Conclusion

The line of work here presented has
allowed on the one hand a preliminary
evaluation of an on-plate screening method for
the identification of transgenic lines altered at
the level of photosynthetic physiology. A large-
scale phenotypic screening strategy had never
been tested on diatoms, and the development
and refinement of such a protocol would be a
useful tool for the search for photosynthesis
mutants in these algae. The results generated
on transgenic lines presenting a construct for
RNA silencing gave mixed results. A relatively
high percentage of lines demonstrated
phenotypic alterations at the level of growth or
quantum yield of photosynthesis in plate-
grown cells, for which confirmation in liquid
was possible for the *bHLH2* line. These
limitations may be attributed in part to the fact
that growth on solid medium is not
physiologically optimal for *P. tricornutum*, as
demonstrated by the variability in
photosynthetic capacity shown by multiple
independent control lines only containing the
resistance cassette for phleomycin antibiotic.
While these preliminary tests have
demonstrated the possible limitations of a
plate screen in *P. tricornutum*, they also
provide useful information that can serve to
refine this technique in the future to make it
eventually applicable.

Secondly, the results reported here on
the *bHLH2* RNAi lines are consistent with an
implication of this protein in managing the
dynamic response of the plastid to light
changes and irradiation exposures capable of
affecting PSII stability. Physiological
perturbations of *bHLH2* RNAi lines converge
toward the description of *bHLH2* as a
regulator of *P. tricornutum* plastid physiology,
possibly via the modulation of PSII repair and
plastid protein resynthesis. Given that
predictions of cellular localization indicate that
*bHLH2* is targeted to the nucleus and not to
the plastid, regulation of photosynthetic

activity likely passes through regulation of
nuclear genes subsequently targeted to the
thylakoid. What these genes are and the more
general structure of the regulatory gene
network managed by *bHLH2* remain
unexplored. The light intensity dependence of
the observed phenotypes is probably
attributable to the fact that the role of *bHLH2*
is particularly important under conditions of
light stress, where an impairment of the repair
of photosynthetic complexes is more relevant
in the face of more rapid photodegradation.
Involvement in such a function is consistent
with the expression profiles shown by the
*bHLH2* gene in Wt lines, which is repressed
by the activity of the photosynthetic apparatus
and maximized following dark exposure. A
similar type of regulation agrees with a role for
*bHLH2* in the response to transitions from
dark to light, where the photosynthetic
apparatus must be more ready to respond to a
sudden change in received irradiance. *bHLH2*
could also act as a negative regulator of
proteins implied in the photosynthetic
processes and its repression in the light by
plastid signalling could be necessary for a fine
modulation of chloroplast activity during
variable light stress conditions.

At present, it has not been possible to
characterize in detail the photosynthetic
properties of *bHLH2* RNAi lines during these
dark-to-light transitions also because of the
lack of a clear deregulation profile of gene
expression in these lines under these
conditions, which would impede to properly
correlate possible phenotypes with the mRNA
level itself. Although this lack of alteration of
transcript levels has already been observed in
other RNAi lines for other genes (Lavaud et
al., 2012), this aspect has prevented us from
proposing a clear correlation between *bHLH2*
expression levels and the observed
phenotypes. For this reason, the newly
generated KO lines will represent an
extremely useful tool to verify the results
obtained on RNAi lines and to subsequently
delineate more precisely the effects of
obliteration of this gene during dark-to-light
transitions, in which bHLH2 likely plays its
role. Should the KO lines lead to confirmation
of the above data, bHLH2 would result as the
first example of a bHLH-PAS protein
implicated in the regulation of photosynthetic
processes. This family of transcription factors
is in fact absent in primary endosymbiont
phototrophs and therefore probably originates
from the heterotrophic ancestor from which
*Stramenopila* emerged. This would underline
how the regulation of chloroplast activity in

secondary endosymbionts is not based only on
the action of genes from the endosymbiont but
has also co-opted genes already present in the
host before the symbiosis. Such a discovery
would therefore open up new questions and
new lines of study on the complex evolution of
the regulation of photosynthesis in
*Stramenopila* and other secondary
endosymbionts.
Bibliography

Almagro Armenteros, J.J., Sønderby, C.K., Sønderby, S.K., Nielsen, H., Winther, O., 2017. DeepLoc: prediction of protein subcellular localization using deep learning. Bioinformatics 33, 3387-3395. https://doi.org/10.1093/bioinformatics/btx431

Bailleul, B., Rogato, A., de Martino, A., Coesel, S., Cardol, P., Bowler, C., Falciatore, A., Finazzi, G., 2010. An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light. Proc. Natl. Acad. Sci. 107, 18214-18219. https://doi.org/10.1073/pnas.1007703107

Bajhaiya, A.K., Ziehe Moreira, J., Pittman, J.K., 2017. Transcriptional Engineering of Microalgae: Prospects for High-Value Chemicals. Trends Biotechnol. 35, 95-99. https://doi.org/10.1016/j.tibtech.2016.06.001

Brameier, M., Krings, A., MacCallum, R.M., 2007. NucPred Predicting nuclear localization of proteins. Bioinformatics 23, 1159-1160. https://doi.org/10.1093/bioinformatics/btm066

Buck, J.M., Sherman, J., Bártulos, C.R., Serif, M., Halder, M., Henkel, J., Falciatore, A., Lavaud, J., Gorbunov, M.Y., Kroth, P.G., Falkowski, P.G., Lepetit, B., 2019. Lhcx proteins provide photoprotection via thermal dissipation of absorbed light in the diatom Phaeodactylum tricornutum. Nat. Commun. 10, 4167. https://doi.org/10.1038/s41467-019-12043-6

Campbell, D.A., Tyystjärvi, E., 2012. Parameterization of photosystem II photoinactivation and repair. Biochim. Biophys. Acta BBA - Bioenerg. 1817, 258-265. https://doi.org/10.1016/j.bbabio.2011.04.010

Chauton, M.S., Winge, P., Brembu, T., Vadstein, O., Bones, A.M., 2013. Gene Regulation of Carbon Fixation, Storage, and Utilization in the Diatom *Phaeodactylum tricornutum* Acclimated to Light/Dark Cycles. Plant Physiol. 161, 1034-1048. https://doi.org/10.1104/pp.112.206177

Daboussi, F., Leduc, S., Maréchal, A., Dubois, G., Guyot, V., Perez-Michaut, C., Amato, A., Falciatore, A., Juillerat, A., Beurdeley, M., Voytas, D.F., Cavarec, L., Duchateau, P., 2014. Genome engineering empowers the diatom Phaeodactylum tricornutum for biotechnology. Nat. Commun. 5, 3831. https://doi.org/10.1038/ncomms4831

De Riso, V., Raniello, R., Maumus, F., Rogato, A., Bowler, C., Falciatore, A., 2009. Gene silencing in the marine diatom Phaeodactylum tricornutum. Nucleic Acids Res. 37, e96-e96. https://doi.org/10.1093/nar/gkp448

Eberhard, S., Finazzi, G., Wollman, F.-A., 2008. The Dynamics of Photosynthesis. Annu. Rev. Genet. 42, 463-515. https://doi.org/10.1146/annurev.genet.42.110807.091452

Falciatore, A., Casotti, R., Leblanc, C., Abrescia, C., Bowler, C., 1999. Transformation of Nonselectable Reporter Genes in Marine Diatoms. Mar. Biotechnol. 1, 239-251. https://doi.org/10.1007/PL00011773

Giordano, B., Epistole italiane. Epistole italiane. Epistole italiane.

Giovagnetti, V., Jaubert, M., Shukla, M.K., Ungerer, P., Bouly, J.-P., Falciatore, A., Ruban, A.V., 2022. Biochemical and molecular properties of LHCX1, the essential regulator of dynamic photoprotection in diatoms. Plant Physiol. 188, 509-525. https://doi.org/10.1093/plphys/kiab425

Gruber, A., Rocab, G., Kroth, P.G., Armbrust, E.V., Mock, T., 2015. Plastid proteome prediction for diatoms and other algae with secondary plastids of the red lineage. Plant J. 81, 519-528. https://doi.org/10.1111/tpj.12734
Gschloessl, B., Guermeur, Y., Cock, J.M., 2008. HECTAR: A method to predict subcellular targeting in heterokonts. BMC Bioinformatics 9, 393. https://doi.org/10.1186/1471-2105-9-393

Guillard, R.R.L., 1975. Culture of Phytoplankton for Feeding Marine Invertebrates. Cultures of Marine invertebrate Animals.

Horton, P., Park, K.-J., Obayashi, T., Fujita, N., Harada, H., Adams-Collier, C.J., Nakai, K., 2007. WoLF PSORT: protein localization predictor. Nucleic Acids Res. 35, W585-W587. https://doi.org/10.1093/nar/gkm259

Huang, W., Daboussi, F., 2017. Genetic and metabolic engineering in diatoms. Philos. Trans. R. Soc. B Biol. Sci. 372, 20160411. https://doi.org/10.1098/rstb.2016.0411

Kautsky, H., 1960. Chlorophyll-fluorescenz und Kohlensaureassimilation. XIII. Mitteilung. Die Fluorescenzkurve und die Photochemie der Pflanze. Biochem Z 332, 277-292.

Kilian, O., Kroth, P.G., 2004. Identification and characterization of a new conserved motif within the presequence of proteins targeted into complex diatom plastids: Protein targeting into diatom plastids. Plant J. 41, 175-183. https://doi.org/10.1111/j.1365-313X.2004.02294.x

Kleine, T., Voigt, C., Leister, D., 2009. Plastid signalling to the nucleus: messengers still lost in the mists? Trends Genet. 25, 185-192. https://doi.org/10.1016/j.tig.2009.02.004

Kyle, D.J., 1985. THE 32000 DALTON Q B PROTEIN OF PHOTOSYSTEM II. Photochem. Photobiol. 41, 107-116. https://doi.org/10.1111/j.1751-1097.1985.tb03456.x

Lavaud, J., Goss, R., 2014. The Peculiar Features of Non-Photochemical Fluorescence Quenching in Diatoms and Brown Algae, in: Demmig-Adams, B., Garab, G., Adams III, W., Govindjee (Eds.), Non-Photochemical Quenching and Energy Dissipation in Plants, Algae and Cyanobacteria, Advances in Photosynthesis and Respiration. Springer Netherlands, Dordrecht, pp. 421-443. https://doi.org/10.1007/978-94-017-9032-1_20

Lavaud, J., Materna, A.C., Sturm, S., Vugrinec, S., Kroth, P.G., 2012. Silencing of the Violaxanthin De-Epoxidase Gene in the Diatom Phaeodactylum tricornutum Reduces Diatoxanthin Synthesis and Non-Photochemical Quenching. PLoS ONE 7, e36806. https://doi.org/10.1371/journal.pone.0036806

Lavaud, J., Six, C., Campbell, D.A., 2016. Photosystem II repair in marine diatoms with contrasting photophysiologies. Photosynth. Res. 127, 189-199. https://doi.org/10.1007/s11120-015-0172-3

Lepetit, B., Sturm, S., Rogato, A., Gruber, A., Sachse, M., Falciatore, A., Kroth, P.G., Lavaud, J., 2013. High Light Acclimation in the Secondary Plastids Containing Diatom *Phaeodactylum tricornutum* is Triggered by the Redox State of the Plastoquinone Pool. Plant Physiol. 161, 853-865. https://doi.org/10.1104/pp.112.207811

Levitan, O., Dinamarca, J., Zelzion, E., Lun, D.S., Guerra, L.T., Kim, M.K., Kim, J., Van Mooy, B.A.S., Bhattacharya, D., Falkowski, P.G., 2015. Remodeling of intermediate metabolism in the diatom *Phaeodactylum tricornutum* under nitrogen stress. Proc. Natl. Acad. Sci. 112, 412-417. https://doi.org/10.1073/pnas.1419818112

Lin, J., Hu, J., 2013. SeqNLS: Nuclear Localization Signal Prediction Based on Frequent Pattern Mining and Linear Motif Scoring. PLoS ONE 8, e76864. https://doi.org/10.1371/journal.pone.0076864

Livak, K.J., Shcmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2^ΔΔCT^ method.pdf. METHODS 25, 402-408.

Long, M., Peltekis, A., González-Fernández, C., Hégaret, H., Bailleul, B., 2021. Allelochemicals of Alexandrium minutum: Kinetics of membrane disruption and photosynthesis inhibition in a co-occurring diatom. Harmful Algae 103, 101997. https://doi.org/10.1016/j.hal.2021.101997

Matthijs, M., Fabris, M., Broos, S., Vyverman, W., Goossens, A., 2016. Profiling of the Early Nitrogen Stress Response in the Diatom *Phaeodactylum tricornutum* Reveals a Novel Family
of RING-Domain Transcription Factors. Plant Physiol. 170, 489-498.
https://doi.org/10.1104/pp.15.01300

Maxwell, K., Johnson, G.N., 2000. Chlorophyll fluorescence—a practical guide. J. Exp. Bot. 51, 659-668.

Metz, J.G., Pakrasi, H.B., Seibert, M., Arntzer, C.J., 1986. Evidence for a dual function of the herbicide-binding D1 protein in photosystem II. FEBS Lett. 205, 269-274.
https://doi.org/10.1016/0014-5793(86)80911-5

Moustafa, A., Beszteri, B., Maier, U.G., Bowler, C., Valentin, K., Bhattacharya, D., 2009. Genomic Footprints of a Cryptic Plastid Endosymbiosis in Diatoms. Science 324, 1724-1726.
https://doi.org/10.1126/science.1172983

Müller, P., Li, X.-P., Niyogi, K.K., 2001. Non-Photochemical Quenching. A Response to Excess Light Energy. Plant Physiol. 125, 1558-1566. https://doi.org/10.1104/pp.125.4.1558

Nguyen Ba, A.N., Pogoutse, A., Provart, N., Moses, A.M., 2009. NLStradamus: a simple Hidden Markov Model for nuclear localization signal prediction. BMC Bioinformatics 10, 202.
https://doi.org/10.1186/1471-2105-10-202

Niyogi, K.K., Bjorkman, O., Grossman, A.R., 1997. Chlamydomonas Xanthophyll Cycle Mutants Identified by Video Imaging of Chlorophyll Fluorescence Quenching. Plant Cell 9, 1369-1380.
https://doi.org/1369-1380

Nymark, M., Valle, K.C., Hancke, K., Winge, P., Andresen, K., Johnsen, G., Bones, A.M., Brembu, T., 2013. Molecular and Photosynthetic Responses to Prolonged Darkness and Subsequent Acclimation to Re-Illumination in the Diatom Phaeodactylum tricornutum. PLoS ONE 8, e58722.
https://doi.org/10.1371/journal.pone.0058722

Oudot-Le Secq, M.-P., Grimwood, J., Shapiro, H., Armbrust, E.V., Bowler, C., Green, B.R., 2007. Chloroplast genomes of the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana: comparison with other plastid genomes of the red lineage. Mol. Genet. Genomics 277, 427-439.
https://doi.org/10.1007/s00438-006-0199-4

Ozaki, H., Ikeuchi, M., Ogawa, T., Fukuzawa, H., Sonoike, K., 2007. Large-Scale Analysis of Chlorophyll Fluorescence Kinetics in Synechocystis sp. PCC 6803: Identification of the Factors Involved in the Modulation of Photosystem Stoichiometry. Plant Cell Physiol. 48, 451-458.
https://doi.org/10.1093/pcp/pcm015

Perin, G., Bellan, A., Segalla, A., Meneghesso, A., Alboresi, A., Morosinotto, T., 2015. Generation of random mutants to improve light-use efficiency of Nannochloropsis gaditana cultures for biofuel production. Biotechnol. Biofuels 8, 161.
https://doi.org/10.1186/s13068-015-0337-5

Phaeodactylum_tricornutum - Ensembl Genomes 52 [WWW Document], n.d. URL
http://protists.ensembl.org/Phaeodactylum_tricornutum/Info/Index (accessed 3.3.22).

Platt, T., Gallegos, C.L., Harrison, W.G., 1980. Photoinhibition of photosynthesis in natural assemblages of marine phytoplankton. J. Mar. Res. 38, 9.
https://doi.org/10.1093/pasj/57.2.341

Ralph, P.J., Gademann, R., 2005. Rapid light curves: A powerful tool to assess photosynthetic activity. Aquat. Bot. 82, 222-237.
https://doi.org/10.1016/j.aquabot.2005.02.006

Rayko, E., Maumus, F., Maheswari, U., Jabbari, K., Bowler, C., 2010. Transcription factor families inferred from genome sequences of photosynthetic stramenopiles. New Phytol. 188, 52-66.
https://doi.org/10.1111/j.1469-8137.2010.03371.x

Serif, M., Dubois, G., Finoux, A.-L., Teste, M.-A., Jallet, D., Daboussi, F., 2018. One-step generation of multiple gene knock-outs in the diatom Phaeodactylum tricornutum by DNA-free genome editing. Nat. Commun. 9, 3924.
https://doi.org/10.1038/s41467-018-06378-9
Siaut, M., Heijde, M., Mangogna, M., Montsant, A., Coesel, S., Allen, A., Manfredonia, A., Falciatore, A., Bowler, C., 2007. Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum. Gene 406, 23-35. https://doi.org/10.1016/j.gene.2007.05.022

Smith, S.R., Gillard, J.T.F., Kustka, A.B., McCrow, J.P., Badger, J.H., Zheng, H., New, A.M., Dupont, C.L., Obata, T., Fernie, A.R., Allen, A.E., 2016. Transcriptional Orchestration of the Global Cellular Response of a Model Pennate Diatom to Diel Light Cycling under Iron Limitation. PLOS Genet. 12, e1006490. https://doi.org/10.1371/journal.pgen.1006490

Sperschneider, J., Catanzariti, A.-M., DeBoer, K., Petre, B., Gardiner, D.M., Singh, K.B., Dodds, P.N., Taylor, J.M., 2017. LOCALIZER: subcellular localization prediction of both plant and effector proteins in the plant cell. Sci. Rep. 7, 44598. https://doi.org/10.1038/srep44598

Taddei, L., Stella, G.R., Rogato, A., Bailleul, B., Fortunato, A.E., Annunziata, R., Sanges, R., Thaler, M., Lepetit, B., Lavaud, J., Jaubert, M., Finazzi, G., Bouly, J.-P., Falciatore, A., 2016. Multisignal control of expression of the LHCX protein family in the marine diatom *Phaeodactylum tricornutum*. J. Exp. Bot. 67, 3939-3951. https://doi.org/10.1093/jxb/erw198

Trebst, A., 2007. Inhibitors in the functional dissection of the photosynthetic electron transport system. Photosynth. Res. 92, 217-224. https://doi.org/10.1007/s11120-007-9213-x

Valle, K.C., Nymark, M., Aamot, I., Hancke, K., Winge, P., Andresen, K., Johnsen, G., Brembu, T., Bones, A.M., 2014. System responses to equal doses of photosynthetically usable radiation of blue, Green, and red light in the marine diatom *phaeodactylum tricornutum*. PLoS ONE 9, 1-37. https://doi.org/10.1371/journal.pone.0114211

van Kooten, O., Snel, J.F.H., 1990. The use of chlorophyll fluorescence nomenclature in plant stress physiology. Photosynth. Res. 25, 147-150. https://doi.org/10.1007/BF00033156
# Chapter IV:

Conclusions
and
perspectives
In spite of diatom ecological relevance, very little is known on the basic regulatory processes controlling gene expression in these organisms. *In silico* predictions of transcription factors have already been carried out in various diatom species (Rayko et al., 2010). However, a functional characterization of these elements is largely missing so far, with the exception of few studies (Matthijs et al., 2017, 2016). Likewise, the specific binding sites of diatom TFs, their interaction in gene regulatory networks (GRN) and the mechanisms behind the precise temporal regulation of gene expression are mostly undetermined. Furthermore, this kind of information cannot be easily extrapolated by comparative genomic analysis considering the divergence of diatom genomes in respect to other established plant and animal model systems (Blaby-Haas and Merchant, 2019; Keeling, 2013). With the work presented here, I contributed to address these questions by partaking in the functional characterization of three putative transcription factors of the model diatom *P. tricornutum*, named bHLH1a (also known as RITMO1), bHLH1b and bHLH2. The common elements shared by these proteins are the presence in their structure of a bHLH and a PAS domains (Rayko et al., 2010). Notably, before being identified in *Stramenopila*, transcription factors containing both bHLH and PAS were thought to be a peculiarity of metazoans. The work currently carried out therefore represents the first effort ever made to study this family of proteins in a photosynthetic organism.

Specifically, the main body of my thesis work focused on studying the role of bHLH1a in regulating the biological rhythmicity of *P. tricornutum*. At the time I began my research work in the lab, the study of the molecular regulators that control the internal clock of diatoms was still lacking in literature. The totality of studies carried out on rhythmic processes in these algae had previously focused on the analysis of

physiological and metabolic processes, such as migration in sediments in the case of benthic intertidal diatoms or metabolic processes associated with photosynthesis (Brand, 1982; Happey-Wood and Jones, 1988; Palmer et al., 1964; Palmer and Round, 1967). An in-depth overview of these studies and similar works on other marine algae is provided in Chapter I and Chapter II.a. However, these studies were very few and, although they had begun to provide initial indications that diatoms possess an endogenous oscillator capable of maintaining free-running biological rhythmicity, no study had ever addressed the regulators underlying these phenomena. Understanding how the molecular life of diatoms is synchronized with environmental rhythms is, however, essential both from an ecological point of view and more generally to be able to make the most of these organisms in basic research. For this reason, before my arrival, the host laboratory had already conducted a preliminary search for transcription factors that present the typical features of the components of a transcriptional clock and that could therefore represent good candidate components of such a system. These works led to identify bHLH1a as a first candidate and to demonstrate how its deregulation leads to an alteration of gene expression rhythmicity in *P. tricornutum*, the reason why this protein has been renamed RITMO1. In this early work, reported in Chapter II.b, one of my roles was to characterize how overexpression of this protein influences the expression of a number of target genes under light-dark cycles. Additional steps were still needed to prove RITMO1 function as a regulator of cellular rhythmicity. Indeed, once a possible molecular clock component has been identified, it is critical to implement a thorough characterization of how its mutation or deregulation impacts cellular rhythms. One of the most commonly exploited strategies for this purpose is to transform the organism under study with a luciferase placed under the control of a rhythmic promoter and to follow
its expression by imaging its bioluminescence. Such a protocol is the basis of major work on clock characterization in organisms such as *Arabidopsis thaliana* (Millar et al., 1995), *Chlamydomonas reinhardtii* (Matsuo et al., 2008), *Ostreococcus tauri* (Corellou et al., 2009) or, more recently, *Nannochloropsis spp.* (Poliner et al., 2019). To date, however, attempts to obtain similar transformed lines to follow *in vivo* luciferase activity have been successful only using strong constitutive promoters for the expression of luciferase (Seo et al., 2015), but have never been reproduced with promoters of rhythmic genes. Thus, it became necessary to develop alternative techniques to be able to follow the cellular rhythmicity in these algae effectively and with minimal human intervention. The possibility to follow long time courses with a non-labor-intensive set up enormously facilitated the characterization of possible clock mutants. One of the first important results of my work was thus to optimize the strategy based on the analysis of cellular fluorescence via flow cytometry. This system showed to be an efficient and straightforward method to collect and study large amounts of data for prolonged circadian experiments, all requiring minimal human intervention for the sampling process. Globally, chlorophyll-related fluorescence clearly emerged as a useful tool for studying daily rhythmicity in *P. tricornutum* and became the backbone of the characterisation of the clock presented in Chapters II.b, II.c and II.d.

This technique also allows fluorescence to be used as a proxy for cell division (Chapter II.b and II.c) and had me demonstrate how *P. tricornutum* adapts its division to the growth photoperiod, setting the timing of division based on the length of light periods as reported in Chapter II.c. I was also able to show that rhythmic cellular fluorescence persists following release into continuous light, indicating that cellular fluorescence, as well as likely cell cycle regulation, are controlled by an endogenous

clock. In unicellular organisms that exhibit a growth rate close to one division per day, such persistence of a circadian rhythmicity of cell division can be attributed to a simple persistence of this daily division rate following release into continuous light, without it actually being regulated by an endogenous pacemaker. In such a case, one would expect that in continuous light the doubling time and the free run period of cell division (or, in this case, fluorescence oscillations) should coincide perfectly. As reported in Chapter II.c the persistence of cellular fluorescence however has a free running period of 29h in continuous light even when the doubling time is less than 24h. This might imply that the internal circadian clock rhythmically defines the time when cells have the opportunity to divide, both in the presence of dark light cycles and after release into continuous light. Importantly, when light gets too high, the persistence of rhythms is lost, possibly because the metabolic inputs play a role in making the oscillations of the clock visible. On the contrary, when light is too low, oscillations are equally lost, simply because rhythms cannot be present if cells are not dividing. Such a regulation of the circadian phase in which cell division is "allowed" has already been observed in the past for unicellular algae with an infradian doubling periodicity, both eukaryotic (e.g. *Chlamydomonas reinhardtii*, (Goto and Johnson, 1995), *Euglena gracilis*, (Carré and Edmunds, 1993) or *Ostreococcus tauri*, (Moulager et al., 2007) and prokaryotic cyanobacteria (see Mori et al., 1996). Although the data collected so far point in this direction, at the moment it is not yet possible to definitively conclude whether indeed the endogenous clock of *P. tricornutum* guarantees such a gating of cell division. In order to obtain a conclusive answer, the next analysis that will be important to perform in this direction is a phase response curve of cell division as a function of transitions from dark to light, similar to what has already been done in *C. reinhardtii* (Goto and Johnson, 1995) or in *O. tauri* (Moulager et al., 2007).
Unfortunately, due to a lack of time it has not yet been possible for me to perform this test, but this analysis will address if under specific conditions the timing of cell division in *P. tricornutum* is dependent on clock control and not on a simple metabolic response to light exposure.

However, cellular fluorescence analysis was the kickstarter for the characterization of the *P. tricornutum* clock and is the tool that allowed us to demonstrate how RITMO1 is directly implicated in the regulation of cellular rhythmicity. Analysis of over-expressing lines before (Chapter II.b) and KO lines after (Chapter II.c) for this protein allowed me to confirm how its perturbation radically alters the persistence of fluorescence oscillations in free run. Once these data were obtained, it was then possible to further characterize the rhythmic physiology in these cell lines with more targeted techniques.

In particular, one of the most important results of my thesis was the demonstration that perturbation of RITMO1 expression almost completely disrupts the rhythms of gene expression in cells released in free run. Although several papers had already probed the rhythmicity of gene expression in *P. tricornutum* cells subjected to different rhythms of light and dark (Chauton et al., 2013; Smith et al., 2016), at the beginning of my Ph.D. it was still unexplored whether such gene expression oscillations could persist following release in continuous light. With my work I not only demonstrated the existence of such persistence, but I was also able to show how RITMO1 plays a crucial role in this phenomenon. This reflects the function of RITMO1 as a transcription factor In addition, the generalized deregulation of gene transcription rhythmicity observed implies that RITMO1 regulates cellular rhythmicity over a broad spectrum, not solely controlling cellular fluorescence, thus favoring its place at the heart of the endogenous regulator. At the

moment, the results obtained have been limited to a reduced set of genes selected and analyzed by real time qRT-PCR, but the generalized alteration of transcription profiles observed indicate that this deregulation is to be referred to cellular gene rhythmicity in a broad sense. In addition, these analyses allowed to determine the optimal conditions to conduct a genome-wide transcriptomic study that will attest the effect of the mutation of RITMO1 on the global transcriptome. Such an analysis is being planned and will involve sampling of Wt and KO lines at time points comparable to those used for the experiments in Chapter II.c.

Another important result was the observation of how the rhythmic photosynthetic activity of *P. tricornutum*, is also affected by RITMO1 mutagenesis, while in Wt lines it maintains a periodicity following free-run release, as reported in Chapter II.c. These results demonstrate the importance, also in diatoms, of controlling the timing of plastid activity, a crucial factor to better exploit ambient light in a natural context. It will be interesting, in the future, to conduct more in-depth molecular and biophysical analyses to determine which step(s) of photosynthetic metabolism are actually regulated by the transcriptional clock in which RITMO1 is involved. In plants, it has been widely demonstrated that the clock regulates plastid metabolism through the cyclic expression of genes involved in both light-dependent and biochemical phases of photosynthesis such as the Calvin Benson Bassham cycle (Espinoza et al., 2010; Farré and Weise, 2012; Filichkin et al., 2011).

In plants, the circadian clock has also been shown to be involved in the modulation of the cyclic expression of thioredoxins f and m, fundamental redox-dependent regulators of Calvin Benson Bassham cycle activity, to the point of being considered among the possible central mediators of the interaction between clock and metabolism (Barajas-
López et al., 2011). These enzymes are ubiquitous in all organisms and diatoms possess several copies of them, some of which are targeted in the plastid (Weber et al., 2009). It will be relevant to verify if a similar regulation of gene expression is conserved in *P. tricornutum* and at which level the mutation of RITMO1 alters its profile. An additional interesting aspect of the results obtained is that, in the wild-type line, oscillations at the level of photosynthetic activity as well as gene expression seem to present a period close to 24 hours in free running light. On the contrary, the oscillations of cellular fluorescence present a period of about 28-29 hours in the same conditions as shown in chapter II.c. The analyses carried out so far do not allow to calculate precisely the free running period of these two other clock outputs, as they could be conducted only over a day and a half for technical limitations. If future analyses confirm them, these observations could indicate that a different level of control to the circadian clock influences fluorescence oscillations and, potentially, cell division.

More in general, studies on the rhythmic regulation of metabolism in diatoms are still at their beginnings, but some studies allow us to advance some initial hypotheses. It has already been observed, in fact, that as in plants also in *P. tricornutum* the genes associated with the tricarboxylic acids (TCA) cycle have an important rhythmic regulation (Matthijs et al., 2017). Remarkably, it has been observed that a transcription factor of the bZIP family, bZIP14, is involved in the regulation of carbon metabolism via the TCA cycle and that it also shows a cyclic expression (Matthijs et al., 2017). Under Light-Dark cycles, this protein is expressed at the end of the day and peaks its expression after dusk, so that it's regulation has been proposed to be modulated by the endogenous oscillator (Matthijs et al., 2017). It is therefore plausible that the regulation of metabolism via the circadian clock has a key node in the cycle of

tricarboxylic acids. The importance of probing the links between circadian clock and carbon metabolism is also marked by the fact that bZIP14 and RITMO1 appear to be the transcription factors most induced in response to nitrogen deprivation (Matthijs et al., 2017), a condition in which the TCA cycle is itself upregulated. At present, we have not yet been able to explore this subject, but it will certainly be relevant in the future.

Overall, from what has been observed, it is clear that RITMO1 is the first identified component of the endogenous diatom clock. However, it is fundamental to consider that in transcriptional clock systems proteins play a key role in gene expression regulation in physical interaction networks, with protein complexes assembled during the daily cycle and acting in coordination to activate or repress transcription (Creux and Harmer, 2019; Takahashi, 2017; Wijnen and Young, 2006). With my work I hence contributed to expand our comprehension on the molecular clock of diatoms by starting the identification of RITMO1 partners. As presented in Chapter II.d, I used a Yeast two Hybrid approach to demonstrate how RITMO1 can interact with its paralogous protein bHLH1b through their bHLH fold thus adding a new piece to the puzzle of *P. tricornutum* timekeeper. The implication of this second bHLH-PAS protein in the clock system was further confirmed by the generation of KO mutants for this protein which showed a deregulation of fluorescence persistence in free run similar to that presented by mutants lacking RITMO1. However, the phenotypes manifested by bHLH1b KO mutants appeared to be weaker than in RITMO1 mutants, possibly implying that these two proteins have non completely superposing functions. As reported in Chapter II.b and II.c, the perturbation of *RITMO1* expression led to an alteration of *bHLH1b* transcription, meaning that these two proteins control their own expression, possibly via their heterodimer. This phenomenon is logical
considering the feedback loop structure of
clock machinery. It will be relevant in future to
verify to what extent the obliteration of
bHLH1b leads to similar deregulation of its
RITMO1 counterpart.

The identification of bHLH1b as a partner of RITMO1 clearly represents only the first step toward the delineation of the *P. tricornutum* clock structure. It is plausible that proteins that physically interact with these two TFs to regulate their activity are numerous, and their identification represents a strategy for the future discovery of novel components of the endogenous clock. As seen in Chapter II.d, RITMO1 is capable of autonomously recruiting the transcriptional machinery in *Saccharomyces cerevisiae* and thereby inducing expression of the *His3* reporter. This makes a screening strategy for yeast two hybrids of RITMO1 partners inapplicable. Furthermore, although Y2H strategies have already been exploited in the past on other organisms for characterizing the interactome of transcription factors (e.g. (Gunther et al., 2000; Mitsuda et al., 2010), this technique is limited by the fact that it's really labor intensive and the fact that the interactions are observed in a yeast model and not *in vivo* in the species under study. Indeed, analysis of protein interactions would be limited by the impossibility to control post translational modifications, which can play a relevant role in determining dimerization properties, as already discussed in Chapter II.d. For this reason, alternative *in vivo* proteomics methods will be used for this purpose. Specifically, it is planned to perform a co-immunoprecipitation analysis to define which other proteins physically interact with these two transcription factors. To this end, KO mutants of RITMO1 have been complemented by transformation with an exogenous copy of the gene modified with an HA tag and, in independent lines, with fluorescent protein tags. These complemented lines will be used to efficiently precipitate RITMO1 and its interaction partners by commercial antibodies directed

against the tag, all in a genetic background
deprived of the endogenous copy of the gene.
The same lines are also currently being
analyzed to see if the exogenous copy of
RITMO1 restores proper cellular rhythmicity.
Such confirmation would further confirm the
involvement of RITMO1 in the *P. tricornutum* clock. Similarly, it is planned to
perform complemented transgenic lines and
similar analysis also in bHLH1b mutants, so
as to conduct in parallel the characterization of
the two transcription factors.

The fact that RITMO1 and bHLH1b
mutants both exhibit a free-run period
shortening outlines their possible partial
redundancy in the generation of endogenous
rhythms in *P. tricornutum*. The same is given
by the fact that the two genes coding for these
proteins share their 5' regulatory regions,
suggesting likely co-modulation of gene
expression by other transcriptional regulators.
Which are these regulators, however, still
remains an unanswered question. Recent
transcriptomics data generated on
aureochrome 1a KO mutants show that the
expression of RITMO1 and bHLH1b is
strongly induced upon exposure to blue light,
in a response profoundly altered by the
mutation of the photoreceptor (Mann et al.,
2020). This suggests that the two proteins are
likely implied in the regulatory network
involving aureochrome 1a. Whether this
occurs directly or indirectly remains to be
defined. The picture is made even more
complex to delineate because of the large
number of genes whose expression is
impacted by the aureochrome 1a mutation,
suggesting that the response to blue light
mediated by this photoreceptor is
fundamental to the physiology of *P. tricornutum* beyond the regulation of the
circadian clock (Mann et al., 2020). For this,
the laboratory is currently collaborating with
Peter Kroth's research group, Konstanz
University, to probe this point. Specifically, a
Yeast one Hybrid assay is being used to test
whether aureochrome 1a recognizes the
promoter sequences of RITMO1 and
bHLH1b, as well as other genes possibly
involved in the clock network. Such a setup
will give in the near future an answer to this
question. Protein binding microarrays,
Chromatin Immunoprecipitation and Yeast
one hybrid techniques could also be used to
identify the binding sites of RITMO1 and
bHLH1b transcription factors. This set of data
would shed a light on their targets, helping to
outline the GRN they are involved in.

Returning to the possible overlapping
role of RITMO1 and bHLH1b, similar partial
redundancy of factors involved in the clock
mechanisms have already been widely
demonstrated and studied in other organisms,
most notably in land plants. CCA1 and LHY
or the Pseudo Response Regulators 7 and 9
are the best-described cases of paralogous
proteins that exhibit similar co-implication in
the core of the endogenous oscillator (Alabadi
et al., 2002; Farré et al., 2005; Mizoguchi et al.,
2002; Salomé and McClung, 2005). Similar to
what I observed in KO mutants of RITMO1
and bHLH1b, single gene KOs for these
genes also manifest only partial alteration of
free-running rhythmicity, whereas dual CCA1-
LHY (Alabadi et al., 2002; Mizoguchi et al.,
2002) or PRR7-PRR9 (Farré et al., 2005;
Salomé and McClung, 2005) mutagenesis is
required to achieve large clock disruption and
possibly arrhythmicity. At present, a similar
characterization has not yet been conducted in
P. tricornutum, but mutagenesis techniques
for CRISPR/Cas9 are now well mastered
within the laboratory and KO mutant lines for
both these genes will make the generation of
double mutants straightforward.

Although the redundancy of function
of RITMO1 and its paralogue bHLH1b is
likely, it remains possible that these two
proteins have a set of gene targets that are not
totally overlapping and may therefore present
a different regulatory role. Indeed, as seen, I
was able to demonstrate not only

heterodimerization of RITMO1 with
bHLH1b, but also homodimerization of the
latter. The homodimerization of RITMO1 is
also possible, although it was not possible to
demonstrate it for the moment because of the
self-activation phenomenon shown by this
protein. The possibility that the different
dimers show a different preference for DNA
binding sites remains open, although this
remains purely speculative. Also, the results
obtained on the model species *P. tricornutum*
also allow for a broader understanding of the
entire diatom family. Indeed, the fact that
genes homologous to RITMO1 and bHLH1b
have been found in other diatoms (Bilcke et
al., 2021) allows us to assume that their
function is potentially conserved in other
species. The search for functional
homologues of RITMO1 and bHLH1b in
other diatoms will necessarily pass through a
careful analysis of the daily transcription of
these proteins, so as to look for those that
present profiles compatible with the
components of an oscillator. First studies in
this sense are beginning to emerge, as in the
case of a recent work on *Seminavis robusta*
that identified multiple candidate functional
homologues for these proteins with similar
expression profiles (Bilcke et al., 2021).

To conclude this overview on the
characterization of the diatom pacemaker, it is
important to emphasize that there are still
numerous aspects that need to be explored to
fully dissect its functioning. It will be
interesting to characterize the clock response
to light of different colors, an analysis that will
be useful to determine which photoreceptor(s)
are involved in the entrainment via the input
pathways. This study can be conducted with
two alternative strategies. On the one hand, it
will be possible to test which colors can entrain
the clock and ensure persistence of oscillations
after free run release. The cellular
fluorescence results reported in Chapter II.b
have already shown that Wt cells are capable
of maintaining proper cellular fluorescence
oscillation when released in free-running blue
light, but in these experiments the cells were
initially entrained in white light during light-
dark cycles. To determine precisely which
wavelengths affect the input pathways and
which do not, it will be necessary to perform
analyses using monochromatic light already.
In a second strategy, dissection of input
pathways could be accomplished by analyzing
endogenous rhythmicity in lines mutated for
different photoreceptors. The laboratory has
already developed KO lines for phytochrome
and equally possesses KO lines for the
different aureochromes of *P. tricornutum*.
These transgenic lines will allow a rapid
evaluation of the role of these proteins by
using the techniques already applied on
RITMO1 and bHLH1b mutants. Similar
analyses may also be performed on mutants of
other *P. tricornutum* photoreceptors (e.g.
CPF1) currently under development.
Through the analysis of gene expression
presented in Chapter II.c I have already
highlighted how these proteins are involved in
the regulatory network managed by RITMO1
and it will therefore be necessary to verify how
they, in turn, are able to influence the activity
of the clock.

Also, during my thesis work it was not
possible for me to verify the temperature
compensation properties of the oscillator of *P. tricornutum*. The maintenance of the correct
periodicity of endogenous rhythms under a
wide range of temperatures is, as presented in
the introduction, Chapter I.4.a, one of the
fundamental characteristics of the clock. This
property has already been recently analyzed in
*Nannochloropsis spp.*, also belonging to the
*Stramenopila*, where the rhythmicity of gene
expression persists as expected over a wide
range of temperatures (Poliner et al., 2019). A
complete clock dissection of *P. tricornutum*
will require similar analyses.

Also, the techniques and knowledge
gained so far pave the way for the study of the
ecological role of the clock in diatoms. As
reported in Chapter II.c, *P. tricornutum* is

capable of synchronizing its cellular
fluorescence rhythms in different
photoperiods. Correct response to different
light-dark cycles is a key factor for seasonal
adaptation of cellular physiology. However,
the analyses conducted so far have been
carried out on only one ecotype of *P. tricornutum*, *Pt1*, originating from temperate
regions (Blackpool, UK, 54°N). A
comparative analysis of cellular rhythmicity in
different ecotypes from different latitudes,
already available in the laboratory, will permit
to highlight possible geographical adaptations
to the seasonal photoperiods observed in
different environments. The characterization
of ecogeographic adaptations of the clock at
the level is a subject that is gaining increasing
interest in the field of chronobiology, in
animal as well as in plant, e.g. see (Beauchamp
et al., 2018; Greenham et al., 2017).
Moreover, diatoms are cosmopolitan algae
found at all latitudes and are the dominant
form of phytoplankton in the poles. In these
regions, the extreme variations of photoperiod
during the year open many questions about
the possible role and functioning of a circadian
clock and it will be therefore relevant if and
how the endogenous rhythmicity is maintained
in diatoms under these conditions. As
reported in Chapter II.b, homologs of
RITMO1 and bHLH1b are found in several
diatom species. Expanding the functional
study of its role in other species with different
geographic origins and lifestyles will help to
elucidate the properties of the timekeeper in
these algae.

Still, the use of the homologs of
RITMO1 and bHLH1b as entry points for the
potential study of circadian rhythms in other
diatom species will have to be conducted with
caution. Indeed, as shown extensively in
Chapter III, the functions of the bHLH-PAS
family members are not limited to the
regulation of endogenous rhythmicity. The
second important result of my thesis work was
in fact to characterize a set of transgenic lines
containing a construct for RNA mediated
silencing of the third member of the bHLH-PAS family of *P. tricornutum*, bHLH2. These lines exhibit a strong perturbation of photosynthetic activity. Similar alterations of photosynthesis were not found in RNAi lines in which silencing is directed toward the other two bHLH-PAS proteins, indicating that the phenotypes are specific to bHLH2. Thus, by further characterizing these transgenic lines, I was able to show that anti-bHLH2 antisense results in reduced electron transfer rates at the thylakoid level, with a defect that differential absorbance spectroscopy analysis suggests to be localized upstream of the PSI. The perturbations of the photosynthetic apparatus shown by these lines appear to be dependent on the intensity of light irradiation and lead to a defect in growth rate as light intensity increases. The main limitation of this work is that it has been so far impossible to demonstrate a significant and homogeneous reduction of the bHLH2 transcript level in RNAi transgenic lines, which prevents to date to definitively correlate the observed phenotypes to this protein. Therefore, the KO lines deprived of bHLH2 and described at the end of Chapter III will represent the important tool to confirm these results and definitively define bHLH2 as a modulator of photosynthetic activity. However, it will be important to determine whether complete obliteration of the activity of this protein is lethal under high light growth conditions or following dark to bright light transitions. As seen, *bHLH2* RNAi lines could not be grown under irradiation more intense than 200 µmol photons m⁻²s⁻¹. If the effects of KO are even more intense, this could lead to lower tolerance by the mutants of strong light levels. All of this will obviously need to be verified. Such a confirmation, would be of primary relevance for understanding chloroplast regulation in diatoms and this for multiple reasons. First, as already seen in Chapter I, Paragraphs I.3.a and I.3.b, the study of photosynthetic activity in these algae has focused essentially on proteins that are directly implicated in photochemical processes and

thylakoid photoprotection mechanisms. Although there are still many questions to be solved, an important amount of information has in fact been acquired, for example, on LHCX proteins and their implication in non-photochemical quenching (Bailleul et al., 2010; Taddei et al., 2016), on the structure and spatial distribution of antenna complexes (Flori et al., 2017, 2016; Wang et al., 2019) or on the control of carbon fixation at the level of the Calvin Benson Bassham cycle (Matsuda et al., 2017; Tsuji et al., 2017). What are the gene regulatory networks underlying this modulation of plastid activity remains an almost unexplored field. Therefore, bHLH2 would represent one of the first cases of diatom transcription factors directly involved in these processes and would thus provide a crucial entry point for the dissection of such networks.

Secondly, this protein presents an expression profile totally distinct from that shown by RITMO1 and bHLH1b and that allows us to exclude that it is predominantly controlled by the endogenous oscillator. bHLH2 shows in fact an expression that responds directly to light exposure and does not present an anticipation of transitions from light to dark or from dark to light. As I demonstrated in my analyses of dark-adapted cells subsequently treated with DCMU, bHLH2 expression is inhibited by the action of the photosynthetic apparatus in a retrograde signaling mechanism already observed in other *P. tricornutum* nuclear genes and probably mediated by the oxidoreductive state of the thylakoid plastoquinone pool (Lepetit et al., 2013; Taddei et al., 2016). For this, bHLH2 also represents an important starting point to dissect how these retrograde signaling mechanisms mechanically influence nuclear activity in diatoms. Although this is merely a speculation for the moment, given its expression profile inhibited by light and its function as a candidate transcription factor, bHLH2 could indeed represent a key link in this process and potentially be a central
element in this communication between the
two cellular compartments. For the same
reason, it will be of great interest to test
whether homologs of bHLH2 in other
diatoms exhibit the same expression profile
and, potentially, the same function. The
regulation of cell physiology in the dark and its
preparation for re-exposure to light is of
utmost importance for these algae. As
mentioned above, diatoms are abundant in
Polar Regions, where they are exposed to
prolonged darkness for months during polar
nights. Understanding how they regulate their
plastid activity upon dark to light transitions
may shed light on their ecological success.

As a final point, these results open
new questions on the evolution of
photosynthesis regulation in diatoms and,
more generally, in secondary endosymbionts.
Indeed, if the bHLH-PAS proteins of diatoms
originated from the heterotrophic ancestor
and not from the algae that gave rise to their
plastid, the function of bHLH2 would imply

that these organisms have co-opted genes
originally not involved in photosynthesis for
the control of the chloroplast, in a complex
process of evolution of gene regulation.
Whether bHLH2 is an isolated case or just an
example of a larger such process is an open
question. As already described for circadian
oscillator proteins, KO mutants of bHLH2
may also be used for genome-wide
transcriptomics, chromatin
immunoprecipitation and protein co-
immunoprecipitation analysis, so to identify
the genes whose expression is affected by its
obliteration and its binding partner proteins.

Overall, the work done opened a
window on bHLH-PAS containing proteins
never examined before in diatoms, which have
been demonstrated to be involved in the
control of two processes of crucial importance
for the life of these organisms and their
ecological success in the environment.
Bibliography

Alabadi, D., Yanovsky, M.J., Más, P., Harmer, S.L., Kay, S.A., 2002. Critical Role for CCA1 and LHY in Maintaining Circadian Rhythmicity in Arabidopsis. Curr. Biol. 12, 757-761.
https://doi.org/10.1016/S0960-9822(02)00815-1

Bailleul, B., Rogato, A., de Martino, A., Coesel, S., Cardol, P., Bowler, C., Falciatore, A., Finazzi, G., 2010. An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light. Proc. Natl. Acad. Sci. 107, 18214-18219.
https://doi.org/10.1073/pnas.1007703107

Barajas-López, J. de D., Serrato, A.J., Cazalis, R., Meyer, Y., Chueca, A., Reichheld, J.P., Sahrawy, M., 2011. Circadian regulation of chloroplastic f and m thioredoxins through control of the CCA1 transcription factor. J. Exp. Bot. 62, 2039-2051. https://doi.org/10.1093/jxb/erq394

Beauchamp, M., Bertolini, E., Deppisch, P., Steubing, J., Menegazzi, P., Helfrich-Förster, C., 2018. Closely Related Fruit Fly Species Living at Different Latitudes Diverge in Their Circadian Clock Anatomy and Rhythmic Behavior. J. Biol. Rhythms 33, 602-613.
https://doi.org/10.1177/0748730418798096

Bilcke, G., Osuna-Cruz, C.M., Silva, M.S., Poulsen, N., Bulankova, P., Vyverman, W., Veylder, L.D., Vandepoele, K., 2021. Diurnal transcript profiling of the diatom Seminavis robusta reveals adaptations to a benthic lifestyle. Plant J. 107. https://doi.org/10.1111/tpj.15291

Blaby-Haas, C.E., Merchant, S.S., 2019. Comparative and Functional Algal Genomics. Annu. Rev. Plant Biol. 70, 605-638. https://doi.org/10.1146/annurev-arplant-050718-095841

Brand, L.E., 1982. Persistent diel rhythms in the chlorophyll fluorescence of marine phytoplankton species. Mar. Biol. 69, 253-262. https://doi.org/10.1007/BF00397491

Carré, I.A., Edmunds, L.N., 1993. Oscillator control of cell division in Euglena: cyclic AMP oscillations mediate the phasing of the cell division cycle by the circadian clock. J. Cell Sci. 104, 11. https://doi.org/10.1242/jcs.104.4.1163

Chauton, M.S., Winge, P., Brembu, T., Vadstein, O., Bones, A.M., 2013. Gene Regulation of Carbon Fixation, Storage, and Utilization in the Diatom *Phaeodactylum tricornutum* Acclimated to Light/Dark Cycles. Plant Physiol. 161, 1034-1048.
https://doi.org/10.1104/pp.112.206177

Corellou, F., Schwartz, C., Motta, J.-P., Djouani-Tahri, E.B., Sanchez, F., Bouget, F.-Y., 2009. Clocks in the Green Lineage: Comparative Functional Analysis of the Circadian Architecture of the Picoeukaryote *Ostreococcus*. Plant Cell 21, 3436-3449.
https://doi.org/10.1105/tpc.109.068825

Creux, N., Harmer, S., 2019. Circadian Rhythms in Plants. Cold Spring Harb. Perspect. Biol. 11, a034611. https://doi.org/10.1101/cshperspect.a034611

Espinoza, C., Degenkolbe, T., Caldana, C., Zuther, E., Leisse, A., Willmitzer, L., Hincha, D.K., Hannah, M.A., 2010. Interaction with Diurnal and Circadian Regulation Results in Dynamic Metabolic and Transcriptional Changes during Cold Acclimation in Arabidopsis. PLoS ONE 5, e14101. https://doi.org/10.1371/journal.pone.0014101

Farré, E.M., Harmer, S.L., Harmon, F.G., Yanovsky, M.J., Kay, S.A., 2005. Overlapping and Distinct Roles of PRR7 and PRR9 in the Arabidopsis Circadian Clock. Curr. Biol. 15, 47-54.
https://doi.org/10.1016/j.cub.2004.12.067

Farré, E.M., Weise, S.E., 2012. The interactions between the circadian clock and primary metabolism. Curr. Opin. Plant Biol. 15, 293-300. https://doi.org/10.1016/j.pbi.2012.01.013

Filichkin, S.A., Breton, G., Priest, H.D., Dharmawardhana, P., Jaiswal, P., Fox, S.E., Michael, T.P., Chory, J., Kay, S.A., Mockler, T.C., 2011. Global Profiling of Rice and Poplar Transcriptomes Highlights Key Conserved Circadian-Controlled Pathways and cis-Regulatory Modules. PLoS ONE 6, e16907. https://doi.org/10.1371/journal.pone.0016907

Flori, S., Jouneau, P.-H., Bailleul, B., Gallet, B., Estrozi, L.F., Moriscot, C., Bastien, O., Eicke, S., Schober, A., Bártulos, C.R., Maréchal, E., Kroth, P.G., Petroutsos, D., Zeeman, S., Breyton, C., Schoehn, G., Falconet, D., Finazzi, G., 2017. Plastid thylakoid architecture optimizes photosynthesis in diatoms. Nat. Commun. 8, 15885. https://doi.org/10.1038/ncomms15885
Flori, S., Jouneau, P.-H., Finazzi, G., Maréchal, E., Falconet, D., 2016. Ultrastructure of the Periplastidial Compartment of the Diatom Phaeodactylum tricornutum. Protist 167, 254-267. https://doi.org/10.1016/j.protis.2016.04.001

Goto, K., Johnson, C.H., 1995. Is the cell division cycle gated by a circadian clock? The case of Chlamydomonas reinhardtii. J. Cell Biol. 129, 1061-1069. https://doi.org/10.1083/jcb.129.4.1061

Greenham, K., Lou, P., Puzey, J.R., Kumar, G., Arnevik, C., Farid, H., Willis, J.H., McClung, C.R., 2017. Geographic Variation of Plant Circadian Clock Function in Natural and Agricultural Settings. J. Biol. Rhythms 32, 26-34. https://doi.org/10.1177/0748730416679307

Gunther, M., Laithier, M., Brison, O., 2000. A set of proteins interacting with transcription factor Sp1 identified in a two-hybrid screening. Mol. Cell. Biochem. 210, 12. https://doi.org/10.1023/A:1007177623283

Happey-Wood, C.M., Jones, P., 1988. Rhythms of vertical migration and motility in intertidal benthic diatoms with particular reference to Pleurosigma angulatum. Diatom Res. 3, 83-93. http://dx.doi.org/10.1080/0269249X.1988.9705018

Keeling, P.J., 2013. The Number, Speed, and Impact of Plastid Endosymbioses in Eukaryotic Evolution. Annu. Rev. Plant Biol. 64, 583-607. https://doi.org/10.1146/annurev-arplant-050312-120144

Lepetit, B., Sturm, S., Rogato, A., Gruber, A., Sachse, M., Falciatore, A., Kroth, P.G., Lavaud, J., 2013. High Light Acclimation in the Secondary Plastids Containing Diatom *Phaeodactylum tricornutum* is Triggered by the Redox State of the Plastoquinone Pool. Plant Physiol. 161, 853-865. https://doi.org/10.1104/pp.112.207811

Mann, M., Serif, M., Wrobel, T., Eisenhut, M., Madhuri, S., Flachbart, S., Weber, A.P.M., Lepetit, B., Wilhelm, C., Kroth, P.G., 2020. The Aureochrome Photoreceptor PtAUREO1a Is a Highly Effective Blue Light Switch in Diatoms. iScience 23, 101730. https://doi.org/10.1016/j.isci.2020.101730

Matsuda, Y., Hopkinson, B.M., Nakajima, K., Dupont, C.L., Tsuji, Y., 2017. Mechanisms of carbon dioxide acquisition and CO₂ sensing in marine diatoms: a gateway to carbon metabolism. Philos. Trans. R. Soc. B Biol. Sci. 372, 20160403. https://doi.org/10.1098/rstb.2016.0403

Matsuo, T., Okamoto, K., Onai, K., Niwa, Y., Shimogawara, K., Ishiura, M., 2008. A systematic forward genetic analysis identified components of the Chlamydomonas circadian system. Genes Dev. 22, 918-930. https://doi.org/10.1101/gad.1650408

Matthijs, M., Fabris, M., Broos, S., Vyverman, W., Goossens, A., 2016. Profiling of the Early Nitrogen Stress Response in the Diatom *Phaeodactylum tricornutum* Reveals a Novel Family of RING-Domain Transcription Factors. Plant Physiol. 170, 489-498. https://doi.org/10.1104/pp.15.01300

Matthijs, M., Fabris, M., Obata, T., Foubert, I., Franco-Zorrilla, J.M., Solano, R., Fernie, A.R., Vyverman, W., Goossens, A., 2017. The transcription factor bZIP14 regulates the TCA cycle in the diatom *Phaeodactylum tricornutum*. EMBO J. 36, 1559-1576. https://doi.org/10.15252/embj.201696392

Millar, A.J., Carré, I.A., Strayer, C.A., Chua, N.-H., Kay, S.A., 1995. Circadian Clock Mutants in *Arabidopsis* Identified by Luciferase Imaging. Science 267, 1161-1163. https://doi.org/10.1126/science.7855595

Mitsuda, N., Ikeda, M., Takada, S., Takiguchi, Y., Kondou, Y., Yoshizumi, T., Fujita, M., Shinozaki, K., Matsui, M., Ohme-Takagi, M., 2010. Efficient Yeast One-/Two-Hybrid Screening Using a Library Composed Only of Transcription Factors in Arabidopsis thaliana. Plant Cell Physiol. 51, 2145-2151. https://doi.org/10.1093/pcp/pcq161

Mizoguchi, T., Wheatley, K., Hanzawa, Y., Wright, L., Mizoguchi, M., Song, H.-R., Carré, I.A., Coupland, G., 2002. LHY and CCA1 Are Partially Redundant Genes Required to Maintain Circadian Rhythms in Arabidopsis. Dev. Cell 2, 629-641. https://doi.org/10.1016/S1534-5807(02)00170-3

Mori, T., Binder, B., Johnson, C.H., 1996. Circadian gating of cell division in cyanobacteria growing with average doubling times of less than 24 hours. Proc. Natl. Acad. Sci. 93, 10183-10188. https://doi.org/10.1073/pnas.93.19.10183
Moulager, M., Monnier, A., Jesson, B., Bouvet, R., Mosser, J., Schwartz, C., Garnier, L., Corellou, F., Bouget, F.-Y., 2007. Light-Dependent Regulation of Cell Division in *Ostreococcus*: Evidence for a Major Transcriptional Input. Plant Physiol. 144, 1360-1369.
https://doi.org/10.1104/pp.107.096149

Palmer, J.D., Livingston, L., Zusy, Fr.D., 1964. A Persistent Diurnal Rhythm in Photosynthetic Capacity. Nature 203, 1087-1088. https://doi.org/10.1038/2031087a0

Palmer, J.D., Round, F.E., 1967. PERSISTENT, VERTICAL-MIGRATION RHYTHMS IN BENTHIC MICROFLORA. VI. THE TIDAL AND DIURNAL NATURE OF THE RHYTHM IN THE DIATOM HANTZSCHIA VIRGATA. Biol. Bull. 132, 44-55.
https://doi.org/10.2307/1539877

Poliner, E., Cummings, C., Newton, L., Farré, E.M., 2019. Identification of circadian rhythms in *Nannochloropsis* species using bioluminescence reporter lines. Plant J. 99, 112-127.
https://doi.org/10.1111/tpj.14314

Rayko, E., Maumus, F., Maheswari, U., Jabbari, K., Bowler, C., 2010. Transcription factor families inferred from genome sequences of photosynthetic stramenopiles. New Phytol. 188, 52-66.
https://doi.org/10.1111/j.1469-8137.2010.03371.x

Salomé, P.A., McClung, C.R., 2005. PSEUDO-RESPONSE REGULATOR 7 and 9 Are Partially Redundant Genes Essential for the Temperature Responsiveness of the Arabidopsis Circadian Clock. Plant Cell 17, 791-803. https://doi.org/10.1105/tpc.104.029504

Seo, S., Jeon, H., Hwang, S., Jin, E., Chang, K.S., 2015. Development of a new constitutive expression system for the transformation of the diatom Phaeodactylum tricornutum. Algal Res. 11, 50-54. https://doi.org/10.1016/j.algal.2015.05.012

Smith, S.R., Gillard, J.T.F., Kustka, A.B., McCrow, J.P., Badger, J.H., Zheng, H., New, A.M., Dupont, C.L., Obata, T., Fernie, A.R., Allen, A.E., 2016. Transcriptional Orchestration of the Global Cellular Response of a Model Pennate Diatom to Diel Light Cycling under Iron Limitation. PLOS Genet. 12, e1006490. https://doi.org/10.1371/journal.pgen.1006490

Taddei, L., Stella, G.R., Rogato, A., Bailleul, B., Fortunato, A.E., Annunziata, R., Sanges, R., Thaler, M., Lepetit, B., Lavaud, J., Jaubert, M., Finazzi, G., Bouly, J.-P., Falciatore, A., 2016. Multisignal control of expression of the LHCX protein family in the marine diatom *Phaeodactylum tricornutum*. J. Exp. Bot. 67, 3939-3951. https://doi.org/10.1093/jxb/erw198

Takahashi, J.S., 2017. Transcriptional architecture of the mammalian circadian clock. Nat. Rev. Genet. 18, 164-179. https://doi.org/10.1038/nrg.2016.150

Tsuji, Y., Nakajima, K., Matsuda, Y., 2017. Molecular aspects of the biophysical CO2-concentrating mechanism and its regulation in marine diatoms. J. Exp. Bot. 68, 3763-3772.
https://doi.org/10.1093/jxb/erx173

Wang, W., Yu, L.-J., Xu, C., Tomizaki, T., Zhao, S., Umena, Y., Chen, X., Qin, X., Xin, Y., Suga, M., Han, G., Kuang, T., Shen, J.-R., 2019. Structural basis for blue-green light harvesting and energy dissipation in diatoms. Science 363, eaav0365.
https://doi.org/10.1126/science.aav0365

Weber, T., Gruber, A., Kroth, P.G., 2009. The Presence and Localization of Thioredoxins in Diatoms, Unicellular Algae of Secondary Endosymbiotic Origin. Mol. Plant 2, 468-477.
https://doi.org/10.1093/mp/ssp010

Wijnen, H., Young, M.W., 2006. Interplay of Circadian Clocks and Metabolic Rhythms. Annu. Rev. Genet. 40, 409-448. https://doi.org/10.1146/annurev.genet.40.110405.090603
# Alessandro MANZOTTI

## Curriculum vitae

### Personal informations

Name Alessandro Manzotti

Date of birth 03/06/1994

E-mail alessandro.manzotti@protonmail.com

Phones +33(0)766334971

+39 340 7955 601

Address 15, rue Fernand Enguehard Bagneux, 92220

Professional references Angela Falciatore (Ph.D. director, falciatore@ibpc.fr), Jean-Pierre Bouly (Ph.D. co-director, jean-pierre.bouly@upmc.fr)

### Work experiences

Ph.D. research candidate (2018-2022)
Ph.D. project in molecular and cell biology at the Institut de Biologie Physico Chimique - UMR7141 (Ecole Doctorale Complexité du Vivant ED515, Sorbonne Université). Project title: "*Response to light stimuli in the marine diatom Phaeodactylum tricornutum: Involvement of bHLH/PAS proteins in the circadian clock and plastid physiology*"

Teaching (2019-2021)
Sorbonne Université - Campus Pierre et Marie Curie - Teaching experience in biochemistry and molecular biology

### Studies

University Ongoing

University Sorbonne Université

### Master

Date Juin 2018

Title Double degree master program in Molecular Biology and Genetics -

University Università degli Studi di PADOVA (Italie) et Université Paris Diderot Paris VII

Evaluation 110/110 *cum laude*

### Bachelor

Date 26/09/2016

Title Università degli Studi di PADOVA

University Biologia molecolare (Biologie Moleculaire)

Evaluation 110/110 *cum laude*

### Trainings

> Statistics course in « R » - Ecole Doctorale du MNHN (30 hrs)

> Course « Multidimensional data analysis in R » - Agro Campus Ouest (25 hrs)

> Course « Scientific integrity for researchers » - Université de Bordeaux (1.5 hrs)

> Summer school « Climate action for sustainable development » - Radboud University, Nijmegen (Netherlands) (1 week, full time)

> Course « Biodiversity and global changes », Université Virtuelle Environnement et Développement Durable (12hrs)

> Course « Pedagogy and teaching in Biology for University » - Sorbonne Université (11hrs)
## Publications

Published :

* Rossella Annunziata, Andrés Ritter, Antonio Emidio Fortunato, **Alessandro Manzotti**, Soizic Cheminant-Navarro, Nicolas Agier, Marie J. J. Huysman, Per Winge, Atle M. Bones, François-Yves Bouget, Marco Cosentino Lagomarsino, Jean-Pierre Bouly, and Angela Falciatore; **bHLH-PAS protein RITMO1 regulates diel biological rhythms in the marine diatom Phaeodactylum tricornutum**, PNAS 116 (26) 13137-1314, 2019.
doi: https://doi.org/10.1073/pnas.1819660116

Submitted

* Sören Häfker*, Gabriele Andreatta*, **Alessandro Manzotti***, Angela Falciatore, Florian Raible and Kristin Tessmar-Raible. **Rhythms and clocks in marine organisms**, Submitted to Annual Reviews of Marine Science at the end of February 2022.
*: co-first authors

In preparation :

* **Alessandro Manzotti**, Raphael Monteil, Antoine Hoguin, Soizic Cheminant-Navarro, Denis Jallet, Francois-Yves Bouget, Fayza Daboussi, Marianne Jaubert, Jean-Pierre Bouly & Angela Falciatore*
Physiological rhythms in the marine diatom *Phaeodactylum tricornutum* are regulated by the circadian clock transcriptional regulator *RITMO1*, in preparation, to be submitted in April 2022

* **Alessandro Manzotti**, Soizic Cheminant-Navarro, Marianne Jaubert, Antonio Emidio Fortunato, Andres Ritter
Alessandra Bellan, Tomas Morosinotto, Jean-Pierre Bouly & Angela Falciatore, **bHLH2 is a light-intensity dependent modulator of photosynthesis and growth in the marine diatom Phaeodactylum tricornutum**, in preparation

**Languages**

<table><tr><td>English</td><td>C2</td></tr><tr><td>French</td><td>DALF C1</td></tr><tr><td>Italian</td><td>Mother tongue</td></tr></table>

**Informatics and programming**

<table><tr><td>GraphPad Prism</td><td>Excellent</td></tr><tr><td>Zotero</td><td>Excellent</td></tr><tr><td>Adobe Photoshop + Lightroom</td><td>Excellent</td></tr><tr><td>Adobe Illustrator</td><td>Really good</td></tr><tr><td>InkScape</td><td>Good</td></tr><tr><td>R</td><td>Discrete</td></tr></table>

**Distinctions**

Scholarship awarded to the best university students of the city of Novara by the Nobile Collegio Caccia di Novara (2013-2018)
Gold medal for merit from the Nobile Collegio Caccia di Novara (2018)

**Additional activities**

**Students representative**

Università degli Studi di Padova (2013 - 2017)

Member of the Student Council as a representative of the students of the School of Biology

**Popularization of science**

Coordination, organization and implementation of the conference cycle for scientific dissemination "L'ombra di Galileo" at the University of Padua (2014-2017).
# Abstract

Response to light stimuli in the marine diatom *Phaeodactylum tricornutum*: Involvement of bHLH-PAS proteins in the circadian clock and plastid physiology

Diatoms are eukaryotic microalgae that represent one of the most successful groups of phytoplankton. Despite their ecological relevance, little information is available on the regulatory mechanisms controlling gene expression in these algae. Through the use of the model marine diatom *Phaeodactylum tricornutum*, the work presented highlighted the role of bHLH-PAS family transcription factors in the regulation of two critically important light-dependent processes. On the one hand, it was shown how the bHLH1a protein, later renamed RITMO1, is involved in the modulation of endogenous rhythmicity regulated by light-dark cycles. Overexpression and knock-out mutation of this gene led to a disruption of daily cellular fluorescence oscillations, photosynthetic activity, and gene expression under constant light condition, identifying RITMO1 as the first characterized component of the diatom circadian clock. It was also shown that a paralogous transcription factor, bHLH1b, dimerizes with RITMO1 and a mutation of this protein led to similar disruptions of cellular fluorescence rhythmicity, indicating its co-implication in the endogenous timekeeper. In parallel, the third *P. tricornutum* bHLH-PAS protein, bHLH2, has been shown to be involved in the modulation of photosynthetic activity in a light intensity-dependent mechanism not controlled by the endogenous clock. Cell lines presenting a construct for RNA interference against bHLH2 showed reduced electron flow at the level of the thylakoid photosynthetic chain and a subsequent deficit in growth. It has been shown that the expression of this protein is controlled by a retrograde signalling pathway, thus highlighting a crosstalk between nucleus and plastid. This study represented the first characterization of bHLH-PAS proteins in a photosynthetic organism.

Keywords: diatoms, *Phaeodactylum tricornutum*, light, circadian clock, photosynthesis, bHLH-PAS proteins

# Résumé

Réponse aux stimuli lumineux chez la diatomée marine *Phaeodactylum tricornutum* : implication des protéines bHLH-PAS dans l'horloge circadienne et la physiologie du plaste

Les diatomées sont des microalgues eucaryotes qui représentent l'un des groupes de phytoplancton les plus répandus. Malgré leur importance écologique, peu d'informations sont disponibles sur les mécanismes de régulation contrôlant l'expression des gènes chez ces algues. En utilisant la diatomée marine modèle *Phaeodactylum tricornutum*, les travaux présentés ont mis en évidence le rôle des facteurs de transcription de la famille bHLH-PAS dans la régulation de deux processus critiques dépendant de la lumière. D'une part, il a été montré quela protéine bHLH1a, renommée RITMO1, est impliquée dans la modulation de la rythmicité endogène régulée par les cycles lumière-obscurité. La surexpression et la mutation KO de cette protéine conduisent à une perturbation des oscillations journalières de la fluorescence cellulaire, de l'activité photosynthétique et de l'expression génique en condition de lumière continue, l'identifiant comme le premier composant caractérisé de l'horloge circadienne des diatomées. Il a également été montré que RITMO1 dimérise avec son paralogue bHLH1b et que la mutation de cet autre facteur de transcription mène à des perturbations similaires de la rythmicité de la fluorescence cellulaire, indiquant sa co-implication dans l'horloge endogène. En parallèle, il a été démontré qu'un troisième facteur de transcription de type bHLH-PAS, bHLH2, est impliqué dans la modulation de l'activité photosynthétique dans un mécanisme dépendant de l'intensité lumineuse et non contrôlé par l'horloge endogène. Les lignées cellulaires transformées avec des ARN interférents contre bHLH2 montrent un flux d'électrons réduit au niveau de la chaîne photosynthétique thylakoidale et un déficit de croissance subséquent. L'expression de cette protéine s'est avérée dépendre d'un système de signalisation rétrograde, mettant ainsi en évidence une communication croisée entre le noyau et le plaste. Cette étude représente la première caractérisation des protéines bHLH-PAS dans un organisme photosynthétique.

Mots-clés : diatomées, *Phaeodactylum tricornutum*, lumière, horloge circadienne, photosynthèse, protéines bHLH-PAS.