles of three core clock genes (CCA1, LHY, and TOC1) in S. costatum and P. tricornutum showed clear diel oscillations (Fig. 3). The oscillatory period of CCA1 was similar in both diatom species and lasted about 24 h. During the day, the expression level of CCA1 increased and peaked 2 h after cells had entered the dark cycle, and then began to fall, reaching a minimum 1 h after entering the photoperiod (Figs 3a-1, b-1). The LHY gene had an oscillation period of about 24 h in both diatoms (Figs 3c-1, d-1). In the first cycle, LHY expression began to increase almost 7 h after entering the light cycle, peaking about 1 h before the dark phase. Then, 1 h after entering the dark cycle, the expression level of LHY began to decrease and reached a minimum level almost 1 h before the next cycle. At the beginning of the second cycle, the expression level of LHY began to increase and peaked 2 h after the beginning of the dark cycle (Figs 3c-1, d-1). The TOC1 gene also exhibited a diel oscillatory pattern that was similar to those of LHY and CCA1 (Figs 3e-1, f-1).
3.3 Expression of core clock genes under different light, N and P supply conditions
Although the expression phases of the three core clock genes CCA1, LHY, and TOC1 in S. costatum and P. tricornutum varied under different light, N, and P supply conditions, a clear circadian pattern could still be detected (Fig. 3). Under continuous light, the peak of CCA1 expression in S. costatum (Fig. 3a-2) was not significantly altered, whereas two peaks of CCA1 expression could be detected in P. tricornutum (Fig. 3b-2). The oscillation period lasted about 24 h in both diatoms. The peak of LHY expression in the two diatom species moved forward under the continuous light regime; the period was shortened in S. costatum, but remained at 24 h in P. tricornutum (Figs 3c-2, d-2). The peak of TOC1 expression was delayed for almost 3 h and 12 h in S. costatum and P. tricornutum, respectively, but the period remained unchanged (Figs 3e-2, f-2). Under continuous dark conditions, the peak of CCA1 expression moved forward 12 h and 9 h in S. costatum and P. tricornutum, respectively, but the oscillation period remained unchanged (Figs 3a-3, b-3). The peak expression of LHY moved forward 3 h and 9 h in S. costatum and P. tricornutum within a 24 h oscillation period, respectively (Figs 3c-3, d-3). The peak of TOC1 expression in S. costatum moved forward 3 h but remained unchanged in P. tricornutum (Figs 3e-3, f-3).
Compared with the control, the expression of CCA1 was increased only in the N-depleted S. costatum cells (Fig. 3a-4), and the peak phase moved forward after N resupply (Fig. 3a-5); however, the peak of CCA1 expression moved forward in the N-deplete P. tricornutum cells (Fig. 3b-4), showing high expression during the daytime, but returned to the evening with N resupply (Fig. 3b-5). The peaks of expression of both LHY and TOC1 moved forward in the N-deplete cells; however, the oscillation period remained at about 24 h. The peaks of LHY and TOC1 expression in the N-deplete S. costatum cells moved forward 6 h and 9 h (Figs 3c-4, e-4), respectively, while those of the N-deplete P. tricornutum cells moved forward 9 h and 6 h (Figs 3d-4, f-4), respectively. After N resupply, the peaks of LHY and TOC1 expression were again delayed in the two diatoms (Figs 3c-5, d-5, e-5 and f-5).
The expression patterns of CCA1 and LHY in the P-deplete S. costatum cells remained unchanged compared with those of the control (Figs 3a-6, c-6), while the peak of TOC1 expression was delayed for 3 h (Fig. 3e-6). However, the peaks of CCA1, LHY, and TOC1 expression in P. tricornutum moved forward, and these genes were highly expressed during the day (Figs 3b-6, d-6 and f-6). After P resupply, CCA1 expression in S. costatum exhibited two peaks within an oscillation period (Fig. 3a-7), LHY was highly expressed during the day (Fig. 3c-7), and TOC1 expression returned to a normal pattern (Fig. 3e-7).
3.4 Genetic evolution of core clock genes in S. costatum and P. tricornutum
To examine the evolutionary status of core clock genes in S. costatum and P. tricornutum, we used the neighbor-joining method to construct phylogenetic trees. The amino acid sequences for CCA1, LHY, and TOC1 in Brassica campestris, B. napus, B. juncea, B. oleracea, B. carinata, Noccaea caerulescens, Arabidopsis thaliana, Arabidopsis lyrata, Arabidopsis halleri, Ananas comosus, Picea abies, Nicotiana tabacum, Helianthus annuus, S. costatum, P. tricornutum, and Chlamydomonas reinhardtii were selected for phylogenetic analysis (Fig. 4). The genes were well clustered into their relevant phylogenetic branches and were located at the same branchpoint as Helianthus annuus, Nicotiana tabacum, Picea abies, and Arabidopsis. However, they were clustered in different evolutionary branches from that of Chlamydomonas reinhardtii (Figs 4a–c). The LHY phylogenetic tree indicated that the two subclasses were well clustered into their relevant phylogenetic branches. The LHY genes of S. costatum and P. tricornutum were clustered together, while the LHY genes of Vigna radiata, Gossypium hirsutum, and Cucumis melo were clustered into one branch; these were then clustered together with Anthurium amnicola and A. thaliana (Fig. 4b). The TOC1 genes of S. costatum and P. tricornutum were clustered at the same branchpoint as Schizosaccharomyces pombe. Further analysis showed that the TOC1 gene of Betula platyphylla was clustered with those of Populus tremula, Phasedus vulgaris, Ipomoea nil, A. thaliana, Cryptomeria japonica, and Hordeum vulgare, and was clustered with nematodes to form another branch (Fig. 4c).
3.5 A speculative molecular model for the circadian clock in marine diatoms
Based on the circadian clock genes identified in S. costatum and P. tricornutum, we established a speculative molecular model of the circadian clock in marine diatoms by referring to the model previously created for A. thaliana (McClung, 2001). According to our model, five red-light receptor PHY genes (PHYA, PHYB, PHYC, PHYD, and PHYE), a blue-light receptor gene CRY1, and three downstream clock regulator genes ZTL, LKP2, and FKF1 are located in the input pathway of the circadian clock of marine diatoms, and these factors transmit environmental signals to the core oscillator (Fig. 5). Nine genes are located in the core oscillator and form a complex multiple feedback regulatory network that includes a core loop based on CCA1/LHY-TOC1, a morning loop featuring CCA1/LHY and PRR7/PRR9, and an evening loop under the control of the TOC1 core gene (Fig. 5). Signals from the input pathway are passed to the complex regulatory network formed by the core oscillator to create new signals that exhibit regular diel variation. However, only the CAB2 gene was identified in the output pathway (Fig. 5).
4 Discussion
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Studies investigating the circadian clock have largely focused on the identification, expression, and regulation of clock genes in the cyanobacteria Synechococcus (Kondo et al., 1994) and the higher plant A. thaliana (Millar et al., 1995). In this study, circadian clock genes were identified in two diatom species, S. costatum and P. tricornutum, that were similar to those of A. thaliana, but completely different from those of the prokaryote Synechococcus. This suggests that marine diatoms have evolved a circadian clock system that is similar to that of higher plants.
During their growth and development, marine diatoms often undergo stress as a result of a range of abiotic factors (Irigoien et al., 2004). Light is the most important of these factors, not only affecting cell growth, but also representing the primary environmental input for the circadian clock (McClung, 2001). Input pathway genes such as PHYs, CRY, ZTL, and the valve effector gene LUX were identified in both diatom species (Table 1). Members of the phytochrome red-light photoreceptor PHY family, cryptochrome blue-light photoreceptor CRY family, and genes encoding the ZTL family of LOV domain-containing proteins, are involved in converting light signals into transcriptional regulatory signals and transmit light via LKP2 and FKF1 (Kevei and Nagy, 2003; Lin and Shalitin, 2003; Demarsy and Fankhauser, 2009). LUX acts as a valve effector and controls the strength of input signals (Hazen et al., 2005). When red or far-red light input reaches a critical value, it will be prevented from entering the circadian clock system and possibly damaging it (Hazen et al., 2005). Five genes of the phytochromes (PHY) family (PHYA, PHYB, PHYC, PHYD, and PHYE) were identified in S. costatum, whereas only three (PHYC, PHYD, and PHYE) were identified in P. tricornutum (Table 1). Moreover, only three of these genes (PHYA, PHYB, and PHYC) have been identified in rice (Dehesh et al., 1991; Basu et al., 2000). The LKP2 and FKF1 genes, known to play roles in light signaling, were also identified in both S. costatum and P. tricornutum (Table 1). In A. thaliana, overexpression of LKP2 results in hypocotyl elongation and a late-flowering phenotype under long-day conditions. Furthermore, by regulating the downstream expression of CAB2 under continuous light or darkness, such plants show disorder in their circadian rhythm (Sato et al., 2002). The same genes were identified in the photoreceptor and light-transport pathways of S. costatum and P. tricornutum, suggesting that the input pathway adopted by these species is similar to that adopted by higher plants. However, PHYA and PHYB were not identified in P. tricornutum, indicating that circadian clock input pathway genes in marine diatoms might differ from those of higher plants.
In the core oscillator, we identified genes involved in the core loop, morning loop, and evening loop in both diatom species, including three core genes (CCA1, LHY, and TOC1) (Table 1). The three genes are the main components of the core oscillator and together form a regulatory feedback loop. The LHY and CCA1 gene products bind to the evening element (AAATATCT) on the TOC1 promoter and inhibit its expression, while TOC1 also acts as a transcriptional repressor for CCA1 and LHY (Alabadí et al., 2001). The clock signal integrated by the core oscillator in marine diatoms exerts its effects on a range of physiological and biochemical processes by regulating the expression of the downstream gene CAB2. The morning loop, consisting of CCA1/LHY and PRR9/PRR7/PRR5, and the evening loop, consisting of TOC1 and LUX, might also be present in marine diatoms. The CCA1 gene is known to be involved in regulating photoperiod sensitivity, breaking seed dormancy, seed germination, and adaptation to cold stress, while also promoting hypocotyl elongation in A. thaliana (Hotta et al., 2007). Under continuous light, LHY and CCA1 promote plant flowering by reducing the expression of the short vegetative phase (SVP) gene (Fujiwara et al., 2008). Homologs of the TOC1 gene have been identified in a range of plants, including rice, alfalfa (Medicago sativa), soybean (Glycine max) and maize (Zea mays), demonstrating strong functional conservation in terms of protein structure and evolution (Murakami et al., 2003; Boxall et al., 2005; Liu et al., 2009). In A. thaliana, overexpression of TOC1 leads to a longer circadian cycle and dysregulation of the circadian rhythm (Salter et al., 2003; Derveaux et al., 2010). In addition, TOC1 also participates in photomorphogenesis in A. thaliana. However, TOC1 mutants flower early under short-day conditions (Más et al., 2003). Among the genes involved in the morning cycle, PRR5, PRR7, and PRR9 were identified in P. tricornutum; however, PRR9 was not present in S. costatum. The PRR protein family has a CCT domain at the C-terminus, which is unique to plants, and includes a nuclear import signal (Matsushika et al., 2007). Arabidopsis PRR7 and PRR9 mutants exhibit a longer circadian clock period, while PRR5 mutants have a shorter cycle; PRR5 and PRR7 can control the sensitivity of circadian clock photoresponses (Kaczorowski and Quail, 2003; Michael et al., 2003). In addition, PRR3 was also identified in both diatom species (Table 1). The expression of the PRR3 protein in the vascular bundles of leaves affects the stability of the TOC1 protein, while a ZTL competitor preferential interacts with TOC1 (Para et al., 2007). TOC1 and LUX were identified in both diatom species, while PRR9 was identified only in P. tricornutum; however, GIGANTEA (GI), EARLY FLOWERING 3 (ELF3), and EARLY FLOWERING 4 (ELF4) were not detected in either species (Table 1). This indicated that the two marine diatom species retained the core components of the circadian clock during evolution, and that the genes missing from the evening loop might have been lost due to evolutionary selection.
The GI, CONSTANS (CO), and FLOWERING LOCUS T (FT) genes of the output pathway were not detected in either of the diatom species, with only CAB2 being identified (Table 1). In etiolated monocotyledons or dicotyledons, the phytochrome promotes the transcriptional accumulation of CAB2, and the CAB2 promoter is also known to be induced by light and inhibited in the dark (Meyer et al., 1989). We also identified CK2 in both diatom species (Table 1). In A. thaliana, CCA1 and LHY are phosphorylated by CK2 (Lu et al., 2011). CK2 regulates the circadian clock by influencing the DNA binding of CCA1 (Mulekar and Huq, 2014). The circadian rhythm of plants may have originated in green algae (Matsuo et al., 2008). The fact that we identified the same circadian clock genes in S. costatum and P. tricornutum as those found in higher plants indicated that marine diatoms and higher plants have evolved a similar biological clock mechanism.
The expression pattern of CCA1 in S. costatum and P. tricornutum showed clear diel variation with a cycle of almost 24 h (Fig. 3). In P. tricornutum, LHY also exhibited a diel expression pattern that was similar to that of CCA1, while LHY oscillated for about 27 h in S. costatum (Fig. 3). In higher plants such as A. thaliana, rice, and wheat, CCA1 and LHY were expressed in the morning; expression peaks before and after illumination, decreased during the daytime, and begins to increase again after darkness (Izawa et al., 2003; Murakami et al., 2007). However, in both diatom species, the expression of CCA1 and LHY increased at the onset of darkness, and peaked before dawn, earlier than in higher plants such as A. thaliana and rice. The expression pattern of CCA1 in the two diatoms was similar to that in the green alga Ostreococcus tauri (Dong et al., 2011). Moreover, the expression of TOC1 in the two diatom species was similar to that in A. thaliana and other higher plants, and peaked during the night (Dong et al., 2011). The peak expression of TOC1 in the two diatom species moved backward when compared with higher plants (Alabadí et al., 2001). Moreover, we found that variations in ambient light and levels of N and P affected the expression patterns of the three core clock genes in S. costatum and P. tricornutum, indicating that light, N and P might improve their environmental adaptability by adjusting the circadian clock. However, the expression patterns between the two diatoms under conditions of changing light and N and P availability were different, indicating species-specific responses to environmental change. Flowering plants live in a terrestrial environment while diatoms live in an aquatic environment, and these habitats differ in both light composition and intensity. These factors are likely to have played important roles in driving the differentiation of circadian clock genes during evolution.
Evolutionary analysis of the core clock genes revealed that, in S. costatum, P. tricornutum, and Chlamydomonas reinhardtii, CCA1 was located in different evolutionary branches, and had diverged from that of A. thaliana and B. campestris, but was closely related to that of Helianthus annuus, Nicotiana tabacum, Picea abies, and Ananas comosus. In the two diatom species, LHY was located in evolutionary branches different from those of Vigna radiata, Gossypium hirsutum, Cucumis melo, Anthurium amnicola, and A. thaliana, indicating that LHY in the two diatoms had diverged from those of other species. The TOC1 genes of S. costatum, P. tricornutum, and Schizosaccharomyces pombe were clustered at a common branchpoint and had a close evolutionary relationship. We speculate that the physiological function of the marine diatom circadian clock may have differentiated throughout evolution. That marine diatoms and higher plants possess the same circadian clock genes suggests that functional differentiation may have occurred before the emergence of terrestrial plants.
5 Conclusions
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In this study, the circadian clock genes and their expression patterns in marine diatoms were investigated for the first time. We identified circadian clock genes in S. costatum and P. tricornutum that have already been described in A. thaliana and other higher plants. Furthermore, most of the clock genes were the same in the two diatom species, with some exceptions. Our results indicated that marine diatoms have evolved circadian clock mechanisms suited to their environment. Based on the circadian clock genes identified in marine diatoms and the established circadian clock model for higher plants, we constructed a speculative molecular model for the circadian clock in marine diatoms, including the input pathway, core oscillator, output pathway, and valve effector (Fig. 5). The core clock genes of the two diatom species, CCA1, LHY, and TOC1, showed circadian oscillations for periods of approximately 24 h, but changes in ambient light and N and P availability induced phase shifts of these genes which might adjust the circadian clock performance to adapt to the environmental changes. However, their expression patterns differed from those of terrestrial plants. Homology and cluster analyses further showed that, in S. costatum and P. tricornutum, the three core clock genes had evolved separately from those of other plants, even though they shared the same genetic clock components. Future efforts should be devoted to investigating circadian clock genes in other diatom species and characterizing their expression patterns under different environmental conditions, which should significantly advance our understanding of how the circadian clock allows marine diatoms to respond and adapt to environmental changes.
Funding
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  • The National Natural Science Foundation of China under contract Nos 41425021 and 41706131; the National Key Research and Development Program of China under contract No. 2017YFC1404302; the “Ten Thousand Talents Program” for Leading Talents in Science and Technological Innovation to Dazhi Wang.
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Year 2022 volume 41 Issue 8
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doi: 10.1007/s13131-021-1962-4
  • Receive Date：2021-10-06
  • Online Date：2025-11-21
  • Published：2022-08-25
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  • Received：2021-10-06
  • Accepted：2021-11-18
Funding
The National Natural Science Foundation of China under contract Nos 41425021 and 41706131; the National Key Research and Development Program of China under contract No. 2017YFC1404302; the “Ten Thousand Talents Program” for Leading Talents in Science and Technological Innovation to Dazhi Wang.
Affiliations
    1 State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen 361102, China
    2 College of the Environment and Ecology, Xiamen University, Xiamen 361102, China
    3 Key Laboratory of Marine Ecology & Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China

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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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