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Horloge ! dieu sinistre, effrayant, impassible,
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Charles Baudelaire - L'horloge - Les Fleurs du mal
Chapter II.b:
bHLH-PAS protein RITMO1
regulates diel biological rhythms
in the marine diatom
*Phaeodactylum tricornutum*

Rossella Annunziata<sup>1,2,1</sup>, Andrés Ritter<sup>1,2</sup>, Antonio Emidio Fortunato<sup>2,3</sup>, Alessandro Manzotti<sup>3</sup>,
Soizic Cheminant-Navarro<sup>2,4</sup>, Nicolas Agier<sup>1</sup>, Marie J. J. Huysman<sup>1,5</sup>, Per Winge<sup>1</sup>, Atle M. Bones<sup>1</sup>, François-
Yves Bouget<sup>1</sup>, Marco Cosentino Lagomarsino<sup>1</sup>, Jean-Pierre Bouly<sup>2,3</sup>, and Angela Falciatore<sup>2,4</sup>

'Laboratory of Computational and Quantitative Biology, Sorbonne Université, CNRS, Institut de
Biologie Paris-Seine, F-75005 Paris, France

''Integrative Marine Ecology, Stazione Zoologica Anton Dhorn, 80121 Napoli, Italy

'Laboratoire de Biologie du chloroplaste et perception de la lumière chez les microalgues UMR7141,
CNRS, Sorbonne Université, Institut de Biologie Physico-Chimique, 75005, Paris, France

'Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium

'Vlaams Instituut voor Biotechnologie Center for Plant Systems Biology, B-9052 Ghent, Belgium

'Cell Molecular Biology and Genomics Group, Department of Biology, Norwegian University of Science
and Technology, 7491 Trondheim, Norway

'Sorbonne Université, CNRS, UMR 7621, Laboratoire d'Océanographie Microbienne, Observatoire
Océanologique, 66650 Banyuls-sur-Mer, France

Keywords: diatom, circadian rhythms, bHLH-PAS, gene expression, cellular fluorescence
<figure><img src="image_22.png" /></figure>

# bHLH-PAS protein RITMO1 regulates diel biological rhythms in the marine diatom *Phaeodactylum tricornutum*

Rossella Annunziata<sup>a,b,1</sup>, Andrés Ritter<sup>a,1,2</sup>, Antonio Emidio Fortunato<sup>a,3</sup>, Alessandro Manzotti<sup>a,c</sup>, Soizic Cheminant-Navarro<sup>a,c</sup>, Nicolas Agier<sup>a</sup>, Marie J. J. Huysman<sup>a,d,e</sup>, Per Winge<sup>f</sup>, Atle M. Bones<sup>f</sup>, François-Yves Bouget<sup>g</sup>, Marco Cosentino Lagomarsino<sup>a</sup>, Jean-Pierre Bouly<sup>a,c</sup>, and Angela Falciatore<sup>a,c,4</sup>

<sup>a</sup>Laboratory of Computational and Quantitative Biology, Sorbonne Université, CNRS, Institut de Biologie Paris-Seine, F-75005 Paris, France; <sup>b</sup>Integrative Marine Ecology, Stazione Zoologica Anton Dhorn, 80121 Napoli, Italy; <sup>c</sup>Laboratoire de Biologie du chloroplaste et perception de la lumière chez les microalgues UMR7141, CNRS, Sorbonne Université, Institut de Biologie Physico-Chimique, 75005, Paris, France; <sup>d</sup>Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium; <sup>e</sup>Vlaams Instituut voor Biotechnologie Center for Plant Systems Biology, B-9052 Ghent, Belgium; <sup>f</sup>Cell Molecular Biology and Genomics Group, Department of Biology, Norwegian University of Science and Technology, 7491 Trondheim, Norway; and <sup>g</sup>Sorbonne Université, CNRS, UMR 7621, Laboratoire d'Océanographie Microbienne, Observatoire Océanologique, 66650 Banyuls-sur-Mer, France

Edited by Susan S. Golden, University of California, San Diego, La Jolla, CA, and approved May 10, 2019 (received for review November 27, 2018)

Periodic light–dark cycles govern the timing of basic biological processes in organisms inhabiting land as well as the sea, where life evolved. Although prominent marine phytoplanktonic organisms such as diatoms show robust diel rhythms, the mechanisms regulating these processes are still obscure. By characterizing a *Phaeodactylum tricornutum* bHLH-PAS nuclear protein, hereby named RITMO1, we shed light on the regulation of the daily life of diatoms. Alteration of RITMO1 expression levels and timing by ectopic overexpression results in lines with deregulated diurnal gene expression profiles compared with the wild-type cells. Reduced gene expression oscillations are also observed in these lines in continuous darkness, showing that the regulation of rhythmicity by RITMO1 is not directly dependent on light inputs. We also describe strong diurnal rhythms of cellular fluorescence in wild-type cells, which persist in continuous light conditions, indicating the existence of an endogenous circadian clock in diatoms. The altered rhythmicity observed in RITMO1 overexpression lines in continuous light supports the involvement of this protein in circadian rhythm regulation. Phylogenetic analysis reveals a wide distribution of RITMO1-like proteins in the genomes of diatoms as well as in other marine algae, which may indicate a common function in these phototrophs. This study adds elements to our understanding of diatom biology and offers perspectives to elucidate time-keeping mechanisms in marine organisms belonging to a major, but under-investigated, branch of the tree of life.

diatom | circadian rhythms | bHLH-PAS | gene expression | cellular fluorescence

The Earth's rotation means that life evolved under a daily cycle of alternate light and dark periods. Most living organisms have developed rhythms of many fundamental biological processes, ranging from physiology to behavior, such that they occur at optimal times of the day, which can enhance fitness (1). These rhythms are the product of the coordinated action of signals from endogenous biological clocks, together with environmental and metabolic inputs (2–4). In most eukaryotes, biological rhythms are controlled by interconnected transcriptional-translational feedback loops involving transcription factors (TFs). Although this regulatory framework is conserved among eukaryotes, the regulators orchestrating these rhythms seem to have emerged several times through evolution (5, 6).

Robust diel rhythms in growth, gene expression, pigment synthesis, phototactic movements, and bioluminescence have been observed in a variety of phytoplanktonic organisms, including diatoms (7–10). Diatoms represent the most species-rich group of algae in the ocean and populate a wide range of aquatic environments (11). These algae of the Stramenopila lineage, derived by secondary endosymbiosis events (12), show peculiar genomic,

metabolic, and cellular features and have an impressive capacity to deal with environmental changes (e.g., refs. 12–16). Genome-wide analyses have also shown that 25% of the diurnal transcriptome is influenced by light–dark cycles in the centric diatom *Thalassiosira pseudonana* (17). Additional studies in the pennate diatom *Phaeodactylum tricornutum* have highlighted strong synchronization of the cell cycle with day–night cycles and a strict temporal separation of transcriptional gene networks and metabolism (18–21), as observed in other algae (22, 23). However, the molecular mechanisms orchestrating these processes are still unknown in diatoms and many other phytoplanktonic organisms, except for the green alga *Ostreococcus tauri*, which possesses a plant-like circadian clock (24). Indeed, no clear homologs of the circadian clock components discovered in bacteria, fungi, animals, or

## Significance

Most organisms experience daily light–dark changes and show rhythms of basic biological processes such that they occur at optimal times of the day. Rhythms are also observed in a multitude of marine organisms, but their molecular foundations are still largely unknown. Here, we report daily oscillations of gene expression and cell fluorescence in the diatom *Phaeodactylum tricornutum*, which persist in the absence of external timing cues. We demonstrate that the protein RITMO1, encoded by a *bHLH-PAS* gene, which is widely represented in algal genomes, regulates these rhythms. By demonstrating circadian regulation in the most species-rich algal group in the ocean, this study unveils critical features of diatom biology, thus advancing the field of diurnal and circadian rhythms in marine algae.

Author contributions: R.A., A.R., A.E.F., J.-P.B., and A.F. designed research; R.A., A.R., A.E.F., A.M., S.C.-N., M.J.J.H., and A.F. performed research; P.W., A.M.B., F.-Y.B., and M.C.L. contributed new reagents/analytic tools; R.A., A.R., A.E.F., N.A., M.C.L., J.-P.B., and A.F. analyzed data; and R.A., A.R., A.E.F., and A.F. wrote the paper.

This article is a PNAS Direct Submission.

This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

Data deposition: Code set information and raw nCounter data are available from the Gene Expression Omnibus database, https://www.ncbi.nlm.nih.gov/geo/ (Series GSE112268).

¹R.A. and A.R. contributed equally to this work.

²Present addresses: Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium; and Vlaams Instituut voor Biotechnologie Center for Plant Systems Biology, B-9052 Ghent, Belgium.

³Present address: ImmunRise Technologies, 75005 Paris, France.

⁴To whom correspondence may be addressed. Email: angela.falciatore@upmc.fr.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1819660116/-/DCSupplemental.
plants have been found in the diatom genomes except for crypto-
chromes and casein kinases (13).

In this work, by characterizing gene transcription and cell
fluorescence rhythms in *P. tricornutum*, we uncover the existence
of circadian rhythms in diatoms and demonstrate the involvement
of the bHLH-PAS protein *bHLH1a*, here named RITMO1, in the
regulation of these processes. Notably, the bHLH-PAS domains
feature in proteins involved in the regulation of rhythmic pro-
cesses in animals (5). Phylogenetic analyses reveal a wide dis-
tribution of RITMO-like homologs in the genomes of diatoms
and other marine algae. Thus, RITMO1 represents a key mo-
lecular entry point for the identification of the timekeeper
components in diatoms and paves the way for a deeper under-
standing of marine rhythms and their evolutionary and ecological
relevance.

Results

Transcriptome Profiling Identifies Potential Regulators of Diurnal Rhythms in *P. tricornutum*. To identify potential regulators of cellular rhythmicity in *P. tricornutum*, a publicly available diurnal transcriptomic dataset (18) was analyzed. One hundred and four genes with robust diel oscillating expression were selected, including eight photoreceptors (19, 25-27), 66 TFs (28), and 30 potential output genes implicated in diel rhythmic processes (pigment synthesis, cell cycle regulation, and photosynthesis) (*SI Appendix*, Table S1). The transcriptional dynamics of the selected genes were further examined in a 16:8-h light:dark (L:D) photocycle for 32 h. Hierarchical clustering analysis revealed four main clusters of coexpressed genes, termed I–IV (Fig. 1 A and B). Cluster I phased at dawn, at around Zeitgeber Time 0 h (ZT: hours after illumination), suggesting a transcriptional anticipation of the light onset. This cluster comprised 18 genes including 14 TFs, mostly belonging to the Heat Shock Transcription Factor family (HSF), the two DNA repair enzymes CPD photolyases (*CPD2* and *CPD3*), and one carotenoid synthesis enzyme (*PDS1*). Cluster II phased around ZT7 and encompassed 36 genes, including the *dsCYC2* gene controlling the onset of cell division (19). Cluster II also contained 18 TFs, of which 8 were sigma factors putatively involved in the regulation of chloroplast transcription, three genes implicated in photoprotection (*LHCX1*, *Vdl2*, and *Zep1*), and the chlorophyll synthesis *POR1* gene. Such active transcription of genes involved in chloroplast activity during the light period has been shown previously (18, 29). The blue light sensors *Aurochrome1b* and the cryptochromes *CPF1* and *CryP-like* also belonged to cluster II and showed a strong expression following light onset in accordance with previous observations (26, 30). Cluster III phased around ZT9 and comprised 9 TFs and 10 metabolism-related genes. Finally, cluster IV phased just before dusk, at around ZT15, and included 23 TF genes, likely contributing to preparing cells for light-to-dark transition. Cluster IV also contained the *CPF4* and the far-red light sensing phytochrome (*DPH1*), the peak expression of which at the end of the light period has been observed previously (26, 31).

To explore transcription dynamics in the absence of light in-
puts, the expression of these rhythmic genes was further analyzed
in cells exposed to continuous darkness (D:D) for 30 h. The
analysis of transcript profiles revealed that around 20% of the
analyzed genes showed persistent oscillating expression in D:D,
although some profiles displayed reduced amplitudes and/or
shifted phases of expression compared with the L:D condition (*SI
Appendix*, Fig. S1). In particular, we identified 18 genes, including
putative TFs, pigment-related enzymes, and cell cycle-related
genes, showing the highest amplitudes of expression in both L:D
and D:D. Conversely, the other 80% of the analyzed expression
profiles did not have persistent oscillating patterns in darkness and
instead had strongly reduced amplitudes or altered expression
timing compared with the L:D condition (*SI Appendix*, Fig. S2).

Rhythmic *bHLH1a* and *bHLH1b* Expression Is Adjusted in a Photoperiod-
Dependent Manner and Persists in Continuous Light and Continuous Dark
Conditions. Our analysis identified two TFs—*bHLH1a* (Phatr3_J44962)
belonging to cluster IV and *bHLH1b* (Phatr3_J44963) belonging

to cluster III—which each have a Per-ARNT-Sim (PAS) domain
in conjunction with a bHLH DNA-binding domain. Because
bHLH-PAS proteins have been shown to be involved in the reg-
ulation of rhythmic processes in animals (5), the expression pro-
files of *bHLH1a* and *bHLH1b* were further examined in *P.
tricornutum* cells grown under different photoperiods. *bHLH1a*
expression peaked at ZT8 in the 12L:12D photoperiod and at
ZT12 in the 16L:8D photoperiod, 4 h before the end of the light
period in both cases, and then gradually decreased to below de-
tection limits at ZT0 (Fig. 1C). Transcription of *bHLH1b*
appeared to start earlier than that of *bHLH1a*. In cells entrained
in 12L:12D cycles, *bHLH1b* expression peaked at ZT8, whereas it
peaked between ZT8 and ZT12 in 16L:8D photoperiods (Fig.
1C). Rhythmic expression was maintained in continuous light
(L:L), although amplitude was reduced and period elongated for
both genes. In D:D (Fig. 1C), *bHLH1a* and *bHLH1b* showed an
increase in relative transcript abundance during the subjective day
that was comparable to that observed in L:D, but the decrease
during the subjective night was less pronounced (Fig. 1C and *SI
Appendix*, Fig. S1). Because iron metabolism is diurnally regulated
in *P. tricornutum* and iron starvation strongly affects rhythmic gene
expression (32), we further examined the *bHLH1a* and *bHLH1b*
expression profiles in cells grown in iron replete and deplete
conditions by using public transcriptome datasets (32). In this
analysis, *bHLH1a* and *bHLH1b* showed similar expression
patterns in both control and iron starvation conditions, with
peak expression at ZT9 in cells grown in 12L:12D photocycles
(*SI Appendix*, Fig. S3). Together, these results demonstrate the
robust control of *bHLH1a* and *bHLH1b* expression timing. Of
these proteins, we decided to further investigate the possible
involvement of *bHLH1a* in the regulation of *P. tricornutum*
rhythms.

Cellular Localization of bHLH1a Protein. To examine bHLH1a cel-
lular localization, we generated transgenic lines expressing bHLH1a
fused to a YFP-tag at the C-terminal under the regulation of the
*Lhcf2p* promoter (33). Confocal laser scanning microscopy of
bHLH1a-YFP-expressing cells showed colocalized signals of YFP
with the Hoechst 33342 (DNA tracker) stain in the nucleus (Fig.
2A). This confirmed the predicted in silico localization (*SI Appen-
dix*) of bHLH1a in the nucleus of *P. tricornutum*, further supporting
its possible role as a transcriptional regulator.

bHLH1a Regulates Pace of Diel Gene Expression. To explore possible
functions of bHLH1a in diel rhythmicity, we generated cell lines
expressing HA-tagged bHLH1a under the regulation of the *Lhcf2p*
promoter, which drives gene expression 3 h after the light onset
(33). Immunoblot analysis performed at ZT7 and ZT12 ina 16L:8D
regime revealed three independent bHLH1a-HA overexpressing
lines, hereafter named OE1, OE2, and OE3 (Fig. 2B). The ectopic
protein was detected at ZT7 and ZT12 in these lines. Analysis of
*bHLH1a* total transcripts, both endogenous (*bHLH1a*) and trans-
genic (*bHLH1a-HA*) mRNAs, also showed earlier expression in the
OE lines compared with the wild type (WT) (Fig. 2 C and D).

To investigate the effect of bHLH1a deregulation on diel gene
expression, WT and OE lines were grown in 16L:8D photocycles
and sampled for RNA analysis. The expression of selected genes
exhibiting a diurnal rhythmic expression pattern (Fig. 1) was an-
alyzed in WT and OE1 sampled every 3 h over 24 h (Fig. 2C and
*SI Appendix*, Fig. S4A). As described above, total *bHLH1a* tran-
script levels were higher in the OE line compared with the WT
(Fig. 2 C and D), but a decrease in endogenous *bHLH1a* tran-
scripts was also observed (Fig. 2 C and D). A similar pattern was
also reported for the *bHLH1b* gene while *bHLH3* showed earlier
expression in OE1 compared with the WT (Fig. 2 C and D). In
addition to the *bHLH* genes, altered expression patterns in OE1
were also found for other putative TF genes and cell cycle regu-
lators (*SI Appendix*, Fig. S4A). To test a possible function of
bHLH1a in the maintenance of diurnal rhythms in the absence of
light inputs, the expression of genes found to be rhythmic in the
WT in D:D (*SI Appendix*, Fig. S1) was analyzed also in OE1. Ten
<figure><img src="image_23.png" /><figcaption>Fig. 1. Diurnal expression of rhythmic *P. tricornutum* genes. (A) Hierarchical clustering of gene expression profiles in 16L:8D, analyzed by nCounter analysis. Arrowheads highlight *bHLH1a* and *bHLH1b*; asterisks indicate the transcript profiles oscillating in D:D, shown in *SI Appendix*, Fig. S1. (B) Polar plot showing average phases of expression of the four clusters. N: number of genes within each cluster; Av. Phase: average phases ± SD. (C) *bHLH1a* and *bHLH1b* expression in 12L:12D, 16L:8D, L:L, and D:D (mean ± SEM, n = 3). In L:L and D:D, cells entrained under 16L:8D cycles were released to continuous light or dark at ZT16. White and gray regions represent light and dark periods; dashed regions represent subjective nights in L:L and D:D. The expression value of each gene is relative to its maximum expression.</figcaption></figure>

of 11 genes displayed reduced amplitudes and shifts in the phase of expression in OE1, compared with WT (Fig. 2E and SI Appendix, Fig. S4B). Analysis of *bHLH1a*, *bHLH1b*, and *bHLH3* in OE2 and OE3 at ZT7 and ZT12 in L:D (Fig. 2D) and at DD7 and DD12 in D:D (Fig. 2F) suggested a similar deregulation of gene expression in independent transgenic lines.

The Ectopic Overexpression of *bHLH1a* Results in Altered Circadian Rhythms. We next questioned if, beside gene expression, other physiological rhythms were regulated by *bHLH1a*. With this aim, daily cellular fluorescence was analyzed using the flow cytometer channel FL3-A that estimates chlorophyll *a* cellular content (8, 29). Cellular fluorescence displayed strong oscillations in 16L:8D grown cultures with a periodicity of ~24 h (Fig. 3A and SI Appendix, Table S2). Cell fluorescence in WT cultures increased during daytime to phase around ZT12 (Fig. 3A) and then started to decrease before night onset. Fluorescence progressively declined during the night period, reaching a trough in the early morning at ZT0 (Fig. 3A). In synchronized cells, the increase of FL3-A fluorescence is concomitant with the increase in the proportion of G2/M cells, and a decrease in the FL3-A relates to an increase in cell concentration (29) (SI Appendix, Fig. S5), likely reflecting chloroplast partitioning to daughter cells during cell division. Despite maintaining period rhythmicity in the cellular fluorescence dynamics, all three OE lines displayed less synchronized phenotypes, resulting in higher variability of waveforms among replicates compared with WT (Fig. 3A). Phase-time estimations using the FFT-NLLS method identified significant shifts of ~2 h in the maximum fluorescence timing in OE1 and OE2 compared with the WT (Fig. 3A and B and SI Appendix, Fig. S6 and Table S2). We then questioned if these oscillations were maintained in continuous blue light (L:L) as observed in other microalgae (24, 34). First, we found that under these conditions *bHLH1a* gene expression showed a stronger rhythmic pattern on the second subjective day (SI Appendix, Fig. S7), compared with the white L:L condition (Fig. 1C). WT cells also showed persistent fluorescence oscillations from LL33, with a period of ~27 h. These rhythms lasted to at least the fifth day of continuous light (Fig. 3C), supporting the hypothesis that they are self-sustained by an endogenous clock. Patterns of cell fluorescence oscillations in L:L were strongly altered in all three OE lines compared with WT (Fig. 3C and D and SI Appendix, Figs. S8 and S9) although similar growth rates were observed (SI Appendix, Fig. S10). Analysis via the Fast Fourier Transform Nonlinear Least Squares (FFT-NLLS) method showed that all lines maintained residual rhythmicity, but with a strong phase shift compared with the WT (Fig. 3C and D) and a reduced amplitude, with the OE2 being the less affected for the latter one (Fig. 3D and SI Appendix, Figs. S8 and S9 and Table S3). Additionally, OE lines also presented shorter periods than the WT, although OE1 lines lacked a significant difference most likely due to the variability introduced by the replicates with high relative amplitude error (SI Appendix, Supplementary Methods, Fig. S8C and D, and Table S3). Fluorescence oscillations were similar between WT and an independent transgenic line expressing only the antibiotic resistance cassette (SI Appendix, Fig. S11), excluding a generic alteration of rhythmicity due to transgenesis. Based on these results, we named *bHLH1a* "RITMO1" (Italian word for "rhythm") after its role as regulator of diatom diel rhythms.

RITMO1-Like Proteins Are Widely Represented in the Genome of Marine Algae. The bHLH-PAS proteins were thought to be restricted to the animal (Opisthokonta) lineage (35) until various sequencing projects revealed bHLH-PAS family members in microalgae (diatoms and *Nannochloropsis*) (36). Diatom proteins show peculiar features including a single predicted PAS domain, whereas animal bHLH-PAS proteins have two and an N-terminal extension that is absent in their animal counterparts (Fig. 4A). Through searching transcriptomic and genomic databases from Archieplastida, Cryptophyta, Stramenopila, Alveolata, and basal Opisthokonta organisms (SI Appendix, Table S4), we discovered about 90 bHLH-PAS proteins. With one exception, the identified
<figure><img src="image_24.png" /><figcaption>Fig. 2. bHLH1a localizes in the nucleus, and its overexpression alters rhythmic diel gene expression. (A) Confocal fluorescence microscopy of *P. tricornutum* cells expressing the bHLH1a-YFP protein under the control of the *LhcF2p* promoter. (Scale bar: 15 µm.) (B) Immunoblot analysis of bHLH1a-HA protein at ZT7 and ZT12 in 16L:8D entrained WT and OE lines, using the anti-HA and anti-D2 (loading control) antibodies. (C) qRT-PCR analysis of *bHLH1a*, *bHLH1b*, and *bHLH3* in WT and OE1 cultures sampled in L:D every 3 h over 24 h (mean ± SEM, *n* = 2). (D) qRT-PCR analysis of the same genes at ZT7 and ZT12 in WT and OE1, OE2, and OE3 lines (mean ± SEM, *n* = 3). *Inset* in the *bHLH3* graph magnifies the ZT7 time point. (E) *n*Counter analysis of the *bHLH1a*, *bHLH1b*, and *bHLH3* over 24 h of continuous dark (D:D) in WT and OE1 lines (mean ± SEM, *n* = 3). (F) qRT-PCR analysis of the same genes at DD7 and DD12 in WT and OE1, OE2, and OE3 lines (mean ± SEM, *n* = 3). *bHLH1a-total* includes endogenous *bHLH1a* and *bHLH1a-HA* transgene transcripts; for all of the other genes, we refer to endogenous transcripts. The expression value of each gene is relative to its maximum expression. \* *P* < 0.05, \*\* *P* < 0.01, \*\*\* *P* < 0.001, *t* test.</figcaption></figure>

proteins showed a single predicted PAS domain, short C-terminal extensions, and N-terminal regions of variable length, similar to the predicted structure of diatom bHLH-PAS (Fig. 4A). Notably, we identified the first Archaeoplastida bHLH-PAS that possesses two PAS domains like the animal proteins in *Galdieria sulphuraria* (Rhodophyta).

All of the identified sequences, including selected bHLH-PAS from Opisthokonta lineages, were used to perform a detailed phylogenetic analysis of the protein family using the bHLH and PAS domains. This analysis revealed that the majority of microalgal bHLH-PAS proteins fall into three separate clades, the first containing 9 TFs from diatoms and *Ectocarpus siliculosus*, the second comprising RITMO1 together with 35 proteins from diatoms and Alveolata (Dinoflagellata), and the third comprising 41 proteins from Alveolata (Ciliophora and Dinoflagellata) and diatoms, including bHLH1b (Fig. 4B). Interestingly, domain organization and branching positions of proteins from basal Opisthokonta (*Monosiga brevicollis*) and secondary endosymbiotic microalgae [*Guillardia theta* (Cryptophyta) and *Nannochloropsis* species (Stramenopila)] (Fig. 4B) support a possible common origin for this TF family from a heterotrophic ancestor (37) featuring single bHLH and PAS domains. However, the basal position and domain organization of the *G. sulphuraria* bHLH-PAS protein hint at a more complex scenario possibly involving endosymbiotic (horizontal) gene transfer, gene duplication/loss, and convergent evolution in the diversification of this family.

Discussion

This study shows that diatoms integrate light signals from the environment as well as from an endogenous circadian clock to tightly regulate diel cellular activities. It also unveils the *P. tricornutum* RITMO1 protein of the bHLH-PAS transcription factor family as the first regulator of circadian rhythms to be found in these algae. *RITMO1* displays robust diel expression patterns, which

are adjusted in a photoperiod-dependent manner and are unaffected by iron deficiency. As for other circadian clock-regulated genes in algae (24), the rhythmic expression of *RITMO1* persists in cells exposed to continuous blue light, a dominant waveband in the ocean (27). However, *RITMO1* transcription shows reduced oscillation amplitude in constant darkness, likely due to the lack of critical light time setting signals and alteration of metabolism due to the fact that photosynthesis is not dispensable in *P. tricornutum*. Thus, the *RITMO1* expression timing is tightly regulated and likely subjected to multiple levels of control from different input regulators, conceivably blue light photoreceptors (27) and metabolism, as already observed for other clock genes in plants and animals (3, 4).

RITMO1 is involved in the transcriptional regulation of diel rhythms, as supported by consistent deregulation of transcript diurnal oscillations for several genes in RITMO1 OE lines in L:D, this pattern being accentuated in D:D. These results suggest, on one hand, that multiple light-driven processes participate in the regulation of daily gene expression, partially masking RITMO1's contribution to this process in cyclic environments and, on the other hand, reveal a key role for RITMO1 in the maintenance of rhythms in the absence of light-dark inputs. Furthermore, the observed down-regulation of the endogenous *RITMO1* transcription in OE lines compared with the WT may reflect negative feedback regulation of RITMO1 controlling its own transcription. This would be compatible with this gene being part of a transcriptional feedback loop operating over the daily cycle (2). We also report robust oscillations of diatom cellular fluorescence in L:D cycles, as observed for gene expression. These rhythms persisted for at least five subjective days in L:L, supporting the existence of a circadian clock regulating diatom physiology. The oscillations in L:L have a period slightly longer than 24 h under free running conditions, a common feature in circadian clock-regulated processes (1, 2). The deregulation of
<figure><img src="image_25.png" /><figcaption>Fig. 3. bHLH1a overexpression alters circadian rhythms of cellular fluorescence. (A) Diurnal oscillation of chlorophyll fluorescence (FL3-A parameter) in WT and OE lines entrained under 16L:8D over 3 d (n ≥ 8). (B) Phase-time calculation of the FL3-A value in WT and OE lines (mean ± SD, n ≥ 8, **P < 0.01, ***P < 0.001, t test). (C) Circadian oscillation of chlorophyll fluorescence in representative WT and OE lines under continuous blue light (L:L) over 4 subjective days (n = 8). (D) Plot of phase against period estimates in L:L (n = 15). Dots of different color indicate independent replicate cultures. White and gray regions represent light and dark periods; black dashed regions represent subjective nights. Brown lines in plots represent the fitted curves (lowess fit) of the average FL3-A.</figcaption></figure>

RITMO1 expression in transgenic lines shifts the phase of fluorescence rhythms in L:D compared with the WT. In constant blue light (L:L), all of the independent OE lines showed altered rhythmicity if compared with the WT and a transgenic control line, with a strongly altered phase, reduced amplitude, and the period also being remarkably perturbed. Although multiple factors may account for the slight phenotypic differences observed between OE lines (e.g., chromosomal position effects on the transgenes, deregulation of other genetic or epigenetic processes due to the overexpression), the consistent deregulation of rhythmicity is coherent with a role of RITMO1 in the regulation of circadian rhythms. Because all of the OE lines show a normal growth rate compared with WT, the observed altered rhythmicity is likely due to a defect in retrieving synchrony in chloroplast ontogeny and cell cycle progression, which are closely coupled in diatoms (20).

Together, our results support the hypothesis that RITMO1 is one component of the still-uncharacterized endogenous circadian clock in diatoms. RITMO1 contains bHLH and PAS protein domains that are also present in the CLOCK and BMAL proteins, components of the mammalian central circadian oscillator (2, 5, 38). Interestingly, previous studies have shown that the *P. tricornutum* animal-like blue light sensor Cpfl can repress the transcriptional activity of these proteins in a heterologous mammalian cell system (25), suggesting at least partial conservation in the regulatory program generating rhythmicity in animals and diatoms.

<figure><img src="image_26.png" /><figcaption>Fig. 4. bHLH-PAS protein family structure and phylogeny. (A) bHLH-PAS protein domain schematic architecture in eukaryotes. Dotted line indicates possible absence of the second PAS in some Opisthokonta species; gray patterns represent the variations in N-terminal and C-terminal length in different groups. (B) Maximum likelihood (ML) phylogenetic tree of the bHLH-PAS family (outgroup: the Opisthokonta clade) midpoint rooted. Numbers refer to bootstrap values of the basal nodes using ML (RAxML, 1,000 bootstraps) and Bayesian inference (MrBayes). Symbols indicate the position of *P. tricornutum* RITMO1 and bHLH1b (arrows) and *T. pseudonana* bHLH1 (square).</figcaption></figure>
We have reported several other putative TFs showing altered diel expression patterns in *RITMO1* OE cells (i.e., *bHLH1b*, *bHLH3*, *bZIP7*, *HSF1g*, *HSF4.7b*), which represent direct or indirect targets of *RITMO1* activity and possible additional components of the network generating circadian rhythms. *RITMO1* might also act downstream of signal transduction cascades activated by the diatom photoreceptors (27). Therefore, *RITMO1* now represents a key molecular entry point for the identification of other diatom timekeeper components and the input-output pathways interacting with the clock.

Biological rhythms are still poorly understood at the molecular level in many phyla of marine algae such as Stramenopila. Our phylogenetic analysis revealed a wide distribution of *RITMO1*-like *bHLH-PAS* proteins in diatoms, as well as in other algae. The similarities in the timing of expression between *RITMO1* and its homolog in the diatom *T. pseudonana* (*TpHLH1*) (17) suggest that these proteins may play a similar role in the regulation of cellular rhythmicity. Regulators such as *RITMO1* may have played a critical role for diatom prominence in marine ecosystems by synchronizing cellular activities in optimal temporal programs and maximizing the diatoms' ability to anticipate and adapt to cyclic environmental variations.

*bHLH-PAS* *RITMO1*-like proteins might have independently acquired a function in rhythm regulation by convergent evolution (6). However, the existence of this function in an ancient heterotrophic marine ancestor that subsequently acquired plastids via endosymbiosis events (12) and before colonization of land cannot be excluded. Thus, further characterization of *RITMO1*-like proteins is expected to provide insights into the evolution of biological rhythms and their significance for life in the marine environment.

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## Methods

Culture Conditions. WT *P. tricornutum* (*Pt1* 8.6) cells and transgenic lines were grown in f/2 Guillard media, as described in *SI Appendix*.

Gene Expression Analyses. To select genes with rhythmic expression, we used data from (18, 39). Total RNA was extracted as described in ref. 31 and analyzed by qRT-PCR and nCounter analysis as described in *SI Appendix*. Proteins were analyzed as previously described (25).

Generation of the *bHLH1a* Transgenic Lines. Diatom cell lines overexpressing *bHLH1a-HA* and *bHLH1a-YFP* were obtained by cotransformation of the Nourseothricin resistance plasmid (pNAT) together with the pDEST-C-*HA-bHLH1a* and the pDEST-C-YFP-*bHLH1a* plasmids, respectively. Details of vectors and transformation are provided in *SI Appendix*.

Microscopic Analysis. Images were produced with the confocal laser scanning microscope Leica SP8× as described in *SI Appendix*.

Data Mining, Protein Sequence, and Phylogenetic Analysis. Detailed information about data mining, protein sequence, and phylogenetic analysis is provided in *SI Appendix*.

ACKNOWLEDGMENTS. We thank M. Jaubert, L. De Veylder, R. Dorrell, and P. Oliveri for critical suggestions; D. Petroutsos and G. Finazzi for support in monitoring cell physiology; G. Benvenuto for assistance in microscopy; and the Institut de Biologie Paris-Seine imaging core facility for help with flow cytometry. This work was funded by Human Frontier Science Program Grant RGY0082/2010; the Gordon and Betty Moore Foundation Grant GBMF 4966; the European Union H2020 EMBRIC Grant G.A. 654008; and the Fondation Bettencourt Schueller (A.F.). M.J.J.H. is currently an employee of the European Research Council Executive Agency. The views expressed are purely those of the writer and may not in any circumstances be regarded as stating an official position of the European Commission.

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# SI APPENDIX

## SUPPLEMENTARY METHODS

### Culture and irradiation conditions

Wild-type *P. tricornutum* (*Pt*1 8.6; CCMP2561) cells and transgenic lines were grown in f/2 Guillard media (1). For experiments in different photoperiods, cultures were pre-adapted to the different L:D cycles for 2 weeks before starting the experiment. For L:D experiments, cells were illuminated at 40 µmol photons m⁻² s⁻¹ of white light (Philips TL-D De Luxe Pro 950) either for 16 or 12 hours. For gene expression experiments in continuous darkness and continuous light, cells were pre-adapted in 16L:8D photocycles for 2 weeks, then transferred to D:D or L:L at the start of the experiment. For fluorescence measurement and growth analysis experiments in free running L:L conditions, cells were grown in 16L:8D cycles, then transferred to 15 µmol photons m⁻² s⁻¹ of blue light from ZT12 onwards. Cell concentrations were measured using a MACSQuant Analyser flow cytometer (Miltenyi Biotec, Germany) by counting the cells based on the R1-A (630 nm excitation, 670-700 nm emission) versus the R1-H parameters. Growth rates (µ) were calculated during the exponential phase of growth over four days. Cellular fluorescence rhythmicity assays were carried out by measuring the flow cytometer FL3-A parameter (488 nm excitation, 655-730 nm emission).

### Analysis of the rhythmic processes

For the analysis of rhythmic parameters, each FL3-A fluorescence dataset was amplitude and baseline detrended, then normalized to the maximum value. For graphical representations, data were aligned to the mean values. Fitted curves of the average FL3-A fluorescence were calculated using the lowess fit function in R (span = 0.35). Circadian period, amplitudes and phases were calculated by Fast Fourier nonlinear least square algorithm (FFT-NLLS), evaluated within a period range of 20 and 30 h using the Biodare2 tool (biodare2.ed.ac.uk, (2)). This analysis allows to formally identify differences in rhythm profiles using the Relative Amplitude Errors (RAE) as a reliable proxy for rhythmic coherence. The RAE is a measure of goodness-of-fit to a theoretical sine wave. RAE is defined as the ratio of the amplitude error to the most probable derived amplitude magnitude. The RAE value ranges from 0.0 to 1.0, with 0.0 meaning a rhythmic component has an infinite precision and with 1.0 meaning the rhythm is not significant (error exceeds the most probable amplitude magnitude) (3). For L:L analyses, periods, phases and amplitudes were estimated starting from 33 h after the last dark/light transition. To evaluate if cell lines presented altered rhythmic traits, the obtained periods and
phases were plotted against the RAE (Table S3 and Fig. S9) so to provide a measure of how well the observed outcomes are replicated by the fitted cos waves, as previously described (4). Eight to nine independent biological replicates were considered for the analysis in 16L:8D conditions (Table S2), while fifteen biological replicates where considered for the analysis in L:L conditions (Table S3). Three periods of oscillations were considered for each replicate in all period estimation analyses. Two-tailed t-test was used to determine statistical differences between mean phases, amplitudes, periods and RAEs (Fig 3, Fig. S8, Table S2 & S3).

## RNA extraction and transcript analyses

For qRT-PCR analysis, *RPS* and *TBP* were used as reference genes. Each independent replicate of the qRT-PCR data was normalized against the maximum expression value of each gene (i.e., gene expression range lies between 0 and 1 across the time series). Average expression and standard error were then calculated and plotted. The full list of oligonucleotides used in this work can be found in Table S5. For the nCounter analysis, gene specific probes (Table S1) were designed and screened against the *P. tricornutum* annotated transcript database (JGI, genome version 2, Phatr2) for potential cross-hybridization. Total RNA extracts (100 ng) from three biological replicates were used for hybridization. Transcript levels were measured using the nCounter analysis system (Nanostring Technologies) at the UCL Nanostring Facility (London, UK) and at the Institut Curie technical platform (Paris, France) as previously described (5). Expression values were first normalized against the internal spike-in controls, then against the geometric mean of the reference genes *RPS* and *TBP*. The 12L:12D and 16L:8D experiments of Fig. 1 were analysed by qPCR, while L:L and D:D experiments were analysed by nCounter. All sequence data in this publication have been deposited in National Center for Biotechnology Information Gene Expression Omnibus (6).

## Immunoblot analysis

Proteins were extracted as previously described (7). Membranes were blocked with PBS-T 5% milk and incubated overnight at 4° C with α-HA (Covance) (1:2000) antibody and an α-D2 (1:10000) antibody used as loading control. Following incubation with HRP-conjugated 521 secondary antibodies (Promega), proteins were detected with Clarity reagents (Bio-Rad) and imaged with a G:Box camera (Syngene, UK).
## bHLH1a subcellular localization

bHLH1a protein sequence was scanned using two online tools for subcellular localization prediction: ELSpred (8) and DeepLoc (9). For microscopic analysis, chlorophyll autofluorescence and YFP fluorescence were excited at 510 nm and detected at 650-741 nm and 529-562 nm respectively. Hoechst 33342 (Life Technologies) was used at a final concentration of 5µg/ml to stain nuclear DNA and stained cells were visualized by illumination at 405 nm and detection at 424-462 nm.

## Generation of the *bHLH1a* transgenic lines

The full length *bHLH1a* coding sequence was obtained by PCR amplification with the specific oligonucleotides *PtbHLH1a-DraI-Fw* and *PtbHLH1a-XhoI-Rv* on cDNA template using the Phusion high fidelity DNA polymerase (Thermo Fisher, USA). The PCR fragment was inserted into the pENTR1A vector (Invitrogen, USA) using the DraI/XhoI restriction sites, and recombined with the pDEST-C-HA vector or the pDEST-C-YFP vector (10) both driving expression of the transgenes under the control of the *Light harvesting complex protein family F2* promoter (*Lhcf2p*). Transgenic lines were generated as in (11). Transformed cells were tested for the presence of the transgene by PCR and qRT-PCR analysis (see Table S5 for oligonucleotide sequences).

## Selection of rhythmic transcripts and clustering analysis

For the selection of genes with rhythmic expression in the light-dark cycle, we used microarray data from (12). First, we identified all the genes belonging to the TFs, photoreceptors, cell cycle and metabolism-related categories (pigment synthesis and photosynthesis). Then, transcripts were ranked based on a defined t-value for each time point (mean gene expression of the replicate/(1+s.d.)) and those showing t-value >+0.7 or <-0.7 across the time series, were retained. The nCounter expression data was normalized against the maximum expression value of each gene, in a similar way to qRT-PCR expression data. This normalization was applied to the 3 replicates independently and for each condition (L:D and D:D) with the average expression value used for the clustering analysis. Hierarchical clustering analysis was performed with MeV 4.9 (13) using Pearson correlation. Peak analysis was performed using the MFourfit curve-fitting method defining average expression phases for each cluster (https://biodare2.ed.ac.uk, (2)).

For the selection of rhythmic transcripts in D:D, expression values were normalized using *RPS*, *TBP* and *ACTIN12* as reference genes. The genes with the highest values of standard
deviation from the average expression over the two time courses (16L:8D and D:D) were
selected. A threshold equal to 1 was set using the published *P. tricornutum* diurnal microarray
dataset (12) as background. Gene expression profiles were further empirically examined and
false positives eliminated.

Data mining, protein sequence and phylogenetic analysis

The *P. tricornutum* bHLH1a (Phatr3_J44962) protein sequence was used as the query for BlastP analyses on the JGI, NCBI and MMETSP public database (14). The Pfam database was also searched for proteins possessing both the HLH and PAS domains. The retrieved sequences were analyzed using the batch search tool on the CDD (Conserved Domain Database) NCBI server to retrieve proteins presenting at least one HLH and one PAS domain only. We identified 100 HLH-PAS proteins from 71 marine algal species which were aligned using MAFFT (15), along with 22 HLH-PAS proteins from relevant metazoan (*Homo sapiens*, *Mus musculus* and *Drosophila melanogaster*) and unicellular Opisthokonta (*Monosiga brevicollis* and *Capsaspora owczarzaki*). As previously reported (16), no bHLH-PAS proteins were identified in Archaeoplastida organisms, with the exception of the Rhodophyta alga *Galdieria sulphuraria*. Preliminary phylogenies were produced with MEGA 7 (17) to eliminate ambiguously aligned sequences, refining the alignment to 107 sequences and a final length of 198 aa (<5% gap per position). The best amino acid model to fit the data was estimated with ProtTest 3.4.2 (18). Phylogenetic analyses were performed with RAxML (1000 bootstraps) and MrBayes 3.2.6 (2.5 million generations, 2 runs, 25% burn-in) on the CIPRESS gateway (19). The final tree was edited in Figure Tree 1.4 (http://tree.bio.ed.ac.uk/software/Figure_tree/). GenBank accession codes of the genes utilized in the bHLH-PAS phylogenetic analysis are reported in Table S4.

Cell cycle analysis

*P. tricornutum* Wt and transgenic cells were synchronized in the G1 phase with prolonged
darkness for 40h. After re-illumination, the FL3-A parameter was measured in cultures on an
hourly basis for 12h using a flow cytometer (Miltenyi Biotec, Germany). At the same time,
samples were harvested for cell cycle analysis. Cells were pelleted by centrifugation (4000 rpm,
15 minutes, 4°C), fixed in 70% EtOH and stored in the dark at 4°C until processing. Fixed cells
were then washed three times with PBS, stained with 4',6-diamidino-2-phenylindole (at a final
concentration of 1 ng/ml) on ice for 30', then washed and resuspended in PBS. After staining,
samples were immediately analyzed with a MACSQuant Analyser flow cytometer (Miltenyi
Biotec, Germany). For each sample 30,000 cells were analysed and G1 and G2 proportions
were inferred by calculating the 2c and 4c peak areas at 450 nm (V1-A channel) using the R
software. A peak calling method was applied to the resulting histogram, based on a 1st derivative
approach (20). The locations of G1 and G2 peaks were first determined using G1 and G2
reference samples and then used to identify G1 and G2 cells in the experimental samples. The
area under each peak was used as a proxy for the proportion of cells in each population.
<figure><img src="image_27.png" /><figcaption>**Fig. S1.** nCounter expression analysis of genes maintaining rhythmic expression in D:D condition and 16L:8D condition in Wt cells. Data represent the average expression of biological triplicates ±SD and are normalized using the *RPS*, *TBP* and *ACTIN12* reference genes. Expression values are given relative to the maximum expression for each gene, where '1' represents the highest expression value of the time series. Results for cells grown in 16L:8D cycle are shown in orange (L:D); results for cells in constant darkness (following 16L:8D adaptation) are shown in grey (D:D). For the *bHLH1a* and *bHLH1b* profiles in D:D, the asterisks on the last time points indicate that the expression at DD32 is significantly decreased with respect to the peak of expression of each gene (DD24 for *bHLH1a* and DD28 for *bHLH1b*) (*P*<0.05, t-test).</figcaption></figure>
<figure><img src="image_28.png" /><figcaption>Fig. S2. nCounter expression analysis of representative genes with altered rhythmic expression in Wt cells in D:D condition compared to 16L:8D condition. Expression values represent the average of three biological triplicates ±SD and are normalized using the RPS, TBP and ACTIN12 reference genes. Expression values are given relative to the maximum expression for each gene, where ‘1’ represents the highest expression value of the time series. Results for cells grown in 16L:8D cycle are shown in orange (L:D); results for cells in constant darkness (following 16L:8D adaptation) are shown in grey (D:D).</figcaption></figure>
<figure><img src="image_29.png" /><figcaption>Fig. S3. Diel expression patterns of *bHLH1a* and *bHLH1b* genes under Fe-depletion conditions in 12L:12D photoperiods. Diel expression patterns of *bHLH1a* and *bHLH1b* in normal (400 pM Fe') and iron depletion conditions (40 and 20 pM Fe') were obtained using transcriptome data extracted from (21). Light and dark periods are represented by white and black rectangles. Expression values are given relative to the maximum expression for each gene, where '1' represents the highest expression value of the time series.</figcaption></figure>
A

Wt OE1

<figure><img src="image_30.png" /></figure>

B

Wt OE1

<figure><img src="image_31.png" /></figure>

**Fig. S4. Analysis of selected rhythmic gene expression profiles in L:D and D:D in Wt and bHLH1a OE1 cells.** Cells were entrained in 16L:8D cycles, then kept in L:D or transferred to D:D and collected every 3 hours for 24 hours. Expression values represent the average of three biological replicates (n=3) ± s.e.m (black bars) and have been normalized using *RPS* and *TBP* genes. A: qRT-PCR analysis; B: nCounter analysis.
<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

**Fig. S5. Cell cycle progression dynamics of Wt *P. tricornutum*.** A-B) Progression of chlorophyll fluorescence (FL3-A parameter) and cell size (FWS-A parameter) measured by flow cytometry each hour over 12h of illumination following dark synchronization. C) Proportion of cells in the G2 phase measured by flow cytometry each hour over 12h of illumination following dark synchronization. D) Hourly measures of cell density in re-illuminated cultures. The trend curve represents the third-degree polynomial regression. Results are representative of three biological replicates ± s.e.m (black bars).
<figure><img src="image_36.png" /></figure>

Fig. S6 Diurnal oscillations of chlorophyll fluorescence (FL3-A parameter) in Wt and *bHLH1a* OE lines. Lowess fitted curves of the mean FL3-A values in Wt and OE lines entrained under 16L:8D over 3 days (n ≥ 8).
<figure><img src="image_37.png" /></figure>

**Fig. S7. Analysis of *bHLH1a* gene expression in continuous blue light.** Cells were entrained in 16L:8D cycles, then transferred to constant blue light (L:L) and sampled every 4 hours for 28 hours. *bHLH1a* gene expression was analysed by qRT-PCR starting from L:L33. Expression values represent the average of two biological replicates (n=2) ± s.e.m (black bars) and have been normalized using *RPS* and *TBP* genes. Dotted region represents the subjective night in L:L.
<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>

<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /></figure>

<figure><img src="image_43.png" /></figure>

<figure><img src="image_44.png" /></figure>

<figure><img src="image_45.png" /><figcaption>Fig. S8. Circadian oscillation of chlorophyll fluorescence in Wt and *bHLH1a* OE lines. A)</figcaption></figure>

Solid lines represent the average diurnal FL3-A fluorescence oscillation under continuous blue light (L:L) of Wt and OE lines replicates reported in Table S3 (n=15). Colored ribbons represent the standard deviation. B) Phase, C) period, D) relative amplitude error and E) amplitude of the FL3-A oscillations in Wt and OE lines (mean ± s.e.m., n=13 to 15, two tailed t-test between transgenic lines and *Wt*: *P*<0.05, **P*<0.01, ***P*<0.001). The OE1 line also showed shorter average periods than the Wt, although it lacked a significant difference most likely due to the high variability introduced by the replicates with high relative amplitude error (RAE) (Table S3).
<figure><img src="image_46.png" /><figcaption>Fig. S9. Analysis of rhythmicity in Wt and *bHLH1a* OE lines under blue L:L. Relative amplitude error plotted against A) Period and B) Phase estimate for individual cell cultures growing in L:L (n=13 to 15, Table S3). Multiple dots of each color indicate independent replicate cultures.</figcaption></figure>
<figure><img src="image_47.png" /><figcaption>Fig. S10. Growth rate of Wt and *bHLH1a* OE lines grown in blue L:L. Cells were grown in 16L:8D conditions, then transferred to continuous blue light (L:L) and followed during five subjective days. Growth rates were calculated over four days. Results represent the average of three biological replicates ± s.e.m (black bars). *p* values were calculated by comparing Wt to the indicated OE lines via two tailed t-test. No significant difference among lines was detected.</figcaption></figure>
<figure><img src="image_48.png" /><figcaption>Fig. S11. Circadian oscillation of chlorophyll fluorescence in Wt and a transgenic control line (pNAT) under blue (L:L). Cells were grown in 16L:8D conditions, then transferred to continuous blue light (L:L) and followed during four subjective days (n=8). Colors in dots represent independent replicate cultures. White bars represent subjective days and dotted bars represent subjective nights. Brown lines in plots represent the fitted curves (lowest fit) of the average FL3-A fluorescence.</figcaption></figure>
<table><caption>Table S1: Accession codes and sequence probes used in the nCounter analysis.</caption><thead><tr><th>Category</th><th>Gene Name</th><th>NCBI Accession</th><th>Phatr3 Accession</th><th>nCounter Probe Target Sequence</th></tr></thead><tbody><tr><td>Reference Gene</td><td>Actin12</td><td>XM_002182185.1</td><td>Phatr3_j29136</td><td>ATGTAGCCTGCAGCCACTGAGGACTTGTGCTCGTAACCCTGATTTTCGATATTCCAAAATGCGCTCACTAAAAAGACATCGTAGTCCAGTGCCAGTCCC</td></tr><tr><td>Photoreceptor</td><td>Aureo1b</td><td>#N/A</td><td>Phatr3_j49458</td><td>TTATTGCCGAGTGCTGTACCTCGAGTCCGTTGTTTGAAGAAATGGACGGCATAGATCAAACGAAAGGGGCCAATCTGGAACGTGCCGATTTTTCATTGAT</td></tr><tr><td>Photoreceptor</td><td>Aureo2</td><td>XM_002183279.1</td><td>Phatr3_j15468</td><td>AGACGTTACTGTCACTCTCATGAACTACACTGCCGATGGTACACCATTTTGGAAACAGCTCTTTATTGCCGCATTGCGTGACGCGCAAATAAACATTGTC</td></tr><tr><td>TF</td><td>bHLH1a_PAS</td><td>XM_002179032.1</td><td>Phatr3_j44962</td><td>TACTTTGGTCAATATCAAAGTCAGTTTGGTACGAACAGCTCAGCATAGCCCTCGGTTTTTCAATGTGGCGTTGGTGCCATCAGACGATGCAGCGAAGCTG</td></tr><tr><td>TF</td><td>bHLH1b_PAS</td><td>XM_002178812.1</td><td>Phatr3_j44963</td><td>AGGAATTGACTACCGTGCTGTTTTCAATCATTGTCCATATGCCATGGGGTGGCTTCATTGGACGGAAGAATACTGGTCTGCAAACAGCTCTTTGCAATCT</td></tr><tr><td>TF</td><td>bHLH3</td><td>XM_002176560.1</td><td>Phatr3_j42586</td><td>ATCACTAGCGACACATTCAGATTCATCTGCGGGAGAAGGACGGTCTATTCGTCAGTAGTTGGTACAAGTGTTAGCGGTCAAGTGCCTCTGGGAACTGC</td></tr><tr><td>TF</td><td>bHLH5</td><td>XM_002177858.1</td><td>Phatr3_j43365</td><td>ACCCTCTTGAGCGAGCAATCATTCGCTCCCGCATACGACTATGGGTCGATTTCTCAATGAACGCCATTCTTTTCCTCAGACCTTTCGCTCTCGCTGGCGT</td></tr><tr><td>Cell cycle</td><td>BUB1/MAD3</td><td>XM_002178639.1</td><td>Phatr3_j10954</td><td>CGTGTGCTGCATGTATGCGGACAAAACCGATCGTCCACTGGAAGTTTTCCAGCATCTACATCAGCAAAGGATCGGGAGCGATATTGCCGTGTTCTGGGATG</td></tr><tr><td>TF</td><td>bZIP10</td><td>XM_002177776.1</td><td>Phatr3_j43744</td><td>AAGCTGCTCGGGAATCGCGTCGGCGGAAGAGGTTATGATTGAAGAACTTCAACGCGAGCGTGATTTCTTCTCGCGTGCCAACGGAACCCTCAAAACAACA</td></tr><tr><td>TF</td><td>bZIP13</td><td>XM_002181635.1</td><td>Phatr3_j47278</td><td>CGCGGCGGCTTCGCGTGCTAAGATTCGATGCCGCATCACCGAACTTGAAACCGAGGTTAGTGTTGGGAAGGATAAATACACACAAGCAATGGAACGCTTG</td></tr><tr><td>TF</td><td>bZIP14</td><td>XM_002179477.1</td><td>Phatr3_EG02108</td><td>ATGCAGAGCTTGAGGGTCGAGCTACGGATCTCAAGCATGAGCAAATTCGTTTAAAAACAGATCATTAAACGAAAGAAGAACACGGCGAACATTCCTGGTGGGGC</td></tr><tr><td>TF</td><td>bZIP16</td><td>XM_002181462.1</td><td>Phatr3_j47279</td><td>CTTTTGAAGCAGTCACAAAATCCGCAAACATGAACAGTTCCAACGCCAGGAGAAGTCGCTCGTCAACTGAACGAAGATCCTGCGCTCCTGCGGAAGCGTCG</td></tr><tr><td>TF</td><td>bZIP18</td><td>XM_002176556.1</td><td>Phatr3_j42577</td><td>ACTCTTCTATAGCGGAATCCGAGGCCGATTTCAAAACTATAGCTCAGGCTGCTGTTTCGAATCTGATCATGTCGGCTGGAACTACCAAGGTTGAATCTGG</td></tr><tr><td>TF</td><td>bZIP19</td><td>XM_002183101.1</td><td>Phatr3_j48701</td><td>CCTGCACCTTCTCAAATCAGCTGTGCAAACGAGTGCAAACAAGAAGAAATATTGGCTGCCGAGGCATTACCTACACCTTTTATAGCGGTTTTCGCGATTGGC</td></tr><tr><td>TF</td><td>bZIP24</td><td>XM_002184633.1</td><td>Phatr3_j49887</td><td>TTGTCACGGGGCAACAGACTATCTGGAAAGATCACTTCTTGCCCAAAGAGAAGCGGAAAGAAACTCGTTCGGCTTTGTCAAGCATCGCGCTTCGGGGGAA</td></tr><tr><td>TF</td><td>bZIP25</td><td>XM_002185584.1</td><td>Phatr3_j46647</td><td>TCGTCGTTCACGATGGCATCTGTACCAACTCACCTAGTAGGAAATACTGTCAATCACTGATACCAGTTATCAGCCAGTTTCGTCGCTATTACCTGCAGCGG</td></tr><tr><td>TF</td><td>bZIP26</td><td>XM_002182641.1</td><td>Phatr3_EG02494</td><td>GATTTCTTGACGACTCAAGGAGCGACAACTTGTCAATCGACATCTCAGGAGCTTTCGGTGCAGACAGGAAAAGGTCAAGCCGCTTTGAAATCGAAACCTC</td></tr><tr><td>TF</td><td>bZIP5_PAS</td><td>XM_002179145.1</td><td>Phatr3_j45142</td><td>CCCTTGTTCCGAACAGCGCATTCGATCCGCAAACAAACGCTAGCAACAACGCAACAATCACAGTCGCAAATGATGCAGAACGTGGCTATTATGAGTG</td></tr><tr><td>TF</td><td>bZIP6_PAS</td><td>XM_002184884.1</td><td>Phatr3_j50039</td><td>CGCGCAATCTAATACACAATATCAGCTAGACAGCCCACAACATCCCGAAAATACGATGTCGTCAGTGTAGACCGCAGCAGGTTCAATAATTCGCGAAC</td></tr><tr><td>TF</td><td>bZIP7_PAS</td><td>XM_002183297.1</td><td>Phatr3_j48800</td><td>CGTGAGACACCTTGCCATCGTAATCAAGGATTAGAGGCATTAGCGCGGATTGCGACAGCAAGCTCGCTCTTACGGGCACACGTTTAGTACCAACACGAAT</td></tr><tr><td>Metabolism</td><td>CaThioredoxin</td><td>XM_002184396.1</td><td>Phatr3_j49634</td><td>CTGGAGAGCCCTACCGTGCTAGTGTCCGTGTATTGGAAAAATGTCGTTGTTTTCGCGTTCACGCTAGAAGACATTCCTGACAGCTCATTCTGAGTCTGAA</td></tr><tr><td>TF</td><td>CCCH4</td><td>XM_002181104.1</td><td>Phatr3_j13664</td><td>CATGTGCCGCCTTGGAGCGTCGCACATTAACATGTCTACTGGCGAAAACATTCGAAAGGACATTGATGGTCCGAAGCCCAAGCCAATCTTTAAATACTCTA</td></tr><tr><td>TF</td><td>CCCH8</td><td>XM_002186091.1</td><td>Phatr3_j44042</td><td>TCCGAAAAATATGTGCGAAGAGTCCCACTTGTGCGTCGATGTTCTTGGCTCATACGAGTATTGCGCCGACGGAAGTGGAAAGAGATTGAGTTTCGGCGTGA</td></tr><tr><td>TF</td><td>CCHH1_CCHC20</td><td>XM_002179821.1</td><td>Phatr3_EG02317</td><td>TAACTACAACACGTTCCTCAGCTAAACGGTTTGAACCATCTGCCTTCTAAGAAACCTATCCGACTTACATTCCCACTATCAAGATCATAGGAGTCCAAAC</td></tr><tr><td>TF</td><td>CCHH14</td><td>#N/A</td><td>Phatr3_j34600</td><td>GCCCAAGTTCTGCGGAGGATCCCGTTCAATCCGCGATCTGAGTTACGAAACGCACTACATGGCGTACCAAAAAGTTTACTGATGCCCGAGTTGTC</td></tr><tr><td>TF</td><td>CCT3</td><td>XM_002185983.1</td><td>Phatr3_j43850</td><td>AAACGCTGCCGCCGCGTTTGAATAAGAAGATTCGCTACGGTTGCCGTAAAAAATTTTAGCAGACCGCCGTTGCGCGTGAAAGGGCGATTGCGTGAAACGTT</td></tr><tr><td>TF</td><td>CCT4</td><td>XM_002178133.1</td><td>Phatr3_j44285</td><td>TATGAATGTCAGTCACCGGGAATTGAGTTTCCATCAAACAACAGAGCTAAGCTGGTCGAGCACACCAGCACGGCCTTTCGCCCATTTCTTTCACGATAGTG</td></tr><tr><td>Cell cycle</td><td>cdc20</td><td>XM_002180546.1</td><td>Phatr3_j12783</td><td>TTACGACGGAATGCTTCTTGAAGAAATCAGAACACTTCATGGGCACACAGGCCGAATATCGTCGTTGGGATGGAACCAACACTGGTTGAGTTCAGGCGCA</td></tr><tr><td>Photoreceptor</td><td>Cpd2</td><td>XM_002179343.1</td><td>Phatr3_j54342</td><td>TCTGGTGTGCGGCAATACGCGGACAGTGTGTTTCGGAGCTCTGTAAAAATTGCTTCTGAGATTGGCGCGAGTGCAGTCTATTGGAATCGCGAGATGACACCT</td></tr><tr><td>Photoreceptor</td><td>Cpd3</td><td>XM_002180035.1</td><td>Phatr3_j51952</td><td>CTCTGATTTTCTCCGATACGCGAATACCGTCAATGGATGGAACTCCAGGCCGTACCTATCTTGAGGAAGGCAAGGTCCCGTTTTATCAGGTTGATGCC</td></tr><tr><td>Photoreceptor</td><td>Cpf1</td><td>XM_002180059.1</td><td>Phatr3_j27429</td><td>GGATGCGTGCCAAAAATCCGGAATCACTCCTTCCGATTTACGTAGTCGACCCTGAATTTCCCTTCGCGCAAACTGCTGGTGGCCGCGCCGGTACAAATTCGT</td></tr><tr><td>Photoreceptor</td><td>Cpf4</td><td>XM_002184521.1</td><td>Phatr3_j55091</td><td>CTCGATCCCTTGTTGCGCGCGCAAACGAGACTGCATTTCCTCGTAATGGGTCTACCTAATGATTTTTGTAGACTCCATTGTCGAGGCAGCGTTTGAAGCAGCTG</td></tr><tr><td>Photoreceptor</td><td>CryP-like</td><td>XM_002178853.1</td><td>Phatr3_j34592</td><td>CACTTTATGGTGGCGAAACAGCCGGACTCGCACGATTGTTTGAAGGATTACTTCGAAACGCGCAACGGGATGCTGGGCCGAACTATTCGACCAATTCA</td></tr><tr><td>TF</td><td>CSF2</td><td>XM_002185522.1</td><td>Phatr3_j41601</td><td>GAACAGAACGAAAAGATTCGGGACGCTTATGCATTCCAACAGACGAAAATCGACGAGGCTCATCAACTTATTGTCAATTTGGGCATGGACGTTGACAGACT</td></tr><tr><td>Cell cycle</td><td>CYCB1</td><td>XM_002180361.1</td><td>Phatr3_j46095</td><td>TTAGAGAAAAGTCAATCGCAAATCCTGAGCTCCGAGCGGTGAATAAAAAAGTACAGCGGTCATCGATACGCGGAGTTGCTTCGACCGTTCTGGTATTTGA</td></tr><tr><td>Cell cycle</td><td>CYCH1</td><td>XM_002185709.1</td><td>Phatr3_j36892</td><td>TTTGCGATGCTCGCGTTTGACGTTTCGTTTCCCATCCGCACCGAGGAGTTGCCGCAATCGTCAACCATCTCGTGGATTGCTCACCTCAATGGTTTGGC</td></tr><tr><td>Photoreceptor</td><td>Dph</td><td>XM_002179026.1</td><td>Phatr3_j54330</td><td>CAACAATTCCATTACAACCAAAGAGCTGACGGAATGTGATCGTGAGCCTGTGCACTTGATCGCAAACGTACAAGGGGGTACCGGCCATTGTTGTTTCATT</td></tr><tr><td>Cell cycle</td><td>dsCYC2</td><td>XM_002179247.1</td><td>Phatr3_j34956</td><td>GATATTGCCGTTACCGAACGCGAATAATGACTGCTCTTTCCCTGGCACTTGCATCCTCCAACCGCTATTGGCTTTTCACGAATGTACTGGTCTCTGCTGG</td></tr><tr><td>TF</td><td>E2F_Dp2b</td><td>XM_002181452.1</td><td>Phatr3_EG02016</td><td>TGATTGAATAGAGCTGTGCAGGAAATGAGGGTACAAAAGCGCCGGATTTATGACATTACCAACGTTTTGGAAGGTATTGGGCTGATTACCAAGGATAGC</td></tr><tr><td>Cell cycle</td><td>E2F1</td><td>XM_002176842.1</td><td>Phatr3_j43065</td><td>GTGTGGGTCTCATCGAAAACGTACCAAAAACACAGTCGCTTGGAAGGGAGTGAGCTTCTTCTTGGCTCGTCCTTTTCAAGTGCCGCCAAGCAGAGAAT</td></tr><tr><td>Cell cycle</td><td>FtsZ</td><td>XM_002185088.1</td><td>Phatr3_j42361</td><td>GAAAAGGAGAATGAAGCAAGCGATTGCCGCGATTGAGAGGTTGCGAGAGAATGTGGATACGGTCATCGTAGTGTCCAACGACAGACTGCTCGAGATTAT</td></tr><tr><td>Metabolism</td><td>GapC1</td><td>XM_002182255.1</td><td>Phatr3_j22122</td><td>TGAAGGGATTCCTCGGATACTCCGACGAACCGTTGGTCTCCACCGACTTTGAAGGTGACTTGCGCTCCTCCATCTTTGATGCCGATGCCGGTATCATGCT</td></tr></tbody></table>
<table>
  <tbody>
    <tr>
      <td>Metabolism</td>
      <td>Gsat</td>
      <td>XM_002180931.1</td>
      <td>Phatr3_J36347</td>
      <td>ATTCGTTGGTCCGCGCACACACGGGTCGGGAAAGGTCATCAAGTTCGAAGGATGCTACCACGGACACGCCGATTCATTTTTGGTCCAAGCCGGATCCG</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>Hox1</td>
      <td>XM_002184935.1</td>
      <td>Phatr3_EG02213</td>
      <td>AACAAACATCGGTTCACATACTGCGTCATACCGGACAAGTCCCGACAGTCGAAGATGTTACGATCCTTACCAATGTTCCGGCGGAACGCATTCTCCGAAC</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>HSF1.3b</td>
      <td>XM_002179894.1</td>
      <td>Phatr3_J35419</td>
      <td>CTCCAGGGAACAACTCGATACCAGAGTTCCTCTATCAGCTAACAAAGATGCTGACGGATAACACCGAGATATTATTGAATGGACAAATGCAAGATTGA</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>HSF1a</td>
      <td>XM_002177362.1</td>
      <td>Phatr3_J43051</td>
      <td>CCACCAACGTCGGATAGCTCCGACTGGGGTTCTCCAATCCAATCATCCAAGCCAGCTGTTGATCTCTTAACAGGAGTATGCCGCAATGGACAAATATAC</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>HSF1b</td>
      <td>XM_002181395.1</td>
      <td>Phatr3_J47181</td>
      <td>TTGTAGTGGACACTTCGATGCTCGAAGAGGTCATCAACGAAACGCAGCCACTCGTTGGAGACAAAGGTAGCTTCTCCCTCTTGACAAATAGAGCACCACTG</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>HSF1d</td>
      <td>XM_002187673.1</td>
      <td>Phatr3_J44750</td>
      <td>GTCACCATTCCAGCATCGGATGCGGCCTCTTACGCCATTTCTCCGAAACCTCACCGAGAGACTTGACTCTGAGACACAAACGAAATTCCCAACCTTGATG</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>HSF1g</td>
      <td>XM_002177016.1</td>
      <td>Phatr3_J42514</td>
      <td>ATAGAGAATCTGCAAGCACTAGGGACCATGACGACTACTTCATCGAAGCGTAGCATTACAGAGCGAAGATAGTTCCTGTCAGACTCAGCATCAGGAG</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>HSF2.1a</td>
      <td>XM_002181681.1</td>
      <td>Phatr3_J47360</td>
      <td>AGCCCTTGCCGCTGGAATTTTGGGCTCGTTGTCGCCTCAAGTCAACCAACCACAGCGTGGATGATCCAACACGCCCTGTCATCAATCGCTCAAGCGTTAGTT</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>HSF2.2c</td>
      <td>XM_002185405.1</td>
      <td>Phatr3_J50481</td>
      <td>GGACTGGAAACTGTAAGCGACGATATATTCGCCAGCGCCTTCTAGCGATTTTGAGCGAATCATCCCTATCTGACATTATCACTTGGCTTCACCATGGACG</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>HSF3.2b</td>
      <td>XM_002186124.1</td>
      <td>Phatr3_J44099</td>
      <td>TTGACTCCTCGCGACCAGCAAGCACTTAGCGGTTTCAACAGTCCCTCGGTGCATCCGAGAGTCAGTTCAAGTCTATGAGCTTTTCCACCAACTCCTC</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>HSF3.2e</td>
      <td>XM_002179404.1</td>
      <td>Phatr3_J45206</td>
      <td>TTGGAACGGGAACGCTTTCCTCAGATTAGCAATCCGCAACCTTCGCCTACTCCTCCAAGTTCGACGATGGTAAATACTTCGGACTTAGCAAGACTACTT</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>HSF3.3a</td>
      <td>XM_002179738.1</td>
      <td>Phatr3_J45393</td>
      <td>TCTGGGCAAATCTTCGTATGGAAGGTTTGGCGACGCCGAGTAGTCAGGGAGTTGGCGTAACCACAGGCAGTTTGCCGCTTCGAGACCAAGACGCTGTTA</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>HSF3.3f</td>
      <td>XM_002179735.1</td>
      <td>Phatr3_J45389</td>
      <td>CATGCCAATTGAACACCCAATTCAGCCATACATGGAAGAGCTTAAGAGGGTCCATCTTGCAGCGCGGCGGGAAGTCTGCAAATGCGCTTCGAATTTTG</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>HSF4.2c</td>
      <td>XM_002182762.1</td>
      <td>Phatr3_J48361</td>
      <td>CGCACGTATATCACGATTTCCGTCACAAGTGGCGCCCGACGCGGATCCCATGATGATCGTCCGAAAAAAAGACTGGAGGAGTCACTCAGCCATTTCCCGAAAA</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>HSF4.2j</td>
      <td>XM_002177548.1</td>
      <td>Phatr3_J43363</td>
      <td>GTCGGCGGACGGGCCTTATCATTCACAAGCCTGACAAGTCTTTAAGGATATAGTGCCGCTATATTTTCGCCAGTCGCGGCTGAGTTCATTCACAAGCGGC</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>HSF4.3a</td>
      <td>XM_002184368.1</td>
      <td>Phatr3_J49594</td>
      <td>CGCGAACACTCGGTTCCAATGTCGCGGAAGAACCTTCCAAGCGAACCTTTGTTTCAGCATATTTACCATACCATATGAACGACCTGAGCAGGTGGATG</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>HSF4.3b</td>
      <td>XM_002183012.1</td>
      <td>Phatr3_J48558</td>
      <td>CTGTCTTGAGCCTATCGCAGCAGTGGCCGCAATTCTACGCTATGCCAACTACTGAAGGTGGAGACAAAGACCAATTATTCCTGATCCAAATACGTGG</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>HSF4.3c</td>
      <td>XM_002183081.1</td>
      <td>Phatr3_J48667</td>
      <td>AAACTGTCAAGTCATGCTTGACCATGTAAAAAGCCGCAGGATTGGACGACGTTATTCTTGGGCCAGTCATGGCCGCTGTTTTAGCATTCACAACCCAGACC</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>HSF4.4b</td>
      <td>XM_002184371.1</td>
      <td>Phatr3_J55070</td>
      <td>CCCATCCGGTAAACCGGTTTCCAACTTGGCGGTATCCAGAGCGCCTAGTTTTGGTAAGCTCGAACCAAGCCAGCCAATTCGCTGCTTATGGCGGTTGTATGA</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>HSF4.6a</td>
      <td>XM_002184347.1</td>
      <td>Phatr3_J49557</td>
      <td>CTCAAACACTGGCGATTGCCGTATCGCCTCGTTCGCCATCCAGTGCACGCCAAACATCGTGAATCCTTCGCACTTTGCGCTCTGTCTCCAATAACGGGCAC</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>HSF4.7a</td>
      <td>XM_002185271.1</td>
      <td>Phatr3_EG00092</td>
      <td>CGGACATCCCGGGGCGGATTTGAGATCGACGCGATAGTGTTCATCTTGCGTTCCAAGAGTATTCATCGCAACTCTACATTATCGGTGGTGACGGTACTCACC</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>HSF4.7b</td>
      <td>XM_002184277.1</td>
      <td>Phatr3_J49596</td>
      <td>CTCGAGGTGGTGTGGGAACTCCTTTTCCAACTAAGCTGCACGAAATGCTTCAAAATGTCGTACAAGACGGCTACGCTCACATTGTATCGTGGCAGCCTCA</td>
    </tr>
    <tr>
      <td>Metabolism</td>
      <td>Lhcx1</td>
      <td>XM_002179724.1</td>
      <td>Phatr3_J27278</td>
      <td>AATCCTTGAGAATCTTCAGGGTTAAAGAGTGCATCCATCCAAAAACTGGAGGTATGGCTGGTCACATGCACAGAGTTAACACCAATCCCTTTTCAAGCGA</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>Myb1R_SHAQKYF4</td>
      <td>XM_002178425.1</td>
      <td>Phatr3_J44331</td>
      <td>CGTATCTGCCGAATCGGTCTCTGGTGCAAATCAAGTCCCACGCCCAAAAAGTGCTCAAGCGCATCGATCAAGGCGAACACGTCCTTCCGACGTATCGAGGA</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>Myb1R_SHAQKYF5</td>
      <td>XM_002181623.1</td>
      <td>Phatr3_J47256</td>
      <td>ACGCTTCACCTGGCCCGAAGACTTACACGGGATTTGTATCGGCAATTTTTGATGTCGGCTTGAAACAGTCGTCACCGTCCACCATTTTGGAAACCATG</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>Myb1R_SHAQKYF6</td>
      <td>XM_002180835.1</td>
      <td>Phatr3_J46535</td>
      <td>TAACGACGAAGCGCAAATCCTTACCGACAAACTTGCCGTCCCGCAAAAAGGGTAAGACACGAAAGTCTGCCGCTTCCTGGTACAGCGTAATACTTCAGTTTC</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>Myb1R10</td>
      <td>XM_002181361.1</td>
      <td>Phatr3_J37257</td>
      <td>CATATCCCTCTTCGGGGCCCTCACTCCCTGTACAAGTGAATCTTTGGGGGTGAAAGCGAATCGTAGAAAGAAACGCTGAATGGACGAGACCTCGGTTCT</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>Myb1R4</td>
      <td>XM_002181410.1</td>
      <td>Phatr3_J47205</td>
      <td>AAAGTTCGTTGCAGGTTTAGACGGAACGTCTACCGCATTGTCAATGGGAACAATCCGGCGAGTAGGATTCACCCATATGCGATTGAACTTCTGGAAGCCG</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>Myb2R3a</td>
      <td>XM_002181911.1</td>
      <td>Phatr3_J47726</td>
      <td>TCCATCTTCCTTTGAGCGACTGTGACGACCTGTGGAGGGAGAGTGATTTTTGTCACTGTGAAGTTGTCGGTACTCCGGCACTCGGCAGGTAAAAATACTC</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>Myb2R3b</td>
      <td>XM_002182076.1</td>
      <td>Phatr3_EG02275</td>
      <td>GCGCACAAACGCGTTTAAAGTGATTCGGGATTCATTCAACAGTCAATGGCTAATTGGCCTCCATCGTTCACTGGTTGACATTTGCGATCGGCCCAACACT</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>Myb3R1</td>
      <td>XM_002182738.1</td>
      <td>Phatr3_J15016</td>
      <td>GTATGATCCTGGAGTGCATTTGACGTTGGGAAACCGCTGGGCCGAAATTGCCAAGCGTCTACCGGAAAGACTGATAACGCTATTAAAAAATCACTGGAA</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>Myb3R2</td>
      <td>XM_002180449.1</td>
      <td>Phatr3_J6839</td>
      <td>CGAATTCTCATAGGAGCACAGGACACAATGGGAAATCGATGGGCTGAGATTTGCCAAGCGCTGCCAGGACGTACGGACAATGCGATAAAGAATCGCTGGA</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>Myb3R4</td>
      <td>XM_002180741.1</td>
      <td>Phatr3_J36337</td>
      <td>GACAGATGCCGAAGACGCAATGTATAATGGATGCGGTCAATTCAGCTCGGAGCAACCACTTATCACTTGGTGGCAGACTTGGCGAACGTCTACCCGGACGT</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>Myb3R5</td>
      <td>XM_002184658.1</td>
      <td>Phatr3_J7959</td>
      <td>GACGTCGTTATCTGAATTGCTTGACCGAACAAATCCGAAAAGACGAATGGACGGAGCAAGAGAAAGAAGCTTTGTTGATGCACAGCGGCGCATGGGAAA</td>
    </tr>
    <tr>
      <td>TF</td>
      <td>Myb5R</td>
      <td>XM_002185245.1</td>
      <td>Phatr3_J50365</td>
      <td>TCAACACCACATAAACGCTCGTTTCAGCTTTGGATGTGTTAGTGAACGCGTTGGTTTTCATCGAGAACAAATGACCAATGAAGCAGACGAAGATGT</td>
    </tr>
    <tr>
      <td>Cell cycle</td>
      <td>PCNA</td>
      <td>XM_002182225.1</td>
      <td>Phatr3_J29196</td>
      <td>AATGCAGGAACCGTCGAACTCACCTTTGCCCTGCGCTACCTCAACTTTTTTACCAAGGCGACTCCGTTGAGTGGACACGTCATTATCTCCATGCACCG</td>
    </tr>
    <tr>
      <td>Metabolism</td>
      <td>Pds1</td>
      <td>XM_002180135.1</td>
      <td>Phatr3_J35509</td>
      <td>AGCATGGCCAAGGCACTCGATTTCATTGATCCCGACAAATTGAGTATGACGGTCGTTTTGACGCCCATGAATCGCTTTTTGAACGAAGACAACGCGCCTCC</td>
    </tr>
    <tr>
      <td>Metabolism</td>
      <td>petA</td>
      <td>GI:118411009</td>
      <td>#N/A</td>
      <td>TCTCTGGATCAGGAGCCATAACAGGGAAAATTAACTCTTGATGAGTCTTTCCTGCAATTGGTCCAACTACTAAGATATTATCAAATTCACTACTATACGG</td>
    </tr>
    <tr>
      <td>Metabolism</td>
      <td>Pgr5</td>
      <td>XM_002178672.1</td>
      <td>Phatr3_J44748</td>
      <td>GTAACGTCGCGCTTCTGATCGCTACCGCTGCCGCGTTTCGCGTGGCGGGCGGAGCCACCGGGGTTCTCCTTTGTGCTTCCCGACGAACCAGCATCG</td>
    </tr>
    <tr>
      <td>Metabolism</td>
      <td>PgrL</td>
      <td>XM_002177043.1</td>
      <td>Phatr3_J42543</td>
      <td>CCACAAACAGTAGCGTTTCCAATTAGACAAGACTACGAAACGATGCGAGTCAACTCTGTGGTATTTGGCCTTTGGTGTTCCTCGGCCATCGAGAT</td>
    </tr>
    <tr>
      <td>Metabolism</td>
      <td>Por1</td>
      <td>XM_002179653.1</td>
      <td>Phatr3_J12155</td>
      <td>TCGGTGGTGGAGATTTTTGAGAATCAGCAGTCCGACGCAGTACGGGATCTTCCTACCGCCAAGAAAATGTGGAAACTGAGTAGGGAAGCAGTTGGTCTTT</td>
    </tr>
    <tr>
      <td>Metabolism</td>
      <td>Por2</td>
      <td>XM_002180956.1</td>
      <td>Phatr3_J13001</td>
      <td>AATCACCACTATTCCGTGAAAAGCCCGTGGTTCCGTAAATACTTTCCCATCTTTATGAAGTTTATTACGGGAGATGACGTTGGAGAGCACGAAGCCGG</td>
    </tr>
    <tr>
      <td>Metabolism</td>
      <td>Por3</td>
      <td>XM_002177432.1</td>
      <td>Phatr3_J43164</td>
      <td>CGCGTTCTCAACACGAGGAAAAGCGGTGAGACTCATGATTCGTGATCCGACCAGTAGTCGTATCGAGGCACACCGTAGCGTGGGAGACGTAGACGTTCCC</td>
    </tr>
    <tr>
      <td>Metabolism</td>
      <td>psaA</td>
      <td>GI:118410964</td>
      <td>#N/A</td>
      <td>GCTAAGAAGAACGCCCAAGTAGTTCCAATACCACCAATAAATAATGAGCTAAACCAACTGCACGACCCTGAGTAATGCTTAATGCACGAGTTGGATTG</td>
    </tr>
    <tr>
      <td>Metabolism</td>
      <td>psaD</td>
      <td>GI:118410999</td>
      <td>#N/A</td>
      <td>TTAAAATGTACGTAATTGGTTCCCAATGCTAAACATTGCTCTTTGCGCGCTAAGTATAATAAATTTTCTCCACTACGCATGATAGCTGCTCCACCAATTG</td>
    </tr>
  </tbody>
</table>
<table><thead><tr><th>Metabolism</th><th>psbC</th><th>HQ912250.1</th><th>#N/A</th><th>GGCCCAACAGGTCCCTGAGGCTTCACAAGCACAAGCATTCACGTTTTTAGTTCGAGATCAACGTTTAGGTGCTAATGTTTCATCAGCACAGGGTCCAACTG</th></tr></thead><tbody><tr><td>Metabolism</td><td>Psy1</td><td>XM_002178740.1</td><td>Phatr3_EG02349</td><td>ACCTGTCGTGTCGGGAATTGCGATTGGAGCGTTTGTGGCAATACGGACAAGTACAGGATGTCTTTGACCTGTGCTTGCTAGATCTTCGAATCCAGTATCC</td></tr><tr><td>Reference Gene</td><td>Rps</td><td>XM_002178225.1</td><td>Phatr3_j10847</td><td>CTCCGCGCATTTTTGCCCGTTCCCATTTGACCGGACAGTTGCCCGACGAAGATCTCATTGGAAACAACTTGCACTTAAATTCCCTGAAGTCAACCAGG</td></tr><tr><td>TF</td><td>Sigma70.1a</td><td>XM_002182291.1</td><td>Phatr3_j14599</td><td>GACAATTAGGGGTATCTCGGGATCGGATTCGCCTTGTCGAACCCGAGCACTGAACAAATTGCGACACCCGCAACAAATTACAAGCTGCAGTCTTACGT</td></tr><tr><td>TF</td><td>Sigma70.1b</td><td>XM_002182124.1</td><td>Phatr3_j3388</td><td>TTCTGGTCATTGGAAGAAACGGCCAAACAACTAGGGATGTCTCGGGATCGGATTCGCCTTGTCGAAGCCCGAGCACTGAATAATTGCGACGTCCACAA</td></tr><tr><td>TF</td><td>Sigma70.2</td><td>XM_002178682.1</td><td>Phatr3_j5537</td><td>ACGTGGATTGCGATTCCGCCACCTACGCCACTTACTGGGTCACCAATTCCGTACGCTTCTGCTTCCAAACCGCATCCACCGGATGTTTGCGGGTACCGGTA</td></tr><tr><td>TF</td><td>Sigma70.3</td><td>XM_002185461.1</td><td>Phatr3_j17029</td><td>TATGATGGTTCCTGATCTAAAGAAGCAAATTCGTGCTCGCTTCGCGCCAAGGCTGCCCTGACGGAAATCAAATATTCGACTCGTGGTATCCATTGCGAAA</td></tr><tr><td>TF</td><td>Sigma70.4</td><td>XM_002177232.1</td><td>Phatr3_j9312</td><td>ACCCGAAAGATCTCGGTCACGAAAGTCACGATTTGCTAGGGGACACAATCTCCGCCAGTAGTGCTCTTTTTGACGAAAGCACACCCGAAAAACGAGTCG</td></tr><tr><td>TF</td><td>Sigma70.5</td><td>XM_002182854.1</td><td>Phatr3_j14908</td><td>AAGAACTCATACAGGAAGGCAGTTTGGGATTGTTGCGCGCGCCGAATTGTTTGATCCCGAGCGAGGACTGCGATTCAGCACTTACGCGGTAGTGTGGAT</td></tr><tr><td>TF</td><td>Sigma70.6</td><td>XM_002178042.1</td><td>Phatr3_j9855</td><td>CTGGAAAAATCAATATGGAAAGCTATTTCGGCAGACCATTGAAGAAGGACTGGAAGCCAAAAATCAGCTAGTCACTTCGAATCTGCGGATGGTACAGAGTGT</td></tr><tr><td>TF</td><td>Sigma70.7</td><td>XM_002185047.1</td><td>Phatr3_j50183</td><td>AATGATGACGCTCCTCTCGCATACGGTGGAACTCCGTCGTATCAAGCAAATCGAAGCTGATCTTGCTTTGACGTATCCATCTTCCCGGAAACCCACTACT</td></tr><tr><td>TF</td><td>Taz2</td><td>XM_002178935.1</td><td>Phatr3_j44807</td><td>CCTAATGTAGAGACTGTAGTGGTAAAAGCGAAGCGCGCAGCCTCGACTATTTGGACTTTACTTCATGCACAGAATTGCGTTTTGGGGACAGATGCCCAC</td></tr><tr><td>Reference Gene</td><td>Tbp</td><td>XM_002186285.1</td><td>Phatr3_j10199</td><td>CTGATTTTTCCAGCGGTAAAAATGTGCGTGACCGGAGTCAAGAGCACACACAACGCCAATCTGGCGCGGAAAAAGTTTGCCTACATTGTGGAGCGCGTCG</td></tr><tr><td>Metabolism</td><td>Vde</td><td>XM_002178607.1</td><td>Phatr3_j51703</td><td>GGCGATGACGGATCATATCCCGTTCCCGCACCCGAAATCGTCGTACCCAAGTTTGACACCAAGTTCTTCGATGGACGACTCTACATCTCAGCCGACAA</td></tr><tr><td>Metabolism</td><td>Vd1l</td><td>XM_002180599.1</td><td>Phatr3_j36048</td><td>AAGGCTCTCTTTAGCAATCCGCGTGGAAATCAAAGGTGTCTCCTGTTTGGGACGCTGCAAGGGCGAGCAATCGTGTGCGACGCGGTGTTTTGCCGAATTCG</td></tr><tr><td>Metabolism</td><td>Vd12</td><td>XM_002180015.1</td><td>Phatr3_j45846</td><td>ACACACCGCTTCGGCAATGAATATCCAATTCCGGAGCCAGACGAATCACTCCATGTCTGGGACCACGTACGAAACATCGGTAAAGTGTGTACCGGGTT</td></tr><tr><td>Metabolism</td><td>Vdr</td><td>XM_002177477.1</td><td>Phatr3_j43240</td><td>GTCGCTTGCGGTGCCAACGAAGCCTATGATAAGTTTCCTTCGCAGAACCAACTCTTATCCCGCTGCTCGCGGTCGAGATTTGTGGTACGATCCCGTTT</td></tr><tr><td>Metabolism</td><td>Zds</td><td>XM_002184417.1</td><td>Phatr3_j30514</td><td>CGGCGTCAACGAATCCATGCGGGACACCGCCAACGTACTCGCCCGTTTTAATTCACTCATTCCTCGCCCGAGTCTTTGGACACGCCGACGTCACGGAACTC</td></tr><tr><td>Metabolism</td><td>Zep1</td><td>XM_002179586.1</td><td>Phatr3_j45485</td><td>TTCCCAATGAAGTTTCTACCACGGTGTATCGCGACTGTTCCTGATCGCGTCGGTATCGACCACGACTTCCTTCACACCGTCCAGTCCTTTGGCGTC</td></tr><tr><td>Metabolism</td><td>Zep3</td><td>XM_002178331.1</td><td>Phatr3_j10970</td><td>CCAAGGTTTGACCAAGATGCCCACCATGGATGTTACCGTACTGGAAACAAACGTCCGAATTCAACGGTTCGGTGGACCCATCCAGCTCGCTAGTAACGC</td></tr></tbody></table>
<table><caption>**Table S2:** Calculated periods, phases, amplitudes and relative amplitude errors of the Wt and bHLH1a OE lines growing in 16L:8D. P-values obtained via t-test between Wt and the OE lines are shown. Amp., amplitude; RAE, Relative Amplitude Error.</caption><thead><tr><th>Data</th><th>Period</th><th>Period P value</th><th>Phase</th><th>Phase P value</th><th>Amp.</th><th>Amp. P value</th><th>RAE</th><th>RAE P value</th></tr></thead><tbody><tr><td>Wt</td><td>23.9</td><td></td><td>13.44</td><td></td><td>0.1</td><td></td><td>0.15</td><td></td></tr><tr><td>Wt</td><td>23.71</td><td></td><td>12.41</td><td></td><td>0.12</td><td></td><td>0.12</td><td></td></tr><tr><td>Wt</td><td>23.86</td><td></td><td>11.95</td><td></td><td>0.13</td><td></td><td>0.13</td><td></td></tr><tr><td>Wt</td><td>24.58</td><td></td><td>12.21</td><td></td><td>0.11</td><td></td><td>0.1</td><td></td></tr><tr><td>Wt</td><td>25.71</td><td></td><td>10.32</td><td></td><td>0.12</td><td></td><td>0.21</td><td></td></tr><tr><td>Wt</td><td>24.79</td><td></td><td>12.15</td><td></td><td>0.12</td><td></td><td>0.12</td><td></td></tr><tr><td>Wt</td><td>24.24</td><td></td><td>12.81</td><td></td><td>0.15</td><td></td><td>0.19</td><td></td></tr><tr><td>Wt</td><td>23.89</td><td></td><td>12.27</td><td></td><td>0.12</td><td></td><td>0.15</td><td></td></tr><tr><td>Wt</td><td>23.64</td><td></td><td>12.89</td><td></td><td>0.11</td><td></td><td>0.19</td><td></td></tr><tr><td>Wt average</td><td>24.26</td><td></td><td>12.27</td><td></td><td>0.12</td><td></td><td>0.15</td><td></td></tr><tr><td>OE1</td><td>23.87</td><td></td><td>15.24</td><td></td><td>0.07</td><td></td><td>0.24</td><td></td></tr><tr><td>OE1</td><td>24.53</td><td></td><td>16.17</td><td></td><td>0.07</td><td></td><td>0.23</td><td></td></tr><tr><td>OE1</td><td>23.29</td><td></td><td>16.53</td><td></td><td>0.06</td><td></td><td>0.37</td><td></td></tr><tr><td>OE1</td><td>23.18</td><td></td><td>18.16</td><td></td><td>0.07</td><td></td><td>0.52</td><td></td></tr><tr><td>OE1</td><td>24.17</td><td></td><td>15.23</td><td></td><td>0.08</td><td></td><td>0.28</td><td></td></tr><tr><td>OE1</td><td>24.67</td><td></td><td>12.19</td><td></td><td>0.14</td><td></td><td>0.09</td><td></td></tr><tr><td>OE1</td><td>24.41</td><td></td><td>12.14</td><td></td><td>0.12</td><td></td><td>0.09</td><td></td></tr><tr><td>OE1</td><td>23.77</td><td></td><td>13.32</td><td></td><td>0.09</td><td></td><td>0.16</td><td></td></tr><tr><td>OE1</td><td>23.64</td><td></td><td>12.63</td><td></td><td>0.08</td><td></td><td>0.22</td><td></td></tr><tr><td>OE1 average</td><td>23.95</td><td>0.2940</td><td>14.62</td><td>0.0078</td><td>0.09</td><td>0.0042</td><td>0.24</td><td>0.0653</td></tr><tr><td>OE2</td><td>23.65</td><td></td><td>15.47</td><td></td><td>0.11</td><td></td><td>0.19</td><td></td></tr><tr><td>OE2</td><td>24.04</td><td></td><td>14.8</td><td></td><td>0.11</td><td></td><td>0.21</td><td></td></tr><tr><td>OE2</td><td>24.23</td><td></td><td>14.78</td><td></td><td>0.11</td><td></td><td>0.22</td><td></td></tr><tr><td>OE2</td><td>24.18</td><td></td><td>14.98</td><td></td><td>0.11</td><td></td><td>0.23</td><td></td></tr><tr><td>OE2</td><td>23.34</td><td></td><td>14.22</td><td></td><td>0.09</td><td></td><td>0.21</td><td></td></tr><tr><td>OE2</td><td>22.82</td><td></td><td>15.05</td><td></td><td>0.08</td><td></td><td>0.24</td><td></td></tr><tr><td>OE2</td><td>21.91</td><td></td><td>14.14</td><td></td><td>0.08</td><td></td><td>0.32</td><td></td></tr><tr><td>OE2</td><td>22.42</td><td></td><td>13.71</td><td></td><td>0.07</td><td></td><td>0.38</td><td></td></tr><tr><td>OE2</td><td>23.6</td><td></td><td>12.24</td><td></td><td>0.08</td><td></td><td>0.33</td><td></td></tr><tr><td>OE2 average</td><td>23.35</td><td>0.0206</td><td>14.38</td><td>0.0002</td><td>0.09</td><td>0.0021</td><td>0.26</td><td>0.0007</td></tr><tr><td>OE3</td><td>24.14</td><td></td><td>14.05</td><td></td><td>0.13</td><td></td><td>0.13</td><td></td></tr><tr><td>OE3</td><td>24.49</td><td></td><td>13.73</td><td></td><td>0.12</td><td></td><td>0.18</td><td></td></tr><tr><td>OE3</td><td>24.55</td><td></td><td>12.29</td><td></td><td>0.11</td><td></td><td>0.21</td><td></td></tr><tr><td>OE3</td><td>24.31</td><td></td><td>11.32</td><td></td><td>0.1</td><td></td><td>0.25</td><td></td></tr><tr><td>OE3</td><td>24.43</td><td></td><td>11.19</td><td></td><td>0.11</td><td></td><td>0.23</td><td></td></tr><tr><td>OE3</td><td>23.74</td><td></td><td>11.96</td><td></td><td>0.11</td><td></td><td>0.17</td><td></td></tr><tr><td>OE3</td><td>23.74</td><td></td><td>11.97</td><td></td><td>0.12</td><td></td><td>0.17</td><td></td></tr><tr><td>OE3</td><td>24.77</td><td></td><td>12.31</td><td></td><td>0.11</td><td></td><td>0.21</td><td></td></tr><tr><td>OE3 average</td><td>24.17</td><td>0.9607</td><td>12.58</td><td>0.8640</td><td>0.11</td><td>0.3037</td><td>0.20</td><td>0.0364</td></tr></tbody></table>
<table><caption>**Table S3:** Calculated periods, phases, amplitudes and relative amplitude errors of the Wt and bHLH1a OE lines growing in L:L. P-values obtained via t-test between Wt and the OE lines are shown. Amp., amplitude; RAE, Relative Amplitude Error.</caption><thead><tr><th>Data</th><th>Period</th><th>Period P value</th><th>Phase</th><th>Phase P value</th><th>Amp.</th><th>Amp. P value</th><th>RAE</th><th>RAE P value</th></tr></thead><tbody><tr><td>Wt</td><td>25.99</td><td></td><td>22.33</td><td></td><td>2.41</td><td></td><td>0.38</td><td></td></tr><tr><td>Wt</td><td>25.24</td><td></td><td>20.04</td><td></td><td>1.68</td><td></td><td>0.35</td><td></td></tr><tr><td>Wt</td><td>24.7</td><td></td><td>16.92</td><td></td><td>1.69</td><td></td><td>0.36</td><td></td></tr><tr><td>Wt</td><td>25.81</td><td></td><td>22.1</td><td></td><td>2.43</td><td></td><td>0.19</td><td></td></tr><tr><td>Wt</td><td>24.54</td><td></td><td>21.18</td><td></td><td>2.55</td><td></td><td>0.18</td><td></td></tr><tr><td>Wt</td><td>23.83</td><td></td><td>19.07</td><td></td><td>2.14</td><td></td><td>0.21</td><td></td></tr><tr><td>Wt</td><td>28.19</td><td></td><td>21.4</td><td></td><td>1.51</td><td></td><td>0.44</td><td></td></tr><tr><td>Wt</td><td>27.27</td><td></td><td>22.62</td><td></td><td>1.59</td><td></td><td>0.39</td><td></td></tr><tr><td>Wt</td><td>27.29</td><td></td><td>21.93</td><td></td><td>1.37</td><td></td><td>0.31</td><td></td></tr><tr><td>Wt</td><td>28.1</td><td></td><td>21.28</td><td></td><td>1.88</td><td></td><td>0.18</td><td></td></tr><tr><td>Wt</td><td>28.18</td><td></td><td>21.11</td><td></td><td>1.59</td><td></td><td>0.26</td><td></td></tr><tr><td>Wt</td><td>27.25</td><td></td><td>19.18</td><td></td><td>1.21</td><td></td><td>0.35</td><td></td></tr><tr><td>Wt</td><td>27.78</td><td></td><td>19.1</td><td></td><td>1.52</td><td></td><td>0.17</td><td></td></tr><tr><td>Wt</td><td>27.97</td><td></td><td>21.6</td><td></td><td>1.4</td><td></td><td>0.21</td><td></td></tr><tr><td>Wt</td><td>28.44</td><td></td><td>21.69</td><td></td><td>2.12</td><td></td><td>0.23</td><td></td></tr><tr><td>Wt average</td><td>26.71</td><td></td><td>20.77</td><td></td><td>1.81</td><td></td><td>0.28</td><td></td></tr><tr><td>OE1</td><td colspan="8">FAILED Could not find cos waves that fit the data</td></tr><tr><td>OE1</td><td>30.3</td><td></td><td>2.89</td><td></td><td>0.44</td><td></td><td>0.85</td><td></td></tr><tr><td>OE1</td><td>29.04</td><td></td><td>5.7</td><td></td><td>0.46</td><td></td><td>0.93</td><td></td></tr><tr><td>OE1</td><td>20.62</td><td></td><td>11.08</td><td></td><td>0.94</td><td></td><td>0.68</td><td></td></tr><tr><td>OE1</td><td>20.72</td><td></td><td>7.28</td><td></td><td>1.12</td><td></td><td>0.8</td><td></td></tr><tr><td>OE1</td><td>20.54</td><td></td><td>12.1</td><td></td><td>1.1</td><td></td><td>0.64</td><td></td></tr><tr><td>OE1</td><td>20.48</td><td></td><td>12.48</td><td></td><td>1.55</td><td></td><td>0.41</td><td></td></tr><tr><td>OE1</td><td>20.53</td><td></td><td>13.2</td><td></td><td>1.33</td><td></td><td>0.41</td><td></td></tr><tr><td>OE1</td><td>21.05</td><td></td><td>10.95</td><td></td><td>1.09</td><td></td><td>0.43</td><td></td></tr><tr><td>OE1</td><td>22.85</td><td></td><td>9.96</td><td></td><td>1.01</td><td></td><td>0.38</td><td></td></tr><tr><td>OE1</td><td>22.57</td><td></td><td>7.02</td><td></td><td>1.04</td><td></td><td>0.35</td><td></td></tr><tr><td>OE1</td><td>22.1</td><td></td><td>6.82</td><td></td><td>0.61</td><td></td><td>0.4</td><td></td></tr><tr><td>OE1</td><td>36.12</td><td></td><td>11.2</td><td></td><td>0.72</td><td></td><td>0.97</td><td></td></tr><tr><td>OE1</td><td colspan="8">FAILED Could not find cos waves that fit the data</td></tr><tr><td>OE1</td><td>32.87</td><td></td><td>23.41</td><td></td><td>0.86</td><td></td><td>0.95</td><td></td></tr><tr><td>OE1 average</td><td>24.6</td><td>0.17</td><td>10.31</td><td>&lt; 0.0001</td><td>0.94</td><td>&lt; 0.0001</td><td>0.63</td><td>&lt; 0.0001</td></tr><tr><td>OE2</td><td>27.17</td><td></td><td>9.3</td><td></td><td>0.99</td><td></td><td>0.29</td><td></td></tr><tr><td>OE2</td><td>24.79</td><td></td><td>7.42</td><td></td><td>1.19</td><td></td><td>0.21</td><td></td></tr><tr><td>OE2</td><td>24.17</td><td></td><td>6.82</td><td></td><td>1.14</td><td></td><td>0.26</td><td></td></tr><tr><td>OE2</td><td>26.06</td><td></td><td>5.4</td><td></td><td>1.25</td><td></td><td>0.36</td><td></td></tr><tr><td>OE2</td><td>23.86</td><td></td><td>7.11</td><td></td><td>1.17</td><td></td><td>0.43</td><td></td></tr><tr><td>OE2</td><td>25.79</td><td></td><td>5.61</td><td></td><td>1.71</td><td></td><td>0.35</td><td></td></tr><tr><td>OE2</td><td>23.02</td><td></td><td>6.88</td><td></td><td>1.23</td><td></td><td>0.4</td><td></td></tr><tr><td>OE2</td><td>23.4</td><td></td><td>7.1</td><td></td><td>0.88</td><td></td><td>0.51</td><td></td></tr><tr><td>OE2</td><td>24.96</td><td></td><td>5.71</td><td></td><td>1.41</td><td></td><td>0.36</td><td></td></tr><tr><td>OE2</td><td>23.27</td><td></td><td>6.01</td><td></td><td>1.35</td><td></td><td>0.39</td><td></td></tr><tr><td>OE2</td><td>22.7</td><td></td><td>6.71</td><td></td><td>1.74</td><td></td><td>0.28</td><td></td></tr><tr><td>OE2</td><td>23.44</td><td></td><td>6.26</td><td></td><td>1.56</td><td></td><td>0.31</td><td></td></tr><tr><td>OE2</td><td>25.69</td><td></td><td>4.43</td><td></td><td>1.02</td><td></td><td>0.49</td><td></td></tr><tr><td>OE2</td><td>31.21</td><td></td><td>1.1</td><td></td><td>0.85</td><td></td><td>0.73</td><td></td></tr><tr><td>OE2</td><td>20.75</td><td></td><td>4.91</td><td></td><td>0.54</td><td></td><td>0.77</td><td></td></tr><tr><td>OE2 average</td><td>24.69</td><td>0.0108</td><td>6.05</td><td>&lt; 0.0001</td><td>1.2</td><td>0.0002</td><td>0.41</td><td>0.0118</td></tr><tr><td>OE3</td><td>8.28</td><td></td><td>1.3</td><td></td><td>0.55</td><td></td><td>0.86</td><td></td></tr><tr><td>OE3</td><td>8.23</td><td></td><td>2.07</td><td></td><td>0.46</td><td></td><td>0.9</td><td></td></tr><tr><td>OE3</td><td>28.39</td><td></td><td>6.07</td><td></td><td>0.72</td><td></td><td>0.59</td><td></td></tr><tr><td>OE3</td><td colspan="8">FAILED Could not find cos waves that fit the data</td></tr><tr><td>OE3</td><td>19.75</td><td></td><td>12.92</td><td></td><td>0.56</td><td></td><td>0.98</td><td></td></tr><tr><td>OE3</td><td>24.48</td><td></td><td>9.18</td><td></td><td>1.73</td><td></td><td>0.43</td><td></td></tr><tr><td>OE3</td><td>23.12</td><td></td><td>10.81</td><td></td><td>1.51</td><td></td><td>0.41</td><td></td></tr><tr><td>OE3</td><td>22.1</td><td></td><td>7.37</td><td></td><td>2.15</td><td></td><td>0.28</td><td></td></tr><tr><td>OE3</td><td>27.83</td><td></td><td>4.63</td><td></td><td>1.08</td><td></td><td>0.45</td><td></td></tr><tr><td>OE3</td><td>28.74</td><td></td><td>3.28</td><td></td><td>1.2</td><td></td><td>0.51</td><td></td></tr><tr><td>OE3</td><td>23.56</td><td></td><td>10.1</td><td></td><td>1.41</td><td></td><td>0.23</td><td></td></tr><tr><td>OE3</td><td>23.1</td><td></td><td>9.3</td><td></td><td>1.46</td><td></td><td>0.2</td><td></td></tr><tr><td>OE3</td><td>22.81</td><td></td><td>10.22</td><td></td><td>1.11</td><td></td><td>0.24</td><td></td></tr><tr><td>OE3</td><td>22.37</td><td></td><td>4.43</td><td></td><td>0.86</td><td></td><td>0.63</td><td></td></tr><tr><td>OE3</td><td>21.58</td><td></td><td>10.38</td><td></td><td>0.76</td><td></td><td>0.49</td><td></td></tr><tr><td>OE3 average</td><td>21.74</td><td>0.0062</td><td>7.29</td><td>&lt; 0.0001</td><td>1.11</td><td>0.0004</td><td>0.51</td><td>0.0024</td></tr></tbody></table>
<table><caption>**Table S4:** Accession numbers of the proteins utilized in the bHLH-PAS phylogenetic analysis.</caption><thead><tr><th>SPECIES</th><th>ABBREVIATION</th><th>ACCESSION</th><th>ACCESSION(Alt)</th></tr></thead><tbody><tr><td>Amphiprora sp., Strain CCMP467</td><td>Amph</td><td>CAMPEP_0168730192</td><td></td></tr><tr><td>Asterionellopsis glacialis, Strain CCMP134</td><td>Agla2</td><td>CAMPEP_0195290872</td><td></td></tr><tr><td>Asterionellopsis glacialis, Strain CCMP1581</td><td>Agla3</td><td>CAMPEP_0197142484</td><td></td></tr><tr><td>Astrosyne radiata, Strain 13vi08-1A</td><td>Arad1</td><td>CAMPEP_0116831938</td><td></td></tr><tr><td>Astrosyne radiata, Strain 13vi08-1A</td><td>Arad2</td><td>CAMPEP_0116837632</td><td></td></tr><tr><td>Attheya septentrionalis, Strain CCMP2084</td><td>Asep</td><td>CAMPEP_0198303966</td><td></td></tr><tr><td>Capsaspora owczarzaki ATCC 30864</td><td>Cowc1</td><td>XP_004345696</td><td></td></tr><tr><td>Capsaspora owczarzaki ATCC 30864</td><td>Cowc2</td><td>XP_004343694</td><td></td></tr><tr><td>Chaetoceros debilis, Strain MM31A-1</td><td>Cdeb</td><td>CAMPEP_0194099558</td><td></td></tr><tr><td>Chaetoceros dichaeta, Strain CCMP1751</td><td>Cdic</td><td>CAMPEP_0198277646</td><td></td></tr><tr><td>Chaetoceros neogracle, Strain CCMP1317</td><td>Cneo2</td><td>CAMPEP_0195415676</td><td></td></tr><tr><td>Chaetoceros neogracle, Strain CCMP1317</td><td>Cneo3</td><td>CAMPEP_0195453836</td><td></td></tr><tr><td>Chaetoceros sp., Strain GSL56</td><td>Chae</td><td>CAMPEP_0176495412</td><td></td></tr><tr><td>Corethron hystrix, Strain 308</td><td>Chys</td><td>CAMPEP_0113330172</td><td></td></tr><tr><td>Corethron pennatum, Strain L29A3</td><td>Cpen</td><td>CAMPEP_0194280324</td><td></td></tr><tr><td>Cyclophora tenuis, Strain ECT3854</td><td>Cten1</td><td>CAMPEP_0116540656</td><td></td></tr><tr><td>Cyclophora tenuis, Strain ECT3854</td><td>Cten2</td><td>CAMPEP_0116579710</td><td></td></tr><tr><td>Cyclotella meneghiniana, Strain CCMP 338</td><td>Cmen</td><td>CAMPEP_0172279412</td><td></td></tr><tr><td>Cylindrotheca closterium</td><td>Cclo</td><td>CAMPEP_0113624952</td><td></td></tr><tr><td>Dactyliosolen fragilissimus</td><td>Dfra</td><td>CAMPEP_0184871870</td><td></td></tr><tr><td>Detonula confervacea, Strain CCMP 353</td><td>Dcon</td><td>CAMPEP_0172306268</td><td></td></tr><tr><td>Ditylum brightwellii, Strain Pop2 (SS10)</td><td>Dbri</td><td>CAMPEP_0181012646</td><td></td></tr><tr><td>Drosophila melanogaster</td><td>dmClock</td><td>AAC62234</td><td>FBpp0099478</td></tr><tr><td>Drosophila melanogaster</td><td>dmCycle</td><td>NP_524168</td><td>FBpp0074693</td></tr><tr><td>Drosophila melanogaster</td><td>dmSim</td><td>AAC64519</td><td>FBpp0082178</td></tr><tr><td>Drosophila melanogaster</td><td>dmTango</td><td>NP_731308</td><td>FBpp0081483</td></tr><tr><td>Drosophila melanogaster</td><td>dmTrh</td><td>AAA96754</td><td>FBpp0293065</td></tr><tr><td>Durinskia baltica, Strain CSIRO CS-38</td><td>Dbal</td><td>CAMPEP_0170381008</td><td></td></tr><tr><td>Ectocarpus siliculosus</td><td>Esil1</td><td>Ec-02_004780 PAS domain (681) ;mRNA; f:5082087-5086119</td><td></td></tr><tr><td>Eucampia antarctica, Strain CCMP1452</td><td>Eant</td><td>CAMPEP_0197830682</td><td></td></tr><tr><td>Extubocellulus spinifer, Strain CCMP396</td><td>Espi1</td><td>CAMPEP_0178587872</td><td></td></tr><tr><td>Extubocellulus spinifer, Strain CCMP396</td><td>Espi2</td><td>CAMPEP_0178723458</td><td></td></tr><tr><td>Fragilariopsis kerguelensis, Strain L26-C5</td><td>Fkue1</td><td>CAMPEP_0170867208</td><td></td></tr><tr><td>Fragilariopsis kerguelensis, Strain L26-C5</td><td>Fkue2</td><td>CAMPEP_0170907188</td><td></td></tr><tr><td>Fragylariopsis cylindrus</td><td>FcbHLH1a</td><td>jgi|Fracy1|204663</td><td></td></tr><tr><td>Fragylariopsis cylindrus</td><td>FcbHLH4</td><td>jgi|Fracy1|241747</td><td></td></tr><tr><td>Fragylariopsis cylindrus</td><td>FcbHLH7</td><td>jgi|Fracy1|172104</td><td></td></tr><tr><td>Galdieria sulphuraria</td><td>Gsul</td><td>XP_005709404</td><td></td></tr><tr><td>Glenodinium foliaceum, Strain CCAP 1116/3</td><td>Gfol1</td><td>CAMPEP_0167841300</td><td></td></tr><tr><td>Grammatophora oceanica, Strain CCMP 410</td><td>Goce</td><td>CAMPEP_0194036250</td><td></td></tr></tbody></table>
<table>
  <tr>
    <td>Guillardia theta CCMP2712</td>
    <td>Gthe1</td>
    <td>XP_005835918</td>
    <td></td>
  </tr>
  <tr>
    <td>Homo sapiens</td>
    <td>hsArnt</td>
    <td>AAA51777</td>
    <td></td>
  </tr>
  <tr>
    <td>Homo sapiens</td>
    <td>hsbMal1</td>
    <td>NP_1284651</td>
    <td></td>
  </tr>
  <tr>
    <td>Homo sapiens</td>
    <td>hsClock</td>
    <td>AAB83969</td>
    <td></td>
  </tr>
  <tr>
    <td>Homo sapiens</td>
    <td>hsNpas1</td>
    <td>AAH39016</td>
    <td></td>
  </tr>
  <tr>
    <td>Homo sapiens</td>
    <td>hsSim2</td>
    <td>NP_5060</td>
    <td></td>
  </tr>
  <tr>
    <td>Kryptoperidinium foliaceum, Strain CCMP 1326</td>
    <td>Kfol1</td>
    <td>CAMPEP_0176099000</td>
    <td></td>
  </tr>
  <tr>
    <td>Leptocylindrus danicus var. danicus, Strain B650</td>
    <td>Ldan1</td>
    <td>CAMPEP_0116039394</td>
    <td></td>
  </tr>
  <tr>
    <td>Leptocylindrus danicus var. danicus, Strain B651</td>
    <td>Ldan2</td>
    <td>CAMPEP_0116053596</td>
    <td></td>
  </tr>
  <tr>
    <td>Leptocylindrus danicus, Strain CCMP1856</td>
    <td>Ldan3</td>
    <td>CAMPEP_0196809694</td>
    <td></td>
  </tr>
  <tr>
    <td>Licmophora paradoxa, Strain CCMP2313</td>
    <td>Lpar</td>
    <td>CAMPEP_0202478474</td>
    <td></td>
  </tr>
  <tr>
    <td>Minutocellus polymorphus, Strain RCC2270</td>
    <td>Mpol</td>
    <td>CAMPEP_0185804732</td>
    <td></td>
  </tr>
  <tr>
    <td>Monosiga brevicollis</td>
    <td>Mbre2</td>
    <td>jgi|Monbr1|26507|</td>
    <td></td>
  </tr>
  <tr>
    <td>Mus musculus</td>
    <td>mmArnt</td>
    <td>AAA56717</td>
    <td></td>
  </tr>
  <tr>
    <td>Mus musculus</td>
    <td>mmbMal1b</td>
    <td>BAD26600</td>
    <td></td>
  </tr>
  <tr>
    <td>Mus musculus</td>
    <td>mmbMal2</td>
    <td>ABC50103</td>
    <td></td>
  </tr>
  <tr>
    <td>Mus musculus</td>
    <td>mmClock</td>
    <td>AAC53200</td>
    <td></td>
  </tr>
  <tr>
    <td>Mus musculus</td>
    <td>mmNpas1</td>
    <td>NP_32744</td>
    <td></td>
  </tr>
  <tr>
    <td>Mus musculus</td>
    <td>mmSim2</td>
    <td>AAA91202</td>
    <td></td>
  </tr>
  <tr>
    <td>Nannochloropsis gaditana CCMP526</td>
    <td>Ngad_2</td>
    <td>Naga_100016g14</td>
    <td></td>
  </tr>
  <tr>
    <td>Nannochloropsis gaditana CCMP526</td>
    <td>Ngad_3</td>
    <td>Naga_100165g4</td>
    <td></td>
  </tr>
  <tr>
    <td>Nannochloropsis oceanica CCMP1779</td>
    <td>Noce</td>
    <td>NannoCCMP1779_9983-mRNA-1</td>
    <td></td>
  </tr>
  <tr>
    <td>Nannochloropsis oceanica CCMP1779</td>
    <td>Noce_2</td>
    <td>NannoCCMP1779_1600-mRNA-1</td>
    <td></td>
  </tr>
  <tr>
    <td>Nitzschia punctata, Strain CCMP561</td>
    <td>Npun</td>
    <td>CAMPEP_0178827522</td>
    <td></td>
  </tr>
  <tr>
    <td>Nitzschia sp.</td>
    <td>Nitz</td>
    <td>CAMPEP_0113465330</td>
    <td></td>
  </tr>
  <tr>
    <td>Phaeodactylum tricornutum</td>
    <td>PtbHLH1a</td>
    <td>jgi|Phatr2|44962</td>
    <td>Phatr3_J44962</td>
  </tr>
  <tr>
    <td>Phaeodactylum tricornutum</td>
    <td>PtbHLH1b</td>
    <td>jgi|Phatr2|44963</td>
    <td>Phatr3_J44963</td>
  </tr>
  <tr>
    <td>Phaeodactylum tricornutum</td>
    <td>PtbHLH2</td>
    <td>jgi|Phatr2|54435</td>
    <td>Phatr3_J54435</td>
  </tr>
  <tr>
    <td>Proboscia alata, Strain PI-D3</td>
    <td>Pala</td>
    <td>CAMPEP_0194393728</td>
    <td></td>
  </tr>
  <tr>
    <td>Pseudo-nitzschia arenysensis, Strain B593</td>
    <td>Pare</td>
    <td>CAMPEP_0116139930</td>
    <td></td>
  </tr>
  <tr>
    <td>Pseudo-nitzschia australis, Strain 10249 10 AB</td>
    <td>Paus1</td>
    <td>CAMPEP_0168282354</td>
    <td></td>
  </tr>
  <tr>
    <td>Pseudo-nitzschia australis, Strain 10249 10 AB</td>
    <td>Paus2</td>
    <td>CAMPEP_0168310394</td>
    <td></td>
  </tr>
  <tr>
    <td>Pseudo-nitzschia delicatissima, Strain B596</td>
    <td>Pdel1</td>
    <td>CAMPEP_0116091770</td>
    <td></td>
  </tr>
  <tr>
    <td>Pseudo-nitzschia delicatissima, Strain B596</td>
    <td>Pdel2</td>
    <td>CAMPEP_0116094064</td>
    <td></td>
  </tr>
  <tr>
    <td>Pseudo-nitzschia fraudulenta, Strain WWA7</td>
    <td>Pfra1</td>
    <td>CAMPEP_0201229664</td>
    <td></td>
  </tr>
  <tr>
    <td>Pseudo-nitzschia fraudulenta, Strain WWA7</td>
    <td>Pfra2</td>
    <td>CAMPEP_0201232896</td>
    <td></td>
  </tr>
  <tr>
    <td>Pseudo-nitzschia heimii, Strain UNC1101</td>
    <td>Phei1</td>
    <td>CAMPEP_0197189528</td>
    <td></td>
  </tr>
  <tr>
    <td>Pseudo-nitzschia heimii, Strain UNC1101</td>
    <td>Phei2</td>
    <td>CAMPEP_0197199168</td>
    <td></td>
  </tr>
  <tr>
    <td>Pseudo-nitzschia multiseries</td>
    <td>PmbHLH1a</td>
    <td>jgi|Psemu1|26622</td>
    <td></td>
  </tr>
  <tr>
    <td>Pseudo-nitzschia multiseries</td>
    <td>PmbHLH7</td>
    <td>jgi|Psemu1|228145</td>
    <td></td>
  </tr>
  <tr>
    <td>Pseudo-nitzschia pungens, Strain cf. pungens</td>
    <td>Ppun</td>
    <td>CAMPEP_0172413298</td>
    <td></td>
  </tr>
  <tr>
    <td>Rhizosolenia setigera, Strain CCMP 1694</td>
    <td>Rset</td>
    <td>CAMPEP_0178942232</td>
    <td></td>
  </tr>
  <tr>
    <td>Skeletonema costatum, Strain 1716</td>
    <td>Scos1</td>
    <td>CAMPEP_0113408658</td>
    <td></td>
  </tr>
</table>
<table><tr><td>Skeletonema dohrnii, Strain SkeIB</td><td>Sdoh</td><td>CAMPEP_0195221452</td><td></td></tr><tr><td>Skeletonema japonicum, Strain CCMP2506</td><td>Sjap</td><td>CAMPEP_0201725346</td><td></td></tr><tr><td>Skeletonema marinoi, Strain FE7</td><td>Smar1</td><td>CAMPEP_0180846198</td><td></td></tr><tr><td>Skeletonema marinoi, Strain UNC1201</td><td>Smar2</td><td>CAMPEP_0197231700</td><td></td></tr><tr><td>Skeletonema menzelii, Strain CCMP793</td><td>Smen</td><td>CAMPEP_0183679140</td><td></td></tr><tr><td>Stauroneis constricta, Strain CCMP1120</td><td>Scon1</td><td>CAMPEP_0119548954</td><td></td></tr><tr><td>Stauroneis constricta, Strain CCMP1120</td><td>Scon2</td><td>CAMPEP_0119572288</td><td></td></tr><tr><td>Staurosira complex sp., Strain CCMP2646</td><td>Stau</td><td>CAMPEP_0202487246</td><td></td></tr><tr><td>Synedropsis recta cf, Strain CCMP1620</td><td>Srec1</td><td>CAMPEP_0119009808</td><td></td></tr><tr><td>Synedropsis recta cf, Strain CCMP1620</td><td>Srec2</td><td>CAMPEP_0119029558</td><td></td></tr><tr><td>Thalassionema frauenfeldii, Strain CCMP 1798</td><td>Tfra1</td><td>CAMPEP_0178899126</td><td></td></tr><tr><td>Thalassionema frauenfeldii, Strain CCMP 1798</td><td>Tfra2</td><td>CAMPEP_0178923098</td><td></td></tr><tr><td>Thalassionema nitzschioides, Strain L26-B</td><td>Tnit1</td><td>CAMPEP_0194218124</td><td></td></tr><tr><td>Thalassionema nitzschioides, Strain L26-B</td><td>Tnit2</td><td>CAMPEP_0194240022</td><td></td></tr><tr><td>Thalassiosira antarctica, Strain CCMP982</td><td>Tant</td><td>CAMPEP_0202006238</td><td></td></tr><tr><td>Thalassiosira gravida, Strain GMp14c1</td><td>Tgra</td><td>CAMPEP_0201684800</td><td></td></tr><tr><td>Thalassiosira miniscula, Strain CCMP1093</td><td>Tmin</td><td>CAMPEP_0183747574</td><td></td></tr><tr><td>Thalassiosira oceanica</td><td>Toce2</td><td>EJK65393</td><td></td></tr><tr><td>Thalassiosira pseudonana</td><td>TpbHLH1</td><td>jgi|Thaps3|24007</td><td></td></tr><tr><td>Thalassiosira pseudonana</td><td>TpbHLH2</td><td>jgi|Thaps3|20899</td><td></td></tr><tr><td>Thalassiosira pseudonana</td><td>TpbHLH7</td><td>jgi|Thaps3|23208</td><td></td></tr><tr><td>Thalassiosira sp., Strain FW</td><td>Tfw</td><td>CAMPEP_0172354094</td><td></td></tr><tr><td>Thalassiosira weissflogii, Strain CCMP1010</td><td>Twei</td><td>CAMPEP_0203515806</td><td></td></tr><tr><td>Thalassiothrix antarctica, Strain L6-D1</td><td>Txnt</td><td>CAMPEP_0194143228</td><td></td></tr><tr><td>Tiarina fusus, Strain LIS</td><td>Tfus1</td><td>CAMPEP_0117003500</td><td></td></tr><tr><td>Tiarina fusus, Strain LIS</td><td>Tfus3</td><td>CAMPEP_0117046192</td><td></td></tr></table>
<table><caption>**Table S5:** List of the oligonucleotides used in this work.</caption><thead><tr><th>Gene</th><th>Phatr3 Accession</th><th>Oligo name</th><th>Sequence</th><th>Type</th></tr></thead><tbody><tr><td rowspan="6">bHLH1a</td><td rowspan="6">Phatr3_J44962</td><td>bHLH1a-endogenous-QFw</td><td>TTATGTCTTTCGGCGACGGG</td><td rowspan="2">QPCR</td></tr><tr><td>bHLH1a-endogenous-QRv</td><td>AGCAACGAATGCATGCAAGG</td></tr><tr><td>bHLH1a-total-QFw</td><td>ATTCTTGGTCCCACCCGGTA</td><td rowspan="2">QPCR</td></tr><tr><td>bHLH1a-total-QRv</td><td>ACGCCACATTGAAAAACCGAG</td></tr><tr><td>Pt-bHLH1a-Dral-Fw</td><td>GATTTTAAAATGAATAAGCCAGGACAGCG</td><td rowspan="2">Full length cloning</td></tr><tr><td>Pt-bHLH1a-Xhol-Rv</td><td>TTGCTCGAGCACAGCTTCGCTGCATCGTC</td></tr><tr><td rowspan="2">bHLH1b</td><td rowspan="2">Phatr3_J44963</td><td>bHLH1b-QFw</td><td>TGCGATCTCAACGGCTAATA</td><td rowspan="2">QPCR</td></tr><tr><td>bHLH1b-QRv</td><td>CGCAAAACGAGGCTAATTTC</td></tr><tr><td rowspan="2">bHLH3</td><td rowspan="2">Phatr3_J42586</td><td>bHLH3-QFw</td><td>CACTCTCATCATGCGGGAAT</td><td rowspan="2">QPCR</td></tr><tr><td>bHLH3-QRv</td><td>GCGCGTTGTCTTCCTCTATC</td></tr><tr><td rowspan="2">bZIP7</td><td rowspan="2">Phatr3_J48800</td><td>bZIP7-QFw</td><td>CCTTATTGATATTCAAGATTCCAAGG</td><td rowspan="2">QPCR</td></tr><tr><td>bZIP7-QRv</td><td>GTTTCGGAACCTGCATAGGA</td></tr><tr><td rowspan="2">bZIP5</td><td rowspan="2">Phatr3_J45142</td><td>bZIP5-QFw</td><td>ACCGGGTACAAAGAAGTCGC</td><td rowspan="2">QPCR</td></tr><tr><td>bZIP5-QRv</td><td>GACCCTCCAAGCTCTGCATT</td></tr><tr><td rowspan="2">CYCH1</td><td rowspan="2">Phatr3_J36892</td><td>CYCH1-QFw</td><td>ATACACGACGAGCGGATAC</td><td rowspan="2">QPCR</td></tr><tr><td>CYCH1-QRv</td><td>AACAAGACGCCGTAAATCG</td></tr><tr><td rowspan="2">CYCB1</td><td rowspan="2">Phatr3_J46095</td><td>CYCB1-QFw</td><td>TCCTGGTCCGCTACTTGAAAG</td><td rowspan="2">QPCR</td></tr><tr><td>CYCB1-QRv</td><td>GCTGGCTGGGAAGATAACGC</td></tr><tr><td rowspan="2">HSF1d</td><td rowspan="2">Phatr3_J44750</td><td>HSF1d-QFw</td><td>TGCTCCATCAAGATACCACCAAT</td><td rowspan="2">QPCR</td></tr><tr><td>HSF1d-QRv</td><td>GACGACACCCGAAGCTTGTT</td></tr><tr><td rowspan="2">HSF1g</td><td rowspan="2">Phatr3_J42514</td><td>HSF1g-QFw</td><td>AGTTCCTGTCAGACTCAGC</td><td rowspan="2">QPCR</td></tr><tr><td>HSF1g-QRv</td><td>ACACTTTCGCCTCTACTGTCA</td></tr><tr><td rowspan="2">HSF3.3a</td><td rowspan="2">Phatr3_J45393</td><td>HSF3.3a-QFw</td><td>CCTTCGAATTCCACTGCACC</td><td rowspan="2">QPCR</td></tr><tr><td>HSF3.3a-QRv</td><td>CGTAGCCTTCCATCCGTAGT</td></tr><tr><td rowspan="2">HSF4.7b</td><td rowspan="2">Phatr3_J49596</td><td>HSF4.7b-QFw</td><td>GACGACACCCGAAGCTTGTT</td><td rowspan="2">QPCR</td></tr><tr><td>HSF4.7b-QRv</td><td>ACG AAGCGATCTTGGACAGT</td></tr><tr><td rowspan="2">Tbp</td><td rowspan="2">Phatr3_J10199</td><td>Tbp-QFw</td><td>ACCGGAGTCAAGAGCACACAC</td><td rowspan="2">QPCR</td></tr><tr><td>Tbp-QRv</td><td>CGGAATGCGCGTATACCAGT</td></tr><tr><td rowspan="2">Rps</td><td rowspan="2">Phatr3_J10847</td><td>Rps-QFw</td><td>CGAAGTCAACCAGGAAACCAA</td><td rowspan="2">QPCR</td></tr><tr><td>Rps-QRv</td><td>GTGCAAGAGACCGGACATACC</td></tr></tbody></table>
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Chapter II.c:

Physiological rhythms in the
marine diatom *Phaeodactylum*
*tricornutm* are regulated by the
circadian clock transcriptional
regulator RITMO1

Alessandro Manzotti¹, Raphael Monteil¹, Antoin Hoguin¹, Cheminant Navarro¹, Danis Jallet², Francois-Yves Bouget³, Fayza Daboussi²,⁴, Marianne Jaubert¹, Jean-Pierre Bouly¹ and Angela Falciatore¹*

'Laboratoire de Biologie du Chloroplaste et perception de la lumière chez les micro-algues,
UMR7141,
10 CNRS, Sorbonne Université, Institut de Biologie Physico-Chimique, Paris, 75005,
France

Toulouse Biotechnology Institute (TBI), Université de Toulouse, CNRS, INRAE, INSA,
Toulouse, France

Sorbonne Université, UMR 7621, Laboratoire d'Océanographie Microbienne,
Observatoire Océanologique, Banyuls-sur-Mer, France

Toulouse White Biotechnology (TWB), INSA, Toulouse, France

*corresponding author

Keywords: circadian clock, diatom, rhythms, cell division, gene expression, photosynthesis, bHLH-PAS

Manuscript in preparation
Abstract

Circadian clocks are sophisticated molecular devices of critical importance to the lives of most organisms, as they synchronize biological processes with periodic environmental cycles. Biological rhythms have been described in a variety of marine organisms in laboratory conditions and in the natural environment. However, the timekeepers controlling marine rhythms remain largely uncharacterized, primarily due to the limited genomic resources and tools to study rhythmicity. Here we address circadian regulation in prominent phytoplanktonic organisms, diatoms, by characterizing the *Phaeodactylum tricornutum* bHLH-PAS protein RITMO1, previously proposed as a genetic regulator of diel rhythmicity. We report rhythms in critical physiological processes such as cell fluorescence, photosynthesis and gene expression which persist in continuous light conditions. These rhythms are disrupted in lines in which RITMO1 has been genetically obliterated by CRISPR/Cas9 as well as in ectopic overexpressing lines, supporting a direct role of this protein in the circadian regulation. By analysing the expression of selected genes of interest, we also show that RITMO1 plays a wide role in the circadian transcriptional regulation, acting as a component of the diatom endogenous clock oscillator. RITMO1 represents a key entry point for the molecular exploration of other components of the circadian clock system of diatoms and lays the basis for assessing the relevance of circadian regulation to the life of these organisms in the marine environment.
Introduction

Diatoms are prominent unicellular
algae found in all aquatic environments (Field
et al., 1998; Pierella Karlusich et al., 2020).
With an estimated number of species close to
100,000 (Malviya et al., 2016), diatoms
represent the most species-rich of algae,
playing a central role in trophic networks and
biogeochemical cycles of the oceans (Bar-On
et al., 2018). Although being photosynthetic,
diatoms are not directly related to the
terrestrial plants and green algae of the
Archaeoplastida clade, which are derived by a
primary endosymbiosis event. Diatoms belong
to the *Stramenopila* and acquired their plastid
through multiple secondary endosymbiosis of
a eukaryotic red alga and likely a green alga by
a heterotrophic host (Burki et al., 2020;
Keeling, 2013). Traces of the complex
evolutionary history are found in their genome
(Armbrust, 2004; Bowler et al., 2008) and in
the peculiar gene repertoire derived by the
different heterotroph and phototroph
ancestors as well as by bacteria via horizontal
gene transfer (Dorrell et al., 2017; Vancaester
et al., 2020). Comparative analysis of several
diatom genomes has also highlighted a
significant portion of core diatom-specific
genes and species-specific genes mostly of
unknown function, which may represent
relevant genetic innovations for the diatom
ecological success in the marine environment
(for a review, see Falciatore et al., 2020).

It is well established that diatoms are
able to successfully grow in extremely variable
environments and are able to cope with
unpredictable light changes thanks to
sophisticated acclimation mechanisms
(Lepetit et al., 2022). There is also
considerable evidence that diatoms have
adapted their physiology and metabolism to
the periodic daily changes in light and
darkness, experienced by most organisms on
Earth. Diatoms show robust diurnal rhythms
in critical processes such as photosynthesis

(Brand, 1982), pigment synthesis (Ostgaard
and Jensen, 1982; Owens et al., 1980; Ragni
and d'Alcalà, 2007), gene expression
(Chauton et al., 2013; Smith et al., 2016) and
cell cycle progression (Chisholm and Brand,
1981; Gaidarenko et al., 2019; Nelson and
Brand, 1979; Ostgaard and Jensen, 1982;
Williamson, 1980) so that they occur at
optimal times of the day. Recent metagenomic
analyses also detected strong diurnal patterns
of gene expression in natural diatom
communities (Coesel et al., 2021; Kolody et
al., 2019). Still, the mechanisms controlling
the temporal synchronization and segregation
of cellular and metabolic events during light
and dark periods are still largely unknown in
diatoms as well as in most phytoplanktonic
organisms.

The circadian clock is a powerful
timing system generating biological rhythms of
24-hour cycle, extensively characterised at the
molecular level in terrestrial plants and
animals. Formally defined as an endogenous
oscillator generating rhythmicity, the circadian
clock is entrained by periodic light changes,
which represent the primary time giver
(Zeitgeber, ZT, Roenneberg and Foster,
1997). Photoreceptors play a key role in the
input pathways which synchronize the
circadian clock with light (Dunlap, 1999).
Although different taxa use different
molecular players to generate circadian
rhythms, the timekeeping system share
common features. In eukaryotes, they are
structured around interlocked negative
transcriptional-translational feed-back loops
(ITFL) that uses transcription regulatory
proteins to control the expression of their
cognate genes (Dunlap, 1999). This regulatory
loop is self-sustained, which means that the
oscillations of the endogenous clock, as well as
the physiological processes it controls, are
maintained after transition to a constant
environment (e.g. in continuous light) in a
process called free-run persistence. In
terrestrial plants, the circadian clock has been
shown to play a crucial role in the rhythmic
regulation of genes involved in photosynthesis,
carbon assimilation or cell division as well as
in the regulation of photoperiodic-regulated
processes such as the flowering (Dodd et al.,
2005; Fung-Uceda et al., 2018; Zagotta et al.,
1996). In marine phytoplankton, the
molecular components of the clock system
have been so far elucidated only in the green
alga *Ostreococcus tauri*, which contain a
minimalist plant-like clock with two
components, OtCCA1 and OtTOC1,
Corellou et al., 2009). The existence and the
nature of an endogenous circadian oscillator in
diatoms remains still enigmatic for two main
reasons: Although diurnal rhythms have been
described in many diatom species for many
years now, the persistence of these rhythms in
free-running has been little characterized and,
secondly, diatom genomes lack the canonical
components of the circadian systems
characterised in bacteria, algae, plants or
animals. Recently, however, a new regular of
cellular rhythmicity named RITMO1 has
been identified in the model diatom
*Phaeodactylum tricornutum*, in a survey of the
most rhythmic diurnal transcription factors
(Annunziata et al., 2019). RITMO1 possesses
bHLH-PAS domains, also found in the
animal central clock proteins (Peschel and
Helfrich-Förster, 2011; Takahashi, 2017). In
*P. tricornutum*, it has been shown that
alteration of *RITMO1* timing and expression
levels by ectopic overexpression resulted in
cells with altered gene expression profiles in
L:D cycles but also in continuous darkness,
showing that the regulation of rhythmicity by
*RITMO1* is not directly dependent on light

inputs. Initial analysis of cellular fluorescence
rhythms, which in diatoms reflect synchrony in
the closely coupled chloroplast ontogeny and
cell cycle progression (Hunsperger et al.,
2016; Ragni and d'Alcalà, 2007), also revealed
that ectopic expression of RITMO1 can affect
cellular rhythmicity (Annunziata et al., 2019).
Although these altered phenotypes identified
RITMO1 as a genetic modifier of diel
rhythmicity, they did not allow to assess its
direct implication in the still cryptic diatom
circadian clock system as misexpression was
achieved in a wild-type background, under the
control of a non-constitutive promoter.

To get a more insight into the function
of RITMO1 in circadian regulation of
diatoms, in this study we have performed an
extended characterization of light-depended
biological rhythms, by assessing features of
these rhythms under different photoperiodic
and light intensity conditions. We have
generated and exploited knock-out (KO)
mutants of *RITMO1* by CRISPR/Cas9
proteolistic bombardment to test the direct
consequence of the obliteration of this protein
on the circadian rhythmicity of *P. tricornutum*,
in parallel with the ectopic over-expressing
lines. Our analyses reveal a molecular control
of temporal events in key biological processes
such as cell fluorescence and photosynthetic
activity and support a role of RITMO1 in this
regulation. The expression analysis of selected
genes of interest also demonstrated a strong
transcriptional control of circadian processes
in *P. tricornutum*, and establish RITMO1 as
the first identified component of the
transcriptional clock oscillator of diatoms.
# Materials and Methods

## Cultures and irradiation conditions

Transgenic and control lines of *P. tricornutum* used in this study have a Pt1 8.6; CCMP2561 genomic background. Cell cultures were grown in batch culture in F/2 Guillard media (Guillard, 1975) at 18°C under white light (Bridgelux BXRV-DR-1830H-3000-A-13 LEDs) of different intensities and photoperiods, as described in the text. Cells were preadapted at least two weeks to the specific growth condition before performing analysis of rhythmicity. For circadian experiments, cultures were grown in incubators with orbital agitation and were not diluted during the whole course of the cellular fluorescence analysis. Final refresh of culture was performed 3 days before the release to free-run conditions to have cultures in exponential phase of growth during the experiment and ensure minimal perturbation of physiology due to culture refreshment. Cell concentrations were measured using a Z2 Coulter Particle Count and Size Analyser (Beckman Coulter). Minimal and maximal size thresholds were set respectively to 3µm and 11µm. Growth rates were calculated during the exponential phase of growth over four days and expressed as number of divisions per day. The formula used for the calculation of divisions per day is $division \ per \ day = (ln(c_4) - ln(c_0))/(4 * ln(2))$, where $c_0$ and $c_4$ represent the culture concentrations at day 0 and day 4.

## Generation of RITMO1 Knock-Out (KO) strains

RITMO1 KO mutants were generated by biolistic delivery of the CRISPR-Cas9 ribonucleoproteins according to Serif et al., 2018. Selection of transformed lines with the riboprotein complex was accomplished by co-targeting the *APT* gene (Phatr3_J6834) to

induce resistance to 2-fluoroadenine (2-FA). A total of three and two independent sgRNAs were co-transformed to target *RITMO1* and *APT*, respectively. The sequences of the sgRNAs and the targeted region in the genome are reported in Table ST1. A total of eight 2-FA resistant colonies were sequenced by Sanger sequencing with allele specific primers to detect biallelic mutants (Fig. S1). Of the eight colonies, four appeared to bear mutations (single nucleotide insertion, deletions or inversions of gene fragments) on the two alleles of *RITMO1* gene and were named KO1 to KO4. An additional colony showed no mutation in *RITMO1* alleles and was therefore chosen as control of the effect of proteolistic bombardment (CTR line).

## Western Blot Analysis

$1 \times 10^8$ cells were pelleted, quickly frozen in liquid nitrogen and stored at -80°C until use. After thawing, pellets were added with 50 µL of lysis buffer (Tris HCl pH 6.8 50 mM, 2% sodium dodecyl sulfate (SDS) and Protease Cocktail Inhibitor solution 1x (SIGMA Aldrich), solubilized at room temperature for 30 minutes, and then centrifuged at 15,000 rpm at 4°C for 30 minutes. Total proteins were extracted and analyzed by western blot as described previously (Taddei et al., 2016). Proteins were analyzed by loading on gel equal amounts of protein (30 µg), quantified with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). RITMO1 protein was detected via purified rabbit antiserum polyclonal antibody (Agrisera Antibodies, 1:10,000 dilution in PBS 5% milk) designed against a RITMO1 specific peptide (NQDHMQDVFGAMRDMLRRPFPGENH₂) corresponding to the amino acids from position 428 to 450. Photosystem II D2 protein was used as charge control and
detected via rabbit anti-D2 antibody (1:10,000 dilution in 5% milk PBS). Following incubation with HRP-conjugated anti-rabbit secondary antibodies (Promega), proteins were detected with Clarity reagents (Bio-Rad) and a ChemiDoc imaging system (Bio-Rad) imaging system.

## Cellular fluorescence analysis

In circadian experiments, culture samples were automatically collected every 2 hours via a BVP standard ISM444 peristaltic pump (Ismatec) controlled by a FC204 fraction collector (Gilson). Previous to cytometry analysis, cells were stored at 4°C in the dark for at least 2 hours to a maximum of 24hours. Cellular fluorescence rhythmicity analyses were performed using a MACSQuant Analyser flow cytometer (Miltenyi Biotec, Annunziata et al., 2019). B3-A parameter (488 nm excitation, 655-730 nm detection) was used to track chlorophyll fluorescence. Between 10,000 and 30,000 cells were analysed per each time point per each culture replicate and median B3-A value was used for following rhythmicity analysis.

## Analysis of the rhythmic processes

The analysis of B3-A fluorescence dataset rhythmicity was performed using the BioDare2 webtool (https://biodare2.ed.ac.uk/, Zielinski et al., 2014). For graphical representation, data traces were baseline detrended, normalized to the maximum value and aligned to the mean values. Data traces were first analysed using the Enright Periodogram (EPR) algorithm, which was used as an arrhythmicity test (Zielinski et al., 2014). Tracks not identified as rhythmic by EPR were used for graphical representations of average fluorescence, but were not further analysed for period, amplitude and Relative Amplitude Errors (RAE). Period, amplitude and RAE were calculated via Fast Fourier nonlinear least

square algorithm (FFT-NLLS), within a period range of 18 and 34 hours. For fluorescence traces of cells grown in photoperiod, the acrophase relative to dark onset was also calculated via the same algorithm. At least three full oscillations were considered for the analysis. RAE was used as a measure of goodness-of-fit to a theoretical sine wave of the empirical traces. RAE is defined as the ratio of the amplitude error to the most probable derived amplitude magnitude (Plautz et al., 1997). Statistical differences in rhythmicity parameters among lines were evaluated via One-way analysis of variance against Wt with Bonferroni post-test for multiple testing correction.

## Cell cycle analysis

Cell cycle analysis were performed either in *P. tricornutum* cells grown under 16L:8D cycles or on cultures synchronized in G1 phase with prolonged darkness for 40h. Under diurnal conditions, 15 mL of samples in exponential growth (1*10⁶ to 2*10⁶ cells per mL) were collected by centrifugation (2500 rpm, 15 minutes, 4°C) every 3 hrs for 24 hours. In dark synchronised experiments, the samples were collected every 3 hrs for 30 hours after re-illumination (25 µmol photons m⁻² s⁻¹). Cell pellets were fixed in cold 70% EtOH and stored for at least 24hrs in the dark at 4°C until processing. Fixed cells were then washed once in cold EtOH 70%, once in PBS and stained in the dark for 45 minutes at room temperature with DAPI (4',6-diamidino-2-phenylindole) at a final concentration of 0.5ng/ml. After staining, cells were washed with PBS and kept on ice in the dark until the flow cytometry analysis. 50,000 cells per each sample were analysed using the MACSQuant Analyser flow cytometer. V1-A channel (408 nm excitation, 450/50 nm detection) was used to evaluate the content of DAPI-stained DNA. 2c and 4c peaks were used to infer the fraction of cells in G1 and S/G2/M phases, using a dedicated R script (Agier and Fischer, 2016). The area under each peak was used as a proxy
for the proportion of cells in each phase. Two-way analysis of variance was performed to verify statistical differences among lines along the time series. Bonferroni post-test was used to compare replicate means by rows, comparing each line to Wt.

## Analysis of photosynthetic parameters

The analyses were performed on cultures (three biological replicates) adapted to 16L:8D (25µmol photons m⁻² s⁻¹) and then either maintained in 16L:8D 25 µmol photons m⁻² s⁻¹ or transferred to L:L 17 µmol photons m⁻² s⁻¹. For the analysis, samples were collected every 4 hours over 36 hours starting from the second free run day (LL32). 15 mL of samples in exponential growth (1*10⁶ to 2*10⁶ cells per mL) were concentrated 10 folds in their own supernatant by centrifugation (2,500 rpm, 10 minutes). For chlorophyll fluorescence analysis, the cells were left under dim light (<5 µmol photons m⁻² s⁻¹) in order to allow full relaxation of Photosystem II (PSII) reaction centers before Rapid Light Curve (RLC) analysis (Ralph and Gademann, 2005). Samples were then exposed to one minute of dark, followed by increasing intensity steps of actinic light up to 1450 µmol photons m⁻² s⁻¹ to obtain the RLCs. Each light step lasted 30 seconds and illumination was performed with red LEDs. At the end of each light step, chlorophyll fluorescence was measured with a blue light measuring pulse and a 250 ms red light saturating pulse (1700 µmol photons m⁻² s⁻¹) was used to induce PSII maximal fluorescence (Fm'). Fluorescence of light adapted PSII (Fs) was measured just prior to saturating light application. Chlorophyll fluorescence detection was performed using a Paradigme Camera Speedzen system (Beambio). PSII quantum yield φPSII was calculated as (Fm'-Fs)/Fm' (Maxwell and Johnson, 2000). Non-Photochemical Quenching (NPQ) was calculated as (Fm'-

Fm)/Fm', where Fm is the maximal fluorescence of dark adapted PSII (Maxwell and Johnson, 2000). NPQ values were obtained at the last 1,450 µmol photons m⁻² s⁻¹ light step. For each light step, the relative Electron Transfer Rate through the photosynthetic chain (rETR) was calculated as φPSII x Light intensity. The RLC resulting from plotting rETR as a function of light intensity was fitted to a double exponential decay function: $ETR = ETR_{max} * (1 - e^{-\alpha * \frac{E}{ETR_{max}}}) * (e^{-\beta * \frac{E}{ETR_{max}}})$ following Platt et al., 1980. By RLC fitting, the maximum value of rETR (rETRmax) and the angular coefficient of the function in the light limiting region (α) were calculated. Fitting was performed via Origin software version 9.1.

## RNA extraction and RT-qPCR analysis

For RNA purification, 10⁸ cells from exponential phase cultures were filtered onto Whatman filter 589/2. Filters were then washed with 25mL of PBS 1X, cut in two to have technical replicates, flash frozen in liquid nitrogen and stored at -80°C until use. RNA was extracted with TriPure Isolation Reagent (Roche) following the protocol provided by the company and further purified by an ammonium acetate precipitation. RNA purity and integrity were checked on samples by assessing the 260nm/230nm and 260nm/280nm OD ratios and by gel electrophoresis. 500ng of RNA were retro-transcribed via an oligo-dT and random primers mix using the QuantiTect Reverse Transcription Kit (Quiagen). For RT-qPCR, ribosomal protein small subunit 30S (RPS) and TATA box Binding Protein (TBP) were used as reference genes (Siaut et al., 2007). For each sample, geometrical average of Ct was used as reference to evaluate the expression of genes of interest via the ΔΔCt Livak method (Livak and Shcmittgen, 2001). For each gene, data was normalized against the maximum
expression value among the different strains,
so that expression values are all between 0 and
1. This normalization method allows to
compare the relative gene expression among
different cell lines. All RT-qPCR analyses

were performed on two biological replicates
obtained in distinct experiments. All the
primers used for RT-qPCR analysis are listed
in Table ST5.
# Results

<figure><img src="image_49.png" /><figcaption>B</figcaption></figure>

<table><caption>C</caption><thead><tr><th>Strain name</th><th>Type of mutation</th></tr></thead><tbody><tr><td>KO1</td><td>Allele1: insertion (A) in the gHLH3 region at position 832-nt<br>Allele2: deletion of 338bp from position 489-nt between gHLH2 and gHLH3 regions</td></tr><tr><td>KO2</td><td>Allele1: deletion of 289bp from position 205-nt between gHLH1 and gHLH2 regions<br>Allele2: 336bp inversion between the gHLH2 and gHLH3 regions (from position 489-nt to position 824-nt)</td></tr><tr><td>KO3</td><td>Allele1: insertion (G) in the gHLH3 sequence at position 832-nt<br>Allele2: 338bp inversion between the gHLH2 and gHLH3 regions (from position 488-nt to 825-nt)</td></tr><tr><td>KO4</td><td>Allele1: insertion (A) in the gHLH3 region at position 832-nt<br>Allele2: 336bp inversion between the gHLH2 and gHLH3 regions (from 488-nt to 823-nt)</td></tr></tbody></table>

<figure><img src="image_50.png" /><figcaption>Figure 1. Generation of knock-out (KO) mutants for *P. tricornutum* RITMO1.</figcaption></figure>

(A) Schematic representation of the *RITMO1* locus of *P. tricornutum* on Chromosome 5 (Phatr3_J44962). Exons are represented as black boxes, the intron as a white box. Blue and light blue boxes represent the relative location of the bHLH and PAS domains respectively. Red, orange and yellow boxes highlight the localisation of the target sequences (underlined) of the three gRNAs used for RITMO1 CRISPR-Cas9 mutagenesis. Letters in bold represent the protospacer adjacent motif associated with each target site. (B) Description of the mutations introduced on each allele of the *RITMO1* locus on the four KO mutants as identified by Sanger sequencing. (C) Immunoblot analysis in Wt, KO and CTR lines of RITMO1 protein at ZT9 in cells adapted to 12L:12D of 25 µmol photons m⁻² s⁻¹. An antipeptide antibody recognizing the RITMO1 protein downstream the targeted mutation sites (corresponding to the amino acids from position 428 to 450 in the wild-type sequence) was used for the detection of the RITMO1 protein. An α-D2 antibody was used as loading control.

## Generation of knock-out mutants for the *P. tricornutum* RITMO1 by CRISPR-Cas9

In order to characterize more thoroughly the role of *P. tricornutum* RITMO1 within the diatom timekeeper machinery, we generated knock-out lines for *RITMO1* by proteolistic bombardment of the CRISPR-Cas9 protein (Serif et al., 2018). A set of three *RITMO1*-specific sgRNAs (Fig. 1A) were co-transformed together with two sgRNAs for the endogenous counter-selectable markers *PtAPT* (*Adenine Phosphoribosyl Transferase*), whose mutation allows the selection of KO lines via the resistance to 2-fluoroadenine (2-FA) (Table S1). Four knock-out lines presenting mutations on both alleles of the *RITMO1* gene were generated (Figure 1 B and Fig. S1),

hereafter named KO1-4. A line resistant to 2-FA but not presenting any mutation for *RITMO1* was also generated and chosen as independent transformation control (CTR). Wild-type (Wt), Kos and CTR lines were analysed by western blot by using an anti-peptide recognizing the RITMO1 protein downstream of the targeted mutation sites. The analysis shown in Fig. 1C demonstrated a loss of the RITMO1-associated band in the four mutant lines, but not in the Wt and CTR lines. The KO1 and KO3 lines were used to further analyse cellular rhythmicity, along with the previously generated RITMO1 over-expressor OE1 line (Annunziata et al., 2019).
Diatoms cellular fluorescence
rhythmicity is adjusted in a
photoperiod-dependent manner
and is not altered in knock-out
mutants for *RITMO1*

Previous studies in *P. tricornutum*
(Annunziata et al., 2019; Hunsperger et al.,
2016; Ragni and d'Alcalà, 2007) grown under
periodic light:dark (L:D) cycles indicated that
the diurnal fluorescence changes monitored
by flow cytometer represent a good proxy of
the chlorophyll a content in the growing
chloroplast and of progression through the cell
cycle. To characterise the features and
robustness of diurnal and circadian processes
in diatoms and the involvement of RITMO1
in these processes, we first performed an
extended characterization of cellular
fluorescence changes in *P. tricornutum* cells
adapted to different photoperiods and light
intensities (Fig. 2). We performed this
comparative analysis on Wt, CTR, KO1, KO3
and OE1 lines. In cells adapted to 16L:8D
cycles of moderate light intensity (25 µmol
photons m⁻²s⁻¹) and showing a cell division per
day (Table S2), cellular fluorescence displayed
strong diurnal oscillations during the L:D, with
an acrophase of fluorescence at around ZT14,
hence anticipating dark transition of 2 hours
(Fig. 2A and F, and Table S3). During the
night period, fluorescence continued to
decrease, reaching a minimum at the end of
the dark. To confirm the correlation between
cell fluorescence changes and cell cycle
progression, cell concentration was analysed in
wild-type cells all three hours over the course
of three days, and the different cell cycle
phased monitored by DAPI staining. We
observed a strong inverse correlation between
the increase in cell number after cytokinesis
(Fig. S1A-C) during the night period and
fluorescence signal, confirming the
fluorescence as a good proxy of the cell cycle.
This is also confirmed by the progressive
reduction of the percentage of cells in G1
along the light period (Fig. S1D).

We further tested *P. tricornutum*
responses to different photoperiods. Cells
adapted to 12L:12D of 25 µmol photons m⁻²s⁻¹
(Fig. 2B) showed oscillations, as for the
16L:8L (Fig. 2A) and a comparable division
rate (Table S2). They exhibited a similar
anticipation of the dark, with an acrophase of
cellular fluorescence anticipating dark
transition of about 1.5 h (Fig. 1F) indicating
that cell rhythmicity was adjusted to the shorter
photoperiod. Differently, in cells acclimated
to 8L:16D photoperiod at 25 µmol photons m⁻²s⁻¹
(Fig. 2C), the fluorescence peak occurred 2
hours after the onset of darkness (Fig. 2F).
Cells showed a reduced growth (around 0.6
division/day, Table S2). To test whether the
loss of night anticipation and the observed
phase shift was due a metabolic control of
growth and synchronization capacity due to
the limiting amount of light, we repeated the
analysis in 8L:16D, but applying twice the light
intensity (50 instead of 25 µmol photons m⁻²s⁻¹)
during the light phases (Fig. 2D). This
increased irradiation resulted in an increase
growth of *P. tricornutum* (~1 division/day,
Table S2). Under these conditions, however,
no significant change of the phase was
observed compared to the analysis done in the
same photoperiod with lower irradiation (Fig.
2F and Table S3). Similarly, when *P. tricornutum*
cells were adapted to 16L:8L with
higher light intensity (75 µmol photons m⁻²s⁻¹,
Fig. 2F), they showed an increased growth (1.5
division/day, Table S2), but a similar phase
compared to cells adapted to lower irradiation
(Fig. 2A and Table S3), suggesting a
synchronization of cell population within L:D
cycles, independently on the light intensity
applied and division rate.

As clearly seen in Fig. 2 and Table S3,
neither the obliteration of RITMO1 in knock-
out lines nor the deregulation of its expression
in OE1 altered cellular fluorescence
rhythmicity in cells adapted to different
photoperiods and light intensities. All the
transgenic lines showed rhythmic fluorescence
trends comparable to that of the Wt and CTR
<figure><img src="image_51.png" /><figcaption>Figure 2. Fluorescence rhythmicity of wild-type and ritmo1 knock-out mutants under different light:dark cycles.</figcaption></figure>

<figure><img src="image_52.png" /></figure>

A-E) Diurnal oscillations of cellular red fluorescence (B3-A parameter) in *P. tricornutum* Wt, CTR, KO1, KO3 and OE1 lines entrained under different photoperiods and light intensities (n=4 for Wt, n=3 for other lines). A= 16L:8D 25µmol photons m⁻² s⁻¹, B= 12L:12D 25µmol photons m⁻² s⁻¹, C=8L:16D 25µmol photons m⁻² s⁻¹, D= 8L:16D 50µmol photons m⁻² s⁻¹, E= 16L:8D 75µmol photons m⁻² s⁻¹, n≥4). Dots represent mean B3-A values, coloured dashed lines represent SD. (F) Phase-time estimations of B3-A oscillations relative to the light to dark transition (mean ±s.e.m. n=4), (G) amplitude and (H) R.A.E. estimations of B3-A fluorescence traces represented in panels A-E.
lines, indicating that RITMO1 does not have
a major impact on cell synchronization as far
as the cells are exposed to periodic L:D cycles.

RITMO1 obliteration affects
fluorescence rhythmicity under free-
running conditions

Previous study in *P. tricornutum*
showed that cells pre-adapted in L:D cycles
and subsequently released under constant light
maintain cell fluorescence rhythms for several
days, suggesting a possible regulation of these
rhythms by a circadian clock (Annunziata et
al., 2019). To test the robustness of these
rhythms, we here first analysed cellular
fluorescence profiles in wild-type cells adapted
to 16L:8D cycles of 25 µmol photons m⁻² s⁻¹ and
then exposed to free-run conditions either of
continuous dark (Fig. 3A), or continuous light
of different light intensities (5, 17 or 25 µmol

photons m⁻² s⁻¹) for four days (Fig 3B-D). The
transition to continuous darkness resulted in
the total flattening of fluorescence oscillations
after the first subjective night, which is
consistent with the arrest of cell cycle
progression observed in *P. tricornutum* cells
exposed to prolonged darkness (Huysman et
al., 2010). In case of the transition to constant
low light condition (from 16L:8D 25 µmol
photons m⁻² s⁻¹ to L:L of 5 µmol photons m⁻² s⁻¹,
Fig. 3B), the cell continued to divide over the
days considered for the analysis, but to a
strongly reduced rate (around 0.22
division/day in L:L compared to 1
division/day before the transition (Table S2).
In none of the biological replicates, the cells
passed the periodicity EPR test (Materials and
Methods), suggesting a possible
desynchronization of the whole population
under light irradiance potentially limiting for
the metabolism. On the contrary, in the case
of cells exposed to 17 µmol photons m⁻² s⁻¹ L:L

<figure><img src="image_53.png" /><figcaption>Figure 3. Characterization of fluorescence rhythmicity of wild-type *P. tricornutum* cells after the transition from L:D cycles to free running conditions.</figcaption></figure>

A-D) Cellular fluorescence profiles after transition from 16L:8D 25µmol photons m⁻² s⁻¹ to different free-run conditions over four subjective days: A= D:D, B= L:L 5µmol photons m⁻² s⁻¹, C= L:L 17µmol photons m⁻² s⁻¹, D= L:L 25µmol photons m⁻² s⁻¹, n≥4). (E) Cellular fluorescence profiles after transition from 16L:8D 75µmol photons m⁻² s⁻¹ to L:L of 50µmol photons m⁻² s⁻¹ (n=7) and (F) to L:L 75µmol photons m⁻² s⁻¹ (L, n=4) over four subjective days. Dots represent mean B3-A values, coloured ribbons represent SD. Gray dashed regions represent subjective nights in free-run conditions.