Marianne Nymark

Marthe Caroline

Grønbech Hafskjold

Charlotte Volpe

Davi De

Miranda Fonseca

Animesh Sharma

Eirini Tsirvouli

Per Winge Manuel Serif

Giovanni Finazzi

Atle Magnar

Marthe Caroline Grønbech Hafskjold

Atle Magnar Bones

Functional studies of CpSRP54 in diatoms show that the mechanism of thylakoid protein insertion differs from plants and green algae

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Introduction

Diatoms (Bacillariophyceae) are a species-rich group of unicellular, photosynthetic eukaryotes found in freshwaters and oceans worldwide, which are of enormous ecological relevance [START_REF] Armbrust | The life of diatoms in the world's oceans[END_REF]. This group of phytoplankton functions as a primary source of food for many aquatic organisms and is responsible for performing similar levels of photosynthetic fixation of carbon as all terrestrial rainforests combined [START_REF] Nelson | Production and dissolution of biogenic silica in the ocean -Revised global estimates, comparison with regional data and relationship to biogenic sedimentation[END_REF][START_REF] Armbrust | The life of diatoms in the world's oceans[END_REF][START_REF] Gilbert | The seasonal structure of microbial communities in the Western English Channel[END_REF]. Diatoms are also promising organisms for a multitude of potential applications within the bio-, nano-and environmental technologies [START_REF] Bozarth | Diatoms in biotechnology: modern tools and applications[END_REF][START_REF] Mishra | All new faces of diatoms: Potential source of nanomaterials and beyond[END_REF][START_REF] Butler | Phaeodactylum tricornutum: A diatom cell factory[END_REF]. However, their role in primary production and potential for industrial applications is not matched by the level of basic knowledge of their photosynthetic machinery.

Although the photosynthetic apparatus of Viridiplantae and Heterokonts such as diatoms share many common features, they are separated from a common ancestor by more than a billion years of evolution [START_REF] Hohmann-Marriott | Evolution of photosynthesis[END_REF]. Diatoms arose by a secondary endosymbiotic event and as a result their chloroplasts are surrounded by four membranes [START_REF] Gibbs | The chloroplasts of some algal groups may have evolved from endosymbiotic eukaryotic algae[END_REF][START_REF] Flori | Ultrastructure of the periplastidial compartment of the diatom Phaeodactylum tricornutum[END_REF]. Instead of having thylakoids organized into grana stacks interconnected by stroma-exposed lamellae typical for land plants and green algae, diatom thylakoids are organized in stacked bands of three thylakoids each, spanning along the entire length of the plastid [START_REF] Gibbs | Comparative ultrastructure of algal chloroplast[END_REF][START_REF] Grouneva | Phylogenetic viewpoints on regulation of light harvesting and electron transport in eukaryotic photosynthetic organisms[END_REF]. Photosystem II (PSII), cytochrome b6f (Cytb6f), photosystem I (PSI) and ATP synthase are multi-subunit protein complexes embedded in the thylakoid membranes and responsible for photosynthetic electron transport and ATP synthesis together with protein-bound and mobile electron carriers [START_REF] Nelson | The complex architecture of oxygenic photosynthesis[END_REF]. Light energy is collected and delivered to PSs by light harvesting complexes (LHCs) consisting of pigments and pigment-binding proteins. While the pigment-binding proteins of diatoms and plants/green algae belong to the LHC protein (LHCP) superfamily, they bind different pigments [START_REF] Grossman | Light-harvesting complexes in oxygenic photosynthesis: diversity, control, and evolution[END_REF][START_REF] Dittami | Chlorophyll-binding proteins revisited-a multigenic family of light-harvesting and stress proteins from a brown algal perspective[END_REF]. The LHCPs of the green lineages (LHCa and LHCb) bind mainly Chl a and Chl b, whereas diatoms contain a different subfamily of LHCPs that in addition to Chl a also bind Chl c and fucoxanthin (Fx) and are therefore called Fx-Chl a/c-binding proteins (FCPs). The photosynthetic apparatus of eukaryotic organisms is composed of proteins encoded by both nucleus and chloroplast localized genes. In Viridiplantae, the nucleus encoded LHCPs and selected chloroplast encoded proteins are guided to thylakoid membranes by the Chloroplast Signal Recognition Particle (CpSRP) pathway (Kirst and Melis, 2014[START_REF] Jeong | Loss of CpSRP54 function leads to a truncated light-harvesting antenna size in Chlamydomonas reinhardtii[END_REF], Ziehe et al., 2017, Ziehe et al., 2018). The CpSRP54 GTPase of the CpSRP pathway has a dual function in plants and targets proteins both post-translationally (LHCPs) and co-translationally (chloroplast-encoded thylakoid membrane proteins) to thylakoids in cooperation with other members of the pathway [START_REF] Pilgrim | Expression of a dominant negative form of cpSRP54 inhibits chloroplast biogenesis in Arabidopsis[END_REF][START_REF] Amin | Arabidopsis mutants lacking the 43-and 54-kilodalton subunits of the chloroplast signal recognition particle have distinct phenotypes[END_REF][START_REF] Rutschow | Quantitative proteomics of a chloroplast SRP54 sorting mutant and its genetic interactions with CLPC1 in Arabidopsis[END_REF][START_REF] Hristou | Ribosome-associated chloroplast SRP54 enables efficient cotranslational membrane insertion of key photosynthetic proteins[END_REF]. In contrast to the universally conserved SRP54 protein that plays an essential role in mediating cotranslational targeting of newly synthesized proteins to cellular membranes across all kingdoms of life, the function of plant CpSRP54 is not dependent on an SRP RNA [START_REF] Träger | Evolution from the prokaryotic to the higher plant chloroplast signal recognition particle: the signal recognition particle RNA is conserved in plastids of a wide range of photosynthetic organisms[END_REF], Ziehe et al., 2017). In the post-translational pathway of plants, CpSRP54 is part of the LHCP-CpSRP43-CpSRP54-CpFTSY complex that guides LHCPs from the chloroplast membrane to the thylakoid membrane, where the ALBINO3 (ALB3) insertase mediates protein insertion (Ziehe et al., 2018). In the co-translational pathway, CpSRP54 interacts with nascent polypeptides and targets translating ribosomes to thylakoid membranes [START_REF] Nilsson | Interactions of ribosome nascent chain complexes of the chloroplast-encoded D1 thylakoid membrane protein with cpSRP54[END_REF][START_REF] Nilsson | Transient interaction of cpSRP54 with elongating nascent chains of the chloroplast-encoded D1 protein; 'cpSRP54 caught in the act[END_REF][START_REF] Piskozub | Ribosome nascent chain complexes of the chloroplast-encoded cytochrome b6 thylakoid membrane protein interact with cpSRP54 but not with cpSecY[END_REF][START_REF] Hristou | Ribosome-associated chloroplast SRP54 enables efficient cotranslational membrane insertion of key photosynthetic proteins[END_REF]. Differences in the function of CpSRP54 have been reported between plants and green algae. The Chlamydomonas reinhardtii CpSRP54 does not form a complex with the plastid specific chaperon CpSRP43 and is not involved in LHCP recognition, but is believed to function downstream of CpSRP43 together with its receptor CpFTSY in the posttranslational pathway [START_REF] Dünschede | Chloroplast SRP54 was recruited for posttranslational protein transport via complex formation with chloroplast SRP43 during land plant evolution[END_REF][START_REF] Jeong | Loss of CpSRP54 function leads to a truncated light-harvesting antenna size in Chlamydomonas reinhardtii[END_REF]. In both plants and green algae cpsrp54 knock out mutants, loss of CpSRP54 causes decreased levels of LHCPs and, consequently, lower pigment levels and paler green coloration than in their wild type counterparts [START_REF] Amin | Arabidopsis mutants lacking the 43-and 54-kilodalton subunits of the chloroplast signal recognition particle have distinct phenotypes[END_REF][START_REF] Rutschow | Quantitative proteomics of a chloroplast SRP54 sorting mutant and its genetic interactions with CLPC1 in Arabidopsis[END_REF][START_REF] Jeong | Loss of CpSRP54 function leads to a truncated light-harvesting antenna size in Chlamydomonas reinhardtii[END_REF][START_REF] Hristou | Ribosome-associated chloroplast SRP54 enables efficient cotranslational membrane insertion of key photosynthetic proteins[END_REF]. Arabidopsis thaliana CpSRP54 co-translationally associates with a range of different subunits of the photosynthetic electron transport chain complexes, but a similar function has not been reported in C. reinhardtii [START_REF] Nilsson | Interactions of ribosome nascent chain complexes of the chloroplast-encoded D1 thylakoid membrane protein with cpSRP54[END_REF][START_REF] Nilsson | Transient interaction of cpSRP54 with elongating nascent chains of the chloroplast-encoded D1 protein; 'cpSRP54 caught in the act[END_REF][START_REF] Piskozub | Ribosome nascent chain complexes of the chloroplast-encoded cytochrome b6 thylakoid membrane protein interact with cpSRP54 but not with cpSecY[END_REF][START_REF] Jeong | Loss of CpSRP54 function leads to a truncated light-harvesting antenna size in Chlamydomonas reinhardtii[END_REF][START_REF] Hristou | Ribosome-associated chloroplast SRP54 enables efficient cotranslational membrane insertion of key photosynthetic proteins[END_REF]. In diatoms, homologues of CpSRP54, CpFTSY and two variants of ALB3 (ALB3a and ALB3b) have been identified, whereas CpSRP43 homologues are absent [START_REF] Träger | Evolution from the prokaryotic to the higher plant chloroplast signal recognition particle: the signal recognition particle RNA is conserved in plastids of a wide range of photosynthetic organisms[END_REF][START_REF] Nymark | Loss of ALBINO3b insertase results in truncated lightharvesting antenna in diatoms[END_REF].

In addition, the LHCP translocation defect protein (LTD), which forwards LHCPs from the chloroplast import machinery to the CpSRP complex in plants and green algae is apparently also missing in diatom genomes [START_REF] Ouyang | LTD is a protein required for sorting light-harvesting chlorophyll-binding proteins to the chloroplast SRP pathway[END_REF][START_REF] Jeong | Deletion of the chloroplast LTD protein impedes LHCI import and PSI-LHCI assembly in Chlamydomonas reinhardtii[END_REF]. A chloroplast encoded SRP RNA is present in most, but not all diatom genomes sequenced to date [START_REF] Träger | Evolution from the prokaryotic to the higher plant chloroplast signal recognition particle: the signal recognition particle RNA is conserved in plastids of a wide range of photosynthetic organisms[END_REF][START_REF] Brembu | The chloroplast genome of the diatom Seminavis robusta: new features introduced through multiple mechanisms of horizontal gene transfer[END_REF]. Information from gene models available at NCBI and The Marine Microbial Eukaryote Meta/transcriptome Sequencing Project (MMETSP) database reveal that the CpSRP54, CpFTSY and ALB3 proteins are widespread in all algae, including red algae, haptophytes and the SAR (Stramenopila, Alveolata, and Rhizaria) clade. The LTD and CpSRP43 related proteins are probably restricted to the green lineage, even though some CpSRP43-like ankyrin-repeat proteins can be found in red algae, haptophytes, and in several species within the SAR clade. The CpSRP43-like ankyrin-repeat proteins are missing in Bolidophyceae and diatoms.

The function of ALB3b was recently investigated in the model diatom Phaeodactylum tricornutum, where loss of the insertase caused a striking change in cell color from brown to green [START_REF] Nymark | Loss of ALBINO3b insertase results in truncated lightharvesting antenna in diatoms[END_REF]. The change in pigmentation was caused by a ~75% decrease of the main group of FCP proteins (LHCFs) and a subsequent lowered level of antenna pigments (including fucoxanthin that provides the brown color of diatoms). The phenotype of the alb3b mutants pointed to a role of this protein in posttranslational insertion of FCPs into the thylakoid membrane. However, the ALB3b protein does not contain the basic Lys-rich C-terminal domain (CTD) known from plants to interact with other members of the CpSRP pathway [START_REF] Falk | The C terminus of the Alb3 membrane insertase recruits cpSRP43 to the thylakoid membrane[END_REF][START_REF] Horn | Structural basis for cpSRP43 chromodomain selectivity and dynamics in Alb3 insertase interaction[END_REF][START_REF] Chandrasekar | Anionic phospholipids and the Albino3 translocase activate signal recognition particle-receptor interaction during light-harvesting chlorophyll a/b-binding protein targeting[END_REF]. Instead, a CTD conserved within the group of diatoms was identified in the ALB3b insertase, but its protein interaction partners are unknown [START_REF] Nymark | Loss of ALBINO3b insertase results in truncated lightharvesting antenna in diatoms[END_REF]. ALB3a does contain the basic Lys-rich CTD, but repeated attempts to create knock out mutants of this insertase were unsuccessful, implying a crucial function in the co-translational CpSRP pathway similar to ALB3 and ALB3.2 of plants and green algae, respectively [START_REF] Sundberg | ALBINO3, an Arabidopsis nuclear gene essential for chloroplast differentiation, encodes a chloroplast protein that shows homology to proteins present in bacterial membranes and yeast mitochondria[END_REF][START_REF] Göhre | One of two Alb3 proteins is essential for the assembly of the photosystems and for cell survival in Chlamydomonas[END_REF][START_REF] Nymark | Loss of ALBINO3b insertase results in truncated lightharvesting antenna in diatoms[END_REF].

Previously published experiments with P. tricornutum cpsrp54 mutants indicated that loss of CpSRP54 causes increased high light sensitivity based on a more profound decrease in photosynthetic efficiency in mutants compared to WT when exposed to high intensity blue light [START_REF] Nymark | A CRISPR/Cas9 system adapted for gene editing in marine algae[END_REF]. However, changes in the cell content of pigments and FCPs were not tested in mutant lines. Knowledge of the roles of members of the diatom CpSRP pathway is very limited, but based on currently available data CpSRP54 has been hypothesized to play a part in the co-translational part of the CpSRP pathway, but not in the post-translational part [START_REF] Nymark | A CRISPR/Cas9 system adapted for gene editing in marine algae[END_REF][START_REF] Nymark | Loss of ALBINO3b insertase results in truncated lightharvesting antenna in diatoms[END_REF]. To achieve further insight into the role of CpSRP54 in the CpSRP pathway, we investigated changes in photosynthetic properties, pigment level, protein abundance and growth characteristics as a response to light stress in P. tricornutum cpsrp54 knockout (KO) lines. We propose a model illustrating the mechanistic differences of thylakoid protein insertion in diatoms, plants and green algae.

Results

Structural features of diatom CpSR54 proteins

The diatom CpSRP54 protein harbors the conserved multidomain structure of SRP54 proteins:

A N-terminal helical bundle domain, a central SRP GTPase (G) domain and a methionine rich M-domain (Figure 1A). The N and G domains form a functional unit called the NG domain that is generally known to be necessary for binding and hydrolyzing GTP and interaction with a similar NG domain in its receptor, the FTSY protein [START_REF] Pool | Signal recognition particles in chloroplasts, bacteria, yeast and mammals (review)[END_REF], Ziehe et al., 2017). The Mdomain contains the conserved SRP RNA binding motif and is also responsible for interaction with ribosomes and signal sequences of cargo proteins [START_REF] Pool | Signal recognition particles in chloroplasts, bacteria, yeast and mammals (review)[END_REF][START_REF] Chandrasekar | Anionic phospholipids and the Albino3 translocase activate signal recognition particle-receptor interaction during light-harvesting chlorophyll a/b-binding protein targeting[END_REF], Ziehe et al., 2017[START_REF] Hristou | Ribosome-associated chloroplast SRP54 enables efficient cotranslational membrane insertion of key photosynthetic proteins[END_REF]. In addition, the CpSRP54 contains a chloroplast targeting peptide (CTP). In plants, a conserved motif within the C-terminal region of the Mdomain, ARRKR, is known to be important for CpSRP43 binding [START_REF] Funke | A unique sequence motif in the 54-kDa subunit of the chloroplast signal recognition particle mediates binding to the 43-kDa subunit[END_REF][START_REF] Dünschede | Chloroplast SRP54 was recruited for posttranslational protein transport via complex formation with chloroplast SRP43 during land plant evolution[END_REF]. This motif is poorly conserved in diatom CpSRP54 proteins and in CpSRP54 from C. reinhardtii which does not bind CpSRP43 (Figure 1D) [START_REF] Dünschede | Chloroplast SRP54 was recruited for posttranslational protein transport via complex formation with chloroplast SRP43 during land plant evolution[END_REF]. These proteins contain a polybasic motif with two conserved arginines (the RR-motif in Figure 1A andD). The GTPase G1-G5 domains, the I-box, "ALL[DE]ADV", "D[SA]RGG", and "RILGMGD" motifs are all present in diatom CpSRP54 proteins. However, CpSRP54 proteins in diatoms have a 24 -26 amino acid insertion between the G1 and G2 domain (Figure 1B). A model based on the crystal structure of Arabidopsis thaliana CpSRP54 and FTSY (PDB ID: 5L3R) using Swiss Model [START_REF] Waterhouse | SWISS-MODEL: homology modelling of protein structures and complexes[END_REF] suggests that this amino acid insert forms a loop-structure that does not directly interfere with the CpSRP54-FTSY interaction surface (Figure 1C).

CRISPR/Cas9 editing of CpSRP54 resulted in frame-shift mutations Three CRISPR/Cas9 derived cpsrp54 mutant lines (cpsrp54-8, cpsrp54-11, cpsrp54-20) originating from single cells from the M2, M4 and M8 lines, respectively [START_REF] Nymark | A CRISPR/Cas9 system adapted for gene editing in marine algae[END_REF] were selected for functional characterization. Frame-shift mutations were confirmed in all cell lines by TOPO cloning and subsequent Sanger sequencing of PCR products spanning the region targeted by the Cas9-gRNA complex. The Cas9/gRNA complex was designed to target a part of the CpSRP54 gene (Phatr2_35185; XM_002179577) encoding the SRP54 N-terminal helical bundle domain of the CpSRP54 protein (Figure 1A). Polymorphisms in the CpSRP54 gene confirmed that both alleles had been amplified and sequenced for cpsrp54-8 and cpsrp54-11 (Figure S1). Both cpsrp54-8 and cpsrp54-11 contained small indels. Cpsrp54-20 contained a 212 bp insertion in one allele consisting of fragments from one of the vectors used for transformation of the P. tricornutum cells (Figure S1). The other allele of the cpsrp54-20 line could not be amplified indicating larger insertions or deletion events preventing amplification by PCR. The frame-shift mutations caused premature stop codons shortly after the indels, resulting in truncated CpSRP54 proteins (Figure 1E).

Cpsrp54 mutants showed no visible coloration phenotype

The alb3b mutants displayed a distinct difference in coloration, lower absorbance in the bluegreen part of the spectrum and a decreased transfer of energy from light harvesting antenna pigments Fx and Chl c to PSII compared to WT [START_REF] Nymark | Loss of ALBINO3b insertase results in truncated lightharvesting antenna in diatoms[END_REF]. If CpSRP54 functions in the same part of the CpSRP pathway as ALB3b, a similar phenotype would be expected.

Cell culture color, in vivo fluorescence excitation and absorbance spectra from cpsrp54 mutants and WT were therefore recorded. No visible color change could be seen in P. tricornutum cpsrp54 mutants compared to WT that would indicate major differences in light harvesting antenna pigments content (Figure 2A). This observation was further supported by the close to identical in vivo fluorescence excitation and absorbance spectra from mutants and WT (Figure 2B-C). These results imply that the ability of mutants and WT to harvest light energy for photosynthesis as well as the different pigment's relative energy transfer efficiency to Chl a in the reaction center of PSII are identical (Figure 2B). The presented results are all from low light (LL, 35 umol photons m -2 s -1 ) acclimated cells.

Cpsrp54 mutants are sensitive to increased light intensities

Based on the lack of a visible phenotype in LL and the indication that the cpsrp54 mutants might be sensitive to increased light intensities [START_REF] Nymark | A CRISPR/Cas9 system adapted for gene editing in marine algae[END_REF], we decided to perform a more thorough investigation of pigment content and photophysiological responses of the cpsrp54 mutants by exposing LL-acclimated cells (0 h) to medium light (ML, 200 µmol photons m -2 s -1 ) intensities for 0.5, 6, 24, 48, and 168 h. Additionally, we calculated the stoichiometry of functional reaction centers, the photosynthetic electron flow and growth rates in LL and ML acclimated cells. As expected from the close to identical color and in vivo fluorescence excitation spectra, the pigment analyses confirmed that the content of Chl a and Fx in cpsrp54 lines was similar to WT at most time points during the time series study (Figure 3A-B). In contrast, the conversion of the xanthophyll cycle pigment diadinoxanthin (Ddx) to the photoprotective pigment diatoxanthin (Dtx) was greatly enhanced in the mutants after the shift from LL to ML conditions as illustrated by the significantly higher Dtx content per cell (Figure 3D) and the elevated de-epoxidation state index (DES; DES = Dtx/(Dtx+Ddx); Figure 3E). Whereas the conversion of Ddx to Dtx peaked at the 0.5 h time point in the WT samples, the DES index reached its maximum after 6 h in ML in the cpsrp54 lines and stayed at a higher level than WT during the entire length of the ML exposure experiment. In addition, the nonphotochemical quenching (NPQ) capacity of the mutants was found to be 20-30% higher than that of the WT samples (Figure 3F). Altogether, these responses point towards a higher level of light stress in the mutants compared to the WT.

Chl a variable fluorescence measured with a pulse-amplitude modulated (PAM) fluorometer was used to monitor the physiological response of LL-acclimated cpsrp54 KO mutants during acclimation to ML intensity. The photosynthetic (PSII) efficiency (Fv/Fm), the maximum lightutilization coefficient (the slope of the photosynthesis versus irradiance curves; α), the photosynthetic capacity (maximum relative electron transport rate, rETRmax) and the light saturation index (Ek= rETRmax/α) were similar in cpsrp54 and WT cultures in LL-acclimated cells (Figure 4A-D). Exposure to ML caused only a minor drop in Fv/Fm in WT cultures, whereas Fv/Fm was significantly lower in cpsrp54 cultures compared to WT at all time points after the shift from LL to ML (Figure 4A). These results imply increased levels of photodamage and/or photoprotection when CpSRP54 is absent. The WT cells showed the expected response to increased light intensities displaying a pronounced increase in rETRmax and Ek as a function of ML exposure time. In contrast, these values decreased or remained at LL levels in the cpsrp54 cultures. The functional status of the photosynthetic apparatus of the cpsrp54 mutants was compared to that of WT cells using the ElectroChromic Shift (ECS) signal. This is the stark effect, i.e. a modification of the absorption properties of membrane embedded photosynthetic pigments following the buildup of a trans-thylakoid membrane potential during photosynthesis [START_REF] Witt | Energy conversion in the functional membrane of photosynthesis. Analysis by light pulse and electric pulse methods. The central role of the electric field[END_REF]. The ECS signal can be used to determine spectroscopically the stoichiometry of functional reaction centers and the photosynthetic electron flow [START_REF] Bailleul | Electrochromism: a useful probe to study algal photosynthesis[END_REF]. We used this approach to compare LL and ML-acclimated cells and found that the fraction of functional PSII reaction centers (RC) per functional PSI RC were less than half in the mutants compared to the WT in ML-acclimated cells, whereas no significant differences were found in LL-acclimated cells (Figure 4E). Similarly, the photosynthetic electron flow was around 50% lower in ML-grown cpsrp54 lines than in WT cells (Figure 4F). The decreased photophysiological fitness of the cpsrp54 mutants in ML is reflected also in an approx. 20% slower growth rate compared to WT at this light intensity (Table 1). The difference in growth rate did not increase further when the cells were grown in high light (HL; 1000 µmol photons m -2 s -1 ).

Decreased rate of PSII repair

The increased susceptibility of the cpsrp54 mutants to ML demonstrated above indicates an enhanced photosensitivity of the mutant strains, which could result from either an increased rate of photodamage to PSII core proteins or a decreased rate of PSII repair. To separate between these two phenomena, we performed an experiment where lincomycin (LINC), an inhibitor of chloroplast translation, was added to LL-acclimated cells before exposure to high light (HL; 1000 µmol photons m -2 s -1 ) for 1 h. In parallel, cells without LINC were exposed to the same light treatment. Addition of LINC removes the possibility to synthesize new chloroplast proteins and PSII repair cannot take place. Fv/Fm was measured before the HLtreatment, after 1 h of HL, and after 30 min of recovery after the HL-treatment as an indicator of PSII functionality. The measurements performed directly after the 1 h HL treatment will be affected both by thermal dissipation of absorbed light energy (qE) and photodamaged PSII reaction centers (qI). 30 min of recovery in very dim light allows for relaxation of the rapidly reversible qE component of NPQ. No significant differences were found between LLacclimated cultures or between cultures exposed to HL after addition of LINC (Figure 5). The HL+LINC combination caused a severe and close to identical decrease in Fv/Fm in WT and cpsrp54 lines. An increase in Fv/Fm from ~25% to ~39 % of LL-levels was detected at the end of the 30 min dark period which can probably be attributed to relaxation of qE, although LINC might slow down the process [START_REF] Bachmann | Effects of lincomycin on PSII efficiency, non-photochemical quenching, D1 protein and xanthophyll cycle during photoinhibition and recovery[END_REF]). Significant differences were found between WT and cpsrp54 lines in HL-treated cells without the addition of LINC (two-tailed Student's t-tests (p<0.05); Figure 5). After relaxation of qE, Fv/Fm recovered to 90% of the LLlevels in WT, whereas the equivalent average value in the cpsrp54 cultures was ca. 70%. It is important to be aware that the use of the Fv/Fm to assess photodamage is liable to artefacts.

Upon strong illumination, long living fluorescence quenching phenomena other than photodamage can contribute to change in fluorescence yield [START_REF] Dall'osto | A mechanism of nonphotochemical energy dissipation, independent from PsbS, revealed by a conformational change in the antenna protein CP26[END_REF]. However, the fact that Fv/Fm recovers comparably in WT and mutants after LINC treatment (i.e. blocking PSII repair cycle; Figure 5) suggests that, if other long living quenching processes than photoinhibition were induced in our conditions, they were of the same extent in both genotypes.

Our combined results are therefore interpreted to indicate that the rate of PSII repair is negatively affected by absence of CpSRP54, whereas the rate of PSII inactivation is highly similar.

Differences at the protein level

To investigate the effect of loss of CpSRP54 on the level of chloroplast proteins, quantitative proteomics of cpsrp54-8 and cpsrp54-11 cells that had been exposed to ML for 6 h was performed and compared to WT samples. The 6 h time point was chosen since the pigment and variable Chl fluorescence measurements described above indicated a high level of light stress at this timepoint in cpsrp54 lines. Only proteins encoded in the chloroplast genome or predicted to contain chloroplast transit peptides sequences were chosen for analysis. Chloroplast proteins that were significantly regulated (FDR <0.01) in the same direction in both cpsrp54 lines, showing log2 ratios ≥ ± 0.5 for at least one of the mutant lines and where at least two unique peptides were detected are included in Table 2. We further investigated the relative protein expression level of selected proteins by western blot. The most interesting findings are listed below.

Effects on the photosynthetic apparatus

Several FCPs involved in light harvesting were moderately affected in the cpsrp54 mutant lines after 6 h of ML exposure, but the regulation was in most cases found to go in opposite direction in cpsrp54-8 and cpsrp54-11 compared to WT, meaning that none of these FCP proteins fulfilled the criteria for being included in Table 2. The significantly, but moderately, regulated FCP proteins were in general expressed at a lower level than WT in cpsrp54-8 and at a higher level in cpsrp54-11 (Supplementary Dataset 1). After 6 h in ML, the diatoms will be in the process of downsizing their light harvesting antenna, and the results might reflect differences in acclimation status. Since the proteomics data recorded for the FCPs was inconclusive with regards to the effect of absence of CpSRP54, western blot analyses were additionally performed for a subgroup of FCPs, the LHCFs. The LHCF subgroup contains the major lightharvesting proteins in diatoms. The expression level of these proteins was investigated in total protein extracts from LL-acclimated cells, cells exposed to 6 h ML and ML-acclimated cells using an antibody predicted to detect LHCF1-11 (Figure 6A) [START_REF] Juhas | A novel cryptochrome in the diatom Phaeodactylum tricornutum influences the regulation of lightharvesting protein levels[END_REF]. The observed differences in LHCF expression levels were very modest between mutants and WT, and additional blots were therefore produced from total protein extracts from LL and MLacclimated cells. Relative abundances of LHCF proteins were estimated based on average ratios between cpsrp54 and WT lines from several blots and two independent experiments (Figure S2). No differences in LHCF content was found between crpsrp54 lines and WT in LL-acclimated cells, whereas prolonged growth in ML seemed to induce somewhat higher expression of LHCF proteins in the mutants than in WT (Figure 6B). These results are in line with the spectral properties (Figure 2) and light harvesting pigment data (Figure 3A-B) described above, indicating that loss of CpSRP54 does not result in a smaller light harvesting antenna in the cpsrp54 lines.

Eleven chloroplast-encoded and one nucleus-encoded subunit of the photosynthetic electron transport (PET) chain complexes PSII, PSI, Cytb6f and ATPase fulfilled the above described criteria and were found to be moderately downregulated in the cpsrp54 lines compared to WT after exposure to 6 h of ML (Table 2). Prolonged exposure to ML caused a decrease of the ratio of functional PSII/PSI in cpsrp54 lines (Figure 4E). Light induced inactivation of PSII is typically caused by damage to the PSII RC core protein D1 (PsbA), but the proteomics data indicated that the lack of CpSRP54 had the strongest negative effect on the other PSII RC core protein D2 (PsbD; Table 2). D2 was among the strongest downregulated PET chain subunits, whereas D1 was only significantly downregulated in one of the mutant lines (Supplementary Dataset 1). To get a better overview of the content of PSII RC proteins in cpsrp54 lines relative to WT, western blot analyses was performed on total protein extracts from LL-acclimated cells, cells exposed to 6 h ML and ML acclimated cells with antibodies specific to D1 and D2. The initial analysis of D1 and D2 supported a generally lower amount of D2 in the mutants compared to WT, whereas no clear effect on the relative abundance of D1 was observed at any light treatment (Figure 6A). As for the LHCF proteins relative quantification of D1 and D2 levels in LL and ML was performed running additional western blots (Figures S3-S4). Average relative abundance values for D1 and D2 are presented in Figure 6C-D. The results indicated that D2 protein levels in cpsrp54 mutants were lowered to around 70% of WT levels in both LL and ML conditions (Figure 6D). The relative content of D1 in mutants compared to WT showed larger variation between experiments, but was close to WT levels on average (Figure 6C; Figure S3). A negative effect of loss of CpSRP54 on the amount of D1 could therefore neither be proven from the proteomics nor the western blot analyses. In contrast to PSII core proteins, the subunits comprising the oxygen evolving complex (OEC) of PSII are mainly nucleus-encoded (except for PsbV). Of the OEC proteins detected by the proteomics data, PSBO, OEE3 and PsbV were unaffected by the loss of CpSRP54, whereas PSBP was slightly upregulated (Table 2). As for the PSII RC protein D1, the PSI RC proteins PsaA and PsaB were also only significantly downregulated in one of the lines, but the abundance of five other chloroplast-encoded PSI core subunits was found at lower levels in both mutant lines than in WT (Table 2; Supplementary Dataset 1). Two ferredoxin NAPD(H) reductases (FNRs), both nuclear-encoded, were among the strongest upregulated proteins in the dataset. FNR mainly catalyzes the final step in linear electron transport, reducing NAPD + to NADPH, but in plants some isoforms might also be involved in cyclic electron flow (CEF) around PSI, resulting in ATP formation [START_REF] Goss | The end of the line: Can ferredoxin and ferredoxin NADP(H) oxidoreductase determine the fate of photosynthetic electrons?[END_REF]. However, the contribution of CEF in ATP production in diatoms is negligible [START_REF] Bailleul | Energetic coupling between plastids and mitochondria drives CO2 assimilation in diatoms[END_REF]. The increased amount of FNR in cpsrp54 mutants might be an attempt to compensate for the lower amounts of electrons available for NADPH formation in the mutants (Figure 4C and4F).

Up-regulation of proteins connected to photoprotective responses

Several proteins known or predicted to be involved in photoprotective mechanisms were detected at moderately higher levels in mutants compared to WT (Table 2). LHCX1 and LHCX2 are FCP proteins important for NPQ and were upregulated in cpsrp54 lines, consistent with the NPQ phenotype of the mutants (Figure 3F). A putative thylakoid bound ascorbate peroxidase (APX) likely to be involved in removal of H2O2 produced during photosynthesis was also expressed at higher levels in the mutants than in the WT. In addition, the expression of a protein annotated as a Cyclophilin type peptidyl-prolyl cis-trans isomerase was increased by around 50% in the mutants (Table 2). Chloroplast-localized cyclophilin type proteins has been found to be important for PSII biogenesis and repair in plants [START_REF] Fu | A chloroplast cyclophilin and maintenance of functions in the assembly photosystem II in Arabidopsis thaliana[END_REF][START_REF] Järvi | Photosystem II repair in plant chloroplasts -Regulation, assisting proteins and shared components with photosystem II biogenesis[END_REF].

Up-regulation of chaperones and possible interaction partners

Chloroplast-localized members of the four major families of ATP-dependent molecular chaperones (chaperonin/Cpn60, Hsp70, Hsp90 and Hsp100 families) and cofactors were detected by the proteomics analyses. Of these, the chloroplast chaperon annotated as DnaK (Hsp70, chloroplast genome), its co-chaperone DNAJ (Hsp40) and the Hsp100 chaperone CLPB were expressed at higher levels in both the investigated mutant lines than in WT, with DnaK being the most upregulated chloroplast protein in our dataset (Table 2). The functions of these chaperons are unknown in diatoms, but HSP70 (DnaK homologs)-and HSP40 (DNAJ homologs)-type chaperons are known to be part of a network of molecular chaperons important for co-translational folding in chloroplasts [START_REF] Ries | Co-translational protein folding and sorting in chloroplasts[END_REF]. In addition to protein folding, other proposed functions of stromal HSP70 chaperones include protein import into chloroplasts, protection/repair of PSII from photoinhibition and (dis-) assembly of vesicleinducing protein in plastids 1 (VIPP1) oligomers [START_REF] Trösch | ATP-dependent molecular chaperones in plastids -More complex than expected[END_REF]. VIPP1 (also called the inner membrane-associated protein of 30 kDa (IM30)) proteins are highly conserved proteins found in cyanobacteria, algae and plants [START_REF] Heidrich | Specific interaction of IM30/Vipp1 with cyanobacterial and chloroplast membranes results in membrane remodeling and eventually in membrane fusion[END_REF][START_REF] Siebenaller | Functional implications of multiple IM30 oligomeric states[END_REF]. The exact function of VIPP1 is unclear, but it is proposed to be multifunctional and to be involved in thylakoid biogenesis/stabilization, coping with chloroplast membrane stress, remodeling of thylakoid membranes during light shifts and co-translational insertions of photosynthetic proteins (Walter et al., 2015a[START_REF] Heidrich | Specific interaction of IM30/Vipp1 with cyanobacterial and chloroplast membranes results in membrane remodeling and eventually in membrane fusion[END_REF][START_REF] Junglas | What is Vipp1 good for?[END_REF][START_REF] Siebenaller | Functional implications of multiple IM30 oligomeric states[END_REF]. The P. tricornutum homolog of VIPP1 was upregulated almost 2-fold in the cpsrp54 mutants (Table 2). VIPP1 is known to dynamically shift between monomeric and oligomeric states, likely with the help of chaperons [START_REF] Siebenaller | Functional implications of multiple IM30 oligomeric states[END_REF]. DnaK-type chaperons and co-chaperones of the DNAJ type have been shown to interact with and impact the oligomeric state of VIPP1 in other photosynthetic organisms [START_REF] Liu | The chloroplast HSP70B-CDJ2-CGE1 chaperones catalyse assembly and disassembly of VIPP1 oligomers in Chlamydomonas[END_REF][START_REF] Gao | α-helical domains affecting the oligomerization of Vipp1 and its interaction with Hsp70/DnaK in Chlamydomonas[END_REF][START_REF] Siebenaller | Functional implications of multiple IM30 oligomeric states[END_REF]. CLPB belongs to the family of Caseinolytic proteases (CLP) that are important for removal of stress-related protein aggregates [START_REF] Mishra | ClpB/Hsp100 proteins and heat stress tolerance in plants[END_REF]. CLPB-type chaperones have been found to collaborate and directly interact with the DnaK system to solubilize and rescue aggregated proteins [START_REF] Doyle | Interplay between E. coli DnaK, ClpB and GrpE during protein disaggregation[END_REF][START_REF] Mogk | Cooperation of Hsp70 and Hsp100 chaperone machines in protein disaggregation[END_REF][START_REF] Mishra | ClpB/Hsp100 proteins and heat stress tolerance in plants[END_REF].

Discussion

CpSRP54 is unlikely to play a role in post-translational targeting of FCPs to thylakoid membranes in diatoms The role of CpSRP54 in the CpSRP pathway has previously been studied in the plant and green algal model organisms A. thaliana and C. reinhardtii, respectively [START_REF] Amin | Arabidopsis mutants lacking the 43-and 54-kilodalton subunits of the chloroplast signal recognition particle have distinct phenotypes[END_REF][START_REF] Nilsson | Interactions of ribosome nascent chain complexes of the chloroplast-encoded D1 thylakoid membrane protein with cpSRP54[END_REF][START_REF] Hutin | Double mutation cpSRP43 -/cpSRP54is necessary to abolish the cpSRP pathway required for thylakoid targeting of the light-harvesting chlorophyll proteins[END_REF][START_REF] Nilsson | Transient interaction of cpSRP54 with elongating nascent chains of the chloroplast-encoded D1 protein; 'cpSRP54 caught in the act[END_REF][START_REF] Rutschow | Quantitative proteomics of a chloroplast SRP54 sorting mutant and its genetic interactions with CLPC1 in Arabidopsis[END_REF][START_REF] Dünschede | Chloroplast SRP54 was recruited for posttranslational protein transport via complex formation with chloroplast SRP43 during land plant evolution[END_REF], Walter et al., 2015b[START_REF] Jeong | Loss of CpSRP54 function leads to a truncated light-harvesting antenna size in Chlamydomonas reinhardtii[END_REF][START_REF] Hristou | Ribosome-associated chloroplast SRP54 enables efficient cotranslational membrane insertion of key photosynthetic proteins[END_REF]. CpSRP54 targets members of the LHCP family to thylakoid membranes in both species through the post-translational pathway, and CpSRP54 KO mutants display a pale green color as a result of having a truncated light harvesting antenna (TLA) [START_REF] Amin | Arabidopsis mutants lacking the 43-and 54-kilodalton subunits of the chloroplast signal recognition particle have distinct phenotypes[END_REF][START_REF] Hutin | Double mutation cpSRP43 -/cpSRP54is necessary to abolish the cpSRP pathway required for thylakoid targeting of the light-harvesting chlorophyll proteins[END_REF][START_REF] Rutschow | Quantitative proteomics of a chloroplast SRP54 sorting mutant and its genetic interactions with CLPC1 in Arabidopsis[END_REF][START_REF] Jeong | Loss of CpSRP54 function leads to a truncated light-harvesting antenna size in Chlamydomonas reinhardtii[END_REF]. In addition to the color change and the lower levels of antenna proteins and pigments, typical features of TLA mutants are increased photosynthetic activity per Chl and a higher light saturation point of photosynthesis (Kirst et al., 2012a, Kirst et al., 2012b, Kirst et al., 2014[START_REF] Jeong | Loss of CpSRP54 function leads to a truncated light-harvesting antenna size in Chlamydomonas reinhardtii[END_REF]. These features were all present in P. tricornutum alb3b mutants, and the TLA phenotype was interpreted as the ALB3b insertase playing an important, but not essential role in integration of FCPs into the thylakoid membrane in diatoms [START_REF] Nymark | Loss of ALBINO3b insertase results in truncated lightharvesting antenna in diatoms[END_REF]. In contrast, the P. tricornutum cpsrp54 mutants did not display any of the above described TLA-features, and they displayed the WT phenotype for almost all measured parameters when grown in LL conditions. Taking into consideration also the CTD of the ALB3b insertase having unknown interaction partners and the absence of key players of the LHCP targeting machinery like the CpSRP43 chaperon and the LTD protein, an essential role for the diatom CpSRP54 in post-translational targeting of FCPs to thylakoid membranes seems highly unlikely. The post-translational part of the CpSRP pathway known to function in plants and green algae is the only SRP-mediated protein targeting process that is not a co-translational process [START_REF] Pool | Signal recognition particles in chloroplasts, bacteria, yeast and mammals (review)[END_REF], Ries et al., 2020). It is tempting to speculate that the diatom CpSRP54 protein has not evolved to function posttranslationally, and that an alternative strategy for targeting of FCPs to thylakoids exists in diatoms.

However, a study performed by Lang and Kroth two decades ago showed that FCP integration into diatom thylakoid membranes is GTP-dependent [START_REF] Lang | Diatom fucoxanthin chlorophyll a/c-binding protein (FCP) and land plant light-harvesting proteins use a similar pathway for thylakoid membrane Insertion[END_REF]. They also showed that LHCPs from peas could be inserted into diatom thylakoids in the presences of GTP and diatom stromal extracts, and that diatom FCPs could be inserted into pea thylakoids under reciprocal experimental conditions, although to a lesser extent. In both experiments a low fraction of LHCPs/FCPs were seemingly also spontaneously integrated into thylakoids [START_REF] Lang | Diatom fucoxanthin chlorophyll a/c-binding protein (FCP) and land plant light-harvesting proteins use a similar pathway for thylakoid membrane Insertion[END_REF].

At the time, the results were interpreted as LHCPs of plants and FCPs of diatoms being integrated through a highly similar CpSRP-dependent machinery. In light of the current knowledge of the CpSRP pathway in diatoms, another possible explanation for their results could be that the LHCPs and FCPs are similar enough to be recognized by two different thylakoid protein targeting machineries.

The phenotype of cpsrp54 mutants indicates a role for the diatom CpSRP54 in the cotranslational CpSRP pathway Phenotypic differences are triggered by increased light intensities One of the aims of this study was to test the hypothesis of CpSRP54 being involved in co-translational insertions of chloroplast encoded proteins into the thylakoid membrane. A shift from LL to ML conditions resulted in photophysiological differences between cpsrp54 and WT that were in support of such a role. Despite stronger induction of photoprotective mechanisms (Figure 3D-F, Table 2) physiological parameters indicated higher levels of PSII inactivation in cpsrp54 mutants compared to WT (Figure 4), likely to be caused by a moderately decreased rate of PSII repair (Figure 5). Similarly, a moderately reduced PSII repair efficiency has also been reported for A. thaliana mutants lacking CpSRP54 (Walter et al., 2015b). Increased light intensities demand a higher rate of PSII repair involving an efficient removal and replacement of chloroplast encoded proteins prone to photodamage by a newly synthesized copy (Järvi et al., 2015, Theis and[START_REF] Theis | Revisiting the photosystem II repair cycle[END_REF]. Loss of CpSRP54 might cause the replacement of damaged PSII core proteins to be less efficient. In plants, a connection between accumulation of non-functional PSII and down regulation of the activity and amount of zeaxanthin epoxidase (ZEP) has been shown [START_REF] Bethmann | The zeaxanthin epoxidase is degraded along with the D1 protein during photoinhibition of photosystem II[END_REF]. ZEP is responsible for the reconversion of zeaxanthin to violaxanthin in plants and Ddx to Dtx in diatoms when the photosynthetic electron transport is no longer saturated either because of acclimation of the photosynthetic apparatus to higher light intensities or by transfer to low light conditions. If a connection between decreased ZEP amount/activity and increased PSII inhibition exists also in diatoms, it could explain the more limited back conversion of Dtx to Ddx in cpsrp54 mutants after prolonged ML-treatment. NADPH is a cofactor of ZEP, and a slower epoxidation of Dtx to Ddx during acclimation to ML might also be caused by insufficient availability of NADPH because of the lower electron flow rate in the cpsrp54 lines [START_REF] Lavaud | The peculiar features of non-photochemical fluorescence quenching in diatoms and brown algae. In Non-photochemical quenching and energy dissipation in plants, algae and cyanobacteria. Advances in photosynthesis and respiration (Including bioenergy and related processes)[END_REF].

Physiological differences cannot be explained by changes in the abundance of PSII RC proteins

Analyses of the proteome after 6 h of ML exposure were performed hoping that this data could provide a molecular explanation for the light-induced phenotypic differences seen at the physiological level. A straightforward explanation for the increased level of photoinactivated PSII in cpsrp54 lines would have been a decrease in the level of PSII RC proteins as a response to the light-treatment.

Photosynthetic organisms typically display a high turnover rate of D1, and to a lesser extent D2, when subjected to increased light intensities [START_REF] Aro | Photoinhibition of Photosystem II. Inactivation, protein damage and turnover[END_REF][START_REF] Jansen | Accelerated degradation of the D2 protein of photosystem II under ultraviolet radiation[END_REF], Aro et al., 2005, Rokka et al., 2005). In contrast, marine diatom species are shown to have more similar turnover rates for D1 and D2 [START_REF] Wu | Distinctive photosystem II photoinactivation and protein dynamics in marine diatoms[END_REF][START_REF] Wu | Photosystem II photoinactivation, repair, and protection in marine centric diatoms[END_REF]. The proteomics data confirmed a lower relative abundance of D2 in both the investigated cpsrp54 lines, whereas the data for D1 was inconclusive. Additional analysis of D1 and D2 in cells acclimated to LL and ML detected roughly 30% lower levels of D2 in cpsrp54 lines than in WT in both light conditions, whereas the relative amount of D1 varied between experiments, but was on average more similar to WT levels. The consistently lower amount of D2 found in all light treatments therefore seems to be a general feature of the cpsrp54 mutants, and not a result of light stress. The photosynthetic performance of cpsrp54 mutants is similar to WT levels in LL, meaning that there is no difference in functionality of the assembled PSII complexes in the mutants and WT, although the relative amount of one of the PSII core subunits seem to be lower. The existence of sub-pools of D1 and D2 that were not a part of active PSII has previously been reported for marine diatoms by Wu and coworkers [START_REF] Wu | Distinctive photosystem II photoinactivation and protein dynamics in marine diatoms[END_REF][START_REF] Wu | Photosystem II photoinactivation, repair, and protection in marine centric diatoms[END_REF]. They also showed that a decrease in PSII activity often did not correlate with changes in D1 and D2 abundance [START_REF] Wu | Photosystem II photoinactivation, repair, and protection in marine centric diatoms[END_REF]. A similar phenomenon seems likely in our study since the relative lower content of D2 compared to WT under the same light conditions is stable, whereas the difference in photosynthetic performance increases when shifted from LL to ML intensities. In support of our observations and previous reports is also a recent study of PSII supercomplexes in P. tricornutum thylakoid membranes, where a subpopulation of inactive/damaged PSII RCs not associated with FCP complexes was observed and suggested to function as PSII repair stations [START_REF] Levitan | Structural and functional analyses of photosystem II in the marine diatom Phaeodactylum tricornutum[END_REF]. The analysis of PSII RC proteins levels carried out in this study were performed on whole cell extracts and will not separate between proteins incorporated in functional PSIIs, proteins in inactivated PSIIs or unassembled D1 and D2 proteins.

Loss of cpsrp54 triggers similar responses on the protein level in diatom and plants

In A. thaliana, there is convincing data supporting that CpSRP54 enhances the efficiency of cotranslational insertion of a selection of chloroplast encoded subunits [START_REF] Amin | Arabidopsis mutants lacking the 43-and 54-kilodalton subunits of the chloroplast signal recognition particle have distinct phenotypes[END_REF][START_REF] Nilsson | Interactions of ribosome nascent chain complexes of the chloroplast-encoded D1 thylakoid membrane protein with cpSRP54[END_REF][START_REF] Nilsson | Transient interaction of cpSRP54 with elongating nascent chains of the chloroplast-encoded D1 protein; 'cpSRP54 caught in the act[END_REF][START_REF] Rutschow | Quantitative proteomics of a chloroplast SRP54 sorting mutant and its genetic interactions with CLPC1 in Arabidopsis[END_REF][START_REF] Piskozub | Ribosome nascent chain complexes of the chloroplast-encoded cytochrome b6 thylakoid membrane protein interact with cpSRP54 but not with cpSecY[END_REF][START_REF] Hristou | Ribosome-associated chloroplast SRP54 enables efficient cotranslational membrane insertion of key photosynthetic proteins[END_REF]. Several studies have shown a reduced content of PSI and PSII RC proteins in developing A. thaliana cpsrp54 mutants [START_REF] Amin | Arabidopsis mutants lacking the 43-and 54-kilodalton subunits of the chloroplast signal recognition particle have distinct phenotypes[END_REF][START_REF] Rutschow | Quantitative proteomics of a chloroplast SRP54 sorting mutant and its genetic interactions with CLPC1 in Arabidopsis[END_REF][START_REF] Hristou | Ribosome-associated chloroplast SRP54 enables efficient cotranslational membrane insertion of key photosynthetic proteins[END_REF], and additional potential targets for CpSRP54 were revealed through a proteomic study, indicating that also other subunits of PSI, PSII and ATP synthase were negatively affected by a lack of CpSRP54 [START_REF] Rutschow | Quantitative proteomics of a chloroplast SRP54 sorting mutant and its genetic interactions with CLPC1 in Arabidopsis[END_REF]. Direct evidence for the interaction of nascent chains of D1 of PSII and PetB of the Cytb6f complex with CpSRP54 also exists [START_REF] Nilsson | Interactions of ribosome nascent chain complexes of the chloroplast-encoded D1 thylakoid membrane protein with cpSRP54[END_REF][START_REF] Nilsson | Transient interaction of cpSRP54 with elongating nascent chains of the chloroplast-encoded D1 protein; 'cpSRP54 caught in the act[END_REF][START_REF] Piskozub | Ribosome nascent chain complexes of the chloroplast-encoded cytochrome b6 thylakoid membrane protein interact with cpSRP54 but not with cpSecY[END_REF]. A recent ribosome profiling study comparing thylakoid membrane association of translating ribosomes between WT and a mutant lacking CpSRP54 further confirmed CpSRP54's role in co-translational targeting of central photosynthetic proteins in plants [START_REF] Hristou | Ribosome-associated chloroplast SRP54 enables efficient cotranslational membrane insertion of key photosynthetic proteins[END_REF]. Similar to the results described above, several chloroplast-encoded subunits of PSI, PSII, Cytb6f and ATP synthase were found at moderately lower levels in the P. tricornutum cpsrp54 mutants. In A.

thaliana, the clearest effects of CpSRP54 depletion were seen on the level of PSI and PSII RC proteins, whereas in P. tricornutum only the PSII RC protein D2 was significantly downregulated in mutant lines. Despite some discrepancies in which subunits that were most severely affected by the loss of CpSRP54, and that we have no evidence for direct interaction between CpSRP54 and elongating thylakoid membrane proteins in P. tricornutum, the combined results from this study do imply that CpSRP54 is involved, but not essential, in targeting of chloroplast-encoded proteins to thylakoids in diatoms as well.

Effect of loss of CpSRP54 is alleviated by compensating mechanisms

The relatively mild phenotype of both the A. thaliana and P. tricornutum cpsrp54 mutants might be partially explained by execution of compensation mechanisms involving the upregulation of chaperones that can alleviate the effects of loss of CpSRP54. Absence of CpSRP54 resulted in upregulation of DnaK homologs cpHSP70-1/2, CPN60 proteins, co-chaperonin CPN21 and ClpC-1/2 in A. thaliana [START_REF] Rutschow | Quantitative proteomics of a chloroplast SRP54 sorting mutant and its genetic interactions with CLPC1 in Arabidopsis[END_REF] and elevated levels of DnaK, DnaJ and ClpB in P. tricornutum.

Upregulations of a range of cytoplasmic chaperons belonging mostly to the same families as the ones found in the chloroplastic responses, have also been reported as a common response to the loss of cytoplasmic SRP54 in yeast and bacteria [START_REF] Arnold | The stress response to loss of signal recognition particle function in Saccharomyces cerevisiae[END_REF][START_REF] Mutka | Multifaceted physiological response allows yeast to adapt to the loss of the signal recognition particle-dependent protein-targeting pathway[END_REF][START_REF] Wickström | Consequences of depletion of the signal recognition particle in Escherichia coli[END_REF], Zhang et al., 2012). Here, upregulation of the chaperone systems is believed to prevent the accumulation of mis-localized and aggregated proteins and might provide alternative pathways to target proteins to its target membranes [START_REF] Arnold | The stress response to loss of signal recognition particle function in Saccharomyces cerevisiae[END_REF][START_REF] Mutka | Multifaceted physiological response allows yeast to adapt to the loss of the signal recognition particle-dependent protein-targeting pathway[END_REF][START_REF] Wickström | Consequences of depletion of the signal recognition particle in Escherichia coli[END_REF], Zhang et al., 2012). Although no functional data is available about roles and interaction partners of the chloroplast-localized DnaK in diatoms, the ~3-fold upregulation of the P. tricornutum DnaK in cpsrp54 lines compared to WT suggests key roles for this multifunctional chaperon in coping with the stress of lacking CpSRP54. The simultaneous upregulation of DnaK, its putative cochaperone DnaJ and of one of their possible interaction partners, the diatom VIPP1 homolog, as a response to the lack of CpSRP54 opens for speculations about an indirect role of DnaK in insertion of thylakoid membrane proteins. As previously mentioned, the exact function of VIPP1 beyond its importance in thylakoid membrane biogenesis/maintenance is unclear, but one of its many suggested roles is connected to insertion of photosynthetic proteins [START_REF] Bryan | Localisation and interactions of the Vipp1 protein in cyanobacteria[END_REF][START_REF] Trösch | ATP-dependent molecular chaperones in plastids -More complex than expected[END_REF][START_REF] Heidrich | Specific interaction of IM30/Vipp1 with cyanobacterial and chloroplast membranes results in membrane remodeling and eventually in membrane fusion[END_REF][START_REF] Gutu | Dynamical localization of a thylakoid membrane binding protein is required for acquisition of photosynthetic competency[END_REF]. In plants, VIPP1 has been detected as a component of D1 intermediates together with CpSRP54, CpFTSY, ALB3 and CpSecY, and found to stimulate insertion of D1 (Walter et al., 2015a). Depletion of the ALB3.2 insertase of the co-translational CpSRP pathway in C. reinhardtii resulted in upregulation of VIPP1, the DnaK homolog HSP70B and its cochaperone [START_REF] Göhre | One of two Alb3 proteins is essential for the assembly of the photosystems and for cell survival in Chlamydomonas[END_REF]. VIPP1 is also upregulated in C. reinhardtti mutants lacking the ALB3.1 insertase mainly involved in the posttranslational insertions of LHCPs, but absence of ALB3.1 also causes lower levels of D1 [START_REF] Bellafiore | Loss of Albino3 leads to the specific depletion of the light-harvesting system[END_REF][START_REF] Theis | VIPP2 interacts with VIPP1 and HSP22E/F at chloroplast membranes and modulates a retrograde signal for HSP22E/F gene expression[END_REF]. In addition, both HSP70B and VIPP1 are high light inducible and indicated to be involved in PSII repair in C. reinhardtii [START_REF] Drzymalla | Light-inducible gene HSP70B encodes a chloroplast-localized heat shock protein in Chlamydomonas reinhardtii[END_REF][START_REF] Nordhues | Evidence for a role of VIPP1 in the structural organization of the photosynthetic apparatus in Chlamydomonas[END_REF][START_REF] Trösch | ATP-dependent molecular chaperones in plastids -More complex than expected[END_REF]. A possible connection between DnaK-type chaperones, VIPP1 and PS biogenesis has been suggested where the chaperones influence the activity of VIPP1 by catalyzing the assembly and disassembly of the VIPP1 oligomers, and where VIPP1 provides local areas where PS biogenesis can take place [START_REF] Liu | The chloroplast HSP70B-CDJ2-CGE1 chaperones catalyse assembly and disassembly of VIPP1 oligomers in Chlamydomonas[END_REF][START_REF] Rütgers | A role of VIPP1 as a dynamic structure within thylakoid centers as sites of photosystem biogenesis[END_REF][START_REF] Bryan | Localisation and interactions of the Vipp1 protein in cyanobacteria[END_REF][START_REF] Trösch | ATP-dependent molecular chaperones in plastids -More complex than expected[END_REF][START_REF] Gutu | Dynamical localization of a thylakoid membrane binding protein is required for acquisition of photosynthetic competency[END_REF]. A similar role in PS biogenesis for the DnaK-type chaperone system and VIPP1 in diatoms is imaginable, but no functional data is currently available to support such speculations.

Conclusion

Previous studies of the effects of loss of CpSRP54 in model organisms representing land plants and green microalgae and the current study of the effect of loss of CpSRP54 in the model diatom P. tricornutum show that although there are some similarities (see overview in Table 3), the mechanisms for targeting proteins to thylakoid membranes are too different for a direct transfer of knowledge from one group of species to another. A schematic representation of the CpSRP pathway for integration of thylakoid membrane proteins in P. tricornutum compared to plants (A. thaliana) and green algae (C. (Kirst andMelis, 2014, Jeong et al., 2017). Truncation of the light harvesting antenna in green microalgae causes improved rates of growth and productivity under high-density and bright sunlight conditions, features that are desired from a commercial point of view (Kirst andMelis, 2014, Jeong et al., 2017). The data presented above clearly show that such a phenotype is not achieved by disrupting the gene encoding CpSRP54 in P. tricornutum, and our data is only in support of a role for the CpSRP54 in the cotranslational insertion of chloroplast-encoded proteins. Targeting of diatom FCP proteins to thylakoid membranes seems to be independent of the CpSRP pathway (Figure 7A, left side), making it unlikely that members of this pathway can be exploited in strain optimization for high density cultivation of diatoms.

Experimental procedures

The axenic P. tricornutum Bohlin clone Pt1 8.6 (CCMP632) culture was obtained from the culture collection of the Provasoli-Guillard National Center for Culture of Marine Phytoplankton, Bigelow Laboratory for Ocean Sciences, USA.

P. tricornutum cpsrp54 knock out mutants

The cpsrp54-8, cpsrp54-11, cpsrp54-20 mutant lines used for this study were isolated from single cells from the M2, M4 and M8 lines, respectively, previously published in Nymark et al. [START_REF] Nymark | A CRISPR/Cas9 system adapted for gene editing in marine algae[END_REF]. The cpsrp54 KO mutants had been created with the aid of the CRISPR/Cas9 technology using a P. tricornutum culture derived from the sequenced clone Pt1 8.6 [START_REF] Bowler | The Phaeodactylum genome reveals the evolutionary history of diatom genomes[END_REF] as a starting point. Knock out mutations were confirmed in the three lines by TOPO cloning and subsequent Sanger sequencing of PCR products spanning the region of the CpSRP54 gene targeted by the Cas9-gRNA complex as described previously [START_REF] Nymark | A CRISPR/Cas9 system adapted for gene editing in marine algae[END_REF][START_REF] Nymark | CRISPR/Cas9 Gene editing in the marine diatom Phaeodactylum tricornutum[END_REF] Growth and experimental light conditions P. tricornutum wild type (WT) cells and cpsrp54 lines (cpsrp54-8, cpsrp54-11, cpsrp54-20) were cultured as described previously [START_REF] Nymark | An integrated analysis of molecular acclimation to high light in the marine diatom Phaeodactylum tricornutum[END_REF]. Cell cultures were grown at 15°C under continuous cool white fluorescent light at scalar irradiance (EPAR) of ~35 µmol photons m -2 s -1 (low light (LL)) or ~200 µmol photons m -2 s -1 (medium light (ML)), unless stated otherwise. High light (HL; ~1000 µmol photons m-2 s-1) was provided by a full spectrum LED lamp (5500 K). The cultures were kept in the exponential growth phase for at least two weeks under LL, ML or HL conditions before conducting experiments.

Growth rates

Growth rates were estimated in batch cultures of WT and cpsrp54 KO lines (three biological replicates) acclimated to LL, ML or HL. The starting concentrations used for determining the growth rates were 200.000 cells/ml for LL-acclimated cells, 100.000 cells/ml for ML-acclimated cells and 50.000 cells/ml for HL-acclimated cells. Cells were fixed in Lugol's solution and counted by using a Bürker-Türk counting chamber or by flowcytometry as described previously [START_REF] Nymark | A CRISPR/Cas9 system adapted for gene editing in marine algae[END_REF]. The average maximum growth rates (cell division/day) for WT and mutant lines were calculated by using the mean of the growth rates from the three biological replicates during the exponential phase.

In vivo fluorescence excitation

In vivo fluorescence excitation spectra were obtained from LL-acclimated WT and cpsrp54 cultures (three biological replicates) using a Hitachi F-3000 spectrofluorometer (Hitachi Corp.) as previously described (Johnsen andSakshaug, 2007, Nymark et al., 2013). Excitation light was provided at 1 nm spectral resolution (5 nm bandwidth) from 400 to 700 nm, registering Chl a fluorescence emission at 730 nm (5 nm band width) as a function of absorbed light at the different wavelengths. 3-(3,4dichlorophenyl)-1,1-dimethylurea (DCMU; 50 µM final concentration) was added 1 minute prior to each measurement to avoid variable fluorescence. All spectra were normalized to the red emission maximum of Chl a of the WT cultures.

Absorbance spectra

Absorption spectra were measured with a Multiscan sky spectrophotometer (Thermo Fisher Scientific, USA) with a resolution of 1 nanometer. Cells were loaded on a multiwell plate and spectra were normalized on the blue absorption peak. Data are representative of 3 biological replicates (3 technical replicates per genotype per experiment).

LL to ML shift time-series experiments

LL acclimated (0 h) cpsrp54 KO lines were exposed to ML for 0.5, 6, 24, 48 h and 168 h. Three biological replicates were included for each line and time point. Cell concentrations were approx. 1

x 10 6 cells/mL at the day of harvesting. Harvested material was used for monitoring of the photophysiological state and pigment concentration per cell in LL and after the shift to ML. The timeseries experiment in the current study and in a previously published study characterizing the P. tricornutum alb3b mutant [START_REF] Nymark | Loss of ALBINO3b insertase results in truncated lightharvesting antenna in diatoms[END_REF] were performed in parallel and the responses of the cpsrp54 and alb3b mutants were therefore compared to the same WT dataset.

Pigment analysis

Pigment analysis were performed by HPLC according to [START_REF] Rodriguez | Photoacclimation in phytoplankton: implications for biomass estimates, pigment functionality and chemotaxonomy[END_REF] using a Hewlett-Packard HPLC 1100 Series system. Pigment values from the HPLC analysis were calculated as femtomol (fmol) pigment per cell. Cell concentrations were determined using a BD Accuri C6 Flow Cytometer as described previously [START_REF] Nymark | Loss of ALBINO3b insertase results in truncated lightharvesting antenna in diatoms[END_REF].

Measurements of photosynthetic parameters

Variable in vivo Chl a fluorescence was measured as described in Nymark et al. [START_REF] Nymark | Loss of ALBINO3b insertase results in truncated lightharvesting antenna in diatoms[END_REF] using a PhytoPAM (System I, Walz, Germany) equipped with a photomultiplier detector (PM-101P, Walz, Germany). The photosynthetic efficiency (Fv/Fm), the photosynthetic capacity (maximum relative electron transport rate (rETRmax), the maximum light utilization coefficient (α), the light saturation index (Ek=rETRmax/α) and NPQ were calculated as described previously [START_REF] Nymark | An integrated analysis of molecular acclimation to high light in the marine diatom Phaeodactylum tricornutum[END_REF][START_REF] Nymark | Loss of ALBINO3b insertase results in truncated lightharvesting antenna in diatoms[END_REF]. NPQ data was derived from light response curves from LL-acclimated cultures. All samples were incubated 3 min in darkness prior to performing measurements. A Peltier cell (US-T/S, Walz) kept the temperature at 15°C (±0.2°C) during the measurements.

The ECS signal in LL and ML-acclimated cells was measured with a Joliot-type spectrophotometer (JTS-10, Biologic) equipped with a laser or xenon lamp as excitation sources, white probing light and interference filters (3-8 nm bandwidth) allowing measurements of the absorption difference signals at selected wavelengths [START_REF] Bailleul | Energetic coupling between plastids and mitochondria drives CO2 assimilation in diatoms[END_REF]. All cultures were upconcentrated to 20 million cells/ml by centrifugation prior to performing the measurements. Measurements of PSII/PSI stoichiometries were measured under single turnover flash regime. ECS kinetics were triggered by excitation with a saturating light flash produced by a laser or xenon lamp causing a single charge separation in all active PSs. Three different phases were observed: a fast rise of the signal corresponding to charge separation in PSI and PSII (<100 µs after laser flash), a slower rise (ms time range) representing electron flow through Cytb6f and a decay phase due to charge leakage through the membrane [START_REF] Bailleul | Electrochromism: a useful probe to study algal photosynthesis[END_REF]. The linear component of the ECS (ECSlin) was used to estimate the PSII/PSI ratio. ECSlin was deconvoluted from superimposed signals by measuring at three different wavelengths (520, 554 and 563 nm) as described in Bailleul et al. [START_REF] Bailleul | Energetic coupling between plastids and mitochondria drives CO2 assimilation in diatoms[END_REF]. Briefly, we first assessed the contribution of c-type cytochromes in the measured signal as Cyt c=

[554] -0.4 x [520] -0.4 x [563],
where [554], [520] and [566] were the measured absorption difference signals at the three different wavelengths. Secondly, we evaluated ECSlin as = [520] -0.25 x Cyt c. PSII contribution was evaluated from the amplitude of the fast ECSlin phase as the decrease in the signal amplitude upon poisoning samples with DCMU (20µM) and hydroxylamine (HA; 1 mM) to irreversibly block PSII charge separation. PSI was estimated as the fraction of the signal that was insensitive to these inhibitors [START_REF] Bailleul | Electrochromism: a useful probe to study algal photosynthesis[END_REF].

The same approach was used to evaluate photosynthetic electron flow (electrons per second) in LL and ML-acclimated WT and cpsrp54 cells. Cells were illuminated with continuous red light (590 µmol photons m -2 s -1 ) until steady state photosynthesis was achieved. Then, light was switched off and the rate of photosynthetic electron flow was measured as described in [START_REF] Bailleul | Electrochromism: a useful probe to study algal photosynthesis[END_REF].

Briefly, in steady state the ECS signal reflects membrane potential generation by the PSs and Cytb6f complex and membrane potential dissipation by the ATP synthase. When light is switched off, PSII and PSI activity stops abruptly, while ATP synthase and the Cytb6f complex activities remain (transiently) unchanged. Therefore, the difference between the slopes of the ECS signal measured in the light and after the light is switched off is proportional to the rate of PSI and PSII photochemistry (i.e. to the rate of electron flow). This rate can be quantified by dividing the slope difference by the amplitude of the fast ECSlin phase measured under single turnover flashes in the presence of DCMU and HA. The latter reflects the ECS absorption changes induced by the transfer of a single charge across the membrane (e.g. one electron per photosynthetic chain).

High light lincomycin experiment

Lincomycin (LINC; Sigma-Aldrich) blocks chloroplast protein synthesis [START_REF] Ridley | Interaction of chloroplasts with inhibitors: Location of carotenoid synthesis and inhibition during chloroplast development[END_REF] and was added to LL-acclimated cultures (3 biological replicates) in the exponential phase at a final concentration of 500 µg mL -1 . Cultures were incubated in darkness for 15 min before exposure to HL (1000 µmol photons m -2 s -1 ) for 1 h. In parallel, cultures without LINC were exposed to the same treatment. Fv/Fm was measured in LL and HL treated cultures using an AquaPen-C (Photon System Instruments). LL acclimated cells were incubated in darkness for 3 min prior to measurements, whereas HL-treated cells were measured after both a 3 min and 30 min very dim light period. The additional measurement after 30 min was included to relax the rapidly reversible qE component of NPQ so that only the photoinihibitory, slowly reversible quenching (qI), caused by damaged PSII reaction centers, would influence the Fv/Fm value.

Proteomics

Proteomic analyses were performed of cpsrp54-8, cpsrp54-11 and WT lines after 6 h exposure to ML. Five biological replicates were included for each line, and cells were harvested by filtration as described previously [START_REF] Nymark | Loss of ALBINO3b insertase results in truncated lightharvesting antenna in diatoms[END_REF]. To each sample, 100 µl of SDC buffer (1% SDC, 10 mM TCEP, 40 mM 2-Chloroacetamide, Roche Protease Inhibitor cocktail, 100 mM Tris pH 8.5) was added. After mixing by pipetting, contents were transferred to LoBind tubes (Eppendorf), the tubes were kept at 95 °C for 5 minutes, before being sonicated at 4 °C in a Bioruptor Pico (Diagenode) for 10 cycles, each with 1 min sonication followed by 30 sec without sonication. Protein concentration was measured in a Direct Detect (Millipore) and a volume containing 50 µg total protein was separated for further sample preparation. The volume was adjusted to 50 µl by addition of water.

Next, a KingFisher Flex (Thermo Scientific) magnetic-bead handling robot performed clean-up of the proteins using HILIC magnetic microparticles (ReSyn Bioscience) followed by tryptic digestion into peptides. In more details, deep well plates were prepared and placed into the KingFisher as: 25 µl of HILIC microparticles (20 mg/ml) and 175 µl Equilibration buffer (15% Acetonitrile, 100 mM Ammonium acetate pH 4.5) at position 1, 500 µl Equilibration buffer at position 2, 50 µl sample and 50 µl Bind buffer (0.5% SDS, 30% Acetonitrile, 200 mM Ammonium acetate pH 4.5) at position 3, 200 µl 95% Acetonitrile at positions 4 and 5, 50 µl Trypsin (0.1 µg/µl in 45 mM Acetic Acid) and 150 µl Digestion buffer (20 mM Ammonium formate pH 8.2) at position 6, comb Tip at position 7.

Then the robot executed the following protocol: picked up the comb tip, collected HILIC magnetic particles, equilibrated HILIC magnetic particles in Equilibration buffer, bound protein to HILIC magnetic particles, washed in high organic content twice, eluted from HILIC and digested protein with Trypsin at 37° for 4 hours, removed HILIC magnetic particles. BindIt Software 3.3.1 (Thermo Scientific) was used to prepare the robot protocol and to run it. Unless otherwise-stated, chemicals were from Sigma.

Subsequently the digested peptides were transferred to new tubes, they were acidified by addition of 2 µl formic acid, then the tubes were spun-down (16,000g for 5 min) and 100 µl of supernatants were transferred to LC-MS vials. Analysis was performed on an EASY-nLC 1200 UHPLC system (Thermo Scientific) interfaced with an Q Exactive HF mass spectrometer (Thermo Scientific) via a Nanospray Flex ion source (Thermo Scientific). Peptides were injected onto an Acclaim PepMap100 C18 trap column (75 μm i.d., 2 cm long, 3 μm, 100 Å, Thermo Scientific) and further separated on an Acclaim PepMap100 C18 analytical column (75 μm i.d., 50 cm long, 2 μm, 100 Å, Thermo Scientific) at 250 nl/min using a 180-minute gradient (145 min 5%-35% B, 15 min 35%-100% B, 20 min 100% B;

where A is 0.1 % formic acid and B is 0.1 % formic acid, 80% acetonitrile). Unless otherwise-stated, chemicals were from Fisher Scientific.

Peptide ions were analysed in positive ion mode under data dependent acquisition using the following parameters: Electrospray voltage 1.9 kV, HCD fragmentation with normalized collision energy 27.

Each MS scan (200 to 2000 m/z, 3e6 AGC target, profile) was acquired at a resolution of 60,000 Maximum cell divisions per day during the exponential phase were calculated from three biological replicates of WT and cpsrp54 KO lines acclimated to LL (35 µmol photons m -2 s -1 ), ML (200 µmol photons m -2 s -1 ) and HL (1000 µmol photons m -2 s -1 ). Values are presented with ±SD. The LHCF antibody is predicted to detect LHCF1-11 [START_REF] Juhas | A novel cryptochrome in the diatom Phaeodactylum tricornutum influences the regulation of lightharvesting protein levels[END_REF]. An antibody recognizing the β-subunit of ATP synthase (AtpB) was used as loading control on each of the individual blots.

  reinhardtii) is given in Figure 7. Major differences exist for the role of CpSRP54 in the three model systems. In P. tricornutum we suggest that CpSRP54, likely in interaction with the CpFTSY receptor, guides translating ribosomes to the ALB3a insertase (and possibly the CpSecY translocase) for cotranslational insertion of chloroplast-encoded thylakoid membrane proteins (Figure 7A, right side) in a similar manner as the CpSRP54 of A. thaliana (Figure 7B, right side) (Ziehe et al., 2018). A. thaliana CpSRP54 has a dual function and targets also LHCPs to thylakoid membranes (Figure 7B, left side). The role(s) of CpSRP54 in P. tricornutum and A. thaliana contrasts with the role of CpSRP54 in C. reinhardtii, which is reported to function exclusively in the post-translational insertion of LHCPs (Figure 7C, left side) (Jeong et al., 2017). CpSRP54 and other members of the CpSRP pathway (CpFTSY and CpSRP43) have been suggested to be suitable universal targets for gene editing with the purpose of minimizing the chlorophyll antenna size in microalgae because of their role in the post-translational part of the CpSRP pathway in C. reinhardtii

Figure 1 .

 1 Figure 1. Structural features of diatom CpSRP54 proteins. a) Schematic view of the conserved domains of CpSRP54. The area of the CpSRP54 protein corresponding to the 20-bp target region for CRISPR/Cas9-based gene editing is located in the N-terminal helical bundle domain (N-domain),

Figure 2 .

 2 Figure 2. Color and spectral properties of WT and cpsrp54 mutants. a) Culture color of LLacclimated WT and cpsrp54 mutants at equal cell densities. b) In vivo fluorescence excitation spectra and c) absorbance spectra of cultures acclimated to LL. Fluorescence emission was measured at 730 nm to allow for full visible range (400 -700 nm) fluorescence excitation. Presented spectra for all lines are representative of three replicates per line.

Figure 3 .

 3 Figure 3. Pigment levels, DES index and NPQ capacity in WT and cpsrp54 mutant cell lines. Cellular pigment contents (fmol/cell) are shown for a) Chl a, b) Fx, c) Ddx, and d) Dtx in WT and cpsrp54 mutant cells as a function of time following a shift from LL conditions (0 h; 35 µmol photons m -2 s -1 ) to ML conditions (200 µmol photons m -2 s -1 ) for 0.5, 6, 24, 48, and 168 h. e) DES index [DES = Dtx/(Dtx + Ddx)] calculated from data shown in C and D. f) Induction of NPQ as a function of increasing light intensity was calculated from rapid light curves derived from LL-acclimated cells. NPQ = (Fm´max/Fm´) -1. Fm´max replaces the commonly used Fm, since Fm´ values frequently occur at low light intensities that are higher than the F m from dark-treated diatom samples (Serôdio et al., 2006). Results are presented as means of three biological replicates ±SD. Black circles indicate individual data points for the replicates. Asterisks describe significant differences between cpsrp54 mutants and WT as indicated by two-way ANOVA with Dunnet's multiple comparison tests (p < 0.05).

Figure 4 .Figure 5 .

 45 Figure 4. Photophysiological responses of WT and cpsrp54 mutant cell lines after a shift from LL to ML conditions. a) Photosynthetic (PSII) efficiency (Fv/Fm), b) Maximum light-utilization coefficient (α), c) Photosynthetic capacity (rETRmax), and d) Light saturation index (Ek) in WT and cpsrp54 mutant cells as a function of time following a shift from LL conditions (0 h; 35 µmol photons m -2 s -1 ) to ML conditions (200 µmol photons m -2 s -1 ) for 0.5, 6, 24, 48, and 168 h. e) In vivo assessment of PSII/PSI reaction centers ratios and f) Photosynthetic electron flow in LL and ML-acclimated cultures. Results are presented as means of three biological replicates ±SD. Black circles indicate individual data points for the replicates. Asterisks indicate the results of two-way ANOVA with Dunnet's multiple comparison test (p < 0.05)

Figure 6 .

 6 Figure 6. Analysis of thylakoid membrane protein accumulation at different light conditions in WT and cpsrp54 mutant lines. a) Abundance of FCP proteins belonging to the LHCF group and PSII core proteins D1 and D2 in LL-acclimated cells, after 6h in ML and in cells acclimated to ML.

  Images have been cropped. Relative quantification of b) LHCF1-11, c) D1 and d) D2 protein accumulation in cpsrp54 lines compared to WT in LL and ML-acclimated cells was performed using ImageJ to estimate band intensity. All values were normalized against AtpB included as a loading control on all blots. Ratios presented are an average of results calculated from two independent experiments and minimum three different blots with 1-3 biological replicates included in each blot. 10 µg total protein was loaded in each well. Black circles indicate individual data points for the replicates.

Figure 7 .

 7 Figure 7. Models of the CpSRP pathway for integration of thylakoid membrane (TM) proteins in P. tricornutum compared to plant (A. thaliana) and green algae (C. reinhardtii) model organisms. a) Schematic overview of the suggested roles of members of the CpSRP pathway in P. tricornutum. CpSRP54, likely in interaction with its receptor CpFTSY, are proposed to guide translating ribosomes to the ALB3a insertase and possibly the CpSecY translocase for cotranslational insertion of chloroplast encoded TM proteins (right side). FCP proteins synthesized by ribosomes on the chloroplast endoplasmic reticulum membrane (cERM) are transported through the four membranes surrounding diatom chloroplasts and eventually delivered to ALB3b in an unknown manner independent of the CpSRP pathway (left side). b) Schematic overview of the roles of members of the CpSRP pathway in A. thaliana. Several members of the CpSRP pathway (CpSRP54, CpFTSY; ALB3) have dual roles and function in both post-translational insertion of LHCPs (left side) and co-translational insertion of chloroplast encoded proteins (right side) into the TM membrane. LTD and CpSRP43 of the post-translational CpSRP pathway are not present in diatom genomes. LTD delivers LHCPs to the CpSRP43/CpSRP54 complex that in the next step interacts with CpFTSY before integration of LHCPs into TMs through ALB3. c) Schematic overview of the roles of members of the CpSRP pathway in C. reinhardtii. The CpSRP pathway of C. reinhardtii comprises mostly the same key molecular players as A. thaliana, but CpSRP54 and CpFTSY are proposed to function exclusively in the post-translational CpSRP pathway. Here CpSRP54 is not complexed to CpSRP43, but CpSRP54 is believed to function further downstream. Similar to diatoms, but in contrast to plants, two different ALB3 insertases function in the post-and co-translational CpSRP pathway. Abbreviations used are: cERM: chloroplast endoplasmic reticulum membrane; PPM: periplastidial membrane; OEM: outer envelope membrane; IEM: inner envelope membrane; FCP: Fx-Chl a/cbinding proteins; LHCP: Light harvesting complex proteins; LTD: LHCP translocation defect protein; Cp: Chloroplast; SRP: signal recognition particle.

  photons m -2 s -1 )

, D., Sweredoski, M.J., Graham, R.L., Hess, S. and Shan, S.O

  FWHM, followed by 15,000 FWHM MS/MS scans (200 to 2000 m/z, 1.2 m/z isolation width, 1e5 476. https://doi.org/10.1104/pp.112.203067 Zhang. (2012) Novel proteomic tools reveal essential roles of SRP and importance of proper membrane protein biogenesis.

	Mol Cell Proteomics, 11, M111 011585. https://doi.org/10.1074/mcp.M111.011585
	Ziehe, D., Dunschede, B. and Schunemann, D. (2017) From bacteria to chloroplasts: evolution of
	the chloroplast SRP system. Biol Chem, 398, 653-661. https://doi.org/10.1515/hsz-2016-0292
	Ziehe, D., Dunschede, B. and Schunemann, D. (2018) Molecular mechanism of SRP-dependent
	light-harvesting protein transport to the thylakoid membrane in plants. Photosynth Res, 138,
	303-313. https://doi.org/10.1007/s11120-018-0544-6

Table 1 . Growth rates of WT and cpsrp54 mutant lines acclimated to different light intensities.

 1

Table 2 .

 2 Proteomics data. Proteins encoded in the chloroplast genome or predicted to contain chloroplast transit peptides sequences that were significantly regulated (FDR <0.01) in the same direction in both cpsrp54-8 and cpsrp54-11 lines compared to WT after 6 h in ML showing log2 ratios ≥ ± 0.5 for at least one of the mutant lines and where at least two unique peptides were detected. Only proteins with a Score sequest HT >10 were included in the table. Ratios were calculated based on results from five biological replicates for each line. Downregulated proteins are marked in bold. †Low molecular weight proteins included despite detection of only one unique peptide.

	The abbreviations

Table 3 . Overview of the effect of loss of CpSRP54 in diatom (P. tricornutum), plant (A.

 3

Table 1 .

 1

		35 µmol s -1 m -2 200 µmol s -1 m -2 1000 µmol s -1 m -2
	WT	1.54 ± 0.24	2.23 ± 0.03	2.03 ± 0.18
	cpsrp54-8	1.41 ± 0.18	1.61 ± 0.14	1.55 ± 0.03
	cpsrp54-11	1.38 ± 0.26	1.75 ± 0.09	1.46 ± 0.15
	cpsrp54-20	1.38 ± 0.28	1.77 ± 0.12	1.60 ± 0.06

(a) (b) (c) (d) (e) (f)

Acknowledgements

We wish to thank Kjersti Andresen for assistance with the HPLC analyses and Professor Claudia 26 Büchel for kindly providing LHCF antibodies. The authors would also like to thank Stoyan Stoychev at ReSyn Bioscience for helpful discussions on the HILIC sample preparation protocol, and Felicity Ashcroft for help with statistical analyses.

Funding

This work was supported by a grant from the Research Council of Norway to A.M.B through funding of the project "Downsizing light harvesting antennae to scale up production potential and valorization from cultivation of marine microalgae" (project no. 267474), to Olav Vadstein through funding of the project Microbially Produced Raw Materials for Aquafeed (project no. 239001) and the NTNU enabling technologies program to P.W.

AGC target, 48 ms maximum IT, centroid) triggered for the 15 most intense ions, with a 25 s dynamic exclusion. Charge exclusion was set to unassigned, 1 and greater than 5.

Proteins were identified and quantified by processing the MS data using Thermo Scientific Proteome Discoverer (Thermo Scientific) version 2.3/4. Preview version 2.3.5 from Protein Metrics Incorporated [START_REF] Kil | Preview: a program for surveying shotgun proteomics tandem mass spectrometry data[END_REF] was used to inspect the raw files to determine optimal search criteria.

Namely, following search parameters were used: enzyme specified as Trypsin with maximum two missed cleavages allowed; Acetylation of Protein N-terminal with Met-loss [START_REF] Bonissone | N-terminal protein processing: a comparative proteogenomic analysis[END_REF], Oxidation of Methionine and Deamidation of Asparagine/Glutamine as dynamic post-translational modification while Carbamidomethylation of Cysteine as static; Precursor mass-tolerance of 20 PPM while Fragment mass-tolerance of 0.02 Dalton. Spectrum file RC, PD's node, was set up to query the raw files against the Phaeodactylum tricornutum (strain CCAP 1055/1) downloaded from Uniprot (https://www.uniprot.org/proteomes/UP000000759) in December 2018 and internal contaminants database to recalibrate and detect features with the Minora node and Sequest [START_REF] Eng | An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database[END_REF] search engines available PD. For downstream analysis of these peptide-spectra-matches (PSM), both protein and peptide identifications/PSM false-discovery-rate (FDR) was set to 1% as high and 5% as medium confidence, thus only unique peptides with these confidences thresholds were used for final protein group identification and label the level of confidence respectively. Each protein group abundance was normalized by the total abundance of all identified peptides/PSM at the FDR mentioned earlier and scaled on all average with Precursor Ion Quantifier node of PD.

Protein isolation, SDS-PAGE and Western blot analysis

Cell harvesting, total protein isolation, determination of protein concentration, SDS-PAGE and Western blot analysis were performed as previously described [START_REF] Nymark | Loss of ALBINO3b insertase results in truncated lightharvesting antenna in diatoms[END_REF] using material from WT and cpsrp54 cultures acclimated to either LL or ML. Proteins were detected with the following antibodies: anti-D1 (AS05 084 Agrisera; 1:20000 dilution), anti-D2 (AS06 146 Agrisera; 1:5000), anti-AtpB (AS05 085, Agrisera; 1:4000) and anti-LHCF1-11 (1:1000) (kind gift from C.

Büchel, University of Frankfurt, Germany; [START_REF] Juhas | A novel cryptochrome in the diatom Phaeodactylum tricornutum influences the regulation of lightharvesting protein levels[END_REF]. Primary antibody incubation was performed overnight at 4°C for all antibodies. Secondary antibody incubation was performed for 2 h at room temperature using Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP conjugate (ThermoFisher, 1:10000). Proteins were detected with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) and the blots were imaged using a G:BOX ChemiXRQ gel doc system (Syngene).

Statistical analysis

The R package ROTS (reproducibility-optimized test statistic) was used for statistical analysis of the proteomics data aiming to identify differentially expressed proteins in cpsrp54-8 and cpsrp54-11 compared to WT after 6 h exposure to ML [START_REF] Suomi | ROTS: An R package for reproducibility-optimized statistical testing[END_REF]. ROTS employs a bootstrapping method to identify the proper t-test statistics based on the data. Prior to the differential expression analysis, the intensity counts were normalized across samples. Additionally, for the validity of the results, the homogeneity in pair-wise and group-wise variances was tested using an F-test and a pvalue of 0.05 as a significance threshold. Once normality and homogeneity in variance were ensured, two independent tests were performed; one for WT against cpsrp54-8 and one for WT against cpsrp54-11. The differential expression analysis was performed using log2 transformed values, while proteins with only one abundance value across the samples were omitted. For both tests, the number of bootstrapping and the number of top-ranked features for reproducibility optimization were set to 1000.

Two-way ANOVA with Dunnet's multiple comparison tests were carried out using GraphPad Prism Software (version 8.4.3) to determine if there were significant differences (p<0.05) between the pigment levels and photosynthetic parameters in cpsrp54 mutants compared to WT.

Accession numbers

The CpSRP54 gene has Draft ID Phatr2_35185 and NCBI accession number XM_002179577.

UniProt accession numbers for differentially expressed proteins discussed in the text are included in Table 2. UniProt accession numbers are provided for all proteins detected by the proteomics analyses and the data are deposited to the ProteomeXchange Consortium as described below in the Data availability statement.

Data availability statement

A complete submission of proteomics data including raw data (raw), peak-list (mzML), identifications (mzID), workflow (pdAnalysis) and annotated results (xlsx) has been deposited to the ProteomeXchange Consortium via the PRIDE [START_REF] Perez-Riverol | The PRIDE database and related tools and resources in 2019: improving support for quantification data[END_REF] partner repository with the dataset identifier PXD020167 and 10.6019/PXD020167. All other relevant information can be found within the manuscript and its supporting information.

Conflict of interest

The authors declare that they have no competing interests.

Supporting information


WORKSHOP REPORT

Open-access paleo databases are of the utmost importance to arrive at a regional, continental, or global understanding of long-term processes and investigate past dynamics of Earth systems. the European Pollen Database (EPD), created alongside the North American Pollen Database more than 25 years ago, serves as the base for numerous analyses of climate, vegetation and land-cover change, while also being an important data repository and educational tool (Fyfe et al. 2009). to function as an upto-date research tool, the database requires both the submission of new pollen data from the community as well as data stewardship and development of the database. the database is a community-wide effort, but there is an inherent risk of a division between data producers and data users. Group and plenary discussions focused on how to improve the database and make it more accessible to other communities, and we reviewed and simplified the rules and regulations. We also reviewed and reorganized the following EPD working groups: the taxonomy working group, coordinated by steffen Wolters, keeps track of synonym names in the database and is developing a hierarchical system based on morphology. Future activities will include non-pollen-palynomorphs.

the Chronologies working group has produced standardized age models for over 800 sites (Giesecke et al. 2014). Coordinated by thomas Giesecke, the group aims to provide the same type of models in addition to bayesian age models for the recently submitted sites.

  to overcome this separation and to generate new momentum in the development of the database, we called for an open EPD meeting and training workshop. Co-funded by PAGEs, it attracted 126 paleoecologists, at all career stages, from 24 countries. the meeting consisted of plenary lectures, training workshops, and group and plenary discussions. the lectures illustrated different research fields that use the database, such as climate and land-cover reconstructions (J. Guiot and M.-J. Gaillard), archeology (R. Fyfe), last glacial vegetation change (M. F. sanchez Goñi), nature conservation (R. Bradshaw), and the postglacial spread of plants (t. Giesecke). Lectures also addressed interactions with the Global Charcoal Database (B. Vannière & D. Colombaroli), problems developing age models beyond the radiocarbon timescale (C. tzedakis), and the new database infrastructure Neotoma (E. Grimm). seven parallel training workshops introduced participants to numerical problems in paleodata analyses and representation: (i) the data management and graphing software tilia and its interaction with Neotoma (E. Grimm); (ii) the use of the modern surface sample dataset in quantitative reconstructions (B. Davis & M. Zanon); (iii) quantitative vegetation reconstructions using the Landscape Reconstruction Algorithm (P. Kuneš & M. theuerkauf); (iv) charcoal software and database (B. Vannière & W. Finsinger); (v) working with the EPD using the data analysis software "R" (A. seddon & J. Chipperfield); and (vi) age modeling (t. Giesecke). Another workshop was dedicated to the identification of non-pollen-palynomorphs (B. Dietre & s. Wolters), which is a field of active development in Quaternary palynology. All workshops were well received by participants, who wished they could be organized on a regular basis.

Figure 1 :

 1 Figure 1: Development of the European Pollen Database since the open meeting in 2007. the number of sites and cores has doubled over the last nine years.